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Patterns of variation in Pacific silver fir (Abies amabilis [Dougl.] Forbes) on Vancouver Island Davidson, Roberta H. 1990

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PATTERNS OF VARIATION IN PACIFIC SILVER FIR (ABIES AMABILIS [DOUGL.] FORBES) ON VANCOUVER ISLAND  by Roberta B.Sc,  H.  Davidson  Simon F r a s e r U n i v e r s i t y ,  A T H E S I S SUBMITTED  1980  I N PARTIAL FULFILLMENT  OF T H E R E Q U I R E M E N T S  F O R THE DEGREE OF  DOCTOR OF P H I L O S O P H Y in THE F A C U L T Y OF GRADUATE S T U D I E S (Faculty  We a c c e p t  this  o fForestry)  thesis  to the required  as conforming standard  THE U N I V E R S I T Y OF B R I T I S H August,  COLUMBIA  1990  © R o b e r t a H. D a v i d s o n ,  1990  In presenting this thesis in partial fulfilment  of the  requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  Fbra  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT  This cone, fir  thesis describes  patterns  seed and s e e d l i n g c h a r a c t e r i s t i c s  (Abies  Island,  amabilis  B.C.  material  The  [Doug.] F o r b e s )  Cone  made a t e i g h t  collections  kept  locations during  f o r the  loci  conformed t o the assumptions  at least  AAT-2  inheritance,  although  distortion.  No l i n k a g e g r o u p s  intensity levels  other  than  selfing  evidence  were  estimates shown  method  o f the extent  todiffer  employed.  heterozygous  than  segregation  based  tree).  on a m u l t i l o c u s i nfive  suggests  o f seven  r e l a t e d matings  V a r i a t i o n among (0.725 < t  correlated with High  levels  seed  (extant)  trees  d i d v i a b l e embryos a n d  <  o f allozyme  within populations  of population  m  size  (95-98%)  differentiation  d e p e n d i n g upon t h e p a r t i c u l a r  Maternal  These  be e s t a b l i s h e d a t t h e  may b e o c c u r r i n g .  found t o e x i s t  loci  variants.  i no u t c r o s s i n g r a t e was e v i d e n t  were  were  o f Mendelian-type  could  (measured b y 1000-seed w e i g h t ) .  and  loci  d i s p l a y e d marked  of inbreeding,  andappears p o s i t i v e l y  variation  were  o f 1983 t o p r o v i d e  a v a i l a b l e (20 s e e d s p e r  and indirect  populations  Vancouver  by tree  two allozyme  o f o u t c r o s s i n g , were d e t e c t e d  populations  from  separate  o f 13 e n z y m e  found t o possess  estimate  sampled  silver  s e e d t i s s u e s o f 87 t r e e s , a n d s e v e n  from  Significant  ofPacific  the f a l l  were  sampling  i nseveral  study.  inheritance patterns  determined  1.0)  ofvariation  appeared  analytic more  populations  sampled  more g e n e t i c a l l y northern  A  on s o u t h e r n  Vancouver  sub-sample  provided  pollinated  behavior  Seed  Abies  eight  zero  growth  pregermination control of  Anomalous  germination  to  sub-optimal  o f mold.  of P a c i f i c  and germinating  from t h e f i r s t  stock  The e f f e c t  count  were grown  silver f i r  seeds  on a  part  on g r o w t h  of the germination  o f growth  i n a greenhouse  alongside  Population differences  of variance  of population  on h e i g h t  but declined t o Populations  rate of seedlings  among o p e n - p o l l i n a t e d  test  f o r measurement  f o r 29 weeks.  w e e k s was s i g n i f i c a n t  rate  open-  basis.  by t h e end o f t h e t e s t .  influence  and  of genetic  of seedlings  accounted f o r a considerable size.  of  was a t t r i b u t e d  by c o l l e c t i n g  Seedlings  production  at  fir.  o p e n - p o l l i n a t e d progeny  variables.  seed  degree  silver  i n production  Germinants  with  Vancouver  test  c o n d i t i o n s and p r o l i f e r a t i o n  family-by-family  each  d o r m a n c y was n o t p r o n o u n c e d  irrespective  a high  i n Pacific  may b e a c h i e v e d  on  Large family d i f f e r e n c e s i n germination  i n one p o p u l a t i o n  Improvement  provided  study.  suggesting  stratification  mid and southern  material f o r a germination  were d e t e c t e d ,  germination  sampled  c o n s i s t i n g o f two p o p u l a t i o n s ,  progeny  treatment,  appeared  Island.  among p o p u l a t i o n s . responses  Island  diverse than d i dpopulations  seven t r e e s , from northern, Island  Vancouver  had  as w e l l .  families  was  i n cone  and  of seedlings virtually negligible Variation i n statistically  significant  but  variation.  S i g n i f i c a n t population  in  root  in  t h i s and  the  f o r only  20%  other biomass v a r i a b l e s  Family  total were  detected  differences  were a t most 20%,  of v a r i a t i o n i n s e e d l i n g  families.  of the  differences  weight of h a r v e s t e d s e e d l i n g s .  majority  within  accounted  growth t r a i t s  with residing  V  TABLE OF CONTENTS P  ABSTRACT  viii  LIST OF FIGURES  xi  ACKNOWLEDGEMENTS  . . . . x i i  INTRODUCTION  1  Study O b j e c t i v e s  2.  MATERIAL COLLECTION AND PROCESSING  3.  ISOZYME INHERITANCE  5.  8 9 13  3.1  Introduction  13  3.2  M a t e r i a l s and Methods  17  3.3  R e s u l t s and D i s c u s s i o n  21  3.4 4.  e  v  LIST OF TABLES  1.1  9  i i  TABLE OF CONTENTS  1.  a  3.3.1  Enzymes C l a s s e d as Monomorphic  ...  21  3.3.2  Enzymes E x h i b i t i n g Polymorphism  ..  28  Conclusion  LINKAGE  40 42  4.1  Introduction  42  4.2  M a t e r i a l s and Methods  44  4.3  R e s u l t s and D i s c u s s i o n  47  4.4  Conclusion  53  MATING SYSTEM  55  5.1  Introduction  55  5.2  M a t e r i a l s and Methods  57  5.3  R e s u l t s and D i s c u s s i o n  62  5.3.1  62  A l l e l e Frequencies  vi Page 5.3.2 5.4 6.  8.  Rates  68  Conclusions  75  ESTIMATES OF ELECTROPHORETIC VARIATION, ITS STRUCTURE AND RELATIONSHIP TO THE MATING SYSTEM 6.1  Introduction  6.2  M a t e r i a l s and Methods  6.3  7.  Outcrossing  76 76 . ...  79  6.2.1  Sample c o l l e c t i o n and e l e c t r o p h o r e t i c assay  79  6.2.2  A n a l y t i c methods  80  Results  86  6.3.1  A l l e l i c variation  86  6.3.2  D i s t r i b u t i o n of v a r i a t i o n  91  6.3.3  Mating system e f f e c t s on v a r i a t i o n .  97  6.4  Discussion  101  6.5  Conclusions  109  SEED GERMINATION  110  7.1  Introduction  110  7.2  M a t e r i a l s and Methods  114  7.2.1  C o l l e c t i o n and t e s t i n g methods  114  7.2.2  A n a l y t i c methods  116  7.3  R e s u l t s and D i s c u s s i o n  120  7.4  Conclusions  136  WIND-POLLINATED PROGENY NURSERY-STAGE GROWTH  ..  138  8.1  Introduction  138  8.2  M a t e r i a l s and Methods  139  8.2.1  Test establishment  and c u l t u r e  8.2.2  Measurement and a n a l y s i s  ...  139 140  vii  Page 8.3  Results 8.3.1  9. 10.  145 Geographic and maternal t r e e variation  147  8.3.2  Geographic i n f l u e n c e s on seed variables  148  8.3.3  Maternal and seed r e l a t i o n s h i p s  8.3.4  Relationships seedlings  ..  among seeds and  148 151  8.4  Discussion  163  8.5  Conclusions  172  SUMMARY AND CONCLUSIONS  173  LITERATURE CITED  176  APPENDICES 1.  S c i e n t i f i c names o f t r e e s p e c i e s  202  2.  B u f f e r s , s o l u t i o n s , g e l p r e p a r a t i o n , running c o n d i t i o n s and s t a i n r e c i p e s f o r h o r i z o n t a l starch gel electrophoresis  203  M u l t i p l e r e g r e s s i o n equations i n v o l v i n g geographic l o c a t i o n o f p o p u l a t i o n s as predictor variables  207  3.  viii LIST OF  TABLES  Table 1.1 2.1  3.1  3.2 4.1  Page Number of seedlings of Abies spp. grown or p l a n t e d i n B.C. from 1978 t o 1986.  5  L o c a t i o n s of p o p u l a t i o n s of A. amabilis from which cone c o l l e c t i o n s were made i n f a l l 1983.  10  Summary of enzyme systems s t u d i e d , nomenclature and numbers of l o c i obtainedfrom e l e c t r o p h o r e s i s .  20  Segregation loci.  31  p a t t e r n s f o r seven v a r i a b l e  R e s u l t s of c h i - s q u a r e t e s t s f o r the d e t e c t i o n of two-point l i n k a g e .  48  5.1  A l l e l e f r e q u e n c i e s f o r maternal o u t c r o s s i n g p o l l e n gene p o o l s .  63  5.2  R a t i o s of d i s c e r n i b l y o u t c r o s s e d (DOC) embryos t o embryos p o s s e s s i n g maternal (homozygous) genotype f o r t h r e e v a r i a b l e loci.  66  S i n g l e - and m u l t i - l o c u s estimates of o u t c r o s s i n g r a t e (± 95% c o n f i d e n c e intervals).  69  P o l l e n a l l e l e f r e q u e n c i e s d e t e c t e d at s i x l o c i i n e i g h t p o p u l a t i o n s of A. amabilis.  87  A c t u a l and " e f f e c t i v e " numbers of per l o c u s .  89  5.3  6.1 6.2 6.3 6.4  6.5  6.6  and  allele  Mean expected h e t e r o z y g o s i t i e s f o r a d u l t and progeny gene p o o l s .  90  Estimates of g e n e t i c d i s t a n c e s and corresponding geographic d i s t a n c e s between p a i r s of A. amabilis stands.  92  Gene d i v e r s i t y estimates f o r s i x and t h i r t e e n l o c i i n e i g h t stands of A. amabilis.  94  A l l e l i c differentiation indices for six variable l o c i .  96  ix Page 6.7 6.8  6.9  Fixation indices gene p o o l s .  f o r maternal  and progeny 98  Comparison o f f i x a t i o n i n d i c e s c a l c u l a t e d u s i n g i n d i r e c t and d i r e c t methods f o r maternal and progeny gene p o o l s .  100  Comparison between amounts of p o p u l a t i o n d i f f e r e n t i a t i o n d e t e c t e d by two d i f f e r e n t measures (G and 5) at s i x l o c i .  104  Average G Abies spp.  106  ST  6.10 7.1a  7.1b  7.2  7.3a  7.3b 7.4  7.5  7.6 7.7  8.1  sx  v a l u e s determined f o r s e v e r a l  R e s u l t s of ANOVA of germination c a p a c i t y (GC) of s t r a t i f i e d seeds from s i x p o p u l a t i o n s of A. amabilis.  120  Apportionment of t o t a l v a r i a t i o n and expected mean squares from ANOVA d e s c r i b e d i n Table 7.1a.  122  T o t a l number of germinants vs. nongerminants, i n d i c a t i n g treatment and cabinet e f f e c t s .  123  Apportionment of v a r i a t i o n f o r GC and germination value (GV) i n u n s t r a t i f i e d seeds.  126  Apportionment of v a r i a t i o n i n s t r a t i f i e d seeds.  f o r GC and  GV 127  Average GC i n s i x p o p u l a t i o n s of A. amabilis, f o r u n s t r a t i f i e d and s t r a t i f i e d seeds.  128  Average GV i n s i x p o p u l a t i o n s of A. amabilis , f o r s t r a t i f i e d and u n s t r a t i f i e d seeds.  129  Mean GC and GV f o r i n d i v i d u a l t r e e s i n a s i n g l e p o p u l a t i o n of A. amabilis.  131  L i n e a r c o r r e l a t i o n s of average p o p u l a t i o n seed s i z e with GC, GV and l a t i t u d e of origin.  133  L e v e l of sampling, number of measurements and v a r i a b l e s measured i n nursery stage growth of A. amabilis.  141  C o r r e l a t i o n s among p o p u l a t i o n , t r e e and v a r i a b l e s .  maternal  Apportionment o f v a r i a t i o n f o r mean cone length, seed weight and cotyledon number i n s i x p o p u l a t i o n s of A. amabilis. Summary s t a t i s t i c s variables.  f o r cone seed  C o r r e l a t i o n s and v a r i a n c e p a t t e r n s f o r seed and s e e d l i n g growth v a r i a b l e s . Summary s t a t i s t i c s variables.  of s e e d l i n g growth  R e s u l t s o f ANOVA of cumulative and incremental s e e d l i n g height growth over 2 weeks. C o r r e l a t i o n s and v a r i a n c e p a t t e r n s f o r seed and s e e d l i n g form v a r i a b l e s . C o r r e l a t i o n s t r u c t u r e among p o p u l a t i o n , maternal t r e e and s e e d l i n g form variables. Ranking o f p o p u l a t i o n mean value f o r v a r i a b l e s d e s c r i b i n g s e e d l i n g form at 29 weeks.  xi LIST OF FIGURES Figure 1.1 2.1 , 3.1 6.1  8.1  Page D i s t r i b u t i o n of P a c i f i c western North America.  silver  f i r in  L o c a t i o n of e i g h t sampled p o p u l a t i o n s Vancouver I s l a n d , B r i t i s h Columbia.  2 on 11  Band p a t t e r n s of seven polymorphic l o c i d e t e c t e d by s t a r c h g e l e l e c t r o p h o r e s i s .  29  Dendrogram r e p r e s e n t i n g g e n e t i c d i s t a n c e s among e i g h t p o p u l a t i o n s of P a c i f i c s i l v e r fir.  93  R e l a t i o n s h i p s of v a r i a b l e s examined i n w i n d - p o l l i n a t e d progeny of 42 f a m i l i e s from s i x l o c a t i o n s .  146  xii ACKNOWLEDGEMENTS I t i s with enormous r e s p e c t  and  g r a t i t u d e I acknowledge  the c o n t r i b u t i o n s of the members of my Drs.  D.G.W. Edwards, Y.A.  and B.J. my  van  0.  Dr.  this dissertation. a d v i s o r Dr. The  El-Kassaby, D.T.  der Kamp, and  supervisor,  J.W.  the experiences and  Sziklai,  J.R.  Maze  i n s i g h t s of  throughout the p r e p a r a t i o n  Thanks are a l s o extended t o my  Wilson, f o r h i s c o n t i n u i n g  the B.C.  of m a t e r i a l was Glenn and  M i n i s t r y of F o r e s t s  i n v a l u a b l e , as was  colleagues  the volume of seeds  at Reid,  encouragement.  f o r the t h e s i s and,  collection  in  Lachlan  processing  required.  along  Rob  Scagel,  who  establish objectives  with Mishtu Banerjee, p a r t i c i p a t e d  i n many d i s c u s s i o n s on how Bohm, who  i n the  C o l l i n s nursery,  arranged a f i e l d t r i p which helped me  best  to reach them.  supported some p r e l i m i n a r y  I a l s o thank work on  needle  flavonoids. I am  g r a t e f u l to Lee  a s s i s t a n c e and work.  of  program  the a s s i s t a n c e of  Thanks are owed to Jack Maze and  B.A.  Lester,  generous support of s e v e r a l f o r e s t product  companies and  Dr.  t h e s i s committee,  Lee  f r i e n d s h i p throughout the course of  and her husband, N e i l ,  accommodation and occasions,  Charleson f o r both her t e c h n i c a l  L e i l a and  opened heart  and  also graciously  this provided  Yousry El-Kassaby, on numerous home to  me.  Many thanks are due t o George Edwards, Doug T a y l o r and Frank P o r t l o c k o f the Canadian F o r e s t r y S e r v i c e f o r h e l p i n mounting  t h e g e r m i n a t i o n and progeny t e s t s a t PFC, V i c t o r i a  and V l a d K o r e l u s and the s t a f f of CP F o r e s t P r o d u c t s f o r e s t r y c e n t r e i n Saanich f o r s u p p o r t i n g much o f my work. I am e s p e c i a l l y g r a t e f u l t o Joy Parkinson f o r her p a t i e n c e and d e d i c a t i o n throughout the isozyme a n a l y s e s . S t a t i s t i c a l a d v i c e was r e c e i v e d from Mishtu Banerjee, Yousry El-Kassaby, D r s . H.-R. P.L. M a r s h a l l , M. Penner w e l l as computing  and K. R i t l a n d , and Rob S c a g e l ; as  a s s i s t a n c e from Rob Davidson,  Emmanuel, Dermot McCarthy, all  Gregorius, M. G r i e g , A. Kozak,  John  Steve M c G i l l i v r a y and Barry Wong;  o f i t much a p p r e c i a t e d . I cannot adequately acknowledge here the c o n t r i b u t i o n s  of my f a m i l y and f r i e n d s , who gave f r e e l y of t h e i r time t o all  aspects o f t h i s work, from d i s s e c t i n g cones t o  b a b y s i t t i n g my daughter. Quay, Pat Johnson,  The t h e s i s was ably typed by Patsy  and, at the e l e v e n t h hour, by L i s a  A l l e n b a c h and Yolanda M c G i l l i v r a y .  I c o u l d not have done  any o f t h i s without the u n f a i l i n g support of my husband, Rob. the  T h i s t h e s i s r e p r e s e n t s the e f f o r t s of many, however, mistakes remain my own.  1  1.  Pacific Forbes)  1  s i l v e r o r a m a b i l i s f i r (Abies amabilis  (Figure 1.1) from the southern end o f t h e  A l a s k a panhandle  (56°N) t o northwestern C a l i f o r n i a  i s restricted  P a c i f i c Ocean  1982,  the  seldom found more than 300 km from  (Schmidt 1957, F o w e l l s 1965, Packee et al.  W o r r a l l 1983).  development  (42°N)  i n i t s eastward d i s t r i b u t i o n t o a  r e l a t i v e l y narrow band, the  [Dougl.]  occurs over a l a r g e p o r t i o n o f the west coast o f  North America  but  INTRODUCTION  The s p e c i e s reaches i t s g r e a t e s t  and commercial p r o d u c t i v i t y on the west s i d e s o f  Olympic and Cascade Mountains, the f o o t h i l l s o f the  Columbia R i v e r and the west coast of Vancouver (Handley  Island  1982).  In B r i t i s h Columbia, P a c i f i c  s i l v e r f i r i s found at  e l e v a t i o n s from s e a - l e v e l t o 1500 m at the 49th p a r a l l e l and to  300 m at i t s n o r t h e r n l i m i t  (Schmidt 1957).  r e g i o n o f g r e a t e s t p r o d u c t i v i t y i t i s codominant western hemlock  (Pojar 1982).  Pacific  In the with  s i l v e r f i r grows t o  h i g h e r e l e v a t i o n s than does D o u g l a s - f i r , grand f i r , S i t k a spruce, western hemlock,  western r e d cedar and A l a s k a  y e l l o w - c e d a r but i s not found as h i g h as mountain hemlock or s u b a l p i n e f i r (Schmidt 1957).  Other t r e e s p e c i e s a r e mentioned i n the t e x t only. S c i e n t i f i c names a r e l i s t e d i n A p p e n d i x 1  by common 1.  name,  2  125°  F i g u r e 1.1  120°  115°  D i s t r i b u t i o n o f P a c i f i c s i l v e r f i r i n western North America ( a f t e r F o w e l l s , 1965).  3  Shade t o l e r a n c e of P a c i f i c s i l v e r f i r i s h i g h e s t f o r e s t t r e e s p e c i e s , i n B.C.,  of a l l  which c o n t r i b u t e s t o r e l a t i v e l y  dense s t o c k i n g because o f t h e low space requirement of the s p i r e - s h a p e d crown  ( K r a j i n a et a i . 1982).  Pacific  silver  f i r t o l e r a t e s and even r e q u i r e s l a r g e q u a n t i t i e s of water, as much as 6650 mm  annual mean t o t a l p r e c i p i t a t i o n ( K r a j i n a  et al. 1982), however s i t e s must be s u f f i c i e n t l y w e l l drained  f o r the s p e c i e s t o produce good growth  1957).  Schmidt  (1957) and Packee et al.  (Schmidt  (1982)  emphasize  the a b i l i t y of the s p e c i e s t o occupy a wide range of c l i m a t i c and s i t e c o n d i t i o n s , s l o p e s may  outgrow D o u g l a s - f i r  other r e p o r t s 1982)  and on c o o l ,  (Handley 1982,  north-facing  (Thornburgh 1969).  Several  Husted and K o r e l u s 1982,  a t t e s t t o the p r o d u c t i v i t y of P a c i f i c  Pojar  s i l v e r f i r , due  t o h i g h e r s t o c k i n g d e n s i t i e s and b e t t e r form but, by v i r t u e of i t s lower wood d e n s i t y , to D o u g l a s - f i r . used i n general  i t produces i n f e r i o r saw timber  Nevertheless, P a c i f i c construction  corewood  i n plywood p r o d u c t i o n  pulpwood  species  Until  s i l v e r f i r i s widely  (known as "hem-bal"), as a and i s a h i g h - y i e l d i n g  (Handley 1982).  r e c e n t l y the importance of P a c i f i c  a commercial s p e c i e s  i n c o a s t a l B.C.  has been  s i l v e r f i r as overshadowed  by the d e s i r a b i l i t y and a v a i l a b i l i t y of D o u g l a s - f i r . S t a t i s t i c s from the B.C.  M i n i s t r y of F o r e s t s Annual Report  (1985-86) show t h a t i n the c o a s t a l p o r t i o n o f the Vancouver Region the stumpage p r i c e f o r Abies  species  ( i n c l u d i n g grand  4  and  subalpine  i s about 50%  f i r and  r e f e r r e d t o c o l l e c t i v e l y as  of t h a t r e c e i v e d f o r D o u g l a s - f i r .  average timber s a l e p r i c e s i n c o a s t a l B.C. 1915-1979 ( c a l c u l a t e d from Handley 1982) 0.5:1.0 f o r balsam t o D o u g l a s - f i r , placed  on these s p e c i e s has  the past  70 years.  f o r the  was  suggesting  In the past  (Husted and Korelus 1982,  not be  (Table  Green et al.  million. reflects  The not  seedlings  1984), or  will  accessed stands of lowers i l v e r f i r i s being industry.  i n the sharp r i s e i n the number of  1.1),  value  or brush-prone  more f a v o r a b l y by the c o a s t a l f o r e s t  s i l v e r f i r seedlings 1986  t h a t the  s i l v e r f i r as a s u i t a b l e  e l e v a t i o n s p e c i e s are logged P a c i f i c  T h i s i s evident  period  (Reuter 1973).  As more and more e a s i l y  considered  grown i n B.C.  nurseries  increased  sowing of P a c i f i c  Pacific  from 1978  from l e s s than 386,000 i n 1978  to  3.4  silver f i r  f o r r e f o r e s t a t i o n but  a l s o represents on the  s i n c e 1966  as a measure t o c o n t r o l the spread of balsam  460,  Plant  Protection  that had  nursery  of a l l t r u e f i r s  Council  i n B.C.,  2  a response  production  in  to  only the apparent need f o r s u i t a b l y adapted  t o the removal of a province-wide b a n  Order  of  approximately  where D o u g l a s - f i r p l a n t a t i o n s are not  successful  ratio  decade, however, i n c r e a s i n g  t r e e f o r r e f o r e s t a t i o n on h i g h e r - e l e v a t i o n  particularly  The  remained v i r t u a l l y the same f o r  a t t e n t i o n has been p a i d t o P a c i f i c  sites  "balsam")  Act,  B.C.  been i n e f f e c t  Reg.  58/66.  5  Table 1.1 Number of Abies spp. s e e d l i n g s grown or p l a n t e d i n B r i t i s h Columbia from 1978 t o 1986.  Year  Thousands  o f s e e d l i n g s grown® or planted®  1986  3, 400  1985 *  3,700  1984  1,400  1983  1, 600  1982  2, 900  1981  3, 400  1980  2, 245  1979  1,984®  1978  396®  ® Information courtesy of M. Pelchat, B.C. Ministry of Forests, S i l v i c u l t u r e Branch, V i c t o r i a , B.C. for 1981-1986. @ Information obtained from B.C. Reports 1978-1980.  @ Includes A. grandis  woolly aphid  and A.  (Adelges piceae  Ministry of Forests Annual  lasiocarpa.  Ratz.).  The present  r e g u l a t i o n s , which are under r e v i e w , attempt t o l i m i t the 3  infestation 3  4  of balsam woolly aphid by r e s t r i c t i n g movement  Order i n Council 44, Plant Protection Act, B.C.  Reg. 7/77.  Peter H a l l , Entomologist, B.C. Ministry of Forests, Protection Branch, V i c t o r i a , B.C. 4  6  of Abies  s p e c i e s grown w i t h i n a s p e c i f i e d quarantine  and e n s u r i n g proper treatments  zone  t o c o n t r o l the aphid are  a p p l i e d at the n u r s e r y . The  r o l e o f P a c i f i c s i l v e r f i r i n f u t u r e f o r e s t s of  c o a s t a l B.C. appears promising, the s p e c i e s a r e l a c k i n g .  but b a s i c g e n e t i c s t u d i e s of  A nine-provenance study was  e s t a b l i s h e d at two t e s t s i t e s on Vancouver I s l a n d i n 1980 by the M i n i s t r y of F o r e s t s .  [To date,  one, t h r e e and s i x year  h e i g h t data have been c o l l e c t e d but not e v a l u a t e d ( C C . Ying, p e r s . comm., February  1990)].  A five-provenance  test  r e p l i c a t e d at four e l e v a t i o n s near Squamish, B.C. was s e t up i n 1978 and 1979 by  the U n i v e r s i t y of B.C. i n c o - o p e r a t i o n  w i t h the M i n i s t r y o f F o r e s t s . assessed  These t r i a l s have been  f o r v a r i a t i o n i n b u d - f l u s h i n g dates and t h r e s h o l d  temperatures  (Worrall 1983).  temperatures were found suggesting  Significantly  lower t h r e s h o l d  i n high e l e v a t i o n provenances,  a d a p t a t i o n t o a short growing season.  P a c i f i c s i l v e r f i r has been the s u b j e c t o f s e v e r a l recent systematic s t u d i e s . v a r i a t i o n i n needle  et al. (1979a) looked at  and twig anatomy among f i v e  growing i n northwestern variability  Parker  B.C. (above 54°N).  populations  Monoterpene  i n c o r t i c a l o l e o r e s i n has been assessed by  Z a v a r i n et al. (1973) as a means of d e t e c t i n g  geographic  d i f f e r e n c e s i n the s p e c i e s , and l e a f and twig o i l terpenes were used t o estimate  r e l a t i v e v a r i a t i o n among t r e e s and  p o p u l a t i o n s f o r 19 l o c a t i o n s i n the P a c i f i c Northwest (von  7  Rudloff  and  Hunt 1977).  i d e n t i f i e d f o r use P a r k e r et al.  Needle f l a v o n o i d s have been  as p o t e n t i a l taxonomic c h a r a c t e r s  (1979b).  Somewhat more work on the ecology and  silvical  c h a r a c t e r i s t i c s of P a c i f i c s i l v e r f i r has been (Schmidt 1957, 1978, al.  Dimock 1958,  Murray and 1982,  Martin  K r a j i n a 1969,  1982,  1979,  1985)  and the b i o g e o c l i m a t i c  Green et al.  K r a j i n a et al.  a l l u d e to or d e s c r i b e which the s p e c i e s  1984).  important t r a i t s .  The  in site  variability o n l y one  growth and  conclusions  work  has  s u r v i v a l of elevations has  been  regarding  i n s e e d l i n g performance were r e s t r i c t e d because  seed source was  used.  s i l v e r f i r were p a r t of a study by Leadem  as c h a r a c t e r i s t i c a l l y other Abies  species.  (1982)  "poor" seed g e r m i n a b i l i t y i n In Leadem's study,  d i f f e r e n c e s were found t o a f f e c t germination in Pacific silver f i r .  Pacific  (1986) which  examined s e v e r a l f a c t o r s a f f e c t i n g what Edwards  t h i s and  studies  component of v a r i a t i o n i n  Seed source e f f e c t s on the germination of  described  Krumlik  conditions  i n the Cascade Range northwest of S e a t t l e , WA, (1978) but  Packee et  (Packee 1974,  P a c i f i c s i l v e r f i r on d i f f e r e n t h a b i t a t s at two  i n v e s t i g a t e d by Kotar  and  units i t  to date, l i t t l e  been done t o determine the g e n e t i c adaptively  1982,  Many of these  the v a r i a b i l i t y  occupies but,  published  Kotar 1,972  Treat  o c c u p i e s have been w e l l c h a r a c t e r i z e d et al.  by  seedlot  significantly  8  1.1  Study O b j e c t i v e s Given t h e i n c r e a s e d u t i l i z a t i o n o f P a c i f i c s i l v e r f i r  f o r r e f o r e s t a t i o n and t h e l a c k o f knowledge r e g a r d i n g t h e nature and extent o f v a r i a t i o n i n t h e s p e c i e s , a c o l l e c t i o n of cones from i n d i v i d u a l t r e e s was made i n t h e f a l l t o p r o v i d e m a t e r i a l f o r the present study.  o f 1983  The o v e r a l l g o a l  of t h e p r o j e c t was t o e l u c i d a t e p a t t e r n s o f v a r i a t i o n i n s e v e r a l b i o c h e m i c a l , m o r p h o l o g i c a l and p h y s i o l o g i c a l c h a r a c t e r s o f P a c i f i c s i l v e r f i r sampled from Vancouver Island. (i)  The s p e c i f i c o b j e c t i v e s were: t o determine  t h e mode o f i n h e r i t a n c e and l i n k a g e  of e l e c t r o p h o r e t i c a l l y d e t e c t a b l e enzyme l o c i , (ii)  t o estimate parameters o f t h e mating system,  ( i i i ) t o estimate indices of genetic v a r i a t i o n using allozyme (iv)  polymorphisms,  t o estimate t h e extent o f g e n e t i c c o n t r o l  over  germination parameters, and (v)  t o d e s c r i b e h a l f - s i b progeny performance over one growing season and t o examine v a r i a b l e i n t e r r e l a t i o n s h i p s , p a r t i c u l a r l y between parent and progeny and c o r r e l a t i o n w i t h geographic  variables.  maternal  2.  MATERIAL COLLECTION AND  PROCESSING  C o l l e c t i o n s of cones from e i g h t p o p u l a t i o n s  distributed  on Vancouver I s l a n d from 49"18' t o 50°42' l a t i t u d e and t o 945 m a l t i t u d e September 1983.  (Table 2.1)  were made i n August  L o c a t i o n s are shown i n F i g u r e  P o p u l a t i o n s w i l l be r e f e r r e d to by t h e i r designation.  150  and  2.1.  "letter"  P o p u l a t i o n s A, B and C each c o n s i s t e d of e i g h t  t r e e s , p o p u l a t i o n F - 10 t r e e s , H, N and R - 13 t r e e s and - 17 t r e e s . trees  The  W  c r i t e r i o n of 30 m s p a c i n g among sampled  (Lines 1967)  was  adhered to f o r p o p u l a t i o n s A, F and  W  where cones were c o l l e c t e d from the ground, however the remaining  sampling  was  done using a cone rake suspended from  a h e l i c o p t e r , and the minimum spacing c o u l d not be However, P a c i f i c s i l v e r f i r was  present  as a codominant  western hemlock i n each of the f i v e stands i n a l l cases was  and the age  with class  mature(150+ y e a r s ) , so i t i s l i k e l y t h a t  s u f f i c i e n t d i s t a n c e between sampled t r e e s was these  verified.  obtained i n  populations. The  c o l l e c t i o n s were made over a four-week p e r i o d , each  p o p u l a t i o n monitored f o r r i p e n e s s by f i e l d Nevertheless, maturity,  t h e r e was  personnel.  a high degree of v a r i a b i l i t y  i n cone  among p o p u l a t i o n s , among t r e e s w i t h i n a p o p u l a t i o n  and even w i t h i n i n d i v i d u a l t r e e s .  Cones were kept  bags at 4°C u n t i l a l l c o l l e c t i o n s were o b t a i n e d .  i n mesh They were  Table  2.1  Location of populations (September 1983) .  Collection site  Reference code  of Pacific  silver  f i r from which cones were c o l l e c t e d  Average s l e v a t i o n (m)  Average age (yr)  f o r present  Biogeoclimatic designation®  study  during  a four-week p e r i o d  Collection Agency®  Latitude  Longitude  A  49'  18'  125*  22'  300  150+  CWH  B®  49-  57'  126*  25'  300  150+  CWH  bl  Canadian F o r e s t  Products  c®  50"  03'  126*  20'  500  150 +  CWH  bl  Canadian F o r e s t  Products  F  48-  39'  124-  06'  710  150+  CWH  Hathaway C r e e k  H®  50*  34'  45"  127"  43'  212  150+  CWH  NE62 (Holberg  N®  50-  43'  30"  128*  0'  215  Ronning Creek  R®  50-  36'  30"  128*  11'  275  Mystery  W  48"  48'  15"  128"  09'  Taylor  River  Sebalhall Maquilla Fleet  Creek Creek  River  Inlet)  Creek  G r e e n e t al.  30"  30"  45"  15"  625  (1984) a n d R.N. G r e e n , R e s e a r c h P e d o l o g i s t ,  Company names a t t h e t i m e o f c o l l e c t i o n , Helicopter collection;  remaining  sites  MacMillan  b3  P a c i f i c Forest  Products  bl  Western F o r e s t  Products  150+  CWHdl  Western F o r e s t  Products  150 +  CWHdl  Western F o r e s t  Products  CWHbl  B.C.  40  b4  B.C. M i n i s t r y o f F o r e s t s , p e r s .  Forest  comm.  September 1983.  collected  f r o m t h e ground  Bloedel  ( t r e e s topped u s i n g  rifle  o r climbed).  Products  Figure  2.1  Location of eight sampled populations s i l v e r f i r on V a n c o u v e r I s l a n d , B.C.  of  Pacific  then a i r - d r i e d at 12 t o 20°C f o r two weeks, d u r i n g time an average o f e i g h t i n t a c t cones were randomly  which selected  f o r 79 t r e e s from which t h e r e were s u f f i c i e n t cones, t o enable seed y i e l d s p e r cone and m o r p h o l o g i c a l measures t o be made. The hand s e p a r a t i o n of seeds from cone s c a l e s and y i e l d a n a l y s e s u s i n g S o f t e x - r a y procedures were c a r r i e d out at the G.S. A l l e n F o r e s t G e n e t i c s - Tree Seed L a b o r a t o r y , F a c u l t y of F o r e s t r y , U n i v e r s i t y of B.C.  Seeds from the remaining  cones were e x t r a c t e d u s i n g the f a c i l i t y A l d e r g r o v e , B.C. seed samples  at Reid, C o l l i n s i n  These seeds were hand-dewinged and f i l l e d  f o r weight d e t e r m i n a t i o n , e l e c t r o p h o r e t i c  procedures and a germination/progeny t e s t were o b t a i n e d u s i n g a vacuum s e p a r a t i o n apparatus (Edwards  1979).  Care  was e x e r c i s e d at a l l stages of seed h a n d l i n g t o a v o i d damage t o the seed coats, as b u r s t i n g of r e s i n v e s i c l e s has been i m p l i c a t e d i n reducing g e r m i n a b i l i t y of Abies ( K i t z m i l l e r et a i . 1975).  species  13  3.  3.1  ISOZYME INHERITANCE  Introduction Most, i f not a l l ,  economically  important  c h a r a c t e r i s t i c s of f o r e s t t r e e s are known to be i n h e r i t e d quantitatively-  These t r a i t s are p o l y g e n i c  l a r g e numbers of l o c i may  not  characters  Phenotypic v a r i a t i o n i n such  as stem height,  size provide  the raw  Estimates  with  i n v o l v e d and where a l l genes may  c o n t r i b u t e e q u a l l y to t h e i r e x p r e s s i o n  A l l a r d 1965).  program.  i n nature,  or  (Wehrhahn  polygenic  wood d e n s i t y and branch angle  material for a forest tree of the extent  to which observed i s genetic i n o r i g i n  thus manipulable though breeding  have t r a d i t i o n a l l y come  from provenance and progeny t e s t s and (Libby et al.  long generation i n t e n s i v e data  and  breeding  variation in quantitative t r a i t s  experiments  and  1969).  common garden  These are, because of  time of c o n i f e r s , o f t e n slow and c o l l e c t i o n methods  and  (Mitton  the  labour-  1983).  F o r e s t g e n e t i c s and t r e e improvement programs have b e n e f i t t e d from advances made i n g e n e t i c analyses  of  agronomic s p e c i e s such as maize, as w e l l as animal ( i n c l u d i n g human) g e n e t i c s t u d i e s . decades isozyme analyses,  For the past  three  i n v o l v i n g the e l e c t r o p h o r e t i c  s e p a r a t i o n of enzyme v a r i a n t s  (Smithies  1955)  combined  histochemical  s t a i n i n g techniques  have p r o v i d e d  an extremely u s e f u l set of molecular  with  (Hunter and Markert 1957), marker  14  loci  t o a l l these f i e l d s o f study.  F e r e t and Bergmann  (1976) d e f i n e d isozymes as e l e c t r o p h o r e t i c a l l y d e t e c t a b l e enzyme v a r i a n t s which possess i d e n t i c a l or s i m i l a r c a t a l y t i c activities.  Once t h e i n h e r i t a n c e p a t t e r n  f o r a g i v e n enzyme  l o c u s i s e s t a b l i s h e d , isozymes may be i n f e r r e d t o as allozymes  (= a l l e l i c  Allozyme l o c i  isozyme).  are c h a r a c t e r i z e d by t h e i r  "Mendelian" i n h e r i t a n c e p a t t e r n s , (heterozygotes  codominant  single-gene, expression  are d i s t i n g u i s h a b l e from homozygotes,  thus,  genotype i s i n f e r r e d d i r e c t l y from phenotype) and t h e i r occurrence i n a wide range o f animal and p l a n t ( H a r t l 1980).  Their u t i l i t y  species  as markers o f gene f u n c t i o n and  v a r i a t i o n i s r e f l e c t e d i n the dozens o f p o p u l a t i o n - b a s e d s t u d i e s o f enzyme polymorphisms which e x i s t  (reviewed by  Selander 1976, G o t t l i e b 1979, Nevo 1978, Hamrick e t al. 1979)  and i n the numerous b r e e d i n g a p p l i c a t i o n s i n f o r e s t  trees  (reviewed by Adams 1983, Paule 1990). P r i o r t o t h e advent o f molecular markers, g e n e t i c i s t s  r e l i e d upon simply  i n h e r i t e d m o r p h o l o g i c a l t r a i t s such as  c o l o u r mutants i n attempts t o a s s o c i a t e marker l o c u s  with  quantitative t r a i t  Single-  v a r i a t i o n (Edwards e t al. 1987).  l o c u s m o r p h o l o g i c a l v a r i a t i o n i s r e l a t i v e l y uncommon i n gymnosperms vegetative genes  (Mitton 1983).  The use o f monoterpene l e v e l s i n  t i s s u e , a l s o shown t o be c o n t r o l l e d by s i n g l e  (Adams 1983), i s hindered  by cumbersome a n a l y t i c a l  15  techniques and complicated by dominance i n t h e i r  expression  ( S q u i l l a c e et al. 1980). Isozyme marker l o c i have been used t o s u c c e s s f u l l y a s s o c i a t e q u a n t i t a t i v e l y i n h e r i t e d t r a i t s i n tomato (Tanksley et al. 1982, Tanksley and Hewitt 1988) and maize (Edwards et al. 1987, Stuber et al. 1987) and a newer c l a s s of m o l e c u l a r  (DNA) markers known as r e s t r i c t i o n fragment  l e n g t h polymorphisms (RFLPs) have a l s o been used t o d e t e c t l i n k e d genes c o n t r o l l i n g q u a n t i t a t i v e v a r i a t i o n i n tomato (Osborn et al. 1987, Tanksley and Hewitt 1988) and maize (Edwards et al., unpublished, c i t e d i n H e l e n t j a r i s 1987). G u s e l l a et al.  (1983) r e p o r t e d a polymorphic DNA  l i n k e d t o Huntington's d i s e a s e i n humans. model f o r u t i l i z i n g  marker  In f a c t , the  RFLPs t o c o n s t r u c t g e n e t i c l i n k a g e s and  l i n k a g e maps i n p l a n t s p e c i e s stemmed from human g e n e t i c studies and  ( B o t s t e i n et al. 1980, H e l e n t j a r i s 1987).  Hewitt  (1988) suggest t h a t once s u f f i c i e n t l y  Tanksley tight  l i n k a g e between genes c o n t r o l l i n g a molecular marker and a quantitative t r a i t  locus  (QTL) i s found, then the  chromosomal segment with the d e s i r e d QTL can be t r a n s f e r r e d into different  l i n e s or v a r i e t i e s .  Helentjaris  (1987)  p r e d i c t s the e v e n t u a l c l o n i n g of such l o c i by chromosome walking or jumping (Poustka et al. 1987)  techniques.  Such advancements i n the breeding of agronomic s p e c i e s have been a i d e d by f e a t u r e s of the organisms' b i o l o g y which have enabled r a p i d c h a r a c t e r i z a t i o n of t h e i r genomes.  The  16  development of h i g h l y i n b r e d l i n e s ,  especially efficient  in  s e l f - p o l l i n a t i n g s p e c i e s such as tomato, and the presence of chromosomal a b e r r a t i o n s  such as d e f i c i e n c i e s ( i . e . ,  n u l l i s o m i c s i n maize) or e a s i l y i d e n t i f i a b l e t r a n s l o c a t i o n s have r e s u l t e d i n w e l l developed l i n k a g e maps f o r these species.  In c o n t r a s t , the c y t o g e n e t i c s  of most f o r e s t t r e e  s p e c i e s are not w e l l known, karyotypes are c h a r a c t e r i z e d and  poorly  c o n s t r u c t i o n of isozyme-based l i n k a g e maps  only b a r e l y begun.  To date even the a l l o c a t i o n of marker  l o c i t o p a r t i c u l a r chromosomes has  not been p o s s i b l e .  With c u r r e n t f o r e s t r y r e s e a r c h being  s t e e r e d toward  " b i o t e c h n o l o g i c a l " development of g e n e t i c a l l y improved s p e c i e s through v a r i o u s s t a t e - o f - t h e - a r t techniques from p l a n t and  animal breeders,  t o implement techniques  there may  pressure  which are p r e d i c a t e d upon knowledge  of the c o n i f e r genome not yet at hand. u t i l i z a t i o n of molecular  be great  borrowed  loci  P r i o r to  the  i n assembling l i n k a g e maps,  t h e i r i n h e r i t a n c e p a t t e r n s must be known (Cheliak and 1985) .  Pitel  . ,  Inheritance  s t u d i e s i n gymnosperms are  particularly  e f f i c a c i o u s owing t o the presence of h a p l o i d , megagametophyte t i s s u e i n seeds, which possesses the same g e n e t i c c o n s t i t u t i o n as the female gamete G i f f o r d 1974). s e l e c t i o n and  Assuming r e g u l a r meiosis,  (Foster no  and  gametic  random sampling of mature seeds,  the  e l e c t r o p h o r e t i c assay of s u f f i c i e n t megagametophytes of  17  mother t r e e s heterozygous f o r a given enzyme l o c u s s h o u l d produce equal numbers of the two a l t e r n a t e A g o o d n e s s - o f - f i t t e s t i s then employed segregation.  (allelic)  forms.  t o v e r i f y the  1:1  Where enzymes are f u n c t i o n a l i n embryo  ( d i p l o i d ) t i s s u e , the quaternary s t r u c t u r e may (El-Kassaby 1980,  Millar  deduced  1985).  In t h i s chapter, isozyme band phenotypes f o r 13 p u t a t i v e l o c i  be  are d e s c r i b e d  i d e n t i f i e d i n P a c i f i c s i l v e r f i r and  compared t o r e l a t e d s p e c i e s , balsam and F r a s e r f i r , other c o n i f e r s .  Mode of i n h e r i t a n c e i s e i t h e r  or, f o r i n v a r i a n t enzymes, i n f e r r e d .  and  demonstrated  Isozyme o r i g i n s  and  d e v i a t i o n s from Mendelian s e g r e g a t i o n are d i s c u s s e d . 3.2  M a t e r i a l s and Methods W i n d - p o l l i n a t e d seeds were o b t a i n e d from cones of 87  P a c i f i c s i l v e r f i r t r e e s c o l l e c t e d i n e i g h t l o c a t i o n s on Vancouver  Island  (as d e s c r i b e d i n Chapter 2 ) .  were s e p a r a t e d by s o f t x-ray and s t o r e d at 2°C. electrophoresis,  Filled  seeds  P r i o r to  seeds were soaked f o r 48 hr i n d e i o n i z e d  water at 20°C and then h e l d at 3°C f o r one t o f o u r days. Embryos were s e p a r a t e d from c o r r e s p o n d i n g megagametophytes and i n d i v i d u a l l y homogenized buffer  i n one drop of e x t r a c t i o n  (El-Kassaby et al. 1982b).  Electrophoretic  t e c h n i q u e s f o l l o w e d those of Conkle et al. two b u f f e r systems: El-Kassaby et al.  'A' - T r i s - c i t r a t e  (1982b) and  (1982) u t i l i z i n g  (pH 7.0)  following  'B' - Sodium b o r a t e (pH 8.0),  18  modified  from Conkle et al. (1982) by d i l u t i o n of the g e l  b u f f e r t o one-half  s t r e n g t h with d e i o n i z e d  Horizontal starch gels systems.  (12.5% w/v) were used f o r both  The enzymes, t h e i r a b b r e v i a t i o n s  commission numbers, and the a p p r o p r i a t e are l i s t e d preparation  water.  i n Table 3.1.  and enzyme  b u f f e r systems used  S t a i n r e c i p e s and s u b s t r a t e  f o l l o w e d Conkle et al. (1982).  Detailed  d e s c r i p t i o n s of e l e c t r o p h o r e t i c procedures and r e c i p e s are given  i n Appendix 2. Where p o s s i b l e , 20 seeds from each t r e e were assayed,  however e i g h t t r e e s had 19 seeds with s u f f i c i e n t a c t i v i t y and i n two cases,  only  enzyme  18 seeds were a v a i l a b l e .  Assuming no e r r o r s i n d e t e c t i o n , the p r o b a b i l i t y of m i s c l a s s i f y i n g a heterozygote at any one locus i s 0.5 for  k megagametophytes ( T i g e r s t e d t 1973).  Eighteen  t o 20  seeds a f f o r d s a l a r g e enough sample t o make t h i s l i k e l i h o o d extremely s m a l l , unless  severe s e g r e g a t i o n  distortion exists  ( M i l l a r 1985). For those l o c i where no v a r i a t i o n was observed, the band phenotypes i n megagametophytes were compared t o embryo t i s s u e and, where p o s s i b l e , t o r e p o r t s of i n h e r i t a n c e i n related species.  Where heterozygous t r e e s were i d e n t i f i e d ,  a l o g - l i k e l i h o o d G-test t e s t the g o o d n e s s - o f - f i t  (Sokal and Rohlf  of the observed s e g r e g a t i o n  t o the 1:1 a l l e l i c segregation to Mendelian e x p e c t a t i o n s  1981) was used t o  predicted for l o c i  ratio  conforming  (ME) and, where more than one t r e e  19  e x h i b i t e d h e t e r o z y g o s i t y at a p a r t i c u l a r l o c u s , a heterogeneity  G-test  (Sokal and Rohlf 1981)  was  performed t o  t e s t the homogeneity of s e g r e g a t i o n among t r e e s . Enzyme l o c i are r e f e r r e d t o by t h e i r abbreviation  (Table 3.1)  three-letter  and where more than one  locus i s  r e s o l v e d f o r a given enzyme system, the most a n o d a l l y m i g r a t i n g i s assigned the number  l.  5  Isozyme d e s i g n a t i o n f o l l o w e d the method of et al.  (1987b).  El-Kassaby  At a given l o c u s , the most f r e q u e n t l y  o c c u r r i n g band was  designated  '1' and bands m i g r a t i n g  slower  and f a s t e r were a s s i g n e d odd and even numbers, r e s p e c t i v e l y . P r e l i m i n a r y experiments a) determined t h a t t h e r e was  no  s u b s t a n t i a l change i n isozyme presence or r e s o l u t i o n i n seeds h e l d at 3°C f o r up to four days and b) e l i m i n a t e d L i t h i u m borate  (pH 8.3)  systems of Conkle et al. reliably  and T r i s - c i t r a t e  (pH 6.2)  (1982), as too few  buffer  l o c i c o u l d be  scored.  T h i s i s t h e m o s t common b u t b y no means s t a n d a r d p r a c t i c e i n inheritance studies. O ' M a l l e y e t al. (1979) u s e d a s c o r i n g s y s t e m w h i c h numbered t h e l e a s t - a n o d a l l o c u s a s 1. H a r r y (1986) a d o p t e d t h e most common s y s t e m i n h i s t e x t b u t showed m i g r a t i o n toward the opposite electrode i n h i s f i g u r e s . This suggests that c a r e must be e x e r c i s e d when c o m p a r i n g p a t t e r n s a c r o s s s p e c i e s a n d researchers.  20 Table 3.1  Enzyme systems, t h e i r a b b r e v i a t i o n s as used i n the t e x t , Enzyme Commission r e f e r e n c e number, b u f f e r system used and the number of l o c i observed and those c o n s i s t e n t l y s c o r a b l e f o r each system. Enzyme Commission Number  Buffer System®  A s p a r t a t e amino t r a n s f e r a s e (AAT)  2.6.1.1  B  Glucose-6-phosphate dehydrogenase (G6P)  1.1.1.49  B  Glutamate dehydrogenase  (GDH)  1.4.1.3  B  Isocitrate dehydrogenase  (IDH)  1.1.1.42  A  Malate dehydrogenase  (MDH)  1.1.1.37  A  6-Phosphogluconate dehydrogenase (6PG)  1.1.1.4 4  A  Phosphoglucose isomerase (PGI)  5.3.1.9  A  Phosphoglucomutase (PGM)  2.7.5.1  A  Superoxide dismutase (SOD)  1.15.1.1  B  Enzyme system (abbr.)  ®  No.  loci  Observed  Scored  Composition of b u f f e r s o l u t i o n s and s t a i n r e c i p e s are given i n Appendix 2.  21  3.3  Results  and D i s c u s s i o n  Nine enzyme systems y i e l d e d c l e a r and c o n s i s t e n t l y s c o r a b l e bands f o r 13 p u t a t i v e l o c i .  An a d d i t i o n a l band  phenotype was d e t e c t e d but c o u l d not be r e l i a b l y  3.3.1  scored.  Enzymes c l a s s e d as monomorphic  Six l o c i  (AAT-1 and 3, GDH, SOD, MDH-3 and 6PG) i n f i v e  enzyme systems d i s p l a y e d i n v a r i a n t band p a t t e r n s . (a)  Aspartate-amino t r a n s f e r a s e (AAT) AAT,  equivalent  (Cheliak and P i t e l migrating  loci.  in a l l trees.  t o glutamate o x a l a c e t a t e  transaminase  1985), was found t o have t h r e e  anodally-  AAT-1 appeared i n v a r i a n t and s i n g l e banded A s i m i l a r locus was d e t e c t e d  i n balsam f i r  (Neale and Adams 1981, Jacobs et al. 1984) and F r a s e r f i r (Jacobs et al. 1984), and found t o be d i a l l e l i c species.  In d i p l o i d heterozygotes,  intermediate,  i n both  the presence of an  h y b r i d band i n a d d i t i o n t o t h e a l l e l i c  forms  c o n t r i b u t e d by both gametes i n d i c a t e s t h a t t h e enzyme i s dimeric  i n structure  (Guries and L e d i g  1978).  Neale and  Adams  (1981) d e t e c t e d t h i s s t r u c t u r e f o r AAT-1 i n balsam  fir.  Shea (1988) found AAT-1 t o be i n v a r i a n t i n a sample o f  subalpine  f i r , but d i d not d e s c r i b e the band p a t t e r n .  This  l o c u s was r e p o r t e d t o be monomorphic i n samples of blue and Engelmann spruce  (Ernst et al. 1987), whitebark  pine  22  ( F u r n i e r et al. Douglas-fir  1986), tamarack  (El-Kassaby  et al.  In c o n t r a s t , AAT-3 was  (Cheliak and P i t e l  1985)  and  1982b).  found i n P a c i f i c s i l v e r f i r to  be double banded and monomorphic over a l l sampled t r e e s . The  bands appeared i n the same p o s i t i o n on the g e l f o r  embryo t i s s u e , but were p o o r l y r e s o l v e d .  In balsam f i r t h i s  l o c u s a l s o d i s p l a y e d a double band p a t t e r n , but two were d e t e c t e d unclear,  (Neale and Adams 1981).  however the s e g r e g a t i o n  alleles  Embryos were a l s o  patterns  of heterozygous  mother t r e e s confirmed s i n g l e - l o c u s Mendelian i n h e r i t a n c e . Double and t r i p l e band phenotypes i n h a p l o i d megagametophyte t i s s u e have been d e t e c t e d conifers  (Cheliak and P i t e l  al.  1986,  has  a l s o been demonstrated  1978).  at t h i s locus i n s e v e r a l  Harry 1986,  AAT-3 was  (Shea 1988)  1985,  E r n s t et al.  M i l l a r 1985, 1987).  (Rudin 1975,  F u r n i e r et  Dimeric  Guries  and  structure Ledig  found t o be monomorphic i n s u b a l p i n e f i r  but the s t r u c t u r e was  El-Kassaby et al.  not  reported.  (1982b) r e p o r t e d a s i n g l e band  phenotype f o r D o u g l a s - f i r at AAT-3, with two detected  other  a fourth, cathodally migrating  t o covary with AAT-3.  O'Malley et al.  alleles  and  zone which was  found  (1979) found a  s i m i l a r p a t t e r n i n ponderosa pine and p o s t u l a t e d t h a t both zones were c o n t r o l l e d by a s i n g l e l o c u s . m i g r a t i n g bands, designated  AAT-1  i n p i t c h pine,  cathodal  and  a third,  the v a r i a t i o n i n AAT-2 e x a c t l y  Two  anodally-  and AAT-2, were zone was  (Guries and  detected  found t o match  Ledig  1978).  23  However, when t h i s s p e c i e s was running c o n d i t i o n s  assayed u s i n g the same  f o r l o b l o l l y pine  (Adams and  t r i p l e banded allozymes were observed. suggest t h a t b u f f e r pH  r e s o l u t i o n and/or p o s i t i o n i n the g e l .  blue  These r e s u l t s  and/or composition as w e l l  e l e c t r o p h o r e t i c running c o n d i t i o n s may  al.  J o l y 1980a),  (1987) observed t r i p l e - b a n d e d  as  a f f e c t band Further,  Ernst  phenotypes f o r AAT-3 i n  and Engelmann spruce h a p l o i d t i s s u e while only  bands were d e t e c t e d both s p e c i e s .  et  i n d i p l o i d embryo and bud  two  t i s s u e s of  They determined that both double and  banded allozymes are i n h e r i t e d as a s i n g l e a l l e l e  tripleand  suggested t h a t some p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n of a s i n g l e allozyme  (apparently  d i f f e r e n t t i s s u e s ) may  d i s s i m i l a r modifications  in  be the cause of such complex  phenotypes. Another p o s s i b l e e x p l a n a t i o n are the loci  r e s u l t of d u p l i c a t e d and  (Harry  1986) .  be t h a t m u l t i p l e bands  necessarily tightly  D u p l i c a t i o n of l o c i and  mutation i s c o n s i d e r e d Scandalios  may  (1975) and  subsequent  by Markert and Whitt Gottlieb  linked  (1968),  (1982) to be the  probable  o r i g i n of many isozymes. (b)  Glutamate dehydrogenase GDH  invariant  was  found to be  (GDH)  s i n g l e banded and  i n t h i s study, although one  considered  t r e e d i s p l a y e d what  appeared to be a f a s t e r - m i g r a t i n g band i n one  of  20  24  megagametophytes assayed.  The m i g r a t i o n d i s t a n c e s under t h e  e l e c t r o p h o r e t i c c o n d i t i o n s of t h i s study were so c l o s e t h a t the band p a t t e r n of the corresponding to characterize. observed  embryo was i m p o s s i b l e  One zone of a c t i v i t y with no v a r i a t i o n was  i n balsam, F r a s e r and s u b a l p i n e f i r (Neale and  Adams 1981, Jacobs et al. 1984, Shea 1988) and s e v e r a l other conifer species 1985,  (El-Kassaby  et al. 1982b, C h e l i a k and P i t e l  M i l l a r 1985, El-Kassaby  1986,  Harry  et al. 1987b, F u r n i e r et al.  1986, Strauss and Conkle 1986).  l o c u s was found t o possess  two a l l e l e s and p o s s i b l y be  m u l t i m e r i c i n Engelmann spruce spruce  (Stewart  However, t h i s  (Ernst et al. 1987),, i n white  and Schoen 1986) and i n b l a c k spruce,  double-banded embryos suggested and Morgenstern 1985).  where  a monomeric s t r u c t u r e (Boyle  F i v e a l l e l e s at GDH and an apparent  m u l t i m e r i c s t r u c t u r e were r e p o r t e d f o r l o b l o l l y p i n e  (Guries  and L e d i g 1978). A l a r g e r sample of P a c i f i c s i l v e r f i r may c o n f i r m a l l e l i s m at t h i s l o c u s a l s o . (c)  Superoxide  dismutase (SOD)  SOD appeared as a s i n g l e , s t a i n e d f o r GDH.  r e v e r s e - s t a i n i n g band on g e l s  I t was monomorphic and o c c u p i e d the same  g e l p o s i t i o n i n both t i s s u e types.  T h i s enzyme was not  s t u d i e d i n balsam or F r a s e r f i r (Neale and Adams 1981, Jacobs et al. 1984).  SOD has been d e t e c t e d i n s e v e r a l  members o f the Pineaceae, bishop p i n e  d i s p l a y i n g one i n v a r i a n t band i n  ( M i l l a r 1985) and white spruce  (King and Dancik  1983), and one zone with t h r e e a l l e l e s i n D o u g l a s - f i r ( E l -  25  Kassaby et al. 1982b). for  Three i n v a r i a n t bands were observed  t h i s enzyme i n whitebark p i n e ( F u r n i e r et al. 1986)  knobcone p i n e (Strauss and Conkle 198 6), t h r e e bands were d e s c r i b e d as f a i n t .  and  although two o f t h e  Since no v a r i a t i o n  e x i s t e d , these bands c o u l d not be c h a r a c t e r i z e d as d i s t i n c t loci  in Pacific silver f i r .  (d)  Malate dehydrogenase (MDH) Three l o c i were r e s o l v e d f o r MDH and t h e s l o w e s t -  migrating locus banded study.  (MDH-3) appeared monomorphic and s i n g l e -  i n t h e megagametophytes o f a l l t r e e s sampled  A c t i v i t y at t h i s locus was observed i n embryos, but  bands d i d not r e s o l v e c l e a r l y enough t o score. Adams (1981) r e p o r t e d only two MDH l o c i s i n g l e banded  and i n v a r i a n t .  i n balsam f i r embryo t i s s u e . the  balsam and F r a s e r f i r s  El-Kassaby  MDH-2 was found t o be i n a c t i v e Two zones were a l s o found i n  sampled by Jacobs et al. (1984).  (1981) r e p o r t e d t h r e e l o c i  and grand f i r .  Neale and  i n balsam f i r ; both  MDH-2 was found t o be polymorphic f o r t h r e e  fir  in this  Three t o four l o c i  t h i s enzyme system i n o t h e r c o n i f e r s  alleles. i n MDH f o r white  are o f t e n observed i n  (Simonsen and  W e l l e n d o r f 1975, O'Malley et al. 1979, El-Kassaby e t a l . 1982b, C h e l i a k e t al. 1984, Boyle and Morgenstern 1985, C h e l i a k and P i t e l  1985, M i l l a r 1985, El-Kassaby et al.  1987b, Harry 1986 and E r n s t et al. 1987).  In a d d i t i o n , a  band d e s c r i b e d as a non-genetic i n t e r - l o c u s heterodimer has  26  been d e t e c t e d i n the MDH complex o f many c o n i f e r s i n El-Kassaby  1981,  Morgenstern 1985,  El-Kassaby  (reviewed  et al. 1982b, Boyle and  C h e l i a k and P i t e l  1985 and E r n s t e t a i .  1987) . In a comparative study o f four genera o f El-Kassaby  (1981) found t h a t t h e t h r e e l o c i  Pineaceae,  o f white and  grand f i r were e q u i v a l e n t t o MDH-1, 2 and 4 o f D o u g l a s - f i r and t h a t t h e l e a s t anodal  locus, designated MDH-4, was not  w e l l r e s o l v e d i n grand f i r . heterodimer assayed.  band was absent  He a l s o found t h a t t h e i n both  s p e c i e s he  Abies  I n t e r e s t i n g l y , no heterodimer  band ( u s u a l l y  forming between t h e second and t h i r d locus) was d e t e c t e d i n Pacific silver f i r . As w e l l as n o n - a l l e l i c i n t e r a c t i o n s i n t h i s enzyme system, genes which appear t o a l t e r t h e m i g r a t i o n of  MDH l o c i have been reported,  incense-cedar  i n maize  (Newton 1979),  (Harry 1983) and bishop pine  Guries and L e d i g  patterns  1985).  (Millar  (1978) determined t h a t i n p i t c h  t h e r e i s l o c a l i z a t i o n o f MDH enzymes such t h a t  pine  mitochondria  c o n t a i n only the MDH-2 l o c u s , while crude t i s s u e p r e p a r a t i o n s y i e l d both MDH-1 and MDH-2. s u b c e l l u l a r compartmentalization  Similar  has been observed  i n maize  where t h r e e u n l i n k e d MDH l o c i produce m i t o c h o n d r i a l and another  two u n l i n k e d l o c i  (Goodman e t al. 1980,  are c y t o p l a s m i c  isozymes  i n origin  Newton and Schwartz 1980).  Enzyme  27  d u p l i c a t i o n and l o c a l i z a t i o n i n p l a n t s has been examined i n an e v o l u t i o n a r y context by G o t t l i e b cited).  (1982  and r e f e r e n c e s  He concluded t h a t where gene products are  produced  i n the same s u b c e l l u l a r compartment, both i n t r a - and  inter-  l o c u s h y b r i d enzymes form, whereas such h y b r i d molecules not form between enzymes of d i f f e r e n t compartments. band phenotypes of the t h r e e MDH  loci  s i l v e r f i r suggest t h a t they may  be c o n t a i n e d w i t h i n  different subcellular organelles. study may In  a d d i t i o n , the presence  of an enzyme l o c u s i n  ( M i l l a r 1985)  (Adams and J o l y 1980a)  suggests d i f f e r e n t i a l e x p r e s s i o n  o n t o g e n e t i c a l l y , which makes the MDH  system  a good candidate  s t u d i e s of gene r e g u l a t i o n . The complexity of the MDH  enzyme system  a l s o by the e x i s t e n c e of multimerism loci  fractionation  r e v e a l i n f o r m a t i o n to support t h i s h y p o t h e s i s .  vice versa  for  The  observed i n P a c i f i c  A cell  megagametophyte but not embryo t i s s u e or  r e p o r t e d i n many c o n i f e r s ,  i s evidenced  at some or a l l of the  however, t h i s cannot  confirmed i n the monomorphic locus found i n P a c i f i c  be silver  fir. (e)  do  6-Phosphogluconate dehydrogenase Gels s t a i n e d f o r 6PG  showed one  (6PG) zone of a c t i v i t y .  Gametophytes were s i n g l e banded and i n v a r i a n t a c r o s s a l l t r e e s sampled i n t h i s study. found i n balsam  A s i m i l a r band p a t t e r n was  f i r by Neale and Adams (1981), with  two  28  a l l e l e s observed. alleles,  were d e t e c t e d  by Jacobs et al. f i r by Shea 6PG 8.0)  However, two  i n populations  (1984) and  (1988).  variable loci,  These two  two  of balsam/Fraser f i r  a single population  of  subalpine  d i f f e r i n g reports resolved  on d i f f e r e n t b u f f e r systems  ( T r i s - c i t r a t e pH  6.2  vs.  pH  which suggests t h a t the d e t e c t i o n of l o c i i n t h i s  enzyme system i s p H - s e n s i t i v e .  T h i s phenomenon was  observed i n tamarack by C h e l i a k and P i t e l Other c o n i f e r s commonly e x h i b i t two (Guries and L e d i g 1978, al.  each with  O'Malley et al.  1982b, King and Dancik 1983,  and M'orgenstern 1985, Schoen 1986  and E r n s t et al.  are o f t e n r e p o r t e d .  zones f o r 1979,  6PG  El-Kassaby et  Harry 1986,  1987), and d i m e r i c  1984,  Boyle  Stewart  and  structures  Three l o c i were r e p o r t e d i n knobcone  pine  (Strauss and Conkle 1986)  pine  (Adams and 3.3.2  (1985).  C h e l i a k et al.  M i l l a r 1985,  also  and  a s i n g l e zone i n l o b l o l l y  J o l y 1980a).  Enzymes' e x h i b i t i n g polymorphism  Seven l o c i i n s i x enzyme systems d i s p l a y e d v a r y i n g band p a t t e r n s under the e l e c t r o p h o r e t i c c o n d i t i o n s of t h i s The  r e l a t i v e p o s i t i o n i n g of the band phenotypes with  study. respect  t o t h e i r o r i g i n a l placement on the s t a r c h g e l i s p i c t u r e d i n Figure  3.1.  29  r-F  AAT  G6P  Aat-2  G6p 2 2 1  3  5  MDH  PGI Mdh-2  Mdh-1  2  Pgi-2  2 1  ^ ™  1 3  1 ™ "  PGM  IDH  Pgm  Idh  2 "™'  3  5  1  2  3  L-0  Figure 3.1 Representations of band patterns observed i n seven polymorphic l o c i found i n trees from eight populations of P a c i f i c s i l v e r f i r on Vancouver Island, B r i t i s h Columbia. Band positions indicate r e l a t i v e migration distance between the o r i g i n (0) and the buffer front (F). For each locus, the band designated *1' occurred most frequently.  30  (a)  Glucose-6-phosphate dehydrogenase  (G6P)  T h i s enzyme e x h i b i t e d a s i n g l e band phenotype w i t h a t o t a l of f o u r allozymes when assayed  in Pacific  silver f i r .  C l e a r l y s t a i n i n g embryos showed t r i p l e band p a t t e r n s i n heterozygotes,  indicating  dimerism.  G6P  was  balsam or F r a s e r f i r (Neale and Adams 1981, 1984).  One  zone of a c t i v i t y with two  been found i n jack p i n e pine  et  al.  to f i v e a l l e l e s  has  (Cheliak et al.  Jacobs  1984), western  (El-Kassaby et a i . 1987b), incense-cedar  b l a c k spruce  (Boyle and Morgenstern  (El-Kassaby et al.  1982b).  r e p o r t e d i n knobcone pine pine  not s t u d i e d i n  (Millar  1985)  Two  1985)  white  (Harry 1986),  and D o u g l a s - f i r  zones of a c t i v i t y were  (Strauss and Conkle  and ponderosa pine  1986), bishop  (O'Malley  et  al.  1979), with only one being v a r i a b l e i n each s p e c i e s . C h e l i a k et al. variation  (1984) observed  some o n t o g e n e t i c  i n t h i s enzyme system i n jack p i n e .  i n i t i a l l y d e t e c t e d i n one  Activity  zone and as germination  was  progressed  (days f o u r t o ten) the a c t i v i t y disappeared from t h a t zone to  a more anodal r e g i o n .  than one  l o c u s may  developmentally  T h i s r e p o r t suggests t h a t more  be present, but may  unstable.  assayed at approximately  a l s o be  Seeds of P a c i f i c  s i l v e r f i r were  the same s t a t e of germination,  a l l t r e e s c o n s i s t e n t l y presented only one  zone of  S e g r e g a t i o n analyses of the four d i f f e r e n t combinations  (Table 3.2)  r e v e a l e d no s i g n i f i c a n t  and  activity.  allelic deviation  31  from 1:1 r a t i o s .  For the two combinations  d e t e c t e d i n more  than one t r e e h e t e r o g e n e i t y t e s t s showed s i g n i f i c a n t  Table 3.2  Observed s e g r e g a t i o n i n megagametophytes o f heterozygous mother t r e e s and G t e s t s o f g o o d n e s s - o f - f i t t o 1:1 r a t i o (df = 1) and h e t e r o g e n e i t y among t r e e s (df i n p a r e n t h e s e s ) .  Locus  Allelic Combinationt  AAT-2  1/2  224:53  113.57**  G6P  1/2  12:8 44 :36 315:321 8:12  0.81 0.80 0.06 0.81  12:8 22: 18  0.81 0.40 1.33  3.69 (1) -  1/2 1/3  116:102 9:11  0.90 0.20  28.02 (10) -  MDH-2  1/2  13:7  1.83  PGI-2  1/3  409:404  0.03  PGM  1/2  8:12 227:210 21:19  0.81 0. 66 0.10  1/3  1/5 2/5 IDH  1/2  1/3 1/5  MDH-1  1/3 1/5  Observed Deviation Segregation G  12:7  Heterogeneity G  t  Most common a l l e l e designated '1'; faster and slower a l l e l e s given even and odd numbers, respectively.  *  S i g n i f i c a n t , P < 0.05.  ** Highly s i g n i f i c a n t , P < 0.01.  22.25 (13)  —  16.75M3) 64.79* (32)  — _  63.09** (39) —  44.02 (21) 0.10 (1)  32  v a r i a t i o n among t r e e s . i n d i c a t e d that alleles  Individual goodness-of-fit  f i v e of the  '1' and  33 t r e e s  '5' d i d not  four trees possessing  s i g n i f i c a n t d e v i a t i o n from 1:1  i n favour of one  allele  heterozygous f o r  conform to ME  alleles  h e t e r o z y g o t e s the deviant  (15%)  '1' and  G tests  and  one  of  '3' a l s o e x h i b i t e d  segregation.  In  Adams and  Joly  a  1/5  r a t i o s were not u n i f o r m l y  .  the  skewed  (1980a) suggest  t h a t when a p a r t i c u l a r genotype d i s p l a y s a d e f i c i e n c y of a l l e l e c o n s i s t e n t l y over s e v e r a l parents i t may t h a t the it,  allele,  or some p o r t i o n of the  i s being selected against.  Stewart and  be  itself,  a cause f o r  d i s t o r t i o n (Cheliak  Schoen 1986).  The  the  et al.  1984,  maternal genotype i s  determined from v i a b l e seeds and, determines the  chromosome l i n k e d to  presents  i n the p o l l e n pool may  v a r i a t i o n i n segregation  indicate  Where no p a r t i c u l a r p a t t e r n  i n the unequal a l l e l e f r e q u e n c i e s heterogeneity  an  s u r v i v o r s h i p of the  i n turn,  embryo v i a b i l i t y  female gametophyte.  Germinable seeds are thus products of a maturation process wherein some form of embryonic s e l e c t i o n might occur, r i s e to a disproportionate  number of favoured  depending upon p o l l e n d i s t r i b u t i o n and Adams and genetic  Joly  alleles,  viability.  (1980a) a l s o suggest t h a t  backgrounds of female gametes may  degrees of i n c o m p a t i b i l i t y among t r e e s .  differing  produce d i f f e r e n t Polyembryony i s  known t o occur i n P a c i f i c s i l v e r f i r (Owens and and  Sorensen  (1982) maintains that  giving  Molder  i n noble f i r the  1977)  presence  33  o f more t h a n one fertilization  ovule allows considerable post-  s e l e c t i o n to take place.  of heterozygous  t r e e s conformed  t o assume t h a t G6P  t o ME,  silver  d e v i a t i o n s suggest t h i s  l o c u s may  (b)  alleles,  t h r e e of which,  distinctly  resolved,  heterozygous,  (IDH)  although very c l e a r l y (Table 3.2).  was  e t al.  f i rinvariant  ( E l - K a s s a b y e t al.  and L e d i g  i n balsam  heterozygous  at t h i s  i n Engelmann s p r u c e  also  ( O ' M a l l e y e t al.  tree  was  samples of  i n IDH  ( E r n s t e t al.  1982b),  f i r (Neale  l o c u s as d i d Shea  Dimerism  black spruce  1985), w h i t e spruce  pine  Embryos,  (1984) f o u n d t h e i r  i n a sample o f s u b a l p i n e f i r .  Morgenstern  staining,  b a n d p a t t e r n when  observed  a l t h o u g h o n l y one  Jacobs  balsam/Fraser  ponderosa  s u b j e c t t o some  showed a t r i p l e  S i m i l a r banding  detected.  fir  be  tree  suggesting a dimeric structure f o r t h i s  a n d Adams 1981)  observed  although single  e x h i b i t e d a s i n g l e band phenotype w i t h f o u r  o c c u r r e d i n v e r y few t r e e s  enzyme.  fir,  i n both  influence.  I s o c i t r a t e dehydrogenase IDH  i t seems r e a s o n a b l e  i s under s i n g l e - l o c u s c o n t r o l  seed t i s s u e s of P a c i f i c  selective  Because the m a j o r i t y  has  1987),  1979)  been Douglas-  (Boyle  ( K i n g and D a n c i k  (1988)  and  1983),  and p i t c h p i n e  (Guries  1978).  M o r e t h a n one i n Engelmann s p r u c e  zone o f a c t i v i t y ( E r n s t e t al.  o f IDH  1987)  and  has b e e n  observed  incense-cedar  34  (Harry 1986). bishop pine  IDH e x h i b i t e d a double band phenotype i n  ( M i l l a r 1985)  and S t r a u s s and Conkle  (1986)  found t h i s enzyme t o be s i n g l e or double banded depending the  on  b u f f e r system employed. F u r t h e r , S t r a u s s and Conkle  two l o c i  (1986) noted t h a t one of  r e s o l v e d i n s t a i n i n g of phosphoglucomutase  produced i d e n t i c a l v a r i a t i o n t o IDH. reported that f a i n t  (PGM)  O'Malley et al.  (1979)  IDH p a t t e r n s appeared on many g e l s  s t a i n e d f o r enzymes r e q u i r i n g NADP (nicotinamide adenine d i n u c l e o t i d e phosphate).  The c i t r i c  a c i d found i n the  b u f f e r used was presumed to be the s u b s t r a t e . fir,  El-Kassaby et al.  f a i n t e r zone on PGM  In Douglas-  (1982b) v e r i f i e d t h a t the second,  g e l s was,  in fact,  IDH,  not a second  PGM  locus. These s t u d i e s suggest that the composition of b u f f e r systems are  s h o u l d be c o n s i d e r e d whenever s p e c i e s  made f o r t h i s enzyme system. Table 3.2  shows that f o r the t h r e e a l l e l i c  d e t e c t e d i n IDH,  t h e r e was  where p o s s i b l e t o t e s t .  e x i s t e d among t r e e s ,  The IDH locus i n P a c i f i c s i l v e r f i r  then be assumed t o e x h i b i t simple, Mendelian  inheritance.  combinations  no d e v i a t i o n from the expected  s e g r e g a t i o n r a t i o and t h a t homogeneity  may  comparisons  35  (c)  Malate dehydrogenase (MDH) MDH-1 was found t o be s i n g l e banded with t h r e e a l l e l e s  in Pacific silver f i r . heterozygous  Eleven t r e e s were found t o be  f o r common and f a s t a l l e l e with homogeneous  s e g r e g a t i o n r a t i o s among t r e e s and no d e v i a t i o n from t h e expected 1:1 s e g r e g a t i o n m i g r a t i n g zone o f a c t i v i t y i n one t r e e o n l y .  (Table 3.2).  A second,  (MDH-2) was found t o be v a r i a b l e  The observed s e g r e g a t i o n f o r t h e common  and f a s t a l l e l e at t h i s l o c u s d i d not d i f f e r 3.2).  slower  For both l o c i ,  from ME (Table  r e s o l u t i o n was not d i s t i n c t enough t o  permit a s t r u c t u r a l i n t e r p r e t a t i o n of embryo band p a t t e r n s . Neale and Adams (1981) and Jacobs et al. (1984) both r e p o r t e d o n l y two s i n g l e banded l o c i i n balsam  f o r t h i s enzyme system  f i r and p o p u l a t i o n s of balsam  and F r a s e r f i r .  MDH-1 was i n v a r i a n t i n both s t u d i e s and, while Jacobs e t al. (1984) found t h r e e a l l e l e s at MDH-2, Neale and Adams (1981) found megagametophytes t o be monomorphic and no a c t i v i t y i n embryo t i s s u e a t t h i s l o c u s . Pacific silver fir,  Three l o c i were observed i n  with, as p r e v i o u s l y noted, very poor  r e s o l u t i o n i n embryos at MDH-3.  The assay o f seeds  from  s e v e r a l A b i e s s p e c i e s under the same e l e c t r o p h o r e t i c conditions  ( s i m i l a r t o El-Kassaby 1981)  a c c u r a t e comparisons  would enable more  across t r u e f i r s p e c i e s and perhaps  shed some l i g h t on the e v o l u t i o n o f t h i s enzyme system r e l a t i v e t o other members of t h e  Pineaceae.  36  (d)  Phosphoglucose isomerase (PGI) Two r e g i o n s  f o r PGI. and  of a c t i v i t y were observed on g e l s s t a i n e d  What may be c o n s i d e r e d  as PGI-1 was very b l u r r y  c o u l d not be r e s o l v e d w e l l enough t o s c o r e .  was e v i d e n t ,  but t h e r e were d i f f u s e bands l e a d i n g and  t r a i l i n g t h e area of dark s t a i n i n g .  This was l e s s apparent  i n embryos, but a l l e l i c v a r i a t i o n s t i l l r e l i a b l y detected.  Inconsistent  l o c u s has been r e p o r t e d  s t a i n i n g at t h i s  putative (Ernst  (Boyle and Morgenstern 1985),  (Cheliak and P i t e l  Kassaby e t al. 1982b).  c o u l d not be  i n blue and Engelmann spruce  et al. 1987), b l a c k spruce tamarack  One band  1985) and D o u g l a s - f i r ( E l -  The locus was r e s o l v e d i n balsam,  F r a s e r and subalpine  f i r as a s i n g l e banded i n v a r i a n t zone  i n a l l three  (Neale and Adams 1981, Jacobs e t al.  1984,  species  Shea 1988).  PGI-1 e x h i b i t e d apparent non-genetic  v a r i a t i o n i n D o u g l a s - f i r c o n t r o l l e d c r o s s progeny al.  (Neale e t  1984). The  present  slower m i g r a t i n g  zone  in Pacific silver f i r .  (PGI-2) had two a l l e l e s Heterozygous embryos,  although somewhat b l u r r y , suggested a d i m e r i c t h i s enzyme. subalpine  Shea  fir.  both s t u d i e s  (1988) found 2 a l l e l e s at t h i s l o c u s i n  T h i s locus was a l s o d e t e c t e d  and F r a s e r f i r ,  structure f o r  with three a l l e l e s ,  i n both balsam  one very r a r e , found i n  (Neale and Adams 1981, Jacobs et al.  1984).  Neale and Adams (1981) were not able t o v e r i f y a d i m e r i c s t r u c t u r e f o r t h i s enzyme although i t i s demonstrated i n  37  s e v e r a l other c o n i f e r s (Guries and L e d i g 1978, al.  El-Kassaby et  1982b, Neale et al. 1984, Boyle and Morgenstern 1985). The p o o l e d  s e g r e g a t i o n data f o r PGI-2 (Table 3.2)  showed no d e v i a t i o n from the expected heterogeneity  1:1 r a t i o  among t r e e s was s i g n i f i c a n t .  although  Individual  g o o d n e s s - o f - f i t G t e s t s r e v e a l e d t h a t only s i x o f 40 heterozygous t r e e s e x h i b i t e d deviant s e g r e g a t i o n r a t i o s  and  t h e r e was no t r e n d t o one a l l e l e or t h e other among t r e e s . T h i s p a t t e r n o f s e g r e g a t i o n suggests  s i n g l e - l o c u s c o n t r o l of  PGI-2 i n P a c i f i c s i l v e r f i r . (e)  Phosphoglucomutase (PGM) PGM  appeared as a c l e a r ,  s i n g l e banded r e g i o n o f  a c t i v i t y with f o u r a l l e l e s d e t e c t e d i n P a c i f i c s i l v e r f i r . Double-banded phenotypes from heterozygous embryo t i s s u e i n d i c a t e a monomeric s t r u c t u r e f o r t h i s enzyme.  Two zones  of a c t i v i t y were found by Neale and Adams (1981) i n balsam fir.  PGM-1 was s i n g l e banded with two a l l e l e s and monomeric  s t r u c t u r a l l y whereas PGM-2 was double banded and i n v a r i a n t . Two  a l l e l e s were d e t e c t e d at PGM-1 i n s u b a l p i n e f i r but t h e  PGM-2 l o c u s was t o o f a i n t t o be scored white spruce,  (Shea 1988).  t h i s second locus was observed  vegetative tissue  (Stewart  and Schoen 1986).  In  i n seed but not Neale et al.  (1984) determined t h a t PGM-2 i n D o u g l a s - f i r i s a d i s t i n c t , independent l o c u s when r e s o l v e d at pH's higher than t h a t  38  used by El-Kassaby et al.  (1982b), where IDH bands no l o n g e r  cover the f a i n t e r PGM-2 bands. Table 3.2 shows no d e v i a t i o n from the expected r a t i o for  any o f the t h r e e a l l e l i c combinations and no  h e t e r o g e n e i t y among t r e e s where p o s s i b l e t o t e s t . r e s u l t s suggest t h a t , under the e l e c t r o p h o r e t i c of  t h i s experiment, PGM appears t o e x h i b i t  These  conditions  Mendelian  inheritance i n Pacific s i l v e r f i r . (f)  Aspartate-animo t r a n s f e r a s e (AAT) AAT-2 was the only band among t h r e e r e s o l v e d on g e l s  s t a i n e d f o r AAT t h a t was v a r i a b l e with two a l l e l e s d e t e c t e d in P a c i f i c s i l v e r f i r .  Heterozygous embryos d i d not s t a i n  c l e a r l y enough t o permit any i n f e r e n c e on s t r u c t u r e at t h i s locus.  Neale and Adams  (1981) r e p o r t a s i n g l e band with two  a l l e l e s and d i m e r i c s t r u c t u r e at AAT-2 i n balsam f i r . s i m i l a r p a t t e r n was d e t e c t e d by Jacobs et al. balsam/Fraser f i r .  Of t h r e e l o c i  A  (1984) i n  resolved i n subalpine f i r ,  only AAT-2 d i s p l a y e d a l l e l i c v a r i a t i o n  (Shea  1988).  Three zones of a c t i v i t y have been observed f o r AAT i n s e v e r a l other c o n i f e r s al. et  (O'Malley et al.  1982b, C h e l i a k and P i t e l al.  1987b, F u r n i e r et al.  and E r n s t et al.  1987).  embryos o f b l a c k spruce O'Malley et al.  1979, El-Kassaby et  1985, M i l l a r 1985, El-Kassaby 1986, S t r a u s s and Conkle 1986  AAT-2 was found t o be i n a c t i v e i n (Boyle and Morgenstern 1985) and  (1979) suggested isozyme l o c a l i z a t i o n i n •  39  s p e c i f i c t i s s u e s f o r t h i s enzyme system. a c t i v i t y l e v e l s of AAT pine AAT  Tissue-specific  isozymes were d e t e c t e d i n l o d g e p o l e  ( P i t e l et a i . 1984)  and o r g a n e l l e - s p e c i f i c a c t i v i t y of  isozymes has been observed i n other p l a n t s p e c i e s  et al.  1976,  Hart and Langston  In AAT-2, two a l l e l e s ,  (Huang  1977).  common and f a s t , were observed  t o segregate i n 14 of the 87 t r e e s sampled.  Segregation  r a t i o s were found t o be homogeneous among t r e e s  (Table 3.2).  When pooled, however, these data e x h i b i t e d a h i g h l y s i g n i f i c a n t d e v i a t i o n from the h y p o t h e s i z e d 1:1 was  ratio.  It  found t h a t 13 of the 14 t r e e s showed s i g n i f i c a n t  i n d i v i d u a l d e v i a t i o n s from ME and those i n d i v i d u a l  ratios  were u n i f o r m l y and h e a v i l y skewed i n favour of the common allele  (216:43).  S e g r e g a t i o n d i s t o r t i o n i n AAT-2 has been r e p o r t e d i n several conifers and F e r e t 1978,  (Rudin 1975,  Rudin and Ekberg 1978,  O'Malley et al.  1979,  Witter  Boyle and Morgenstern  1985, Adams and J o l y 1980a, Harry 1986,  Strauss and Conkle  1986).  l i m i t p l a c e d on the  Although t h e r e i s a s t a t i s t i c a l  magnitude  of d e v i a t i o n from ME a sample  i s able to detect  as small as 20 seeds  (Mulcahy and Kaplan 1979), these data  suggest s t r o n g l y that e i t h e r there are s e l e c t i v e between the two isozymes themselves 1986, allele  C h e l i a k et al.  1984)  (Strauss and Conkle  or a d i s t o r t e d or  (Strauss and Conkle 1986)  may  differences  "selfish"  be l i n k e d t o t h i s  l o c u s , r e s u l t i n g i n an o v e r - r e p r e s e n t a t i o n of the common  40  a l l e l e i n sampled megagametophytes.  The apparent  s e g r e g a t i o n d i s t o r t i o n was not c o n f i n e d t o t r e e s o f j u s t one or even a few p o p u l a t i o n s i n t h i s study. it  From t h i s evidence  appears t h a t AAT-2 i n P a c i f i c s i l v e r f i r samples  Vancouver  I s l a n d does not e x h i b i t t h e c l a s s i c a l  inheritance pattern.  The homogeneity  from  Mendelian  among t r e e s f o r t h e  d i r e c t i o n o f d i s t o r t i o n tends t o support some g e n e t i c mechanism such as l i n k a g e t o a s o - c a l l e d s e l f i s h  allele,  which a c t s t o i n c r e a s e i t s own frequency and those t o which i t may be l i n k e d  (Strauss and Conkle 1986) , however non-  g e n e t i c causes o f the observed d i s t o r t i o n cannot be r u l e d out without f u r t h e r study. 3.4  Conclusions  (a)  Band p a t t e r n s o f i n v a r i a n t enzyme l o c i  in Pacific  s i l v e r f i r (AAT-1 and 3, GDH, SOD, MDH-3 and 6PG) appear t o be s i m i l a r t o other c o n i f e r s , but may not be "good" Mendelian markers  without v e r i f i c a t i o n o f  inheritance. (b)  The polymorphic enzymes IDH, MDH-1, MDH-2 and PGM appear t o e x h i b i t s e g r e g a t i o n p a t t e r n s conforming t o Mendelian e x p e c t a t i o n s , while G6P, PGI-2 and AAT-2 show some s e g r e g a t i o n d i s t o r t i o n . whether s e g r e g a t i o n d i s t o r t i o n gametophytes  only  (i.e.,  I t cannot be a s c e r t a i n e d i s due t o female  gametic s e l e c t i o n ) o r t o t h e  heterogeneous d i s t r i b u t i o n o f l e t h a l or s e m i - l e t h a l  41  a l l e l e s i n the p o l l e n p o o l .  But, f o r G6P and PGI-2,  where t h e r e i s no apparent t r e n d i n s e g r e g a t i o n distortion, represent (c)  MDH  i t i s assumed t h a t these l o c i  also  Mendelian markers.  and AAT possess m u l t i p l e l o c i ,  which may be  e x p l a i n e d by gene d u p l i c a t i o n and subsequent mutation. There i s a l s o i n d i r e c t compartmentalization  evidence f o r  f o r these enzymes.  42  4.  4.1  LINKAGE  Introduction Enzyme polymorphisms have proven t o be u s e f u l markers  for  t h e study  o f p o p u l a t i o n s t r u c t u r e (reviewed  i n Hamrick  1982), g e n e t i c d i v e r s i t y  (reviewed  mating systems  i n Adams and B i r k e s 1990) i n f o r e s t  trees.  (reviewed  i n El-Kassaby 1990) and  The use o f isozymes i n the e s t i m a t i o n o f g e n e t i c  parameters r e q u i r e s c e r t a i n assumptions, one o f which b e i n g t h a t they d i s p l a y r e g u l a r Mendelian i n h e r i t a n c e p a t t e r n s (Rudin  1976).  When isozymes are employed i n e s t i m a t i n g  parameters o f the mating system and  (Shaw et  al.  El-Kassaby 1985) , i t i s a l s o necessary  are u n l i n k e d .  Linkage i s d e f i n e d by H a r t l  1981, R i t l a n d t o assume they (1980) as a l a c k  of independent assortment due t o l o c i being same chromosome.  The g r e a t e r the d i s t a n c e between two l o c i ,  the more f r e q u e n t l y w i l l maximum frequency statistical  l o c a t e d on the  c r o s s o v e r events occur.  o f recombination  (RF) w i l l  The  be 0.5, the  e q u i v a l e n t of l o c i behaving as i f they s o r t  independently. Recombination f r e q u e n c i e s between l o c i are a l s o important  i n e s t a b l i s h i n g l i n k a g e maps, as mapping the  l o c a t i o n o f allozyme l o c i  i s the p r e l i m i n a r y step i n the use  of isozymes as markers of q u a n t i t a t i v e l y c o n t r o l l e d characters  ( V a l l e j o s and Tanksley  1983).  43  S t u d i e s o f l i n k a g e i n f o r e s t t r e e s have a l s o insights into t h e i r evolutionary r e l a t i o n s h i p s .  provided Harry  (1986) noted t h a t t i g h t l i n k a g e between AAT and PGI has been reported Picea,  (Pinus,  f o r s e v e r a l genera o f the Pinaceae  Pseudotsuga  and a l s o Larix  and although the data  Abies,  [ C h e l i a k and P i t e l  1985]),  i s i n s u f f i c i e n t t o conclude t h a t t h e  same AAT and PGI genes a r e i n v o l v e d i n a l l cases,  Harry's  d i s c o v e r y o f the same l i n k a g e group i n incense-cedar, suggests the maintenance o f t h i s gene b l o c k divergence  o f the Pinaceae and Cupressaceae.  of l i n k a g e support karyology 1978,  since before the Other  reports  t h e n o t i o n t h a t gene arrangements and  o f c o n i f e r s are h i g h l y conserved  King and Dancik 1983, Strauss  Roberds and B r o t s c h o l  (Guries e t al.  and Conkle 1986).  (1986) contend t h a t t h e r e i s  s u b s t a n t i a l evidence t h a t e v o l u t i o n a r y processes  involve  m u l t i p l e l o c u s a s s o c i a t i o n s and t h a t i t may be i n a p p r o p r i a t e t o use s i n g l e l o c u s measures o r means t o d e s c r i b e e f f e c t s over s e v e r a l l o c i .  The i d e n t i f i c a t i o n o f non-random  a s s o c i a t i o n s may p r o v i d e  c l u e s t o t h e mechanism(s)  r e s p o n s i b l e f o r the maintenance o f l i n k a g e groups ( E l Kassaby e t al. populations  1982a) and b a l a n c e d polymorphisms i n n a t u r a l  (Adams and J o l y 1980b).  i n t h i s chapter  The r e s u l t s  summarize attempts t o d e t e c t  allozyme l o c i i n Abies  amabilis  presented  l i n k a g e among  sampled on Vancouver I s l a n d .  44  4.2  M a t e r i a l s and Methods L i n k e d l o c i may  ( B a i l e y 1961, 1986),  Hill  be uncovered  1974,  Wright  i n a number of ways  1976,  S q u i l l a c e and  however, l i n k a g e s t u d i e s of allozyme l o c i  Swindel  in conifers  can be conducted without the n e c e s s i t y of c o n t r o l l e d c r o s s i n g experiments  (Adams and J o l y 1980b) because  megagametophytes of a t r e e found heterozygous at two r e p r e s e n t m e i o t i c products analogous t o d i p l o i d phenotypes  from a double backcross  mated t o a double r e c e s s i v e ,  haploid loci  progeny  (a doubly-dominant  [ c f . B a i l e y 1961]).  parent  Linkage i s  d e t e c t e d when d e v i a t i o n from the 1:1:1:1 j o i n t s e g r e g a t i o n of  the f o u r a l l e l e  assumption (Hattemer  combinations, expected under the  of independent 1979).  between l o c i may combinations  assortment,  In a d d i t i o n ,  i s significant  recombination d i s t a n c e s  be c a l c u l a t e d from the two  ( B a i l e y 1961,  Rudin and Ekberg  smallest  allele  1978).  Of 87 t r e e s s u b j e c t e d t o s t a r c h g e l e l e c t r o p h o r e s i s ,  as  d e s c r i b e d i n Chapter 3, 42 t r e e s were found t o be heterozygous at two or more of the seven l o c i polymorphic.  found t o be  Segregation a n a l y s e s c o n f i r m i n g s i n g l e - l o c u s  i n h e r i t a n c e i n those t r e e s was  a l s o p r e s e n t e d i n Chapter  Of 21 p o s s i b l e two-locus l i n k a g e groups,  16 are r e p r e s e n t e d  i n t h i s study by at l e a s t one doubly-heterozygous t r e e 12 are r e p r e s e n t e d by more than one t r e e , homogeneity among t r e e s t o be made. be confirmed, the data may  3.  and  a l l o w i n g t e s t s of  Where homogeneity can  be pooled over t r e e s t o produce a  45  more powerful  a n a l y s i s (Zar 1984), thus i n c r e a s i n g t h e  p r e c i s i o n o f l i n k a g e estimates C h e l i a k and P i t e l Linkage  1985, Muona et al.  1987).  t e s t s o f s e v e r a l p a i r s of l o c i  than one a l l e l i c combination, polymorphic  (Neale and Adams 1981,  i n v o l v e d more  because these l o c i  f o r more than two a l l e l e s .  were  In these cases, the  a l l e l e s at each l o c u s were t r e a t e d as two c l a s s e s (e.g., A and A') p r i o r t o a n a l y s i s , Chi-square to  f o l l o w i n g Adams and J o l y  (1980b).  t e s t s of h e t e r o g e n e i t y were performed a c c o r d i n g  Zar (1984),  where the observed  two-locus  segregation  r a t i o s were t e s t e d a g a i n s t the n u l l h y p o t h e s i s t h a t t h e r e i s a 1:1:1:1 r a t i o of a l l e l i c  segregants  i n the e n t i r e  p o p u l a t i o n from which the sampled t r e e s came.  I f samples  are homogeneous, then the t o t a l of the c h i - s q u a r e from i n d i v i d u a l t r e e s (X n 2  degrees o f freedom  values  (df) equal t o  the number o f t r e e s i n the sample m u l t i p l i e d by t h r e e independent c l a s s e s ) should be of s i m i l a r magnitude as the c h i - s q u a r e c a l c u l a t e d from the pooled t o t a l s of each o f the four a l l e l i c  combinations  (x , 2  p  df = 3) .  The d i f f e r e n c e  between these two chi-square values i s i t s e l f c a l l e d the h e t e r o g e n e i t y chi-square Should X H 2  D  e  not  (X , df = d f 2  H  s i g n i f i c a n t , then p e r f o r m i n g  t e s t on t h e p o o l e d data  a chi-square, ( T )  - df  ( p )  ).  a goodness-of-fit  (Sokal and Rohlf 1981, Zar 1984) i s  justified. Partitioned,  s i n g l e degree of freedom g o o d n e s s - o f - f i t  t e s t s , with X A t e s t i n g the s e g r e g a t i o n of a l l e l e s at l o c u s 2  46  /  X B t e s t i n g the r a t i o at l o c u s B and %  A  2  t e s t i n g f o r the  2 h  e x i s t e n c e of l i n k a g e , were c a r r i e d out u s i n g the formulae of Bailey  (1961) f o r the analogous double backcross  linkage.  Bailey  (1961) notes t h a t the %  i s s t i l l v a l i d even i f one  of the two  d e v i a t e s from the expected 1:1 The  (G) was  here,  (%)  Schoen 1986), use  Cochran  the use  and  of  the  Although r e p o r t s of G  v a l u e s d e r i v e d from s e g r e g a t i o n  Stewart and  despite i t s  c l a s s with a value of zero,  f r e q u e n c i e s have been r e p o r t e d  not  t e s t s (Sokal  2  1981), because i t s c a l c u l a t i o n p r e c l u d e s  observed frequency  2  single-locus ratios  presented  l o g a r i t h m of which i s undefined.  in x  test for linkage  log-likelihood ratio test s t a t i s t i c  t h e o r e t i c a l advantages over chi-square  any  h  segregation.  employed i n the l i n k a g e analyses  Rohlf  2  model of  c l a s s e s with zero  cell  (Boyle and Morgenstern of j}  t e s t s was  1985,  chosen.  (1954) e s t a b l i s h e d t h a t i n order t o a v o i d b i a s  c a l c u l a t i o n s where sample s i z e s are s m a l l , no more  than 20%  of the expected c e l l  than 5.0.  frequencies  should be  T h i s c o n d i t i o n i s encountered f o r any  less  sample of  l e s s than twenty seeds.  For the purpose of d e t e c t i n g  l i n k a g e , t r e e s which had  l e s s than 20 megagametophytes  a v a i l a b l e were e l i m i n a t e d from the a n a l y s i s . The  criteria  t h i s study  f o r the d e t e c t i o n of l i n k a g e adopted f o r  are e s s e n t i a l l y those  of Strauss and  (1986) , namely t h a t l o c i would be c o n s i d e r e d  Conkle  linked i f % L 2  47  was  s i g n i f i c a n t i n more than one  s i n g l e t r e e c o u l d be confirmed F u r t h e r , Rudin and Ekberg  t r e e , or i f r e s u l t s from a  by r e p o r t s i n other c o n i f e r s .  (1978) note t h a t , f o r the  s i z e employed i n t h i s study  (20 seeds per  sample  doubly  heterozygous t r e e ) , the maximum degree of l i n k a g e t h a t be r e l i a b l y uncovered corresponds to an RF of 0.30. X  2  tests reveal significant  l i n k a g e , i t was  c a l c u l a t e d RF values i n excess of 0.30 4.3  R e s u l t s and  Should  decided t h a t  would be  any  discounted.  Discussion  Of the 12 l i n k a g e p a i r s represented tree,  may  by more than  10 d i s p l a y e d homogeneity of s e g r e g a t i o n  e n a b l i n g data t o be pooled w i t h i n each p a i r  one  among t r e e s ,  (Table  Heterogeneous data and  linkage p a i r s represented  t r e e w i l l be d i s c u s s e d  separately.  4.1).  by only  one  I t i s e v i d e n t t h a t i n a d d i t i o n to being homogeneous, the s e g r e g a t i o n  data f o r 7 of the 10 p a i r s  (PGI-2:G6P,  PGI-  2:PGM, PGI-2:IDH, G6P:PGM, G6P:IDH, G6P:MDH-1, and PGM:MDH1) when pooled n u l l hypothesis  yielded X  2  values too small to r e j e c t the .  of independent j o i n t  4.1).  Partitioned %  pooled  and  well.  None of these  2  segregation  (Table  values were a l s o n o n - s i g n i f i c a n t when  i n d i v i d u a l t r e e s were l a r g e l y n o n - s i g n i f i c a n t as combinations were found t o covary  the balsam f i r s t u d i e d by Neale and Adams (1981), so comparisons w i t h i n the genus are p o s s i b l e .  in  no  In D o u g l a s - f i r ,  El-Kassaby et a l . (1982a) found weak l i n k a g e between G6P  and  Table  4.1.  C h i - s q u a r e a n a l y s e s f o r p a i r - w i s e c o m b i n a t i o n s o f l o c i i n A. amabilis. X t e s t s h o m o g e n e i t y o f l i n k a g e among t r e e s ( w h e r e a p p l i c a b l e ) , X tests d e v i a t i o n from n u l l h y p o t h e s i s o f independent assortment. Partitioned % t e s t s ( f o r m u l a e o f B a i l e y ( 1 9 6 1 ) ) t e s t d e v i a t i o n a t l o c u s A, B a n d j o i n t d e v i a t i o n f r o m 1:1:1:1 s e g r e g a t i o n {% ) . 2  2  P  H  2  2  h  Loci  combination  No. o f t r e e s  X2 (df)/x  2  H  p  (df=3)  X (df=l) 2  A  X (df=D 2  B  % (df=l) 2  L  (42)  0 . 65  0.33  0.21  (30)  0 .00  0. 66  0.66  (3)  0 . 90  0.40  0. 90  (24)  0 .20  0.36  0.80  (3)  0 .40  0.00  0.40  (12)  1 .00  1.44  1.44  (6)  1 .07  1.67  0.07  0 . 60  19.27*  0.07  (12)  1 .44  38.44*  3.24  (3)  0 .10  19.60*  0.90  7  53.20 1.20 39.09 1.31 4 . 60 2.20 35. 64 2.36 5. 60 0.80 19. 68 3.52 9.20 2.80 1. 90 32.90* 11.00 39.60* 1. 60 20.60* 37.15  (18) *  -  -  -  MDH-1:AAT-2  2  10.00  (3) *  -  -  -  PGI-2:MDH-2 PGI-2:IDH PGM:MDH-2 IGH:MDH-1  1 1 1 1  20* 80 80 20  7 .20* 0 .20 0 .20 3 .20  1.80 1.80 1.80 1.80  0.20 1.80 0.80 0.20  PGI-2:G6P  15  PGI-2:PGM  11  PGI-2:IDH  2  G6P:PGM  9  G6P:IDH  2  G6P:MDH-1  5  PGM:MDH-1  3  PGI-2:AAT-2  3  G6PD:AAT-2  5  PGM:AAT-2  2  MDH-1:PGI-2  * = (P < 0.05) - = test not applicable.  -/9. -12. -12. -/5.  (6)  49  IDH  (RF = 0.33  ± 0.04)  and O'Malley et al.  t i g h t e r l i n k a g e between these two confidence 0.26).  interval  The  l o c i i n p i t c h pine  (CI) f o r recombination  l i n k a g e between G6P  i n c o n c l u s i v e i n p i t c h pine al.  (1986) d i s c o v e r e d  and PGM  (95% CI,  (95%  frequency,  was  0.20  considered  0.33-0.42; O'Malley et  1986). The  remaining  t h r e e p a i r s where data d i s p l a y e d among-  t r e e homogeneity i n v o l v e p o s s i b l e l i n k a g e s with AAT-2 2:AAT-2, G6P:AAT-2, and PGM:AAT-2). pooled x  2  for joint  segregation  < 0.05), s u g g e s t i n g partitioned %  2  linkage  analyses  d i s t o r t i o n at l o c u s B t o the d e v i a n t %  P  The X H v a l u e s 2  significant  (df = 3) was  (Table 4.1).  significant  (P  segregation  (AAT-2 i n a l l three cases) c o n t r i b u t e s as X i 2  (df = 1) v a l u e s i n d i c a t e  unlinked. f o r PGI-2:MDH-1 and MDH-1:AAT-2 are  and c o n c l u s i o n s as to t h e i r p o s s i b l e l i n k a g e s In the  i n s t a n c e , two.of seven t r e e s showed a s i g n i f i c a n t (df = 3) value and,  i n the p a r t i t i o n e d x  same t r e e s showed d e v i a t i o n s , one other at MDH-1.  2  The  first  individual  a n a l y s i s , these  at the PGI-2 l o c u s ,  These d e v i a t i o n s appear u n d i r e c t e d  c o n s i d e r i n g the small sample s i z e , may chance.  the  However, the  must be i n f e r r e d from i n d i v i d u a l t r e e data.  2  (PGI-  In a l l t h r e e cases,  s t r o n g l y suggest t h a t  value,  2  t h a t the p a i r s are  X  -  the  and  be a t t r i b u t e d to  f a c t t h a t none of the seven t r e e s e x h i b i t e d a  s i g n i f i c a n t X L suggests t h a t PGI-2 and MDH-1 2  are  unlinked  50  at t h i s l e v e l o f sampling. j o i n t l y vary  Only two t r e e s were found t o  f o r MDH-1 and AAT-2 but examination o f t h e  partitioned x  2  values  r e v e a l s t h a t unequal s e g r e g a t i o n a t  l o c u s B (AAT-2 i n t h i s case) may be the cause o f d i s t o r t e d j o i n t segregation  between these two l o c i ,  r a t h e r than a c t u a l  linkage. Of t h e four l i n k a g e - p a i r s found i n one t r e e only (PGI2:MDH-2, PGI-2:IDH, PGM:MDH-2 and IDH:MDH-1), only PGI2:MDH-2 y i e l d e d a s i g n i f i c a n t x  (df = 3) value  2  The  p a r t i t i o n e d a n a l y s i s revealed  that  (Table 4.1).  l i n k a g e was not as  much a f a c t o r as the s i n g l e - l o c u s d e v i a t i o n a t PGI-2, which may be a t t r i b u t e d t o chance.  The remaining t h r e e  pairs  showed no evidence o f i n d i v i d u a l - l o c u s d e v i a t i o n o r l i n k a g e . Weak l i n k a g e  (RF = 0.36 ± 0.05) has been r e p o r t e d  i n Norway  (Muona e t al.  spruce f o r MDH-2 and PGM-2 i n one t r e e only 1987). Contrary  t o many p u b l i s h e d  l i n k a g e was not evident between and  reports  i n other c o n i f e r s ,  i n t h i s sample o f P a c i f i c s i l v e r f i r  (PGI-2) and (AAT-2).  Tight l i n k a g e between AAT-2  PGI-2 (also r e f e r r e d t o as GOT-2 and GPI-2) was d e t e c t e d  i n balsam f i r by Neale and Adams f i r by El-Kassaby e t al. PGI-2 has been r e p o r t e d spruce  o f AAT l o c i  Linkage between AAT-1 and  f o r several species  (reviewed i n C h e l i a k  screening Pitel  (1982a).  (1981) and a l s o i n Douglas-  and P i t e l  o f pine and  1985).  Simultaneous  i n f i v e c o n i f e r genera by C h e l i a k and  (1985) r e v e a l p o s s i b l e homologies between AAT-1 and  51  AAT-2 and suggest the e v o l u t i o n of the AAT  enzyme system by  means of gene d u p l i c a t i o n . O b s e r v a t i o n of l i n k a g e between AAT  and PGI  in several  s p e c i e s and genera has strengthened arguments which  suggest  t h a t gymnosperm e v o l u t i o n has been very c o n s e r v a t i v e . G u r i e s et al.  (1978) note t h a t p i t c h and ponderosa p i n e ,  although separate s p e c i e s f o r at l e a s t two m i l l i o n years, appear t o possess the AAT:PGI l i n k a g e group. a l s o found c l o s e l i n k a g e between these two perhaps not i d e n t i c a l l o c i ,  Harry  (1986)  enzymes, but  i n incense-cedar,  s u g g e s t i n g the  maintenance of t h i s l i n k a g e s i n c e b e f o r e the divergence of the Pinaceae ago.  and Cupressaceae,  or some 160 m i l l i o n  years  Recently, however, data have been p u b l i s h e d which show  an order of magnitude d i f f e r e n c e i n recombination between these two ( B a r r e t t et al. d i f f e r e n c e may  loci  1987).  frequency  i n d i f f e r e n t samples of b l a c k I t was  spruce  s p e c u l a t e d t h a t the l a r g e  be due t o the c o l l e c t i o n l o c a t i o n of the  samples, w i t h marginal p o p u l a t i o n s expected t o show more v a r i a b i l i t y and have l o o s e r l i n k a g e than p o p u l a t i o n s sampled i n the c e n t e r of a s p e c i e s ' range, c o u l d not be r u l e d out. significant and 144  i n B a r r e t t et al.  seeds.  Morgenstern  The two  errors  t r e e s f o r which l i n k a g e  was  (1987) had sample s i z e s of 51  The o t h e r p u b l i s h e d r e p o r t (Boyle and  1985)  w i t h an average  however, sampling  e s t a b l i s h e d l i n k a g e based on e i g h t t r e e s  of 12 seeds per t r e e .  The d i s c r e p a n c y i n RF  52  values  may be a r e s u l t of d i s s i m i l a r sample s i z e s ,  than t r u e p o p u l a t i o n  differences.  Rudin and Ekberg  (1978) c a u t i o n t h a t because c o n i f e r s  have l o n g chromosomes, ordinary %  2  l o c i may be so widely separated  a n a l y s i s w i l l not uncover l i n k a g e s unless  s i z e s are very estimating  rather  large.  Alternate  sample  (Bayesian) methods f o r  recombination f r e q u e n c i e s  Nordheim et al.  that  have been proposed by  (1983), but they a l s o note that " f o r  recombination values  c l o s e t o 0.5 d e t e c t i o n of l i n k a g e and  good e s t i m a t i o n  r e q u i r e l a r g e sample s i z e s  will  regardless  of s p e c i f i c methodology." Perhaps the s t r e n g t h o f evidence f o r h i g h l y l i n k a g e groups l i e s not only  i n the measurement  conserved o f RF's f o r  i n d i v i d u a l t r e e s u s i n g s u b s t a n t i a l sample s i z e s , but i n the d e t e c t i o n o f l i n k a g e of s i m i l a r magnitude i n a l a r g e proportion  o f t r e e s heterozygous f o r a given p a i r o f l o c i .  Homogeneity o f l i n k a g e can only be observed where permit the s c r e e n i n g  of s u f f i c i e n t t r e e s t o detect  resources enough  double heterozygotes f o r among-tree v a r i a t i o n i n recombination frequency t o be r e l i a b l y estimated.  Larger-  s c a l e s t u d i e s c o u l d begin t o address the causes of population  d i f f e r e n c e s i n l i n k a g e such as those observed by  B a r r e t t et al. Ekberg  (1987) f o r AAT and PGI, and by Rudin and  (1978) f o r LAP ( l e u c i n e amino peptidase) and AAT.  More c o n v i n c i n g  data may r e v e a l apparent d i f f e r e n c e s i n  l i n k a g e among t r e e s and p o p u l a t i o n s  of the same s p e c i e s i n  53  f a c t have a g e n e t i c b a s i s  (i.e.,  genetic v a r i a t i o n i n  frequency o f c r o s s i n g over) and/or are s u b j e c t t o environmental i n f l u e n c e s  ( i . e . , temperature a t time o f  m e i o t i c a c t i v i t y ; Rudin and Ekberg 1978).  Such  information  c o u l d prove u s e f u l i n f u t u r e mapping of q u a n t i t a t i v e t r a i t loci  i n conifers. In the present  by X  2  analyses  study, no c o n c l u s i v e l i n k a g e i s r e v e a l e d  i n any o f the 16 l o c u s - p a i r s t e s t e d .  Although t h r e e  l i n k a g e s with AAT-2 i n d i c a t e d s i g n i f i c a n t  d e v i a t i o n from independent assortment, suggesting  linkage,  p a r t i t i o n e d X t e s t s showed the d e v i a t i o n t o be i n f l u e n c e d 2  e n t i r e l y by s e g r e g a t i o n  d i s t o r t i o n at AAT-2.  previously,  distortion  segregation  i t s e l f does not  i n v a l i d a t e t h e t e s t o f independent assortment however, i t can cause b i a s i n t h e e s t i m a t i o n o u t c r o s s i n g r a t e s and p o l l e n pool a l l e l e (Cheliak et  al.  polymorphism estimation  1984).  As mentioned  ( B a i l e y 1961), o f both  frequencies  For t h i s reason, t h e apparent  observed i n AAT-2 w i l l not be u t i l i z e d  i n the  o f parameters o f t h e mating system o f P a c i f i c  silver f i r . 4.4  Conclusions  (a)  Independence  amongst the 16 l o c u s - p a i r s a v a i l a b l e f o r  t e s t i n g c o u l d not be r u l e d out, thus p r o v i d i n g s i x polymorphic l o c i on which mating system parameters may be estimated  (IDH, MDH-1, MDH-2, PGM, G6P and PGI-2.) .  Linkages with AAT-2 were not confirmed, but severe segregation d i s t o r t i o n  (30% i n excess of the  e x p e c t a t i o n f o r the most common a l l e l e ) this  1:1  observed at  l o c u s , suggests i t not be used as a marker l o c u s  i n a study of the mating system  in Pacific  silver f i r .  55  5. 5.1  MATING SYSTEM  Introduction The  nature  and extent o f g e n e t i c v a r i a t i o n i n a s p e c i e s i s  l a r g e l y determined by i t s p a t t e r n o f b r e e d i n g . mating c o n s t i t u t e s t h e l i n k ..between s u c c e s s i v e  I t s system o f generations  whereby g e n e t i c i n f o r m a t i o n i s t r a n s f e r r e d , o r g a n i z e d and d i s t r i b u t e d among progeny  (Clegg 1980).  P l a n t s p e c i e s which  p r a c t i s e a h i g h degree o f c r o s s - f e r t i l i z a t i o n are more g e n e t i c a l l y v a r i a b l e than s p e c i e s which reproduce v i a s e l f fertilization particular,  (Hamrick et al. 1979).  Forest trees, c o n i f e r s i n  a r e c o n s i d e r e d t o be among t h e most heterozygous  p l a n t s known, a t l e a s t at the l e v e l o f enzyme v a r i a t i o n (Hamrick 1982). t o be high,  Mating system s t u d i e s have shown most c o n i f e r s  although  not o b l i g a t e , o u t c r o s s e r s  (see Adams and  B i r k e s 1990 f o r a review).  Because t h e r e p r o d u c t i v e process o f  many e c o n o m i c a l l y  c o n i f e r s i s c h a r a c t e r i z e d by some  important  n a t u r a l s e l f - f e r t i l i z a t i o n and most e x h i b i t l a r g e amounts o f growth d e p r e s s i o n Sorensen 1982), become important  as a r e s u l t o f i n b r e e d i n g  accurate estimates i n planning  ( F r a n k l i n 1970,  of l e v e l s o f i n b r e e d i n g  and implementation o f t r e e  improvement programs. The mating systems of gymnosperms have been s t u d i e d e x t e n s i v e l y i n recent years, e l e c t r o p h o r e t i c single-gene  owing t o the a v a i l a b i l i t y o f markers and the unique s t r u c t u r e of  c o n i f e r seeds which e l i m i n a t e s the n e c e s s i t y o f c o n t r o l l e d  56  matings  (Brown et al. 1975, Shaw and A l l a r d 1982a).  l e v e l estimates  of o u t c r o s s i n g based on allozyme l o c i  exceed 90% (Adams and B i r k e s 1990), although p o p u l a t i o n estimates  Speciesgenerally  individual  were found t o be as low as 54% (tamarack,  Knowles e t al. 1987) and 78% (balsam f i r , Neale and Adams 1985b).  As w e l l , i n d i v i d u a l t r e e o u t c r o s s i n g r a t e v a r i a t i o n  has been r e p o r t e d  (60 t o 100% i n a n a t u r a l p o p u l a t i o n o f  western white pine, El-Kassaby et al. 1987b; 20 t o 100% i n an o r c h a r d p o p u l a t i o n o f D o u g l a s - f i r , El-Kassaby e t al. 1986 and 78-100% i n a c l o n a l orchard white spruce Schoen 1988).  population,  Both environmental and g e n e t i c f a c t o r s have been  shown t o i n f l u e n c e mating systems ' (Clegg 1980). r a t e s were r e p o r t e d t o vary with stand d e n s i t y Mitton  Denti and  Outcrossing  ( F a r r i s and  1984, Shea 1987), e l e v a t i o n ( P h i l l i p s and Brown 1977,  Neale and Adams 1985b) and p o p u l a t i o n  substructuring (Ritland  and El-Kassaby 1985). The  mating system of P a c i f i c s i l v e r f i r i s of i n t e r e s t  given i t s p a r t i c u l a r s i l v i c a l The  and e c o l o g i c a l c h a r a c t e r i s t i c s .  h i g h shade t o l e r a n c e o f P a c i f i c s i l v e r f i r permits the  development o f f a m i l y s t r u c t u r e w i t h i n p o p u l a t i o n s .  The  presence o f f a m i l y s t r u c t u r e i s conducive t o mating among r e l a t i v e s w h i l e the cone-bearing  h a b i t of t h e s p e c i e s  (females  r e s t r i c t e d t o the t o p 20 t o 30% of the canopy, most male cones i n t h e lower p o r t i o n o f the crown) promotes c r o s s - f e r t i l i z a t i o n ( F r a n k l i n and R i t c h i e 1970, Owens and Molder 1977).  Noble f i r  has been shown t o possess r e l a t i v e l y high s e l f - f e r t i l i t y  among  57  conifers  (Sorensen et al. 1976).  Although no c o n t r o l l e d mating  s t u d i e s have been r e p o r t e d f o r P a c i f i c s i l v e r f i r , the s p e c i e s is  c h a r a c t e r i z e d by very low y i e l d s of f i l l e d  1974,  Owens and Molder In  seed  (Franklin  1977).  t h i s chapter, estimates of mating system parameters of  seven p o p u l a t i o n s of P a c i f i c s i l v e r f i r on Vancouver u t i l i z i n g allozyme markers are r e p o r t e d .  Island  P o s s i b l e causes and  i m p l i c a t i o n s of o u t c r o s s i n g r a t e v a r i a t i o n are d i s c u s s e d . A s p e c t s of the r e p r o d u c t i v e b i o l o g y and e c o l o g y of P a c i f i c s i l v e r f i r which may  p l a y a r o l e i n d e t e r m i n i n g mating  b e h a v i o u r are a l s o c o n s i d e r e d . 5.2  M a t e r i a l s and Methods Cones c o l l e c t e d from seven p o p u l a t i o n s o f P a c i f i c  fir  on Vancouver  Island  ( l o c a t i o n s g i v e n i n Table 2.1,  e x c l u d i n g p o p u l a t i o n F) were used i n t h i s study. w e r e sampled  Eight  as d e s c r i b e d i n Chapter 2.  During cone  and seed p r o c e s s i n g , the i d e n t i t y of each mother t r e e Two  seed samples were randomly  t h r e e t o s i x (average 4.7) x-rayed  cones per t r e e .  was  drawn from each of These samples were  ( d e s c r i b e d i n Chapter 2) and the percentage of  seeds determined.  Seed s i z e was  trees.  filled  i n d i r e c t l y estimated using  1000-seed weight measures based on samples of f i l l e d individual  trees  i n p o p u l a t i o n s A, B and C, 13 i n N, 11 i n H and R  and 17 i n p o p u l a t i o n W,  maintained.  silver  seeds from  S t a r c h g e l e l e c t r o p h o r e s i s o f megagametophyte h a p l o i d g e n e t i c composition  as t h e egg) and  (same  corresponding  embryo t i s s u e s was conducted a c c o r d i n g t o methods o u t l i n e d i n s e c t i o n 3.2 and d e t a i l e d i n Appendix 2. G6P, PGM,  although  and l i n k a g e s t u d i e s  3 and 4) which i n d i c a t e d they were polymorphic, not i n a l l p o p u l a t i o n s ,  Maternal allozymes  (PGI-2,  IDH and MDH-1) were s e l e c t e d f o r e s t i m a t i o n o f matin  system parameters based on s e g r e g a t i o n (Chapters  F i v e enzymes  and i n h e r i t e d  independently.  genotypes were i n f e r r e d from t h e s e g r e g a t i o n o f  i n megagametophyte t i s s u e of 18 t o 20 seeds per t r e e  The p r o b a b i l i t y o f i n c o r r e c t l y c l a s s i f y i n g a heterozygote any  one l o c u s i s ( 0 . 5 )  k_1  at  where k = number o f seeds ( T i g e r s t e d t  1973), and f o r a sample of t h i s s i z e , very c l o s e t o zero. Tandem assay o f megagametophyte and d i p l o i d embryo r e v e a l s the pollen contribution directly.  The s e g r e g a t i o n p a t t e r n s of  marker l o c i i n these progenies  provide the data upon which  estimates  o f t h e extent  o f apparent o u t c r o s s i n g a r e based.  S i n g l e - and m u l t i - l o c u s p o p u l a t i o n estimates  of outcrossing  rate  frequencies  ( t and t ) and o u t c r o s s e d p o l l e n a l l e l e s  m  (p)  were c a l c u l a t e d u s i n g the maximum l i k e l i h o o d procedure o f R i t l a n d and El-Kassaby  (1985).  The procedure i s based on a  m u l t i - l o c u s , mixed mating system model which was shown t o be s t a t i s t i c a l l y more e f f i c i e n t than 'observed o u t c r o s s ' models (e.g., Shaw and A l l a r d 1982a), e s p e c i a l l y when r e l a t i v e l y few l o c i are assayed.  A m u l t i - l o c u s estimate  c o n s i d e r e d more accurate  of outcrossing i s  and l e s s s e n s i t i v e t o v i o l a t i o n s of  59  model assumptions because a g r e a t e r number o f o u t c r o s s e s may be i d e n t i f i e d with c e r t a i n t y ( i . e . the p o l l e n a l l e l e i s d i s t i n g u i s h a b l e i n i t s g e n e t i c o r i g i n - see Shaw e t al. 1981 f o r more d e t a i l ) .  A d d i t i o n a l l y the model takes advantage o f  information contained  i n the o f f s p r i n g of mothers which a r e  heterozygous f o r a given  locus.  The mixed mating model i s u s u a l l y employed t o study p a t t e r n s o f mating i n c o n i f e r s because i t s attendant assumptions a r e c o n s i d e r e d more b i o l o g i c a l l y merely assume panmixis  (Schoen and Clegg  reasonable  than t o  1984), where t h e  p r o b a b i l i t y o f mating between i n d i v i d u a l s of a p a r t i c u l a r genotype i s s t r i c t l y equal t o the product frequencies  i n the population  (Hedrick  of t h e i r  1983) .  individual  The model  assumes t h a t (i)  f e r t i l i z a t i o n events are a mixture of random o u t c r o s s i n g and s e l f - f e r t i l i z a t i o n ;  (ii)  t h e r e i s no s e l e c t i o n between f e r t i l i z a t i o n and census;  ( i i i ) t h e r a t e of o u t c r o s s i n g i s independent of the genotype of the mother t r e e ; (iv)  a l l e l e s at d i f f e r e n t  l o c i a c t independently ( f o r  m u l t i - l o c u s estimates) and (v)  allele  frequencies  i n the o u t c r o s s i n g p o l l e n p o o l  are i d e n t i c a l over the p o p u l a t i o n o f mother t r e e s (Fyfe and B a i l e y 1951, Shaw et al. 1981).  60  The  l a s t assumption i s one t h a t , although  (1980) as "fundamental", may stands  of t r e e s .  viewed by  not be e a s i l y met  Clegg  in natural  The d i s t r i b u t i o n o f p o l l e n i n a g i v e n  p o p u l a t i o n i s most l i k e l y s u b j e c t t o both p h e n o l o g i c a l  and  s p a t i a l v a r i a t i o n , which c o u l d r e s u l t i n the p o l l e n genotypes r e c e i v e d by maternal t r e e s b e i n g c o r r e l a t e d (Schoen and  Clegg  1984) . Population F  (9 t r e e s ) was  study but no estimates  o r i g i n a l l y included i n t h i s  f o r o u t c r o s s i n g r a t e s c o u l d be  u s i n g the model of R i t l a n d and El-Kassaby  (1985).  procedure f o r e s t i m a t i n g t and p i s i t e r a t i v e and, of F,  f a i l e d t o produce convergent v a l u e s .  El-Kassaby  case  R i t l a n d and  ( i n the Appendix) t h a t  do not always converge i f parents  orchard  "estimates  are heterozygous at s e v e r a l  y i e l d i n g too great a number of n o n - d i s c e r n a b l e  mating  For the three l o c i which were v a r i a b l e among a l l  populations  of P a c i f i c s i l v e r f i r ,  F had the  p r o p o r t i o n of heterozygous mothers at two G6P).  i n the  f a i l e d t o converge f o r 15-25% o f t h e i r  D o u g l a s - f i r sample and note  events.  The  (1985) found t h a t i n d i v i d u a l female t r e e o u t c r o s s i n g  r a t e estimates  loci",  obtained  T h i s may  greatest  of them  (PGI-2  have c o n t r i b u t e d to the f a i l u r e o f the model t o  produce e s t i m a t e s  of t and p f o r t h i s  population.  A l t e r n a t i v e l y , the nonconvergence of estimates i n d i c a t i v e of a f a i l u r e of one (Schoen 1988;  and  may  be  or more model assumptions  Dr. K. R i t l a n d , p e r s . comm., March 1989).  mixed mating model assumes t h a t " s u c c e s s i v e o u t c r o s s  The  events  61  w i t h i n a f a m i l y a r i s e from independent draws of p o l l e n from t o t a l population  of male p l a n t s "  previously stated, phenological p o l l e n d i s t r i b u t i o n may  (Schoen and  Clegg 1984).  modify the f e r t i l i z a t i o n p r o b a b i l i t i e s  females are c o r r e l a t e d t o some degree. l i k e l y t o occur when r e l a t i v e l y  few  (Schoen 1988).  This scenario  Population  F i s the  (1970) observed c l o s e  before  cloud.  synchrony•in•pollen  seed cone r e c e p t i v i t y i n P a c i f i c s i l v e r f i r ,  a l s o noted c o n s i d e r a b l e majority  highest  act to reduce  the number of parents c o n t r i b u t i n g to the p o l l e n  d i s p e r s a l and  i s most  amounts of  p r e c i p i t a t i o n and/or a short growing season may  Ritchie  t r e e to t r e e v a r i a t i o n , where  but  "the  of P a c i f i c s i l v e r f i r t r e e s began shedding p o l l e n  female s t r o b i l i were r e c e p t i v e ,  and  one  tree actually  shed the bulk of i t s p o l l e n s e v e r a l days p r i o r to t h i s Further  by  males are c o n t r i b u t i n g t o  e l e v a t i o n sample i n t h i s study, where high  F r a n k l i n and  As  and/or s p a t i a l v a r i a t i o n i n  of p o l l e n genotypes such t h a t p o l l e n genotypes r e c e i v e d  the p o l l e n c l o u d  the  stage".  support f o r non-uniform p o l l e n d i s t r i b u t i o n i n t h i s  population  comes from the  sampled i n t h i s stand but f i l l e d seed and  was  f a c t that ten t r e e s were o r i g i n a l l y one  t r e e y i e l d e d l e s s than  eliminated.  Because estimates of t < 1.0 actual inbreeding  0.1%  may  be due  to some amount of  or non-homogeneous d i s t r i b u t i o n of p o l l e n  (Ennos and Clegg 1982), a t e s t f o r i n t r a p o p u l a t i o n p o l l e n a l l e l e heterogeneity  (Brown et al.  mother t r e e s i n each p o p u l a t i o n  was  1975)  u s i n g homozygous  conducted f o r the  three  62  commonly v a r i a b l e l o c i heterozygous  progeny  (PGI-2, G6P and PGM).  The number o f  (= d i s c e r n i b l y outcrossed)  and the number  of progeny w i t h genotypes i d e n t i c a l t o t h e maternal t r e e ( i n c l u d e s u n d e t e c t a b l e o u t c r o s s e s p l u s any s e l f s ) were compared f o r each l o c u s and p o p u l a t i o n u s i n g contingency % When forms o f i n b r e e d i n g other than d i r e c t the amount of s e l f i n g w i l l be o v e r e s t i m a t e d 1982a).  2  tests.  selfing  occur,  (Shaw and A l l a r d  Since t h e m u l t i - l o c u s method i s l e s s s e n s i t i v e t o  v i o l a t i o n s o f the assumptions of the mixed mating model (Shaw et al. 1981), when consanguineous matings occur, the m u l t i A  l o c u s e s t i m a t e s are presumed t o be l e s s b i a s e d , and thus t A.  m  A  would exceed t . s  A comparison of t  m  and the minimum v a r i a n c e A  mean s i n g l e l o c u s o u t c r o s s i n g r a t e e s t i m a t e  (t ) p r o v i d e s an s  i n d i c a t i o n of t h e extent of mating other than s e l f i n g A l l a r d 1982a, El-Kassaby 5.3  (Shaw and  et al. 1987b).  R e s u l t s and D i s c u s s i o n 5.3.1  A l l e l e frequencies  Estimates of a l l e l e both t h e maternal  f r e q u e n c i e s at f i v e enzyme l o c i f o r  (ovule) and o u t c r o s s e d p o l l e n gene pools (p)  over seven p o p u l a t i o n s of P a c i f i c s i l v e r confidence i n t e r v a l s , common a l l e l e  are l i s t e d  i s reported.  along with 95%  i n Table 5.1.  Only the most  D i f f e r e n c e s between gene pools a t  any one l o c u s were determined confidence i n t e r v a l s  fir,  by comparing t h e o v e r l a p of  (P <0.05; Jones and M a t l o f f 1986, E l -  Table 5.1  A l l e l i c frequencies (most common a l l e l e ) and their 95% confidence intervals for the maternal (Ovule, 0) and outcrossing pollen (P) gene pools for the seven P a c i f i c s i l v e r f i r populations on Vancouver Island.  Population Locus  PGI-2  G6P  PGM  IDH  MDH-1  Gene  16 +  16  B  16 Z 159  26  22  N  26 R 258  34 W 335  pool  A  0  0.750 ± 0.212  0.812 ± 0.191  0.750 ± 0.212 0.769 ± 0.162 0.773 ± 0;175  0.538 ± 0.192  0.559 ± 0.167  P  0.786 ± 0.064  0.825 ± 0.059  0.887 ± 0.049 0.868 ± 0.051 0.694 ± 0.061  0.605 ± 0.060  0.704 ± 0.049  0  0.750 ± 0.212  0.875 ± 0.162  0.812 ± 0.191 0.769 ± 0.162 0.727 ± 0.186  0.923 ± 0.102  0.676 ± 0.157  P  0.736 ± 0.067  0.775 ± 0.065  0.817 ± 0.060 0.822 ± 0.047 0.827 ± 0.050  0.818 ± 0.047  0.803 ± 0.043  0  0.812 ± 0.191  0.937 ± 0.119  1.000 ± 0.000 0.808 ± 0.151 0.909 ± 0.120  0.923 ± 0.102  0.647 ± 0.161  P  0.672 ± 0.073  0.944 ± 0.036  0.905 ± 0.046 0.837 ± 0.049 0.808 ± 0.052  0.845 ± 0.044  0.716 ± 0.048  0  1.000 ± 0.000  0.962 ± 0.073  P  0.994 ± 0.012  1.000 ± 0.000  159  160  H  258  219  O  0.962 ± 0.073 0.964 ± 0.078  0.923 ± 0.102  P  0.996 ± 0.008 1.000 ± 0.000  0.988 ± 0.013  t S u p e r s c r i p t s r e p r e s e n t the number o f maternal genes sampled and s u b s c r i p t s represent the number o f embryos sampled.  64  Kassaby e t al.  1987b).  With the e x c e p t i o n  o f the  PGM  l o c u s i n p o p u l a t i o n C, no s i g n i f i c a n t d i f f e r e n c e s between the two  gene p o o l s were observed, i n d i c a t i n g t h a t  the  o u t c r o s s e d p o l l e n p o o l i s r e p r e s e n t a t i v e of the maternal p o p u l a t i o n , or conversely,  t h a t the maternal t r e e s are  r e p r e s e n t a t i v e o f the stands (Brown et al.  1975,  i n which they were c o l l e c t e d  El-Kassaby et al.  noteworthy t h a t the confidence  1987b).  i n t e r v a l s of p o l l e n a l l e l e  f r e q u e n c i e s are s m a l l e r than t h a t of ovule frequencies.  It i s also  allele  T h i s i s expected because of v a r i a t i o n i n sample  size  (Brown et al.  1975)  pool  (with l a r g e r 'n')  and r e i n f o r c e s use  estimates  of the p o l l e n  i n population genetic studies  where the sample o f maternal parents  i s limited  (El-Kassaby  1990) . The  penetrance of a l l e l e s  apparent from Table 5.1.  across p o p u l a t i o n s  Populations  most v a r i a b l e i n a l l e l i c composition p o p u l a t i o n W f o r G6P  and PGM.  i s also  R and W tend t o be  the  f o r the PGI-2 l o c u s  and  These two  stands  n e a r l y 2 degrees l a t i t u d e , with W b e i n g one  differ  by  of the most  s o u t h e r l y c o l l e c t i o n s and R the most n o r t h e r n .  The  of the p o p u l a t i o n s possess q u i t e s i m i l a r a l l e l e  frequencies.  T h i s p a t t e r n has been d e s c r i b e d i n other Abies frequency fir  d i f f e r e n c e s at e i g h t l o c i  remainder  species.  i n four stands  Allele  of balsam  l o c a t e d along a steep e l e v a t i o n a l t r a n s e c t were viewed by  Neale and Adams (1985b) as being s m a l l . and Adams' study  The  stands  i n Neale  spanned a d i s t a n c e of l e s s than f o u r km,  but  65  represented  a range i n a l t i t u d e of some 610  m.  The  apparent  l a c k o f v a r i a t i o n i n t h e i r maternal a l l e l e f r e q u e n c i e s  was  c o n t r a s t e d w i t h steep c l i n e s observed on the same t r a n s e c t by F r y e r and L e d i g  (1972) i n s e e d l i n g q u a n t i t a t i v e t r a i t s .  A wider-ranging  study  of balsam f i r by Jacobs et  (1984) a l s o r e v e a l e d l o c i with s i m i l a r allozyme among 12 p o p u l a t i o n s two  populations  o n l y one  frequencies  sampled from North C a r o l i n a to Maine.  of subalpine  In  f i r growing on c o n t r a s t i n g s i t e s ,  of seven l o c i showed s i g n i f i c a n t  frequencies  al.  differences in allele  (Shea 1987).  Table 5.1  a l s o r e v e a l s that not a l l l o c i are v a r i a b l e  a c r o s s a l l seven p o p u l a t i o n s ,  however a l l e l i c  at l e a s t  i n the p o l l e n pool, at PGI-2, G6P  stands.  Vaquero et al.  variation  and PGM  (1989) note that l o c i with  exists,  in a l l relatively  r a r e a l l e l e s c r e a t e a l a r g e number of empty genotypic  classes  i n a r r a y s of progeny from a s i n g l e mother, which i n turn, cause difficulties  i n estimating outcrossing rates.  s i n g l e l o c u s o u t c r o s s i n g r a t e estimates maternal genotype f r e q u e n c i e s . monomorphic  (p  A  recovered  depend on a l l e l e  Marker l o c i t h a t are  > 0.970; Shaw and A l l a r d  i n f o r m a t i o n on o u t c r o s s i n g  P r e c i s i o n of  heterozygotes  i n the progeny) and produce l a r g e v a r i a n c e s  A l l a r d 1982a, R i t l a n d 1983).  virtually  1982a) add very  (because so few  Brown et al.  and  little are  (Shaw and  (1975) p o i n t  out  t h a t the v a r i a n c e of a given s i n g l e locus t value i s minimized when a l l e l e f r e q u e n c i e s are equal.  Given these  m u l t i - l o c u s o u t c r o s s i n g rate estimates  considerations,  were c a l c u l a t e d using  Table  5.2  For three commonly variable l o c i , the r a t i o of discernibly outcrossed (heterozygous) embryos to embryos possessing the maternal genotype, followed by the heterogeneity X (df) value. 2  Population Locus  PGI-2  A  W  1/1 6 : 7 4 7.93M3) 3/3  G6P  1/1 23:57 2.78(3)  PGM  1/1 29:71 3.11(4) 3/3  B  C  H  N  R  30:50  7 : 9 3  2:97  11:148  23:117 1 7 : 4 3  6 . 5 6 (3)  3 . 9 9 (4)  8 . 1 6 (4)  9 . 0 2 (7)  3 . 4 2 (6)  3 5 : 5  1 7 : 3  13:7  8:12  5.71*(1)  (0)  (0)  (0)  15.87*(2) 2 4 : 1 6 10.41MD  26:93  25:94  22:98  21:99 11:89  36:184  2.23(5)  2.79(5)  3.79(5)  2.60(5)  3.47(4)  13.42(10)  28:183  10:130  10:130  20:140  32:143  36:184  7.72(5)  5.80(6)  4.39(6)  5.49(7)  11.32(8)  31.28* (10) t  32:7 4.68*(1)  * Significant  at P <0.05.  t T h i s X v a l u e made s i g n i f i c a n t 2  by one t r e e out of 11  ( > 50% of value of X H ) 2  (TI ON  67  a l l v a r i a b l e l o c i i n each p o p u l a t i o n  ( t ) and as w e l l , u s i n g  o n l y t h e t h r e e most polymorphic l o c i  (PGI-2, G6P and PGM) which  were a l s o common t o a l l p o p u l a t i o n s  m  (t ) .  Some p o l l e n a l l e l i c h e t e r o g e n e i t y  mc  within populations i s  r e v e a l e d by a n a l y z i n g heterozygous'(= d e t e c t a b l y  outcrossed)  genotype f r e q u e n c i e s among homozygous mother t r e e s , l i s t e d i n Table 5.2.  The r a t i o s o f heterozygous t o maternal-type progeny  (summed over t r e e s ) are given f o r the t h r e e commonly v a r i a b l e loci.  R e s u l t s a r e s t r u c t u r e d by l a t i t u d e o f p o p u l a t i o n .  These  data suggest t h e r e i s some v a r i a b l e p e n e t r a t i o n o f p o l l e n a l l e l e s within populations,  however most appear homogeneous  (only s i x o f 24 contingency  % t e s t s were s i g n i f i c a n t ; P <  0.05).  2  These r e s u l t s should be i n t e r p r e t e d w i t h some c a u t i o n  as Cochran  (1954) showed t h a t the y} t e s t i s v a l i d where no  more than 20% o f t h e c l a s s e s have expected f r e q u e n c i e s o f l e s s than 5.0. performed.  T h i s c r i t e r i o n was met i n only f o u r o f t h e 24 t e s t s Brown et a i . (1975) a l s o f a i l e d t o meet t h i s  c r i t e r i o n f o r some o f t h e i r data, however t h e t e s t was s t i l l used t o i d e n t i f y l o c a l p o l l e n h e t e r o g e n e i t y .  Non-uniform  d i s t r i b u t i o n o f p o l l e n a l l e l e s over maternal t r e e s r e s u l t s i n an underestimate o f the extent 1975).  of outcrossing  Intra-population heterogeneity  may be t h e r e s u l t o f  tree-to-tree v a r i a t i o n i n outcrossing rate assumption  (iii)  (Brown et al.  (a v i o l a t i o n o f  o f the mixed mating model) and/or non-  uniformity of pollen a l l e l e d i s t r i b u t i o n  (violating  assumption  68  (v) , l i s t e d  i n s e c t i o n 5.2) but Brown et al. (1985) a s s e r t t h a t  contingency  %  5.3.2  2  t e s t s cannot d i s c r i m i n a t e between causes.  Outcrossing  rates  S i n g l e - and m u l t i - l o c u s o u t c r o s s i n g r a t e estimates a r e listed  i n Table 5.3.  different  S i n g l e locus estimates  were s i g n i f i c a n t l y  from t = 1.0 f o r at l e a s t one l o c u s i n ,five o f seven  populations.  Estimates  o f o u t c r o s s i n g v a r i e d from as low as  48% t o 100% and the PGI-2 locus gave c o n s i s t e n t l y lower estimates  than other l o c i .  V a r i a t i o n i n s i n g l e locus  of o u t c r o s s i n g i s common among c o n i f e r s ( D o u g l a s - f i r , Kassaby e t al. 1981, 1986, 1 9 8 8 ; Shaw and A l l a r d  estimates El-  1982a; Neale  and Adams 1985a; R i t l a n d and El-Kassaby 1985; Yeh and Morgan 1987; al.  balsam f i r , Neale and Adams 1985b; tamarack, Knowles e t 1985; white spruce,  b l a c k spruce, jack p i n e ,  King et al. 1984; C h e l i a k et al. 1985b;  Boyle and Morgenstern 1 9 8 6 ; B a r r e t t et al. 1987;  C h e l i a k et al. 1985a; Snyder et a l . 1985; lodgepole  pine, Epperson and A l l a r d  1984; Perry and Dancik 1986; J e f f r e y  pine, F u r n i e r and Adams 1 9 8 6 ; Western white pine, El-Kassaby et al.  1987b; ponderosa pine, Mitton  Mitton  et al. 1981; F a r r i s and  1984; Scots pine, El-Kassaby et al. 1989; l o b l o l l y  Friedman and Adams 1985).  This v a r i a t i o n i s most  pine,  likely  statistical  (as d i s c u s s e d p r e v i o u s l y ) because even i f a l l e l e  frequencies  (p's)  are the same there w i l l be v a r y i n g genotypes  among mother t r e e s sampled i n each p o p u l a t i o n ,  which w i l l  possess d i f f e r e n t powers of d e t e c t i o n of o u t c r o s s i n g  events.  Table 5.3  Locus  Single-locus and multi-locus estimates of outcrossing rate for seven p o p u l a t i o n s of P a c i f i c s i l v e r f i r from Vancouver Island, B.C. (95% confidence i n t e r v a l s ) .  A  B  Population H  C  N  R  W  PGI-2  0.478 ± 0.206*  0.446 ± 0.213*  0.669 ± 0.234*  0.647 ± 0.175*  0.461 ± 0.154*  0.892 ± 0.167  1.204 ± 0.152  G6P  0.740 ± 0.174*  0.906 ± 0.209  0.913 ± 0.234  0.903 ± 0.178  0.803 ± 0.190*  0.947 ± 0.153  1.066 ± 0.151  PGM  0.853 ± 0.206  0.999 ± 0.220  0.842 ± 0.289  0.870 ± 0.179  0.555 + 0.212  0.999 ± 0.173  1.001 ± 0.107  0.899 ± 0.269  0. 999 ± 0.188  0. 949 ± 0.214  IDH  0.900 ± 0.515  MDH-  0.999 ± 0.175  1  A  t  s  0.696 + 0.112*  0.787 ± 0.120*  0.803 ± 0.144*  0.859 ± 0.083*  0.684 ± 0.091*  0.946 ± 0.087  1.068 + 0.076  t  m  0.762 + 0.115*  0.888 ± 0.130  0.798 + 0.148*  0.869 ± 0.091*  0.725 ± 0.107*  0.993 ± 0.062  1.089 ± 0.073  t  m r  0.762 ± 0.115*  0.867 ± 0.137  0.798 ± 0.148*  0.847 ± 0.100*  0.650 ± 0.116*  0.976 ± 0.080  1.089 ± 0.073  * S i g n i f i c a n t at P < 0.05. t  s  t  m c  - minimum variance mean, t  m  - multi-locus outcrossing rate.  - multi-locus outcrossing rate based on three common l o c i .  70  Statistical  f l u c t u a t i o n i s reduced by adding more l o c i t o t  estimates.  Yeh  and Morgan  (1987) p o s t u l a t e t h a t some e a r l y  z y g o t i c s e l e c t i o n a g a i n s t s e l f e d genotypes may i n o u t c r o s s i n g r a t e s among l o c i . t h a t t h i s may loci,  Brown et al.  create  variation  (1985) suggest  not be a d e s i r a b l e argument f o r e l e c t r o p h o r e t i c  which are c o n s i d e r e d more remote from s e l e c t i o n than  c o n t r o l l i n g morphological  traits  loci  ( R i t l a n d 1983).  Whether the v a r i a t i o n i s s t a t i s t i c a l or l o c i do not f i t assumptions of the mating model, the g r e a t e r degrees o f freedom p r o v i d e d by m u l t i - l o c u s estimates estimate  of outcrossing  from Table 5.3 estimates  (Shaw et al.  1981).  from 1.0  i n a l l populations  biased  I t can be  t h a t o u t c r o s s i n g r a t e s based on  differ  Shaw and A l l a r d  make them a l e s s  multi-locus  except B, R and  (1982a) suggest t h a t where forms o f  ( i . e . mating among r e l a t i v e s ) i n a d d i t i o n t o s e l f i n g then t  m  seen  W.  inbreeding occur,  i s expected t o be h i g h e r than s i n g l e - l o c u s e s t i m a t e s .  The minimum v a r i a n c e mean of s i n g l e locus e s t i m a t e s compared t o corresponding  m u l t i - l o c u s estimates  p o p u l a t i o n of P a c i f i c s i l v e r f i r .  Multi-locus  were  f o r each estimates  A  exceeded means of t C.  s  for a l l populations  except f o r p o p u l a t i o n  D i f f e r e n c e s range from 1% t o n e a r l y 6% but  the l i m i t s of the  95% confidence  intervals.  a l l fall  within  Neale and Adams  (1985b) found a s i m i l a r range of d i f f e r e n c e s i n f o u r populations  of balsam f i r and concluded  s e l f i n g was  not a f a c t o r .  the i n t e r p r e t a t i o n of how  t h a t mating other  than  There seems t o be some v a r i a t i o n i n s u b s t a n t i a l the d i f f e r e n c e i n  71  estimates  must be t o i n f e r consanguineous mating.  A difference  o f 3% was not enough f o r Shaw e t al. (1981) t o a t t r i b u t e any i n b r e e d i n g t o mating among r e l a t i v e s , y e t a 2.5% (also w i t h i n 95% CI's)  d i f f e r e n c e between t  m  and t  s  was c o n s i d e r e d as  i n d i c a t i v e o f i n b r e e d i n g o t h e r than s e l f i n g by El-Kassaby e t al.  (1987b).  heavy seed  In P a c i f i c s i l v e r f i r , h i g h shade t o l e r a n c e and  ( F r a n k l i n 1974) a r e conducive t o the development o f  f a m i l y c l u s t e r s and very l i k e l y  render t h e assumption t h a t a l l  i n b r e e d i n g i s due t o s e l f - f e r t i l i z a t i o n between t  m  invalid.  Differences  and t , w h i l e not g r e a t i n magnitude, are observed s  i n 6 o f 7 sampled p o p u l a t i o n s ,  which suggests t h a t some mating  among r e l a t i v e s may be o c c u r r i n g and some a s s o r t a t i v e mating may be p r a c t i s e d even when complete o u t c r o s s i n g i s apparent (populations B , R and W ) . Table 5.3 a l s o g i v e s m u l t i - l o c u s estimates  f o r each  p o p u l a t i o n based on t h e t h r e e commonly v a r i a b l e l o c i .  These  d i f f e r e n c e s a r e g e n e r a l l y s m a l l and t ' s with a reduced number m  of l o c i are always s m a l l e r , probably i n sample s i z e .  as a r e s u l t o f r e d u c t i o n  The s i g n i f i c a n c e o f d e v i a t i o n from complete  o u t c r o s s i n g does not change. Estimates  o f o u t c r o s s i n g o b t a i n e d by s i n g l e and m u l t i -  l o c u s methods exceed 1.0 i n one o f e i g h t p o p u l a t i o n s ( W ) . These v a l u e s  a r e not reasonable  b i o l o g i c a l l y but are seen  r a t h e r as an e f f e c t o f sampling and not n e c e s s a r i l y a v i o l a t i o n of model assumptions be  (Brown e t al. 1985).  i n t e r p r e t e d as b e i n g  i n f a c t t = 1.0.  These estimates I t i s known t h a t  may  72  c o n s t r a i n i n g estimates  of t t o b i o l o g i c a l l i m i t s  d u r i n g e s t i m a t i o n procedures a l g o r i t h m of C h e l i a k et al.  ( i . e . the 1983)  v a r i a n c e downward (Brown et al. 1985).  Estimates  p r o v i d e unbiased  of t > 1.0  (0-100%)  Expectation-Maximization  b i a s e s estimates 1985,  R i t l a n d and  should be p e r m i t t e d  p o p u l a t i o n - l e v e l estimates  El-Kassaby i n order  of t  o u t c r o s s i n g p o l l e n pool a l l e l e frequencies  of t and i t s  to  and  ( R i t l a n d and E l -  Kassaby 1985). As p r e v i o u s l y noted, non-uniform s p a t i a l d i s t r i b u t i o n  of  p o l l e n a l l e l e s w i t h i n a p o p u l a t i o n w i l l produce g r e a t e r v a r i a t i o n i n the o u t c r o s s e d  progeny genotype d i s t r i b u t i o n s of  sampled f a m i l i e s and thus be d e t e c t a b l e by a % statistic  (Brown et al.  heterogeneity  1975).  2  deviation  I f there i s s u b s t a n t i a l  then a) p o l l e n of d i f f e r i n g a l l e l e frequency  a r r i v i n g at d i f f e r e n t t r e e s and/or b) the p r o p e n s i t y outcross  i s v a r y i n g between t r e e s .  were a cause of t < 1.0, heterogeneity 1981).  then a p o s i t i v e r e l a t i o n s h i p between  and s e l f i n g r a t e would occur  (Mitton et  was  and  low o u t c r o s s i n g r a t e s  found t h a t three of f o u r v a r i a b l e l o c i  = 1.0)  al.  there i s no  c o n s i s t e n t a s s o c i a t i o n between heterogeneous l o c i 5.2)  to  It i s expected t h a t i f (a)  In P a c i f i c s i l v e r f i r p o p u l a t i o n s ,  *'s i n Table  is  ( i n d i c a t e d by  (Table 5.3).  It  i n population R ( t  are s i g n i f i c a n t l y heterogeneous, two  m  of f i v e i n  A  population W p o p u l a t i o n A)  ( t = 1.0) m  and most i m p o r t a n t l y  i n the f i v e p o p u l a t i o n s  inbreeding detected.  The  extent  only one  (PGI-2 i n  where there i s some  to which o u t c r o s s i n g r a t e s  73  vary  among i n d i v i d u a l t r e e s w i t h i n a p o p u l a t i o n  c o u l d not  e s t i m a t e d given the number of progeny a v a i l a b l e t o t h i s (progeny a r r a y s should exceed 30, pool heterogeneity  be  study  R i t l a n d 1983), however p o l l e n  does not appear t o be  i n f l u e n c i n g departures  from panmixis. Given t h a t some amount of i n b r e e d i n g outcrossing  r a t e s < 1.0  i n f i v e of seven p o p u l a t i o n s )  apparent, i t i s p e r t i n e n t to ask a c t u a l amount of i n b r e e d i n g describes  ' t ' as the  whether  is  *t' reflects  taking place.  " e f f e c t i v e " outcross  variable representing  (as i n d i c a t e d by  Ritland  the  (1983)  r a t e , a summary  the net e f f e c t of d e v i a t i o n from panmixis  caused by c o r r e l a t i o n s of maternal and  paternal  genotypes,  v a r i a t i o n i n s e l f - c o m p a t a b i l i t y , gametic s e l e c t i o n and zygotic l e t h a l i t y . 1-t)  represents  s e l f i n g , but  Adams and  Birkes  the p r o p o r t i o n  early  (1990) c a u t i o n that s  of v i a b l e progeny due  to  i s not a measure o f the a c t u a l frequency of  p o l l i n a t i o n , which may  be c o n s i d e r a b l y  greater.  (=  self-  Sorensen  (1982) maintains that s e l f - i n c o m p a t a b i l i t y mechanisms appear to be  l a c k i n g i n c o n i f e r s and  c r o s s i n g experiments i n noble f i r by  Sorensen et a l . (1976) r e v e a l e d high  i n that species.  that r e l a t i v e s e l f - f e r t i l i t y  Embryo a b o r t i o n  i s common i n P a c i f i c  s i l v e r f i r (Owens and Molder 1977), but extent  is  i t i s not  embryos r e s u l t i n g from s e l f i n g are aborted.  known to what The  species  i s known t o produce l a r g e numbers of otherwise normal-appearing empty seeds  ( F r a n k l i n 1974).  seed by  i n noble f i r (Sorensen et al.  31%  S e l f i n g reduced numbers of 1976).  It may  filled be  14  expected then,  t h a t a p o s i t i v e r e l a t i o n s h i p would e x i s t between  percentage of f i l l e d seed and o u t c r o s s i n g r a t e , with high seed y i e l d s c o r r e l a t e d with high l e v e l s of o u t c r o s s i n g . study,  simple  c o r r e l a t i o n between mean seed y i e l d  p o p u l a t i o n and t significant.  In t h i s  m  The  was  very weakly n e g a t i v e  r e l a t i o n s h i p has  sample s i z e s p e r m i t t e d  per  (r = -0.165) and  i n t u i t i v e appeal  outcrossing rate estimates  and  on  not  had  an  i n d i v i d u a l t r e e l e v e l then the c o r r e l a t i o n c o u l d have been based on i n d i v i d u a l v a r i a t i o n r a t h e r than p o p u l a t i o n means. However, El-Kassaby et al. between percent  filled  (1987b) found the  seed per t r e e and  correlation  individual tree  o u t c r o s s i n g r a t e i n a Western white pine p o p u l a t i o n t o be nonsignificant  (r = 0.012).  Seed y i e l d s are s u b j e c t to a number  of environmental f a c t o r s (climate, i n s e c t s , etc.) and relationship,  although  t h e o r e t i c a l l y p l a u s i b l e , may  d e t e c t a b l e from c o l l e c t i o n s i n n a t u r a l Seed s i z e , however, i s c o n s i d e r e d p l a s t i c of p l a n t c h a r a c t e r s  Stoehr  to be one  (Sorensen and  and Farmer 1986).  not  be  stands. of the  least  F r a n k l i n 1977)  known to be under a high degree of g e n e t i c c o n t r o l 1986,  a clear  and  (Khalil  A highly positive correlation  of p o p u l a t i o n mean seed s i z e and m u l t i - l o c u s o u t c r o s s i n g r a t e was  observed i n P a c i f i c s i l v e r f i r (r = 0.712, 0.05  0.10).  Seed s i z e was  estimates. by Shea obtained  represented  < P <  by mean thousand-seed weight  T h i s i s the reverse of t h a t found i n s u b a l p i n e f i r  (1987) where higher than average o u t c r o s s i n g r a t e s were from t r e e s with s m a l l e r  seeds.  75  5.4  Conclusions Although v a r i a t i o n i n a l l e l e frequencies  i s not e x t e n s i v e  over t h e range o f P a c i f i c s i l v e r f i r sampled, t h e r e i s some apparent v a r i a t i o n i n the r a t e o f o u t c r o s s i n g among t h e seven populations  o f P a c i f i c s i l v e r f i r i n t h i s study.  system i s known t o be dynamic  (Hamrick 1982) and g i v e n  s i l v e r f i r ' s e c o l o g i c a l s t a t u s as a climax al.  The mating  species  Pacific  ( K r a j i n a et  1982), i t i s not s u r p r i s i n g t h a t p o p u l a t i o n estimates o f  i n b r e e d i n g were v a r i a b l e (from zero t o as much as 27 p e r c e n t ) . D i f f e r e n c e s i n the magnitudes o f o u t c r o s s i n g o b t a i n e d by s i n g l e and m u l t i - l o c u s e s t i m a t i o n procedures suggest t h a t some r e l a t e d matings a r e o c c u r r i n g , a r e s u l t which i s not unexpected i n l i g h t o f t h e high shade t o l e r a n c e and l i m i t e d seed d i s p e r s a l e x h i b i t e d by t h i s s p e c i e s . t o t h i s study,  At the sampling i n t e n s i t y a v a i l a b l e  d e v i a t i o n from panmixis c o u l d not be a s s o c i a t e d  w i t h mating behaviour however seed s i z e was s t r o n g l y r e l a t e d t o the e x t e n t  o f apparent o u t c r o s s i n g i n P a c i f i c s i l v e r f i r .  76  6. ESTIMATES OF ELECTROPHORETIC VARIATION, STRUCTURE AND RELATIONSHIP TO THE MATING SYSTEM  ITS  6.1  Introduction Prerequisite  tree-breeding  t o t h e achievement  program  i s some k n o w l e d g e  magnitude o f v a r i a t i o n interest. is  few e x c e p t i o n s ,  number o f g e n e t i c  which  exists  virtually  types  determining  document t h e v a r i a t i o n among p h e n o t y p e s " . trees  arenot likely  units,  efficient  estimate  diverse  results  variation  (Guries  i n selective  1987),  as i d e a l ,  1977,  interest  differences of forest  panmictic  s t r a t e g i e s depend upon  and Ledig  rich  i s to  Because n a t u r a l p o p u l a t i o n s t o behave  conifers,  (meaning  "ourfundamental  of genetic  that  sampling  variation  breeders.  o f t h e degree t o which populations  genetically 19B6).  of genetic  as d e f i n e d by Gregorius  describes,  t h e extent  i n any  o f t h e nature and  f o r improvement b u t most  aregenetically  (1983)  Hedrick  gain  i n the species of  devoid  i s , i n t u r n , e x p l o i t e d by  As in  that  obviously not suitable  with in  A species  of genetic  breeding  some  are subdivided  Gregorius  and Roberds  S e l e c t i o n m e t h o d s may t h e n b e e m p l o y e d t o " t a k e  advantage  of actual population  1977).  Where t h e r e  stands,  then  represent  s t r u c t u r e " (Guries  i sextensive  s e v e r a l stands  the species,  differentiation  may b e r e q u i r e d t o  whereas  and Ledig among  adequately  l o w amounts o f among-  77  population  v a r i a t i o n i n a given  character  comparison-tree s e l e c t i o n (Guries and baseline  supports  Ledig  1977).  However,  ( i n d i v i d u a l ) s e l e c t i o n w i l l be more e f f i c i e n t  than  the comparison-tree method i f i n d i v i d u a l s w i t h i n a population  are i n b r e d  (Ledig 1974) .  H i s t o r i c a l l y , methods of measuring g e n e t i c v a r i a t i o n i n f o r e s t t r e e s have f o l l o w e d two  avenues.  The  traditional  approach r e l i e d on the assessment of some m o r p h o l o g i c a l and/or p h y s i o l o g i c a l t r a i t s a c c o r d i n g model which enables among and  to an  experimental  within-population  components of v a r i a t i o n to be estimated.  and  family  Such s t u d i e s  u s u a l l y e n t a i l the growth of progeny from w i n d - p o l l i n a t i o n or c o n t r o l l e d c r o s s i n g i n one al.  1969).  polygenic  The  or more environments  i n nature and  o f t e n e x h i b i t l a r g e amounts of  expensive t o conduct and  1983).  Such s t u d i e s  require considerable  time t o o b t a i n r e l i a b l e data  (Libby et al.  lengths  v a r i a t i o n i n n a t u r a l and  T h e i r ease of d e t e c t i o n , and  codominant e x p r e s s i o n  of  experimental  studies populations  of f o r e s t t r e e s have u t i l i z e d enzyme polymorphisms starch gel electrophoresis  are  1969).  In the past twenty years a l a r g e number of  by  et  t r a i t s which are measured are mostly  environmental v a r i a t i o n (Mitton  estimating  (Libby  revealed  (reviewed by El-Kassaby 1990).  single-gene  inheritance  patterns  make isozymes w e l l - s u i t e d f o r  s t u d i e s of g e n e t i c v a r i a t i o n . e l e c t r o p h o r e t i c a l l y detected  Moreover, i t i s assumed t h a t  gene products are l e s s  78  s u s c e p t i b l e t o changes i n s e l e c t i o n p r e s s u r e and thus r e p r e s e n t a more s t a b l e sample o f the t o t a l genome than metric t r a i t s  (Lewontin  1974, R i t l a n d 1983, Gregorius and  Roberds 1986, P l e s s a s and S t r a u s s  1986).  Most c o n i f e r s e x h i b i t s i m i l a r l i f e h i s t o r i e s .  They are  l o n g - l i v e d , o f t e n occupy heterogeneous environments and n e c e s s a r i l y must possess enable  the c a p a c i t y f o r a d a p t a t i o n s  them t o remain i n subsequent g e n e r a t i o n s  and L e s t e r 1969, M u l l e r - S t a r c k and Gregorius v a r i a t i o n i s maintained  which  (Rehfeldt  1986).  Genetic  by high l e v e l s o f o u t c r o s s i n g and  mechanisms t o promote o u t c r o s s i n g , such as i n b r e e d i n g d e p r e s s i o n and s e l f - i n c o m p a t i b i l i t y , c h a r a c t e r i s t i c o f outbreeding 1984,  species  Lande and Schemske 1985).  true f i r s  are expected  t o be  (Loveless and Hamrick  C o n i f e r s i n g e n e r a l and  i n p a r t i c u l a r are known t o e x h i b i t  inbreeding  d e p r e s s i o n i n growth ( F r a n k l i n 1970, Sorensen et al. 1976). To date, no such s t u d i e s have been conducted with  Pacific  silver f i r . The b i o l o g y and ecology  of P a c i f i c s i l v e r f i r are  unique among western North American c o n i f e r s .  I t s extremely  h i g h shade t o l e r a n c e and l a t e s u c c e s s i o n a l s t a t u s ( K r a j i n a et al. 1982) coupled with p r o d u c t i o n o f heavy seeds which are o f t e n cached by f r u g i v o r e s ( F r a n k l i n 1974) suggest at l e a s t t h e p o t e n t i a l f o r some r e p r o d u c t i v e i s o l a t i o n and population substructure within the species.  F u r t h e r , the  a b i l i t y o f P a c i f i c s i l v e r f i r t o occupy a v a r i e t y o f s i t e s  79  (Schmidt 1957) regimes.  suggests exposure t o v a r i a b l e s e l e c t i o n  Narrow d i s t r i b u t i o n  (east-west) and  island  i n h a b i t a n c e may  a l s o act t o r e s t r i c t gene flow and promote  differentiation  (Loveless and Hamrick  1984).  The o r g a n i z a t i o n of g e n e t i c v a r i a t i o n i n P a c i f i c fir  on Vancouver  I s l a n d was  s t u d i e d u s i n g enzyme  polymorphisms  d e t e c t e d i n seeds sampled from e i g h t  populations.  S e v e r a l analyses based on a l l e l e and  f r e q u e n c i e s were used t o d e s c r i b e the extent apportionment o f v a r i a t i o n .  genotype  and  Parameters o f v a r i a t i o n  t h e i r r e l a t i o n s h i p t o the mating system of P a c i f i c fir  silver  ( d i s c u s s e d i n Chapter 5) are a l s o e x p l o r e d  and  silver  in this  chapter. 6.2  M a t e r i a l s and Methods 6.2.1  Sample c o l l e c t i o n and e l e c t r o p h o r e t i c assay  Samples o f w i n d - p o l l i n a t e d seeds c o l l e c t e d i n 1983 eight populations B.C.  of P a c i f i c s i l v e r f i r on Vancouver  from  Island,  were the source o f t i s s u e s f o r the e l e c t r o p h o r e t i c  assays o u t l i n e d i n Chapter 3 and d e t a i l e d i n Appendix Mendelian i n h e r i t a n c e p a t t e r n s  2.  f o r 13 p u t a t i v e l o c i are  d e s c r i b e d i n Chapter 3 and t h e i r l i n k a g e r e l a t i o n s h i p s i n Chapter 4.  Some evidence f o r s e g r e g a t i o n d i s t o r t i o n i n one  l o c u s , AAT-2, d i d not exclude i t i n e s t i m a t i n g v a r i a t i o n patterns  genetic  (Adams 1983). P o p u l a t i o n F i s a l s o  i n c l u d e d d e s p i t e the f a c t t h a t i t s mating system c o u l d not  80 be  characterized using  Bailey  1951)  and  6.2.2 (a)  the  i t s attendant  assumptions.  e x c e e d nk  = 100  for estimating  (Brown and  number o f m a t e r n a l t r e e s  equals  the  number o f p r o g e n y p e r  consist  of the  ideal  recommended by obtain  El-Kassaby  reliable  r e v i e w by  conifer  many as  tree.  the  Several  to  a population  to  60  although  (1983) i n o r d e r allele  the  17),  pollen allele  eight populations  variation  and  i n determining  d i s t a n c e measures of Nei differentiation allele  (1972,  statistics  frequencies  maternal genotype  the  i n the  data  diversity 1977  of Gregorius are  (known w i t h  d e r i v e d by virtual  of  and  to  as  collections  present  i n analyses  1973,  on  v a r i e d f r o m one  frequency  the  to  frequencies.  number  seed  none  trees  number o f c o n t r i b u t i n g t r e e s unknown. sampled  is  'k'  55 p a p e r s p u b l i s h e d  showed t h a t  represent  Pollen  40  s t u d i e s used bulk  number o f m a t e r n a l t r e e s (eight  study,  of p o p u l a t i o n  variation  and  'n'  T h i s number i s  i n my of  frequencies  where  population  Sziklai  (1990) o f  representing  132.  and  estimates  El-Kassaby  genetic  individuals  sample s i z e  allele  Moran 1981)  i n the  exceeded i n a l l eight populations  with  and  Caveats  should  A  (Fyfe  A n a l y t i c methods  A d e q u a t e sample s i z e  the  m i x e d m a t i n g model  Because study  the  is  were c h o s e n of  allelic  and  genetic  and  1978)  Roberds  to  and  the  (1986).  "subtracting"  c e r t a i n t y from  low  a  the  81  sample of 20 seeds per tree) genotype.  from the d i p l o i d embryo  Pollen a l l e l e frequencies  have been used i n  s e v e r a l s t u d i e s of e l e c t r o p h o r e t i c v a r i a t i o n i n c o n i f e r s because a l a r g e r p o r t i o n of the p o p u l a t i o n represented  ( S t e i n h o f f et al.  Adams 1989,  S u r l e s et al.  1983,  1989).  gene p o o l i s  M i l l a r 1983,  t h e s e two The  were u t i l i z e d ,  s e t s of  embryo a l l e l e  acknowledging the dependency of  data.  o v e r a l l o b j e c t i v e s of t h i s t h e s i s  (outlined in  Chapter 1) d i c t a t e d c e r t a i n sampling c r i t e r i a , populations conditions  (stands) of t r e e s represent and  a range of growing  However, c e r t a i n l i m i t a t i o n s inherent  l i m i t s were set by the b i o l o g y cone crop year.  P a c i f i c s i l v e r f i r and the  collection size.  stratified,  The the  identified.  of the s p e c i e s  i n the Some  i t s e l f as  1983  crop q u a l i t y both l i m i t e d  Sampling of course was  necessarily  as non-conebearing t r e e s were excluded. of  set l i m i t s to the amount of m a t e r i a l which c o u l d  processed.  And,  given  the e x p l o r a t o r y  nature of the  or a l l of the s e l e c t e d c h a r a c t e r s .  be  study,  I c o u l d not be assured that v a r i a t i o n would be d e t e c t e d any  well  a c c e s s i b i l i t y of cones of  In a d d i t i o n to the cost of sampling, the cost analyses  as  f o l l o w i n g IUFRO g u i d e l i n e s  r e s u l t i n g m a t e r i a l c o l l e c t i o n need to be  as the  namely t h a t  i n d i v i d u a l t r e e s be sampled as randomly  p o s s i b l e w i t h i n each p o p u l a t i o n , (Lines 1967).  and  Where between-generation  comparisons were d e s i r e d , maternal t r e e and frequencies  Li  in  Sampling d e c i s i o n s ,  82  then, and  were a f f e c t e d by b i o l o g i c a l and resource  l a r g e l y shaped by p r e v i o u s  true f i r s .  restrictions  s t u d i e s on r e l a t e d s p e c i e s o f  The "Catch-22" dilemma of sampling f o r g e n e t i c  v a r i a t i o n e s t i m a t i o n i s emphasized by A r c h i e e t al. (1989) i n t h a t "...our a b i l i t y t o d e t e c t and estimate  t h e frequency  of v a r i a n t s i s d i r e c t l y r e l a t e d t o the number o f i n d i v i d u a l s sampled... The i m p l i c a t i o n s . . . are t h a t an i n v e s t i g a t o r must c o l l e c t and analyze  l a r g e samples t o d i s c o v e r the p a t t e r n s  of g e n e t i c d i v e r s i f i c a t i o n before  recommendations can be  made as t o how many i n d i v i d u a l s should be sampled". The  sampling p r o p e r t i e s of measures used t o q u a n t i f y  g e n e t i c and e s p e c i a l l y e l e c t r o p h o r e t i c v a r i a t i o n have been s t u d i e d by a number of i n v e s t i g a t o r s (Lewontin and Cockerham 1959, al.  Brown 1970, Smith 1970, Ward and Sing 1970, Brown e t 1975, N e i 1978, M u e l l e r  1979, Curie-Cohen 1982, N e i and  Chesser 1983, Weir and Cockerham 1984, A r c h i e 1985, Simon and A r c h i e 1985, A r c h i e et al. 1989, are some).  T h i s body  of work suggests t h a t c l a s s i c a l methods of h y p o t h e s i s t e s t i n g t o determine how v a r i a b l e i s v a r i a b l e or how different  i s d i f f e r e n t may not be a p p r o p r i a t e because o f  non-normality and i n h e r e n t l y l a r g e v a r i a n c e s a s s o c i a t e d these e s t i m a t e s .  F o r example, t o be 90%  r e j e c t i n g a f i x a t i o n index  confident i n  of 0.0035 (a value  observed i n h i g h l y outcrossed  comonly  species) u s i n g a = 0.05 a  sample of at l e a s t 830,000 i n d i v i d u a l s i s r e q u i r e d Sing 1970).  Shaw and A l l a r d  with  (Ward and  (1982b) acknowledge the l a c k of  83  p r e c i s i o n i n v o l v e d i n s t u d i e s of p o p u l a t i o n v a r i a t i o n i n s a y i n g t h a t t h e i r i n c l u s i o n of standard  e r r o r s i n the  c a l c u l a t i o n of f i x a t i o n i n d i c e s i s intended  as an i n d i c a t o r  of d i s p e r s i o n only, "as i t i s u n l i k e l y t h a t the  criteria  necessary  intervals  f o r the v a l i d formation  from sample standard  of confidence  e r r o r s w i l l be met  variance for a l l observations)". papers such as Kahler estimates assays may estimates  (e.g. homogeneity of  However, p u b l i c a t i o n of  et a l . (1986) suggests t h a t  while  of g e n e t i c v a r i a t i o n based on e l e c t r o p h o r e t i c only be p o i n t estimates  ( t h e i r paper c o n t a i n s  no  of v a r i a n c e f o r e i t h e r f i x a t i o n i n d i c e s or g e n e t i c  d i s t a n c e measures), they p a t t e r n s or trends  are s t i l l  i n the data.  encountered i n the present a n a l y s i s and  study  considered  Similarly,  as evidence  limitations  do not preclude  subsequent i n t e r p r e t a t i o n but  of  data  introduce a  degree of u n c e r t a i n t y which should and w i l l temper  any  conclusions. (b)  A n a l y t i c methods A l l e l i c v a r i a t i o n of sampled p o p u l a t i o n s  s i l v e r f i r was  q u a n t i f i e d i n s e v e r a l ways.  nurober of a l l e l e s per  locus was  l o c i i n each p o p u l a t i o n . reflect allelic  richness  of P a c i f i c  The  actual  averaged over a l l d e t e c t e d  This value  i s considered  (Marshall and Brown 1975)  to but  i n f l a t e s the c o n t r i b u t i o n low-frequency a l l e l e s make to variation. Kimura  A more i n f o r m a t i v e measure i s t h a t of Crow and  (1970) known as the  " e f f e c t i v e " number of a l l e l e s  per  84  locus, n . e  N  e  equals the i n v e r s e of the sum o f squares of  a l l e l e frequencies  (Pi's) at a given l o c u s .  when a l l e l e s are of equal  frequency  r e f l e c t s both presence and frequency  ('v')  i s greatest  e  and i s c l o s e t o one i f Thus, n  only one a l l e l e i s i n very high frequency.  1983).  N  of a l l e l e s  e  (Hedrick  T h i s measure i s i d e n t i c a l t o the d i v e r s i t y measure  proposed by Gregorius  (1987).  The average n i s e  determined by c a l c u l a t i n g the geometric mean of i n d i v i d u a l l o c u s values, The  i n c l u d i n g monomorphic l o c i  quantity 1 - Xpi ,  (Lundkvist  1979).  the t h e o r e t i c a l h e t e r o z y g o s i t y  2  o b t a i n e d under Hardy-Weinberg assumptions of mating, i s very o f t e n used as a measure of g e n e t i c v a r i a t i o n (Hedrick I t i s v a r i o u s l y r e f e r r e d t o i n the p o p u l a t i o n  genetics  l i t e r a t u r e as expected panmictic  heterozygosity  A l l a r d 1982b) or genie d i v e r s i t y  (Brown et al.  Falkenhagen 1985, Muona and Szmidt 1985). expected h e t e r o z y g o s i t y  1983).  (Shaw and 1980,  The average  (H ) i s the sum over a l l l o c i and e  i n d i v i d u a l s o f i n d i v i d u a l locus h e t e r o z y g o s i t i e s . S t a t i s t i c a l b i a s i n t r o d u c e d by small sample s i z e s ( l e s s than 20 i n d i v i d u a l s ) i s found t o be reduced by a p p l y i n g a c o r r e c t i o n f a c t o r of 2N/2N-1 t o H 1974).  e  (Nei and Roychoudhury  T h i s c o r r e c t i o n was a p p l i e d t o estimates  a l l maternal gene pools i n t h i s study. properties of heterozygosity  Further  are c o n s i d e r e d  of H f o r e  sampling  i n s e c t i o n 6.4.  In a d d i t i o n t o the amount of v a r i a t i o n present, i t s d i s t r i b u t i o n among and w i t h i n p o p u l a t i o n s  i s of i n t e r e s t .  A  85  d i s t a n c e measure, analogous to geometric d i s t a n c e , developed by Nei  (1972, 1978).  p o p u l a t i o n X and Y  (D )  The  population differences i n a l l e l e  of D values  averaged over  A c l u s t e r a n a l y s i s of the  f o r a l l p a i r s of p o p u l a t i o n s  a r i t h m e t i c averaging (Wilkinson 1988)  the amount of between-  frequencies,  monomorphic and polymorphic l o c i . matrix  g e n e t i c d i s t a n c e between  represents  xy  was  procedure was  using  an  performed u s i n g SYSTAT  to o b t a i n a g r a p h i c a l r e p r e s e n t a t i o n of the  g e n e t i c r e l a t e d n e s s of the e i g h t sampled stands  of P a c i f i c  silver f i r . The  apportionment of v a r i a t i o n among and  populations  was  d e v i s e d by Nei measure Nei's  ('8')  estimated  within  u s i n g the d i v e r s i t y  (1973, 1977)  and  statistics  a population  developed by Gregorius  and  differentiation  Roberds  " c o e f f i c i e n t of g e n e t i c d i f f e r e n t i a t i o n "  (1986).  (G )  was  ST  c a l c u l a t e d u s i n g only polymorphic l o c i to make a comparison with 8 and  a l s o with the i n c l u s i o n of monomorphic l o c i  allow comparison with d i v e r s i t y s t a t i s t i c s estimated  to  for  other t r u e f i r s and c o n i f e r s i n g e n e r a l . Both G [1978] F ) ST  ST  (which was  shown to be e q u i v a l e n t to Wright's  and 8 were o r i g i n a l l y d e v i s e d to  estimate  p o p u l a t i o n s u b d i v i s i o n or m i c r o d i f f e r e n t i a t i o n but  these  s t a t i s t i c s have been a p p l i e d to d i s c r e t e p o p u l a t i o n s number of s t u d i e s of f o r e s t t r e e s 1979,  F u r n i e r and Adams 1986,  Muller-Starck  and Gregorius  (e.g. O'Malley et a l .  L i and Adams 1989 1986  in a  f o r 8).  The  for  G ; ST  86  d i f f e r e n t i a t i o n estimates  a c c o r d i n g t o the concept  of  Gregorius  and Roberds (1986) were p r o v i d e d by  H.-R.  Gregorius  (pers. comm., March 1989) u s i n g p o l l e n a l l e l e  frequencies. Mating system e f f e c t s on p o p u l a t i o n g e n e t i c s t r u c t u r e were estimated u s i n g Wright's I n d i v i d u a l l o c u s estimates  (F).  (1969) f i x a t i o n index  of F ( = 1 - [observed number of  heterozygous genotypes/expected number of heterozygous genotypes]) were c a l c u l a t e d as w e l l as a minimum v a r i a n c e mean over l o c i  (Shaw and A l l a r d  1982b) f o r maternal  t r e e and  embryo samples i n each p o p u l a t i o n . • T h i s q u a n t i t y i s the same as the ' i n d i r e c t ' estimate (1979).  A ' d i r e c t ' estimate  r e l a t i o n s h i p with s e l f i n g  of F r e f e r r e d t o by Brown  of F u s i n g the e q u i l i b r i u m  ( F = s/2-s) where s i s equal t o e  the r a t e of s e l f - f e r t i l i z a t i o n  (Hedrick 1983) was a l s o  obtained f o r seven p o p u l a t i o n s  ( e x c l u d i n g F) u s i n g the  m u l t i l o c u s estimates  of o u t c r o s s i n g r a t e  (t = 1-s) r e p o r t e d  i n Chapter 5. 6.3  Results 6.3.1  A l l e l i c variation  Pollen a l l e l e  frequency  estimates  for individual  across e i g h t p o p u l a t i o n s are shown i n Table i n c l u s i o n of IDH and MDH-1  6.1.  as polymorphic r e f l e c t  f r e q u e n c i e s where the i n c i d e n c e of the most common  loci  The individual allele  Table 6.1  P o l l e n a l l e l e f r e q u e n c i e s f o r s i x v a r i a b l e l o c i i n e i g h t p o p u l a t i o n s of P a c i f i c s i l v e r f i r on Vancouver I s l a n d , B.C. (Populations are arranged i n order o f i n c r e a s i n g l a t i t u d e and t h e i r l e t t e r d e s i g n a t i o n s are followed by sample s i z e ; represents a n u l l a l l e l e ) .  Population Locus/Allele  F (180)  W(335)  A (159)  B(160)  C (159)  N(219)  R(258)  H(258)  PGI-2  1 2 'n'  0.672 0.328 0.000  0 .704 0.296 0.000  0.786 0.201 0.013  0 .825 0.175 0.000  0.887 0 .113 0.000  0.694 0.306 0.000  0.605 0.395 0.000  0.868 0.132 0.000  G6P  1 2 3 5 •n'  0.572 0.044 0.006 0.378 0.000  0.803 0.003 0.000 0.191 0.003  0.736 0.000 0.031 0.223 0.000  0.775 0.000 0.012 0.213 0.000  0.817 0.000 0.000 0.183 0.000  0.827 0.000 0.000 0.164 0.009  0.818 0.000 0.000 0.182 0.000  0.822 0.000 0.008 0.169 0.000  PGM  1 2 3 5 'n'  0.783 0.011 0.189 0.006 0.000  0.716 0 . 027 0.248 0.009 0.000  0. 672 0.006 0.322 0.000 0.000  0. 944 0.000 0.056 0.000 0.000  0 . 905 0.013 0.076 0.006 0.000  0.808 0. 000 0.187 0.000 0.005  0.845 0.000 0.155 0.000 0.000  0.837 0.000 0.151 0.004 0.008  IDH  1 3  1.000 0.000  0. 997 0.003  1.000 0.000  0. 994 0.006  1.000 0.000  1.000 0.000  1.000 0.000  1.000 0.000  MDH-1  1 2  1.000 0.000  1.000 0.000  1.000 0.000  1.000 0.000  1.000 0.000  1.000 0.000  0. 988 0.012  0.996 0.004  AAT-2  1 2  0. 972 0.028  0.788 0.212  0.874 0.126  0.938 0.162  0. 924 0.076  0. 945 0.055  0.965 0.035  0.984 0.016  88  was l e s s t h a n  0.99 b u t when a v e r a g e d o v e r  e x c e e d s t h e 99% c r i t e r i o n geographic  (Hartl  1980).  There a r e s e v e r a l  trends apparent i n t h e t a b l e .  f o u n d o n l y i n p o p u l a t i o n A was d e t e c t e d whereas a l l o t h e r a l l e l e s populations.  Allele  occurred  a l l individuals  A private allele, a t t h e PGI-2 l o c u s ,  i n a t l e a s t two  '2' i n G6P was d e t e c t e d o n l y i n t h e t w o (F a n d W).  southernmost p o p u l a t i o n s  The n u l l  allele  ('n')  was d e t e c t e d a t t h e PGM l o c u s i n o n l y t w o o f t h e n o r t h e r n m o s t p o p u l a t i o n s , N and H . found only i n t h e northern had  either four or five  loci  V a r i a t i o n a t MDH-1 (R a n d H ) .  samples  varying loci  was  Populations  a n d t h r e e o f t h e o f 11  a s s a y e d were commonly v a r i a b l e (PGI-2, G6P a n d PGM). A contingency  y} a n a l y s i s o f a l l e l e f r e q u e n c i e s ( Z a r  1984) was a t t e m p t e d t o o b t a i n an e s t i m a t e heterogeneity sufficiently estimates  among p o p u l a t i o n s , h o w e v e r o n l y two l o c i h a d l a r g e expected  t o b e made.  heterogeneity  The  cell  Both l o c i  among p o p u l a t i o n s  f o r AAT-2 was 111.06, a = 0.05J  of a l l e l i c  frequencies t o permit showed (%  2  %  2  significant  f o r PGI-2 was 82.3 a n d  compared t o a c r i t i c a l v a l u e  [7 d f ,  o f 14.07) . a v e r a g e a c t u a l a n d e f f e c t i v e number o f a l l e l e s p e r  locus a r e l i s t e d i n Table  6.2.  these  i n p o p u l a t i o n H where, a l t h o u g h  two measures o c c u r s  l a r g e r number o f a l l e l e s l o c u s on a v e r a g e ) , relatively  The g r e a t e s t d i s p a r i t y i n  were d e t e c t e d  some o f t h e s e  alleles  a  (1.73 a l l e l e s p e r occurred at  l o w f r e q u e n c i e s , r e d u c i n g t h e e f f e c t i v e number t o  89 Table  6.2  Population  P o p u l a t i o n s l i s t e d i n order of i n c r e a s i n g l a t i t u d e , sample s i z e , a c t u a l and ' e f f e c t i v e ' numbers of a l l e l e s per locus f o r e i g h t stands of Pacific silver f i r . Latitude  F  48 '39'  W  48 48 15  A  No. o f trees  No. of alleles per l o c u s  E f f e c t i v e no. of a l l e l e s per l o c u s  9  1.73  1.23  17  1. 82  1.22  49 'l8 '  8  1. 64  1.21  B  49* 57 '  8  1.55  1.11  C  50 '03'30"  8  1.55  1.09  H  50* 34 45  13  1.73  1.10  R  50*'3e'3o"  13  1. 45  1.16  N  50* 43'30"  11  ' 1.55  1.16  1 . 64  1.16  >  1  _  Weighted Mean  1  II  11  1  A r i t h m e t i c average f o r a c t u a l number of a l l e l e s ; geometric mean f o r e f f e c t i v e number (Lundkvist 1979). 1  1.10.  A c t u a l a l l e l e counts d i d not show any t r e n d  with  geography, however e f f e c t i v e numbers of a l l e l e s were n e g a t i v e l y c o r r e l a t e d (r = - 0 . 7 1 9 , that, i n general, increasing  a l l e l i c v a r i a t i o n decreases  a l l e l i c d i v e r s i t y with n a l l e l e t o dominate. e  indicating with  latitude.  O v e r a l l , p o p u l a t i o n s possessed  and n  P < 0.05),  estimates  e  values  relatively  low l e v e l s of  showing a tendency f o r one  No p o p u l a t i o n was devoid of v a r i a t i o n  suggest p o p u l a t i o n W i s the most  90  g e n e t i c a l l y d i v e r s e and p o p u l a t i o n C the most depauperate. These p o p u l a t i o n s a l s o had the l a r g e s t and  s m a l l e s t samples,  r e s p e c t i v e l y , however Spearman's rank c o r r e l a t i o n 1984)  f a i l e d t o show any  s i z e and d i v e r s i t y by Gregorius  6.3  Table  6.3  (1987) .  f o r maternal  h e t e r o z y g o s i t i e s (H) are l i s t e d i n t r e e and embryo gene p o o l s .  Weighted  Average expected heterozygosities® f o r a d u l t (maternal tree) and embryo gene pools f o r e i g h t p o p u l a t i o n s of P a c i f i c s i l v e r f i r .  Population  Adult®  Embryo  F  0. 156  0.108  W  0. 177  0.125  A  0. 097  0.091  B  0.068  0.054  C  0.889  0. 052  H  • 0.124  0. 072  N  0.109  0.073  R  0.081  0.073  Mean® ®  s t r o n g r e l a t i o n s h i p between sample  (r = 0.241), as t h e o r e t i c a l l y p r e d i c t e d  Average expected Table  (Zar,  0.118  0.085  Average expected h e t e r o z y g o s i t y = N-m XXHij where r e p r e s e n t s the h e t e r o z y g o s i t y of the i i n d i v i d u a l at the j l o c u s , summed over m l o c i and N i n d i v i d u a l s . t h  t  h  ©  Sample c o r r e c t i o n f a c t o r 2N/2N-1 a p p l i e d to a d u l t estimates (Nei and Roychoudhury 1974).  ®  Weighted by sample s i z e .  91  mean v a l u e s are a l s o g i v e n . showed a s i m i l a r geographic  These measures of d i v e r s i t y t r e n d to n ,  w i t h l a t i t u d e b e i n g n e g a t i v e and a = 0.05  f o r embryos a l s o  correlation  j u s t s i g n i f i c a n t at  (r = -0.643) f o r the maternal  significant  the  e  gene p o o l  (r = -0.755).  and  T h i s i s not  unexpected s i n c e both q u a n t i t i e s are d e r i v e d from data s t r u c t u r e s ( p ^ ' s ) .  Average H's  do d i f f e r between gene  p o o l s , and i n a l l p o p u l a t i o n s maternal i s g r e a t e r than t h a t expected 6.3.2  similar  tree heterozygosity  f o r the embryo p o p u l a t i o n .  D i s t r i b u t i o n of v a r i a t i o n  Estimates of Nei's  (1978) g e n e t i c d i s t a n c e between  p a i r s of P a c i f i c s i l v e r geographic  distances  i n Table 6.4.  f i r p o p u l a t i o n s along with  (measured i n k i l o m e t e r s ) are  their presented  The values of D are a l l very c l o s e t o zero.  O v e r a l l , the average g e n e t i c d i s t a n c e i s 0.0112.  The  s m a l l e s t v a l u e j o i n s p o p u l a t i o n s B and C which are a l s o i n the c l o s e s t geographic  proximity  (9 km).  Another two  stands  (N and R) which are p h y s i c a l l y near each other are a l s o g e n e t i c a l l y very s i m i l a r  (D = 0.00192).  Population F i s  d i s t i n g u i s h a b l e from the r e s t of the stands by being  the  most remote g e n e t i c a l l y and t h i s i s e v i d e n t i n the g r a p h i c a l r e p r e s e n t a t i o n shown i n F i g u r e 6.1.  Two  other subgroups  appear i n the c l u s t e r diagram but grouping s t r o n g geographic (N and R)  t r e n d , as two  of the northernmost  are grouped more c l o s e l y with two  populations  (A & W)  does not f i t any  of the  samples southern  than with the other northern sample  and  Table 6.4 E s t i m a t e s o f g e n e t i c d i s t a n c e s (Nei 1978) below the diagonal and geographic d i s t a n c e s (measured i n k i l o m e t e r s and l i s t e d above the diagonal) between p a i r s o f stands o f P a c i f i c s i l v e r f i r .  POPULATION F  F  —  W  A  B  C  H  28  110  222  225  335  368  363  —  83  195  199  308  341  336  112  116  225  258  254  —  9  113  147  145  —  111  145  142  33  32  —  15  W  0. 01848  A  0. 01420  0 .00480  —  B  0. 01696  0 .01283  0 .01433  C  0. 02133  0 .01517  0 .01316  0 .00178  H  0. 01745  0 .01390  0 .00889  0 .00504  0.00192  —  N  0. 01173  0 .00596  0 .00783  0 .00830  0.00843  0 .00617  R  0. 01165  0 .01080  0 .01589  0 .01332  0.01681  0 .01359  N  0.00201  R  —  10 to  93  0.03  0.02  0.01  0.0  GENETIC DISTANCE  F i g u r e 6.1  Dendrogram from c l u s t e r a n a l y s i s based.on Nei's (1978) g e n e t i c d i s t a n c e s between e i g h t p o p u l a t i o n s of P a c i f i c s i l v e r f i r , r e f e r e n c e d by l e t t e r and d e s c r i b e d i n Table 2.1.  94  the two middle l a t i t u d e  stands  among g e n e t i c and geographic  (B & C ) .  The c o r r e l a t i o n  d i s t a n c e s was not s i g n i f i c a n t ,  with g e o g r a p h i c a l d i s t a n c e , e x p l a i n i n g only 1.4% o f the variation  i n g e n e t i c d i s t a n c e among p a i r s o f p o p u l a t i o n s .  I n d i v i d u a l l o c u s and average v a l u e s of Nei's d i v e r s i t y s t a t i s t i c s are l i s t e d  i n Table  6.5.  (1977)  Only  polymorphic l o c i were used i n order t o emphasize the extent of p o p u l a t i o n d i f f e r e n t i a t i o n  (Brown 1979) and t o make  comparisons with the d i f f e r e n t i a t i o n  Table  6.5  index  of Gregorius and  Gene diversity® estimates f o r s i x polymorphic l o c i averaged over e i g h t p o p u l a t i o n s o f P a c i f i c silver f i r . Mean gene d i v e r s i t y  Locus  Total (H )  PGI-2 G6P PGM IDH MDH-1 AAT-2  0.381 0.349 0.318 0.002 0.005 0.150  T  Within  Population (H ) s  Among P o p u l a t i o n (D ) ST  0.363 0. 340 0.288 0. 002 0. 005 0.140  0.018 0.009 0.030 0.000 0.000 0.010  1  0.039  Mean  (including  monomorphic  loci)  0.018  N e i (1975). Gene d i v e r s i t y based on estimates o f expected h e t e r o z y g o s i t y u s i n g p o l l e n a l l e l e f r e q u e n c i e s H = t o t a l gene d i v e r s i t y over a l l p o p u l a t i o n s based on average a l l e l e f r e q u e n c i e s f o r each l o c u s ; H = average gene d e n s i t y w i t h i n p o p u l a t i o n s ; D = H - H. T  s  ST  ®  ST  0.047 0.026 0. 094 0.000 0.000 0.067  Mean  ®  G ®  T  s  G = r e l a t i v e amount of gene d i v e r s i t y due t o d i f f e r e n c e s among p o p u l a t i o n s ( = D / H) . ST  ST  T  95  Roberds  (1986).  Three l o c i  (PGI-2, G6P  and PGM)  moderate l e v e l s of d i v e r s i t y w i t h i n p o p u l a t i o n s  showed (H ) s  but  the  amounts were a l s o v e r y c l o s e to values shown by a l l populations  (stands) considered t o g e t h e r  (H ) , r e s u l t i n g i n T  d i v e r s i t i e s a t t r i b u t a b l e to d i f f e r e n c e s among p o p u l a t i o n s (D 's) which were not much l a r g e r than D ST  e x h i b i t i n g much lower l e v e l s of d i v e r s i t y AAT-2).  T h i s low  manifest  i n an average G  values f o r l o c i  ST  (IDH,  MDH-1  and  l e v e l of p o p u l a t i o n d i f f e r e n t i a t i o n i s ST  value f o r polymorphic l o c i  of  0.039, or l e s s than 4% of the t o t a l d i v e r s i t y d e t e c t e d i n a l l the samples being a t t r i b u t e d to g e n e t i c d i f f e r e n c e s among p o p u l a t i o n s of P a c i f i c s i l v e r  fir.  m a j o r i t y of a l l e l i c v a r i a t i o n  resides within  individual  (96%)  Thus, the vast  stands.  Another measure of g e n e t i c d i s p a r a t e n e s s proposed by Gregorius  and Roberds  (1986) i s summarized i n Table  6.6.  I n d i v i d u a l l o c u s Dj values, r e p r e s e n t i n g the amount of a l l e l i c d i f f e r e n t i a t i o n of the j  t  h  p o p u l a t i o n from a l l other  p o p u l a t i o n s are given i n the t a b l e as w e l l as 8 v a l u e s f o r each l o c u s . can be  A c c o r d i n g to Gregorius  i n t e r p r e t e d as the p r o p o r t i o n  percentage i n Table elements  6.6)  and Roberds  (expressed as a  of the e f f e c t i v e number of g e n e t i c  ( i n t h i s case genes or a l l e l e s ) by which the j  p o p u l a t i o n d i f f e r s from i t s complement populations pooled). (weighted  (1986) D^  ( a l l other  The value 8 r e p r e s e n t s the mean  by sample s i z e ) percentage of the  effective  t h  96 Table  6.6  W  Locus  F  PGI-2 G6P PGM IDH MDH-1 AAT-2  9.6 23.0 2.8 0.1 0.2 5.5  Mean  6.9  ®  I n d i v i d u a l a l l e l i c d i f f e r e n t i a t i o n v a l u e s (Dj's) f o r each l o c u s and p o p u l a t i o n and t o t a l s (summed over population) and average 8 v a l u e s f o r each varying locus detected i n eight populations of Pacific silver f i r .  A  B  Population H c  6.0 3.7 11.4 0.2 0.2 15.5  4.8 4.8 17.0 0.1 0.2 5.6  7.9 0.9 14.7 0.5 0.2 1.6  15.0 5.2 11.3 0.1 0.2 0.0  6.2  5.4  4.3  5.3  R  N  8 (weighted) <£  12.9 5.9 3.3 0.1 0.2 6.8  7 .1 7 .2 1 .9 0 .1 0 .2 2 .4  17.3 5.3 3.4 0.1 1.1 4.7  10.1 6.7 7.7 0.1 0.3 6.3  4.9  3 .2  5.3  5.2  D i f f e r e n t i a t i o n values (5's) determined by t a k i n g a r i t h m e t i c mean o f Dj's weighted by sample s i z e o f each population.  numbers o f genes by which p o p u l a t i o n s complements.  differ  I t i s apparent from Table  d i s c r i m i n a t e b e t t e r than o t h e r s . l e s s than 1% f o r IDH and MDH-1  from t h e i r  6.6 t h a t some  The 8 v a l u e s  loci  range from  t o over 10% f o r PGI-2.  I n d i v i d u a l Dj's r e v e a l t h a t no s i n g l e l o c u s i s p a r t i c u l a r l y d i s c r i m i n a t i n g , as l a r g e Dj v a l u e s are not c o n s i s t e n t f o r any  one l o c u s across a l l p o p u l a t i o n s .  e s p e c i a l l y evident  This v a r i a t i o n i s  f o r G6P, where Dj's vary  from 0.9 t o  23.0. Comparison o f Dj's f o r each p o p u l a t i o n p o p u l a t i o n F i s the most d i f f e r e n t i a t e d  suggest  ( p r i m a r i l y as a  r e s u l t o f t h e h i g h value at G6P), and p o p u l a t i o n N the least.  C a l c u l a t i o n o f mean Dj v a l u e s  shows p o p u l a t i o n F  differs  from the r e s t o f the p o p u l a t i o n s  at n e a r l y 7% o f . t h e  97  e f f e c t i v e number of genes f o r these s i x l o c i .  Mean Dj  v a l u e s a l s o c o r r e l a t e n e g a t i v e l y with l a t i t u d e P < 0.05)  i n d i c a t i n g t h a t p o p u l a t i o n s are  (r = -0.778;  less  d i f f e r e n t i a t e d i n the n o r t h e r n p o r t i o n of the sampled range of  Pacific silver f i r . 6.3.3  Mating  system e f f e c t s on  variation  I n d i v i d u a l l o c u s f i x a t i o n i n d i c e s and mean v a l u e s (weighted by the i n v e r s e of the a s s o c i a t e d v a r i a n c e e s t i m a t e d a c c o r d i n g t o Rasmussen 1964)  f o r maternal  embryo gene p o o l s i n each p o p u l a t i o n are found  t r e e and  i n Table  6.7.  These v a l u e s were c a l c u l a t e d i n order to q u a n t i f y the extent to  which the observed  number of heterozygotes  p o o l d e v i a t e d from the number expected assumptions. heterozygous  i n each gene  under Hardy-Weinberg  Where F i s g r e a t e r than zero,  fewer  genotypes than p r e d i c t e d are present and,  the v a l u e of F i s l e s s than zero, an excess  of  where  heterozygotes  r e l a t i v e t o e x p e c t a t i o n s i s observed. Table 6.7  shows t h a t s i n g l e l o c u s F's are h i g h l y  v a r i a b l e both among l o c i commonly v a r i a b l e l o c i  and gene p o o l s .  (PGI-2, G6P  and PGM),  e s t i m a t e s are n e g a t i v e , 26 of 48 p o s i t i v e . invariant l o c i , heterozygosity  MDH-1  For the t h r e e  showed a tendency t o  22 of  48  Of the more excess  (eight out of 10 estimates are n e g a t i v e ) .  Comparing gene p o o l s r e v e a l s a somewhat s t r o n g e r t r e n d . Over 75% of the i n d i v i d u a l locus F's f o r  Table 6.7  I n d i v i d u a l locus and minimum variance mean estimates of the f i x a t i o n index f o r maternal tree (M) and embryo (E) gene pools i n eight populations of P a c i f i c s i l v e r f i r .  POPN  F  •w E  M  E  LOCUS  M  PGI-2  -0.275  G6P  -0.228 -0.058 -0.435 -0.092 -0.155  0.149 -0.274  0.034 -0.250  PGM  0.640  IDH  0.009 -0.045 -0.035 -0.011  0.050  0.039 -0.101 -0.154  MDH-1  -0.150 -0.088 -0.060 -0.049  AAT-2  0.128 -0.042  MEAN  -0.011  M  c  B  A E  M  E  0.091 -0.154 -0.056 0.077 -0.068 0.045  M 0.375  0.034 -0.096  0.008 -0.005  H E 0.184  M  R  N E  M  E  M  E  0.258  0.302 -0.192  0.012  0.004 -0.252  0.007 -0.310  0.041 -0.020  0.004  0.024 -0.196  0.063 -0.052  0.117 -0.041  0.123  0.017 -0.052 -0.304 -0.041  0.130  0.165  0.123  0.061 -0.049 -0.013 0.059  0.008 -0.040 -0.157  0.002 -0.052 -0.029 -0.183 -0.007 -0.057 -0.028  0.057  0.064 -0.137  0.002 -0.052 -0.111  0.069 -0.063  0.050 -0.030  0.002 -0.078  0.047  00  99  maternal t r e e s show excess h e t e r o z y g o s i t y ,  while  l e s s than  47% of embryo f i x a t i o n i n d i c e s are n e g a t i v e . In s p i t e of l a r g e i n t e r - l o c u s v a r i a t i o n i n F, mean v a l u e s a l s o r e f l e c t the p a t t e r n of g r e a t e r homozygosity i n embryo gene p o o l s than t h a t of maternal t r e e s . e i g h t maternal t r e e p o p u l a t i o n s  have average  i n d i c e s t h a t are negative while negative obtained  f o r only three embryo  Hedrick  Seven of  fixation  average v a l u e s were  populations.  (1983) c l a s s i f i e s t h i s method of e s t i m a t i n g  f i x a t i o n i n d i c e s (based  on d e v i a t i o n s i n genotypic  p o p u l a t i o n s t r u c t u r e ) an i n d i r e c t one  ( a f t e r Brown  [1979]).  An a l t e r n a t e method, c a l l e d the d i r e c t approach, uses the r e l a t i o n s h i p F = 1-t/l+t, where t i s an estimate  of the  p r o p o r t i o n of o u t c r o s s i n g based on progeny genotypes i d e n t i f i e d from o u t c r o s s i n g events. that populations  Both estimates  assume  have reached i n b r e e d i n g e q u i l i b r i a , meaning  t h a t the same amount of o u t c r o s s i n g r e c o n s t i t u t e s the same l e v e l of heterozygotes  at each round of mating.  assumption, both estimates  should be e q u i v a l e n t  Given t h i s i f genotypic  p r o p o r t i o n s are determined by no e v o l u t i o n a r y f a c t o r s other than t h e mating system. Table  6.8 shows F  m u l t i l o c u s estimate  x  and F  D  ( c a l c u l a t e d u s i n g the  of o u t c r o s s i n g , t , d e r i v e d i n Chapter m  5) compared, where p o s s i b l e , f o r maternal and embryo gene pools.  The d i r e c t estimates  of F, determined from progeny  Table  6.8  Estimates o f average e q u i l i b r i u m f i x a t i o n i n d i c e s based on an i n d i r e c t method (F , a f t e r Brown 1979) and a d i r e c t approach, where F i s d e r i v e d from t h e m u l t i l o c u s extimate of o u t c r o s s i n g , t ( R i t l a n d and El-Kassaby, 1985) . x  D  m  (estimated POPULATION F W A B C H N R ®  from 1-H /H )® D  e  F  D  (estimated  from  MATERNAL  EMBRYO  EMBRYO  -0.011 -0.052 -0.183 -0.057 0.057 -0.063 -0.030 -0.078  0,002 -0.029 -0.007 -0.028 0.069 0.050 0.002 0.047  < 3 > -0.043 0.135 0.059 0.112 0.070 0.159 0.004  l-t /l+t ) m  m  Minimum v a r i a n c e mean v a l u e s , c a l c u l a t e d Table 6.7.  from Shaw and A l l a n d 1982b, and l i s t e d i n  No estimate  f o r p o p u l a t i o n F.  of o u t c r o s s i n g r a t e obtained  101  a r r a y s , are l a r g e l y p o s i t i v e . d e f i c i e n c y of heterozygotes  T h i s suggests  i n the progeny  D  o n l y i n nature  overall  generation.  I n d i r e c t e s t i m a t e s of F, determined f o r both match F  an  generations,  and magnitude f o r p o p u l a t i o n  W.  Embryo p o p u l a t i o n s show l e s s homozygosity than t h a t p r e d i c t e d by the o u t c r o s s i n g r a t e estimate p o s i t i v e than F ) , and maternal  6.4  (F  x  less  t r e e p o p u l a t i o n s show even  D  greater heterozygosity  (F-j's are  maternal  < F  x  embryo < F ) . D  Discussion Given the e c o l o g i c a l amplitude  of P a c i f i c s i l v e r  fir,  and s u c c e s s i o n a l s t a t u s  combined w i t h i t s very h i g h shade  t o l e r a n c e and p r o d u c t i o n of heavy seeds,  the  potential  e x i s t s f o r s u b s t a n t i a l g e n e t i c v a r i a t i o n among p o p u l a t i o n s . However, e s t i m a t e s  of enzyme v a r i a b i l i t y are low a c r o s s  both  p o p u l a t i o n s and gene p o o l s . The p a t t e r n s of v a r i a t i o n which were d e t e c t e d may may  r e f l e c t c e r t a i n b i o l o g i c a l phenomena or  be the r e s u l t o f a l a r g e l y unknown degree of  e r r o r inherent i n estimates  of t h i s k i n d .  sampling  In a d d i t i o n ,  t h e r e i s some d i s p u t e i n the f o r e s t g e n e t i c s l i t e r a t u r e  as  t o which measures are most a p p r o p r i a t e f o r d e s c r i b i n g v a r i a t i o n when i t i s d e t e c t e d M u l l e r - S t a r c k and G r e g o r i u s  Apparent low  1986,  1986).  l e v e l s of within-population v a r i a b i l i t y  were based on e s t i m a t e s and expected  (Gregorius and Roberds  o f e f f e c t i v e number of a l l e l e s  heterozygosity  (H ; r e f e r r e d t o as gene • e  (n ) e  102  d i v e r s i t y by Nei 1973).  While n  i s considered a true  e  d i v e r s i t y measure a c c o r d i n g t o Gregorius  (1987),  reflecting  the number o f d i f f e r e n t g e n e t i c types i n a p o p u l a t i o n , i t s sampling H  e  p r o p e r t i e s are not c o n s i d e r e d  by N e i (1975).  Observed f r e q u e n c i e s of a l l e l e s form t h e  b a s i s f o r both o f these estimates,  so i t i s not unexpected  t h a t they would show s i m i l a r t r e n d s . statistical  "as good" as t h a t o f  Statements as t o the  s i g n i f i c a n c e o f any d i f f e r e n c e s among  p o p u l a t i o n s or between gene p o o l s f o r these values have been avoided because of the l a r g e i n t e r - l o c u s v a r i a n c e s i n average h e t e r o z y g o s i t y estimates and the l i k e l i h o o d t h a t parametric  inherent  (Simon and A r c h i e 1985) s t a t i s t i c a l t e s t s may not  be v a l i d f o r the sample s i z e s a v a i l a b l e and the l e v e l of h e t e r o z y g o s i t y present estimates  i n the data  of d i v e r s i t y do suggest  heterozygous i n the south than  (Archie 1985).  t h a t p o p u l a t i o n s are more  i n the northern p o r t i o n of  the sampled range and t h a t extant p o p u l a t i o n s , by maternal  represented  t r e e samples, appear t o be more heterozygous  than the p o t e n t i a l silver fir.  Point  ( v i a b l e seed) p o p u l a t i o n s of P a c i f i c  Measures o f H  e  fall  i n the range o f average  h e t e r o z y g o s i t i e s f o r a number o f c o n i f e r s (Boyle and Morgenstern 1986) but below the average H  e  (0.207) of 20  c o n i f e r s p e c i e s determined by Hamrick et al. (1981). Contingency % suggest  2  t e s t s of a l l e l e  some evidence  frequency d i f f e r e n c e s  f o r genetic heterogenity  among  p o p u l a t i o n s of P a c i f i c s i l v e r f i r but t h e r e are two c a u t i o n s  103  accompanying t h i s o b s e r v a t i o n .  Only two  of s i x v a r y i n g  loci  c o u l d be t e s t e d by t h i s procedure owing t o the presence of too many expected c e l l remaining  loci  f r e q u e n c i e s of l e s s than f i v e i n the  (Cochran 1954).  Muona and  a l s o p o i n t out t h a t a l l e l e frequency s i g n i f i c a n t even i f they s i z e s are s u f f i c i e n t l y 134  seeds  (268  t o 335  seeds  large.  They c o n s i d e r e d  i n the present  %  2  be  are r e l a t i v e l y s m a l l i f sample  (318 to 670  based on contingency  (1985)  differences w i l l  a l l e l e s ) per p o p u l a t i o n  s i z e s f o r populations  Schmidt  a sample of  "large".  study  Sample  ranged from  159  a l l e l e s ) per l o c u s , so d i f f e r e n c e s t e s t s may  Adams (1985b) found a l l e l e  be exaggerated.  Neale  and  f r e q u e n c i e s among f o u r stands  balsam f i r to be r e l a t i v e l y homogeneous f o r e i g h t l o c i  of  and  s i m i l a r t o another balsam f i r p o p u l a t i o n sampled from a d i f f e r e n t l o c a t i o n and 1984).  i n a d i f f e r e n t year  A c o n s i s t e n t l y low  Despite  et  e f f e c t i v e number of a l l e l e s  a t t r i b u t e d by Bousquet et al. flow among p o p u l a t i o n s  (Jacobs  al. was  (1987) to a high l e v e l of gene  of green a l d e r .  a number of r e f e r e n c e s  g e n e t i c d i s t a n c e measure of Nei at v a l u e s c l o s e to zero  i n d i c a t i n g that  (1972, 1978)  (Nei 1972,  Gregorius  the  performs p o o r l y 1978,  Falkenhagen 1985), i t i s most commonly c i t e d i n a s s e s s i n g inter-population variation in conifers.  Commonly, D's  are  very small and do not c o r r e l a t e w e l l with p h y s i c a l d i s t a n c e s ( L i n h a r t et al.  1981,  suggests i s o l a t i o n per  Boyle se  and Morgenstern 1986), which  i s not r e s p o n s i b l e f o r p o p u l a t i o n  104  differentiation. fir  T h i s p a t t e r n i s observed i n P a c i f i c s i l v e r  and when c o n s i d e r e d  along with the apparent commonality  of a l l e l e s may i n d i c a t e few b a r r i e r s t o gene flow a r e present, sampled  despite considerable e c o l o g i c a l v a r i a t i o n  among  populations.  The  two measures of p o p u l a t i o n d i f f e r e n t i a t i o n  t h i s paper, G purposes  ST  although  (equivalent t o F , but d e r i v e d f o r d i f f e r e n t ST  [Nei 1975]) and 5 a l s o suggest  differentiation  cited in  population  i s not s t r o n g i n P a c i f i c s i l v e r f i r ,  t h e measures themselves do not appear t o be  equivalent. Table  Table  6.9 l i s t s  6.9  individual  locus d i f f e r e n t i a t i o n  values  Comparison of the p r o p o r t i o n o f d i f f e r e n t i a t i o n e x p l a i n e d by v a r i o u s l o c i f o r estimates o f G (Nei 1977) and 8 (Gregorius and Roberds 1986). Values expressed as percentages. ST  8  Locus  G  PGI-2  4.7  10. 1  G6P  2.6  6. 7  PGM  9.4  7.7  IDH  0.0  0. 1  MDH-1  0.0  0. 3  AAT-2  6.7  6. 3  f o r both G if  ST  and 8.  ST  Although d i f f e r e n c e s might be negated  some i n d i c e s of d i s p e r s i o n were known with any  reliability,  i t appears t h a t i n g e n e r a l ,  the ranking o f l o c i  105  i n t h e i r a b i l i t y to detect population d i f f e r e n t i a t i o n i s v e r y c l o s e when a l l e l e f r e q u e n c i e s are extreme  (IDH, MDH-1,  AAT-2) but where a l l e l e f r e q u e n c i e s .are more moderate the rank and magnitude of the c o n t r i b u t i o n of l o c i v a r i e s widely (e.g. the 8 v a l u e of PGI-2 suggests d i s c r i m i n a t i n g as the G  ST  i t i s twice  as  value would i n d i c a t e ) .  Perhaps  t h i s a r i s e s from l a r g e r v a r i a n c e s a s s o c i a t e d with a l l e l e f r e q u e n c i e s which are approximately El-Kassaby  and S z i k l a i 1983).  equivalent  This r e s u l t  (Brown  suggests  1979,  the  importance of i n c l u d i n g as many l o c i as p o s s i b l e i n e s t i m a t i o n of p o p u l a t i o n d i f f e r e n t i a t i o n and t h a t c o n s i d e r a t i o n be given to the degree of polymorphism expressed  by l o c i when s p e c i e s or p o p u l a t i o n comparisons are  desired. In P a c i f i c s i l v e r f i r , average 8 was average G  ST  (using polymorphic l o c i only) of  Comparisons between F p i n e subpopulations populations  T h i s may  S T  3.9%.  (Gregorius and Roberds 1986), beech  (Miiller-Starck  1986)  sub-  and d i s c r e t e p o p u l a t i o n s  and Gregorius  1986)  at l e a s t twice the magnitude of F  S T  and  in a l l  (= G ) . ST  r e f l e c t a g r e a t e r s e n s i t i v i t y of 8 i n d e t e c t i n g  p o p u l a t i o n d i f f e r e n c e s (El-Kassaby result  compared t o  and 8 ave been made f o r ponderosa  (Gregorius et al.  of Scots p i n e cases 8 was  5.2%  1990), or may  of comparing apples and oranges - F  S T  be  and G  ST  the measures  are based upon assumptions of random samples of the genome  106  i n c l u d i n g both mono- and polymorphic defined for variable l o c i  whereas 8 i s  only.  Table 6.10 shows average G species.  loci,  The v a r i a t i o n i n G  ST  ST  values o f several  among r e l a t e d s p e c i e s  p a r a l l e l s r e s u l t s f o r most other c o n i f e r genera El-Kassaby  1990). N e i (1973) s t r e s s e s t h a t G  ST  p o p u l a t i o n - s p e c i f i c and non-comparable u n l e s s systems a r e s i m i l a r .  Abies  (reviewed by  values are breeding  Given t h a t v a r i a t i o n i n mating systems  among p o p u l a t i o n s o f P a c i f i c s i l v e r f i r was d e t e c t e d (see  Table 6.10  Species A.  A.  balsamea  lasiocarpa  Estimates o f G  No. o f Populations  ST  f o r s e v e r a l s p e c i e s o f Abies.  G,ST  References  5  0.012  Neale 1978 ( c i t e d i n G u r i e s and L e d i g [1981])  1  0.015  Grant 1977  8  0.018  Present  and M i t t o n  A.  amabilis  A.  procera  22  0.123  Yeh u n p u b l i s h e d ( c i t e d i n Adams 1983)  A.  grandis  23  0.140  Yeh unpublished ( c i t e d i n Adams 1983)  study  107  Chapter 5) and  i s o f t e n encountered i n other c o n i f e r s (Adams  and B i r k e s 1990)  the concept of a s p e c i e s average G  i n a p p r o p r i a t e , yet Nei Roberds and Conkle a l l e l e f r e q u e n c i e s may s t r u c t u r e may  (1975) r e p o r t s these v a l u e s (1984) maintain  by  may  be  himself.  t h a t even though  not d i f f e r s u b s t a n t i a l l y , p o p u l a t i o n  s t i l l be p r e s e n t .  The mating system  a f f e c t p o p u l a t i o n s t r u c t u r e by promoting or hybridization  ST  ( R i t l a n d 1983).  can  restricting  Mating systems c h a r a c t e r i z e d  some degree of i n b r e e d i n g w i l l e x h i b i t reduced  recombination  and  i n c r e a s e d homozygosity which u l t i m a t e l y  r e s t r i c t s gene flow and decreases the e f f e c t i v e size  ( R i t l a n d 1983,  the present  study  L o v e l e s s and  population  Hamrick 1984).  Results  of  suggest t h a t some e v o l u t i o n a r y f o r c e s i n  a d d i t i o n t o the mating system may of P a c i f i c s i l v e r f i r .  be a c t i n g i n p o p u l a t i o n s  I n d i v i d u a l f i x a t i o n i n d i c e s were  h i g h l y v a r i a b l e over l o c i and p o p u l a t i o n s ,  which  may  i n d i c a t e t h a t some genotypes are favored i n some environments, but not i n o t h e r s . heterozygosity detected  A l s o , t h e r e was  i n most of the p o p u l a t i o n s  expected from the mating system alone  (F  a d d i t i o n gene pools r e p r e s e n t i n g extant populations  T  D  In  Pacific silver f i r  Thus, i t may  there i s s e l e c t i o n favoring heterozygosity as was  < F) .  than  showed g r e a t e r h e t e r o z y g o s i t y than t h a t  e x h i b i t e d by p o t e n t i a l p o p u l a t i o n s .  fir,  more  be  in Pacific  that silver  p o s t u l a t e d f o r balsam f i r by Neale and Adams  108  (1985b),  and l i k e l y s e l e c t i o n p r e s s u r e s are d i f f e r e n t i n  d i f f e r e n t environments Ritland  ( L i n h a r t et al.  (1983) contends  1981).  t h a t the assumption  i n b r e e d i n g e q u i l i b r i a i s not unreasonable  of  in highly  o u t c r o s s e d s p e c i e s but the known i n s e n s i t i v i t y of e s t i m a t e s of f i x a t i o n i n d i c e s t o the d e t e c t i o n of i n b r e e d i n g at b e l i e v e d t o be e x i s t i n g i n most c o n i f e r stands Ward and Sing 1970,  Brown 1979,  (F <  Shaw and A l l a r d  levels  0.10;  1982b),  would weigh i n favour of the i n b r e e d i n g l e v e l s p r e d i c t e d by the m u l t i l o c u s o u t c r o s s i n g r a t e . e s t i m a t e d from t  m  Inbreeding  levels  should be more p r e c i s e f o r the sample  s i z e s a v a i l a b l e t o t h i s study because of i t s g r e a t e r statistical efficiency efficient  statistic  ( R i t l a n d and El-Kassaby  i s d e f i n e d by Zar  when o b t a i n e d from any  1985).  (1984) as one t h a t  s i n g l e sample w i l l be very c l o s e t o  the v a l u e of the parameters being estimated.  The method of  e s t i m a t i n g f i x a t i o n used i n t h i s study possesses relatively  l a r g e inherent b i a s  u n l e s s sample s i z e s are very The  An  f i x a t i o n index based  a  (Weir and Cockerham  1984)  large. on samples of v i a b l e seed i s  by nature a c o n s e r v a t i v e estimate, as i t c o n t a i n s the e f f e c t s of any e a r l y embryonic c o m p e t i t i o n which may  occur  p r i o r t o sampling  reason  (Brown and A l l a r d  alone, i n d i r e c t estimates of F may  1970).  For t h i s  be b i a s e d downward.  109  6.5  Conclusion The  e x p e c t a t i o n of a h i g h degree of p o p u l a t i o n  d i f f e r e n t i a t i o n based on the b i o l o g y of P a c i f i c s i l v e r f i r was  not borne out i n the a n a l y s i s of. e l e c t r o p h o r e t i c  v a r i a t i o n , but t h i s r e s u l t may  not be t h a t  surprising  c o n s i d e r i n g the l i m i t s to d e t e c t i o n p l a c e d on i t by nature  of the v a r i a b l e s being estimated.  are s e v e r a l l i n e s of evidence heterogeneity  Nonetheless t h e r e  which p o i n t to some l i m i t e d  among p o p u l a t i o n s .  Heterozygosity l a t i t u d e s and  appears t o be reduced  i n embryo gene p o o l s .  t o be higher i n extant p o p u l a t i o n s ,  i n northern  Heterozygosity suggesting  a c t i n g t o e l i m i n a t e i n b r e d seeds i n nature. diversity,  the  as measured by n  e  or H , e  differentiated,  selection i s  Greater  genetic  i s seen i n p o p u l a t i o n s  sampled i n southern Vancouver I s l a n d . s o u t h e r l y p o p u l a t i o n , F,  appears  I n t e r e s t i n g l y , a more  i s a l s o the most g e n e t i c a l l y  as r e v e a l e d by both D and 8.  i s a l a c k of agreement between G  ST  However, t h e r e  and 8 i n the d e t e c t i o n of  population genetic structure in P a c i f i c s i l v e r f i r .  Lack of agreement between d i r e c t and of the f i x a t i o n index suggests  indirect  estimates  t h a t other e v o l u t i o n a r y  mechanisms i n a d d i t i o n to the mating system may  be  i n f l u e n c i n g the d i s t r i b u t i o n of genotypes w i t h i n p o p u l a t i o n s of P a c i f i c s i l v e r f i r on Vancouver I s l a n d .  110  7. 7.1  SEED GERMINATION  Introduction At present, r e f o r e s t a t i o n i n B r i t i s h Columbia  almost  relies  e x c l u s i v e l y upon p l a n t i n g stock grown from seeds  ( K o n i s h i et al.  1989).  Ever-increasing costs of  producing  c o n t a i n e r i z e d stock demand the use o f h i g h q u a l i t y Edwards  (1982) r e p o r t e d t h a t seeds o f Abies  seeds.  s p e c i e s are  f r e q u e n t l y of lower q u a l i t y than other c o n i f e r s , the range i n nursery germination b e i n g t y p i c a l l y 20-50% 1974). o f Abies  Although  (Franklin  improvements i n the c o l l e c t i o n and  seeds have i n c r e a s e d germination,  handling  t r u e f i r seeds  are known t o e x h i b i t v a r y i n g degrees o f dormancy, which hampers nursery p r o d u c t i o n  (Leadem 198 6).  A c c o r d i n g to Wang (1981) the components o f seed at the p h y s i o l o g i c a l l e v e l i n c l u d e seed g e r m i n a b i l i t y and v i g o u r .  viability,  The v i a b i l i t y of a seed i s simply  i t s c a p a c i t y f o r growth and development 1978).  quality  (Bewley and  Black  G e r m i n a b i l i t y i s a measure o f the a b i l i t y of a  p o p u l a t i o n of seeds t o germinate, (1978) d e s c r i b e i t ,  or as Bewley and  "the maximum percentage  Black  o f seeds t h a t  w i l l germinate under f a v o r a b l e c o n d i t i o n s " .  Vigour  i s more  p r o b l e m a t i c i n i t s d e f i n i t i o n because of i t s complexity. The v i g o u r of seeds was  seen by Heydecker  many "shades of meaning" as seed q u a l i t y .  (1969) t o have as The A s s o c i a t i o n  of O f f i c i a l Seed A n a l y s t s  (AOSA) s t a t e d t h a t v i g o u r i s  o p e r a t i n g on at l e a s t two  l e v e l s - at the b i o c h e m i c a l  level,  Ill  as the c o o r d i n a t i o n of s e v e r a l m e t a b o l i c events and at the macroscopic l e v e l ,  i n the speed and completeness o f  g e r m i n a t i o n over a range of e n v i r o n m e n t a l c o n d i t i o n s 1976a).  (Anon.  V i g o u r i s c o n t r o l l e d by two major f a c t o r s - one  b e i n g g e n e t i c and the o t h e r c o n s i s t i n g of v a r i o u s e n v i r o n m e n t a l c o n d i t i o n s which may development, 1969,  occur d u r i n g seed  maturation, p r o c e s s i n g and s t o r a g e (Heydecker  Maguire 1977).  Seed v i g o u r i s then the sum of a l l  "those p r o p e r t i e s which determine the p o t e n t i a l f o r r a p i d , u n i f o r m emergence and development  of normal s e e d l i n g s under  a wide range of f i e l d c o n d i t i o n s "  (Bonner  1984).  Germination i n c o n i f e r o u s seeds i s the c u l m i n a t i o n of a complex  o f m e t a b o l i c a c t i v i t y i n v o l v i n g two d i s t i n c t genomes  (the d i p l o i d embryo surrounded by the n u t r i t i o n a l  and  osmotic envelopes of the h a p l o i d megagametophyte) and s p e c i f i c environmental t r i g g e r s .  In a d d i t i o n , germination  responses are l i k e l y c o n d i t i o n e d by environments encountered by seeds throughout t h e i r development The p r o c e s s of seed development  (Rowe 1964). which i n v o l v e s the  accumulation of n u t r i e n t r e s e r v e s and the e v e n t u a l suspension of embryo growth. content can drop d r a m a t i c a l l y B l a c k 1978)  As a seed matures, the water (to about 10%; Bewley  and  whereupon normal metabolism i s d i s r u p t e d .  In  t h i s s o - c a l l e d q u i e s c e n t s t a t e the embryo can o f t e n remain a l i v e f o r extended p e r i o d s of time.  Jann and Amen (1977)  m a i n t a i n t h a t a quiescent seed i s r e a d i l y germinable - that  112  is,  growth w i l l resume when the seed i s exposed t o  favourable conditions. dormant  T h i s i s i n marked c o n t r a s t t o a  seed d e f i n e d by Amen (1963) as one t h a t w i l l not  germinate under c o n d i t i o n s n o r m a l l y c o n s i d e r e d f a v o r a b l e f o r i t s growth.  In dormant  seeds, the temporary suspension o f  growth i s c r e d i t e d t o some endogenous i n h i b i t o r y mechanism(s) triggers  f o r which t h e r e may  (Jann and Amen 1977).  dormancy may  be s p e c i f i c environmental Thus, both g e r m i n a t i o n and  be viewed as gross m a n i f e s t a t i o n s of a seed's  g e n e t i c program being engaged  (or disengaged) i n response t o  the p a r t i c u l a r environment which i t encounters. responses are s u b j e c t t o endogenous c o n t r o l ,  Germination  presumably  through the mediation of hormones c o n t a i n e d i n the seed itself, et al.  as w e l l as e x t e r n a l cues  (Jann and Amen 1977, Wang  1982).  S t r a t i f i c a t i o n - moist, low-temperature storage f o r a few t o s e v e r a l weeks e i t h e r i n some medium ( A l l e n 1941) "naked"  ( A l l e n and B i e n t j e s 1954)  or  - i s a commonly-used  dormancy-breaking treatment i n temperate zone c o n i f e r species  (Wang et al. 1982).  C o n d i t i o n s of s t r a t i f i c a t i o n  (or p r e c h i l l i n g ) are s e t t o approximate the environments t h a t autumn-ripening seeds might f i n d themselves exposed t o upon d i s s e m i n a t i o n dormancy may  The degree of  be expected t o show some v a r i a t i o n r e l a t e d t o  c l i m a t e of o r i g i n and R i t l a n d  (Krugman et al. 1974).  (Levins 1969,  Thompson 1981).  Campbell  (1982) found p o p u l a t i o n s of western hemlock at  113  h i g h e r l a t i t u d e s t o e x h i b i t e a r l i e r and more r a p i d germination,  a p a t t e r n which was  d e t e c t e d i n other  forest  t r e e s p e c i e s a l s o i n h a b i t i n g c l i m a t e s where c o l d temperatures l i m i t the growing season  (see r e f e r e n c e s  therein). S t r a t i f i c a t i o n has been shown t o improve the. germination Abies  ( i n terms of c a p a c i t y and/or speed) of s e v e r a l  s p e c i e s , which i s a l s o taken as evidence  e x i s t s i n these seeds  (Edwards 1962).  t h a t dormancy  P r e c h i l l i n g produces  a m e l i o r a t i n g e f f e c t s on the germination  of grand,  subalpine  and P a c i f i c s i l v e r f i r i n degrees v a r y i n g with both and  seedlot  (Edwards 1982,  Leadem 1986).  S i n g l e - t r e e seed c o l l e c t i o n s a v a i l a b l e t o t h i s make i t p o s s i b l e t o analyze germination below t h a t of the seedlot equivalent to population).  germination  responses on a l e v e l  A germination  t e s t of s i x  designed  to obtain  of the magnitude of f a m i l y v a r i a t i o n i n  response,  c o l l e c t i o n region  r e l a t i v e t o t h a t of p o p u l a t i o n  and  (populations grouped by l a t i t u d e ) ,  and  a g i v e n s t r a t i f i c a t i o n regime might a f f e c t t h e i r structure.  As w e l l , the. s i z e of the t e s t  r e p l i c a t i o n s be s p l i t between two which was  study  (where s e e d l o t i s seen as  p o p u l a t i o n s of P a c i f i c s i l v e r f i r was some estimate  species  variance  required that  germination  cabinets,  a l s o i n c o r p o r a t e d i n t o the germination  model.  how  114  7.2  M a t e r i a l s and Methods 7.2.1  C o l l e c t i o n and t e s t i n g methods  Cones were c o l l e c t e d from e i g h t p o p u l a t i o n s of P a c i f i c s i l v e r f i r as d e s c r i b e d i n Chapter 2.  Owing t o space  r e s t r i c t i o n s f o r g e r m i n a t i o n and subsequent progeny six  testing,  p o p u l a t i o n s , two each from north, middle and southern  l a t i t u d e c o l l e c t i o n r e g i o n s of Vancouver  I s l a n d were chosen  and a subset of seven t r e e s w i t h i n each p o p u l a t i o n were randomly Six tree  s e l e c t e d , where t h e r e were s u f f i c i e n t  random samples of 50 f i l l e d  filled  seeds.  seeds were o b t a i n e d f o r each  (42 t r e e s i n t o t a l ) by X-ray. A double germination t e s t , m o d i f i e d from the  I n t e r n a t i o n a l Seed T e s t i n g A s s o c i a t i o n ' s for  t h e t e s t i n g of A. amabilis  (Anon. 1976b) r u l e s  seeds, was conducted at the  Canadian F o r e s t r y S e r v i c e , P a c i f i c F o r e s t r y Centre, V i c t o r i a B.C.  One h a l f of the seeds were s u b j e c t e d t o a 28-day  stratification  ( p r e c h i l l ) p e r i o d p r i o r t o i n c u b a t i o n of a l l  seeds on February 7, 1985.  P r e c h i l l i n g e n t a i l e d p l a c i n g dry  seed samples i n c l e a r p l a s t i c ,  s e a l e d g e r m i n a t i o n boxes  (12x12x3 cm) on t h r e e l a y e r s of Whatman #1 f i l t e r  paper over  "Kimpak" c e l l u l o s e t o w e l l i n g wetted with 43 ml d i s t i l l e d water. for  Dishes were immediately p l a c e d i n darkness at 1-4° C  28 days.  115  U n s t r a t i f i e d seeds were s e t i n t o germination boxes i n a s i m i l a r manner but p l a c e d immediately seeds i n t o two was  along with p r e c h i l l e d  u p r i g h t germination c a b i n e t s .  The  too l a r g e t o be c a r r i e d out i n one c a b i n e t but  experiment was  s t r u c t u r e d such t h a t t h r e e samples of both p r e c h i l l e d  and  u n t r e a t e d seeds from each t r e e were p l a c e d at random on t r a y s i n each c a b i n e t .  Temperatures were maintained  a l t e r n a t e l y at 30° C f o r e i g h t hours and 20° C f o r 16 with l i g h t  at approximately  the h i g h e r temperature tubes.  1000  was  lux being p r o v i d e d d u r i n g  period using cool-white f l u o r e s c e n t  Germinants were counted e i g h t times d u r i n g the  day germination p e r i o d with two first  hours,  o b s e r v a t i o n s d u r i n g the  week t o assess onset of germination.  extended  The t e s t p e r i o d  and a d d i t i o n a l counts were made at 35 and  days t o reduce the t r u n c a t i o n e f f e c t on seeds from germinating t r e e s .  had reached the l e n g t h of the seed coat  a c c o r d i n g t o ISTA  (Anon. 1976b) r u l e s  T h i s number may  (Edwards 1982).  The  ( i . e . cotyledon  extremely  small  ( l e s s than  have been h i g h e r had counts been  made when a l l "normal" s t r u c t u r e s c o u l d be assessed recommended by ISTA  radicle  t h a t would be c o n s i d e r e d abnormal  emergence, twin r a d i c l e s ) was 0.05%).  42  later-  Germinants were removed when the  number of germinants  28  (Anon 1985).  However, t h i s  as  procedure  r e q u i r e s a l o n g e r p e r i o d of i n c u b a t i o n which would i n c r e a s e the l i k e l i h o o d of both f u n g a l contamination e r r o r s as a r e s u l t of crowding.  and  counting  For the purpose of t h i s  116  experiment,  the few abnormal germinants  observed were  c l a s s e d as normal. Germination  counts were summarized as two  response  variables:  germination c a p a c i t y (GC), the number of  germinants,  expressed as a percentage  of f i l l e d  the end of the t e s t ; and germination value a c c o r d i n g t o Czabator the product of two  (1962).  quantities:  seeds,  at  (GV) , computed  T h i s index of germination i s The mean d a i l y  germination  (MDG), o b t a i n e d by d i v i d i n g the t o t a l number of  germinants  by the l e n g t h of the t e s t p e r i o d ( i n days), and the peak value  (PV) which i s determined  germination percentage  by c a l c u l a t i n g a  cumulative  f o r each s u c c e s s i v e count  and  d i v i d i n g by the number of e l a p s e d days.  The maximum  q u o t i e n t corresponds  x PV.  the value of GV, germination 7.2.2  t o PV,  and GV = MDG  The  higher  the more complete and/or the more r a p i d the  process. A n a l y t i c methods  P r e v i o u s work on p r e c h i l l i n g treatments s i l v e r f i r (Davidson  et al.  1984)  suggested  on  Pacific  t h a t the  s t r a t i f i c a t i o n p r o c e s s would a l t e r germination p a t t e r n s t o the extent t h a t i t would be very u n l i k e l y t h a t homogeneity of v a r i a n c e s between treatment  groups would be  achieved.  Given t h i s p r o b a b i l i t y , p r e l i m i n a r y a n a l y s i s of the germination data was  c a r r i e d out u s i n g a multi-way  contingency t a b l e approach  (Fienberg 1970)  i n order t o get  117  some estimate of the r e l a t i o n s h i p s among the sources of v a r i a t i o n without variance  hypothesized  i n v o k i n g the a n a l y s i s of  (ANOVA) assumptions of h o m o s c e d a s t i c i t y  normality.  and  The method a l s o accommodates censored data  The a n a l y s i s i s based individual c e l l germinants  on f i t t i n g a l o g - l i n e a r model t o  f r e q u e n c i e s , i n t h i s case, the number of  per day.  The r e l a t i v e importance  f a c t o r i n the model i s determined approximate %  2  sets.  of a g i v e n  by o b t a i n i n g an  value f o r the f i t of a p a r t i c u l a r model  c o n t a i n i n g the f a c t o r of i n t e r e s t and then r e f i t t i n g  the  model without t h a t f a c t o r and o b s e r v i n g the change i n % . 2  The magnitude of the d i f f e r e n c e r e f l e c t s the  relative  importance  1970).  of the term of i n t e r e s t  (Schoener  Not a l l  f a c t o r s were t e s t a b l e by t h i s method, however, which i s not uncommon where models c o n t a i n both f i x e d and random e f f e c t s (M. G r i e g , UBC The  Computing Center, p e r s . comm. November 1985).  a n a l y s i s d i d c l e a r l y r e v e a l t h a t the  treatment  was  the g r e a t e s t s i n g l e f a c t o r  stratification affecting  germination p a t t e r n s i n sampled P a c i f i c s i l v e r f i r . analyses were conducted  Further  on s t r a t i f i e d and u n s t r a t i f i e d seeds  separately. R e c o g n i z i n g t h a t employing  censored data would  u l t i m a t e l y l e a d t o an underestimate d e c i s i o n t o use ANOVA was  of e r r o r v a r i a n c e s , a  made because of the  reasonably  h i g h germination e x h i b i t e d by most t r e e s i n the study because ANOVA i s f r e q u e n t l y used i n s t u d i e s of seed  and  source  118  variation i n conifers.  An ad-hoc procedure f o r f i n d i n g  s u i t a b l e t r a n s f o r m a t i o n s t o normalize  the c a l c u l a t e d  response v a r i a b l e s and achieve homogeneity o f v a r i a n c e s (A. Kozak, UBC F a c u l t y o f F o r e s t r y ; p e r s . comm. December 1985) was  utilized  f o r GC and GV o f s t r a t i f i e d  u n s t r a t i f i e d seed.  Box's  seed and f o r GC o f  (1949) t e s t f o r e q u a l i t y of  v a r i a n c e s was used i n c o n j u n c t i o n with an a p p r o p r i a t e power transformation software,  ( a l l performed u s i n g MIDAS s t a t i s t i c a l  Fox and Guire 1976) t o o b t a i n v a r i a b l e s s u i t a b l e  t o ANOVA.  Lack of n o r m a l i t y  heteroscedasticity  (Hicks 1982)  and  (Glass et al. 1972) do not s e r i o u s l y  a f f e c t ANOVA f o r balanced  designs.  Attempts t o achieve  homogeneity by some data t r a n s f o r m a t i o n u s u a l l y improves n o r m a l i t y and a d d i t i v i t y o f e f f e c t s censored  nature  Germination  o f t h e data remains i n h e r e n t i n the study.  value f o r u n s t r a t i f i e d seeds f u l f i l l e d ANOVA  assumptions without was  (Zar 1984), however the  transformation.  Where t r a n s f o r m a t i o n  o f b e n e f i t , r e s u l t s u s i n g untransformed v a r i a b l e s are  i n c l u d e d f o r comparison.  A n e s t e d - f a c t o r i a l a n a l y s i s was  based on t h e f o l l o w i n g model:  i jklm  where  = Ji. + Ri + P  + Tk ( i j ) + C + CR 1  n  ji = o v e r a l l mean germination R  A  P  = c l i m a t i c region  j(i)  Tk(ij)  + CPj l ( i ) + CTk l ( i j )  +  response  (i=l,2,3)  = population within c l i m a t i c region = tree within population  (j = l,2)  (k=l,...,7)  ^ ( i jkl)  119  C  = cabinet  2  (1=1,2)  em(ijki) = e r r o r (m=l,2,3)  A l l e f f e c t s i n the model were c o n s i d e r e d random except C which were deemed t o be  fixed.  Expected  R  and  mean squares were  i n c l u d e d t o i n d i c a t e a p p r o p r i a t e terms f o r s i g n i f i c a n c e t e s t i n g and t o enable  e s t i m a t i o n of v a r i a n c e components.  In t h i s chapter, which may  the r e l a t i v e magnitudes of v a r i a t i o n  be a s c r i b e d t o f a c t o r s i n the model are  presented  as r a t i o s o f the a p p r o p r i a t e v a r i a n c e components t o t h e i r sum  (CV,  expressed  as a p e r c e n t a g e ) .  intraclass correlation  (r  I #  The  coefficient  of  Sokal and R o h l f 1981), which i n  these a n a l y s i s measures the p r o p o r t i o n o f v a r i a t i o n among maternal  trees,, i s a l s o computed.  by F a l c o n e r  (1981, p. 126)  This v a l u e i s r e f e r r e d t o  as r e p e a t a b i l i t y and may  viewed as an upper l i m i t of h e r i t a b i l i t y  be  i n the broad  sense.  In a d d i t i o n , the apportionment of v a r i a t i o n based on a percentage of the t o t a l sums o f squares F i s h e r 1932, method o f  Little  1981,  Hicks  1982)  apportioning v a r i a b i l i t y  (%SS  or e t a , 2  i s presented.  after This  i s a p p e a l i n g because  t h e r e i s no chance of o b t a i n i n g negative v a r i a n c e components when source 1987).  c o n t r i b u t i o n s are very small  Numerically,  r e s p e c t i v e %CV Maze et al.  was  (1989).  ( i . e . Huehn et  the s i m i l a r i t y between %SS  al.  and  shown f o r D o u g l a s - f i r growth v a r i a b l e s by However, when compared with  the  e q u i v a l e n t v a r i a n c e components, r e s i d u a l v a r i a t i o n i s  120  u s u a l l y underestimated u s i n g %SS,  so t h a t the p r o p o r t i o n of  the t o t a l v a r i a t i o n i n the data accounted f o r by o t h e r terms s h o u l d be c o n s i d e r e d maximal 7.3  (Hicks 1982  p.  135).  R e s u l t s and d i s c u s s i o n R e s u l t s of /ANOVA f o r germination c a p a c i t y  (transformed)  of s t r a t i f i e d seeds are p r e s e n t e d i n T a b l e s 7.1a  and  7.1b.  I t i s e v i d e n t t h a t the c a b i n e t e f f e c t and i t s i n t e r a c t i o n s are n e g l i g i b l e .  A s i m i l a r l a c k of s i g n i f i c a n c e was o b t a i n e d  u s i n g response v a r i a b l e s f o r u n s t r a t i f i e d seeds,  prompting  the d e c i s i o n t o remove the c a b i n e t terms from the model, thus improving the e r r o r degrees of freedom. collection was  The r e g i o n of  (R) - northern, mid and southern Vancouver  Island  r e t a i n e d i n the model d e s p i t e i t s s m a l l c o n t r i b u t i o n  because  o f i t s i m p l i c a t i o n f o r seed crop management.  P o p u l a t i o n s w i t h i n r e g i o n s and t r e e s w i t h i n p o p u l a t i o n s account f o r a s u b s t a n t i a l p o r t i o n of t o t a l v a r i a n c e , as r e v e a l e d by e i t h e r %SS or %CV  (Table 7.1b).  The frequency of germinants vs. non-germinating compiled i n Table 7.2  seeds  f u r t h e r i l l u s t r a t e s both c a b i n e t  s t r a t i f i c a t i o n e f f e c t s on germination c a p a c i t y .  There  and was  v e r y l i t t l e d i f f e r e n c e i n the number of germinants of e i t h e r u n s t r a t i f i e d or s t r a t i f i e d seed between the two g e r m i n a t i o n cabinets  ( l e s s than 2% of the t o t a l number of germinants i n  the t e s t ) .  There was,  however, s u b s t a n t i a l d i f f e r e n c e i n  the number of seeds l e f t ungerminated  at the end of the t e s t  Table 7.1a  Sources o f v a r i a t i o n , a s s o c i a t e d degrees of freedom, sum of squares, mean squares, F v a l u e s and a s s o c i a t e d p r o b a b i l i t i e s f o r a mixed e f f e c t s ANOVA model o f g e r m i n a t i o n c a p a c i t y f o r s t r a t i f i e d seeds (transformed v a l u e s ) .  Source o f Variation Region  Degree o f Freedom  (R)  Sum.of Squares  Mean Squares  F  Probability  2  0..515  0,.257  0..155  0 .86  3'  4..991  1,.664  11..228  < 0 .00  36  5,.335  0..148  12..268  < 0 .00  Cabinet (C)  1  0..003  0..003  0..085  0 .78  C X R  2  0.. 053  0..027  0..695  0 .57  C X P (R)  3  0.,115  0..038  2..456  0 .08  36  0,.560  0,.016  1..288  0 .15  Residual  168  2..029  0..012  Total  251  13.. 601  Population Tree  (P(R))  (T(PR))  C X T (PR)  Table 7.1b  Sources of v a r i a t i o n , percentage of t o t a l v a r i a t i o n based on sums of squares (%SS), components of v a r i a n c e (%CV) and expected mean squares based on a mixed e f f e c t s ANOVA model of germination i n P a c i f i c s i l v e r f i r on Vancouver I s l a n d . C a l c u l a t i o n s based on ANOVA d e s c r i b e d i n Table 7.1a.  Source of Variation  %SS  %CV  Region  3. 8  3.3  <Te + 6 o  36. 7  48.8  ° e + 6c  39. 2  28.4  ° e + 60"  < 0. 0  < 0.0  ° e + 3o"  C XR  0. 4  0.7  ° e + 3o  C X P (R)  0. 9  2.2  ° e + 3G  C X T(PR)  4. 1  1.5  <5e + 3o  14 . 9  15.1  100. 0  100.0  (R)  Population Tree  (T (PR) )  Cabinet  Residual Total  (P(R))  (C)  Expected Mean Squares  2  2  T P R  + 42a  T P R  + 42a2  2  2  2  2  TPR  2  2  2  PR  +  pR  :  2  2  2  CTPR  + 21C + 2la  2  CTPR  2la  2  CTPR  2  2  2 CTPR  2 CPR  C P R  C P R  + 126())  c  + 42a  2 CR  Table 7.2  T o t a l number o f germinants and non-germinants i n a l l 42 t r e e s i n v o l v e d i n a p a i r e d g e r m i n a t i o n t e s t of s i x populations of P a c i f i c s i l v e r f i r . One h a l f o f the seeds were s u b j e c t e d t o a 28-day s t r a t i f i c a t i o n p r i o r t o t e s t i n g ; seeds from each pre-treatment were f u r t h e r d i v i d e d between two germination cabinets.  PRETREATMENT  CABINET  No. Germinated  No. Ungerminated.  Unstratified  A  4718  1260  5978  B  4890  1213  6103  TOTAL  9608  2473  12081  A  5429  607  6096  B  5317  705  6022  10746  1372  12188  Stratified  TOTAL  Total  as a r e s u l t of pretreatment, with over 20% o f the seeds remaining f o r u n s t r a t i f i e d compared t o j u s t over 11% f o r seeds s u b j e c t e d t o the 28-day p r e c h i l l .  This  result  suggests t h a t t h i s sample o f P a c i f i c s i l v e r f i r seeds possess some degree o f dormancy.  O v e r a l l means f o r  g e r m i n a t i o n c a p a c i t y were 79.6 ± 1 . 8 % (95% CI) and 89.9 ± 1.8% f o r u n s t r a t i f i e d and s t r a t i f i e d seeds, r e s p e c t i v e l y ( c a l c u l a t e d from untransformed d a t a ) .  The i n f l u e n c e o f  s t r a t i f i c a t i o n on both the t o t a l amount and r a p i d i t y o f g e r m i n a t i o n i s r e f l e c t e d more d r a m a t i c a l l y i n the o v e r a l l i n c r e a s e i n germination value, from an average of 4.42 ±.  124  0.22  t o 11.37  ± 0.44.  These r e s u l t s suggest t h a t dormancy  i s indeed a f a c t o r i n f l u e n c i n g germination and i t i s e v i d e n t t h a t the s t r a t i f i c a t i o n treatment dormancy i h t h i s s p e c i e s .  i s e f f e c t i v e i n overcoming  S t r a t i f i c a t i o n has been found t o  improve t o t a l germination i n s e v e r a l s e e d l o t s of P a c i f i c s i l v e r f i r (Edwards 1980, The  Leadem 1986).  r e s u l t s a l s o show t h a t the v a r i a n c e s a s s o c i a t e d  w i t h average  GC i n u n s t r a t i f i e d and s t r a t i f i e d seeds are  e q u i v a l e n t , and perhaps more r e v e a l i n g , the v a r i a n c e i n GV f o r p r e c h i l l e d seeds i s twice t h a t of seeds not s u b j e c t e d t o prechilling. expect  T h i s i s not the t y p i c a l p a t t e r n one would  from s t r a t i f i c a t i o n .  The u s u a l e f f e c t  of  s t r a t i f i c a t i o n i n c o n i f e r s i s a h a s t e n i n g of germination w i t h a concomitant B i e n t j e s 1954,  reduction i n v a r i a b i l i t y  (Allen  and  Edwards 1969) .  Examination  of the r e s u l t s of h i e r a r c h i c a l ANOVA f o r  both u n s t r a t i f i e d and s t r a t i f i e d seeds  (Table 7.3a  and b  r e s p e c t i v e l y ) r e v e a l s t h a t the l a r g e s t source of v a r i a b i l i t y i n both GC and GV trees.  C o n s i s t e n t l y h i g h v a l u e s of i n t r a - c l a s s  correlations, seeds,  i s a s s o c i a t e d w i t h d i f f e r e n c e s among  o b t a i n e d f o r both u n s t r a t i f i e d and  f u r t h e r emphasize the l a r g e i n t e r - t r e e  stratified  variation.  Regions are i n most analyses r e l a t i v e l y unimportant, major e f f e c t of s t r a t i f i c a t i o n on the apportionment  and of  v a r i a t i o n i s seen i n the s h i f t i n g of n e a r l y h a l f of the r e l a t i v e v a r i a n c e a s s o c i a t e d w i t h i n d i v i d u a l t r e e s t o the  the  125  population l e v e l . approximately  I n t r a - i n d i v i d u a l v a r i a t i o n remains  t h e same ( l e s s than 25% of t o t a l SS).  apparent t h a t seed pretreatment  by s t r a t i f i c a t i o n  days has v a r y i n g e f f e c t s on p o p u l a t i o n s  It i s  f o r 28  w i t h i n t h e same  l a t i t u d i n a l band. There does not seem t o be an immediate b i o l o g i c a l explanation  f o r the apparent change i n the r e l a t i v e  importance o f i n d i v i d u a l and p o p u l a t i o n e f f e c t s from seed pretreatment. dormancy i n Abies  resulting  Seedlot v a r i a t i o n i n the degree o f  i s w e l l known (Edwards 1962) and t h i s  might be a t t r i b u t e d t o the t i m i n g of c o l l e c t i o n ,  as Edwards  (1969) observed i n c r e a s e d dormancy from e a r l y t o l a t e c o l l e c t i o n s o f noble f i r .  Cones from both  populations  w i t h i n any one c o l l e c t i o n r e g i o n i n the present c o l l e c t e d at most one day a p a r t . reports that maturity  study  However, Edwards  were  (1982)  d i f f e r e n c e s can e x i s t among cones  w i t h i n the same t r e e and even among seeds w i t h i n any one cone.  Cones were monitored f o r embryo m a t u r i t y  v i a cone-  c u t t i n g t e s t s but the l o g i s t i c s of c o l l e c t i o n r e q u i r e d t h a t cones be p i c k e d from a l l t r e e s i n a given stand on t h e same day.  Thus i t i s u n l i k e l y t h a t the sampled t r e e s  the same degree o f seed m a t u r i t y  w i t h i n each p o p u l a t i o n .  geographic t r e n d i n dormancy i s not evident Pacific silver f i r . deeply  represent A  i n samples o f  None of the samples c o u l d be c o n s i d e r e d  dormant as average germination  c a p a c i t i e s (Table 7.4)  f o r u n s t r a t i f i e d seeds i n a l l s i x p o p u l a t i o n s  Table 7.3a  The percentages o f t o t a l sums o f squares and e q u i v a l e n t v a r i a n c e components from ANOVA a s s o c i a t e d with each source of v a r i a t i o n f o r g e r m i n a t i o n response v a r i a b l e s i n u n s t r a t i f i e d seeds o f P a c i f i c s i l v e r f i r (TRANS = transformed v a l u e s ) .  Source o f Variation  Degrees o f Freedom  % Sums o f Squares GC  (TRANS)GC  % Components of V a r i a n c e GV  GC  (TRANS)GC  GV  Region  2  1,. 1  1. 0  9.7*®  0..0  0.,0  11..7  Population  3  5,.7  6.. 6  1. 2  0..0  0.,0  0,.0  36  74 .7 ,  69..3*  71. 5*  79..1*  66.,2*  69,.8*  Residual  210  18 .5 ,  23.,1  17 .6  20..9  33,,8  18,.5  Total  251  100..0  100..0  100. 0 '  100..0  100,,0  100,.0  Tree  r®  --  x  ® ®  *, s i g n i f i c a n t  0., 66  at P < 0.05  r , intra-class :  0.,79  correlation  coefficient = 0  2 T P R  / o*  2 TPR  + o  2 e  0,.79  Table 7.3b  The percentages o f t o t a l sums of squares and e q u i v a l e n t v a r i a n c e components from ANOVA a s s o c i a t e d with each source of v a r i a t i o n f o r g e r m i n a t i o n response v a r i a b l e s i n s t r a t i f i e d seeds o f P a c i f i c s i l v e r f i r (TRANS = t r a n s f o r m e d v a l u e s ) .  Source of Variation  Degrees of Freedom  R  2  P(R)  3  T (PR)  % Sums of Squares GC  (TRANS)GC  7,.5  36  GV  % Components of V a r i a n c e  (TRANS)GV  3.,8  4 ,,  8  4 ,, 4  33,. 8*®  36., 7*  29..7*  42 . . 4*  39..2*  GC  (TRANS)GC 0..0  0..0  0,.0  28,;6*  47,. 9*  50,.2*  41..0*  39,.4*  48..5*  50,,1*  36., 6*  31..4*  42..7*  44..3*  16.,3  16,.3  210  16., 3  20.,3  17 .0 .  16..9  15..5  18,.4  Total  251  100., 0  100.,0  100.,0  100.,0  100..0  100,.0  --  r  ® ®  *, s i g n i f i c a n t t  --  0.,70  0,, 63  at P < 0.05  r , intra-class  correlation  (TRANS)GV  0..0  Residual  r®  GV  coefficient  = 0"  2 TPR  / C  2 T P R  + 0  2 E  100..0 • 0.,72  100..0 0.,73  128  are c o n s i s t e n t l y h i g h .  In response t o the s t r a t i f i c a t i o n  regime a p p l i e d i n t h i s study, significantly  (P < 0.05)  i n a l l p o p u l a t i o n s except and germination unstratified germination population W  was  t o t a l germination  (GC)  i n c r e a s e d and v a r i a b i l i t y W.  was reduced  Fewer germinants were o b t a i n e d  more v a r i a b l e f o r s t r a t i f i e d seeds than  seeds i n t h i s p o p u l a t i o n . (inferred  from GV)  (Table 7.5).  The  speed of  improved the l e a s t  in  I t s l a t i t u d i n a l counterpart,  F,  on the o t h e r hand, e x h i b i t s a more c l a s s i c response to stratification.  Table 7.4  Region  Sound seeds,  already  possessing  Average germination c a p a c i t y (±95% c o n f i d e n c e i n t e r v a l ) f o r s i x p o p u l a t i o n s of P a c i f i c s i l v e r f i r f o r both u n s t r a t i f i e d and s t r a t i f i e d seeds.  Population  >  PREGERMINATION TREATMENT No 28-day Stratification Stratification  North  R H  82.2 76.2  (3.7) (4.9)  92.5 91.0  Middle  B C  76.2 79.7  (5.5) (3.7)  89.2 (3.5) 94 .7 (2.4)  So-uth  F W  86.6 76.8  (3.9) (3.7)  97.8 (1.4) 68. 9 (5.9)  a reasonably  h i g h c a p a c i t y f o r germination  exposed only t o the c o n d i t i o n s of c o l l e c t i o n (i.e. relatively  (2.9) (2.9)  a f t e r being and p r o c e s s i n g  non-dormant), show higher, more u n i f o r m  129  g e r m i n a t i o n w i t h a marked i n c r e a s e i n GV a f t e r 28 days.  prechilling  However, t h e v a r i a n c e o f GV i n c r e a s e d f o r  s t r a t i f i e d seeds i n a l l s i x p o p u l a t i o n s , although i n c r e a s e was l e a s t unexpected  this  i n p o p u l a t i o n F (Table 7.5). T h i s  r e s u l t merits further  examination.  Table 7.5 Average g e r m i n a t i o n v a l u e s (Czabator, 1962) f o l l o w e d by 95% c o n f i d e n c e i n t e r v a l f o r s i x populations of P a c i f i c s i l v e r f i r , f o r unstrati f i e d and s t r a t i f i e d seeds.  REGION  POPULATION  PREGERMINATION TREATMENT 28-day No S t r a t ification Stratification  North  R H  4.55 (0.51) 3.93 (0.51)  11.06 11.63  (0.94) (0.96)  Middle  B C  4.36 (0.63) 4.16 (0.43)  11.82 12.83  (1.10) (0.69)  South  F W  5.48 (0.49) 5.34 (0.45)  13.70 (0.59) 7.15 (0.90)  P o p u l a t i o n s R and H, sampled from northern Vancouver Island, exhibit unstratified exhibit  germination v a l u e s f o r both  and s t r a t i f i e d seeds and both p o p u l a t i o n s  v i r t u a l l y the same l e v e l s  (Table 7.5). C),  similar  o f v a r i a b i l i t y among t r e e s  In the m i d - l a t i t u d e c o l l e c t i o n  r e g i o n (B and  germination behaviour p a r a l l e l s t h a t i n the n o r t h ,  although p o p u l a t i o n C appears  t o respond more r a p i d l y and  130  uniformly to s t r a t i f i c a t i o n .  Germination  v a l u e s are h i g h e s t  i n the southernmost c o l l e c t i o n r e g i o n without stratification, lower  suggesting t h a t germination i s more r a p i d at  latitudes.  However, the average  GV  for s t r a t i f i e d  seeds of p o p u l a t i o n W i s c o n s i d e r a b l y l e s s than t h a t of F. T h i s r e s u l t when c o n s i d e r e d along with the  substantial  i n d i v i d u a l t r e e component of v a r i a t i o n i n germination response  (Table 7.3b)  prompted a c l o s e r look at germination  b e h a v i o r w i t h i n p o p u l a t i o n W. The average  GC and GV of s i x 50-seed r e p l i c a t i o n s f o r  each t r e e r e p r e s e n t i n g W are l i s t e d Germination  percentages  i n Table  range from 54 t o 92 f o r u n s t r a t i f i e d  seeds and 38 t o 95 f o r s t r a t i f i e d seed. treatment  appeared  7.6.  The  stratification  t o be d e t r i m e n t a l to seeds from f o u r of  the seven t r e e s i n the sample s i g n i f i c a n t l y reduced).  (mean germination c a p a c i t y  Germination  value was  significantly  improved only i n the t h r e e t r e e s i n which germination c a p a c i t y d i d not d i m i n i s h i n response (Table  to  stratification  7.6).  S e v e r a l s t u d i e s on t r e e seed m a t u r i t y reviewed Edwards  by  (1980) have r e v e a l e d t h a t immature seeds tend t o : a)  be l i g h t e r i n weight;  b) germinate  slowly i f at a l l ;  c) show  reduced germination as a r e s u l t of p r e c h i l l i n g ; and d) more s u s c e p t i b l e t o d i s e a s e . l a s t t o be c o l l e c t e d  be  P o p u l a t i o n s F and W were the  (September 30, vs September 8/9  and C and September 28 f o r R and H) and a l s o produced  for B the  131  Table 7.6  Mean germination c a p a c i t y (GC) and germination value (GV) f o r i n d i v i d u a l t r e e s of p o p u l a t i o n W (each mean based on s i x 50-seed r e p l i c a t i o n s ) and t h e i r a s s o c i a t e d 95% c o n f i d e n c e i n t e r v a l s , and p o p u l a t i o n mean v a l u e s f o r both germination responses (*, s i g n i f i c a n t treatment response, P < 0.05). MEAN GERMINATION CAPACITY  Tree No.  No S t r a t i f i c a t i o n  1 2 3 4 5 6 7 Mean  Stratification  75.. 1 (2..6) 77., 7 (3.. 6) 85..7 (4..2) 54 ..5 (8..1) 77.. 0 (4..3) 92..0 (3..2) 75..5 (3..9)  58..7 64..5 85..2 38..2 64..7 95.. 6 75..7  76..8 (3..7)  68.. 9 (5..9)  (4..6)* (8..9)* (4..5) (7.• D* (7..0)" (1..9) (9..9)  MEAN GERMINATION VALUE Tree No.  No S t r a t i f i c a t i o n 5 .23 5 .86 6. 95 2 .54 5 .54 6.23 5 .03  1 2 3 4 5 6 7  5. 34  ;an  h e a v i e s t seeds  (0 .25) (0 .82) (0 .89) (0 .83) (0 . 65) (0 .39) (0 .48)  (0. 45)  Stratification 5 . 07 6 .99 9.49 2 .36 7 . 15 10 .67 8 .29 7. 15  (0 . 69) (1 .73) (0 .54) * (0 . 81) (1 .62) (0 .51) * (1 .52) *  (0. 90) *  (42.8 ± 5 . 3 and 53.1 ± 12.5 g are the mean  v a l u e s and t h e i r  standard d e v i a t i o n s f o r 1000-seed  weights  of p o p u l a t i o n s F and W, r e s p e c t i v e l y ) of a l l s i x p o p u l a t i o n s  132  ( o v e r a l l average  33.3  ± 13.1  g).  Ackerman and Gorman (1969)  found t h a t l i g h t e r weight seeds had percentages  i n lodgepole p i n e .  lower  germination  Seed weights  of ponderosa  p i n e were a l s o c o r r e l a t e d with germination c a p a c i t y and among stand d i f f e r e n c e s were found t o be s i g n i f i c a n t study by Wang and P a t e l  (1974).  In l o b l o l l y pine, h e a v i e r  seeds were shown t o have b e t t e r germination with f a m i l y d i f f e r e n c e s i n seed s i z e et  al.  1982).  c a p a c i t y was  (Hodgson 1980,  C o r r e l a t i o n of seed weight with a l s o h i g h i n wide-ranging  spruce and seed weight was  in a  significant  c i t e d i n Wang germination  samples of white  a l s o found t o c o r r e l a t e  s i g n i f i c a n t l y with population l a t i t u d e i n t h i s species ( K h a l i l 1986).  The  Douglas f i r was  found t o c o r r e l a t e with a l t i t u d e of o r i g i n  ( B i r o t 1972)  1000-seed weight of provenances of  and i n jack pine, Chalupa  and Durzan  (1972)  found seed s i z e s h i g h l y c o r r e l a t e d with c l i m a t e of seed origin.  Simple  l i n e a r c o r r e l a t i o n s among p o p u l a t i o n mean  seed weight and germination response  v a r i a b l e s and  l a t i t u d e f o r P a c i f i c s i l v e r f i r (Table 7.7) p o p u l a t i o n W, latitude, Without W,  also  indicate that  with i t s l a r g e seeds and more s o u t h e r l y  appears  anomalous i n i t s germination  germination speed  ( i n f e r r e d from GV,  behavior. Table  7.5)  i s c l o s e l y r e l a t e d t o seed weight and given the n e g a t i v e c o r r e l a t i o n of weight with l a t i t u d e , appears  faster  germination  c h a r a c t e r i s t i c of more southern p o p u l a t i o n s , the  r e v e r s e of t h a t found by Campbell and R i t l a n d  (1982) i n  133  Table 7.7  Simple l i n e a r c o r r e l a t i o n s between average seed s i z e per p o p u l a t i o n and germination response v a r i a b l e s and l a t i t u d e of p o p u l a t i o n s , c a l c u l a t e d with and without v a l u e s f o r p o p u l a t i o n W. The c r i t i c a l v a l u e s of r f o r t h r e e and f o u r degrees of freedom are 0.878 and 0.811 (* P < 0.05), respectively. Without W  Seed s i z e and  i ) GC ii) iii)  western  hemlock.  m a t u r i t y per  GV  With W  a) u n s t r a t i f i e d b) s t r a t i f i e d  0.872 0.763  0.238 -0.624  a) u n s t r a t i f i e d b) s t r a t i f i e d  0.959* 0.731  0.921* -0.537  l a t i t u d e of population  -0.872  -0.882*  Thus, c o l l e c t i o n date, seed s i z e and/or  se do not adequately  d i f f e r e n c e s observed  account  f o r the  i n germination b e h a v i o r s between F  and  W. In both u n t r e a t e d and t r e a t e d seeds, germination responses inter-tree  are most s t r o n g l y a s s o c i a t e d with  (family) v a r i a t i o n  and R i e n h o l t  differences in  (1986) observed  (Tables 7.3  and 7.4).  Farmer  large family differences i n  g e r m i n a t i o n responses  of tamarack seeds exposed t o d i f f e r e n t  temperature  combinations,  and l i g h t  and stand and provenance  d i f f e r e n c e s were i n a l l cases n o n - s i g n i f i c a n t ,  prompting  them t o s p e c u l a t e a h i g h degree of g e n e t i c c o n t r o l over q u a l i t y and germination c h a r a c t e r i s t i c s ,  seed  s i n c e environments  were presumed t o be r e l a t i v e l y uniform w i t h i n stands.  134  Significant  f a m i l y v a r i a t i o n was a l s o found  (Barnett and Farmer 1978). germinative  i n yellow  Both germination  c a p a c i t y and  energy were r e l a t e d t o p a r e n t a l genotype i n  c o n t r o l l e d crosses of V i r g i n i a pine  (Bramlett  The m a j o r i t y o f v a r i a t i o n i n germination  et al. 1983).  percentage was  a t t r i b u t e d t o d i f f e r e n c e s among 19 b l a c k spruce Stoehr  poplar  and Farmer  (1986) .  They a l s o observed  c l o n e s by  t h a t a few  c l o n e s with "weak, decay-prone" seed c o n t r i b u t e d h e a v i l y t o t h i s variance.  Leadem  (1986) found v a r i a t i o n among s e e d l o t s  of P a c i f i c s i l v e r f i r i n response t o s t r a t i f i c a t i o n and suggested  d i f f e r e n c e s may be the r e s u l t  d i f f e r e n c e s among seeds. (1986) was t h a t the ISTA  of vigour  An a d d i t i o n a l f i n d i n g o f Leadem (Anon. 1976b) p r e s c r i b e d i n c u b a t i o n  temperature was t o o h i g h f o r P a c i f i c s i l v e r  f i r seeds,  which  germinated b e t t e r at a l t e r n a t i n g temperatures of 15 and 10° C.  T h i s f i n d i n g r e i n f o r c e s r e s u l t s from a s e e d l o t o f  P a c i f i c s i l v e r f i r germinated on a thermogradient f i v e constant temperatures  (Davidson  et al. 1984).  p l a t e at I t was  determined t h a t g r e a t e r germination  o c c u r r e d at and below  21° C due i n p a r t t o the prevalence  of seed coat f u n g i at 24  and 27° C which i n f e c t e d emerging r a d i c l e s . a freguent problem i n germination  Mold growth i s  s t u d i e s o f Abies  species  (Edwards 1969, 1982; K i t z m i l l e r et al. 1973, Adkins 1983, B l a z i c h and H i n e s l e y 1984) and high temperatures i n c r e a s e s u s c e p t i b i l i t y t o fungal attack  (Leadem 1986).  135  Every e f f o r t was  made t o a v o i d damage t o the  resin  v e s i c l e s p r e s e n t i n the seed coats of P a c i f i c s i l v e r f i r o b t a i n e d f o r the present study.  The a c t u a l f u n c t i o n o f the  r e s i n i s unknown i n P a c i f i c s i l v e r f i r but i t was i n h i b i t g e r m i n a t i o n i n seeds of white and r e d ( K i t z m i l l e r et al. development  1975)  1973).  When the cones  c o l l e c t e d from p o p u l a t i o n W a r r i v e d at UBC,  two  firs  and t o be a good medium f o r f u n g a l  ( K i t z m i l l e r et al.  v i s i b l e on cones of two  found t o  of the 17 t r e e s .  molds were  N e i t h e r of these  t r e e s were i n c l u d e d i n the germination t e s t ,  however  molds were p r e s e n t i n every germination d i s h f o r a l l t r e e s i n p o p u l a t i o n W, without e x c e p t i o n , by the f o u r t h week of the g e r m i n a t i o n t e s t .  Molds o c c u r r e d i n other p o p u l a t i o n s  as w e l l , but none were i n f e c t e d t o the l e v e l seen i n p o p u l a t i o n W, p a r t i c u l a r l y  f o r s t r a t i f i e d seed.  The  s t r a t i f i c a t i o n c o n d i t i o n s , employed i n t h i s t e s t moisture, temperature  and d u r a t i o n , may  i n terms of  not have been  o p t i m a l f o r p o p u l a t i o n W, p e r m i t t i n g molds t o f l o u r i s h while i n h i b i t i n g germination.  Still,  t h e i r p r o p e n s i t y t o germinate as seen i n Table  individual trees varied i n  d e s p i t e the presence  7.6.  Although the germination p a t t e r n s r e v e a l e d by study may  of f u n g i ,  this  have r e s u l t e d i n p a r t from seed p r e c o n d i t i o n i n g  p r i o r t o h a r v e s t , as suggested by Campbell and (1982) f o r western  Ritland  hemlock, the l a r g e i n d i v i d u a l t r e e e f f e c t  would argue f o r a s i g n i f i c a n t g e n e t i c component i n germination c h a r a c t e r i s t i c s ,  at l e a s t i n the 1983  cone-crop  136  year.  Sorensen and F r a n k l i n  (1977) found l a r g e year e f f e c t s  on seed weight i n noble f i r , preconditioning  which suggests t h a t any  e f f e c t s a s s o c i a t e d with parent t r e e  environment may be temporally preconditioning populations pointed  out,  of h a n d l i n g  variable.  Environmental  can mimic g e n e t i c d i f f e r e n c e s among  (Campbell and R i t l a n d 1982) and, as A l l e n  (1958)  t h e g e n e t i c component may be masked by the k i n d seeds r e c e i v e .  I t i s not known t o what  extent  these f a c t o r s a f f e c t e d germination performances observed i n this test.  Repeating the c o l l e c t i o n i n another year as w e l l  as v a r y i n g a f t e r - r i p e n i n g and s t r a t i f i c a t i o n  conditions  would help t o f u r t h e r d e f i n e the f a c t o r s i n f l u e n c i n g i n t e r and  7.4  intra-population variation.  Conclusions T h i s study does not r e v e a l any strong geographic  trend  i n dormancy over the sampled range, but, the high degree o f f a m i l y v a r i a t i o n has important i m p l i c a t i o n s f o r nursery germination i n t h a t there may be inadvertent rapid-germinating  f a m i l i e s w i t h i n a given  selection for  seedlot.  In  addition, s t r a t i f i c a t i o n , i s l i k e l y to a f f e c t seedlots d i f f e r e n t l y and i t s e f f e c t i v e n e s s may be a complex f u n c t i o n of the environments o f developing  seed, h a n d l i n g  conditions  as w e l l as i n t e r - and i n t r a - t r e e v a r i a t i o n i n dormancy and germination responses.  137  The  l a r g e f a m i l i a l component of v a r i a t i o n d e t e c t e d i n  t h i s study suggests  t h a t s e e d l i n g s of P a c i f i c s i l v e r f i r may  be more e f f i c i e n t l y o b t a i n e d by s i n g l e t r e e cone c o l l e c t i o n and g e r m i n a t i o n / p l a n t i n g on a f a m i l y b a s i s .  The  apparent  c l i n a l t r e n d i n seed s i z e and i t s r e l a t i o n s h i p t o g e r m i n a t i o n performance c o u l d a l s o have i m p l i c a t i o n s f o r nursery p r a c t i c e .  Separation of f i l l e d  difficult  i n Abies s p e c i e s  but l i g h t  seeds present  and empty seeds i s  (Edwards 1979,  i n bulked  1982)  and  s e e d l o t s c o u l d be  r e s u l t i n g i n u n d e r - r e p r e s e n t a t i o n of some f a m i l i e s .  filled lost,  138  8.  8.1  WIND-POLLINATED PROGENY NURSERY-STAGE GROWTH  Introduction Some k n o w l e d g e o f t h e p a t t e r n  a  species  i nt r a i t s  importance  which  i so f value  appropriate  a r eadaptive  t o breeders  (or population)  collections  have p r o v i d e d  of variation  conifers growth  i n growth  (El-Kassaby  f o r s e v e r a l Abies  1969,  Lester  1978,  Kleinschmit  exception,  1970,  a high  1986,  thesis  proportion  enables  individual  parent  progeny nursery  42 t r e e s e m p l o y e d  C h a p t e r 7.  of northern  V a r i a t i o n i n height  has been e s t i m a t e d Lester  of this  on t h e  (Arbez  e t a l . 1976, L i n e s With  variation  little  has been  populations.  which  trees.  trial  seed  of information  e t al. 1 9 9 0 ) .  f u r t h e r refinement  to a level  t o determine  bulked  family structure o f t h ematerial  variation  the  1976,  Funck  o f commercial  characteristics  species  Hamrick  shown t o r e s i d e w i t h i n The  majority  e t al. 1 9 8 7 a ) .  e x h i b i t e d by  improvement.  tests using the  and/or  i norder  selection strategies for  Provenance  extent  of variation  this  of within-population  accounts To t h i s  collected for  for differences  end,  among  a wind-pollinated  was e s t a b l i s h e d u s i n g i nt h egermination  o f f s p r i n g from  test  reported i n  139  8.2  M a t e r i a l s and 8.2.1  Test  Methods  establishment  and-culture  7An o p e n - p o l l i n a t e d p r o g e n y t e s t polystyrene cavities, with  blocks  65ml p e r  blocks,  with  non-test  discussed of the every  tree.  Germination  i n two  By  day  populations planting was  length  count  of t h e  The  c o u n t was  i n C h a p t e r 7. t o e n s u r e an  i n W.  it  first  adequate had  but  i n o n l y two  i n one  of  the  Germinants  were  delayed  test  until  day  of germinants  one  on  day  i n H,  four and  occurred  12  from of  three  in a l l  t r e e s i n p o p u l a t i o n R.  The  o f e v e n - a g e d g e r m i n a n t s became i m p r a c t i c a b l e ,  and  assumed t h a t p l a n t i n g g e r m i n a n t s o f u n i f o r m  radicle  (ideally,  would  reduce the  age  one  times  length.  The  considered  mean age  t o be  Blocks  the  difference.  several early-germinating  the  Each  perimeter  been o b s e r v e d  had  design  around the  supply  s i x , some g e r m i n a t i o n  tree.  germination  t r e e s i n p o p u l a t i o n B,  160  randomized  plot  seedlings planted  test  test  f o r m e d a row  t o r e d u c e edge e f f e c t s .  from the  in  L t d . , each with  of eight seedlings per  replication  of each block, obtained  Plastics  planted  cavity) i n a completely  four replications  eight-seedling  the  (Beaver  was  one  l e n g t h of the  Nevertheless,  t r e e s had  of the  p l a n t a t i o n on  and  of ideal  establishment  week.  were p l a c e d  B.C.  germinants  a l l exceeded the  in a controlled  Canadian F o r e s t r y S e r v i c e , P a c i f i c  Victoria,  seed)  g r o w t h chamber Forestry  Centre,  exposed t o a growing regime of  high  at  was  140  humidity and 18°C, 14-hour days white  ( l i g h t p r o v i d e d by c o o l -  f l o r e s c e n t tubes at 1000 l u x at t h e c e n t e r o f the  bench) and 10 hours of darkness  at 15°C. At week t h r e e ,  when a l l the seed coats were shed  (or removed),  incandescent  l i g h t s at 200 l u x were used t o extend the p h o t o p e r i o d t o 18 hours.  During week e i g h t , the t e s t was moved t o Canadian  P a c i f i c F o r e s t Products L t d . Products) B.C.  (formerly P a c i f i c F o r e s t  Saanich F o r e s t r y Centre n u r s e r y i n Saanichton,  and s e t up a l o n g s i d e other t r u e f i r s e e d l i n g s b e i n g  grown o p e r a t i o n a l l y .  The experimental  s e e d l i n g s were  exposed t o the same c u l t u r a l regime employed by the n u r s e r y for  c o n t a i n e r p r o d u c t i o n of Abies s p e c i e s , i n c l u d i n g  supplemental  l i g h t i n g u n t i l week 26, whereupon the t r i a l  exposed t o an eight-hour p h o t o p e r i o d  (controlled using  b l a c k - o u t c u r t a i n s ) and moderate moisture encourage bud formation.  The t r i a l  was  stress to  was measured weekly  d u r i n g the b l a c k - o u t p e r i o d , i n an attempt  t o assess  v a r i a t i o n i n bud set, but no buds were d e t e c t e d at week 29 when the t r i a l  8.2.2  was harvested.  Measurement and a n a l y s i s  The v a r i a b l e s s e l e c t e d f o r measurement are l i s t e d i n Table 8.1.  Cotyledon numbers were based  on 40 s e e d l i n g s p e r  f a m i l y , as the two edge t r e e s f o r each row p l o t came from the same f a m i l y .  I n d i v i d u a l s e e d l i n g h e i g h t s were taken  along the main stem above the c o t y l e d o n s t o the t i p s o f the  141 Table 8.1  V a r i a b l e s used i n a n a l y s i s of w i n d - p o l l i n a t e d progeny n u r s e r y growth t r i a l of s i x p o p u l a t i o n s of P a c i f i c s i l v e r f i r from Vancouver I s l a n d , B r i t i s h Columbia.  Sampling L e v e l  (n)  V a r i a b l e s measured (abbreviations i n brackets)  Population  (6)  l a t i t u d e (LAT), l o n g i t u d e e l e v a t i o n (ELEV)  (7)  mean r a c h i s l e n g t h (RACHLEN), mean seed weight (SDWT), observed enzyme h e t e r o z y g o s i t y (ENZHET)  Tree/POPN  (POPN)  Replication/tree/POPN (4) Seedling (6-10/replication)  uppermost mm.  (LONG)  mean shoot dry weight (SHWT), mean root dry weight (RTWT) h e i g h t at 8, 16, 22, 26, 27, 28, and 29 weeks (HT), f i n a l diameter (DIAM), c o t y l e d o n number (COTY), growth r a t e (RATE)  needles (held v e r t i c a l l y ) ,  measured t o the n e a r e s t  Root c o l l a r diameter of each s e e d l i n g  determined at harvest u s i n g m i c r o c a l i p e r s .  was Biomass  measurements were o b t a i n e d a f t e r oven-drying each r e p l i c a t i o n s e p a r a t e l y and c a l c u l a t i n g a mean dry weight per seedling.  Maternal t r e e v a r i a b l e s c o n s i s t e d of average cone  r a c h i s l e n g t h , based on measures of seven t o t e n cones (to the  nearest mm) , average seed weight  (mean of 10 samples of  100 f i l l e d  seeds) and observed enzyme h e t e r o z y g o s i t y based  on 11 l o c i  (reported i n Chapter 3 ) .  P o p u l a t i o n s were  142  characterized longitude,  and  by t h e i r geographic l o c a t i o n ( l a t i t u d e , elevation).  P l o t s of seedling height values against average, a p a t t e r n  transformation, l i n e a r i z e the  In data  X i s time).  Y = In  a + b •In  ( S t e e l and  A X,  logarithmic was  Guire 1976)  data f o r each s e e d l i n g 1219). seedling  s e l e c t e d t o best given  The  c a l c u l a t e d i n a simple l i n e a r r e g r e s s i o n and  that b,  slope,  analysis  was  using  on l o g a r i t h m i c a l l y transformed  s u r v i v i n g t o the end  of the t e s t  These i n d i v i d u a l b v a l u e s were taken t o growth r a t e s  on  where  b  T o r r i e 1980)  measurement i n t e r v a l s were i r r e g u l a r .  MIDAS (Fox  (Y = a • X ,  o f e x p o n e n t i a l growth  Y i s s e e d l i n g h e i g h t and  time showed,  (RATE i n Table 8.1).  s i m i l a r t o t h a t o f Maze et al.  (n =  represent  T h i s approach i s  (1989) f o r a n a l y s i s of branch  growth i n Douglas f i r s e e d l i n g s . The upon the  discussion  of v a r i a b l e i n t e r r e l a t i o n s h i p s i s based  r e l a t i v e magnitudes of c o e f f i c i e n t s o b t a i n e d from  l i n e a r c o r r e l a t i o n and  regression  b i v a r i a t e c o r r e l a t i o n s do not any  analyses.  account f o r i n t e r a c t i o n s  o t h e r v a r i a b l e s w i t h the p a i r of i n t e r e s t  however they serve as a u s e f u l s t a r t i n g p o i n t d e t e r m i n i n g r e l a t i v e magnitudes and, relationships.  Simple  (Zar  of  1984),  for  thus, s t r e n g t h  of  Partial correlation coefficients reflect  c o r r e l a t i o n between any  two  v a r i a b l e s while holding  v a l u e s o f other i n t e r a c t i n g v a r i a b l e s c o n s t a n t .  the  In a  the  143  multiple  c o r r e l a t i o n a n a l y s i s , t h e square o f t h e m u l t i p l e  c o r r e l a t i o n c o e f f i c i e n t , o r the c o e f f i c i e n t o f m u l t i p l e determination  (R ), i s i n t e r p r e t e d as t h e amount o f 2  v a r i a b i l i t y i n one o f M v a r i a b l e s which i s accounted f o r by c o r r e l a t i n g i t w i t h M - 1 other v a r i a b l e s . regression,  R  2  In a m u l t i p l e  r e f l e c t s that p o r t i o n o f the t o t a l v a r i a t i o n  i n t h e dependent v a r i a b l e  (Y) which can be a t t r i b u t e d t o t h e  l i n e a r r e l a t i o n s h i p w i t h the independent or p r e d i c t o r variables  (Xj's).  I f t h e X i ' s are themselves h i g h l y  i n t e r c o r r e l a t e d , then c o n c l u s i o n s s i g n i f i c a n c e may be " s p u r i o u s " Simple and m u l t i p l e  regarding  their  (Zar 1984, p. 338) .  c o r r e l a t i o n analyses assume a  normal d i s t r i b u t i o n o f v a l u e s f o r each v a r i a b l e . v a r i a b l e s employed i n t h i s study  (Table  8.1) meet t h i s  c r i t e r i o n w i t h the e x c e p t i o n o f those d e s c r i b i n g (LAT, that  LONG and ELEV).  The  populations  Although Zar (1984, p. 311) s t a t e s  t e s t i n g t h e h y p o t h e s i s t h a t the p a r a m e t r i c c o r r e l a t i o n  c o e f f i c i e n t i s zero remains v a l i d when only variables multiple  one o f two  i s normally d i s t r i b u t e d , i t was d e c i d e d t h a t regression  a n a l y s i s using  LAT, LONG and ELEV as  independent v a r i a b l e s would more c l o s e l y r e s p e c t t h e structure  o f t h e a v a i l a b l e data.  either multiple  regression  However, r e s u l t s from  or c o r r e l a t i o n a r e s i m i l a r  because t h e s i g n i f i c a n c e t e s t o f the p a r t i a l coefficient  regression  i s " i d e n t i c a l " t o t h a t of t h e c o r r e s p o n d i n g  p a r t i a l c o r r e l a t i o n c o e f f i c i e n t (Zar 1984, p. 340).  144  Because v a r i a b l e s were measured at d i f f e r e n t sampling levels  (Table 8.1),  same v a l u e  data s e t s were c o n s t r u c t e d  of a population-  such t h a t t h e  o r maternal t r e e - l e v e l v a r i a b l e  was p a i r e d many times with v a r i a b l e s measured on i n d i v i d u a l s e e d l i n g s , which r e s u l t e d i n very  low c r i t i c a l v a l u e s o f  c o r r e l a t i o n c o e f f i c i e n t s (r ) , due t o t h e l a r g e number o f c  degrees o f freedom  (df).  Therefore,  correlations  i n t h i s chapter are based on f a m i l y mean v a l u e s  reported  (n = 42 f o r  a l l v a r i a b l e s except RACHLEN where n = 38; d f = n - 2 f o r s i n g l e c o r r e l a t i o n s , n - M f o r p a r t i a l and m u l t i p l e c o r r e l a t i o n s i n v o l v i n g M v a r i a b l e s ) , unless otherwise.  stated  T h i s allows comparison o f the magnitude o f r or  i t s square, t h e c o e f f i c i e n t o f determination of t h e p r o p o r t i o n explained  (r ) , a measure 2  o f v a r i a t i o n i n one v a r i a b l e t h a t can be  by v a r i a t i o n i n another.  ANOVA was used t o a p p o r t i o n h y p o t h e s i z e d sources, maternal t r e e s seedlings  v a r i a t i o n among  namely, p o p u l a t i o n  (families),  (geography),  r e p l i c a t i o n s and i n d i v i d u a l  (where a p p r o p r i a t e ) .  A f u l l y nested random  e f f e c t s ANOVA was based on t h e f o l l o w i n g model: p  i  +  )ii)  F  +  -f^uj)  +  m(ijk) where Y  e  seedling i n the k  ijkm  ijkm  = \i +  i s t h e measure o f t h e m  r e p l i c a t i o n i n the j  th  Y  t  h  family i n the i  population,  (1 the o v e r a l l mean, Pi the e f f e c t a s s o c i a t e d  with the i  population  j  t h  t h  family i n the i  t h  th  t h  ( i = 1,...,6), Fj t h e e f f e c t o f t h e  population  a s s o c i a t e d with t h e k  th  (j = 1,...,7), R  r e p l i c a t i o n i n the j  t  h  k  the e f f e c t  f a m i l y and i  t h  145  population  (k = 1 , . . . , 4 )  term.  number of s e e d l i n g s per r e p l i c a t i o n  The  s u r v i v i n g u n t i l the end (except  and  the r e s i d u a l (seedling) (those  o f the t e s t ) v a r i e d from 6 t o 8  i n the case of the v a r i a b l e COTY where m =  making t h i s d e s i g n unbalanced.  10)  Where components o f v a r i a n c e  were c a l c u l a t e d , c o e f f i c i e n t s were o b t a i n e d u s i n g the method d e s c r i b e d by Sokal and R o h l f  v a r i a b l e s measured at the l e v e l maternal t r e e  (i.e.,  302-305).  (1981, pp.  For  of r e p l i c a t i o n w i t h i n  SHWT) and measures based on mean v a l u e s  f o r each maternal t r e e  (i.e.,  SDWT), the a p p r o p r i a t e  effects  were removed from the model s p e c i f i e d above. El-Kassaby  et al.  (1987a) p o i n t out t h a t estimates  of  g e n e t i c v a r i a n c e and h e r i t a b i l i t y r e q u i r e common gene frequencies across populations.  Where p o p u l a t i o n  d i f f e r e n c e s i n s e e d l i n g growth t r a i t s were not i t was  considered  significant  appropriate to c a l c u l a t e h e r i t a b i l i t y  (narrow-sense) as  h  2  = 4a  2 F / P  / o~  2  + O" R/F/P 2  F/P  f o r ANOVA of growth r a t e , which was  +  °"e-  Except  2  conducted u s i n g  the  v a r i a n c e components approach, a l l apportionment of v a r i a t i o n was  based on a percentage o f the total.sums  of squares  (%SS,  see s e c t i o n 7.2.2). 8.3  Results S e e d l i n g l o s s e s d u r i n g the 29 week t r i a l were low  (9.3%)  and were p r i m a r i l y as a r e s u l t of h a n d l i n g  measurement.  The m a t e r i a l d i d not show any  ,  during  evidence  of  d i s e a s e o r i n s e c t problems and shoot growth appeared l u s h throughout t h e experiment.  The  r e l a t i o n s h i p s among f a c t o r s presumed t o i n f l u e n c e  the growth responses o f t h e o p e n - p o l l i n a t e d progeny sampled are summarized i n F i g u r e 8.1. o f these h y p o t h e s i z e d  sources  The magnitude o f t h e e f f e c t s o f v a r i a t i o n w i l l be  determined by d e s c r i b i n g both c o r r e l a t i o n and v a r i a n c e s t r u c t u r e s and any changes i n these course  o f t h e experiment.  s t r u c t u r e s over t h e  Source e f f e c t s w i l l be d i s c u s s e d  u s i n g t h e numbering scheme i n F i g u r e 8.1.  (1-2) 2. maternal parent —  1. population  (2-3)  (1-3)  3.seed  (2-4)  (1-4) ( 3-4 ) Growth (a) pattern (b) form  L .  F i g u r e 8.1  4. seedling  ._l  R e l a t i o n s h i p s o f t h e v a r i a b l e s examined i n t h e a n a l y s i s o f w i n d - p o l l i n a t e d from 42 f a m i l i e s o f P a c i f i c s i l v e r f i r c o l l e c t e d i n s i x l o c a t i o n s on Vancouver I s l a n d , B r i t i s h Columbia.  147  8.3.1 The the  Geographic and maternal t r e e v a r i a t i o n  c o l l i n e a r i t y among geographic v a r i a b l e s  s i x sampled stands  (Table  overall d i s t r i b u t i o n pattern  describing  8.2:1) c l o s e l y r e f l e c t s the o f the s p e c i e s .  As one moves  n o r t h and west, P a c i f i c s i l v e r f i r i s found at i n c r e a s i n g l y lower e l e v a t i o n s Multiple  (Schmidt 1957, Green et al. 1984).  regression  equations d e s c r i b i n g the c o n t r i b u t i o n s  of l a t i t u d e , l o n g i t u d e heterozygosity  and cone r a c h i s l e n g t h  seedling variables) the m u l t i p l e (Table  and e l e v a t i o n o f p o p u l a t i o n  are r e p o r t e d  t o enzyme  (also seed and  i n Appendix 3.  Although  c o r r e l a t i o n c o e f f i c i e n t s (R's) are s i g n i f i c a n t  8.2:11), t h e s t a n d a r d e r r o r s o f the p a r t i a l  regression  c o e f f i c i e n t s ( l i s t e d beneath the  appropriate  c o e f f i c i e n t s i n Appendix 3) are r e l a t i v e l y l a r g e , which a l s o suggests the presence o f s u b s t a n t i a l i n t e r c o r r e l a t i o n among predictor variables  (Zar 1984, p. 338).  For both enzyme h e t e r o z y g o s i t y (represented by r a c h i s l e n g t h ) ,  and cone s i z e  l a t i t u d e of population  e x h i b i t s the l a r g e s t p a r t i a l c o r r e l a t i o n c o e f f i c i e n t among the v a r i a b l e s d e s c r i b i n g geographic l o c a t i o n The  (Table  8.2:11).  p a r t i a l c o r r e l a t i o n s among ENZHET and RACHLEN i n d i c a t e  t h a t both p r o t e i n v a r i a b i l i t y and s i z e o f cones a r e reduced i n more n o r t h e r l y p o p u l a t i o n s , west.  Associations  from east t o  with e l e v a t i o n are not s i g n i f i c a n t .  Enzyme h e t e r o z y g o s i t y poorly  but i n c r e a s e  intercorrelated  and r a c h i s l e n g t h were themselves ( r = 11%) . 2  One-way ANOVA  (Table  8.3a) showed t h a t  differences  among p o p u l a t i o n s accounted f o r 41% o f the v a r i a t i o n i n Table 8.3b i n d i c a t e s t h a t t h e l a r g e s t cones  rachis length.  are produced i n the southernmost 8.3.2  locations.  Geographic i n f l u e n c e s  on seed v a r i a b l e s  Seed weight and c o t y l e d o n number had t h e same  pattern  o f r e l a t i o n s h i p w i t h geography as d i d maternal t r e e variables  (Table  8.2: 1-3).  The s t r e n g t h  of the  r e l a t i o n s h i p among geographic v a r i a b l e s and seed weight (R  2  = 63%) i s g r e a t e r 47%).  This  ANOVA  (Table  than with t h e number o f c o t y l e d o n s  i s also r e f l e c t e d i n population 8.3a) which e x p l a i n  (R = 2  differences i n  67% o f t h e v a r i a t i o n i n  seed weight and l e s s than 10% i n c o t y l e d o n numbers, s u g g e s t i n g t h a t the number o f c o t y l e d o n s shows f a r g r e a t e r intra-population  v a r i a t i o n than does seed weight.  8.3b shows t h a t h e a v i e r seeds, a s s o c i a t e d  with  Table  larger  numbers o f cotyledons, tend t o come from southern Vancouver Island  (exception:  8.3.3  population  R) .  Maternal t r e e and seed r e l a t i o n s h i p s  Table 8.2  (2-3)  indicates that  weight a r e h i g h l y c o r r e l a t e d ,  rachis length  suggesting that  and seed  large  cones  are p r o d u c i n g h e a v i e r seeds, and t h a t t o a l e s s e r extent, embryos from h e a v i e r seeds produce a l a r g e r number o f c o t y l e d o n s , as i n d i c a t e d by t h e lower c o r r e l a t i o n between rachis length  and c o t y l e d o n number.  Similar trends are  Table 8.2  C o r r e l a t i o n c o e f f i c i e n t s f o r geographic, maternal t r e e and seed progeny variables. C r i t i c a l v a l u e f o r simple c o r r e l a t i o n s a) among LAT, LONG and ELEV i s 0.811 (df=4); b) w i t h RACHLEN i s 0.320, a l l others r = 0.304. For p a r t i a l and m u l t i p l e c o r r e l a t i o n s , r = 0.329 f o r RACHLEN, otherwise r = 0.320 c  c  c  I.  Simple c o r r e l a t i o n s among p o p u l a t i o n and maternal t r e e LAT LONG ELEV  II.  ELEV  0.987 -0.927  RACHLEN ENZHET  -0.933  0.329  P a r t i a l and m u l t i p l e c o r r e l a t i o n c o e f f i c i e n t s f o r p o p u l a t i o n , maternal t r e e and seed v a r i a b l e s (1-2, 1-3): LAT ENZHET RACHLEN SDWT COTY  III.  variables:  LONG  -0 .366 -0 . 649 -0 .507 -0 .22 6  NS  0 .312 0 .57 6 0 .390 0 . 138  ELEV  R  0 . 038NS 0 .202 0. 1 0 5 N 0. 150 NS  S  NS  NS  (multiple) 0.472 0 .802 0.792 0. 688  Simple c o r r e l a t i o n s among maternal t r e e and seed v a r i a b l e s  SDWT COTY  ENZHET  RACHLEN  0.574 0.304  0.798 0. 622  COTY SDWT  0.747  (2-3):  Table 8.3a  Variance s t r u c t u r e s f o r mean cone rachis length (RACHLEN) , mean seed weight (SDWT) and cotyledon number (COTY). Differences among means determined to be s i g n i f i c a n t (*, P < 0.05) based on F t e s t (not presented).  I. RACHLEN: SOURCE  df  Sum of Squares  %SS 41.1' 58.9  POPULATION ERROR  5 33  7358.64 10533.50  TOTAL  38  17892.14  100.0  II. SDWT: SOURCE  III.  df  Sum of Squares  %SS 67.0* 33.0  POPULATION ERROR  5 36  40.738 20.047  TOTAL  41  60.785  COTY: SOURCE  df  POPN TREE/POPN REP/TREE/POPN ERROR  5 36 126 1495  TOTAL  1662  %SS 9.1* 8.5* 8.0* 74.4 100.0  100.0  151 Table  8.3b  P o p u l a t i o n average values of r a c h i s l e n g t h , 1000-seed weight and c o t y l e d o n number, with a s s o c i a t e d 95% confidence i n t e r v a l s . RACHLEN  SDWT  COTY  R  88.7 ± 2.0  3.08 ± 0.22  4.78 + 0.17  H  68.1 ± 3.4  2.33 ± 0.13  4 . 69 + 0.16  B  79.5 ± 1.9  2. 95 ± 0.15  4 .82 ± 0.17  C  76.2 ± 2.1  2.77 ± 0.21  4 . 87 ± 0.18  F  95.3 ± 3.1  4 . 44 + 0.16  5.12 ± 0.18  W  112.5  5. 10 ± 0.33  5.24 ± 0.17  POPULATION  evident  ± 3.1  among p o p u l a t i o n means  heterozygosity  (Table 8.3b).  showed a p o s i t i v e r e l a t i o n s h i p with  weight however, the c o r r e l a t i o n with c o t y l e d o n just significant cotyledon 56%).  (r = r  c  = 0.304).  seed  number i s  Seed weight and  number are themselves p o s i t i v e l y c o r r e l a t e d ( r = 2  A h i e r a r c h i c a l ANOVA  showed s i g n i f i c a n t trait  Enzyme  number  (P < 0.05) f a m i l y d i f f e r e n c e s f o r t h i s  however t h i s source  t o t a l variance.  (Table 8.3a) of c o t y l e d o n  accounts f o r l e s s than 10% o f the  The r e p l i c a t i o n e f f e c t was a l s o s i g n i f i c a n t  (8% o f t h e t o t a l v a r i a n c e ) , which w i l l be c o n s i d e r e d  further  i n s e c t i o n 8.4. 8.3.4 The  R e l a t i o n s h i p s among seeds and s e e d l i n g s  average seed weight  (per t r e e ) among the 42 t r e e s  sampled i n t h i s t e s t v a r i e d from 15.4 t o 70.9 mg, a 360% difference.  Cotyledons were l e s s v a r i a b l e i n number,  152  ranging  from three  v a r i a b l e s and  t o seven.  The r e l a t i o n s h i p s among s e e d  v a r i a b l e s d e s c r i b i n g the (3-4a).  s u m m a r i z e d i n T a b l e 8.4  are  s e e d w e i g h t and  cotyledon  weeks i s h i g h but but  not  derived as  cotyledon  from h e a v i e r  seedlings  negative  and  becomes  a t 29 weeks g r o w t h i n  A v e r a g e g r o w t h r a t e s have a n e g a t i v e w e i g h t and  The c o n t r i b u t i o n o f  number t o s e e d l i n g h e i g h t  drops d r a s t i c a l l y  s i g n i f i c a n t l y so,  growth o f s e e d l i n g s  negative, height.  r e l a t i o n s h i p with  number, i n d i c a t i n g t h a t ( l a r g e r ) seeds d i d not  produced from l i g h t e r  (smaller)  suggests that  seedlings grow a s r a p i d l y seeds.  shorter  seedlings  i n the  e a r l y stages o f  o v e r a l l r a t e s o f growth.  r e l a t i o n s h i p was  reversed  i n d i c a t e d by the  h i g h l y p o s i t i v e c o r r e l a t i o n between  a t 29 weeks and  by the  end  t e s t period, as  b y ANOVA ( T a b l e  8.4).  The g r o w t h r a t e  f i r seedlings  appears t o have very  little  geographic influence  in this  term i s n o n - s i g n i f i c a n t , P < 0.05).  differences within population  with  height  among d i f f e r e n t s o u r c e s  silver  population  This  RATE.  g r o w t h r a t e was a p p o r t i o n e d  estimated  o fthe  e x h i b i t e d by P a c i f i c  for  The  ( r = -0.658)  corresponding growth r a t e s  development possessed higher  was  seed  c o r r e l a t i o n between i n d i v i d u a l s e e d l i n g h e i g h t s a t  e i g h t weeks and  How  a t eight  are  s i g n i f i c a n t but  pattern  test (the Tree-to-tree account  l e s s v a r i a t i o n t h a n do d i f f e r e n c e s among r e p l i c a t i o n s a single tree.  And  by f a r the  greatest  variation in  Table 8.4  C o r r e l a t i o n and v a r i a n c e s t r u c t u r e s f o r v a r i a b l e s d e s c r i b i n g s e e d l i n g growth. ( = n o n - s i g n i f i c a n t ; P < 0.05)  seeds and  NS  I.  Correlation HT  HT RATE  NS  HT SDWT COTY  0. 677  HT  8  of V a r i a t i o n  df  Among p o p u l a t i o n s (P) Among f a m i l i e s  (F/P)  Among r e p l i c a t i o n (R/F/P)  N  (RATE):  SS  %SS®  5  2.19  3. 1  36  12.12  17.3  13.7  G 2 + k 0-2  126  14.60  20.8  17.2  O2 + cy  %vc® NS  0. 8  Among s e e d l i n g s (error)  1051  41.21  58.8  68. 3  TOTAL  1218 . 70.12  100.0  100. 0  ® V a r i a t i o n accounted of squares ® ®  Variation (a  2 p  + a  2 F / p  RATE  29  -0.108 s -0.426 -0 . 117NS -0.302  0.546 0.511  Nested ANOVA f o r growth r a t e o f s e e d l i n g s  Source  (3-4a):  growth v a r i a b l e s  RATE  8  -0 . 049 -0.658  29  II.  among seed and s e e d l i n g  Components of Variance® cy + k«o2  NS  e  c  2  R/F/p  R/F/p  + k G2 5  + k C2 3  + k o  2  6  F/p  f o r by each source expressed as a percentage of the t o t a l  expressed as a percentage o f the t o t a l v a r i a n c e + a  2  R/F/P  2  P  k ^ F / p  + a ,) 2  k = 7.338, k = 6.955, k = 7.346; k = 29.010, k = 29.990, k c a l c u l a t e d a c c o r d i n g t o the method of Sokal and R o h l f (1981) x  F/p  A  3  5  6  = 203.230  sum  154  growth r a t e r e s i d e s among i n d i v i d u a l s e e d l i n g s .  When  separate ANOVA*s o f growth r a t e were conducted on each p o p u l a t i o n , t h e amount of v a r i a t i o n accounted f o r by i n t e r t r e e d i f f e r e n c e s was not the same w i t h i n each p o p u l a t i o n . D i f f e r e n c e s among t r e e s i n p o p u l a t i o n R accounted f o r 3.8% of t h e v a r i a t i o n ,  8.6 i n p o p u l a t i o n H, 7.6 i n B, 25.1 i n C,  20.0 i n F and 21.9 i n W .  T r e e - t o - t r e e e f f e c t s on s e e d l i n g  growth r a t e appear t o be'stronger p o p u l a t i o n s than those  i n more s o u t h e r l y  sampled from northern Vancouver  Island. Table  8.5 l i s t s some p o p u l a t i o n - l e v e l s t a t i s t i c s  r e g a r d i n g height growth.  The o v e r a l l minimum s e e d l i n g  h e i g h t a t e i g h t weeks was 13 mm and the maximum, 60 mm.  The  range i n s e e d l i n g h e i g h t s f o r p o p u l a t i o n H was 13-49 mm and f o r W , 21-60 mm.  A t 29 weeks, the minimum s e e d l i n g h e i g h t  was 65 mm and the maximum was 229 mm.  The range o f s e e d l i n g  h e i g h t s as 29 weeks was 74-228 mm f o r p o p u l a t i o n H, and f o r W,  68-218 mm.  P o p u l a t i o n W had the h e a v i e s t seeds  (5.08 ±  0.15 mg, mean ± 95% C I ) , h i g h e s t number o f c o t y l e d o n s ± 0.09),  (5.25  and h i g h e s t mean s e e d l i n g height a t e i g h t weeks but  not a t 29 weeks, and average growth r a t e was t h e slowest o f all  s i x p o p u l a t i o n s i n the t e s t .  hand, had the l i g h t e s t c o t y l e d o n number  seeds  P o p u l a t i o n H, on the other  (2.32 ± 0.06 mg) , lowest  (4.67 ± 0.09),  lowest  average s e e d l i n g  height a t e i g h t weeks, but showed the second h i g h e s t  height  155  at the end of the t e s t and e x h i b i t e d the f a s t e s t  average  r a t e of growth of a l l p o p u l a t i o n s . Examination of the r e l a t i v e v a r i a b i l i t y  of the s i x  p o p u l a t i o n s i n terms of the t h r e e growth response v a r i a b l e s (Table 8.5)  shows t h a t , whether the v a r i a n c e or range of  v a l u e s i s used t o d e s c r i b e d i s p e r s i o n , some p o p u l a t i o n s show dramatic  changes i n v a r i a b i l i t y over time.  At e i g h t weeks  growth p o p u l a t i o n R i s the l e a s t v a r i a b l e (and second tallest)  i n h e i g h t whereas at 29 weeks i t e x h i b i t s the  greatest v a r i a t i o n .  P o p u l a t i o n F, on the other hand,  behaves i n e x a c t l y the o p p o s i t e manner.  Growth r a t e i s a l s o  l e a s t v a r i a b l e i n p o p u l a t i o n F while v a r y i n g most i n population Table %SS)  C. 8.6  l i s t s the r e l a t i v e c o n t r i b u t i o n s (based  on  of p o p u l a t i o n s and t r e e s w i t h i n each p o p u l a t i o n to the  t o t a l v a r i a t i o n i n s e e d l i n g h e i g h t growth over the d u r a t i o n of the t r i a l . significant  C o n t r i b u t i o n s , u n l e s s noted, (P < 0.05)  magnitude and terms.  are a l l  but of much g r e a t e r i n t e r e s t i s the  s t a b i l i t y of the v a r i a t i o n a s c r i b e d t o  V a r i a t i o n i n cumulative  these  s e e d l i n g height a s s o c i a t e d  w i t h p o p u l a t i o n d i f f e r e n c e s i s most pronounced at e i g h t weeks and d e c l i n i n g to v i r t u a l l y  zero at 29 weeks.  p a t t e r n i s seen i n height increment,  except  27 weeks when p o p u l a t i o n c o n t r i b u t i o n r i s e s .  The  between 26 This i s  same and  156 Table  8.5  Summary s t a t i s t i c s o f s e e d l i n g h e i g h t s a n d r a t e s o f g r o w t h b a s e d o n p o p u l a t i o n m e a n s , f o l l o w e d b y 95% confidence intervals.  Population  Average  HT  Average  a  HT  2 9  Average  RATE ±0.033  R  37.29 ± 0.74  149.05 ± 4.47  1.038  H  33.39 ± 0.84  148.07 ± 4.12  1.127 ± 0.033  B  35.44 ± 0.83  143.37 ± 4.50  1.055 ± 0.033  C  36.55 ± 0.83  146.20 ± 4.43  1.045 ± 0.034  F  36.98 ± 0.88  143.02 ± 3.96  1.025 ± 0.030  W  39.77 ± 0.95  145.79 ± 4.11  0.987 ± 0.031  I N D I C E S OF D I S P E R S I O N RANK (RANGE)  f o r6 populations:  HT  HT  8  29  RATE  1  H  R  C  2  F  C  B  3  B  B  W  4  W  5  C  H  H  6  R  F  F  RANK (VARIANCE)  HT  8  W  R  HT  29  RATE  1  W  B  C  2  F  R  R  3  B  C  B  4  H  W  H  5  C  H  W  6  R  F  F  157  c o i n c i d e n t with the i n i t i a t i o n regime i n the n u r s e r y .  of the " s t r e s s "  culture  The d i f f e r e n c e s among t r e e s w i t h i n  each p o p u l a t i o n account f o r a range o f v a r i a t i o n and appear t o f l u c t u a t e somewhat w i t h time.  A l l f a m i l y d i f f e r e n c e s do  d e c l i n e over time except i n p o p u l a t i o n B and C where the c o n t r i b u t i o n remains more o r l e s s s t a b l e . d i f f e r e n c e s a r e o f more importance  Tree-to-tree  i n incremental height  growth i n the f i r s t h a l f o f the t e s t than i n the l a t t e r half. The r e l a t i o n s h i p s among seeds and s e e d l i n g s i n terms of form or biomass are l i s t e d i n Table 8.7  (3-4b).  Variables  d e s c r i b i n g f i n a l s e e d l i n g form are i n t e r c o r r e l a t e d , the h i g h e s t c o r r e l a t i o n between shoot weight and r o o t - c o l l a r diameter.  Over a l l ,  seed weight and c o t y l e d o n number  account f o r s i m i l a r amounts o f v a r i a t i o n i n shoot and root weight and i n diameter at the r o o t - c o l l a r . ANOVA of s e e d l i n g biomass and diameter measures are a l s o g i v e n i n Table 8.7. biomass  With r e s p e c t t o the a l l o c a t i o n of  ( r a t i o of shoot t o root weight)  large  family  d i f f e r e n c e s are apparent, but p o p u l a t i o n s account f o r much less variation.  T h i s p a r a l l e l s the p a t t e r n f o r top growth  alone, whereas root weight v a r i e s among p o p u l a t i o n t o a g r e a t e r degree.  I n d i v i d u a l s e e d l i n g diameter v a r i e s the  l e a s t among p o p u l a t i o n s , and f a m i l y d i f f e r e n c e s are much less  (10% compared t o an average of 30%) than f o r the other  Table 8.6  Source c o n t r i b u t i o n s p r e s e n t e d as %SS from ANOVA o f s e e d l i n g h e i g h t s over time (cumulative and incremental) among 6 p o p u l a t i o n s o f P a c i f i c s i l v e r f i r and among t r e e s w i t h i n each p o p u l a t i o n . CUMULATIVE SEEDLING HEIGHT df  S O U R C E P O P N  (all)  T R E E S  16 wk  8 wk  5  10.0  6 6 6 6 6 6  22.2 25.8 11.0 18.3 27 . 3 17.3  (H)  (R) (B)  (C) (F) (W)  22 wk  4.4  26 wk  2.5  10 . 9 16. 0 25.7 11. l 20.3 9. 9  9.2* • 15.3 21.7* 22.7* 17.4 10.3  a  a  a  a  27 wk  0. I N S  0. 0  7 . 7* 11 . 1 21 . 6 20 . 9 9 .9 11 . 0  9.0 11.2 21.2 21.0 9.9 12.7  NS  a  28 wk  29 wk  0.0  0.1  NS  9.0 10.7 21.3 20.8 9.7 12.0  a  NS  7.5 11.2 21.6 18.7 8.5 13.4 a  SEEDLING HEIGHT INCREMENTS  SOURCE  df  8-16 wk  POPN ( a l l )  5  3.7  TREES (H) (RJ (B) (C) (F) (W)  6 6 6 6 6 6  9.5 24 . 9 a  12.5  wk  16-22  0 .5  NS  8. 7 9.7  a  10.2  18.5 31 . 2 14 . 8  27 . 2  a  a  8.2  a  a  6.8  22-26  wk  26- 27 wk 3. 1  1.8 0. 6 3.3 10 . 9 8.6 0.0 7.9  NS NSa  NS  0. 7 NS 5. 6  2 .4NSa  3. 8 0. Q M S 9.5  C o n t r i b u t i o n i s not s i g n i f i c a n t a t P < 0.05; a l l remaining s i g n i f i c a n t u s i n g approximate F t e s t s .  N S  a  Homogeneity o f v a r i a n c e not a c h i e v e d  i n the ANOVA.  27-28 wk 0. 6 0.2 21. l  NSa  0. 6  NSa  1 0 . 7* 3.2NS*  a  6.9  O.ONS  "0. 0 N S 1 . 7NSa  4. 3  28-29 '  a  10.8  a  5.9 16.7  values are c o n s i d e r e d  v a r i a b l e s d e s c r i b i n g f i n a l s e e d l i n g form.  The m a j o r i t y o f  v a r i a t i o n i n diameter i s a l s o w i t h i n f a m i l i e s and r e p l i c a t i o n s c o n t r i b u t e s i g n i f i c a n t l y t o the i n t r a - f a m i l y variance. Further  r e l a t i o n s h i p s among p o p u l a t i o n s  and maternal  t r e e s and s e e d l i n g form at 29 weeks development are l i s t e d i n Table 8.8  and Appendix 3.  Geographic i n f l u e n c e s on  s e e d l i n g form are n e g l i g i b l e except f o r root weight  (1-4).  P a r t i a l c o r r e l a t i o n reveals that elevation i s associated w i t h v a r i a t i o n i n root weight, suggesting latitude,  root weight o f s e e d l i n g s  increases with a l t i t u d e .  T h i s i s not borne out by p o p u l a t i o n weights  (Table 8.9),  where o v e r l a p  ranking  o f mean values,  smaller  heterozygosity  (Table  8.9).  of maternal t r e e s  intervals  Based on p o p u l a t i o n  seedlings  e i t h e r r o o t s and shoots) are t e n d i n g n o r t h e r l y populations  o f mean root  o f confidence  obscure any d i f f e r e n c e s among means. ranking  t h a t at a given  ( l e s s mass o f  t o come from more Cone s i z e and enzyme  (Table  8.8:2-4)  appear t o  have a p o s i t i v e but n o n - s i g n i f i c a n t i n f l u e n c e on s e e d l i n g biomass accumulation.  Table 8.7  C o r r e l a t i o n and v a r i a n c e s t r u c t u r e s f o r seed and f i n a l v a r i a b l e s ( s e e d l i n g s harvested at 29 weeks).  V a r i a b l e c o r r e l a t i o n s among (3-4b):  SDWT COTY  II.  SHWT  RTWT  0.304 0.316  0. 355 0. 342  POPN TREE/POPN ERROR  DF 5 36 126  SOURCE  DIAM 0. 253NS .0. 370  RTWT  DIAM  5 36 126 1051  Non-significant Significant  SHWT  RTWT  5 . 7NS 32 .1* 62 .2  12.2* 27.4* 60.4  DF  POPN TREE/POPN REP/TREE/POPN ERROR  N S  seed v a r i a b l e s and s e e d l i n g v a r i a b l e s at harvest  ANOVA o f s e e d l i n g biomass and diameter  SOURCE  s e e d l i n g form  SHWT  DIAM  0. 639 0. 651  0.525  (%SS a s s o c i a t e d with each SOURCE): SHWT:RTWT 7. 3 34 . 9* 57.8  NS  DIAM 1.4NS  9.9* 18.3* 70.4  (P > 0.05)  (P < 0.05)  o  Table 8.8  I.  C o r r e l a t i o n s among p o p u l a t i o n and maternal t r e e v a r i a b l e s with those d e s c r i b i n g f i n a l s e e d l i n g form. C r i t i c a l values are the same as those s p e c i f i e d i n Table 8.2.  SHWT  RTWT N  NS  N  Partial  0.277 0.298  NS NS  (1-4):  and M u l t i p l e C o r r e l a t i o n s LAT  SHWT RTWT DIAM  DI7AM  0 22 6 s 0 10 9 0 264 s 0 146NS  ENZHET RACHLEN  II.  (2-4):  Simple C o r r e l a t i o n s  LONG  -0 .230 -0 .058 -0 .244  NS  NS NS  0. 135NS -0. 164NS -0. 073NS  ELEV  R  -0 . 110 -0 .372 -0 .244  NS  NS  0.358 0.538 0.361  NS  NS  Table 8 . 9  I.  Ranking o f p o p u l a t i o n mean values f o r v a r i a b l e s d e s c r i b i n g biomass accumulation at 2 9 weeks (± 95% CI) .  Rank o f p o p u l a t i o n mean SHWT (±95% CI) 1 2 3 4 5 6  -  W F B H R c  1 . 970 1.880 1.831 1.827 1.814 1.765  ± ± ± ± ± ±  and RTWT(±95% CI) :  0. 097 0.096 0.106 0.113 0.093 0.080  of p o p u l a t i o n mean SHWT:RTWT r a t i o 1 2 3 4 5 6  -------  R W H C F B  1.873 1.857 1.827 1.787 1.782 1.719  ± ± ± ± ± ±  0.076 0.073 0.077 0.054 0.057 0.077  seedling  1 2 3 4 5 6 (±95% 1 2 3 4 5 6  -------  CI) -------  1.069 1.061 1. 0 5 7 0 . 997 0 . 988 0 . 967  B W F H C R  W B F H C R  and  DIAM 2.327 2.292 2.290 2.280 2 .247 2.233  ± ± ± ± ± ±  0.051 0.036 0.053 0.039 0.031 0.028  (±95% C I ) : ± + ± ± + ±  0.035 0.036 0.037 0.038 0.036 0.034  163  8.4  Discussion The v e r y h i g h i n t e r c o r r e l a t i o n among geographic  v a r i a b l e s r e p r e s e n t i n g the s i x p o p u l a t i o n s an a r t i f a c t of sampling. the most important  i n t h i s study i s  However, l a t i t u d e appeared t o  be  p r e d i c t o r among the t h r e e a v a i l a b l e  geographic v a r i a b l e s w i t h r e s p e c t t o v a r i a t i o n i n enzyme heterozygosity,  cone s i z e and  seed weight, whereas only  e l e v a t i o n c o n t r i b u t e d s i g n i f i c a n t l y t o root growth v a r i a t i o n i n harvested  seedlings.  range of t h i s study  and  Acknowledging the r e s t r i c t e d i m p r e c i s i o n i n h e r e n t i n the  v a r i a b l e s d e s c r i b i n g l o c a t i o n of p o p u l a t i o n s ,  i t remains  t h a t the s t r u c t u r e of v a r i a t i o n i n cone, seed and t r a i t s i s not populations in  sample  consistent.  seedling  In p a r t i c u l a r , v a r i a t i o n among  i s marked i n some c h a r a c t e r i s t i c s and  negligible  others. Cone  ( r a c h i s ) l e n g t h showed a h i g h degree of geographic  variation in Pacific silver f o r over 40 percent  fir,  with p o p u l a t i o n s  of the t o t a l v a r i a n c e .  with v a r i a t i o n i n cone l e n g t h i n balsam  accounting  This contrasts  f i r where  geographic l o c a t i o n accounted f o r l e s s than o n e - t h i r d o f the total variation  (Lester 1968).  Rachis  l e n g t h and  cotyledon  number show l i m i t e d c o v a r i a t i o n with enzyme h e t e r o z y g o s i t y  164  (r  2  = 11% and 9% r e s p e c t i v e l y ) .  1000-seed weight  The r e l a t i o n s h i p between  and h e t e r o z y g o s i t y i s s t r o n g e r ( r = 33%) 2  but a l a r g e r sample o f both enzyme l o c i required  and i n d i v i d u a l s i s  t o i n f e r any r e l a t i o n s h i p between cone and seed  s i z e as any other p o l y g e n i c t r a i t  and p r o t e i n  variability  (Chakraborty 1981, Simon and A r c h i e 1985). That c o t y l e d o n number p e r se appears t o be a more v a r i a b l e c h a r a c t e r i s t i c i n P a c i f i c s i l v e r f i r than does the weight  of f i l l e d  seeds was not a s u r p r i s i n g r e s u l t .  Sorensen and F r a n k l i n seed weight  (1977) found the r e s i d u a l v a r i a n c e f o r  i n noble f i r t o be 9% compared t o 49% f o r  c o t y l e d o n number.  Helium  (1968) a l s o found the number o f  c o t y l e d o n s t o be more v a r i a b l e than seed weight  itself in  p o p u l a t i o n s of white spruce and observed t h a t c o t y l e d o n number v a r i e d t o t h e same extent over t r e e s  as among  p o p u l a t i o n s , which he presumed i n d i c a t e d t h a t c o t y l e d o n number i s b e i n g determined by a l a r g e number of p o l l e n parents.  Helium  (1968) a l s o s p e c u l a t e d t h a t d i f f e r e n t  p a r e n t a l c o n t r i b u t i o n may be one of the reasons f o r the o v e r a l l low c o r r e l a t i o n between seed weight and c o t y l e d o n number i n white spruce.  Sorensen and F r a n k l i n  (1977) a l s o  found poor c o r r e l a t i o n between seed weight and number o f c o t y l e d o n s but observed that  c o t y l e d o n number e x h i b i t e d  temporal v a r i a t i o n than d i d seed weight led  i n noble f i r , which  them t o i n f e r t h a t c o t y l e d o n number i s determined  independently o f seed weight.  less  165  I t i s known t h a t seed weight i s i n f l u e n c e d by s i t e environmental v a r i a b l e s ,  c o l l e c t i o n year, t i m i n g of  c o l l e c t i o n and storage c o n d i t i o n s 1981) .  (Baldwin 1942, B j o r n s t a d  Because at l e a s t 80% of t o t a l seed weight i s  composed of t i s s u e d e r i v e d from maternal parent (Buchholz 1946,  Perry 1976), then seed weight c o r r e l a t i o n s w i t h o t h e r  v a r i a b l e s w i l l c o n t a i n a g e n e t i c component t h a t i s i n e x t r i c a b l e from macro- and micro-environmental f a c t o r s i n f l u e n c i n g seed development.  Seed s i z e i s viewed as b e i n g  one o f the l e a s t p l a s t i c r e p r o d u c t i v e t r a i t s  (Palmblad 1968,  Harper et al. 1970), however F i n s and Libby (1982) and Salazar  (1986) are c o r r e c t i n t h e i r c a u t i o n t h a t  environment  genotype-  i n t e r a c t i o n i s confounded i n any progeny  where seeds are formed at d i f f e r e n t  locations.  r e c o g n i z e d by Sorensen and Campbell  (1985) i n a seed-  test  T h i s was  s e e d l i n g s i z e study of D o u g l a s - f i r , where seeds o f d i f f e r i n g mean weights were produced on t h e same t r e e  (and p o s i t i o n  w i t h i n the crown) by m a n i p u l a t i n g the micro-environment o f the d e v e l o p i n g cones.  T h e i r r e s u l t s showed t h a t a 10%  i n c r e a s e i n seed weight was a s s o c i a t e d with a 9% i n c r e a s e i n first  year e p i c o t y l l e n g t h .  They a l s o p o i n t out t h a t seed  weight d i f f e r e n c e s i n s e e d l o t s of most c o n i f e r s are l i k e l y t o be g r e a t e r than 10%, so that the p o t e n t i a l f o r growth d i f f e r e n c e s r e s u l t i n g from seed s i z e d i f f e r e n c e s i s p r e s e n t i n w i n d - p o l l i n a t e d seed c o l l e c t i o n s . d i f f e r e n c e s among t r e e s sampled as 360%.  Sweet and Wareing  Seed weight  i n t h i s t h e s i s was as l a r g e  (1966) r e p o r t d i f f e r e n c e s i n  166  seed weight of 250% from a s i n g l e r a d i a t a p i n e t r e e .  No  doubt the range would have been even l a r g e r i n P a c i f i c s i l v e r f i r had seed weight measures been based on uncleaned seed. Seed weight was  seen t o be p o s i t i v e l y a s s o c i a t e d w i t h  e a r l y h e i g h t growth i n P a c i f i c s i l v e r f i r , but by the end of the  29 week t r i a l ,  non-significant.  the a s s o c i a t i o n had become n e g a t i v e and C a n n e l l et al.  (1978) found t h a t seed  weight of l o b l o l l y p i n e no longer c o r r e l a t e d with s e e d l i n g h e i g h t at two months a f t e r sowing.  Mann  (1979) found seed  weights and s e e d l i n g h e i g h t s at 60 and 120 days f o r s e v e r a l Pinus  s p e c i e s t o be l a r g e l y u n r e l a t e d .  r e p o r t e d by Lavender  (1958) f o r D o u g l a s - f i r .  found no seed weight i n f l u e n c e on f i r s t of  A similar result  grand f i r grown i n B r i t a i n .  Lines  was  (1978)  year s e e d l i n g h e i g h t  However, P e r r y  (1976)  m a i n t a i n e d t h a t seed weight or "maternal" e f f e c t s i n some c o n i f e r s can account f o r a simple p r o p o r t i o n of v a r i a t i o n i n s e e d l i n g s i z e and t h a t t h i s i n f l u e n c e may number of y e a r s .  Arbez  extend f o r a  (1969) found that seed weight  i n f l u e n c e d h e i g h t growth i n European s i l v e r f i r at age but  t h a t h e i g h t was  years. of  Khalil  four,  independent of c o t y l e d o n number at f o u r  (1981) a l s o d e t e c t e d a s i g n i f i c a n t  influence  seed weight on f o u r - y e a r height i n white spruce. F a s t e r growth r a t e s among s e e d l i n g s d e r i v e d from  l a r g e r seeds i s o f t e n a t t r i b u t e d t o h i g h e r n u t r i e n t i n these seeds  (Mikola 1980) .  reserves  However, t h i s study showed  167  t h a t h e a v i e r seeds of P a c i f i c s i l v e r f i r d i s p l a y e d slower r a t e s of growth than l i g h t e r - w e i g h t seeds.  This i s s i m i l a r  to the f i n d i n g s of Sweet and VJareing (1966) i n r a d i a t a p i n e and European  l a r c h , where a slower growth r a t e among  s e e d l i n g s d e r i v e d from h e a v i e r seeds was  attributed to a  r e s t r i c t i o n i n growth caused by c o n t a i n e r s i z e . Wareing  Sweet and  (1966) a l s o contend that any g e n e t i c v a r i a t i o n i n  growth r a t e may  be obscured by seed s i z e d i f f e r e n c e s .  This  would have no doubt o c c u r r e d i n the present study had population  (provenance) based bulk seed c o l l e c t i o n s been  made. G r i f f i n and Ching  (1977) c o n s i d e r e d seed weight,  c o t y l e d o n number, and h y p o c o t y l l e n g t h t o be  inter-related  e x p r e s s i o n s of embryo s i z e i n D o u g l a s - f i r , and advocated the measurement of h e i g h t growth be r e s t r i c t e d t o e l o n g a t i o n above the c o t y l e d o n s (length of e p i c o t y l ) t o reduce the i n f l u e n c e of seed s i z e . present study.  This procedure was  adopted i n the  S e e d l i n g growth i n height was  i n f l u e n c e d by seed s i z e  positively  (r = 0.546) at e i g h t weeks but  was  n e g a t i v e l y c o r r e l a t e d with seed s i z e at 29 weeks (r = 0.108 ). NS  A l s o , o v e r a l l f a m i l y i n f l u e n c e s on  seedling  h e i g h t increments d e c l i n e d over the d u r a t i o n of the t e s t (Table 8.6).  Nonetheless, a s i g n i f i c a n t  relationship  between seed s i z e and average growth r a t e of s e e d l i n g s -0.426) remains.  This r e s u l t has i m p l i c a t i o n s f o r the  (r =  168  i n t e r p r e t a t i o n of h e r i t a b i l i t y ,  which w i l l be d i s c u s s e d  later i n this section. The  fitness,  as measured by r e p r o d u c t i v e success, o f  t r e e s with a cone-bearing  h a b i t such as P a c i f i c s i l v e r  i s l a r g e l y determined by the attainment codominant s t a t u s w i t h i n the stand.  fir,  o f at l e a s t  Suppressed  individuals  o f P a c i f i c s i l v e r f i r are not known t o produce cones ( J . Maze, UBC Department o f Botany, p e r s . comm. June 1990). Growth i n h e i g h t i s c o n s i d e r e d an important  component o f  f i t n e s s i n D o u g l a s - f i r (King et al. 1988). C a n n e l l et al. (1978) found s e e d l i n g growth r a t e s t o r e f l e c t in l o b l o l l y pine.  future yields  Because o f the p o t e n t i a l p r e d i c t i v e use  o f s e e d l i n g growth r a t e , i t s r e l a t i o n s h i p t o o v e r a l l  fitness  (Bush et al. 1987) and because p o p u l a t i o n d i f f e r e n c e s i n growth r a t e appeared n e g l i g i b l e heritability  (see ANOVA i n Table 8.4) the  of growth r a t e f o r t h i s sample o f P a c i f i c  s i l v e r f i r was c a l c u l a t e d as f o l l o w s :  h  2  =  4o2  F / p  /  (o-2  F/P  +  <j2  R / F /  p  +  <5\) ,  from a p p r o p r i a t e v a r i a n c e components l i s t e d i n Table 8.4. H e r i t a b i l i t y o f growth r a t e was estimated  as 0.55.  However,  some i n b r e e d i n g was d e t e c t e d i n four o f the p o p u l a t i o n s in the t r i a l  (Chapter  used  5 ) . Thus, t h e assumption t h a t a l l  o f f s p r i n g from one maternal  t r e e are r e l a t e d as h a l f -  s i b l i n g s i s u n l i k e l y t o be t r u e .  F u r t h e r , given t h e h i g h  shade t o l e r a n c e o f P a c i f i c s i l v e r f i r ,  t h e r e i s a l s o the  169  possibility despite 1967,  t h a t m a t e r n a l p a r e n t s may t h e m s e l v e s b e r e l a t e d ,  sampling attempts t o reduce t h a t  likelihood  F l e t c h e r a n d B a r n e r 1 9 7 8 ) . Thus, 0.55 i s n o d o u b t an  over-estimate  of the heritability  o f s e e d l i n g growth r a t e ,  a l t h o u g h t h e d a t a do s u p p o r t t h e h y p o t h e s i s is  (Lines  adaptive,  populations, favored  as s m a l l e r  rate  s e e d s , c o m i n g f r o m more n o r t h e r l y  e x h i b i t f a s t e r r a t e s o f g r o w t h , w h i c h w o u l d be  i n shorter growing seasons.  in Pacific  that growth  silver  f i r i s often  Given that  l o w , t h e common  germination nursery  p r a c t i c e o f m u l t i p l e s e e d i n g and subsequent t h i n n i n g o f container  stock  maladapted  may i n a d v e r t e n t l y r e s u l t i n t h e s e l e c t i o n o f  seedlings.  Maze e t al. families studied the  (1989) f o u n d t h a t o f f o u r intensively forlateral  family with the highest  Douglas-fir  shoot growth r a t e ,  mean g r o w t h r a t e a l s o h a d t h e  lowest among-individual v a r i a t i o n i n growth r a t e . Pacific  silver  f i r , variance  In  s t r u c t u r e was n o t t h e same f o r  g r o w t h r a t e s among f a m i l i e s w i t h i n e a c h p o p u l a t i o n , analyzed  separately  ( S e c t i o n 8.3.3), and f a s t e r growth  were n o t a s s o c i a t e d w i t h fact,  the opposite  growth r a t e  lower v a r i a b i l i t y  rates  a t any l e v e l .  a p p e a r s t o be t r u e , g i v e n  F i n ranking  population  when  the position of  o f growth r a t e and v a r i a b i l i t y i n  (Table 8.5).  Although the t r i a l  was t e r m i n a t e d  In  p r i o r to the  formation  o f dormant buds, t h e s e e d l i n g  form v a r i a b l e s  displayed  some a s s o c i a t i o n w i t h g e o g r a p h y w h i c h  suggests  170  growth i n terms of biomass i s a l s o adaptive s i l v e r f i r (Table 8.7:11 and Table suggests  in Pacific  8.8). Sorensen  s m a l l e r t o p growth and s m a l l e r shoot:root  r a t i o s a r e a s s o c i a t e d with s i t e s having seasons i n D o u g l a s - f i r .  biomass  s h o r t e r growing  Ranks of p o p u l a t i o n s f o r mean shoot  weight and mean shoot:root Pacific silver f i r .  (1983)  r a t i o do not f i t t h i s t r e n d i n  The h i g h e s t r a t i o of shoot:root  p r o d u c t i o n i s o b t a i n e d i n one of the most n o r t h e r l y d i s t r i b u t e d populations  (R), but given the l a r g e v a r i a b i l i t y  a s s o c i a t e d with these mean values, biomass measures are not d r a s t i c a l l y d i f f e r e n t across the e n t i r e sampling Within-family v a r i a b i l i t y  region.  i n e a r l y s e e d l i n g growth i s  v e r y h i g h i n t h i s sample of P a c i f i c s i l v e r f i r .  T h i s may be  best i l l u s t r a t e d by the magnitude of the r e p l i c a t i o n effects,  found t o be s i g n i f i c a n t  i n a l l models where t h e  term was t e s t a b l e , i n c l u d i n g cotyledon number. l e a s t t h r e e p o s s i b l e reasons  forthis.  There are at  When the t r i a l was  p l a n t e d , t h e f o u r r e p l i c a t i o n s per f a m i l y were randomized throughout 12 styrofoam  b l o c k s , and every e f f o r t was made t o  minimize b i a s by p l a n t i n g rows as encountered i n t h e d e s i g n l a y o u t , r a t h e r than by t r e e , so t h a t i t was u n l i k e l y t h a t the f o u r t h r e p l i c a t i o n was always p l a n t e d with the l e a s t d e s i r a b l e germinants. completely  However, " p l a n t e r ' s c h o i c e " cannot be  r u l e d out as a source of e r r o r .  The 12  s t y r o b l o c k s were re-randomized s i x times d u r i n g the 29-week t e s t t o reduce any systematic e f f e c t of p o s i t i o n  with  171  respect  t o i r r i g a t i o n and  not have been s u f f i c i e n t ,  f e r t i l i z e r regimes, but t h i s i n that i n t e r v a l s were not  spaced throughout the growing season. e f f e c t may  a l s o be  statistical,  due  Perry  to s t o c h a s t i c e f f e c t s  from seed s i z e v a r i a t i o n .  Yeh  effects arising  and Rasmussen  the a l l o c a t i o n of s e e d l i n g s  progeny t e s t s and  each  (1976) proposed t h a t family-row p l o t s  are l i k e l y t o themselves c r e a t e competitive  discussed  equally  The r e p l i c a t i o n  r e s u l t i n g from the small number of s e e d l i n g s w i t h i n replication.  may  to  the p o t e n t i a l l a b i l i t y  (1985)  open-pollinated of  family  variances. In t h i s study, f a m i l y v a r i a n c e s consequent h e r i t a b i l i t y estimates,  f o r growth r a t e s  are  l i k e l y b i a s e d upwards  t o an unknown degree by environmental i n f l u e n c e s on size.  As w e l l the d e t e c t i o n of i n b r e e d i n g  populations  and p o t e n t i a l g e n e t i c  t r e e s themselves suggests t h a t h Cannell  2  relatedness  among maternal  estimates are  f a m i l y d i f f e r e n c e s i n growth r a t e over one of the t o t a l v a r i a n c e ,  in P a c i f i c s i l v e r f i r .  The  inaccuracy  seed  i n some  et a l . ' s (1978) study of l o b l o l l y pine  f o r 30 percent  and  inflated. revealed  that  season accounted  compared t o 14 inherent  in h  percent 2  and  the lower c o n t r i b u t i o n of f a m i l i e s to t o t a l v a r i a t i o n i n growth r a t e observed i n P a c i f i c s i l v e r f i r suggests t h a t i t may  be unwise t o use  the h e r i t a b i l i t y of height  upon among- to w i t h i n - f a m i l y  variance  growth based  as a p r e d i c t o r of  f u t u r e growth performance i n P a c i f i c s i l v e r f i r .  172  8.5  Conclusions  (a) P o p u l a t i o n s  account f o r a l a r g e p r o p o r t i o n o f the t o t a l  v a r i a n c e o f cone s i z e seed s i z e  (estimated by r a c h i s length) and  (estimated by 1000-seed  weight).  (b) Seed weight and cotyledon number decrease with  latitude.  Geographic i n f l u e n c e on v a r i a n c e of s e e d l i n g growth c h a r a c t e r i s t i c s i s much l e s s  evident.  (c) D i f f e r e n c e s among o p e n - p o l l i n a t e d f a m i l i e s are g r e a t e s t f o r s e e d l i n g biomass v a r i a b l e s and l e a s t f o r e l o n g a t i o n (height) v a r i a b l e s . (d) The nature  o f v a r i a t i o n i n o p e n - p o l l i n a t e d progeny  growth t r a i t s ,  the i n t r a c t a b i l i t y of environmental  i n f l u e n c e s on seed s i z e ,  and the l i k e l i h o o d of  i n b r e e d i n g and g e n e t i c c o r r e l a t i o n among parent  trees  p l a c e s some r e s t r i c t i o n s on the u s e f u l n e s s o f heritability  of height growth r a t e among 0-P s e e d l i n g s  of P a c i f i c s i l v e r  fir.  9.  SUMMARY AND  CONCLUSIONS  V a r i a t i o n p a t t e r n s i n s e v e r a l cone, seed and s e e d l i n g c h a r a c t e r i s t i c s were s t u d i e d i n P a c i f i c s i l v e r f i r sampled on a s i n g l e - t r e e b a s i s at e i g h t l o c a t i o n s on Vancouver I s l a n d , B.C. (a)  I t was  found  that:  of 13 enzyme l o c i t h a t c o u l d be r e l i a b l y scored, s i x  e x h i b i t e d no d e t e c t a b l e v a r i a t i o n while seven appeared t o each possess  at l e a s t two  isozyme v a r i a n t s .  The mode of  i n h e r i t a n c e f o r these polymorphic l o c i conformed t o Mendelian e x p e c t a t i o n s ,  although AAT-2 d i s p l a y e d marked  s e g r e g a t i o n d i s t o r t i o n i n 13 of 14 t r e e s i n which v a r i a t i o n at t h i s l o c u s was (b)  observed;  at the sampling  evidence Linkage  i n t e n s i t y a v a i l a b l e t o t h i s study,  of l i n k a g e between p a i r s of enzymes was between AAT  and PGI  was  no  detected.  a n t i c i p a t e d , given that i t  has been d e t e c t e d i n balsam f i r (Neale and Adams 1981)  and  s e v e r a l other c o n i f e r s (Guries et a i . 1978,  et  al.  1982b, Boyle and Morgenstern 1985, 1986,  B a r r e t t et al.  1987).  El-Kassaby  C h e l i a k and  Pitel  1985,  Harry  sizes  (of both t r e e s and number of seeds per t r e e ) might  r e v e a l the presence of t h i s apparently l i n k a g e group i n P a c i f i c s i l v e r (c)  small but s i g n i f i c a n t  m u l t i l o c u s estimate  Much l a r g e r sample  highly-conserved  fir;  l e v e l s of i n b r e e d i n g , based on a  of o u t c r o s s i n g , e x i s t i n f i v e of seven  p o p u l a t i o n s of P a c i f i c s i l v e r f i r i n the 1983  collection  174  year.  Evidence  f o r i n b r e e d i n g other than  s e l f i n g , based on  d i f f e r e n c e s between mean s i n g l e l o c u s and m u l t i l o c u s o u t c r o s s r a t e estimates,  i s weak, but some consanguineous  matings may be o c c u r r i n g i n p o p u l a t i o n s of P a c i f i c fir.  silver  O u t c r o s s i n g r a t e v a r i a t i o n among p o p u l a t i o n s i s  e v i d e n t and appears p o s i t i v e l y c o r r e l a t e d with seed (d)  h i g h l e v e l s o f allozyme  size;  v a r i a b i l i t y existed within  p o p u l a t i o n s of P a c i f i c s i l v e r f i r (95-98%), based on the d i v e r s i t y measures of Nei (1977) and the d i f f e r e n t i a t i o n index o f Gregorius  and Roberds  (1986), a p a t t e r n which i s  very common i n w i l d c o n i f e r p o p u l a t i o n s The  (El-Kassaby  extent o f among-population d i f f e r e n t i a t i o n  depending upon which index Roberds) was employed.  (Nei's G  differed  or 8 of G r e g o r i u s and  ST  P o p u l a t i o n F was shown t o be t h e  most d i f f e r e n t i a t e d g e n e t i c a l l y by both Nei's d i s t a n c e measure and 8 (Gregorious Maternal  1990) .  (1978) g e n e t i c  and Roberds 1986).  t r e e s appeared more heterozygous then d i d v i a b l e  embryos, and southern  p o p u l a t i o n s appeared more g e n e t i c a l l y  d i v e r s e than p o p u l a t i o n s of P a c i f i c s i l v e r f i r sampled from the n o r t h e r n end o f Vancouver I s l a n d ; (e) exist  s t r o n g f a m i l y d i f f e r e n c e s i n germination in Pacific silver fir,  treatment.  i r r e s p e c t i v e of p r e g e r m i n a t i o n  C o l l e c t i o n r e g i o n had n e g l i g i b l e a s s o c i a t i o n  with germination germination  responses  performance except  i n the case of  value, where i t i s evident t h a t more s o u t h e r l y  p o p u l a t i o n s germinate more r a p i d l y without  stratification  175  than p o p u l a t i o n s  at h i g h e r l a t i t u d e s .  Response t o the  p a r t i c u l a r s t r a t i f i c a t i o n c o n d i t i o n s employed i n the germination  t e s t appeared t o have a s u b s t a n t i a l p o p u l a t i o n  component, which was withstand  manifest  fungal i n f e c t i o n .  i m p l i c a t i o n s f o r nursery  as a d i f f e r e n t i a l a b i l i t y These f i n d i n g s have  germination,  s e l e c t i o n f o r more r a p i d - g e r m i n a t i n g  as and  to  important  inadvertent for mold-resistant  f a m i l i e s c o u l d reduce the g e n e t i c base of the r e s u l t a n t planting (f)  stock;  a s i z a b l e c l i n a l t r e n d e x i s t s f o r seed weight i n  Pacific silver fir,  with l i g h t e r - w e i g h t seeds found i n more  northerly populations.  Cotyledon  number a l s o decreases  with  l a t i t u d e , but p o p u l a t i o n d i f f e r e n c e s are much l e s s f o r t h i s v a r i a b l e than f o r seed weight  (9% vs 67%).  Population  i n f l u e n c e s on s e e d l i n g height growth r a p i d l y d e c l i n e over a p e r i o d of 29 weeks while the f a m i l y component remains reasonably  s t a b l e i n four of s i x p o p u l a t i o n s .  The  magnitude  of v a r i a t i o n among f a m i l i e s i s g r e a t e r f o r s e e d l i n g biomass v a r i a b l e s than f o r v a r i a b l e s d e s c r i b i n g e l o n g a t i o n . S e e d l i n g growth r a t e appears h i g h l y h e r i t a b l e , although l a r g e w i t h i n - f a m i l y component of v a r i a n c e m i l i t a t e s  the  against  u s i n g e a r l y s e e d l i n g height growth r a t e as a b a s i s f o r selection.  Larger  seeds were found to produce s e e d l i n g s  with slower r a t e s of growth, a r e s u l t which may  have some  i m p l i c a t i o n f o r nursery p r a c t i c e where there i s d e l i b e r a t e or i n a d v e r t e n t  s e l e c t i o n f o r seed s i z e .  176  10-  LITERATURE CITED  Ackerman, R.F. and J . R. Gorman. 1969. E f f e c t of seed weight on the s i z e of lodgepole p i n e and white spruce c o n t a i n e r - p l a n t i n g stock. Pulp and Pap. Mag. Can. Convention i s s u e , 1969:167-169. Adams, W.T. 1983. A p p l i c a t i o n s of isozymes i n t r e e breeding. In: Isozymes i n P l a n t G e n e t i c s and Breeding, Part A. E l s e v i e r S c i . P u b l i s h i n g Company. (S.D. Tanksley and T . J . Orton, e d s ) . pp. 381-400. Adams, W.T. and D.S. B i r k e s . 1990. E s t i m a t i n g mating patterns i n forest tree populations. 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I I I . Leaf and t w i g o i l a n a l y s i s of a m a b i l i s f i r . Can. J . Bot. 55:3087-3092. Wang, B.S.P. 1981. Measuring q u a l i t y of t r e e seed. In: H i g h - Q u a l i t y C o l l e c t i o n and P r o d u c t i o n of C o n i f e r Seed (R.F. Huber, C o m p i l e r ) . Can. For Serv. NFRC Edmonton, A l b e r t a . I n f . Rep. NOR-X-235. pp. 7378. Wang, B.S.P., J.A. P i t e l and D.P. Webb. 1982. Environmental and g e n e t i c f a c t o r s a f f e c t i n g t r e e and shrub seeds. In: Adv. i n Res. and Technol. of Seeds, p t . 7. Centre f o r A g r i c . P u b l . and D o c , Wageningen, The Netherlands. pp. 87-135. Wang, C.W.  and R.K. P a t e l . 1974. V a r i a t i o n i n seed c h a r a c t e r i s t i c s and s e e d l i n g growth of openp o l l i n a t e d ponderosa p i n e . p r o g e n i e s . Univ. of Idaho, C o l l . of F o r e s t r y , W i l d l i f e and Range S c i . Sta. Pap. No. 15. 10pp.  Ward, R.H.  and C F . Sing. 1970. A c o n s i d e r a t i o n of the power of the % t e s t to d e t e c t i n b r e e d i n g e f f e c t s i n n a t u r a l p o p u l a t i o n s . Amer. Nat. 104:355-363. 2  Wehrhahn,  C. a n d R.W. measurement  Allard. 1965. of the effects  The d e t e c t i o n a n d of i n d i v i d u a l genes  201 i n v o l v e d i n the i n h e r i t a n c e of a q u a n t i t a t i v e c h a r a c t e r i n wheat. Genetics 51:109-119. Weir, B.S. and C.C. Cockerham. 1984. E s t i m a t i n g Fs t a t i s t i c s f o r the a n a l y s i s of p o p u l a t i o n structure. E v o l u t i o n 38:1358-1370. Wilkinson,  L. 1988. SYSTAT: The System f o r S t a t i s t i c s . Evanston, IL:SYSTAT, Inc. 1988.  W i t t e r , M.S. and P.P. F e r e t . 1978. I n h e r i t a n c e o f glutamate o x a l o - a c e t a t e transaminase isozymes i n V i r g i n i a pine megagametophytes. S i l v a e Genet. 27:129-134. Woods, J.H. and J.C. Heaman. 1989. 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Yeh,  F.C. and S. Rasmussen. 1985. H e r i t a b i l i t y of h e i g h t growth i n 10-year o l d S i t k a spruce. Can. J . Genet. C y t o l . 27:729-734.  Zar,  J.H.  1984. B i o s t a t i s t i c a l a n a l y s i s (2nd ed.) P r e n t i c e H a l l . Englewood C l i f f s , N.J.  Z a v a r i n , E., K. Snajberk and W.B. C r i t c h f i e l d . 1973. Monoterpene v a r i a b i l i t y of Abies amabilis cortical resin. Biochem. Syst. 1:87-93.  Appendix 1 Common Name  S c i e n t i f i c Name  Alaska yellow-cedar  Chamaecyparis  Balsam  fir  Abies  balsamea  (L.)  Bishop  pine  Pinus  muricata  D. Don  spruce  Picea  mariana  Mill.  Picea  pungens  Engelm.  Black Blue  spruce  nootkatensis  D. Don  Mill.  Douglas-fir  Pseudotsuga  Engelmann s p r u c e  Picea  engelmannii  European  Fagus  s y l v a t i c a L.  Fraser f i r  Abies  fraseri  (Pursh.)  Poir.  Grand f i r  Abies  grandis  (Dougl.)  Lindl.  Green  Alnus  crispa  beech  alder  Incense  cedar  Calocedrus  menziesii  (Mirb.) Franco  (Perry)  (Ait.)  Pursh.  decurrens  (Torr.)  Jack pine  Pinus  banksiana  Lamb.  Knobcone p i n e  Pinus  attenuata  Lemm.  Loblolly  Pinus  taeda  Lodgepole p i n e  Pinus  contorta  Mountain  Tsuga mertensiana  pine hemlock  Engelm.  Florin  L. var. l a t i f o l i a (Bong.)  Englem.  Carr.  Noble f i r  Abies  procera  Norway s p r u c e  Picea  abies  Pitch pine  Pinus  rigida  Pinus  ponderosa  Picea  sitchensis  (Bong.)  Subalpine f i r  Abies  lasiocarpa  (Hook.) N u t t .  Tamarack  Larix  laricina  (Du R o i ) K. K o c h .  Pinus  virginiana  Tsuga  heterophylla  Ponderosa Sitka  pine  spruce  Virginia Western Western  pine hemlock r e d cedar  Western w h i t e p i n e White f i r White  spruce  Whitebark pine Yellow-poplar  Rehd. (L.) K a r s t  Thuja p l i c a t a Pinus  Mill. Laws.  Donn Dougl. (Gord. & Glend.)  Picea  glauca  Pinus  albicaulis  Liriodendron  Mill. (Raf.) Sarg.  monticola  A b i e s concolor  Carr.  (Moench)  Voss  Engelm.  tulipifer'a  L.  Lindl.  203 APPENDIX 2:  i)  Buffers, solutions, gel preparation, running conditions and stain recipes for horizontal starch gel electrophoresis.  Buffer System ' A ' ; Electrode Buffer ( T r i s - C i t r a t e pH 7.0): 62.97 g TRISMA base (Sigma Chemical C o . , St. Louis, MO). 33.04 g c i t r i c acid Dissolve up to 4L with deionized water; run at 120 mA and 120 4 hr.  v o l t s for  Gel preparation; 60 540 50 10 20  mL electrode buffer mL deionized water g Electrostarch lot 392 (Electrostarch C o . , Madison, WI) g hydrolyzed potato starch (Sigma) g hydrolyzed starch (Connaught L a b s . , Willowdale, Ont.)  Mix starches with 150 mL solution to form lump-free s l u r r y ; heat remaining solution to boiling and add a l l at once to s l u r r y . Cook and remove a i r using vacuum apparatus. Makes two g e l s . i i ) Buffer System ' B ' ; Electrode Buffer (Sodium Borate pH 8.0) 8.0 g sodium hydroxide 37.2 g boric oxide T i t r a t e with AN NaOH to pH 8.0; dissolve up to 4L with deionized water. Run at 120 mA and 100 volts for 4 h r . Gel preparation: 200 mL T r i s - c i t r a t e (pH 8.8)* 400 mL deionized water 35 g Electrostarch 40 g  hydrolyzed potato starch (Sigma)  Mix as instructed for gels i n ' A ' above. * T r i s - c i t r a t e (pH 8.8) 48.4 g TRISMA base (Sigma) T i t r a t e with 0.2M c i t r i c acid solution to pH 8.8; deionized water.  dissolve up to 4L with  204 i i i ) S t a i n Buffer; 96.88 g TRISMA base (Sigma) T i t r a t e to pH 8.0 w i t h concentrated HC1; d i s s o l v e up to 4L with deionized water. iv)  Extraction Buffer; 10 mL T-C electrode buffer (pH 7.0) 80 mL deionized water 5 mL NADP s o l u t i o n 5 mL NAD s o l u t i o n 0.018 g ascorbic a c i d 0.034 g EDTA 0.100 g bovine serum albumin 5 drops 2-mercaptoethanol ( t o bind phenolics) Solutions; ( f o r e x t r a c t i o n b u f f e r and i n d i v i d u a l enzyme s t a i n i n g ) : Solution (aqueous)  Concentration  Pyridoxal 5' phosphate Glucose 1,6 diphosphate MTT MgCl NADP NAD PMS NBT Malic acid G6PDH  1 mg/mL 0.1 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL 10 mg/mL 5 mg/mL 10 mg/mL 0.5 M ( t o pH 7.0 with 10N NaOH) 1000 /20 mL T-C electrode buffer (pH 7.0) and 80 mL deionized water + 5.0 mL NADP + 0.5 mL NAD  2  Stain recipes; AAT (buffer system 'B' ) ; 50 mL Tris-HCl s t a i n buffer 200 mg L - a s p a r t i c a c i d 100 mg - k e t o g h i t o r i c acid 200 mg Fast Blue BB s a l t 1 mL p y r i d o x a l 5' phosphate s o l u t i o n Mix i n tray i n above order.  205  G6P ( b u f f e r system 'B'): 50 200 1 1 1 1  mL mg mL mL mL mL  Tris-HCl s t a i n buffer glucose-6-phosphate NADP s o l u t i o n MgCl2 s o l u t i o n MTT s o l u t i o n PMS s o l u t i o n  Mix In tray i n above order. GDH (and SOD - buffer system ' B * ) : 50 400 1 1 1  mL mg mL mL mL  Trls-HCl s t a i n buffer L-glutamic acid NAD s o l u t i o n MTT s o l u t i o n PMS s o l u t i o n  Mix I n tray In above order (SOD appears as c l e a r l i n e on blue stained gel). IDH (and PGM - buffer system 'A'): 50 100 100 0.5 3 1 1 1 1  mL mg mg mL mL mL mL mL mL  Trls-HCl s t a i n buffer l s o c l t r l c acid glucose-l-phosphate glucose 1,6 diphosphate s o l u t i o n G6PDH s o l u t i o n NADP s o l u t i o n MgCl2 s o l u t i o n MTT s o l u t i o n PMS s o l u t i o n  Mix i n tray i n above order; PGM r e s o l v e s most anodally and without Interference with IDH. MDH ( b u f f e r system 'A'): 25 25 1 1 1  mL mL mL mL mL  Tris-HCl s t a i n buffer DL-malic acid (pH 7.0) NAD s o l u t i o n NBT s o l u t i o n PMS s o l u t i o n  Mix i n tray In above order.  206 6PG (buffer system 'B'): 5 mL Tris-HCl s t a i n buffer 10 mg phosphogluconic acid 1 mL NADP s o l u t i o n 1 mL MgCl2 s o l u t i o n 1 mL MTT s o l u t i o n 0.5 mL PMS s o l u t i o n Mix i n above order i n amber f l a s k ; apply dropwl6e to g e l . PGI (buffer system ' A ' ) : 50 25 1 1 1 1 1  mL mL mL mL mL mL mL  Tris-HCl s t a i n buffer fructose-6-phosphate G6PDH s o l u t i o n NADP s o l u t i o n MgCL£ s o l u t i o n MTT s o l u t i o n PMS s o l u t i o n  Mix i n tray i n above order. Incubate a l l enzyme stains a t 37* f o r 15 to 30 min.  207 APPENDIX 3 M u l t i p l e r e g r e s s i o n equations i n v o l v i n g l a t i t u d e (LAT), l o n g i t u d e (LONG) and e l e v a t i o n (ELEV) as p r e d i c t o r v a r i a b l e s ( a s s o c i a t e d standard e r r o r s p r i n t e d below c o e f f i c i e n t s ; * s i g n i f i c a n t , P < 0.05): 1.  Enzyme h e t e r o z y g o s i t y  (R = 0.22*): 2  ENZHET = -1.74 - 0.229 (LAT)* + 0.105(LONG)* + 0.0004 (ELEV) (2.99) (0.094) (0.052) ' (0.0002) 2.  Cone r a c h i s l e n g t h  (R = 0.64*): 2  RACHLEN = -4 96.1 - 69.2(LAT)* + 31.8(LONG)* (452.5) (13.9) (7.7) 3.  Seed weight  + 0.031(ELEV) (0.026)  (R = 0.63*): 2  SDWT = 2.45 - 3.67 (LAT)* + 1.45 (LONG)* + 0.0012 (ELEV) (32.0) (1.01) (0.56) (0.0018) 4.  Cotyledon  number  (R = 0.48*): 2  COTY = 7.79 - 0.387 (LAT) (8.60) (0.271) 5.  + 0.128 (LONG) (0.149)  NS  S e e d l i n g shoot weight at 29 wk  SHWT = 4.87 - 0.309 (LAT) (6.73) (0.212)  N S  RTWT = 8.18 - 0.028 (LAT) (2.50) (0.079)  N S  N s  -  NS  NS  - 0 . 0003 (ELEV) (0.0004)  N S  (R = 0.29 ): 2  - 0.044 (LONG) (0.043)  NS  + 0.0004 (ELEV) (0.0005)  2  + 0.097 (LONG) (0.117)  S e e d l i n g diameter a t 29 wk  DIAM = 8.20 - 0.04 6 (LAT) (3.54) (0.112)  Ns  (R = 0.13 ):  S e e d l i n g root weight a t 29 wk  7.  Ns  NS  NS  - 0.0003 (ELEV) (0.0002)  (R = 0.13 ): 2  NS  0.028 (LONG) (0.062)  N S  - 0.0003 (ELEV) (0.0002)  N s  NS  Ns  

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