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The genetic consequences of contrasting breeding systems in Plectritis (Valerianaceae) Layton, Charles Robert 1980

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THE  GENETIC CONSEQUENCES OF CONTRASTING BREEDING SYSTEMS IN PLECTRITIS  (VALERIANACEAE)  by CHARLES ROBERT LAYTON B.A., V a n d e r b i l t  U n i v e r s i t y , 1975  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE  in  THE  FACULTY OF GRADUATE STUDIES Department o f Botany  We a c c e p t t h i s t h e s i s as conforming to the r e q u i r e d  THE  standard  UNIVERSITY OF BRITISH COLUMBIA A p r i l 1980  ©  C h a r l e s Robert  Layton, 1980  In p r e s e n t i n g  this thesis i n p a r t i a l fulfillment  o f the requirements  f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree that  t h e L i b r a r y s h a l l make i t f r e e l y  available f o r reference  and study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s  thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my department o r his  representative.  I t i s understood t h a t  copying o r p u b l i c a t i o n o f  t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without written  permission.  The U n i v e r s i t y  o f B r i t i s h Columbia  Vancouver, Canada V6T  1W5  my  ABSTRACT  T h i s study d e s c r i b e s breeding  systems on l e v e l s and  the consequences o f c o n t r a s t i n g  o r g a n i z a t i o n o f isozyme v a r i a t i o n i n  c l o s e l y r e l a t e d t a x a , Pleotvitis P. bvaahystemon  F.  £ M.  oongesta  (Valerianaceae).  c o m p a t i b l e , herbaceous w i n t e r coast  ( L i n d l . ) D.C.  and  Both t a x a are  self-  annuals which occur  from B r i t i s h Columbia t o  along  the  two  Pacific  California.  Nine enzyme systems were e l e c t r o p h o r e t i c a l l y surveyed and  fifteen  l o c i i d e n t i f i e d i n Pleotvitis.  to e x p l a i n the observed v a r i a t i o n . locus o r g a n i z a t i o n , sub:-unit each system. The  Genetic  These models d e s c r i b e the m u l t i -  s t r u c t u r e and  models were t e s t e d w i t h  When progeny c l a s s e s were s u f f i c i e n t l y u s i n g the c h i - s q u a r e  statistic.  differed in their a l l e l i c  models were proposed  a l l e l i c variation within  crosses  and  segregation  large, segregations  Although P.  oongesta  were  and P.  analyzed  bvaahystemon  complements, the d a t a suggest t h a t  m u l t i - l o c u s o r g a n i z a t i o n o f the enzyme systems surveyed;are i n both  analyses.  the identical  species. Outcrossing  o f P. hvaohystemon  and  r a t e s were c a l c u l a t e d f o r n i n e  f i f t e e n populations  o u t c r o s s i n g r a t e s were 2.4±  0.8%  and  o f P.  70.2± 4.8%,  populations  oongesta.  T h e i r mean  r e s p e c t i v e l y , and  the d i f f e r e n c e between these means i s h i g h l y s i g n i f i c a n t  (P<0.01).  A number o f g e n e t i c parameters were measured w i t h i n each taxon t o assess  the e f f e c t t h a t b r e e d i n g  As measured by  system has  a l l parameters, the s e l f e r m a i n t a i n s  less v a r i a t i o n within populations detected  on p o p u l a t i o n  structure.  significantly  than the o u t c r o s s e r  although  l e v e l s o f t o t a l v a r i a t i o n (H ) are not s i g n i f i c a n t l y  their different.  These two  s p e c i e s are i d e a l f o r comparison because:  ( i ) they  are  c l o s e l y r e l a t e d and have a l a r g e p r o p o r t i o n o f t h e i r a l l e l e s i n common, ( i i ) the m u l t i - l o c u s o r g a n i z a t i o n o f the enzyme systems s t u d i e d i s homologous and  (iii)  and h a b i t a t requirements. I s l a n d , t o f i n d the two populations).  they have s i m i l a r l i f e - c y c l e s t r a t e g i e s  I t i s not uncommon, p a r t i c u l a r l y on Vancouver s p e c i e s growing s y m p a t r i c a l l y (i.e.  When the i n f l u e n c e o f s i t e v a r i a t i o n was  by comparing o n l y sympatric  populations  d i f f e r e n c e s remained s i g n i f i c a n t . within populations and  The  o f the s e l f e r was  o f the two  Only 36%  contained  85%  species, a l l genetic  r e f l e c t e d i n the FOn  statistics  the average, P.  oongesta  o f the v a r i a t i o n d e t e c t e d w i t h i n the  o f the v a r i a t i o n d e t e c t e d  within local  controlled  reduced l e v e l o f v a r i a t i o n  i n the a n a l y s i s o f gene d i v e r s i t y .  populations  i n mixed  i n P.  braohystemon  was  species.  maintained  populations. In the s e l f e r , the reduced l e v e l o f g e n e t i c " v a r i a t i o n  within populations differentiation.  was  accompanied by  increased populational  P r e l i m i n a r y d a t a suggest t h a t  p a r t i c u l a r l y i n the s e l f e r , whether t h i s r e p r e s e n t s  can occur over s h o r t d i s t a n c e s . However,  m i c r o - g e o g r a p h i c d i f f e r e n t i a t i o n or  i n small r e p r o d u c t i v e l y l x s o l a t e d populations t h i s taxon, a l l e l e f r e q u e n c i e s f l u c t u a t e d widely populations.  In P.  and  and  oongesta,  brachystemon.  g e n e t i c d i s t a n c e and  i s problematic.  drift In  s i n g l e locus h e t e r o z y g o s i t i e s  d i f f e r e n t a l l e l e s were o f t e n f i x e d i n  adjacent  a l l e l e f r e q u e n c i e s and h e t e r o z y g o s i t i e s  a l s o f l u c t u a t e d among p o p u l a t i o n s i n P.  differentiation,  but not with the amplitude observed  There appears to be no r e l a t i o n s h i p between geographic d i s t a n c e . T h i s i m p l i e s t h a t gene  flow between p o p u l a t i o n s does not s i g n i f i c a n t l y i n f l u e n c e frequencies.  allele  No evidence was.found t o suggest t h a t the observed  isozyme polymorphisms are s e l e c t i v e l y m a i n t a i n e d . However, c o - o r d i n a t e d gene complexes and m i c r o - h a b i t a t s e l e c t i o n would p r o b a b l y not have been d e t e c t e d .  I t i s concluded t h a t the observed d i f f e r e n c e s i n the  p o p u l a t i o n s t r u c t u r e o f P. .bvachystemon  and P. oongesta  the r e s u l t o f t h e i r c o n t r a s t i n g b r e e d i n g systems.  iv.  i s primarily  TABLE OF CONTENTS  PAGE  CHAPTER  ii.  ABSTRACT '  viii.  LIST OF TABLES  x.  LIST OF FIGURES  xiii.  ACKNOWLEDGEMENTS  1.0  1.  INTRODUCTION  1.1 Statement 1.2 Breeding  of the  1.5 Breeding 2.0  v a r i a t i o n in  v a r i a t i o n in system  structure selfers  selfers  and genetic  2.2 Potential  4. 6. 9.  variability  10. 16.  MATERIALS AND METHODS  2.1 C o l l e c t i o n sites  and sampling  sources  of bias  strategy  in the collections  16.  19.  2.2.1  Sample size  19.  2.2.2  Phenology  19.  2.2.3  Seed wing  2.2.4  Wahlund effect  . 1 9 .  2.3 Methods of planting 2.4 Electrophoretic 3.0  1.  system, and population  1.3 Morphological 1.4 Isozyme  problem  20. and growth regime  techniques  INHERITANCE  3.1 Isozyme  3.4 Malate  21. 29.  nomenclature  3.2 Esterase 3.3 Leucine  20.  29. 31.  amine-peptidase dehydrogenase  33. 37.  v.  PAGE  CHAPTER,::  4.0  3.5 Phosphoglucose isomerase  40.  3.6 Phosphoglucomuta.se  50.  3.7 Monomorphic enzyme systems  58.  3.8 Summary  60.  VARIATION AMONG POPULATIONS  62.  4.1 Loci polymorphic with E^  0.400  69.  4.1.1 P. brachystemon  69.  4.1.2 P. congesta  74.  4.2 Loci polymorphic with 0.400>E z.0.050  79.  4.2.1 P. brachystemon  79.  4.2.2 P. congesta  82.  4.3 Slightly  polymorphic or monomorphic with E^^O.050  82.  4.3.1 P. brachystemon  82.  4.3.2 P. congesta  82.  4.4 Summary  83.  5.0 .: ANALYSES AND DISCUSSION 5.1 Analysis  86.  of the breeding system  5.2 Genetic variability 5.3 Analysis  86.  within populations  94.  of gene diversity  5.4 Comparisons of genetic  identity  5.4.1 Levels of inter-population  102. and genetic  differentiation  5.4.2 Comparisons between genetic distance geographic  distance  and  HO. 115. 116.  distance  5.4.3 Taxonomic and evolutionary  relationships  5.4.4 Maintenance of observed isozyme polymorphisms 5.5 Summary: the genetic consequences of contrasting breeding systems vi.  124. 127. 133.  PAGE  CHAPTER  BIBLIOGRAPHY APPENDIX  137.  A  Population locations  B  Enzyme systems which were not adequately  1 4 5  resolved with a v a i l a b l e C  '  -  148.  techniques  Gene and genotype f r e q u e n c i e s w i t h i n p o p u l a t i o n s  151.  C . l P. bvachystemon  152.  C.2 Pteotritis  populations  oongesta  vii.  populations  163.-  LIST OF TABLES  TABLE  TITLE  PAGE  1  Collection sites  17.  2  Running  24.  3  Running b u f f e r used, number o f l o c i res.olved and s t a i n i n g r e f e r e n c e s f o r enzyme systems s t u d i e d i n  buffers  P. brachystemon  and P.  25.  congesta  4  Summary o f enzyme s t a i n i n g procedures  27.  5  Stock s o l u t i o n s  28.  6  A n a l y s i s o f allozyme v a r i a t i o n a t EST-1  34.  7  A n a l y s i s o f allozyme v a r i a t i o n a t EST-2  35.  8  A n a l y s i s o f allozyme v a r i a t i o n a t LAP-1  38.  9  A n a l y s i s o f allozyme v a r i a t i o n a t MDH-1  43.  10  A n a l y s i s o f allozyme v a r i a t i o n a t PGI-2  51.  11  A n a l y s i s o f allozyme v a r i a t i o n a t PGI-3  51.  12  A n a l y s i s o f allozyme v a r i a t i o n a t PGM-1  55.  13  A n a l y s i s o f allozyme v a r i a t i o n a t PGM-2  56.  14  A n a l y s i s o f allozyme var i at ion-rat PGM-3  57.  15  A l l e l e f r e q u e n c i e s and h e t e r o z y g o s i t y v a l u e s f o r e i g h t polymorphic l o c i i n P. brachystemon  63.  16  A l l e l e f r e q u e n c i e s and h e t e r o z y g o s i t y .values f o r e i g h t polymorphic l o c i i n P. congesta  66.  17  Comparison o f mean a l l e l e f r e q u e n c i e s w i t h h e t e r o z y g o s i t y v a l u e s f o r polymorphic l o c i i n  72.  P. brachystemon 18  and P.  congesta  Wright's f i x a t i o n index f o r seven polymorphic l o c i i n P. brachystemon  viii.  88.  TITLE  TABLE  PAGE  19  Wright's f i x a t i o n index f o r e i g h t polymorphic l o c i i n P. oongesta  20  Estimates o f o u t c r o s s i n g frequency f o r t e n p o p u l a t i o n s o f P. braohystemon  92.  21  Estimates o f o u t c r o s s i n g frequency f o r f i f t e e n p o p u l a t i o n s o f P. oongesta  93.  22  Summary o f v a r i o u s g e n e t i c parameters f o r t e n p o p u l a t i o n s o f P. braohystemon  95.  23  Summary o f v a r i o u s g e n e t i c parameters f o r f i f t e e n p o p u l a t i o n s o f P. oongesta  96.  24  A n a l y s i s o f gene d i v e r s i t y and degree o f differentiation forfifteen loci i n P. braohystemon  106.  25  A n a l y s i s o f gene d i v e r s i t y and degree o f .:*. . d i f f e r e n t i a t i o n f o r twelve l o c i i n P. oongesta  107.  G e n e t i c i d e n t i t i e s and s t a n d a r d g e n e t i c d i s t a n c e s among p o p u l a t i o n s o f P. braohystemon  113.  27  G e n e t i c i d e n t i t i e s and s t a n d a r d g e n e t i c d i s t a n c e s among p o p u l a t i o n s o f P. oongesta  114.  28  Summary o f g e n e t i c d i s t a n c e i n P. braohystemon  117.  29  Summary o f g e n e t i c d i s t a n c e i n P. oongesta  118.  30  Inter-specific in Pleotritis  129.  31  A comparison between t h e observed and t h e t h e o r e t i c a l inter-locus variances of h e t e r o z y g o s i t y i n p o p u l a t i o n s o f P. braohystemon  131.  32  A comparison between t h e observed and t h e theoretical inter-locus variance of h e t e r o z y g o s i t y i n p o p u l a t i o n s o f P. oongesta  132.  33  Summary o f g e n e t i c d i f f e r e n c e s between P. braohystemon and P. oongesta  134.  -26  comparison o f g e n e t i c d i s t a n c e  ix.  89.  LIST OF FIGURES  FIGURE  TITLE  PAGE  1  Flowers o f Vtectritis congesta and P. brachystemon i l l u s t r a t i n g differences i n size  2  A map showing the l o c a t i o n o f sampled Plectritis populations  18.  3  The r e l a t i v e m i g r a t i o n o f allozymes  d e t e c t e d a t EST-1  32.  4  The r e l a t i v e m i g r a t i o n o f allozymes  d e t e c t e d a t EST-2  32.  5  Allozyme  6  The r e l a t i v e m i g r a t i o n o f allozymes  7  Allozyme  8  The r e l a t i v e m i g r a t i o n o f allozymes  d e t e c t e d a t MDH-1  41.  9  The r e l a t i v e m i g r a t i o n o f allozymes  d e t e c t e d a t MDH-2  41.  variation  a t EST-1  3.  32. d e t e c t e d a t LAP-1  v a r i a t i o n a t LAP-1 i n P. congesta  36. 36.  10  Allozyme  v a r i a t i o n a t MDH-1  42.  11  Allozyme  v a r i a t i o n a t MDH-1  42.  12  S e g r e g a t i o n o f allozyme v a r i a t i o n a t PGI-2 and PGI-3 i n P. congesta  13  Ah:.allelic interpretation p i c t u r e d i n F i g u r e 12  14  A sample o f t h e allozyme v a r i a t i o n d e t e c t e d a t PGI-2 and PGI-3 i n P. congesta  46.  15  A sample o f t h e allozyme v a r i a t i o n d e t e c t e d a t PGI-2 and PGI-3 i n P. congesta  46.  16  The r e l a t i v e m i g r a t i o n o f allozymes  d e t e c t e d a t PGI-2  48.  17  The r e l a t i v e m i g r a t i o n o f allozymes  d e t e c t e d a t PGI-3  48.  x.  o f t h e allozyme  45.  variation  45.  FIGURE  TITLE  PAGE  18  Allozyme  variation  a t PGI-2 i n P. bvachystemon  48.  19  Allozyme  variation  a t PGI-2 i n P. bvachystemon  49.  20  A comparison o f isozyme v a r i a t i o n P. oongesta and P. bvachystemon  21  The r e l a t i v e m i g r a t i o n o f allozymes  d e t e c t e d a t PGM-1  53.  22  The r e l a t i v e m i g r a t i o n o f allozymes  d e t e c t e d a t PGM-2  53.  23  The r e l a t i v e m i g r a t i o n o f allozymes  d e t e c t e d a t PGM-3  53.  24  Allozyme  25  A comparison o f isozyme v a r i a t i o n P. oongesta and P. bvachystemon  26  Allozyme  a c t i v i t y i n ME  59.  27  Allozyme  a c t i v i t y i n 6PG  59.  28  The d i s t r i b u t i o n o f EST-1 a l l e l e s among p o p u l a t i o n s o f P. bvachystemon  70.  29  The d i s t r i b u t i o n o f MDH-1 a l l e l e s among p o p u l a t i o n s of P. bvachystemon  70.  30  The d i s t r i b u t i o n o f LAP-1 a l l e l e s among p o p u l a t i o n s of P. bvachystemon  71.  31  The d i s t r i b u t i o n o f PGM-1 a l l e l e s among p o p u l a t i o n s o f P. bvachystemon  71.  32  The d i s t r i b u t i o n o f LAP-1 a l l e l e s among p o p u l a t i o n s of P. oongesta  76.  33  The d i s t r i b u t i o n o f MDH-1 a l l e l e s among p o p u l a t i o n s o f P. oongesta  76.  34  The d i s t r i b u t i o n o f PGM-3 a l l e l e s among p o p u l a t i o n s o f P. oongesta  77.  35  The d i s t r i b u t i o n o f EST-2 a l l e l e s among p o p u l a t i o n s of?P. oongesta  77.  variation  i n PGI between  i n PGM i n P. oongesta  xi.  i n PGM between  49.  54. 54.  36  The d i s t r i b u t i o n o f PGM-2 a l l e l e s among p o p u l a t i o n s  o f P. 37  among p o p u l a t i o n s  78.  among p o p u l a t i o n s  80.  The d i s t r i b u t i o n o f PGM-3 a l l e l e s among p o p u l a t i o n s  80.  o f P. 40  among p o p u l a t i o n s  81.  congesta 100.  brachystemon  The r e l a t i o n s h i p between t h e o u t c r o s s i n g r a t e and the expected h e t e r o z y g o s i t y among p o p u l a t i o n s o f  P.  81.  brachystemon  The r e l a t i o n s h i p between t h e o u t c r o s s i n g r a t e and the expected h e t e r o z y g o s i t y among p o p u l a t i o n s o f  P. .43  brachystemon  The d i s t r i b u t i o n o f EST-1 a l l e l e s  o f P. .42  brachystemon  The d i s t r i b u t i o n o f PGI-2 a l l e l e s among p o p u l a t i o n s  o f P. 41  congesta  The d i s t r i b u t i o n o f PGI-3 a l l e l e s  o f P.. 39  78.  congesta  The d i s t r i b u t i o n o f PGM-1 a l l e l e s  o f P. 38  PAGE  TITLE  FIGURE;  100.  congesta  44  A dendrograph d e p i c t i n g t h e g e n e t i c r e l a t i o n s h i p s among t h e sampled P. brachystemon populations  119.  45  A dendrograph d e p i c t i n g t h e g e n e t i c r e l a t i o n s h i p s :among the sampled P. congesta populations  120.  46  A dendrograph d e p i c t i n g t h e g e n e t i c r e l a t i o n s h i p s among t h e sampled P l e c t r i t i s populations  125.  xii.  ACKNOWLEDGEMENTS  I would l i k e t o express  my g r a t i t u d e t o a l l those  who have h e l p e d me w i t h t h i s study and have made i t , i n r e t r o s p e c t , a positive  experience. Dr. F r e d Ganders, my a d v i s o r , i n t r o d u c e d me t o the p l a n t s  and  t o many o f the concepts  from which the t h e s i s e v o l v e d .  his  a d v i c e and t o t a l s u p p o r t ,  Without  t h i s r e s e a r c h would not have been  conducted. I would l i k e t o thank Dr. Tony G r i f f i t h s  for his  interest  i n r e a l p l a n t s , h i s encouragement throughout t h e study and h i s evaluation o f the genetic sections o f t h i s  critical  thesis.  A s p e c i a l thanks t o Dr. W i l f S c h o f i e l d who was d r a f t e d , and  agreed  t o p a r t i c i p a t e i n t h i s p r o j e c t , because o f h i s open mind,  p a t i e n c e and good humor. Dr. F r a n c i s Yeh a d v i s e d and p r o v i d e d t e c h n i c a l a s s i s t a n c e with t h e d a t a a n a l y s i s .  I am g r a t e f u l f o r h i s s u g g e s t i o n s  regarding  the d i s c u s s i o n s o f p o p u l a t i o n s t r u c t u r e . In a d d i t i o n , I would l i k e t o thank: Ken Carey f o r h i s a d v i c e on f i n d i n g , growing who i n i t i a l l y  and h a n d l i n g Pteotvitis;  Sue Krepp Denny,  l e t me muddle about h e r l a b and J e f f A r c h e r and  R i c h a r d O'Brien  who a s s i s t e d w i t h the f i e l d  work.  My deepest g r a t i t u d e belongs t o Diane Layton whose p a t i e n c e , encouragement and a s s i s t a n c e throughout t h e study made t h e process  easier.  1.  1.0  1.1 Statement  of the  problem  T h i s study breeding  systems on  INTRODUCTION  d e s c r i b e s the consequences of. c o n t r a s t i n g  l e v e l s and  o r g a n i z a t i o n o f isozyme v a r i a t i o n i n  c l o s e l y r e l a t e d taxa, P l e o t r i t i s braohystemon  F. § M.  herbaceous w i n t e r  oongesta  (Valerianaceae).  ( L i n d l . ) D.C.  Pleotritis  Both taxa are s e l f - c o m p a t i b l e ,  annuals which occur along  B r i t i s h Columbia to C a l i f o r n i a .  and  two  the P a c i f i c coast  P. braohystemon  has  from  a s l i g h t l y wider  d i s t r i b u t i o n , ranging  from the Queen C h a r l o t t e I s l a n d s southward i n t o  southern  These two  California.  t a x a are m o r p h o l o g i c a l l y  the o n l y s i g n i f i c a n t d i f f e r e n c e s are i n t h e i r f l o r a l P. average 6-10  mm.  oongesta  has  i n l e n g t h , are b r i g h t p i n k  and bear a n e c t a r i f e r o u s  oongesta  s m a l l . They a r e white t o p a l e p i n k s p u r r e d , but  a mere s w e l l i n g . The  and  braohystemon  average only 2-4  i n some p o p u l a t i o n s  are q u i t e mm  at a n t h e s i s . The  have o n l y a v e r y f a i n t f r a g r a n c e  i n length.  the spur i s reduced to  f l o w e r s are not markedly p r o t a n d r o u s .  i n c l o s e p r o x i m i t y t o the anthers  P. braohystemon  may  geitonogamous p o l l i n a t i o n s are p o s s i b l e .  In c o n t r a s t , the f l o w e r s o f P.  be  spur.  i s strongly  However, s i n c e numerous f l o w e r s on the i n f l o r e s c e n c e  be open s i m u l t a n e o u s l y ,  is  corollas  are a c t i v e l y v i s i t e d by a number o f  p o l l i n a t o r s . Although s e l f - c o m p a t i b l e , P.  C o r o l l a s may  similar;  characters.  r . e l a t i v e l y . l a r g e f l o w e r s . The  They are s t r o n g l y f r a g r a n t and  protandrous.  very  The  flowers  stigma of  and p o l l i n a t o r s were  not observed v i s i t i n g the f l o w e r s . T h i s r a r i t y o f i n s e c t v i s i t s been r e p o r t e d e a r l i e r by Dempster (1958), Morey  (1962) and  has  Ganders  2.  et al.  (1977b).  F i g u r e 1 i l l u s t r a t e s the s i z e d i f f e r e n c e between  f l o w e r s o f the two The  taxa.  differences i n f l o r a l  c h a r a c t e r s s t r o n g l y suggest  c o n t r a s t i n g b r e e d i n g s t r a t e g i e s . T h i s was confirmed by Ganders et al.  investigated  (1977a, 1977b) who  and  u t i l i z e d a seed wing  dimorphism, p r e s e n t i n both t a x a , to e s t i m a t e o u t c r o s s i n g r a t e s .  The  i n h e r i t a n c e o f the seed wing i s c o n t r o l l e d by a simple one  two  a l l e l e system w i t h the winged a l l e l e e x p r e s s i n g dominance et al.,  1977a).  they found P.  Employing  congesta  almost  (Ganders  the progeny t e s t method o f Harding  t o be p r i m a r i l y o u t c r o s s e d ; a v e r a g i n g  o u t c r o s s i n g over s e v e n r p o p u l a t i o n s Ganders, i n p r e s s ) .  gene,  (Ganders et al.,  In c o n t r a s t , P. brachystemon  (1970), 70%  1977a; Carey  was  and  found t o be  e x c l u s i v e l y s e l f - p o l l i n a t e d , a v e r a g i n g o n l y 2% o u t c r o s s i n g  (Ganders et al.,  1977b).  These two  t a x a are c l o s e l y r e l a t e d ; P. brachystemon  be a d e r i v a t i v e o f P. congesta.  Stebbins  (1957) argues  are p r o b a b l y always d e r i v e d from o u t c r o s s e r s .  may  that s e l f e r s  Many examples o f the  e v o l u t i o n o f autogamous t a x a from o u t c r o s s e r s have been demonstrated. Moore and  Lewis  (1965) p r e s e n t e d c y t o l o g i c a l evidence t h a t n o r m a l l y  o u t c r o s s i n g , p i n k f l o w e r e d , Clarkia  xantiana  has  given r i s e to  d i s t i n c t s m a l l f l o w e r e d autogamous p o p u l a t i o n s ; one w i t h p i n k the o t h e r with white  flowers. Antonovics  evolution of s e l f - f e r t i l i t y Agrostis  tenuis  i n heavy metal  and' Anthoxanthum  n o n - t o l e r a n t p o p u l a t i o n s . Arroyo Limnanthes  floccosa  has  O r n d u f f and C r o v e l l o ,  (1968) i n v e s t i g a t e d  odoratum  and the  tolerant populations of from n o r m a l l y o u t c r o s s i n g  (1973) concluded  t h a t autogamous  e v o l v e d from the o u t c r o s s e r , L. alba  1968).  two  (see a l s o  3.  F i g u r e 1. Flowers o f (A) Weotritis oongesta and (B) P. braohystemon i l l u s t r a t i n g d i f f e r e n c e s i n s i z e and i n the degree o f s e p a r a t i o n o f the stigma and anthers i n the two s p e c i e s . The i l l u s t r a t i o n shows P. oongesta a f t e r the s t y l e has elongated when the stigma i s r e c e p t i v e .  4.  P.  braohystemon  and' P.  oongesta  have s i m i l a r geographic  ranges, l i f e - c y c l e s t r a t e g i e s and h a b i t a t p r e f e r e n c e s . sympatric  {i.e.  mixed) p o p u l a t i o n s  species by all  and  S e l a n d e r (1974) e l e c t r o p h o r e t i c a l l y  p a i r s o f e d a p h i c a l l y r e s t r i c t e d and concluded t h a t  widespreadcplant  l e v e l s o f g e n e t i c v a r i a b i l i t y are  e c o l o g i c a l amplitude. R e c e n t l y ,  Hamrick et al.  p r i o r t o June, 1978.  influenced  (1979) reviewed  genetically interpretable, higher-plantallozyme  published  fact,  are not uncommon, at l e a s t on  s o u t h e r n Vancouver I s l a n d . Babbel and surveyed two  In  literature  Using m u l t i v a r i a t e techniques,  they  attempted to c o r r e l a t e twelve l i f e - h i s t o r y c h a r a c t e r i s t i c s w i t h l e v e l s o f allozyme v a r i a t i o n among 100  p l a n t taxa.  The  life-history  variables  which were i d e n t i f i e d as s i g n i f i c a n t l y a f f e c t i n g l e v e l s - o f g e n e t i c variabilitycamong  the s t u d i e d t a x a were: mating system, p o l l i n a t i o n  mechanism, g e n e r a t i o n range and  l e n g t h , chromosome number, f e c u n d i t y ,  successional  s t a t u s . S i n c e P. oongesta  are c l o s e l y r e l a t e d {i.e.  p o s s i b l y as p r o g e n i t o r  and P. and  geographic  braohystemon  derivative)  and  have s i m i l a r l i f e - h i s t o r y c h a r a c t e r i s t i c s , they o f f e r a unique opportunity  t o study the g e n e t i c  consequences o f c o n t r a s t i n g b r e e d i n g  s t r a t e g i e s w h i l e c o n t r o l l i n g f o r g e n e t i c background and which a l s o i n f l u e n c e the g e n e t i c  1.2 Breeding  system  and population  Populations generally considered which may  variables  populations.  structure  of a predominantly outcrossed  taxon  are  t o c o n s i s t o f a l a r g e number o f b i o t y p e s ,  be r e p r e s e n t e d  by o n l y a s i n g l e i n d i v i d u a l .  m a i n t a i n e d w i t h i n the p o p u l a t i o n population  structure of  other  The  each o f  l e v e l of v a r i a t i o n .  gene p'ool depends on the e f f e c t i v e  s i z e (a f u n c t i o n o f the s i z e o f the b r e e d i n g  population  5.  and the o u t c r o s s i n g : r a t e ) , mutation r a t e , gene flow and the modes  and  severity of selection. In c o n t r a s t , the t r a d i t i o n a l populations  i s one  Each o f these  types may  peak and be r e p r e s e n t e d -by a few  (DuRietz,  1930  and  isolated  Stebbins,  occupy a s l i g h t l y d i f f e r e n t  1950,  adaptive  o r thousands o f i n d i v i d u a l s . S u c c e s s f u l  b i o t y p e s are t r a d i t i o n a l l y c o n s i d e r e d to m a i n t a i n homozygous l i n e s  o f autogamous  o f a number o f d i s c r e t e homozygous b i o t y p e s  from each o t h e r by b r e e d i n g h a b i t 1957).  concept  themselves as  f o r p o t e n t i a l l y l a r g e numbersiof g e n e r a t i o n s .  these r e p r o d u c t i v e l y i s o l a t e d sympatric  lines,  Besides  the p o p u l a t i o n s may  also  c o n s i s t o f numerous h y b r i d s which are the r e s u l t o f o c c a s i o n a l c r o s s e s between these predominant i n b r e d b i o t y p e s . The o f these  i n t e r m i t t e n t c r o s s - p o l l i n a t i o n s i s v i s u a l i z e d as the source  g r e a t e v o l u t i o n a r y a c t i v i t y w i t h i n the p o p u l a t i o n -  1957).  The  s e v e r i t y o f s e l e c t i o n . The  heterozygous.  These h y b r i d s may  o r may  of  1950,  inter-racial  depend on the l e v e l o f o u t c r o s s i n g i n the p o p u l a t i o n  the modes and  and  F^ h y b r i d s w i l l be h i g h l y  not be more v i g o r o u s  e i t h e r o f the p a r e n t a l t y p e s . I f they s u r v i v e and  than  s e l f , the  generation  show a wide range o f s e g r e g a t i o n . S u c c e s s i v e g e n e r a t i o n s r . w i i l "  continue  to produce new  segregants,  t o e i g h t g e n e r a t i o n s . The fit  (Stebbins,  p r o p o r t i o n o f the p o p u l a t i o n composed o f these  hybrids w i l l  will  occurrence  i n d i m i n i s h i n g numbers f o r seven  g r e a t m a j o r i t y o f these types w i l l be  than e i t h e r o f the o r i g i n a l p a r e n t s .  But,  s h o u l d a new  less  biotype  w i t h s u p e r i o r f i t n e s s be s y n t h e s i z e d , i t can be the p r o g e n i t o r o f a l i n e . As Stebbins biotypes  v i s u a l i z e s autogamous p o p u l a t i o n s , the predominant  are almost t o t a l l y homozygous and  are ephemeral.  new  their inter-racial  hybrids  T h i s model p r e d i c t s t h a t the v a s t m a j o r i t y o f the  v a r i a t i o n w i t h i n the p o p u l a t i o n w i l l be d i s t r i b u t e d between i n b r e d l i n e s  '.Ci'.e.  families).  Consequently,  the l e v e l o f v a r i a t i o n  w i t h i n the p o p u l a t i o n w i l l be determined This w i l l  maintained  by the number o f i n b r e d  depend on the e f f e c t i v e p o p u l a t i o n s i z e , mutation  lines.  r a t e , gene  flow, h a b i t a t d i v e r s i t y and modes and s e v e r i t y o f s e l e c t i o n .  I f out-  c r o s s i n g r a t e s are low and h y b r i d s are ephemeral,.the two most  important  f a c t o r s i n f l u e n c i n g the l e v e l s o f v a r i a t i o n w i t h i n p o p u l a t i o n s s h o u l d be h a b i t a t d i v e r s i t y and modes and s e v e r i t y o f s e l e c t i o n . Baker  (1959)  p r e d i c t s t h a t under severe s e l e c t i o n o n l y one b i o t y p e , or a v e r y w i l l e x i s t and autogamous p o p u l a t i o n s w i l l be u n i f o r m , at  few,  i f present  all.  1.3 Morphological  v a r i a t i o n in  selfers  A number o f s t u d i e s have i n v e s t i g a t e d predominantly p o l l i n a t e d t a x a t o determine proposed  self-  i f the t r a d i t i o n a l p o p u l a t i o n model, as  by Stebbins and o t h e r s , agrees w i t h q u a n t i t a t i v e  phenotypic  d a t a and o b s e r v a t i o n s o f m o r p h o l o g i c a l gene markers. J a i n and  Allard  (1960) s t u d i e d v a r i a t i o n at n i n e m o r p h o l o g i c a l gene l o c i w i t h i n e x p e r i m e n t a l b a r l e y p o p u l a t i o n s . They concluded t h a t the m a i n t a i n e d w i t h i n these autogamous p o p u l a t i o n s was  not a s s o c i a t e d  e n t i r e l y w i t h sympatric homozygous l i n e s , and t h a t l e v e l s outcrossing  lower than those f r e q u e n t l y observed  variability  of  in selfers, i f  accompanied by s e l e c t i o n f o r h e t e r o z y g o t e s , c o u l d m a i n t a i n c o n s i d e r a b l e amounts o f v a r i a b i l i t y .  S i m i l a r c o n c l u s i o n s were reached when these same  b a r l e y p o p u l a t i o n s were a n a l y z e d f o r q u a n t i t a t i v e g e n e t i c c h a r a c t e r s ( A l l a r d and J a i n , 1962).  A l l a r d and Workman (1963) d i s c o v e r e d t h a t ,  i n l i m a beans, wide s e a s o n a l f l u c t u a t i o n s i n f i t n e s s were a s s o c i a t e d with c e r t a i n m o r p h o l o g i c a l markers. However, mean s e l e c t i v e v a l u e s  indicat  7.  that heterozygotes homozygote.  left  an average  o f 20% - 30% more progeny than  C o n t i n u i n g t h i s work, Harding, A l l a r d and Smeltzer  found t h a t s e l e c t i o n f o r h e t e r o z y g o t e s was  either  (1966)  frequency-dependent.  When  h e t e r o z y g o t e s were numerous, as i n e a r l y g e n e r a t i o n s f o l l o w i n g an r a c i a l h y b r i d i z a t i o n , they had they were r a p i d l y reduced and an e q u i l i b r i u m was  little  or no s e l e c t i v e v a l u e .  Consequently,  i n number by i n b r e e d i n g . T h i s p r o c e s s  reached when h e t e r o z y g o t e s comprised  inter-  slowed  approximately  7% o f the e x p e r i m e n t a l p o p u l a t i o n . When t h e i r frequency was  below t h i s  l e v e l , t h e i r s e l e c t i v e v a l u e s i n c r e a s e d and a t v e r y low f r e q u e n c i e s h e t e r o z y g o t e s produced homozygotes.  When  frequency o f 2%,  approximately t h r e e times as many progeny as  h e t e r o z y g o t e s were a r t i f i c i a l l y  the 7% e q u i l i b r i u m v a l u e was  g e n e r a t i o n and t h i s was with Avena fatua,  reduced t o a  restored within a single  m a i n t a i n e d i n the s u c c e s s i v e g e n e r a t i o n . Working  Imam and A l l a r d  (1965) d i s c o v e r e d t h a t  families,  d e r i v e d from s i n g l e p l a n t s c o l l e c t e d from n a t u r a l p o p u l a t i o n s , were h i g h l y heterogeneous In  f o r most measured p h e n o t y p i c c h a r a c t e r s .  a d d i t i o n , f a m i l i e s d e r i v e d from:the  of means f o r v a r i o u s c h a r a c t e r s . t h i s predominantly  same s i t e showed a wide  range  They concluded t h a t p o p u l a t i o n s o f  s e l f - p o l l i n a t i n g taxon c o n s i s t o f l a r g e numbers, o f  b i o t y p e s and t h a t many i f n o t a l l i n d i v i d u a l s i n n a t u r a l p o p u l a t i o n s are heterozygous  at numerous l o c i .  Kannenberg and A l l a r d  (1967) s t u d i e d  q u a n t i t a t i v e v a r i a t i o n i n eleven morphological characters w i t h i n t a x a o f the Festuoa  microstachys  complex.  T h i s complex c o n s i s t s  e i g h t s p e c i e s which f r e q u e n t l y o c c u r i n mixed p o p u l a t i o n s and  of  are  almost e x c l u s i v e l y s e l f - p o l l i n a t i n g . S i n c e chasmogamous f l o w e r s are r a r e , Kannenberg and A l l a r d e s t i m a t e d t h a t i t was even one w i l d p l a n t i n 1000  would be an F  7  unlikely  that  h y b r i d and i t was  probable  8.  t h a t the r a t e o f o u t c r o s s i n g was lower than 1/10,000.  However, when the  observed v a r i a t i o n was p a r t i t i o n e d , a l a r g e p r o p o r t i o n o f t h e t o t a l g e n e t i c v a r i a n c e i n a l l measured c h a r a c t e r s was a t t r i b u t e d t o w i t h i n p o p u l a t i o n d i f f e r e n c e s . Species w i t h i n s i t e s . a n d f a m i l i e s w i t h i n accounted f o r 10% and 30%, r e s p e c t i v e l y , o f t h e t o t a l variance i n t h e i r experimental  species  phenotypic  p l o t s . Furthermore, when t h e means o f  f a m i l i e s w i t h i n s i t e s were compared f o r each o f t h e e l e v e n measurement c h a r a c t e r s , i t was found t h a t t h e r e were few f a m i l i e s a t any one s i t e which d i d n o t d i f f e r ,  s i g n i f i c a n t l y with r e s p e c t t o a t l e a s t one  measurement c h a r a c t e r .  In other words, any s i n g l e s i t e  p l a n t s o f many d i f f e r e n t genotypes. characters  contained  They then compared d a t a on t h r e e  t o measurements c o l l e c t e d f o r two o t h e r s p e c i e s which had  contrasting breeding and Avena fatua  systems; an o b l i g a t e o u t c r o s s e r Lolium  multiflovvm  i n which o u t c r o s s i n g v a r i e s between 1% and 10%. D e s p i t e  the d i f f e r e n c e s : i n o u t c r o s s i n g r a t e s , t h e r e were no c o n s i s t e n t d i f f e r e n c e s i n t h e l e v e l s o f w i t h i n - p o p u l a t i o n v a r i a t i o n among three  taxa. These s t u d i e s o f m o r p h o l o g i c a l  s i g n i f i c a n t because they within populations  was h i g h e r than e x p e c t e d :  1962;  Imam and A l l a r d ,  Clegg  and A l l a r d ,  Arroyo,  v a r i a t i o n i n s e l f e r s are  c o n s i s t e n t l y found t h a t the l e v e l s o f v a r i a t i o n  s e v e r a l i n v e s t i g a t o r s (e.g. Jain  and  these  and A l l a r d ,  These r e s u l t s have l e d 1960; A l l a r d and J a i n ,  1965; Kannenberg and A l l a r d ,  1972; Hamrick and A l l a r d ,  1975; S o l b r i g and R o l l i n s ,  1967; S o l b r i g , 1972;  1972; A l l a r d et dl. , 1972;  1977; L e v i n , 1978; K e e l e r , 1978  Hamrick and Holden, 1979).to q u e s t i o n whether p o p u l a t i o n s o f  autogamous t a x a are as g e n e t i c a l l y u n i f o r m  as t r a d i t i o n a l l y b e l i e v e d .  Heterozygote advantage, p a r t i c u l a r l y i f i t i s frequency-dependent,  9.  insures  that'':, i n h i g h l y i n b r e d p o p u l a t i o n s where i n t e r - r a c i a l  crosses  are r a r e , b u r s t s o f s e g r e g a t i o n may be c o n s i d e r a b l y more p e r s i s t a n t than t h e c l a s s i c a l model p r e d i c t s . given h e t e r o s i s , that populations  A l l a r d and J a i n  (1962)  suggest,  i n which o u t c r o s s i n g i s common  (they  do n o t s p e c i f y how common) might c o n s i s t l a r g e l y o f h e t e r o z y g o t e s a s s o c i a t e d w i t h numerous h y b r i d swarms o v e r l a p p i n g i n time. p o p u l a t i o n s would be h i g h l y v a r i a b l e and would n o t d i f f e r from p o p u l a t i o n s o f predominantly  1.4 Isozyme  v a r i a t i o n in  outcrossed  Such  significantly  taxa.  selfers  More r e c e n t s t u d i e s have u t i l i z e d g e l e l e c t r o p h o r e s i s i n order t o i n v e s t i g a t e the d i s t r i b u t i o n o f genetic v a r i a b i l i t y w i t h i n autogamous t a x a and t o e v a l u a t e t h e e f f e c t s o f b r e e d i n g and o r g a n i z a t i o n o f g e n e t i c v a r i a b i l i t y . Allard  (1972) found  l o c i governing  that a l l e l e  morphological  system.;on l e v e l s  In Avena barbata,  C l e g g and  f r e q u e n c i e s , a t f i v e enzyme loci.'.and two  t r a i t s , were d i s t r i b u t e d  in•significantly  non-random, macro-geographic p a t t e r n s which were c l o s e l y a s s o c i a t e d w i t h t h e environment. A l l a r d et al. (1972) found  a large proportion o f  t h i s v a r i a t i o n t o be a s s o c i a t e d with two co-adapted a l l e l e Populations  which o c c u p i e d  combination  o f a l l e l e s a t f i v e enzyme l o c i .  complexes.  x e r i c s i t e s were monomorphic f o r a s p e c i f i c Populations  were monomorphic f o r ah a l t e r n a t e combination  a t mesic  o f allozymes  sites  while  populations  i n i n t e r m e d i a t e h a b i t a t s were u s u a l l y polymorphic at a l l  five  Therefore,  loci.  a l l e l e s were important A.'..barbata. Holden  they concluded  t h a t e p i s t a t i c i n t e r a c t i o n s among  i n t h e maintenance o f g e n e t i c v a r i a b i l i t y i n  In a d d i t i o n , Hamrick and A l l a r d  (1979) have found  t h a t these  (1972) and Hamrick and  co-adapted  allele  complexes i n  10.  A. barbata  are a s s o c i a t e d with m i c r o - g e o g r a p h i c a l  populations. A. barbata  K a h l e r et al.  in Israel.  variation within  (1980) s t u d i e d isozyme v a r i a t i o n i n  They found  t h a t : each p o p u l a t i o n c o n t a i n e d many  isozyme phenotypes, p o p u l a t i o n s were d i f f e r e n t i a t e d by t h e i r  phenotypic  a r r a y s , isozyme v a r i a t i o n was d i s t r i b u t e d i n mosaic p a t t e r n s ,  and  particular:.phenotypes were c o r r e l a t e d w i t h c e r t a i n temperature m o i s t u r e - r e l a t e d v a r i a b l e s . Crawford and Wilson  (1977) a n a l y z e d  isozyme v a r i a t i o n at s i x l o c i i n f o r t y p o p u l a t i o n s o f fremontii. observed  and  Chenopodivm  Most p o p u l a t i o n s were monomorphic and the frequency heterozygotes  was  extremely  low  (i.e.  d e t e c t e d i n o n l y t h i r t e e n p l a n t s , each one, locus).  Keeler  Veronica  peregrina.  heterozygosity  of was  heterozygous at a s i n g l e  (1978) s t u d i e d f i v e enzyme systems i n the h e x a p l o i d No  g e n e t i c a n a l y s e s were attempted because o f  the p o l y p l o i d i n h e r i t a n c e . However, through progeny t e s t i n g ,  individual  p l a n t s were found  that hexaploid  t o be h i g h l y v a r i a b l e .  i n h e r i t a n c e allowed  o u t c r o s s i n g r a t e s and weed Hordeum jubatum.  analyzed  observed  Babbel and  1.5 Breeding  Wain (1977) e s t i m a t e d  l o c i were surveyed  and  h e t e r o z y g o s i t y at i n d i v i d u a l l o c i was  low.  were f i x e d h e t e r o z y g o t e s  tetraploid  the  p o p u l a t i o n s were c a l c u l a t e d to be  a m a j o r i t y o f the f a m i l i e s surveyed l o c i as a r e s u l t  despite  the p o p u l a t i o n s t r u c t u r e o f the  Four allozyme  o u t c r o s s i n g r a t e s w i t h i n two The  concluded  the maintenance o f i n d i v i d u a l v a r i a t i o n  almost complete s e l f - p o l l i n a t i o n .  3%.  I t was  1%  and  However, at  two  of polyploidy.  system and genetic A relatively  variability  l a r g e number o f p u b l i s h e d s t u d i e s have  examined l e v e l s o f g e n e t i c v a r i a t i o n i n e i t h e r s e l f e r s or o u t c r o s s e r s . However, o n l y a few  i n v e s t i g a t i o n s have attempted to d e s c r i b e the  effects  of d i f f e r e n t breeding  systems on t h e o r g a n i z a t i o n o f g e n e t i c v a r i a t i o n  i n r e l a t e d s p e c i e s . S o l b r i g (1972) compared t h r e e  self-incompatible  and  f o r l e v e l s and  four self-compatible  species  o f Leavenwovthia  o r g a n i z a t i o n o f enzyme v a r i a b i l i t y . i n h e r i t a n c e s t u d i e s with  S o l b r i g d i d not attempt any  the enzyme systems surveyed. Consequently,  observed v a r i a t i o n c o u l d n o t be i n t e r p r e t e d with r e s p e c t t o a l l e l e frequencies  and number o f l o c i surveyed. However, based on band  f r e q u e n c i e s , he concluded t h a t the s e l f - c o m p a t i b l e w i t h i n - f a m i l y and w i t h i n - p o p u l a t i o n incompatible  both enzyme data segregating  data on f r u i t  v a r i a t i o n than d i d the s e l f -  s p e c i e s . S o l b r i g and R o l l i n s (1977) s t u d i e d t h e e v o l u t i o n  o f autogamy i n Leavenwovthia  and  s p e c i e s showed l e s s  (see a l s o L l o y d , 1965) .  They  (i.e. j u s t i n terms o f band presence o r absence versus  n o n - s e g r e g a t i n g f a m i l i e s ) and  morphological  c h a r a c t e r s . G e n e r a l l y , wider ranges o f v a r i a t i o n were  found i n t h e s e l f - i n c o m p a t i b l e t a x a . species, within-population  However, i n one s e l f - c o m p a t i b l e  variability i n fruit  c h a r a c t e r s was g r e a t e r  than t h a t found i n two o f t h e s e l f - i n c o m p a t i b l e s p e c i e s . concluded t h a t t h e b r e e d i n g f a c t o r determining Levin  population  structure.  (1977) analyzed  self-compatible  They  system was the s i n g l e most important  allozyme v a r i a t i o n a t twenty  i n two s e l f - i n c o m p a t i b l e and one s e l f - c o m p a t i b l e The  s p e c i e s P. euspidata  the f i e l d o r greenhouse and s e t s a f u l l the maximum l i k e l i h o o d e s t i m a t o r  species  s e l f s automatically compliment o f seed.  o f Brown and A l l a r d  and found t o be t = 0.22.  compared, P. euspidata  loci  o f Phlox. i n either Using  (1970), an  o u t c r o s s i n g r a t e was c a l c u l a t e d f o r a " t y p i c a l " p o p u l a t i o n species  analyzed  of this  When l e v e l s o f allozyme v a r i a t i o n were  had l e s s t o t a l and w i t h i n - p o p u l a t i o n  variability  12.  than e i t h e r o f the s e l f - i n c o m p a t i b l e s p e c i e s .  Rick et al.  s t u d i e d g e n e t i c v a r i a t i o n at 11 enzyme and  morphological  Lyoopevsioon  pimpinellifolium.  which o u t c r o s s i n g  (i.e.  v a r i e d between zero and variability  (i.e.  anther  pollination appearing  40%.  In t h i s taxon a l l measures o f p o p u l a t i o n  h e t e r o z y g o s i t y and mean number o f a l l e l e s (P<0.01) with  (i.e.  estimated  characters  by the p r o p o r t i o n o f n o n - p a r e n t a l  In c o n t r a s t , A r r o y o  o f the s e l f e r Linmanthes  t h a t although  the s e l f e r L. floooosa  has  genetic structures. c a u t i o u s l y s i n c e no  However, these  c o u l d not be  degree o f autogamy among She  slightly  the two  conclusions  also  less  concluded  within-population  s p e c i e s have s i m i l a r s h o u l d be  i n h e r i t a n c e s t u d i e s were conducted.  the number o f loci•-surveyed was  not determined and  accepted Consequently,  enzyme v a r i a b i l i t y  i n t e r p r e t e d i n terms o f a l l e l i c v a r i a t i o n . In a d d i t i o n ,  (1978) c i t e s u n p u b l i s h e d  isozyme d a t a which show t h a t the  i s much l e s s v a r i a b l e w i t h i n p o p u l a t i o n s  o u t c r o s s e r L.  than  the  system on  organization of genetic v a r i a b i l i t y i n r e l a t e d plant species r e s u l t e d i n some c o n f l i c t i n g  conclusions.  Arroyo  c l e a r r e l a t i o n s h i p between o u t c r o s s i n g r a t e and  i n two  Linmanthes  Avena barbata  selfer  alba. I n v e s t i g a t i o n s o f the e f f e c t s o f b r e e d i n g  no  alleles  (1975) d e t e c t e d no c l e a r  floooosa.  v a r i a t i o n than the o u t c r o s s e r L. alba,  L. floooosa  floral  per  l e n g t h and stigma e x s e r t i o n ) and p r o p o r t i o n o f c r o s s -  r e l a t i o n s h i p between enzyme v a r i a b i l i t y and  Jain  in  measured as the p r o p o r t i o n o f c r o s s - p o l l i n a t i o n )  i n progenies).  populations  loci  This i s a self-compatible species i n  l o c u s ) were c o r r e l a t e d p o s i t i v e l y (i.e.  two  (1977)  species.  A l l a r d et al.  the  has  (1975) d e t e c t e d level of v a r i a t i o n  (1975) r e p o r t e d  i s g e n e t i c a l l y l e s s v a r i a b l e than A. fatua  that  although  their  o u t c r o s s i n g r a t e s a r e 0.1 - 7.5% and 0.1 - 1.5%, r e s p e c t i v e l y . J a i n (1978) found no r e l a t i o n s h i p between l e v e l s o f a l l o z y m e v a r i a t i o n and o u t c r o s s i n g r a t e s among p o p u l a t i o n s Levin  (1978),  o f Limnanth.es alba.  S o l b r i g (1972) and S o l b r i g and R o l l i n s  (1977) found  differences i n levels of v a r i a t i o n within self-compatible s e l f - i n c o m p a t i b l e s p e c i e s o f Phlox  and Leavenwovthia,  F u r t h e r m o r e , i n s t u d i e s o f Leavenwovthia 1977)  and Lyoopevsioon  species  versus  respectively.  ( S o l b r i g and R o l l i n s ,  p i m p i n e l l i f o l i u m (Rick e t al.  concluded t h a t the breeding  In contrast,  1977) i t was  3  system was t h e s i n g l e most  important  factor influencing population structure. J a i n (1976) emphasizes t h a t comparisons o f t h e g e n e t i c s t r u c t u r e between r e l a t e d s p e c i e s r e q u i r e q u a n t i t a t i v e d e s c r i p t i o n s of the breeding  systems i n n a t u r e and an e v a l u a t i o n o f t h e s i g n i f i c a n c e  o f parameters used t o d e s c r i b e t h e g e n e t i c v a r i a t i o n . I n a comparison o f s e l f - c o m p a t i b l e and s e l f - i n c o m p a t i b l e s p e c i e s o f Phlox,  Levin  (1978)  c a l c u l a t e d o u t c r o s s i n g i n o n l y one p o p u l a t i o n o f t h e s e l f - c o m p a t i b l e s p e c i e s P. euspidata.  A l t h o u g h he c o n s i d e r e d  t h i s p o p u l a t i o n t o be  " t y p i c a l " , i t may n o t d e s c r i b e a d e q u a t e l y t h e b r e e d i n g species. Outcrossing  r a t e s among p o p u l a t i o n s  of self-compatible  o f t e n v a r y o v e r wide l i m i t s ; e.g. zero t o 40% i n p i m p i n e l l i folium  species  taxa  Lyoopevsioon  ( R i c k et al., 1977); 48%-80% i n Pleetvitis  (Ganders et al., 1977a) and 43%-97% i n Limnanthes Outcrossing  system o f t h i s  alba  f r e q u e n c i e s were n o t c a l c u l a t e d f o r t h e  oongesta  ( J a i n , 1978). Leavenwovthia  ( S o l b r i g , 1972; S o l b r i g and R o l l i n s , 1977) and t h e b r e e d i n g  systems i n Limnanthes degree o f p r o t a n d r y  ( A r r o y o , 1975) were " e s t i m a t e d " by t h e i r and t h e a b i l i t y t o s e t seed i n a p o l l i n a t o r - f r e e  environment.  In a d d i t i o n , no  i n h e r i t a n c e s t u d i e s o f enzyme v a r i a t i o n  were conducted i n e i t h e r Leavenworthia  or Limnanthes.  v a r i a t i o n i n enzyme s t a i n i n g a c t i v i t y c o u l d not be r e s p e c t t o t h e number o f l o c i populations. Therefore, c o r r e l a t e breeding  Consequently,  i n t e r p r e t e d with  s c o r e d and a l l e l e f r e q u e n c i e s  within  most o f the s t u d i e s which have attempted to  system with p o p u l a t i o n s t r u c t u r e i n r e l a t e d t a x a  have f a i l e d t o e i t h e r : ( i ) q u a n t i f y the mating s y s t e m ( s ) , the e f f e c t s of the b r e e d i n g  system from o t h e r  p o p u l a t i o n s t r u c t u r e (e.g.  genetic"background and  ( i i ) isolate  f a c t o r s which i n f l u e n c e life-history  c h a r a c t e r i s t i c s ) and/or ( i i i ) d e s c r i b e the o r g a n i z a t i o n o f v a r i a b i l i t y i n g e n e t i c a l l y s i g n i f i c a n t terms (e.g. In an e f f o r t to c l a r i f y present  study analyzes  on the  l e v e l s and  brachystemon  Populations,  sampling methods and  which were adapted f o r P l e c t r - i t i s  the  are  described  In Chapter 3, the i n t e r p r e t a t i o n o f enzyme m o b i l i t y  i s discussed.  G e n e t i c models which e x p l a i n the  v a r i a t i o n are proposed and statistic.  i s s u e s , the  o r g a n i z a t i o n o f isozyme v a r i a t i o n i n P l e c t r i t i s  e l e c t r o p h o r e t i c techniques  patterns  some o f these  loci).  the consequences o f c o n t r a s t i n g b r e e d i n g s t r a t e g i e s  and P. congesta.  i n Chapter 2.  a l l e l e s at  Allelic  these  are t e s t e d u s i n g the  v a r i a t i o n at f i f t e e n  loci  loci  chi-square  is identified.  g e o g r a p h i c d i s t r i b u t i o n o f a l l e l e s among p o p u l a t i o n s contributions of various  and  to the g e n e t i c d i v e r s i t y  o f each s p e c i e s are d e s c r i b e d i n Chapter 4.  observed  the  The relative  (Nei,  1975)  In Chapter 5, the  analyses  o f the isozyme d a t a are d i s c u s s e d .  Both the o u t c r o s s i n g  c a l c u l a t e d f o r each p o p u l a t i o n ,  the mean o u t c r o s s i n g r a t e s f o r  each taxon are compared. The  and  frequencies,  l e v e l o f v a r i a b i l i t y maintained  within  each p o p u l a t i o n and the h i e r a r c h i c a l organization.o£ v a r i a t i o n i n P.  braohystemon..:and P.  oongesta  are q u a n t i f i e d .  Differences  the two s p e c i e s are i n t e r p r e t e d w i t h r e f e r e n c e to t h e i r strategies.  In a d d i t i o n t o the e f f e c t s o f b r e e d i n g  between  breeding  system 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 , a number o f a d d i t i o n a l problems are addressed. Comparisons o f g e n e t i c d i s t a n c e are used t o make i n f e r e n c e s r e g a r d i n g the l e v e l s o f gene flow between p o p u l a t i o n s r e l a t i o n s h i p o f P.  oongesta  and P.  and t h e taxonomic  braohystemon.  L a s t l y , the f a c t o r s  which i n f l u e n c e the maintenance o f the observed isozyme polymorphisms are  discussed.  16.  2.0  2.1  Collection  sites  MATERIALS AND METHODS  and sampling  strategy  Isozyme v a r i a t i o n was a n a l y z e d i n t e n c o l l e c t i o n s o f Plectritis  bvachystemon  and f i f t e e n c o l l e c t i o n o f P.  congesta.  W i t h i n each l o c a l p o p u l a t i o n , seeds were c o l l e c t e d from in  a randomized f a s h i o n .  individuals  Seeds from each p l a n t were kept s e p a r a t e and  enzyme v a r i a t i o n was s c o r e d i n t h e progeny grown from these seeds. An e f f o r t was made t o c o l l e c t seeds from an unbiased  sample o f  i n d i v i d u a l s that represented the genetic v a r i a b i l i t y in  present  each l o c a l p o p u l a t i o n . In t h e p r e s e n t study, a l o c a l p o p u l a t i o n i s  d e f i n e d as a group o f i n d i v i d u a l s o f the same taxon growing w i t h i n a s p e c i f i e d area:: a topodeme i n t h e t e r m i n o l o g y (1939).  When P. bvachystemon  and P. congesta  o f Gilmour and Gregor grow i n t e r m i x e d o r i n  c l o s e p r o x i m i t y , t h i s i s d e f i n e d as a sympatric p o p u l a t i o n .  The term  p o p u l a t i o n does n o t n e c e s s a r i l y imply t h a t these aggregations o f i n d i v i d u a l s a r e p a n m i c t i c u n i t s (i.e. gamodemes). Table  1 lists  c o l l e c t i o n s i t e s , s p e c i e s p r e s e n t , number o f  i n d i v i d u a l s sampled and date o f c o l l e c t i o n . The l o c a t i o n :of p o p u l a t i o n s i s presented  i n Appendix A. S i t e s a r e l i s t e d i n t h e o r d e r i n which they  appear i n a l l t a b l e s . In o r d e r t o i l l u s t r a t e p o s s i b l e macro-geographic p a t t e r n s w i t h i n each taxon, mainland p o p u l a t i o n s a r e l i s t e d  first,  then Vancouver I s l a n d p o p u l a t i o n s b e g i n n i n g w i t h t h e V i c t o r i a and moving n o r t h . F i g u r e 2 shows t h e l o c a t i o n o f a l l sampled  area sites.  17.  Table  1.  Sample designation  Collection sites •  Locality  Species  1  Sumas Mountain  P.b.  2(1)  Anacortes, Washington  2(2)  Number o f p l a n t s collected  Date collected  300  16 June 1977  P.O.  36  17 June 1976  Anacortes, Washington  P.O.  81  17 June 1976  13  William Head  P.b.  100  10 June 1977  3  John Dean Park  P.O.  99  20 June 1976  5  M i l l H i l l Park  P.c.  40  21 June 1976  4  T h e t i s Lake Park  P.O.  47  21 June 1976  9  F r a n c i s Park  P.b.  46  10 June 1977  9  Francis Park  P.O.  40  10 June 1977  10  Prospect  Lake Road  P.b.  32  10 June 1077  10  Prospect  Lake Road  P.O.  68  10 June 1977  11  Viaduct Avenue  P.a.  100  10 June 1977  7(D  Malahat Drive  P.b.  55  21 June 1976  7(2)  Malahat Drive  P.O.  50  21 June 1976  7(3)  Malahat Drive  P.b.  100  11 June 1977  8  Crofton E x i t  P.O.  60  22 June 1976  14(1)  Jack's Point  P.O.  90  12 June 1977  14(2)  Jack's Point  P.O.  90  12 June 1977  15(1)  Nanoose H i l l  P.b.  29  22 June 1976  15(1)  Nanoose H i l l  P.O.  22  22 June 1976  15(2)  Nanoose H i l l  P.b.  56  12 June 1977  15(2)  Nanoose H i l l  P.a.  37  12 June 1977  16  L i t t l e Qualicum F a l l s  P.b.  100  12 June 1977  17  N i l e Creek  P.O.  61  24 June 1976  18  Elk F a l l s Park  P.b..  50  12 June 1977  18.  F i g u r e 2. The l o c a t i o n o f sampled Tlectritis populations: • designate P . brachystemon p o p u l a t i o n s , • d e s i g n a t e P . congesta p o p u l a t i o n s and • i d e n t i f y s i t e s where b o t h s p e c i e s were c o l l e c t e d .  19.  2.2  Potential  sources  2.2.1 Sample  of bias  in the  collections  size  Given random mating, a sample s i z e o f t h i r t y  individuals  per p o p u l a t i o n i s s u f f i c i e n t t o be 95% c o n f i d e n t o f d e t e c t i n g a l l e l e s w i t h i n p o p u l a t i o n s which a r e p r e s e n t difficult  at f r e q u e n c i e s >0.05.  It i s  t o determine what c o n s t i t u t e s an adequate, and comparable,  sample i n P l e c t r i t i s because these two t a x a have d r a m a t i c a l l y d i f f e r e n t o u t c r o s s i n g r a t e s and n e i t h e r s p e c i e s experiences  complete random  mating. However, t h i s sample s i z e compares f a v o r a b l y w i t h most s t u d i e s o f isozyme v a r i a t i o n i n p l a n t p o p u l a t i o n s , r e g a r d l e s s o f the b r e e d i n g  system. T h e r e f o r e , an attempt was made t o sample a t  l e a s t 30 i n d i v i d u a l s p e r p o p u l a t i o n .  Although  most c o l l e c t i o n s were  s u f f i c i e n t l y l a r g e t o achieve t h i s l e v e l o f sampling,  poor  germination,  combined w i t h t e c h n i c a l problems, produced v a r i a t i o n i n sample s i z e s among p o p u l a t i o n s and between l o c i w i t h i n p o p u l a t i o n s .  2.2.2  Phenology P o p u l a t i o n s were sampled o n l y once i n a g i v e n  consequently,  o n l y those p l a n t s w i t h mature seed  year,  a t t h e time o f  c o l l e c t i o n were i n c l u d e d i n the sample.  2.2.3 Seed wing As  seeds mature i t i s p o s s i b l e t h a t t h e r e are d i f f e r e n c e s  i n t h e r e t e n t i o n o f winged versus w i n g l e s s  seed w i t h i n i n f l o r e s c e n c e s .  In p o p u l a t i o n s polymorphic f o r t h e seed wing, t h i s would s y s t e m a t i c a l l y b i a s f r e q u e n c i e s o f t h e winged and wingless  a l l e l e s i n the sample.  20.  Whether t h i s would b i a s a l l e l e  f r e q u e n c i e s at the enzyme  surveyed  l i n k a g e a n a l y s i s was  i s not known s i n c e no  2.2.4  Wahlund Effect.-, as the result  an apparent  of sampling  conducted.  deficiency  stratified  loci  in  heterozygotes  populations  At many s i t e s , , p l a n t e s were d i s t r i b u t e d over a wide area t h a t encompassed a number o f d i s c r e t e m i c r o - h a b i t a t s . . A s  a result  o f automgamy, v i c i n i s m and p o s s i b l e m i c r o - s i t e s e l e c t i o n , i t i s p o s s i b l e t h a t topodemes may  represent  aggregates o f s u b - p o p u l a t i o n s  neighborhoods), each with unique a l l e l e f r e q u e n c i e s . workers have demonstrated t h a t p o p u l a t i o n s distances  (Antonovics,  and A l l a r d ,  1972;  1968;  Antonovics  (i.e.  Several  d i f f e r e n t i a t e over s m a l l  and  Bradshaw, 1970;  Hamrick  Hamrick and Holden, 1979). Bradshaw (1972) s t a t e s  t h a t e f f e c t i v e p o p u l a t i o n s i z e s h o u l d be measured i n meters not kilometers.  I t was  assumed, f o r the purposes o f the p r e s e n t  that a l l populations  o f each taxon were homogeneous and were comparable  g e n e t i c u n i t s . However, lumping samples from s u b d i v i d e d biases, c a l c u l a t e d a l l e l e frequencies o f expected h e t e r o z y g o s i t y  2.3  Methods  study,  of planting  and produces  populations  over-estimates  (assuming Hardy-Weinberg e q u i l i b r i u m ) .  and growth  Enzyme v a r i a t i o n was  regime  surveyed  i n the progeny grown from  the c o l l e c t e d seeds. A l l p l a n t s were grown under u n i f o r m  conditions i n  o r d e r to e l i m i n a t e p o s s i b l e genotype - environment i n t e r a c t i o n s which might modify isozyme phenotypes.  P l a n t s were grown i n a s t a n d a r d p o t t i n g s o i l m i x t u r e i n nursery f l a t s fertilizing  (254 mm.  x 508 mm.).  A l l f l a t s were g i v e n an  initial  (10 g. H i - S o l 20-20-20/1800 ml. w a t e r ) , a l l o w e d  to  d r a i n f o r 24 h r s . and then p l a n t e d . Seeds were p l a n t e d 5 mm. deep, 200 seeds per f l a t . (i.e.  t o 10  mm.  Wooden semi-micro g r i d l o c u s markers  t o o t h p i c k s ) were p l a c e d next t o each seed t o i d e n t i f y  individuals.  P l a n t e d f l a t s were p l a c e d i n a growth chamber s e t on a " l o n g d a y - c o o l " regime: 16 h r s . o f l i g h t a t 12°C d a r k n e s s a t 7°C w i t h 50% r e l a t i v e h u m i d i t y . was  and 8 h r s . o f  T h i s growth regime  chosen as i t promoted g e r m i n a t i o n and p r o l o n g e d  r o s e t t e s t a g e o f development. to two weeks and developed  the b a s a l  Seeds germinated w i t h i n t e n days  f o u r t r u e l e a v e s t h r e e t o f o u r weeks a f t e r  g e r m i n a t i o n . S i n g l e b a s a l r o s e t t e l e a v e s s e r v e d as the enzyme source m a t e r i a l .  2.4  Electrophoretic  techniques  G e l s were r u n h o r i z o n t a l l y and were p r e p a r e d (Otto H i l l e r l o t #307) a t a 12.5% was  w/v  using Electrostarch  c o n c e n t r a t i o n . Sucrose (10%  w/v)  added t o the g e l s t o improve the r e s o l u t i o n o f c e r t a i n enzymes  and t o toughen g e l s f o r ease o f h a n d l i n g . G e l s were p r e p a r e d a f t e r n o o n p r e c e d i n g a r u n . S t a r c h was p l e x i g l a s s g e l molds (150 mm. to c o o l t o room temperature, overnight at  6°C.  x 200 mm.  the  cooked, degassed and poured i n t o x  6 mm.).  wrapped i n p l a s t i c and  These were a l l o w e d stored  Samples were p r e p a r e d  j u s t p r i o r t o use as enzyme a c t i v i t y  d i m i n i s h e d upon f r e e z i n g and thawing.  Leaves were removed f r o m . ,  p l a n t s and p l a c e d on spot p l a t e s f o r manual g r i n d i n g .  These p l a t e s  were p l a c e d on i c e t o i n h i b i t enzyme a c t i v i t y w h i l e samples were prepared  f o r assay. One o r two b a s a l l e a v e s were ground w i t h two  drops o f g r i n d i n g b u f f e r (0.1 M T r i s - H C l , pH 7.5 w i t h 0.5% 2m e r c a p t o - e t h a n o l ) and an equal amount o f i n s o l u b l e PVP ( i . e . p o l y v i n y l polypyrrolidone).  The crude homogenate was then absorbed onto  f i l t e r paper w i c k s  (4 mm.  x 10 mm.,  were i n s e r t e d i n t o s l o t s c u t 20 mm.  Whatman 3 MM paper) and these from one end o f the s t a r c h g e l .  Runs were s t a n d a r d i z e d by m o n i t o r i n g t h e m i g r a t i o n o f a t r a c k i n g dye  ( d i l u t e r e d food c o l o r i n g ) which was absorbed onto  wicks and p l a c e d i n t h e o u t e r sample s l o t s o f each g e l . s t a n d a r d s ] (i. e.  Genotype  i d e n t i f i e d homozygotes) were run,, when needed, t o  check t h e m o b i l i t y o f isozyme v a r i a n t s . Once t h e samples were l o a d e d , a l l b u t 10 mm a t e i t h e r end o f t h e g e l s were covered w i t h p l a s t i c t o p r e v e n t d e s i c c a t i o n d u r i n g t h e r u n . Gels were then p l a c e d on the a p p r o p r i a t e e l e c t r o d e t r a y s w i t h the o r i g i n ( i . e . samples) a t the cathode. E l e c t r o d e wicks  (Handi-wipes) were p l a c e d on the unwrapped  ends o f the g e l and c u r r e n t a p p l i e d u n t i l the t r a c k i n g dye had migrated  the a p p r o p r i a t e d i s t a n c e . E l e c t r o p h o r e s i s was conducted  i n s i d e a l a r g e g l a s s f r o n t r e f r i g e r a t o r t h a t was m a i n t a i n e d a t 0°-4°C.  This prevented  o v e r h e a t i n g which d i s t o r t s t h e m i g r a t i n g  f r o n t and u l t i m a t e l y denatures  the p r o t e i n s .  23.  Two e l e c t r o p h o r e t i c b u f f e r systems were used: I  Discontinuous  L i - b o r a t e / T r i s - c i t r a t e b u f f e r , pH 8.0  (adapted from S c a n d a l i o s , II  1969);  Continuous T r i s - c i t r a t e  b u f f e r , pH 7.0 ( S i c i l i a n o and  Shaw, 1976). The  composition  o f these b u f f e r s i s g i v e n  i n Table  2.  E l e c t r o p h o r e s i s was  conducted a t t h e f o l l o w i n g v o l t a g e s : I 350 V. (~60mA) u n t i l t r a c k i n g dye migrated 50 mm., approximately 4 h r s . ; II 250 V. (~60mA) u n t i l t r a c k i n g dye migrated 110 mm., a p p r o x i m a t e l y 4.5-5.5 h r s . Upon completion o f t h e r u n , g e l s were removed, sample wicks p u l l e d and t h e g e l s trimmed and s l i c e d strips fishing  f o r staining. Plexiglass  1.5 mm. t h i c k were used as s l i c i n g guides. line  (2 l b . t e s t ) was used t o s l i c e t h e g e l .  weight on t o p t o p r e v e n t b u c k l i n g , s l i c i n g s t r i p s e i t h e r s i d e o f the g e l . The f i s h i n g strips  and drawn through the g e l .  another p a i r o f s t r i p s  placed  Once a s l i c e had been made,  I t was p o s s i b l e t o o b t a i n s i x s l i c e s  i n the a p p r o p r i a t e  The o t h e r s ,  from  as needed, were  enzyme s t a i n i n g s o l u t i o n s and i n c u b a t e d a t  37°C u n t i l the banding p a t t e r n s  running  were p l a c e d on  l i n e was then h e l d a g a i n s t the  The t o p s l i c e was d i s c a r d e d .  enzymes s c o r e d ,  With a  were p l a c e d on e i t h e r s i d e o f t h e g e l and  the procedure r e p e a t e d . a gel.  Monofilament  c o u l d be s c o r e d .  Table  3 l i s t s the  t h e number o f l o c i r e s o l v e d , the a p p r o p r i a t e  b u f f e r and the s t a i n i n g r e f e r e n c e . The number f o l l o w i n g t h e  24.  Table  I.  2 .  Running  Buffers  DISCONTINUOUS Li-BORATE/TRIS-CITRATE BUFFER A:  .03 M 1.20 g 11.89 g H 0 to 2  BUFFER B:  BUFFER  pH 8.0  LiOH-.19M B o r i c A c i d L i t h i u m h y d r o x i d e (monohydrate) Boric Acid 1 l i t e r , a d j u s t pH t o 8.0  .05 M T r i s - .008 M C i t r i c A c i d 6.2 g T r i s 1.6 g C i t r i c a c i d (monohydrate) H 0 t o 1 l i t e r , a d j u s t pH t o 8.0 2  ELECTRODE: BUFFER A GEL  : 1:9 M i x t u r e o f B u f f e r s A § B REFERENCE: adapted from S c a n d a l i o s , J.G. (1969) and S e l a n d e r , e t a l . (1971).  II.  CONTINUOUS TRIS-CITRATE ELECTRODE:  BUFFER  pH 7.0  .13 M T r i s - .043 M C i t r i c A c i d 16.35 g T r i s 9.04 g C i t r i c a c i d (monohydrate) H 0 t o 1 l i t e r , a d j u s t pH t o 7.0 2  GEL  : .013 M T r i s - .0043 M C i t r i c A c i d 1:10 d i l u t i o n o f E l e c t r o d e B u f f e r ( m o d i f i e d reference)  from  REFERENCE: adapted from S i c i l i a n o , M.J. § C.R. Shaw, (1976)  Table 3 .  Running Buffer Used, Number o f Loci Resolved and S t a i n i n g References f o r Enzyme Systems Studied i n P. bvaahystemon and P. oongesta. # LOCI SCORED  ENZYME  RUNNING BUFFER  STAIN REFERENCE Gottlieb  ESTERASE EST E.C. 3.1.1.1  (1973a)  Allendorf,  II  ISOCITRATE DEHYROGENASE IDH E.C. 1.1. 1.42  e t a l . (1977)  Roose 6 G o t t l i e b  LEUCINE AMINO PEPTIDASE LAP E.C. 3.4.11.1  Roose 5 G o t t l i e b  MALIC ENZYME ME E.C. 1.1.1.40 PHOSPHOGLUCOSE ISOMERASE PGI E.C. 5.3.1.9  3  I or II  II  PHOSPHOGLUCOMUTASE PGM E.C. 2.7.5.1 6-PHOSPHOGLUCONIC DEHYDROGENASE 6PG E.C. 1.1.1.44  Roose 5 G o t t l i e b  Allendorf,  o f the o r i g i n a l reference  Due to problems with i n t e r p r e t a t i o n , t h i s enzyme was not scored f o r P.  (1976)  e t a l . (1977)  Discontinuous L i - B o r a t e / T r i s - C i t r a t e Buffer ph 8.0 Continuous T r i s - C i t r a t e Buffer pll 7.0  Most of the s t a i n i n g techniques used are m o d i f i c a t i o n  (1976)  G o t t l i e b (1973ad  SUPEROXIDE DISMUTASE SOD E.C. 1.15.1.1  I II  (1976)  S i c i l i a n o § Shaw (1976)  II  MALATE DEHYDROGENASE MDH E.C. 1.1.1.37  Running Buffers:  2  congesta.  26.  enzyme a b b r e v i a t i o n i s i t s Enzyme Commission code number and S t o t z , 1973).  (Florkin  T a b l e 4 g i v e s the s t a i n i n g p r o c e d u r e s and T a b l e  5 p r o v i d e s a l i s t o f s t o c k s o l u t i o n s . I n a d d i t i o n , Appendix B l i s t s enzyme assays and r u n n i n g b u f f e r s which were attempted but p r o v e d t o be u n s u c c e s s f u l w i t h  Plectritis.  G e l s were s c o r e d i m m e d i a t e l y a f t e r s t a i n i n g when the r e s o l u t i o n was b e s t . i n e i t h e r a 1:4:5 et al.,  When s t a i n i n g was  complete, g e l s were f i x e d  a c e t i c acid-methanol-water s o l u t i o n  1977) or a 1:1 g l y c e r i n e - w a t e r s o l u t i o n  and Shaw, 1976).  (Allendorf  (Siciliano  The f i r s t f i x a t i o n m i x t u r e toughened and p r e s e r v e d  g e l s as semi-permanent r e c o r d s b e t t e r than the l a t t e r . However, the g l y c e r i n e - w a t e r f i x b e t t e r p r e s e r v e d the r e s o l u t i o n o f c e r t a i n enzymes (i.e. MDH,  PGI and PGM)  f o r g e l s t h a t were t o be photographed  and proved later.  superior  TABLE 4 .  ENZYME  Summary o f Enzyme S t a i n i n g Procedures.  STAIN BUFFER  1  NAD  NADP/MgCl  NBT/PMS  2  3  2  MTT/PMS  OTHER COMPONENTS  3  1. EST  2  60 mg a-napthyl acetate 30 mg g-napthyl acetate 100 mg Fast Blue RR  2. IDH  1  3. LAP  3  4. MDH  1  5. ME  4  /  6. PGI  1  /  /  2 ml .018 M Fructose-6phosphate 20 u n i t s G6PDH  7. PGM  1  /  /  75 Na a-D-Glucose-1phosphate 3 ml .00017 M K a-D-Glucose1-phosphate 20 u n i t s G6PDH  /  /  200 mg D L - i s o c i t r i c acid 30 mg l-leucyl-g-napthylamide ( d i s s o l v e d i n 5 ml N, N-dimethyl formamide) 25 mg. Black-K-salt added a f t e r 30 min, i n c u b a t i o n .  25 mg  / /  5 ml IM Na-L-Malate 140 mg L-Malic  m  g  Acid  2  2  8- 6PG  1  /  /  9- SOD  ; !  1  20 mg 6-Phosphogluconic Acid (Naj S a l t ) Develops i n l i g h t on gels s t a i n e d f o r ME, PGI, PGM.  S t a i n Buffers: 1. .2M T r i s - H C l pH 8.0 i 2. .IM K-P0 pH 6.4 3. . IM Tris-Maleate pH 6.5 4. .IM T r i s - H C l pH 8.6 " '  10 mg NADP + 1 ml 0.5 M MgCl  2  4  3  10 mg NBT or, MTT + 5 mg PMS  Table  Stock S o l u t i o n s  5 .  CONG.  SOLUTION  ADDITIONAL  STORAGE  STAINING BUFFERS 1. 2. 3. 4.  T r i s - H C l pH 8.0 K-P0 pH 6.4 Tris-Maleate pH 6.5 T r i s - H C l pH 8.6 4  ,2M , IM ,1M ,1M  R R R R  DYES, COFACTORS, ENZYMES § SUBSTRATES 12.15g N a C 0 H 0 i n 50 ml. H 2 P Keep C o o l , add 13.4g L - M a l i c a c i d H 0 t o 100 ml.  IM  Na-L-Malate  2  3  2  2  MgCl  .5M  2  G6PDH (Glucose-6-phosphate NAD NADP NBT MTT PMS R: r e f r i d g e r a t e RT: room temperature *: p r o t e c t from l i g h t  RT  10'units/ml,  R  D i s s o l v e i n .005M c i t r a t e pH 7.5  10 mg/ml  R  D i s s o l v e i n .005M c i t r a t e pH 7.5  10 mg/ml  R  D i s s o l v e i n .005M c i t r a t e pH 7.5  10 mg/ml  R*  10 mg/ml  R*  5 mg/ml  R*  dehydrogenase)  29.  3.0 INHERITANCE  3.1  Isozyme  nomenatatuve  Fifteen loci, i n Pteotritis.  coding f o r n i n e enzymes, were i d e n t i f i e d  These enzymes a r e l i s t e d i n T a b l e 3. I n t e r p r e t a t i o n s  of enzyme m o b i l i t y p a t t e r n s on g e l s a r e p r e s e n t e d and g e n e t i c models t o e x p l a i n t h e observed v a r i a t i o n a r e proposed. Two terms, isozyme  (isoenzyme) and allozyme need t o  be d e f i n e d . Isozymes a r e m u l t i p l e m o l e c u l a r forms o f enzymes. Allozyme  d e s i g n a t e s isozymes which a r e coded f o r by a l l e l e s a t  a s i n g l e l o c u s . The isozyme nomenclature adopted was  for P l e a t r i t i s  m o d i f i e d from A l l e n d o r f and U t t e r (1979). . An enzyme l o c u s i s  i d e n t i f i e d by t h e enzyme's a b b r e v i a t i o n , p l u s a hyphenated numeral i f t h e system i s c o n t r o l l e d by m u l t i p l e l o c i . migrated  A l l isozymes  surveyed  toward the anode. The locus coding f o r t h e most a n o d a l l y  m i g r a t i n g p r o t e i n s was d e s i g n a t e d one, t h e next as two and so on. Allozymes,  and t h e i r c o r r e s p o n d i n g a l l e l e s ,  a r e i d e n t i f i e d by a  s u p e r s c r i p t a c c o r d i n g t o t h e i r r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t y . One allele,  g e n e r a l l y the most common, was a r b i t r a r i l y d e s i g n a t e d 100.  Other allozymes  were a s s i g n e d n u m e r i c a l v a l u e s a c c o r d i n g t o t h e i r  m i g r a t i o n r e l a t i v e t o t h e 100 form.  F o r example, t h e a l l e l e a t the  most anodal e s t e r a s e (EST) l o c u s , which codes f o r t h e allozyme  migrating  114 14% f u r t h e r than t h e common form was d e s i g n a t e d EST-1 coding f o r allozymes  w i t h no s t a i n i n g a c t i v i t y  .  (i.e. n u l l s ) were  d e s i g n a t e d as lOOn (e.g. PGI-3'''^^ ) ,i w h i l e allozymes w i t h n  staining a c i t i v i t y designation.  Alleles  reduced  a r e i d e n t i f i e d by an r f o l l o w i n g t h e m o b i l i t y  30.  As population present  a p r e l u d e to the i n h e r i t a n c e s t u d i e s , an  survey was  separately,  selfed.  i n P.  braohystemon  and  P.  congesta  to i n s u r e t h a t the mode o f i n h e r i t a n c e was  i n each s p e c i e s .  r a t i o using chi-square  bvachystemon  detected.  identical  H e t e r o z y g o t e s were t e n t a t i v e l y i d e n t i f i e d  S e g r e g a t i o n r a t i o s were then compared t o the  were s u f f i c i e n t l y i n P.  of v a r i a t i o n  i n both s p e c i e s . Whenever v a r i a t i o n e x i s t e d , enzyme  systems were a n a l y z e d  1:2:1  conducted to sample the extent  initial  expected  a n a l y s i s whenever progeny c l a s s e s  l a r g e t o v a l i d l y apply  this s t a t i s t i c .  were extremely r a r e and  Therefore,  and  Heterozygotes  a t some l o c i were never  to v e r i f y a l l e l i c v a r i a n t s , c r o s s e s  between  i n d i v i d u a l s presumed homozygous f o r a l t e r n a t i v e a l l e l e s were attempted. Because o f the bvachystemon,  floral  s t r u c t u r e and  f l o w e r s had  t o be t e a s e d  p r i o r t o c r o s s i n g . Plectvitis  open w i t h f o r c e p s  produces achenes and  w i t h i n an i n f l o r e s c e n c e mature s e p a r a t e l y weeks. In consequence, i t i s not  i n P.  timing of anthesis  the  and  flowers  over a p e r i o d o f a  o f t e n a problem. As  segregation  sample s i z e s are r e s p o n s i b l e  r a t i o s which d e v i a t e  homozygotes were a l s o s e l f e d and genetic  the p a r e n t a l  for  It i s the  from the expected. Presumed the progeny a n a l y z e d  i n t e r p r e t a t i o n s . Without e x c e p t i o n ,  progeny e x p r e s s i n g  and  a r e s u l t , progeny  c l a s s e s i n the i n h e r i t a n c e s t u d i e s were g e n e r a l l y s m a l l . assumed t h a t the s m a l l  few  an i d e a l organism f o r i n h e r i t a n c e  s t u d i e s . S u c c e s s f u l p o l l i n a t i o n s produced a s i n g l e seed p o l l e n c o n t a m i n a t i o n was  emasculated  genotype.  to  confirm  a l l such s e l f s produced  Each enzyme system surveyed to  i s described with respect  i t s m u l t i l o c u s o r g a n i z a t i o n , sub-unit  s t r u c t u r e and a l l e l i c v a r i a t i o n .  (i.e.  Polymorphic  quaternary) enzymes a r e d i s c u s s e d  f i r s t f o l l o w e d by t h e monomorphic systems.  3.2 Esterase  (EST) E.C. 3.1.1.1 Two major zones o f a c t i v i t y appeared on g e l s s t a i n e d  for  EST.  V a r i o u s numbers o f l i g h t l y s t a i n i n g bands were a l s o  p r e s e n t but t h e i r i n h e r i t a n c e c o u l d not be determined. zones o f s t a i n i n g were i n t e r p r e t e d t o be s e p a r a t e d e s i g n a t e d EST-1 and EST-2.  t h e t h r e e EST-1 a l l e l e s were  However, i n c o n t r a s t t o t h e h i g h  o f h e t e r o z y g o s i t y i n P. brachystemon, at  t h i s l o c u s i n P. congesta  v a r i a t i o n was r a r e l y  (Table 17).  i d e n t i f i e d a t EST-2 i n P. congesta P. brachystemon  These were  Three a l l e l e s were d e t e c t e d a t each  l o c u s ( F i g u r e 3 and F i g u r e 4 ) . detected i n both s p e c i e s .  loci.  The major  level detected  Three a l l e l e s were  while a l l populations of  were monomorphic f o r E S T - 2 ^ ^ (Table 17) .  Heterozygotes  a t EST-1 and EST-2 were i d e n t i f i e d by  t h e i r double banded phenotypes. T h i s was i n t e r p r e t e d as t h a t EST i n Plectritis  i s a monomeric p r o t e i n .  evidence  T h i s o b s e r v a t i o n agrees  w i t h t h e s u b - u n i t s t r u c t u r e o f EST r e p o r t e d i n o t h e r t a x a : Cucurbita-  spp. ( W a l l and W h i t a k e r ,  1971), oats  b a r l e y ( K a h l e r and A l l a r d , 1970), Norway spruce Scots p i n e Gaura 1979).  (Clegg and A l l a r d , ( B a r t e l s , 1971),  (Rudin and Rasmuson, 1973), Stephanomeria  ( G o t t l i e b and P i l z ,  1976) and Drosophila  1973),  ( G o t t l i e b , 1973a),  subobscura  (Loukas et al.,  F i g u r e 5 i s a photograph o f two g e l s s t a i n e d f o r EST.  32.  EST-1 EST-2  Figure 3. The r e l a t i v e migration of allozymes detected at EST-1.  Figure 4. The r e l a t i v e migration of allozymes detected at EST-2.  EST-1 EST-2 EST-1 EST-2 1  15  28  Figure 5. Allozyme v a r i a t i o n at EST-1; gel 1 - individuals 1-14 are the progeny from a selfed 1,100 homozygote, i n d i v i d u a l s 15-29 are the F\ progeny from the cross of a l l ! 4 homozygote X l89 homozygote (single banded l H 4 individuals are the r e s u l t o f accidental s e l f s ) ; gel 2- individuals 1-15 demonstrate 1:2:1 segregation i n the progeny of a selfed 100/114 heterozygote, individuals 16-29 are the progeny of a selfed llOO homozygote. EST-2 d i d not photograph well but i t i s uniformly monomorphic i n both gels.  33.  It demonstrates the t h r e e a l l e l e s d e t e c t e d at EST-1 the method used to i n t e r p r e t  isozyme p a t t e r n s .  EST-2 d i d not photograph w e l l i n these  but  and  illustrates  Unfortunately,  i t i s u n i f o r m l y monomorphic  gels. EST-1  was  not s c o r e d i n P.  ( L i t t l e Qualicum F a l l s ) .  braohystemon  population  S t a i n i n g a c t i v i t y at t h i s  locus  was  i n h i b i t e d as a r e s u l t o f s p r a y i n g the p l a n t s with M a l a t h i o n combat an.aphid prevent  infestation.  the.expression  suggests  of allozymes  d i f f e r e n t f u n c t i o n a l classes of  EST-1  to  However, the i n s e c t i c i d e d i d not at EST-2.  t h a t the isozymes coded by these two  Segregation  16  analyses  This loci  observation represent  EST. are p r e s e n t e d  i n Table 6 f o r  and T a b l e 7 f o r EST-2.  3-3 ' Leucine' One  aminopeptidase l o c u s was  (LAP)  E.C.  3.4.11.1  r e s o l v e d on g e l s s t a i n e d f o r LAP.  In  a d d i t i o n , o t h e r l i g h t l y s t a i n i n g bands were sometimes observed. was  a h i g h l y polymorphic enzyme i n both  Seven a l l e l e s were s c o r e d i n P.  species  oongesta  and  (Table  This  17).  a l l but one  of  these  80 (LAP-1  ) were d e t e c t e d Heterozygotes  i n P.  braohystemon  (Figure 6).  were i d e n t i f i e d by t h e i r two-banded p a t t e r n .  T h i s agrees w i t h the monomeric s t r u c t u r e o f LAP corn  (Beckman et al.,  knobcone p i n e  (Conkle,  1964)., Cuourbita  spp.  1971), Norway spruce  Scots, p i n e  (Rudin,  1977), avocado  subobsoura  (Loukas et al.,  1979).  i n other  (Wall and  Whitaker, 1971),  (Lundkvist,  (Torres et al.,  organisms:  1974),  1978a.) and  Drosophila  34.  Table 6.  Analysis  o f allozyme v a r i a t i o n at EST-1  P. brachystemon:  S e l f e d heterozygotes 2  Individual  Genotype  Observed allozyme segregation i n progeny A /A 1  A /A  1  1  A /A  2  2  77-1-14C  89/114  5  7  3  77-1-26C  100/114  0  2  3  77-1-73C  100/114  3  11  1  P. brachystemon: Individual  2  Crosses  Genotype  A  1'1 /  A  A  1^ 2 A  A  2'2 /  A  77-l-13a x 77-1-1  89/89 x 100/100  0  11  0  77-18-186 x 7 7 - 1 - l l l a  89/89 x 114/114  0  8  0  77-18-19C x 77-1-llOc  89/89 x 114/114  0  10  0  P. oohgesta: Individual  s e l f e d homozygotes  Genotype  Number o f  76-4-5  100/100  7  77-9-8b  100/100  12  77-9-41b  100/100  . 8  77-9-43b  100/100  8  77-9-53c  100/100  11  77-9-54a  100/100  9  77-9-60c  100/100  77-9-64b  100/100  8  77-9-65a  100/100  9  77-9-84a  100/100  13  * sample s i z e i n s u f f i c i e n t f o r a v a l i d X  !  1  4  test of significance  ** a l l progeny expressed the maternal genotype  x  (2)  35.  Table 7. P. Individual  Analysis  o f allozyme v a r i a t i o n at EST-2  congesta.:  S e l f e d heterozygotes  Observed allozyme segregation i n progeny  Genotype  V i A  A /A  V 2 A  2  2  2  77-9-8b  74/100  0  9  3  *  77-9-41b  74/100  2  3  3  *  77-9-64b  74/100  1  4  3  77-9-68b  74/100  0  4  2  77-9-78b  74/100  2  4  1  77-9-81a  74/100  1  3  4  77-9-83a  74/100  7  1  0  77-9-95a  74/100  2  3  3  15  31  19  E  P. congesta: Individual  S e l f e d homozygotes  Genotype  Number o:  77-9-45C  74/74  7  77-9-60C  74/74  14  77-9-45a  100/100  6  77-9-45b  100/100  8  77-9-53C  100/100  11  77-9-64a  100/100  13  77-9-65a  100/100  9  76-4-5  126/126  20  * sample s i z e i n s u f f i c i e n t  for a valid x  test of significance  ** a l l progeny expressed the maternal genotype  * * * * * * 0.631  36.  LAP-1  o  ,——  -jlOO  1  Figure 80/95, 80/80, 80/91,  -j112  _jl05  ;  ^95  13  ^1  .85  .80  26  7. A l l o z y m e v a r i a t i o n a t LAP-1 i n P. congesta: genotypes 95/95, b l a n k , 95/100, b l a n k , 95/100, 85/85, 100/100, 95/100 91/91, 95/95, 100/100, 95/95, 80/80, 95/95, 80/100, b l a n k , ' 91/95, 80/100, 100/100, 80/95, 85/95, 100/100, 100/100.  37.  Figure 7 i l l u s t r a t e s T h i s g e l was  of P.  t y p i c a l o f the genotype a r r a y s d e t e c t e d i n p o p u l a t i o n s  congesta. The  presented  3.4  a sample o f the v a r i a t i o n p r e s e n t i n P l e c t r i t i s .  r e s u l t s o f c r o s s e s and s e g r e g a t i o n a n a l y s e s  i n Table  Malate  are  8.  dehydrogenase  (MDH)  E.C.I.1.1.37  There were t h r e e zones o f a c t i v i t y on g e l s s t a i n e d f o r MDH.  These were a r b i t r a r i l y d e s i g n a t e d as zones A,  A b e i n g the most anodal  zone ( F i g u r e s 10 and  B and C w i t h  11). As was  i n maize (Longo and S c a n d a l i o s , 1969), and p i t c h p i n e L e d i g , 1978), these t h r e e s t a i n i n g  zones may  a s s o c i a t e d with d i f f e r e n t c e l l u l a r f r a c t i o n s  represent (i.e.  found  (Guries  and  isozymes  cytoplasm  and  mitochondria). The band which may Zone B was  zone c l o s e s t t o the o r i g i n , C, was o r may  a poorly resolved  not r e p r e s e n t a c t i v i t y a t a s i n g l e l o c u s .  composed o f f i v e to seven w e l l d e f i n e d bands. Most  commonly, i n d i v i d u a l s possessed  f i v e bands. The middle band s t a i n e d  d a r k e s t , the o u t e r two bands were much l i g h t e r and the o t h e r bands were i n t e r m e d i a t e i n both p o s i t i o n and observed  i n the number, the p o s i t i o n and  i n t e n s i t i e s o f bands. at  staining. Variation  least three l o c i  The banding  c o d i n g f o r MDH  the. r e l a t i v e  p a t t e r n s suggest  was  staining  that there  isozymes which migrate  to  are  this  zone. The r e l a t i v e s t a i n i n g o f t h i s s e r i e s o f bands i m p l i e s dosage e f f e c t s caused  by the f o r m a t i o n o f h y b r i d m u l t i m e r i c isozymes which  have o v e r l a p p i n g m o b i l i t i e s . MDH  has been found  to be d i m e r i c i n o t h e r  38.  Table 8.  A n a l y s i s o f allozyme v a r i a t i o n at LAP-1  P. braohystemon: Individuals  Crosses Genotypes  Observed allozyme segregation i n progeny A /A 1  77-l-13a x 77-18-25C 91/91x100/100  P. oongesta: Individual  A /A  1  1  0  A /A  2  2  12  2  0  S e l f e d heterozygotes 2 Observed allozyme segregation i n progeny X(- )  Genotype  2  A /A x  A /A  x  x  2  A /A 2  2  77-9-84a  80/91  1  7  4  *  77-9-64a  80/105  3  6  4  *  77-9-83a  85/100  5  2  0  *  77-9-65a  91/100  0  7  1  *  77-9-78b  91/100  4  0  3  *  P. congesta: Individual  S e l f e d homozygotes Genotype  Number o f progeny**  77-9-75b  80/80  10  76- 4-5  91/91  . 20  77- 9-45a  .91/91  6  77-9-60C  91/91  14  77-9-53C  91/91  11  77-9-86  95/95  12  77-9-95a  100/100  13  * sample s i z e i n s u f f i c i e n t  for a valid  x  test of significance  ** a l l progeny expressed the maternal genotype  39.  organisms: et al.,  horseshoe  crab  1975), p i t c h p i n e  (O'Malley et al., lodgepole p i n e  (Selander et al.,  1970), Eucalyptis  (Brown  (Guries and L e d i g , 1978), ponderosa p i n e  1979), Douglas f i r (El-Kassaby, unpublished) and  (Layton and Yeh,  unpublished).  U n f o r t u n a t e l y , more work i s needed t o determine t h e i n h e r i t a n c e o f v a r i a t i o n i n zone B, consequently,  these l o c i were  not i n c l u d e d i n t h e a n a l y s i s . The v a r i a t i o n observed be d e s c r i b e d by the e x i s t e n c e o f two l o c i  i n zone A can  d e s i g n a t e d MDH-1 and MDH-2.  V a r i a t i o n a t MDH-1 was common i n both s p e c i e s w h i l e a t MDH-2, no v a r i a t i o n was d e t e c t e d i n P. bvachystemon r a r e i n P. congesta.  The presence  and v a r i a t i o n was extremely  o f bands o f i n t e r m e d i a t e m o b i l i t y  between allozymes w i t h d i f f e r e n t .migrations i n d i c a t e t h a t t h e a c t i v e enzymes coded by MDH-1 and MDH-2 are dimers  (i.e.  composed o f two  s u b - u n i t s ) . Isozymes o f m u l t i m e r i c enzymes a r e formed by t h e random a s s o c i a t i o n o f p r o t e i n s u b - u n i t s . An i n d i v i d u a l , heterozygous  for  a l l e l e s A and B a t a locus which codes f o r a d i m e r i c enzyme, s y n t h e s i z e s t h r e e allozymes AA,.AB and BB i n a 1:2:1 r a t i o . heterodimers  Electrophoretically,  ( h y b r i d d i m e r i c enzymes) can be r e c o g n i z e d because they  have m o b i l i t i e s i n t e r m e d i a t e between the homodimeric allozymes and they s t a i n d a r k e r , t h e o r e t i c a l l y e x h i b i t i n g twice t h e a c t i v i t y o f t h e homodimers. The i n t e r p r e t a t i o n o f v a r i a t i o n a t MDH-1 and MDH-2 was c o m p l i c a t e d . The observed  s t a i n i n g p a t t e r n s were c o n s i s t a n t w i t h a  model which p r e d i c t s t h a t isozyme s u b - u n i t s s p e c i f i e d by both  loci  a s s o c i a t e w i t h each o t h e r forming both i n t r a - and i n t e r - l o c u s heterodimers.  The f o r m a t i o n o f i n t e r - l o c u s heterodimers  has been observed pine  i n ponderosa p i n e (O'Malley, et al.,  (Layton and Yeh,  unpublished)  o f MDH 1979),  lodgepole  and Douglas f i r (El-Kassaby,  unpublished).  40.  F i g u r e 8 and F i g u r e 9 d e p i c t t h e r e l a t i v e m i g r a t i o n o f allozymes  s c o r e d a t MDH-1 and MDH-2 r e s p e c t i v e l y . M D H - 1 ^ and M D H - l ^ 1  were d e t e c t e d  i n P. braohystemon  were found i n P. congesta.  w h i l e MDH-1  113  , MDH-1  MDH-1"^ and M D H - h a v e  125  11  139 and MDH-1  identical  m o b i l i t i e s . The e x i s t e n c e o f MDH-2*^ was deduced from t h e p r e s e n c e of i n t e r - l o c u s heterodimers  and c o n f i r m e d  with the discovery o f the  87 r a r e MDH-2  variant.  F i g u r e 10 and F i g u r e 11 a r e photographs  o f g e l s s t a i n e d f o r MDH.  The bands v i s i b l e i n zone A o f  F i g u r e 10 a r e t h e i n t e r - l o c u s h e t e r o d i m e r s s u b - u n i t s coded f o r by MDH-1 and MDH-2.  formed between enzyme Heterodimers g e n e r a l l y  s t a i n t w i c e as d a r k l y as homodimers and o f t e n , the MDH-1 homodimeric a l l o z y m e s  were n o t v i s i b l e . However, MDH-1 homodimers  are v i s i b l e i n F i g u r e 11. Data from c r o s s e s and s e g r e g a t i o n a n a l y s e s are presented  f o r MDH-1  i n T a b l e 9.  3.5 Phosphoglucose  isomerase  (PGI) E.C.5.3.1.9  There were two zones o f a c t i v i t y on g e l s s t a i n e d f o r PGI.  These were a r b i t r a r i l y l a b e l e d  as .zone A and zone B. V a r i a t i o n  i n t h e most anodal zone (A) appeared t o be c o n t r o l l e d by a s i n g l e l o c u s which was designed  PGI-1.  A two l o c u s model s u f f i c i e n t l y  e x p l a i n e d the v a r i a t i o n observed i n zone B and t h e c o r r e s p o n d i n g l o c i were d e s i g n a t e d  PGI-2 and PGI-3 ( F i g u r e 12).  o r g a n i z a t i o n o f PGI i n Pleetritis Gottlieb  The m u l t i l o c u s  i s i d e n t i c a l t o t h a t r e p o r t e d by  (1977b) f o r the more advanced s e c t i o n o f t h e genus  .Clarkva.  41.  M_DH-1 Zone-A •——  Zone-B  •B— '  __•  •—•  tarn  -j139  ^125  -j113  MDH-2  1 0 0  F i g u r e 8. The r e l a t i v e m i g r a t i o n o f allozymes d e t e c t e d a t MDH-1. The v a r i a t i o n i n Zone A i s determined by two l o c i , . MDH-1 and MDH-2. MDH-1 and MDH-2l°° have i d e n t i c a l m o b i l i t i e s . The h o l l o w b a r s represent the p o s i t i o n o f heterodimers (hybrid dimeric isozymes). In MDH, t h e s e bands a r e i n t e r p r e t e d t o be i n t e r - l o c u s h e t e r o d i m e r s , formed by t h e random a s s o c i a t i o n o f p r o t e i n s u b - u n i t s coded f o r by a l l e l e s a t both MDH-1 and MDH-2. The v a r i a t i o n i n Zone B i s determined by an unknown number o f l o c i and was n o t i n t e r p r e t e d i n the present study. 100  MDH-1  1 0 0  MDH-2  8 7  F i g u r e 9. The r e l a t i v e m i g r a t i o n o f allozymes d e t e c t e d a t MDH-2.  42.  1  13  27  F i g u r e 10. Allozyme v a r i a t i o n a t MDH-1; genotypes - 100/100, 100/100, 100/100, 139/139, 125/125, 125/139, 125/139, 113/113, 125/139, 125/125, 125/139, 125/139, 125/139, 100/100, 125/125, 125/125, 125/139, 125/139, 125/139, 100/100, 100/113, 100/113, 100/100, 100/113, 100/113, 100/113, 100/100.  1  11  28  F i g u r e 11. Allozyme v a r i a t i o n a t MDH-1; a l l i n d i v i d u a l s a r e 100/100 homozygotes a t MDH-2; note v a r i a t i o n i n zone B f o r p l a n t s 11 and 28.  43. Table 9.  A n a l y s i s o f allozyme v a r i a t i o n at MDH-1 P. braohystemon: Crosses  Individuals  Genotypes  Observed allozyme segregation i n progeny A' /A 1  A /A  1  1  A /A  2  2  100/100x113/113  0  12  0  7 7 - 1 - l l l a x 77-18-18b 100/100x113/113  0  8  0  77-1-llOc x 77-18-19C 100/100x113/113  0  10  0  77-l-13a x 77-18-25c  2  P. braohystemon: S e l f e d homozygotes Individual  Genotype  77-1-26C  100/100  6  77-l-73a  100/100  14  P. oongesta: Individual  Genotype  Number o f progeny**  S e l f e d heterozygotes Observed allozyme segregation i n progeny  V i A  A /A  V 2  2  A  x  (2)  2  76-4-5  125/139  4  9  6  0.474  77-9-95a  125/139  3  4  7  *  77-9-43b  125/139  2  3  2  *  77-9-45c  125/139  1  4  0  *  77-9-60C  125/139  3  9  1  *  77-9-64a  125/139  4  6  2  *  77-9-75b  125/139  3  4  3  *  77-9-78b  125/139  5  2  0  *  25  41  21  1  1  1  Z 77-9-84a  113/125  P. oongesta:  S e l f e d homozygotes Number o f progeny**  Individual  Genotype  77-9-8b  125/125  12  77-9-45a  125/125  6  77-9-45b  125/125  11  77-9-53c  125/125  11  77-9-54a  125/125  9  77-9-70a  125/125  5  76-4-5  139/139  7  * sample s i z e i n s u f f i c i e n t f o r a v a l i d X  2  test of s i g n i f i c a n c e  ** a l l progeny expressed the maternal genotype  P  0.655  *  0.75-0.90  0.50-0.75  44.  In P.  congesta, an o c c a s i o n a l p l a n t appeared to be  double banded at PGI-1.  T h i s suggests  t h a t allozymes  at t h i s  locus are e i t h e r f u n c t i o n a l as monomers or t h a t u n l i k e  sub-units  do not d i m e r i z e .  as  However, t h i s c o u l d not be confirmed  m o b i l i t y d i f f e r e n c e s between v a r i a n t s were s m a l l , r e s o l u t i o n was not always adequate and  the a p p r o p r i a t e s e g r e g a t i o n analyses  were  not performed. V a r i a t i o n at PGI-2 and  PGI-3 i n P. congesta produced  complex banding p a t t e r n s which were d i f f i c u l t locus model, w i t h i n t r a - l o c u s and  to i n t e r p r e t .  A  i n t e r - l o c u s heterodimers,  p r e d i c t e d a l a r g e percentage o f the s t a i n i n g p a t t e r n s observed segregation analyses  ( F i g u r e 13).  organisms: r a b b i t s (Noltmanri, 1964), f i s h 1976),:Clarkia  et al., 1978b),. p i t c h p i n e ( M i t t e n et al., As  (Avise and  studies i n other K i t t o , 1973), Gaura  ( G o t t l i e b , 1977b), Citrus  (Guries and  L e d i g , 1978)  proposed to e x p l a i n the observed  and ponderosa p i n e  the number o f  of heterodimers  by T o r r e s et al., 1978b) and  alleles  v a r i a t i o n became unwieldy.  Minor v a r i a t i o n s i n m o b i l i t y and/or a c t i v i t y ,  inconsistancy i n  ( a l s o r e p o r t e d f o r AAT  in  Citrus  the presence of n u l l s at both  made i t i m p o s s i b l e to c o n f i d e n t l y genotype i n d i v i d u a l s . 14 and  (Torres  1979) .  more p o p u l a t i o n s were surveyed,  the f o r m a t i o n  in  T h i s model agrees, with the d i m e r i c  s t r u c t u r e o f PGI'which has been i n f e r r e d fromiisozyme  ( G o t t l i e b and P i l z ,  two  loci  Figures  15 i l l u s t r a t e the v a r i a t i o n i n banding phenotypes observed  P. congesta.  in  45.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  Figure 12. Segregation of alloyzme variation at PGI-2 and PGI-3 i n P. congesta: this i s the Fj progeny of a selfed plant, heterozygous at PGI-2 and PGI-3 (individual 12 i s P.  brachystemon).  Figure 13. An a l l e l i c interpretation of the allozyme v a r i a t i o n pictured i n Figure 12 (hollow bars represent the p o s i t i o n of heterodimers).  46.  p&r  log-1  ••••• • • • • • f . r # U * t - f c - * 15 23 26 27 Figure 14. A sample o f the allozyme v a r i a t i o n detected at PGI-2 and PGI-3 i n P. oongesta ( i n d i v i d u a l s 15, 23, 26 and 27 are P. braohystemon). 1  Al  -  t  »  15 Figure 15. A sample o f the allozyme v a r i a t i o n detected at PGI-2 and PGI-3 i n P. oongesta ( i n d i v i d u a l 15 i s P. braohystemon).  47.  I t was determined t h a t more i n h e r i t a n c e work was t o c o n s i s t e n t l y i d e n t i f y PGI-2 and PGI-3 a l l o z y m e s . t h i s system was not s c o r e d i n P. congesta.  Consequently,  However, P.  proved t o be much l e s s p o l y m o r p h i c than P. congesta  i n c l u d e d i n the p o p u l a t i o n s u r v e y s o f t h i s  brachystemon  and the proposed  i n h e r i t a n c e model e x p l a i n e d the observed v a r i a t i o n . was  required  T h e r e f o r e , PGI  species.  No v a r i a t i o n was d e t e c t e d a t PGI-1 i n P.  brachystemon.  However, the a l l e l e f i x e d i n t h i s s p e c i e s was not found i n P.  congesta.  T h i s l o c u s was used throughout the s t u d y as one o f the e l e c t r o p h o r e t i c markers which i d e n t i f y the s p e c i e s . d i f f e r e n t i a t e d from P. congesta  P. brachystemon  can be  i n F i g u r e s 12, 14, 15, 19 and  20 by the p r e s e n c e o f t h i s s l o w l y m i g r a t i n g a l l o z y m e .  F i g u r e s 16  and 17 d e p i c t the v a r i a t i o n observed a t PGI-2 and PGI-3 i n P. brachystemon.  Four a l l e l e s were i d e n t i f i e d a t PGI-2 and 100  two a l l e l e s were found a t PGI-3. 94 p i c t u r e d i n F i g u r e 18.  PGI-2  PGI-2 was  94  and PGI-2  extremely r a r e .  are . I t was  d e t e c t e d i n the h e t e r o z y g o u s s t a t e i n o n l y t h r e e i n d i v i d u a l s ,  two  from p o p u l a t i o n 16 and one from p o p u a l t i o n 9. S i n c e h e t e r o z y g o t e s i n P. brachystemon  were r a r e and t h e s e same i n d i v i d u a l s  appeared  t o be h e t e r o z y g o u s a t PGM-3, t h e s e bands may be a r t i f a c t s . This v a r i a n t was t e n t a t i v e l y d e s i g n a t e d as a PGI-2 a l l o z y m e because o f i t s s t a i n i n g p r o p e r t i e s . However, i n h e r i t a n c e s t u d i e s would be needed 42 t o c o n f i rm t h i s .  F i g u r e 19 shows PGI-2  heterozygous s t a t e .  i n b o t h the homozygous and  T h i s a l l o z y m e i s o f i n t e r e s t because i t was  u n i q u e . t o Nanoose H i l l and y e t , i n p o p u l a t i o n s 15(1) and 1 5 ( 2 ) , i t s f r e q u e n c y was  0.15.  48.  PGI-2  PGM  PGI-2  2ioo  2  9 4  2  8 1  1 0 0  -  •>42  Figure 1 6 . The r e l a t i v e migration Figure 1 7 . The r e l a t i v e migration of allozymes detected at PGI-2 i n of allozymes detected at PGI-3 i n  P. braohystemon.  P. braohystemon: the hollow bar  represents an inter-locus heterodimer formed between PGI-2l°° j PGI-333r. a n (  ?CrV U 7 - 2  )100  ,33r  8  9  15  Figure 1 8 . Allozyme variation at PGI-2 i n P. braohystemon: individuals 8 and 9 are 94 / 1 0 0 heterozygotes, a l l other plants are homozygous for PGI-2 ; individual 1 5 i s homozygous for PGI-3 (the low mobility band i s a 2 / 3 inter-locus heterodimer), a l l other plants are PGI-3 lOOn homozygotes, 100  33r  1 0 0  3  49.  118-3 <m 4p <•  >100  • 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Figure 19. Allozyme v a r i a t i o n at PGI-2 i n P. brachystemon: individuals 7 and 9-13 are homozygous f o r PGI-2l°0; i n d i v i d u a l s 14 and 15 are homozygous f o r PGI-2 ; plant 8 i s a 100/42 heterozygote; individuals 1, 2, 3, 5 and 6 are P. congesta (note the fast PGI-1 allozyme); a l l P. brachystemon plants are homozygous f o r P G I - 3 except i n d i v i d u a l 4 which has the PGI-3 3r allozyme. 42  1 0 0 n  3  PGrl * \ 0 4  1  14 20 23 27 Figure 20. A comparison of isozyme v a r i a t i o n i n PGI between P. congesta and P. brachystemon: plants 1-19, except 14, are P. congesta (note the fast PGI-1 allozyme); plants 20-28 are P. brachystemon, the F i progeny of a cross between plants homozygous for PGI-3l00 and ?GI-2>3ZT, the progeny are a l l heterozygotes except for 20, 23 and 27 which are the r e s u l t o f accidental s e l f s . n  50.  was o r i g i n a l l y assumed t o have normal s t a i n i n g p r o p e r t i e s but a m o b i l i t y i d e n t i c a l t o P G I - 2 * ^ .  This migration distance  was d e s i g n a t e d as r e l a t i v e m o b i l i t y 100. A s l o w l y m i g r a t i n g and f a i n t l y s t a i n i n g allozyme, PGI-3  33r  was l a t e r d e t e c t e d . The r e l a t i v e 33r  o f t h e conspicuous between PGI-3"^ heterodimer  r  band a s s o c i a t e d w i t h PGI-3  mobility  . [%7.e. i n t e r m e d i a t e  and P G I - 2 ^ ^ ) , combined w i t h t h e absence o f any  a s s o c i a t e d with P G I - 3 ^ ^ , supported n  t h e assumption  that  PGI-S'''^  and P G I - 2 ^ ^ had i d e n t i c a l m o b i l i t i e s . However, w i t h t h e 42 100 d e t e c t i o n o f PGI-2 homozygotes which show no s t a i n i n g at the PGI-3 11  position  ( F i g u r e 19), i t was p o s s i b l e t o i d e n t i f y PGI-3''"^^  11  a l l e l e and n o t a n o r m a l l y s t a i n i n g v a r i a n t w i t h a m o b i l i t y to PGI-2^^.  Null alleles  as a n u l l identical  a r e n o t uncommon i n e l e c t r o p h o r e t i c  and t h e i r f r e q u e n c i e s tend t o be underestimated  surveys  because they a r e  i  g e n e r a l l y n o t r e c o g n i z e d i n h e t e r o z y g o t e s . However, i t i s uncommon f o r the most common a l l e l e i d e n t i f i e d a t a locus t o be a n u l l . The r e s u l t s o f c r o s s e s and s e g r e g a t i o n a n a l y s e s f o r PGI-2 and PGI-3 i n P. braohystemon  a r e p r e s e n t e d i n T a b l e 10 and  T a b l e 11.  3.6 Phosphogluoomutase  (PGM) E.C. 2.7.5.1  Gels s t a i n e d f o r PGM d i s p l a y e d two zones o f a c t i v i t y (A and B). I n d i v i d u a l s had e i t h e r one o r two bands i n t h e most anodal  zone (A) and two t o f o u r bands i n t h e s l o w l y m i g r a t i n g  zone ( F i g u r e 24).  Bands i n zone A segregated  as a l l e l e s a t a  s i n g l e l o c u s and was d e s i g n a t e d PGM-1. V a r i a t i o n i n zone B was determined  t o be c o n t r o l l e d by two l o c i ,  PGM-2 and PGM-3.  51.  Table 10.  A n a l y s i s of allozyme v a r i a t i o n at PGI-2  P. brachystemon:  S e l f e d homozygotes  Individual  Genotype  Number of progeny**  77-l-13a  100/100  14  77-l-14c  100/100  IS  77-l-73a  100/100  14  77-9-llc  100/100  7  ** a l l progeny expressed the maternal  genotype  Table 11. A n a l y s i s o f allozyme v a r i a t i o n at PGI-3  P. brachystemon: Individuals  Crosses  Genotypes  Observed allozyme s e g r e g a t i o n i n progeny A /A 1  77-l-13a x 77-18-25c  lOOn/lOOn x 33r/33r  P. brachystemon:  A /A  1  x  0  12  S e l f e d homozygotes  Individual  Genotype  77-9-llc  33r/33r  7  77-l-13a  lOOn/lOOn  14  77-1-14C  lOOn/lOOn  15  77-l-73a  lOOn/lOOn  14  ** a l l progeny  2  expressed the maternal  Number o f progeny**  genotype  A /A 2  0  2  Heterozygptes  were i d e n t i f i e d by t h e presence  were a s s o c i a t e d w i t h a s i n g l e l o c u s . Plectritis  o f two bands which  T h i s suggests  t h a t PGM i n  i s a monomeric enzyme which agrees w i t h t h e s u b - u n i t  s t r u c t u r e observed  f o r PGM i n o t h e r organisms:  ( S e l a n d e r et al., 1970), Citrus  horseshoe crab  (Torres et a l . , 1 9 7 8 b ) , ponderosa  p i n e ( M i t t e n et al., 1979), Drosophila  subobsoura (Loukas et al.,  1979) and Douglas f i r ( E l - K a s s a b y , u n p u b l i s h e d ) . Four a l l e l e s were d e t e c t e d a t PGM-1 and PGM-2.  Their  r e l a t i v e ' m i g r a t i o n s a r e p r e s e n t e d i n F i g u r e s 21 and 22, r e s p e c t i v e l y . S i x a l l e l e s were found a t PGM-3 ( F i g u r e 2 3 ) .  A l l f o u r PGM-1  a l l e l e s were d e t e c t e d i n P. congesta b u t o n l y two, PGM-1 ^ and 10  PGM-1 °° were found i n P. brachystemon.  A t PGM-2, t h r e e a l l e l e s  were found i n P.. congesta w h i l e P. brachystemon p o p u l a t i o n s were e s s e n t i a l l y monomorphic f o r P G M - 2  100  .  The o n l y v a r i a n t a l l e l e  d e t e c t e d i n P. brachystemon was a s i n g l e i n d i v i d u a l homozygous f o r a n u l (i.e.  PGM-2  1 0 0 n  ) . PGM-3 i s o f p a r t i c u l a r i n t e r e s t because t h e two  s p e c i e s were c o m p l e t e l y d i f f e r e n t i a t e d a t t h i s l o c u s . Two a l l e l e s , 115 PGM-3  182 and PGM-3  a l l e l e s , PGM-3  100  i n P. congesta.  were d e t e c t e d i n P. brachystemon w h i l e f o u r  , PGM-3  135  , PGM-3  77  and PGM-3 , were 49  observed  T h i s was t h e o n l y l o c u s a n a l y z e d i n t h e s t u d y  i n which t h e two s p e c i e s had no a l l e l e s i n common. F i g u r e s 24 and 25 i l l u s t r a t e t h e v a r i a t i o n observed  i n PGM.  The r e s u l t s o f c r o s s e s and s e g r e g a t i o n a n a l y s e s i n b o t h s p e c i e s a r e p r e s e n t e d f o r PGM-1 i n T a b l e 12, PGM-2 i n Table 13.and PGM-3 i n T a b l e 14.  53.  PGM-1  PGM-2  PGM-3  Figure 21. The r e l a t i v e migration of allozymes detected at PGM-1.  Figure 22. The r e l a t i v e migration of allozymes detected at PGM-2; (PGM-2l00n shown). n o t  PGM-3 P brachystemon  P congesta PGM-1  PGM-2  311s  2<82  2  100  3  135  2  77  3  4 9  Figure 23. The r e l a t i v e migration o f allozymes detected at PGM-3.  54.  pertA ,106  U8-Z Zon  —  ^00  2  2  3  2  3  118 1 0 0 __>  •  135 — •  81  B  —*  100  Figure 24. Allozyme v a r i a t i o n i n PGM i n P.  1  15  congesta.  21  24  28  Figure 25. A comparison of isozyme v a r i a t i o n i n PGM between P. conge and P. brachystemon: plants 1-20, except 15, are P. congesta; plants 21-29 are P. brachystemon, the F j progeny of a cross between plants homozygous f o r d i f f e r e n t a l l e l e s , a l l plants are 100/106 heterozygot at PGM-1 and 115/182 heterozygotes at PGM-3 except plants 21, 24 and 28 which are the r e s u l t of accidental s e l f s .  55.  Table 12. A n a l y s i s o f allozyme v a r i a t i o n at PGM-1 P.  brachystemon:  Individuals  Crosses  Genotypes  Observed segregation o f allozymes i n progeny  V i A  V 2 A  A  2  / A  77-l-13a x 77- 18-25 c  100/100 x 106/106  0  12  0  7 7 - 1 - l l l a x 77 -18-18b.  100/100 x 106/106  0  8  0  77-1-llOc x 77 -18-19C  100/100 x 106/106  0  10  0  P.  brachystemon:  S e l f e d homozygotes  Individual  Genotype  77-9-llc  100/100  7  77-l-14c  106/106  15  77-l-26c  106/106  5  77-l-73a  106/106  14  P. Individual  congesta:  Genotype  Number o f progeny**  S e l f e d heterozygotes  Observed segregation o f allozymes i n progeny x  V i A  A /A :  A /A  2  2  2  P  2  1.200 0.50-0.75  76-4-5  100/106  7  8  5  *  77-9-45a  100/106  1  3  2  *  77-9-53C  100/106  1  9  1  *  77-9-64b  100/106  6  1  1  77-9-68b  100/106  4  1  1  *  77-9-78b  100/106  3  3  0  *  77-9-81a  100/106  1  5  2  *  77-9-83a  100/106  2  3  4  *  77-9-84a  100/106  3  10  0  *  28  43  16  E  P.  congesta:  S e l f e d homozygotes Number o f progeny**  Individual  Genotype  77-9-45b  100/100  11  77-9-60c  100/100  14  77-9-64a  100/100  13  77-9-75b  100/100  10  77-9-65a  106/106  9  * sample s i z e i n s u f f i c i e n t  2  f o r a v a l i d x" t e s t o f s i g n i f i c a n c e  ** a l l progeny expressed the maternal genotype  3.322 0.10-0.05  56.  Table 13. A n a l y s i s o f allozyme v a r i a t i o n at PGM-2 : Selfed P. braohystemon  homozygotes  Individual  Genotype  Number o f progeny**  77-1-14C  100/100  15  77-l-26c  100/100  6  77-l-73a  100/100  14  77-9-llc  100/100  7  P.  oongesta:  Individual  Genotype  S e l f e d heterozygotes Observed segregation o f allozymes i n progeny x  V  i  A  A /A 1  A /A 2  2  .2 (2)  2  77-9-45a  81/100  0  2  4  *  77-9-68b  81/100  1  3  2  *  77-9-78b  81/100  0  1  5  *  77-9-81a  81/100  1  3  4  *  76-4-5  81/118  7  9  4  P.  Selfed oongesta:  homozygotes Number o f progeny**  Individual  Genotype  77-9-64a  81/81  13  77-9-45b  100/100  11  77-9-53c  100/100  11  77-9-60C  100/100  14  77-0-75b  100/100  10  77-0-84a  100/100  13  77-9-95a  100/100  14  * sample s i z e i n s u f f i c i e n t f o r a v a l i d  x " test of significance  ** a l l progeny expressed the maternal genotype  1. 100  57. Table 14. A n a l y s i s o f allozyme v a r i a t i o n at PGM-3  P. brachystemon: Individuals  Crosses  Genotypes  Observed segregation o f allozymes i n progeny A /A 1  77-l-13a x 77-18-25c  115/115 x 182/182  0  7 7 - 1 - l l l a x 77-18-18b 115/115 x 182/182  0  77-1-llOc x 77-18-19c  0  P.  brachystemon:  A /A  1  :  0  S e l f e d homozygotes  115/115  15  77-l-26c  115/115  6  77-l-73a  115/115  14  77-9-llc  115/115  7  Genotype  0 10  77-l-14c  Individual  Number o f progeny**  S e l f e d heterozygotes  Observed segregation o f allozymes i n progeny  V i  A /A  A  :  A /A 2  2  77-9-45a  100/135  0  4  2  77-9-45b  100/135  7  4  0  77-9-64b  100/135  5  0  3  77-9-65a  100/135  4  4  1  77-9-75b  100/135  2  7  1  77-9-78b  100/135  1  3  2  77-9-81a  100/135  1  4  3  77-9-83a  100/135  0  4  3  77-9-95a  100/135  4  9  1  24  39  16  I  P. Individual  congesta:  2  0  . 8  Genotype  congesta:  2  12  Individual  P.  A /A  2  l  1.633  S e l f e d homozygotes Genotype  Number o f progeny*  77-9-53c  100/100  11  77-9-60c  100/100  14  77-9-64a  100/100  13  77-9-68b  100/100  6  77-9-84a  135/135  13  * sample s i z e i n s u f f i c i e n t f o r a v a l i d x" t e s t o f s i g n i f i c a : ** a l l progeny expressed the maternal genotype  (2)  2  0.25-0.50  58.  3.7 Monomorphic enzyme systems Four enzymes were surveyed  and t h e l o c i r e s o l v e d were  found t o be monomorphic f o r t h e same a l l o z y m e i n both s p e c i e s o f Plectritis. E.C.  These enzymes were i s o c i t r a t e dehydrogenase (IDH:  1.1.1.42), m a l i c enzyme (ME:E.C. 1.1.1.40), 6-phosphogluconic  dehydrogenase (6PG:E.C. 1.1.1.44) and s u p e r o x i d e  dismutase  (S0D:E.C.1.15.1.1). Gels s t a i n e d f o r IDH d i s p l a y e d two areas o f a c t i v i t y , a s i n g l e monomorphic band and a more s l o w l y m i g r a t i n g , d i f f u s e l y s t a i n i n g zone.  Only t h e monomorphic band was s c o r e d as t h e  s l o w e r zone was n o t r e s o l v e d . horseshoe crabs  IDH has been shown t o be d i m e r i c i n  ( S e l a n d e r et al., 1970), Drosophila  (Loukas et al., 1979), ponderosa p i n e  subobscura  (O'Malley et al., 1979) and  Douglas f i r ( E l - K a s s a b y , u n p u b l i s h e d ) . G e l s s t a i n e d f o r ME had a d a r k l y s t a i n i n g monomorphic band p l u s one o r two l i g h t l y s t a i n i n g and more s l o w l y m i g r a t i n g bands ( F i g u r e 26).  V a r i a t i o n was observed  i n the l i g h t l y  s t a i n i n g bands b u t s e g r e g a t i o n s c o u l d n o t be i n t e r p r e t e d a c c o r d i n g to s i m p l e M e n d e l i a n scored.  i n h e r i t a n c e . Consequently,  o n l y ME-1 was  ME appears t o be a t e t r a m e r i c enzyme i n  Drosophila  subobscura (Loukas et al., 1979). A g e l s t a i n e d f o r 6PG i s p i c t u r e d i n F i g u r e 27. A s i n g l e monomorphic band was observed  i n a l l populations  6PG has a d i m e r i c s t r u c t u r e i n humans (Zouros, 1976),  screened. Drosophila  subobsaura (Loukas et al., 1979) and Douglas f i r ( E l - K a s s a b y , unpublished).  59.  Figure 26. Allozyme a c t i v i t y i n ME: note the v a r i a t i o n i n the number of l i g h t l y s t a i n i n g bands.  Figure 27.  Allozyme a c t i v i t y i n 6PG.  60.  SOD,  which i s not i l l u s t r a t e d ,  appeared as a s i n g l e mono-  morphic white band on g e l s s t a i n e d with t e t r a z o l i u m dye. i s o f t e n r e p o r t e d i n the l i t e r a t u r e because, i n v i t r o ,  i t prevents  to b l u e formazan. L i p p i t t TO was  a c t u a l l y SOD.  T h i s enzyme  as t e t r a z o l i u m o x i d a s e  (TO)  the o x i d a t i o n o f t e t r a z o l i u m dyes  and F r i d o v i c h (1973),  Superoxide  dismutase  demonstrated t h a t  (or TO)  has been shown t o  be a d i m e r i c enzyme i n the f o l l o w i n g organisms: humans (Beckman, 1973); dogs  (Brewer, 1967); p o t a t o e s  s p e c i e s o f Drosophila houseflies  ( O e l s c h l e g a l § Stahmann, 1971);  (Richmond and P o w e l l , 1970;  (McDonald et at.,  1975)  A y a l a et  two  aZ.j.1972);  and Douglas f i r (El-Kassaby,  unpublished).  3.8 Summary Nine enzyme systems were surveyed identified  i n Vteotritis.  the observed  and  fifteen  loci  G e n e t i c models were proposed to e x p l a i n  v a r i a t i o n . These models d e s c r i b e d the m u l t i - l o c u s  o r g a n i z a t i o n , s u b - u n i t s t r u c t u r e and a l l e l i c v a r i a t i o n w i t h i n each system.  The models were t e s t e d with c r o s s e s and  segregation  a n a l y s e s . When progeny c l a s s e s were s u f f i c i e n t l y l a r g e , s e g r e g a t i o n s were a n a l y z e d u s i n g the c h i - s q u a r e s t a t i s t i c . model was and  proposed t h a t e x p l a i n e d observed  PGI-3 i n P .  congesta.  s e g r e g a t i o n s at PGI-2  However, the l e v e l o f polymorphism,  i n c o n s i s t a n c y i n the f o r m a t i o n o f heterodimers n u l l s prevented  A  a l l e l i c v a r i a t i o n a t these  c o n f i d e n t l y s c o r e d . Although P.  congesta  and the presence  l o c i 'from b e i n g  and P.  braohystemon  of  differed  i n t h e i r a l l e l i c complements,  the d a t a suggest  that  the m u l t i l o c u s o r g a n i z a t i o n o f the systems a r e i d e n t i c a l i n b o t h species.  U l t i m a t e l y , f i f t e e n l o c i were s c o r e d i n P.  and t w e l v e l o c i i n P. congesta.  brachystemon  The isozyme v a r i a t i o n d e t e c t e d  w i t h i n each p o p u l a t i o n i s p r e s e n t e d i n Appendix C.  The l e v e l o f  polymorphism a t each l o c u s and the d i s t r i b u t i o n o f a l l e l e s among p o p u l a t i o n s i s d e s c r i b e d i n Chapter 4.  62.  4.0  VARIATION AMONG POPULATIONS  A l l e l e f r e q u e n c i e s , expected h e t e r o z y g o s i t y (h) and observed h e t e r o z y g o s i t y (h ) were c a l c u l a t e d f o r each locus and a r e p r e s e n t e d i n T a b l e 15 f o r P. brachystemon P. congesta.  and T a b l e 16 f o r  The expected p r o p o r t i o n o f h e t e r o z y g o t e s at each locus was  c a l c u l a t e d u s i n g t h e f o l l o w i n g formula; 2  h = 1-Ex ( N e i , i  1975)  where x^ i s t h e frequency o f t h e i  allele  at t h e l o c u s i n q u e s t i o n .  T h i s i s e q u i v a l e n t t o t h e expected frequency o f h e t e r o z y g o t e s i f the p o p u l a t i o n i s i n Hardy-Weinberg  equilibrium.  C a l c u l a t i n g expected h e t e r o z y g o s i t y , u s i n g the mean a l l e l e f r e q u e n c i e s f o r each s p e c i e s , y i e l d e d the f o l l o w i n g sequence o f l o c i ranked a c c o r d i n g t o t h e i r t o t a l gene d i v e r s i t y ( i . e . P. brachystemon  H^, N e i ,  1973):  - EST-1 (0.659), MDH-1 (0.484), LAP-1 (0.422),  PGM-1 (0.412), PGI-3 (0.226), PGM-3 (0.196), PGI-2 (0.096), EST-2 (0.000), IDH-1 (0.000), MDH-2 (0.000), ME-1 (0.000), PGI-1  (0.000), PGM-2 (0.000), SOD (0.000), 6PG (0.000);  P. congesta  - LAP-1 (0.792), MDH-1 (0.509), PGM-3 (0.504),  EST-2 (0.502), PGM-2 (0.439), PGM-1 (0.431), EST-1 (0.020), IDH-1 6PG  (0.000, MDH-2 (0.000), ME-1 (0.000), SOD (0.000), (0.000).  In o r d e r t o emphasize d i f f e r e n c e s i n t h e r e l a t i v e  levels  o f v a r i a t i o n at homologous l o c i and f o r convenience o f d i s c u s s i o n ,  loci  surveyed i n t h e two t a x a are c o n s i d e r e d under t h e f o l l o w i n g t h r e e a r b i t r a r y categories:  Table 15. A l l e l e Frequencies and Heterozygosity Values* f o r E i g h t Polymorphic Loci i n PZectritis brachystemon.  Locus  Allele  1  13  9  114  .45  .00  .00  .00  1.00  .32  .72  1.00  .00  100  .53  .20  .97  1.00  .00  .12  .22  .00  .00  89  .02  .80  .03  .00  .00  .56  .06  .00  1.00  h  .516  .320  .058  .000  .000  .570  .430  .000  .000  h  .046  .029  .023  .000  .000  .000  . Ill  .000  .000  0  10  7(1)  7(3)  15(1)  15(2)  16  18  112  .00  .00  .00  .00  .00  .00  .00  .08  .00  .22  105  .00  .00  .00  .05  .00  .00  .00  .39  .00  .00  100  .11  .94  1.00  .95  .95  .40  1.00  .53  1.00  .61  95  .00  .00  .00  .00  .05  .60  .00  .00  .00  .00  91  .84  .06  .00  .00  .00  .00  .00  .00  .00  .00  85  .05  .00  .00  .00  .00  .00  .00  .00  .00  .17  80  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  h  .280  .113  .000  .095  .095  .480  .000  .561  .000  .551  .000  .000  .000  .000  .000  .000  .000  .000  .000  .111  139  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  125  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  113  .00  .00  .00  1.00  .45  .50  .11  .00  1.00  1.00  100  1.00  1.00  1.00  1.00  .00  .00  h 0  .00  .55  .50  .89  h  .000  .000  .000  .000  .495  .500  . 196  .000  .000  .000  h  .000  .000  .000  .000  .000  .000  .000  .000  .000  .000  0  Table Locus  15.  (Continued)  Allele 100  1  13  9  10  1 .00  1 .00  .99  1 .00  7(1)  7(3)  15(1)  15(2)  1 .00  .88  .85  .84  16  18  .95  1 .00  94  .00  .00  .01  ;oo  .00  .00  .00  .00  .05  .00  81  .00  .00  .00  .00  .00  .12  .00  .00  .00  .00  42  .00  .00  .00  .00  .00  .00  . 15  .16  .00  .00  h  .000  .000  .020  .000  .000  .211  .255  .269  .095  .000  h  .000  .000  .017  .000  .000  .000  .000  .029  .091  .000  0  lOOn 33 h h  1 .00  1 .00  .81  1 .00  1 .00  .00  .38  1 .00  1.00  .54  .00  .00  .19  .00  .00  .00  .62  .00  .00  .46  .000  .000  .497  .000  .000  .308  .000  .000  .000  .471  .000  .000  .000  .000  .000  .000  .000  .000  .000  .000  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  o  117 106  1 .00  .48  .03  1 .00  .00  .16  .23  100  .00  .52  .97  .00  1 .00  .84  .77  94  .00  .00  .00  1 .00  1.00  1 .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  h •  .000  .499  .058  .000  .000  .269  .354  .000  .000  .000  h  .000  .042  .000  .000  .000  .000  .000  .000  .000  .000  0  118  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  100  1. , 0 0  1. .00  1.00  1 .00  1 .00  .96  1.00  1, . 0 0  1.00  1 .00  81  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  lOOn  .00  .00  .00  .00  .00  .04  .00  .00  .00  .00  Table  15 . (Continued)  1  13  10  7(1)  7(3)  15(1)  15(2)  16  18  h  .000  .000  .000  .000  .000  .077  .000  .000  .000  .000  h  .000  .000  .000  .000  .000  .000  .000  .000  .000  .000  .00  .00  .00  .03  .05 • 1.00  0  182  .00  .00  .01  .00  135  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  115  1.00  1.00  .99  1.00  1.00  1.00  1.00  .97  .95  .00  100  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  77  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  49  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  h  .000  .000  .020  .000  .000  .000  .000  .058  .095  .000  h  .000  .000  .023  .000  .000  .000  .000  .054  .091  .000  0  2  * h=l-Ex. 1  h  9  = observed heterozygosity  Table 16. A l l e l e Frequencies and Heterozygosity Values* f o r Eight Polymorphic  Plectritis congesta.  Loci i n  POPULATIONS Locus  Allele  2(1)  2(2)  114  .00  .00  100  1.00  1.00  3 .00 .99  5  4  9  10  11  .00  .00  .00  .00  .00  1.00  1.00  .98  1.00  1.00  7(2)  8  14(1)  14(2) 15(1) 15(2)  .00  .00  .00  .01  .00  .01  .89  1.00  1.00  1.00  .99  1.00  .99  .00  .00  .00  .00  .00  .08  .00  .02  .00  .00  .03  .00  .000  .000  .039  .000  .000  .201  .000  .000  .000  .020  .000  .020  .000  .000  .011  .000  .000  .193  .000  .000  .000  .021  .000  .028  .04  .00  .00  .00  .00  .00  .08'  .09  .07  .00  .06  .00  .02  .69  . 19  .63  .68  .51  .55  .16  .86  .78  .89  .13  .17  .94  .83  .04  89  .00  .00  .01  .00  h  .000  .000  .020  h  .000  .000  .009  126  .00  .00  100  .94  .93  0  .76  .05  .15  .11  .81  .495  .390  .250  .364  .196  .323  .282  .114  .436  .385  .219  .310  .152  .333  .333  .114  .00  .00  .00  .00  .00  .00  .00  .00  .00  .05  .03  .00  .08  .10  .02  .00  .00  .03 .01  .07  .27  .81  .37  .32  .49  .45  .113  .130  .450  .308  .466  .435  .500  .118  .143  .415  .231  .467  .341  .380  .04  .00  .00  .00  .00  .00  .00  .03  .00  74  .06  h h 0  112 105  .23  .17  .00  100  .15  .28  . 13  .46  .50  .45  .30  .22  .24  .33  .25  .29  .02  .95  95  .04  .07  .57  .21  .14  .00  . 29  .24  .38  .16  .26  .17  .29  .00  .26  .51  .05  91  .23  .09  .23  .21  .20  .45  .18  .33  .11  .07  85  .27  .26  .03  .12  . 10  .07  .16  .10  .13  .05  .20  .22  .01  .00  .47  80  .04  .13  .04  .00  .03  .03  .02  .08  .24  .27  .12  .04  .17  .00  .07  .765  .768  .723  .770  .801  .769  .626  .095  .620  .375  .486  .477  .531  .467  .276  .396  .100  .278  h li  o  .790  .796  .603  .686  .662  .589  .615  .478  .392  .583  .467  .267  * h = l-jx? h  .03 . .39  .02  = observed heterozygosity  Table 16 .  (Continued) POPULATIONS  Locus  Allele  2(1)  2(2)  139  .65  .38  125  .32  113  .03  100 h  .471  h  3  5  4  9  10  11  7(2)  8  .68  .67  .47  .27  .64  .47  .64  .57  .31  .33  .50  .51  .36  .53  .34  .05  .01  .00  .03  .22  .00  .00  .02  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .474  .528  .441  .442  .528  .619  .461  .498  .474  .372  .300  .431  .667  .467  .367  .500  .385  .269  .281  .77  14(1) 14(2)  15(1)  15(2)  17  .57  .60  .29  .43  .19  .00  .00  .21  .84  .65  .71  .18  .16  .35  .05  .00  .00  .00  .00  .00  .00  .00  .269  .455  .412  .490  .560  .241  .367  .385  .571  .514  0  100  1.00  1.00  87  .00  .00  h  .000  .000  h  .000  .000  o  1.00  1.00  1.00  1.00  1.00  1.00  .95  1.00  1.00  1.00  .99  1.00  1.00  .00  .00  .00  .00  .00  .00  .05  .00  .00  .00  .01  .00  .00  .000  .000  .000  .000  .000  .000  .095  .000  .000  .000  .020  .000  .000  :000  .000  .000  .000  .000  .000  .075  .000  .000  .000  .026  .000  .000  117  .00  .00  .12  .00  .00  .14  .03  .00  .00  .02  .00  .00  .00  .00  .00  106  .03  .06  .38  .08  .37  .23  .35  .28  . 17  .73  .20  . 15  .31  .42  .43  100  .97  .94  .49  .92  .63  .63  .62  .72  .83  .25  .80  .85  .69  .58  .57  94  .00  .00  .01  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  h  .058  .113  .601  .147  .466  .531  .492  .403  .282  .404  .320  .255  .428  .487  .490  .059  .042  .604  .154  .200  .288  .291  .410  . 185. .375  .200  .000  .333  .231  .441  h 0  Table  16.  (Continued) POPULATIONS  Locus  Allele  118  2(1)  2(2)  .03  .10  3  5  4  9  10  .00  .00  .00  .03  .03  11  .05  7(2)  .00  8  .00  14(1)  14(2) 15(1)  15(2)  17  .00  .00  .02  .00  .03  1.00  .62  .71  .51  100  .79  .67  .75  .46  .80  .60  .46  .60  .57  .81  .96  81  .18  .23  .25  .54  .20  .37  .51  .35  .43  .19  .04  .00  .36  .29  .46  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .486  .412  .527  lOOn  .00  .00  .00  .00  .00  .343  .488  .375  .497  .320  .502  .527  .515  .490  .309  .077  .000  .294  .375  .321  .583  .133  .403  .560  .513  .338  .313  .036  .000  .426  .286  .559  182  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  135  .53  .35  .65  .50  .37  .25  .51  .67  .65  .64  .70  .53  .55  .61  .72  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .39  .28  h h  o  .00  115  .00  .00  .00  .00  ..45  100  .47  .63  .35  .50  .47  .75  .49  .33  .35  .36  .28  .47  77  .00  .02  .00  .00  .16  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .00  .02  .00  .00  .00  .00  49  .00  .00  .00  .00  .00  .00  .00  .00  h  .498  .480  .455  .500  .617  .375  .500  .442  .455  .461  .431  .498  .495  .476  .403  .353  .375  .283  .455  .400  .282  .300  .359  .353  .469  .571  .455  .383  .357  .441  h  o  ON OO  69.  H > 0.400, 0 . 4 0 0 > H 2 0.050 and H_<0.050. T  T  4.1 Loci polymorphic 4.1.1  with  E^y 0.400  P. brachystemon  - Four l o c i ; EST-1 (0.659), MDH-1  (0.484), LAP-1 (0.422) and PGM-1 (0.412) a r e c o n s i d e r e d w i t h i n t h i s category.  Allele  29, 30 and 31. in  f r e q u e n c i e s at these  At each o f these  a l l populations.  loci  are p l o t t e d i n F i g u r e s 28,  l o c i , no s i n g l e a l l e l e i s predominant  However, L A P - 1  and P G M - 1  1 0 0  100  occur w i t h mean  f r e q u e n c i e s o f 0.75 and 0.71 r e s p e c t i v e l y (Table 17). None o f t h e a l l e l e s d e t e c t e d at EST-1, LAP-1 and PGM-1 are unique t o P. brachystemon  although  EST-1  1 1 4  , EST-1  1 0 0  , LAP-1  1 0 0  and L A P - 1  s i g n i f i c a n t l y d i f f e r e n t f r e q u e n c i e s i n t h e two s p e c i e s At these t h r e e l o c i ,  have  85  (Table 17).  a l l e l e f r e q u e n c i e s and h e t e r o z y g o s i t y v a l u e s . o  f l u c t u a t e over wide ranges w i t h .no d i s c e r n i b l e p a t t e r n i n the d i s t r i b u t i o n o f a l l e l e s . 2.00  LAP-1 a l l e l e s  - •  On t h e average, 1.89 EST-1 a l l e l e s ,  and 1.40 PGM-1 a l l e l e s were d e t e c t e d w i t h i n each  population. MDH-1 i s o f p a r t c u l a r i n t e r e s t , as t h i s taxonomically  d i a g n o s t i c . Two a l l e l e s , MDH-1  100  l o c u s proved t o be  and MDH-1  113  were d e t e c t e d  w i t h mean f r e q u e n c i e s o f 0.59 and 0.41 r e s p e c t i v e l y (Table 17). MDH-1 113 unique t o P. brachystemon , i n P. congesta. population. what suggests of  w h i l e MDH-1  was a low frequency  100  was  allele  An average o f 1.30 a l l e l e s were found w i t h i n each  The d i s t r i b u t i o n o f these two a l l e l e s clinal variation.  MDH-1  the t h r e e s o u t h e r n Vancouver I s l a n d  100  ( F i g u r e 29) some-  was f i x e d at Sumas Mtn.  populations.  and two  The Malahat  D r i v e and one o f the Nanoose H i l l p o p u l a t i o n s were p o l y m o r p h i c w h i l e  70...  EST-1  Population  F i g u r e 28. The d i s t r i b u t i o n o f EST-1 a l l e l e s among p o p u l a t i o n s o f P. braohystemon.  MDH-1  >>  u  c <b 3  .60  J  <u u  u. 0)  Population  F i g u r e 29. The d i s t r i b u t i o n o f MDH-1 o f P. braohystemon.  a l l e l e s among  populations  • .71,  LAP-1  1  13  9  10  7(1)  7(3)  15(1)  15(2)  16  18  16  18  Population F i g u r e 30. The d i s t r i b u t i o n o f LAP-1 a l l e l e s among p o p u l a t i o n s o f P. brachystemon.  PGM-1  BO J  cr ii  i_  LL  JD  .40  .  tt)  < .20  J  1  13  9  10  7(1)  7(3)  15(1)  15(2)  Population F i g u r e 31. The d i s t r i b u t i o n o f PGM-1 a l l e l e s among p o p u l a t i o n s o f P. brachystemon.  72.  Table 17. Comparison o f Mean A l l e l e Frequencies with Heterozygosity Values f o r Polymorphic Loci i n p. braohystemon and P. oongesta.  Locus EST.!  Allele 114  .39+.143  .01+.005  .34±.134  .99+.007  "T  LAP-1  .27+.133  .00+.002  .659  .020  -  .03+.009  -3.33***  1.00±.000  .60+.079  5.06***  .37+.079  -4.68***  74  -  Hj.  -000  .502  112  .03+.022  .00+.003  1.36  105  .04+.039  .05±.018  - .23  100  .75±.100  .30+.059  95  .06±.060  .19+.041  -1.78  91  .091.084  .22+.038  -1.41  85  .03±.017  .1S±.032  -3.33***  80  -  .09±.022  -4.09***  139  .792  .60+.038  -15.79***  -  .36±.033  -10.91***  113  .41+.142  100  .591.142  100  1.001.000  87  -  Hj,  3.88***  - .  .484  PGI-2  2.03  126  125  MDH-2  2.66* -4.85***  100  .422  MDH-1  P. oongesta  100 89  EST-2  P. braohystemon  .04±.019 .509  1.001.003  .00  .001.003  .00  -000  .000  100  .951.021  NOT SCORED  94  .011.005  81  .011.012  42  .031.021 .096  2.59* 4.15***  t  73.  Table 17. (Continued)  Locus  Allele  PGI-3  lOOn  .874.072  33r  .13±.072  *T PGM-1  P. brachystemon P. congesta  .226  117  .02+.012  106  .29±.128  .28+.047  .07  .71+.128  .70+.050  .07  .00+.001  .00  «T  .412  118 100  lOOn "T 182  .431  .02+.007 1.00±.004  81  PGM-3  -1.67  100 94  PGM-2  NOT SCORED  7.21***  .29+.041  -7.07***  .00+.004 .000  .00 .439  .11+.099  135  -2.86**  .69±.043  1.11 .55±.036  -15.28***  100  .44+.033  -13.33***  77  .01±.011  - .91  49  .00+.001  .00  115  "T  »T  *P <.05 **P <.01 ***P <.005  .89+.099  8.99***  .196  .504  .166±.058  .266±.083  74.  MOrl-l*^  River,,  r e p l a c e d MDH-1*^ at L i t t l e Qualicum F a l l s However, t h e r e were n o t a b l e e x c e p t i o n s  10 (Prospect Lake) was f i x e d f o r MDH-1 was monomorphic f o r MDH-1^^.  113  and Campbell  t o the p a t t e r n s : populat  while  15(2) (Nanoose H i l l )  In a d d i t i o n , two p l a n t s c o l l e c t e d at 113  Cape Perpetua,  Oregon, were homozygous f o r MDH-1  .  Unfortunately,  based on t h e p r e s e n t d a t a , i t i s i m p o s s i b l e t o determine i f t h e r e i s a s e l e c t i v e b a s i s f o r t h i s p a t t e r n or i f i t i s c o i n c i d e n t a l  and t h e r e s u l t  of stochastic forces. 4.1.2 P. congesta  - S i x l o c i , LAP-1 (0.792), MDH-1  (0.509),  PGM-3 (0.504) , EST-2 (0.502), PGM-2 (0.439) and PGM-1 (0.431) f a l l within this  category. A l l e l e  f r e q u e n c i e s f o r these  loci  are p l o t t e d i n  F i g u r e s 32 through 37,, No s i n g l e a l l e l e was predominant i n a l l populations  at any o f these  l o c i . However, PGM-2^^ was t h e most  common a l l e l e i n a l l b u t two p o p u l a t i o n s the h i g h e s t frequency  and PGM-1"^^ o c c u r r e d  i n fourteen o f the f i f t e e n populations  with  (Table 16) on  Seven a l l e l e s were d e t e c t e d w i t h a mean  frequency  P. bvachystemon populations  o f 0.09, was t h e o n l y a l l e l e not d e t e c t e d i n  (Table 17).  surveyed.  at LAP-1 ( F i g u r e 32). LAP-1 ,  LAP-l"^  and LAP-1^* were d e t e c t e d i n a l l  On t h e average, 5.33 a l l e l e s were  maintained  w i t h i n each p o p u l a t i o n . Three a l l e l e s were d e t e c t e d a t MDH-1 i n 139 P. congesta  ( F i g u r e 33).  The two most common a l l e l e s , MDH-1  arid MDH-1'''^ were not found  i n P. bvachystemon.  On the average,  l o c a l p o p u l a t i o n s maintained 2.53 a l l e l e s . A l l p o p u l a t i o n s were 139 125 113 polymorphic f o r MDH-1 and MDH-1 . A d d i t i o n a l l y , MDH-1 was d e t e c t e d i n e i g h t o f the f i f t e e n p o p u l a t i o n s surveyed.  75.  PGM-3 was a t a x o n o m i c a l l y Four a l l e l e s were i d e n t i f i e d  d i a g n o s t i c locus  at t h i s  l o c u s , none o f which was d e t e c t e d  i n P. braohystemon (Table 17). Two a l l e l e s , were found  i n a l l populations while  e i t h e r absent was  (Figure 34).  PGM-3  135  and PGM-3  t h e other two a l l e l e s  o r o c c u r r e d a t low frequency.  100  ,  were  An average o f 2.20 a l l e l e s  d e t e c t e d w i t h i n each p o p u l a t i o n . Three a l l e l e s were s c o r e d at t h e EST^2 locus ( F i g u r e 3 5 ) .  EST-2  7 4  and E S T - 2  1 2 6  were n o t d e t e c t e d i n P. braohystemon.  common a l l e l e , E S T - 2 different  frequencies  for EST-2  1 0 0  populations  1 0 0  The most  , was d e t e c t e d i n both t a x a but at s i g n i f i c a n t l y  (Table 17). A l l p o p u l a t i o n s were p o l y m o r p h i c  and E S T - 2  7 4  while E S T - 2  and always with  1 2 6  a frequency  average, 2.40 a l l e l e s were m a i n t a i n e d  was d e t e c t e d i n o n l y s i x o f l e s s than 0.10.  within local  On t h e  populations.  Three PGM-2 a l l e l e s were d e t e c t e d i n P. oongesta ( F i g u r e 36) Two o f these  a l l e l e s were not found  common a l l e l e , PGM-2 significantly 14(2)  100  i n P. braohystemon, w h i l e t h e most  , was d e t e c t e d i n both s p e c i e s b u t a t  d i f f e r e n t frequencies  (Table 17).  (Jack's P o i n t ) were p o l y m o r p h i c f o r PGM-2  A l l populations 100  except  and PGM-2 . 81  118 PGM-2  was a r a r e a l l e l e . I t was d e t e c t e d i n only seven p o p u l a t i o n s  and w i t h t h e e x c e p t i o n o f p o p u l a t i o n 2(2) ( A n a c o r t e s , never p r e s e n t w i t h a frequency  g r e a t e r than 0.05.  WA ) , i t was  The average number  of a l l e l e s d e t e c t e d p e r p o p u l a t i o n was 2.40. Four a l l e l e s were s c o r e d PGM-1  100  and P G M - 1  These a l l e l e s were identical  106  at PGM-1 ( F i g u r e 3 7 ) .  were common, o c c u r r i n g i n a l l sampled p o p u l a t i o n s .  d e t e c t e d a l s o i n P. braohystemon with  frequencies  Alleles  almost 117  (Table 17). The two r a r e a l l e l e s , PGM-1  94 and PGM-1  , were not found  i n P. braohystemon.  On t h e average,  76..  LAP-1 1.00  _  2(1)  2(2)  3  3  4  9  10  11  7(2)  8  14(1)  14(2)  15(1)  15(2)  17  Population  • F i g u r e 32. The d i s t r i b u t i o n o f LAP-1 a l l e l e s among p o p u l a t i o n s o f P.  oongesta.  MDH-1 1.00 _  2(1)  2(2)  3  3  4  9  10  11  7(2)  8  14(1)  14(2)  15(1)  15(2)  17  Population  F i g u r e 33. The d i s t r i b u t i o n o f MDH-1 a l l e l e s among p o p u l a t i o n s o f P.  oongesta.  77.  PGM-3 1.00  §  CT  _  .60  1) <_  u.  a;  .40  135 100 • 77 v 49 •  <  _  v  ~T  T  -V—f 2(1)  2(2)  10  3  7 11  T 7(2)  T' 8  T—-T 14(1)  14(2)  T  T  1511)  16(2)  T17  Population  F i g u r e 34. The d i s t r i b u t i o n o f PGM-3 a l l e l e s among p o p u l a t i o n s  o f P.  congesta.  EST-2 1.00  126 • 100 • 74 2(1)  2(2)  3  5  4  9  10  11  7(2)  8  14(1)  14(2)  15(1)  15(2)  17  Population  F i g u r e 35. The d i s t r i b u t i o n o f EST-2 a l l e l e s "among p o p u l a t i o n s  o f P.  congesta.  78.  PGM-2  118" 100 • 81 * 2(1)  2(2)  3  S  4  S  10  11  7(2)  8  14(1)  14(2)  15(1)  15(2)  17  Population  F i g u r e 36. The d i s t r i b u t i o n o f PGM-2 a l l e l e s among o f P. oongesta.  populations  PGM-1  F i g u r e 37. The d i s t r i b u t i o n o f PGM-1 o f P. oongesta.  a l l e l e s among p o p u l a t i o n s  79.  2.33 a l l e l e s were d e t e c t e d w i t h i n l o c a l  4.2 Loci  -polymorphic  with  0.400>H >.0.050 T  4.2.1 P. brachystemon  - Three l o c i ;  (0.196) and PGI-2 (0.096) were found diversity. in  At these  populations.  loci,  PGI-3 (0.226), PGM-3  t o have t h i s  l e v e l o f t o t a l gene  one a l l e l e was g e n e r a l l y predominant  a l l populations. Allele  f r e q u e n c i e s f o r PGI-3 a r e p l o t t e d i n F i g u r e 38.  33r PGI-3  was found  i n o n l y 3 p o p u l a t i o n s : 9 ( F r a n c i s P a r k ) , 15(1)  (Nanoose H i l l ) and 18 (Campbell R i v e r ) . However, i n those  populations  i t was d e t e c t e d at f r e q u e n c i e s o f 0.19, 0.62 and 0.46 r e s p e c t i v e l y (Table 15).  T h i s locus averaged 1.30 a l l e l e s p e r p o p u l a t i o n . The  P. brachystemon  two a l l e l e s d e t e c t e d at PGM-3 were unique t o and were t h e r e f o r e d i a g n o s t i c o f the s p e c i e s .  39 d e s c r i b e s t h e d i s t r i b u t i o n fixed  o f these  alleles.  PGM-3  115  Figure  was e i t h e r  o r o c c u r r e d with a f r e q u e n c y 2 0 . 9 5 i n nine o f t h e t e n p o p u l a t i o n s . 182  surveyed.  P o p u l a t i o n 18 (Campbell R i v e r ) was monomorphic f o r PGM-3  Since no o t h e r p o p u l a t i o n s from as f a r n o r t h as Campbell R i v e r were.-rsurveyed, i t was i m p o s s i b l e t o determine i f t h i s p a t t e r n represents, a l l e l e r e p l a c e ment along a l a t i t u d i n a l a simple  c l i n e because  were a l s o found f u r t h e r study,  gradient.  However, t h i s  t h e two p l a n t s c o l l e c t e d 182  t o be homozygous f o r PGM-3  .  does not appear t o be  a t Cape Perpetua,. Oregon,  Unfortunately,  one can only s p e c u l a t e whether., t h e r e i s a  b a s i s f o r t h i s polymorphism o r i f i t i s merely t h e r e s u l t effect  or d r i f t  without  selective o f founder  i n a small population.  Four a l l e l e s were s c o r e d  at PGI-2 ( F i g u r e 40). P G I - 2  was the most common a l l e l e i n a l l p o p u l a t i o n s w h i l e t h e o t h e r  1 0 0  three  80,  PGI-3  1  13  9  10  7(1)  7(3)  13(1)  16(2)  Population  F i g u r e 38. The d i s t r i b u t i o n o f PGI-3 a l l e l e s among p o p u l a t i o n s o f P. braohystemon.  PGM-3 100  _  .80  U c <D  rj 0) L  .60  J  U. ^  .40  4)  7(1)  7(3)  15(1)  Population  F i g u r e 39. The d i s t r i b u t i o n o f PGM-3 a l l e l e s among p o p u l a t i o n s o f P. braohystemon.  16  18  8 1 .  PGI-2  1  13  9  7(1)  10  7(3)  15(1)  15(2)  16  Population  •Figure 40. The d i s t r i b u t i o n o f PGI-2 a l l e l e s among p o p u l a t i o n s o f P. brachystemon.  EST-1 .80  J  .40  J  114  -  100«  89"  —1  -i 14(1)  1—-••—»-—i 14(2)  15(1)  15(2)  17  7(2)  Population  F i g u r e 41. The d i s t r i b u t i o n o f EST-1 a l l e l e s among p o p u l a t i o n s o f P. congesta.  18  82.  a l l e l e s were r a r e or l o c a l l y endemic. were maintained  within local  4.2.2,P. oongesta  On t h e average, 1.50 a l l e l e s  populations.  - None o f t h e l o c i surveyed  had t h i s  level  of gene d i v e r s i t y .  4.3  S l i g h t l y polymorphic  or monomorphic  4.3.1 P. braohystemon  with H^,< 0.050  - E i g h t l o c i were monomorphic or n e a r l y  so: PGM-2, EST-2, MDH-2, IDH-1, ME-1, PGI-1, SOD and 6PG. was d e t e c t e d at any o f these p o p u l a t i o n 7(3) (Malahat Although  l o c i except  PGM-2.  No v a r i a t i o n  A single plant i n  Dr.) was homozygous f o r a n u l l  allele.  PGM-2, EST-2 and MDH-2 were not monomorphic i n P.  congesta,  the a l l e l e s which were f i x e d i n P. braohystemon  were t h e most common  alleles  A l l populations  at t h e homologous l o c i i n P. oongesta.  o f both s p e c i e s , were f i x e d f o r t h e same a l l e l e s  surveyed  at IDH-1, ME-1,  SOD and 6PG.  4.3.2 P. oongesta  - Two l o c i , EST-1 and MDH-2, were s l i g h t l y  polymorphic w h i l e f o u r l o c i , IDH-1, ME-1, SOD and 6PG showed no v a r i a t i o n . A l l e l e f r e q u e n c i e s at EST-1 are p l o t t e d i n F i g u r e 41. T h i s l o c u s was o f p a r t i c u l a r i n t e r e s t because both alleles.  taxa share t h e same t h r e e  However, EST-1 was t h e most v a r i a b l e locus i n P.  braohystemon  (Hj, = 0.659) w h i l e i t was one o f t h e l e a s t v a r i a b l e l o c i i n P. (Hp = 0.020).  On t h e average, p o p u l a t i o n s maintained  oongesta  1.40 a l l e l e s .  87 At MDH-2, a r a r e a l l e l e , MDH-2 7(2)  (Malahat  was d e t e c t e d  D r i v e ) and 15(1) (Nanoose H i l l ) w i t h  i n populations  frequencies o f  83.  0.05 and 0.01 r e s p e c t i v e l y at  IDH-1, ME-1,  (Table 16).  MDH-2  1UU  and the a l l e l e s  fixed  SOD and 6PG were a l s o f i x e d i n P. brachystemon.  4.4 Summary T a b l e 17 compares t h e mean a l l e l e f r e q u e n c i e s and t o t a l gene d i v e r s i t y v a l u e s (H^,) f o r a l l polymorphic  loci  surveyed.  IDH-1,  ME-1, SOD and 6PG were a l s o surveyed but were found t o be monomorphic i n both s p e c i e s . P. brachystemon  In a d d i t i o n , no v a r i a t i o n was d e t e c t e d a t PGI-1 i n although i t was polymorphic i n t h e o t h e r s p e c i e s .  U n f o r t u n a t e l y , PGI c o u l d not be s c o r e d c o n s i s t e n t l y i n P.  congesta  and was t h e r e f o r e e x c l u d e d from.the study. In P. congesta, t h i r t y - t h r e e at t h e twelve l o c i  a l l e l e s were d e t e c t e d  surveyed, an average o f 2.75 a l l e l e s p e r l o c u s . In  P. brachystemon, t h i r t y  a l l e l e s were d e t e c t e d a t f i f t e e n  l o c i , an  average o f 2.00 a l l e l e s p e r l o c u s . When o n l y t h e twelve l o c i s c o r e d i n b o t h s p e c i e s a r e compared, twenty-three a l l e l e s were d e t e c t e d i n P. brachystemon; an average o f 1.92 a l l e l e s p e r l o c u s . P. congesta had a p p r o x i m a t e l y 30% more a l l e l e s P. brachystemon  at the twelve l o c i  surveyed.  Fourteen of i t s t h i r t y -  t h r e e a l l e l e s , 42%, were not s h a r e d w i t h P. brachystemon. alleles  than  can be d e f i n e d as common, r a r e o r endemic.  These unique  Common a l l e l e s  were those d e t e c t e d i n at l e a s t f i v e p o p u l a t i o n s o r w i t h a mean frequency >:0.05.  Rare a l l e l e s were those which were found i n l e s s  than f i v e p o p u l a t i o n s and had a mean frequency < 0.05.  Endemics were  a l l e l e s d e t e c t e d at low frequency i n a s i n g l e p o p u l a t i o n . Nine isozymes  84.  (EST-2 PGM-3  1 2 6  , E S T - 2 , L A P - 1 , MDH-1 7 4  and P G M - 3  135  8 0  100  139  , MDH-1  125  , PGM-2  118  ) , 27% o f the a l l e l e s s c o r e d i n P.  were common b u t unique t o t h i s s p e c i e s . 77 and PGM-3 ) , 9%, were r a r e and unique.  , PGM-2 , 81  oongesta,  87 117 (MDH-2 , PGM-1 , 94 49 (PGM-1 and PGM-3 ) ,  Three a l l e l e s Two a l l e l e s  6%, were endemics. In P. braohystemon,  .four o f i t s twenty-three  a l l e l e s , 17%,  100 were n o t found and  PGM-3  115  i n P. congesta.  ) were common.  Three o f these  The f o u r t h , ( P G M - 2  d e t e c t e d i n o n l y a s i n g l e p l a n t . Nineteen species.  1 0 0 n  , PGM-3  ) , was an endemic  a l l e l e s were p r e s e n t  i n both  However, seven o f t h e s e , 37%, were d e t e c t e d at s i g n i f i c a n t l y  d i f f e r e n t f r e q u e n c i e s i n t h e two t a x a Populations and  (MDH-1  182  a l l e l e frequency  (Table 17).  o f t h e two s p e c i e s were d i f f e r e n t i a t e d by a l l e l e s  d i f f e r e n c e s at three  loci  (EST-2, MDH-1 and PGM-3).  These l o c i were c o n s i d e r e d t a x o n o m i c a l l y d i a g n o s t i c . The two s p e c i e s c o u l d a l s o be d i f f e r e n t i a t e d by t h e i r PGI a c t i v i t y patterns.  In a d d i t i o n , with t h e e x c e p t i o n o f a s i n g l e p l a n t , PGM-2  was monomorphic i n a l l P. braohystemon i n a l l b u t one P. oongesta were d e t e c t e d i n both present  populations.  P. braohystemon.  and polymorphic  None o f the PGM-3 a l l e l e s  s p e c i e s and a l l e l e s unique t o each taxon  a t MDH-1, PGM-2 and EST-2.  i n P. congesta  populations  were  G e n e r a l l y , the most common a l l e l e  was e i t h e r f i x e d o r a l s o the most common a l l e l e i n However, t h i s was not t h e case at EST-1, MDH-1 and  PGM-3. In P. braohystemon,  a l l e l e frequencies.and  single  locus  h e t e r o z y g o s i t i e s f l u c t u a t e d widely:among p o p u l a t i o n s . However, t h e r e were no d i s c e r n a b l e macro-geographic p a t t e r n s t o a l l e l e  distributions,  85.  except  p o s s i b l y MDH-1  than i n P. congesta  and PGM-3.  and d i f f e r e n t  In c o n t r a s t t o P. brachystemon, P. congesta  P o p u l a t i o n s maintained  were m a i n t a i n e d  fewer  alleles  a l l e l e s were often, f i x e d i n p o p u l a t i o n s .  a majority o f the a l l e l e s detected i n  w i t h i n each l o c a l p o p u l a t i o n .  Allele  f r e q u e n c i e s and s i n g l e locus h e t e r o z y g o s i t i e s f l u c t u a t e d among p o p u l a t i o n s but not with t h e amplitude suggest  found  t h a t i n P. brachystemon,  populations which occurs  and/or r e c o m b i n a t i o n i n P.  congesta.  i n P. brachystemon.  These  observations  the l e v e l s o f gene flow among within populations  a r e lower than t h a t  86.  5.0  ANALYSES AND  DISCUSSION  This chapter c o n s i s t s of f i v e s e c t i o n s . s e c t i o n , analyses  and  t h e i r r a t i o n a l e are e x p l a i n e d , d a t a  and the r e s u l t s d i s c u s s e d . the b r e e d i n g  W i t h i n each  S e c t i o n 5.1  presented  d e s c r i b e s the a n a l y s i s o f  system and the c a l c u l a t i o n o f o u t c r o s s i n g r a t e s i n  P. braohystemon  and P. oongesta.  S e c t i o n 5.2  discusses  levels  o f g e n e t i c v a r i a b i l i t y w i t h i n p o p u l a t i o n s , c o n t r a s t i n g the species.  S e c t i o n 5.3  analyzes  v a r i a t i o n w i t h i n each taxon. and i n t e r - s p e c i f i c e s t i m a t e s T h i s s e c t i o n has  the h i e r a r c h i c a l d i s t r i b u t i o n S e c t i o n 5.4  of  compares i n t r a - s p e c i f i c  o f g e n e t i c i d e n t i t y and g e n e t i c d i s t a n c e .  f o u r s u b - s e c t i o n s which u t i l i z e these i n d i c e s  t o make i n f e r e n c e s r e l e v a n t t o v a r i o u s taxonomic and questions.  two  L a s t l y , s e c t i o n 5.5  summarizes the d a t a  evolutionary presented  i n the p r e v i o u s f o u r sections;, i e m p h a s i z i n g d i f f e r e n c e s between the two s p e c i e s and  d e s c r i b i n g the g e n e t i c consequences o f  t h e i r c o n t r a s t i n g breeding s t r a t e g i e s .  5.1 Analysis  of the breeding The b r e e d i n g  system  system o f any taxon can be .estimated  W r i g h t ' s f i x a t i o n index, F (Wright,  1965).  The  fixation  using  index  measures the e x t e n t t o which observed g e n o t y p i c d i s t r i b u t i o n s from Hardy-Weinberg p r o p o r t i o n s  ; ' (i.e.  F = 1- 5 * 2pqn  2 2 p , 2pq, q ) .  - '  where H i s the observed number o f h e t e r o z y g o t e s expected number o f h e t e r o z y g o t e s  deviate  and  2pqn i s the  assuming Hardy-Weinberg e q u i l i b r i u m .  87.  V a l u e s o f F range from -1 t o +1. percentage o f heterozygotes  Negative values i n d i c a t e a  i n excess o f e x p e c t a t i o n s and  p o s i t i v e values represent a d e f i c i e n c y o f heterozygotes.  Wright's  e q u i l i b r i u m law d e s c r i b e s observed genotype d i s t r i b u t i o n s i n terms o f a l l e l e f r e q u e n c i e s and t h e f i x a t i o n index F: genotype frequency  AA  2 - p +pqF  Aa  aa  2pq(l-F)  2 q +pqF.  When F=0, t h e genotype d i s t r i b u t i o n i s d e s c r i b e d by a HardyWeinberg e q u i l i b r i u m .  D e v i a t i o n s from Hardy-Weinberg p r o p o r t i o n s  (F) a r e t h e r e s u l t o f t h e j o i n t e f f e c t o f a l l f a c t o r s a c t i n g on a l l e l e s and genotypes a t a g i v e n l o c u s : non-random m a t i n g , s e l e c t i o n , gene f l o w and g e n e t i c d r i f t .  mutation,  When i n b r e e d i n g i s t h e  o n l y f a c t o r c a u s i n g d e v i a t i o n s from e x p e c t a t i o n s , F i s i d e n t i c a l to the c l a s s i c a l inbreeding c o e f f i c i e n t .  F analysis  measures t h e p r o b a b i l i t y t h a t u n i t i n g gametes, t a k e n a t random from a p o p u l a t i o n , w i l l be r e l a t e d by d e s c e n t .  This value i s  d e f i n e d as t h e c o r r e l a t i o n between u n i t i n g gametes. F i x a t i o n indexes were c a l c u l a t e d f o r each p o l y m o r p h i c l o c u s w i t h i n each p o p u l a t i o n . These v a l u e s a r e p r e s e n t e d i n T a b l e 18 f o r P. brachystemon a d d i t i o n , t h e s t a t i s t i c F.  and i n T a b l e 19 f o r P. congesta.-. In was c a l c u l a t e d f o r each l o c u s .  This  is  v a l u e i s d e f i n e d as t h e average c o r r e l a t i o n between u n i t i n g gametes r e l a t i v e t o t h e i r own l o c a l p o p u l a t i o n . of F^  s  ( i . e . few h e t e r o z y g o t e s )  Large v a l u e s  i n d i c a t e that w i t h i n l o c a l  p o p u l a t i o n s , a l a r g e p e r c e n t a g e o f t h e u n i t i n g gametes a r e r e l a t e d  88.  TABLE. 18. Wrights F i x a t i o n .Index  (F)  1  f o r Seven Polymorphic L o c i i n  P. brachystemon. POPULATION  EST-1  LAP-1  1  .911  1.000  13  .911  1.000  9  .599  Locus PGI-2  PGI-3  PGM-1  1.000  1.000  PGM-2  .917  1.000  10 7(1) 1.000  7(3)  1.000  1.000  1.000  1.000 1.000  .742  15(1)  1.000  1.000  1.000  1.000  not scored  16  1.000  .798  18  =  .865  .988  1.000  1.000  .894  1.000  15(2)  F? is  MDH-1  1.000  .949  1.000  .968  1.000  H F = 1F.  : H = observed number of heterozygotes; 2pqn = expected number. 2pqn = weighted mean of F f o r a l l populations  89.  TABLE  19. Wright's F i x a t i o n Index ( F ) f o r Eight Polymorphic  Loci  1  in P. oongesta.  POPULATION  EST-1  -  2(1) 2(2)  9  PGM-3  .042  .143  .291  .631  .232  .219  -.005  .145  .378  -.174  .091  .116  .583  .352  .408  -  .571 .458  .198  .249  -.085  -  .409  -.063  .400  .367  .228  -  - .018  .004  .188  .014  .340  .433  .345  .310  .224  -  .125  .310  .244  -  .072  -.011  -.017  -  .147  .417  .103  .375  .072  -.326  .040  8  PGM-2  -.047  -  7(2)  PGM-1  -  .709  11  MDH-2  .022  -  10  MDH-1  -.508  5 4  LAP-1  -  .057  3  EST-2  -.041  .221  .007  -.099  .399  .432  .078  .350  .251  .ISO  -.001  .295  .215  .547  .240  .510  .119  .215  14(2)  -  .227  .641  .194  -  -  .083  15(1)  -.064  -.032  .368  .067  -.282  .221  .124  .226  15(2)  -  -.182  -.053  -.166  -  .526  .307  .250  -.389  -.003  .552  .082  -  .100  -.060  -.095  .398  .155  14(1)  17  F? is  =  1  F = 1  2  F ^ = weighted mean o f F f o r a l l populations s  .180  .096'  .032  1.000  .300  .128  .188  - : H = observed number o f heterozygotes; 2 pqn = expected number. 2pqn  90.  by d e s c e n t . are s t r i k i n g .  The  d i f f e r e n c e s i n F^  s  At a l l p o l y m o r p h i c  where v a r i a t i o n was  v a l u e s f o r the two l o c i surveyed,  (LAP-1).  except MDH-2  r a r e , l o c a l p o p u l a t i o n s o f P. congesta  an average d e f i c i e n c y o f h e t e r o z y g o t e s 40%  species  between 10%  In c o n t r a s t , l o c a l p o p u l a t i o n s o f P.  had an average d e f i c i e n c y o f h e t e r o z y g o t e s  show  (EST-2) and brachystemon  between 86%  (EST-1)  and 100%  (MDH-1, PGI-3 and PGM-2).  observed  l e v e l s o f h e t e r o z y g o s i t y w i t h i n p o p u l a t i o n s o f the  two s p e c i e s suggests  T h i s d i f f e r e n c e i n the  t h a t i n P. brachystemon,  a much l a r g e r  p e r c e n t a g e o f the u n i t i n g gametes are r e l a t e d . When p o p u l a t i o n s are a t e q u i l i b r i u m and non-random mating i s the o n l y f a c t o r a f f e c t i n g the d i s t r i b u t i o n o f genotypes a t a l o c u s , the f i x a t i o n i n d e x o u t c r o s s i n g frequency F=  — 1+t  (F) i s r e l a t e d t o the  ( t ) a c c o r d i n g t o the r e l a t i o n s h i p  (Nei and Syakudo, 1958).  O u t c r o s s i n g r a t e s can be c a l c u l a t e d from F by t r a n s f o r m i n g  the  above e q u a t i o n t o t=  (Nei and Syakudo, 1958).  1+  r  T h i s method assumes t h a t p o p u l a t i o n s are a t e q u i l i b r i u m and t h e r e f o r e i s not a r i g o r o u s e s t i m a t o r o f t ( J a i n , 1979). S i n c e t i s a f u n c t i o n o f F, s i n g l e l o c u s e s t i m a t e s o f t are s e n s i t i v e to e v o l u t i o n a r y f o r c e s , b e s i d e s b r e e d i n g system, which a f f e c t genotype f r e q u e n c i e s w i t h i n p o p u l a t i o n s  (Workman, 1969).  However,  b r e e d i n g system s h o u l d be the o n l y f a c t o r which a f f e c t s a l l l o c i within a population equally.  Consequently,  when F i s averaged  over  91.  a number of l o c i and t h i s F i s used t o c a l c u l a t e o u t c r o s s i n g ,  the  r e s u l t i n g t , i f not an a b s o l u t e e s t i m a t o r , i s c e r t a i n l y a r e l a t i v e e s t i m a t o r o f o u t c r o s s i n g among p o p u l a t i o n s . d e v i a t i o n from Hardy-Weinberg p r o p o r t i o n s  The  average net  (F) and the  outcrossing  r a t e ( t ) were c a l c u l a t e d f o r each p o p u l a t i o n and are p r e s e n t e d T a b l e 20 f o r P. braohystemon  and T a b l e 21 f o r P.  in  oongesta.  W r i g h t ' s f i x a t i o n i n d e x can be c a l c u l a t e d o n l y f o r polymorphic l o c i .  C o n s e q u e n t l y , the number o f l o c i c o n t r i b u t i n g  t o the o u t c r o s s i n g e s t i m a t e s f r e q u e n c y was  not  v a r i e s among p o p u l a t i o n s . An  l i s t e d f o r P. braohystemon  p o p u l a t i o n 16  Qualicum F a l l s ) because the o n l y v a r i a t i o n s c o r e d was  detected  PGM-3).  i n two p l a n t s , h e t e r o z y g o u s at two  i n the  loci  u n r e a l i s t i c a l l y high f o r t h i s species The  between 0 and  estimates  population  (PGI-2 and  7.4%  the e s t i m a t e s  (t=0.988).  of o u t c r o s s i n g f o r P. braohystemon  (Francis Park).  the n i n e p o p u l a t i o n s  was  2.4  The  ± 0.8%  ranged from a low o f 42%  P o i n t -2 ) t o a h i g h of 100%  These e s t i m a t e s  were  average (± s t a n d a r d  error)  o u t c r o s s i n g . In P.  oongesta  ( F r a n c i s Park and  ( M i l l H i l l Park).  The  Jack's  average  o u t c r o s s i n g f r e q u e n c y f o r the f i f t e e n p o p u l a t i o n s was  et al.  (Little  T h i s apparent anomaly produced an o u t c r o s s i n g r a t e  which was  for  outcrossing  70.2  ±4.8%.  are i n e x c e l l e n t agreement w i t h those o f Ganders  (1977a, 1977b) and Carey and Ganders ( i n p r e s s ) , who .  u t i l i z e d a seed wing dimorphism o f known i n h e r i t a n c e t o o u t c r o s s i n g i n both taxa.  U s i n g t h i s polymorphism and  t e s t method of H a r d i n g (1970),  they found o u t c r o s s i n g  estimate the progeny  92.  TABLE  20.  E s t i m a t e s o f O u t c r o s s i n g Frequency f o r Ten P o p u l a t i o n s o f P.  POPULATION  braohystemon.  F  f .• 2  1  1  .922  .041  13  .924  .040  9  .863  .074  10  1.000  .000  7(1)  1.000  .000  7(3)  1.000  .000  15(1)  .959  .021  15(2)  .948  .027  16  -  -  .011  .978  18  .024±.008  AVERAGE  F = w e i g h t e d mean o f F f o r a l l p o l y m o r p h i c 2  t = 1-P — 1+F  loci  93. TABLE  21.  E s t i m a t e s o f O u t c r o s s i n g Frequency f o r F i f t e e n of  POPULATION  P.  congesta.  ......F  .  t  2  2(1)  .105  .801  2(2)  .307  .530  3  .146  .745  5  -.037  4  .319  .516  9  .407  .421  10  .252  .597  11  .146  .745  7(2)  .244  .608  8  .121  .784  14(1)  .137  .759  14(2)  .406  .422  15(1)  .090  .835  15(2)  .151  .738  17  .028  .946  1.077  AVERAGE:  F = weighted mean o f F f o r a l l p o l y m o r p h i c 2  t  =  l _ F 1+F  Populations  .702±.048  loci  94.  i n P. bvaahystemon t o average 2% and o u t c r o s s i n g i n P. congesta t o range from 48% t o 80% w i t h a mean o f 70%. The  d i f f e r e n c e between t h e mean o u t c r o s s i n g r a t e s f o r  t h e s e two c l o s e l y r e l a t e d s p e c i e s was h i g h l y s i g n i f i c a n t but n o t s u r p r i s i n g .  The observed d i v e r g e n c e  i n breeding  (PiO.Ol) strategies  had been a n t i c i p a t e d on t h e b a s i s o f f l o r a l morphology and f i e l d observations  and confirmed  the e a r l i e r outcrossing estimates i n  these t a x a .  5.2 Genetic vaviability  within  populations  In o r d e r t o e v a l u a t e t h e consequences o f c o n t r a s t i n g breeding  s t r a t e g i e s on t h e p o p u l a t i o n s t r u c t u r e o f t h e two t a x a ,  f i v e g e n e t i c parameters were measured w i t h i n each p o p u l a t i o n . The  s t a t i s t i c s used t o d e s c r i b e l e v e l s o f g e n e t i c v a r i a b i l i t y w i t h i n  l o c a l populations  are the percentage o f l o c i that are polymorphic ,  average number o f a l l e l e s p e r l o c u s , average number o f a l l e l e s p e r p o l y m o r p h i c l o c u s , expected p e r c e n t a g e o f h e t e r o z y g o u s  loci  per i n d i v i d u a l and t h e observed p e r c e n t a g e o f h e t e r o z y g o u s l o c i per individual. then  These v a l u e s were c a l c u l a t e d s e p a r a t e l y f o r each p o p u l a t i o n  averaged o v e r a l l p o p u l a t i o n s  (± s t a n d a r d  error)  f o r P. bvachystemon and P. congesta i n T a b l e s 22 and 23 respectively. The  proportion of l o c i  that are polymorphic p e r p o p u l a t i o n  v a r i e d i n P. bvachystemon between 6.67% ( P r o s p e c t  Lake) and 40%  (Malahat D r i v e - 2 ) w i t h an average o f 20.76 ± 3.49%. P. congesta was n o t as g r e a t .  P o p u l a t i o n 14(2)  The range i n  (Jack's  Point)  95.  Table 22.  Population  Summary o f Various Genetic Parameters  of Plectritis brachystemon.  Percentage Loci Polymorphic  1  Average Number Alleles/ Locus  Average Number Alleles/ Polymorphic Locus  f o r Ten Populations  Expected Percentage o f Heterozygous Loci/ Individual (H) 2  Observed Percentage o f Heterozygous Loci/ Individual  1  13 .33  1..27+ .18  3 .00+ .00  5.3113.79  .311 .31  13  20 .00  1 .20+...11  2 .00± .00  6.2113.82  .471 .33  9  33 .33  1 .33+.,22  2 .001 .00  3.0912.05  .421..23  10  6 .67  1..07+..07  2,.00+,.00  0.631.63  .001,.00  7(1)  13 .33  1..13±..09  2 .00+,.00  3.9313.32  .001..00  7(3)  40..00  1,.47±..17  2,. 17±, .17  14.0515.49  .001,.00  15(1)  33..33  1,.40+..16  2.,20±,.20  11.37+4.58  .741.,74  15(2)  20..00  1.,27+.,15  2.,33±.,33  5.9214.01  .551.,40  16  14..28  1..14+.,10  2.,00±.,00  1.361.92  13..33  1..20±. 14  2,.501..17  6.9914.77  .741.,74  1.251.04  2.221.10  5.89+1.33  .451.13  18  3  AVERAGE 20.76+3.49  1  2  1.301. 88  The frequency o f the most common a l l e l e i s < .99. tjT f °  rr  l o c i ; h=l-Ex?) expressed as a percentage.  3  A l l estimates with t h i s p o p u l a t i o n based on a sample o f 14 l o c i (EST-1 Excluded)  96.  Table 23.  Percentage Loci Polymorphic  Population  •  Summary o f Various Genetic Parameters f o r F i f t e e n Populations o f Plectritis oongesta.  2(1)  50 .00  1  Average Number Alleles/ Locus  2 .08± .50  Average Number Alleles/ Polymorphic Locus  Expected Percentage o f Heterozygous Loci/ Individual 2  CH)  3 .17+ .79  18 .97±7 .78  Observed Percentage o f Heterozygous Loci/ Individual v  obs.  IS .9216.30  2(2)  50 .00  2 .08± .43  3,.17+ .60  21 .13+8 .14  14 .2815.35  3  58,.33  2 .171 .39  3 .00± .44  24 .5417 .52  20 .4616.50  5  50,.00  1 .67± .26  2 .33+ .33  21 .5017 .41  22,.2817.85  4  SO,.00  2 .00+..43  3 .00+ .63  26 .49+8,.05  17 .78+6.11  9  58..33  2 .00+..30  2 .71+ .29  25 ,75±7,.78  16,.3314.98  10  50..00  2 .00±,.43  3,.00±,.63  27 .04±8,.44  20..0516.40 21..58+6.61  11  50..00  1,.92+,.42  2,,83±,.65  26,.01+8..23  7(2)  66.,67  2,,17±..34  2,,75±,.37  25,.92+7.12  18.,9615.02  8  50. 00  2..08±..43  .60 3., 17±.  21.,38±7.,37  18.,2315.96  14(1)  50. 00  2.,00+..43  3..00±..63  18..85±7.,36  15.,21+5.92  14(2)  41. 67  1..7S±.,41  2.,80±.,80  18.,1117. 57  10. 4214.85  15(1)  66. 67  2.08±. 34  2..63±.,38  23..4117. 14  19. 19+5.60  15(2)  50. 00  1.,50±.,15  2.,00+.,00  18. 6816. 42  IS. 6515.61  17  58. 33  2. 17±. 42  3. 001. 53  22. 78±7. 63  19. 7916.67  1.98+. OS  2.84±. 08  22. 641.79  17. 741.81  AVERAGE  53.33+ 1.78  iThe frequency o f the most common a l l e l e i s < 99. 2(H=  =  lj  f  o  r  l o c i ; h = 1- x?) expressed as a percentage.  97.  w i t h 41.67%, had t h e lowest p e r c e n t a g e o f l o c i t h a t were p o l y m o r p h i c . P o p u l a t i o n s 7(2) (Malahat D r i v e ) and 15(1) (Nanoose H i l l ) w i t h 66.67%, had t h e h i g h e s t p e r c e n t a g e o f l o c i t h a t were p o l y m o r p h i c . average  f o r t h e f i f t e e n p o p u l a t i o n s was 5.3.33 ± 1.78%.  The  The  d i f f e r e n c e between t h e means f o r t h e two s p e c i e s was h i g h l y significant  (P_0.01).  The  average number o f a l l e l e s d e t e c t e d p e r l o c u s i n  P. brachystemon ranged between 1.07 ( P r o s p e c t Lake) and 1.47 (Malahat D r i v e - 3 ) w i t h a mean o f 1.25 t 0.04.  The v a l u e s i n  P. congesta were c o n s i s t e n t l y h i g h e r w i t h a low o f 1.67 ( M i l l and a h i g h o f 2.17 (John Dean Park, Malahat D r i v e and N i l e  Hill)  Creek).  The mean f o r P. congesta was 1.98* 0.05 and t h i s was s i g n i f i c a n t l y l a r g e r than t h e mean observed  i n P. brachystemon  (P^O.01).  When average number o f a l l e l e s p e r p o l y m o r p h i c  l o c u s was  c a l c u l a t e d , P. congesta was a g a i n c o n s i s t e n t l y more v a r i a b l e than P. brachystemon.  The range i n t h e s e l f e r was between 2.00,  found i n f i v e p o p u l a t i o n s , and 2.33 (Nanoose H i l l - 2 ) . P. brachystemon averaged 2.22 t 0.10 a l l e l e s . p e r p o l y m o r p h i c  locus.  The  corresponding  v a l u e s i n t h e o u t c r o s s e r were from 2.00 (Nanoose H i l l - 2 ) t o 3.17 ( A n a c o r t e s - 1 , -2 and C r o f t o n e x i t ) . 2.84 ± 0 . 0 8 ,  and again t h e d i f f e r e n c e between t h e two t a x a was  highly significant The individual  .The mean f o r P. congesta. was  (P<0.01).  expected p e r c e n t a g e o f h e t e r o z y g o u s l o c i p e r  (H) was c a l c u l a t e d from t h e observed  w i t h i n each p o p u l a t i o n .  allele  frequencies  This i s the best s i n g l e s t a t i s t i c , f o r  describing levels of genetic v a r i a b i l i t y w i t h i n populations  98.  (Nei  and Roychoudhury, 1974)  and i s d e f i n e d i n the next s e c t i o n .  P. brachystemon, H ranged between 0.63%  In  (Pospect Lake) and  14.05% (Malahat D r i v e - 3 ) w i t h an average v a l u e o f 5.89  - 1.33%.  In P. congesta, expected h e t e r o z y g o s i t y v a r i e d between 18.11% ( J a c k ' s P o i n t - 2 ) and 27.04% 22.64 _ 0.79%.  ( P r o s p e c t Lake) f o r an aver.age o f  T h i s v a l u e was  +  s i g n i f i c a n t l y h i g h e r than  mean c a l c u l a t e d f o r the s e l f e r  the  (PiiO.Ol).  A l t h o u g h the a b s o l u t e v a l u e o f h e t e r o z y g o s i t y was i n P. brachystemon, the r e l a t i v e range o f v a l u e s was than t h a t observed  i n P. congesta.  collections  T h i s was  much l a r g e r  The magnitude o f the f l u c t u a t i o n s  among p o p u l a t i o n s o f the s e l f e r suggests h e i g h t e n e d differentiation.  lower  population  p a r t i c u l a r l y s t r i k i n g when P. brachystemon  from s y m p a t r i c s i t e s  7(1):7(3)  15(1):15 (2) (Nanoose H i l l ) were compared.  (Malahat D r i v e )  and  These s y m p a t r i c  p a i r s had  large d i f f e r e n c e s i n t h e i r l e v e l s of detected  variation  (H) and y e t t h e y were s e p a r a t e d by d i s t a n c e s o f o n l y  t h i r t y meters a t Malahat D r i v e and no g r e a t e r than one hundred meters at Nanoose H i l l  (the e x a c t d i s t a n c e i s unknown).  The  magnitude  o f t h e s e observed d i f f e r e n c e s i s i n t e r p r e t e d t o be a r e s u l t o f the b r e e d i n g system.  Autogamy r e s t r i c t s r e c o m b i n a t i o n and gene f l o w  v i a p o l l e n v e c t o r s and t h e r e f o r e enhances the r e l a t i v e c o n t r i b u t i o n s o f founder e f f e c t  and d r i f t i n d e t e r m i n i n g p o p u l a t i o n s t r u c t u r e .  The observed percentage individual  (H ^ Q  S  ) was  o f heterozygous  c a l c u l a t e d by d i r e c t  loci  count and  per the  d i f f e r e n c e s between the two s p e c i e s was h i g h l y s i g n i f i c a n t  99.  (P<0.01). The range i n P. braohystemon  was from zero  (Prospect  Lake, Malahat D r i v e - 1 and -3) t o 1.30% ( L i t t l e Qualicum F a l l s ) w i t h a mean o f o n l y 0.45 * 0.13%.  I n P. oongesta,  H  ^  v a r i e d between 10.42% ( J a c k ' s P o i n t - 2 ) and 22.28% ( M i l l  Hill)  w i t h a mean o f 17.74 - 0.81%. Outcrossing  r a t e s and l e v e l s o f expected  heterozygosity  (H) were compared among p o p u l a t i o n s w i t h i n each s p e c i e s . T h i s i s p l o t t e d i n F i g u r e 42 f o r P. braohystemon P. oongesta. and Pinus  and F i g u r e 43 f o r  However, as was found i n Limnanthes  oontorta  alba  ( J a i n , 1978),  (Yeh and L a y t o n , 1979), t h e r e was no c l e a r  r e l a t i o n s h i p between e s t i m a t e d o u t c r o s s i n g r a t e s and g e n e t i c v a r i a b i l i t y among p o p u l a t i o n s o f t h e same t a x o n .  Intuitively,  one might p r e d i c t a p o s i t i v e r e l a t i o n s h i p between these two parameters and t h e r e c o u l d be s e v e r a l reasons f o r t h e l a c k o f c o r r e l a t i o n . Sample s i z e s w i t h i n p o p u l a t i o n s may have been t o o s m a l l t o d e t e c t differences. Yeh  and Layton  Jain  (1978) genotyped 20-30 f a m i l i e s p e r p o p u l a t i o n ,  (1979) genotyped 15 f a m i l i e s p e r p o p u l a t i o n and  from 15 t o 60 p l a n t s were genotyped p e r p o p u l a t i o n of Secondly,  Pleotritis.  t h e range o f o u t c r o s s i n g r a t e s among p o p u l a t i o n s o f  the same s p e c i e s may be t o o s m a l l t o be a c c u r a t e l y measured w i t h the e s t i m a t o r s used.  Although  the technique  employed by J a i n  (1978)  does n o t assume t h a t t h e p o p u l a t i o n s a r e i n Hardy-Weinberg e q u i l i b r i u m , t h e method used t o c a l c u l a t e o u t c r o s s i n g i n l o d g e p o l e p i n e (Yeh and L a y t o n , 1979) and Pleotritis Any d e v i a t i o n s between t h e observed heterozygotes  makes t h a t assumption.  and expected  frequencies of  are a t t r i b u t e d s o l e l y to the rate of outcrossing.  .10.0,. 30.0-  ^  o  25.0  >> 20.04  ."t_  'to  O  O) 15.0  >> N  O fr (D  (D x:  10-0  5.0  .01  .02  .03  .04  — i —  .05  .06  — i — .07  outcrossing rate  ~08~  .09  —i  .90  1.00  .10  F i g u r e 42. The r e l a t i o n s h i p between t h e o u t c r o s s i n g r a t e and t h e e x p e c t e d h e t e r o z y g o s i t y among p o p u l a t i o n s of* "P. brachystemon.  .10  .20  .30  .40  .50  .60  outcrossing rate  .70  .80  F i g u r e 4.3. The r e l a t i o n s h i p between t h e o u t c r o s s i n g r a t e and t h e expected h e t e r o z y g o s i t e among p o p u l a t i o n s o f P. congesta.  101.  T h i s assumption a l l o w s the.combined i n f l u e n c e o f o t h e r f a c t o r s such as m i c r o - h a b i t a t s e l e c t i o n , h e t e r o s i s , Wahlund e f f e c t and s t o c h a s t i c f o r c e s t o m i n i m i z e b r e e d i n g system d i f f e r e n c e s . L a s t l y , t h e i n d i c a t o r used t o measure l e v e l s o f v a r i a t i o n among p o p u l a t i o n s p r e s e n t s problems because e s t i m a t e s o f h e t e r o z y g o s i t y have a l a r g e s t a n d a r d e r r o r . T h i s occurs because each l o c u s does n o t contribute equally to a species' heterozygosity.  Some l o c i  are monomorphic w h i l e o t h e r s a r e h i g h l y p o l y m o r p h i c .  Consequently,  w i t h o u t a v e r y l a r g e sample o f l o c i , t h e S.E. a s s o c i a t e d w i t h the e s t i m a t e o b l i t e r a t e s s u b t l e d i f f e r e n c e s i n t h e l e v e l s o f v a r i a b i l i t y between p o p u l a t i o n s o f t h e same taxon  (Figures  42 and 4 3 ) . Although  no r e l a t i o n s h i p was d e t e c t e d between o u t c r o s s i n g  r a t e s and l e v e l s o f h e t e r o z y g o s i t y among p o p u l a t i o n s o f t h e same s p e c i e s , t h e d a t a suggest determinant  t h a t t h e b r e e d i n g system i s a major  of population structure.  The c o n t r a s t i n t h e  o u t c r o s s i n g r a t e s o f these two t a x a i s p a r a l l e l e d by  observed  d i f f e r e n c e s i n l e v e l s o f v a r i a t i o n maintained w i t h i n populations. As measured by a l l p a r a m e t e r s , p o p u l a t i o n s o f P. congesta m a i n t a i n significantly  higher levels of genetic v a r i a b i l i t y  than do  p o p u l a t i o n s o f i t s autogamous r e l a t i v e , P. bvachystemon. s i t e v a r i a t i o n was c o n t r o l l e d by comparing o n l y those  When  populations  o f b o t h t a x a growing s y m p a t r i c a l l y , a l l d e t e c t e d d i f f e r e n c e s remained significant.  102.  5.3  Analysis  of gene  diversity  The l e v e l o f genie v a r i a b i l i t y and i t s h i e r a r c h i c a l o r g a n i z a t i o n can be q u a n t i f i e d w i t h t h e a n a l y s i s o f gene d i v e r s i t y ( N e i , 1973, 1975).  This a n a l y s i s permits genetic v a r i a t i o n t o  be p a r t i t i o n e d between d i f f e r e n t h i e r a r c h i c a l l e v e l s o f p o p u l a t i o n structure  (i.e. w i t h i n v e r s u s between l o c a l o r s u b - p o p u l a t i o n s ) .  This technique  i s a m o d i f i c a t i o n o f Wright's F - s t a t i s t i c s  expanded t o m u l t i p l e a l l e l e s . However, u n l i k e F - a n a l y s i s , i t i s not .dependent on t h e d e t e c t i o n o f genotype f r e q u e n c i e s because i t does n o t measure d e v i a t i o n s between observed and expected levels of heterozygosity. The b a s i s o f t h e a n a l y s i s i s t h e c a l c u l a t i o n o f expected,  or t h e o r e t i c a l , heterozygosity per locus (h): h = l-Ix , l 2  where x^ i s t h e frequency question.  of the i ^  T h i s i s designed  a l l e l e at the locus i n  t o measure t h e amount o f g e n e t i c  v a r i a t i o n a t a locus w i t h i n a population.  In t h i s use, the  terms h e t e r o z y g o s i t y and gene d i v e r s i t y a r e synonymous. The 2 concepts o f h e t e r o z y g o s i t y and homozygosity (j=Ex^) i n p o p u l a t i o n s were o r i g i n a l l y d e v e l o p e d w i t h r e s p e c t t o random mating p o p u l a t i o n s . I f a p o p u l a t i o n  i s i n Hardy-Weinberg e q u i l i b r i u m ,  the expected h e t e r o z y g o s i t y equals t h e observed h e t e r o z y g o s i t y (h= h ) . Q  When e q u i l i b r i u m c o n d i t i o n s a r e n o t met, as when m a t i n g  i s non-random, h does n o t equal t h e frequency i n the population.  o f heterozygotes.  I n o r d e r t o a v o i d c o n f u s i o n when s t u d y i n g  p o p u l a t i o n s w i t h non-random mating systems, N e i (1973) proposed  103.  the term "gene d i v e r s i t y " f o r t h e h e t e r o z y g o s i t y s t a t i s t i c (H) and "gene i d e n t i t y " f o r t h e homozygosity s t a t i s t i c ( J ) . The expected h e t e r o z y g o s i t y o f a p o p u l a t i o n i s e s t i m a t e d by H =  I  h  k/ r  k=l which i s t h e average o f h over t h e r l o c i sampled.  T h i s can be  i n t e r p r e t e d as t h e average p r o p o r t i o n o f h e t e r o z y g o t e s p e r l o c u s o r t h e p r o p o r t i o n o f l o c i a t which an i n d i v i d u a l can be expected t o be h e t e r o z y g o u s . The o r g a n i z a t i o n o f gene d i v e r s i t y w i t h i n a taxon can be d e s c r i b e d by t h e r e l a t i o n s h i p H  T  =  H  s sr +D  The t o t a l gene d i v e r s i t y  (FLjJ can be p a r t i t i o n e d i n t o t h e gene  d i v e r s i t y w i t h i n l o c a l p o p u l a t i o n s (Hg) and t h a t between l o c a l p o p u l a t i o n s (Dg^).  These v a l u e s a r e c a l c u l a t e d f o r each l o c u s  over a l l p o p u l a t i o n s sampled, then averaged over a l l l o c i t o g e t grand e s t i m a t e f o r t h e t a x o n s t u d i e d . ILj, e s t i m a t e s t h e t o t a l genie v a r i a t i o n sampled over a l l p o p u l a t i o n s as i t i s a f u n c t i o n o f mean a l l e l e f r e q u e n c i e s ; H  T  =  1  " ^ i -  i s t h e mean f r e q u e n c y o f t h e i " * 1  populations.  1  a l l e l e over a l l sampled  T h i s s t a t i s t i c was c a l c u l a t e d from t h e d a t a i n T a b l  17. H  i s an e s t i m a t e o f t h e average amount o f g e n e t i c  v a r i a t i o n maintained w i t h i n l o c a l populations. I f n populations are s u r v e y e d , t h e average gene 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 a t a  104.  p a r t i c u l a r locus i s h  n  H„ = E S  k = 1  k/ n  where h^ i s the expected h e t e r o z y g o s i t y i n t h e k  local  population. I f a l l p o p u l a t i o n s a r e members o f a s i n g l e l a r g e p a n m i c t i c u n i t and t h e r e i s no s e l e c t i o n f o r l o c a l a d a p t a t i o n , then a l l a l l e l e s w i l l be e q u a l l y d i s t r i b u t e d over t h e e n t i r e range o f t h e taxon and H  w i l l equal H .  However, i n n a t u r e  this  does not o c c u r . N a t u r a l p o p u l a t i o n s tend t o d i f f e r e n t i a t e time i n t o s u b - p o p u l a t i o n s random d r i f t H^. by D  as a r e s u l t o f m u t a t i o n ,  and r e s t r i c t e d gene f l o w .  over  selection,  Therefore, H  i s a subset  The l e v e l o f d i f f e r e n t i a t i o n between p o p u l a t i o n s i s measured , the gene d i v e r s i t y between p o p u l a t i o n s , and i s  g i v e n by D  ST  =  H  T  " SH  The g r e a t e r the l e v e l o f s u b - d i v i s i o n w i t h i n t h e t a x o n , t h e l a r g e r the v a l u e o f Dg^The r e l a t i v e amount o f g e n e t i c d i f f e r e n t i a t i o n between l o c a l p o p u l a t i o n s (G G  ) can be e s t i m a t e d by  O 1  ST  = °ST/ H ' /  T  T h i s v a l u e v a r i e s from zero t o one and i s termed t h e c o e f f i c i e n t o f gene d i f f e r e n t i a t i o n .  The s a m p l i n g v a r i a n c e o f Gg^ can be  c a l c u l a t e d ( C h a k r a b o r t y , 1974), a s t a n d a r d e r r o r o b t a i n e d and the s i g n i f i c a n c e o f the g e n e t i c d i f f e r e n t i a t i o n  assessed.  105.  The a n a l y s e s o f gene d i v e r s i t y i n P. brachystemon and P. congesta a r e p r e s e n t e d i n T a b l e s 24 and 25 r e s p e c t i v e l y .  There  i s a l a r g e amount o f i n t e r - l o c u s v a r i a t i o n i n h e t e r o z y g o s i t y i n b o t h s p e c i e s which i s r e f l e c t e d by .the magnitude o f t h e s t a n d a r d e r r o r s o f t h e means.  S i m i l a r r e s u l t s have been o b t a i n e d i n most  o t h e r organisms t^hat . have been e x t e n s i v e l y surveyed f o r electrophoretic v a r i a b i l i t y .  The s t a n d a r d e r r o r s a r e o f t e n one-  f i f t h t o t w o - f i f t h s as l a r g e as t h e mean h e t e r o z y g o s i t i e s ( L e w o n t i n , 1974).  S i n c e a l l l o c i do n o t c o n t r i b u t e e q u a l l y t o a s p e c i e s  average gene d i v e r s i t y , i t i s i m p o r t a n t t o s u r v e y as l a r g e a number l o c i as p o s s i b l e i n o r d e r t o r e a l i s t i c a l l y (Nei  and Roychoudhury,  e s t i m a t e t h i s parameter  1974).  The v a l u e s o f H^, for" P. brachystemon were between zero and 0.659 w i t h a mean o v e r a l l l o c i o f 0.166+0.058. I n P. congesta, t h e v a l u e s ranged between zero and 0.792 w i t h a mean o f 0.266+0.083. A l t h o u g h P. brachystemon g e n e r a l l y h a s l e s s t o t a l gene d i v e r s i t y at  each l o c u s t h a n P. congesta, t h e p a t t e r n o f h e t e r o z y g o s i t y among  l o c i o f each s p e c i e s i s s i m i l a r . exceptions. yet  However, t h e EST l o c i a r e n o t a b l e  Both s p e c i e s share t h e same t h r e e a l l e l e s a t EST-1  t h i s l o c u s had t h e l a r g e s t H^ v a l u e o f a l l l o c i s u r v e y e d i n  P. brachystemon and one o f t h e lowest v a l u e s observed i n P. congesta In..contrast, EST-2 had t h r e e a l l e l e s and was e x t r e m e l y  heterozygous  i n P. congesta w h i l e i t was monomorphic i n P. brachystemon. E s t e r a s e s a r e a l a r g e and heterogeneous c l a s s o f enzymes which a r e i d e n t i f i e d e l e c t r o p h o r e t i c a l l y s o l e l y by t h e i r a b i l i t y  to react  106.  Table 24 .  A n a l y s i s o f Gene D i v e r s i t y and Degree o f D i f f e r e n t i a t i o n f o r 15 L o c i i n Pleotritis  Locus  T o t a l Gene Diversity (Hp)  braohystemon.  Gene D i v e r s i t y Within Populations  ay  Proportion o f I n t e r p e l l a t i o n Gene Differentiation (G ) ST  EST-1  .659  .210  .681  EST-2  .000  .000  .000  IDH-1  .000  .000  .000  LAP-1  .422  .218  .483  MDH-1  .484  .119  .754  MDH-2  .000  .000  .000  ME -1  .000  .000  .000  PGI-1  .000  .000  .000  PGI-2  .096  .085  .115  PGI-3  .226  .128  .434  PGM-1  .412  .118  .714  PGM-2  .000  .008  .000  PGM-3  .196  .017  .913  SOD  .000  .000  .000  6PG  .000  .000  .000  Combining A l l L o c i : .166±.058  .060±.021  ,639±.053  107.  T a b l e . 2 5 . A n a l y s i s o f Gene D i v e r s i t y and Degree o f D i f f e r e n t i a t i o n f o r 12 L o c i i n Pleotritis congesta.-  ..Locus  T o t a l Gene Diversity (H ) T  Gene D i v e r s i t y Within Populations CH ) S  Proportion of I n t e r p o p u l a t i o n Gene Differentiation (G ) S T  EST-1  .020  .020  .000  EST-2  .502  .321  .361  IDH-1  .000  .000  .000  LAP-1  .792  .671  .153  MDH-1  .509  .468  .081  MDH-2  .000  .008  .000  ME-1  .000  .000  .000  PGM-1  .431  .365  . 153  PGM-2  .439  .391  .109  PGM-3  .504  .472  .063  SOD  .000  .000  .000  6PG  .000  .000  .000  Combining A l l  Loci:  .266±.083  .226±.071  . 1501.039  108.  w i t h a c l a s s o f s y n t h e t i c . s u b s t r a t e s . The d i f f e r e n t i a l l o s s o f allozyme a c t i v i t y a t EST^l f o l l o w i n g the a p p l i c a t i o n o f M a l a t h i o n s u g g e s t s t h a t a l l o z y m e s a t EST-1 and EST-2 may r e p r e s e n t d i f f e r e n t f u n c t i o n a l c l a s s e s . o f EST.  I f ' t h e s e polymorphisms a r e a d a p t i v e ;'  (Johnson, 1976), t h i s , c o n t r a s t i n g o r g a n i z a t i o n o f d i v e r s i t y i n EST may r e p r e s e n t an e v o l u t i o n a r y two  divergence o f adaptive s t r a t e g i e s i n the  s p e c i e s . However, whether t h i s observed d i f f e r e n c e l i s o f  p h y s i o l o g i c a l s i g n i f i c a n c e or the r e s u l t o f genetic d r i f t i s purely speculative. The was  d i f f e r e n c e between t h e mean H^ v a l u e s o f t h e two s p e c i e s  compared u s i n g a t - t e s t w i t h 25 d.f. and found t o be n o n - s i g n i f i c a n t  (P<0.40). The n o n - s i g n i f i c a n c e  o f t h e d i f f e r e n c e between H  T  averaged  o v e r a l l l o c i may be a t t r i b u t e d p a r t i a l l y t o t h e l a r g e s t a n d a r d e r r o r o f t h e means. i t s high  However, t h i s comparison demonstrates t h a t  despite  l e v e l o f i n b r e e d i n g , P. brachystemon m a i n t a i n s a l e v e l o f  t o t a l v a r i a t i o n which i s somewhat r e d u c e d , b u t comparable t o t h e p r e d o m i n a n t l y o u t c r o s s e d P. congesta. I t i s t h e gene 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 , r e f l e c t s t h e c o n t r a s t i n g b r e e d i n g systems.  H , which  Inter-locus v a r i a t i o n i n .  Hg was a g a i n l a r g e . V a l u e s ranged from zero t o 0.218 i n P. brachystemon and  zero t o 0.671 i n P. congesta.  When averaged over a l l l o c i , t h e  mean g e n e t i c d i v e r s i t y w i t h i n l o c a l p o p u l a t i o n s  was 0.060±0.021 and  0.226±0.071 f o r P. brachystemon and P. congesta r e s p e c t i v e l y . The mean Hg i s e q u a l t o t h e mean e x p e c t e d h e t e r o z y g o s i t y over a l l p o p u l a t i o n s .  Consequently, H  q  (H) averaged  can be i n t e r p r e t e d as the  109.  average p r o p o r t i o n o f h e t e r o z y g o t e s  per locus or the p r o p o r t i o n  o f l o c i a t which an i n d i v i d u a l can be expected the average, P. oongesta P. braohystemonwas  t o be h e t e r o z y g o u s .  was h e t e r o z y g o u s a t 23% o f i t s l o c i  h e t e r o z y g o u s a t o n l y 6%.  On  while  Despite the i n t e r - l o c u s  v a r i a t i o n , t h e d i f f e r e n c e between t h e means was s i g n i f i c a n t  (P<0.05).  T h i s i l l u s t r a t e s t h a t , the l e v e l o f p o p u l a t i o n s u b - d i v i s i o n i n t h e s e l f e r i s much l a r g e r than t h e l e v e l i n t h e o u t c r o s s e r . d i v e r s i t y w i t h i n populations {i.e.  (H ) d e c r e a s e s ,  differentiating).populations  As t h e gene  t h e gene d i v e r s i t y between  (Dg-jO i n c r e a s e s .  Accordingly,  the r e l a t i v e magnitude, of- gene d i f f e r e n t i a t i o n between p o p u l a t i o n s (Gg^)  a l s o i n c r e a s e s because i t i s d e s c r i b e d by t h e r e l a t i o n s h i p % r  =  D  S.T/_  When G P. braohystemon respectively. (P<0.01).  HT  was averaged over a l l l o c i , t h e v a l u e s f o r  and P. oongesta  were 0.639+0.053 and 0.150+0.039  The d i f f e r e n c e , between t h e means was h i g h l y s i g n i f i c a n t  I n P. braohystemon,  64% o f t h e t o t a l gene d i v e r s i t y c o u l d be  a t t r i b u t e d t o i n t e r - p o p u l a t i o n a l d i f f e r e n c e s . T h i s compared t o o n l y 15% i n . P . congesta:-  I n o t h e r words, p o p u l a t i o n s o f P.  congesta  m a i n t a i n , on t h e average, 85% o f t h e v a r i a t i o n d e t e c t e d over t h e sampled range o f t h e s p e c i e s . I n c o n t r a s t , s i n g l e p o p u l a t i o n s o f P. braohystemon  m a i n t a i n an average o f o n l y 36% o f t h e s e l f e r ' s  a r r a y o f detected, v a r i a t i o n . Although  G O  i s a good measure o f t h e r e l a t i v e amount 1  o f gene d i f f e r e n t i a t i o n between p o p u l a t i o n s , i t s v a l u e i s h i g h l y dependent on  ( N e i , 1975). When  i s low,  may o v e r e s t i m a t e  the magnitude o f i n t e r p o p u l a t i o n a l gene d i f f e r e n t i a t i o n .  Although  110.  FLj, was n o t found t o be s i g n i f i c a n t l y d i f f e r e n t between t h e two t a x a , a n o t h e r measure o f p o p u l a t i o n d i f f e r e n t i a t i o n was c a l c u l a t e d . S t a n d a r d g e n e t i c distance., D ( N e i , 1972) e s t i m a t e s codon d i f f e r e n c e s p e r l o c u s between p o p u l a t i o n s . o f t h e gene 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 - a n d  t h e number o f n e t I t i s independent  t h e r e f o r e can be used  f o r comparing t h e degree o f gene d i f f e r e n t i a t i o n i n d i f f e r e n t organisms.  The average g e n e t i c d i s t a n c e (D ± i n t e r - l o c u s S.E.) among  p o p u l a t i o n s o f P. brachystemon and P. congesta was 0.137+0.083 and 0.052±0.034 r e s p e c t i v e l y . t h e r e were a p p r o x i m a t e l y  I n o t h e r words, f o r every 100 l o c i ,  5.2 codon d i f f e r e n c e s between p o p u l a t i o n s  o f P. congesta and 13.7 codon d i f f e r e n c e s between p o p u l a t i o n s o f P. brachystemon.  Although  t h e D between p o p u l a t i o n s o f t h e s e l f e r was  more than two and a h a l f times g r e a t e r than t h e c o r r e s p o n d i n g  value  i n t h e o u t c r o s s e r , t h e . d i f f e r e n c e was n o n - s i g n i f i c a n t (P<"0.40). However, t h i s l a c k o f s i g n i f i c a n c e can be a t t r i b u t e d t o t h e i n t e r locus v a r i a t i o n i n heterozygosity.  5.4  Comparisons of genetic Nei's  identity  and genetic  distance  (1972, 1975) i n d e x o f g e n e t i c i d e n t i t y  ( I ) and  s t a n d a r d g e n e t i c d i s t a n c e (D) were c a l c u l a t e d f o r a l l . ' . i n t r a - s p e c i f i c and i n t e r - s p e c i f i c p a i r w i s e c o m b i n a t i o n s o f p o p u l a t i o n s .  There were  four objectives i n t h i s a n a l y s i s : i.  t o assess t h e r e l a t i v e l e v e l s o f i n t e r - p o p u l a t i o n a l d i f f e r e n t i a t i o n w i t h i n each s p e c i e s and c o r r e l a t e t h i s with the breeding  system;  111.  ii.  t o determine i f g e n e t i c d i s t a n c e r e f l e c t s  geographic  d i s t a n c e between p o p u l a t i o n s ; iii.  t o c a l c u l a t e t h e i n t e r - s p e c i f i c s i m i l a r i t y o f t h e two t a x a i n o r d e r t o e v a l u a t e t h e i r taxonomic  relationsip  . and i v . to determine i f sympatric p o p u l a t i o n s are g e n e t i c a l l y more s i m i l a r than o t h e r i n t e r - s p e c i f i c  combinations.  The purpose o f t h i s comparison was t o t e s t f o r t h e possible selective  maintenance o f t h e observed  isozyme  polymorphisms. N e i ' s g e n e t i c i d e n t i t y s t a t i s t i c compares p o p u l a t i o n s f o r b o t h t h e presence/absence o f i d e n t i c a l a l l e l e s and t h e i r occurrence  a t s i m i l a r f r e q u e n c i e s . T h i s measure i s c a l c u l a t e d f o r  each l o c u s :  ^'(P ) )(£(P ) ) 2  2  j x  where P_.^ and  j y  are the frequencies o f the j  a l l e l e i n populations  X and Y r e s p e c t i v e l y . The numerator i s c a l c u l a t e d f o r a l l a l l e l e s at t h e l o c u s and then d i v i d e d by t h e geometric mean o f i d e n t i c a l homozygote f r e q u e n c i e s .  Two p o p u l a t i o n s which have i d e n t i c a l  f r e q u e n c i e s a t a l o c u s , have a g e n e t i c i d e n t i t y o f one.  allele  Their  g e n e t i c i d e n t i t y i s zero i f they have no . a l l e l e s in-.common.  When  a l l gene l o c i a r e c o n s i d e r e d , t h e g e n e t i c i d e n t i t y between two p o p u l a t i o n s i s c a l c u l a t e d by  112.  /"IXEp?  1  ij  ) (EEp^  * j  x  YL ' 1  i j ijy  The numerator, I , i s an i n d e x o f g e n e t i c s i m i l a r i t y  ( S o k a l and  s  Sneath,  1963): LK I  =  EE P. . . P. . . s L i j - IJX ijy In b o t h the numerator and denominator, P.. T-  and P..  IJX  corresponding  f r e q u e n c i e s o f the j  p o p u l a t i o n s X and Y.  are  the  ijy  ^ a l l e l e a t the i ^  n  locus i n  L i s the number o f l o c i surveyed.  Spiess  (1977)  p r e s e n t s a more d e t a i l e d d i s c u s s i o n o f the t h e o r y and c a l c u l a t i o n o f this  statistic. The  s t a n d a r d g e n e t i c d i s t a n c e between p o p u l a t i o n s X and  i s d e f i n e d as D = - l o g I . e  This i s approximately  e q u a l t o 1-1.  Y  This  v a l u e e s t i m a t e s the accumulated number o f gene s u b s t i t u t i o n s per l o c u s , between p o p u l a t i o n s , assuming the r a t e o f gene s u b s t i t u t i o n s is identical for a l l loci. The  g e n e t i c i d e n t i t i e s and s t a n d a r d g e n e t i c d i s t a n c e s between  p o p u l a t i o n s o f P. braohystemon Tables  and P. oongesta  are p r e s e n t e d  26 and.27 r e s p e c t i v e l y . A l l c a l c u l a t i o n s were based on  f r e q u e n c i e s from f i f t e e n l o c i i n P. braohystemon  in allele  and t w e l v e l o c i i n  P. oongesta.  P o p u l a t i o n 16  was  from a l l g e n e t i c d i s t a n c e c a l c u l a t i o n s because a l l e l e  excluded  f r e q u e n c i e s f o r EST-1  ( L i t t l e Qualicum F a l l s ) o f P.  were not  available.  braohystemon  T a b l e 26.  Genetic i d e n t i t y  ( I ) and s t a n d a r d g e n e t i c d i s t a n c e (D)  between p o p u l a t i o n s o f P.'.^brachystemon 1 1  13  9  10  7(1)  7(3)  15(1)  15(2)  18  0.9030  0.8665  0.8687  0.8468  0.8709  0.8690  0.8708  0.6856  0.9422  0.8687  0.9113  0.9451  0.9291  0.9112  0.8174  0.8657  0.9167  0.9136  0.9466  0.9154  0.7830  0.8433  0.8671  0.8366  0.7818  0.7721  0.9492  0.9577  0.9731  0.8174  0.9180  0.9317  0.8617  0.9523  0.8031  13  0. 1020  9  0. 1433  0.0595  10  0. 1408  0.1408  0.1442  7(1)  0. 1663  0.0929  0.0868  0.1704  7(3)  0. 1382  0.0565  0.0904  0.1426  0.0521  15(1)  0. 1404  0.0735  0.0549  0.1784  0.0432  0.0856  15(2)  0. 1384  0.0930  0.0884  0.2461  0.0273  0.0707  0.0489  18  0. 3775  0.2016  0.2446  0.2586  0.2016  0.1488  0.2193  0.2646  I pop.  0. 8451  0.9026  0.8923  0.8372  0.9002  0.9066  0.8999  0.8850  0.7869  D pop.  0. 1683  0.1025  0.1140  0.1777  0.1051  0.0981  0.1055  0.1222  0.2396  E s t i m a t e s o f I above t h e d i a g o n a l  I = 0.8720  E s t i m a t e s o f D above t h e d i a g o n a l  D = 0.1370 + 0.0825  0.7675  T a b l e 27.  Genetic i d e n t i t y  ( I ) and s t a n d a r d g e n e t i c d i s t a n c e (D) between p o p u l a t i o n s  o f P. oongesta.  2(1) 2(1)  2(2) .9930  3  5  4  9  10  11  7(2)  8  14(1) 14(2)  15(1)  15(2)  17  .9576  .9269 .9705  .9534  .9555  .9673  .9203  .9396  .9859  .9956  .9133  .8877  .9677  .9427  .9256 .9777  .9723  .9549  .9647  .9154  .9247  .9593  .9820  .8976  .8940  .9479  .9372 .9750  .9403  .9792  .9819  .9510  .9721  .9779  .9701  .9537  .9178  .9578  .9623  .9464  .9859  .9748  .9917  .8831  .9261  .9232  .9802  .9732  .9019  .9857  .9849  .9892  .9528  .9623  .9698  .9831  .9493  .9686  .9462  .9667  .9684  .9185  .9218  .9220  .9541  .9293  .9317  .9359  .9922  .9777  .9488  .9559  .9534  .9730  .9579  .9566  .9741  .9454  .9648  .9687  .9737  .9513  .9606  .8995  .9389  .9219  .9803  .9569  .9031  .9662  .9541  ;?030  .9067  .9586  .9924  .9255  .9116  .9557  .9188  .9076  .9550  .9393  .9097  2(2)  .0070  3  .0433  .0590  5  .0759  .0773  .0649  4  .0299  .0226  .0253  .0384  9  .0477  .0281  .0616  .0551  10  .0455  .0461  .0210  .0142 .0152  .0339  11  .0332  .0359  .0183  .0255 .0109  .0321  .0078  7(2)  .0831  .0884  .0502  .0083 .0484  .0850  .0226  .0262  8  .0623  .0783  .0283  .1243 .0384  .0814  .0526  .0561  ..1059  14(1)  .0142  .0416  .0223  .0768 .0307  .0812  .0451  .0358  .0630  .0344  14(2)  .0044  .0182  .0304  .0799 .0170  .0470  .0477  .0318  .0813  .0470  .0076  15(1)  .0907  .1080  .•0474  .0200 .0520  .0733  .0274  .0267  .0199  .1020  .0774  '.'0847  15(2)  .1191  .1121  .0858  .0272 .0319  .0707  .0430  .0499  .0441  .0979  .0925  .0970  .0626  17  '."0328  .0535  .0431  .1033 .0553  :0663  .(0444  .0402  .1019  .0423  .0453  .0460  .0946  .1418  I pop-i. .9520  .9461  .9580  .9451 .9697  .9460  .9672  .9698  .9425  .9344  .9534  .9553  .9387  .9261  .9370  D pop. :.0492  .0554  .0429  .0565 .0308  .0555  .0333  .0307  .0592  .0679  .0477  .•0457  .0633  .0767  .0651  .0144  E s t i m a t e s o f I above t h e d i a g o n a l  I = 0.9493  E s t i m a t e s o f D above t h e d i a g o n a l  D = 0.0520 ± 0.0344  .8678  115.  5,.4.1 Levels of inter-population  differentiation  Levels of i n t e r - p o p u l a t i o n a l d i f f e r e n t i a t i o n w i t h i n t a x a can.be compared b y . c a l c u l a t i n g mean g e n e t i c i d e n t i t i e s p a i r w i s e combinations  ( I ) from a l l  o f p o p u l a t i o n s . On the average, p o p u l a t i o n s  P. brachystemon were g e n e t i c a l l y more d i s t i n c t  (I = 0.8720 than  o f P. conge,sta ( t = 0.9493). Based on a sample o f t w e n t y - e i g h t  of  populations species,  G o t t l i e b (1977a) e s t i m a t e d the mean i d e n t i t i e s o f i n t r a - s p e c i f i c p l a n t p o p u l a t i o n s t o be 0.951.02.  He t h e r e f o r e concluded  that i n general,  a sample o f one o r a few p o p u l a t i o n s c o n s t i t u t e an adequate sample of a species v a r i a b i l i t y , . is i n  While the v a l u e o f I f o r P.  congesta  e x c e l l e n t agreement w i t h G o t t l i e b ' s e s t i m a t e , the c a l c u l a t e d  v a l u e f o r P'. brachystemon/is  much lower.  In f a c t , i t i s lower  than  e s t i m a t e s o f I between f i v e c o n g e n e r i c s p e c i e s p a i r s ( G o t t l i e b , 1977a) : Clarkia  biloba  and C. lingulata  (1=0.88), Gaura longiflora  G. demareei ( I = 0.99), Hymenopappus scabrosaeus and H. (I = 0.90), Oenothera strigosa  i n Plectritis  artemisiaefolius  and 0. biennis '(I = 0.97), and  Stephanomeria exigua subsp. coronaria The observed  and  and S. malheurensis  ( I = 0.94).  d i f f e r e n c e s i n the mean g e n e t i c i d e n t i t i e s  r e f l e c t s the r e l a t i v e degree o f p a r t i t i o n i n g  p o o l o f each s p e c i e s . In o r d e r f o r I t o be l a r g e , l o c a l must m a i n t a i n a l a r g e p r o p o r t i o n o f the taxon's  i n the gene  populations  array of v a r i a t i o n .  On the a v e r a g e , 85% o f the v a r i a t i o n i n P. congesta- was  detected  w i t h i n each p o p u l a t i o n . I n c o n t r a s t , P. brachystemon p o p u l a t i o n s c o n t a i n e d o n l y 34% o f the v a r i a t i o n o f the s p e c i e s . Large f l u c t u a t i o n s i n a l l e l e f r e q u e n c i e s between p o p u l a t i o n s were much more p r e v a l e n t in  the s e l f e r than the o u t c r o s s e r ( F i g u r e s 27-40).  The  '..  116.  d i s t r i b u t i o n o f many a l l e l e s i n P.•brachystemon might b e s t be d e s c r i b e d as a "random w a l k . "  This pattern of a l l e l e d i s t r i b u t i o n i s explained  most e a s i l y t o be a consequence o f t h e b r e e d i n g  system. Autogamy r e s t r i c t s  gene f l o w and lowers t h e e f f e c t i v e p o p u l a t i o n s i z e . T h i s enhances t h e importance o f s t o c h a s t i c f o r c e s i n d e t e r m i n i n g  a l l e l e frequencies w i t h i n  p o p u l a t i o n s . T h i s i s n o t t o s a y t h a t founder e f f e c t and g e n e t i c are n o t i m p o r t a n t  w i t h i n predominantly  drift  outcrossing taxa, only that  these  f o r c e s become r e l a t i v e l y more i m p o r t a n t when gene f l o w i s reduced.  5.4.2 Comparisons between genetic distance  and geographic  distance Genetic distance estimates  a r e summarized i n T a b l e 28 f o r  P. brachystemon and i n T a b l e 29 f o r P. congesta. I n a d d i t i o n , dendrographs (McCammon, 1968) were g e n e r a t e d u s i n g t h e e s t i m a t e s these a r e p r e s e n t e d  i n Figures.44  r e s p e c t i v e l y . Although  o f g e n e t i c i d e n t i t y and  and 45 f o r P. brachystemon and P. congesta  c e r t a i n comparisons suggest a r e l a t i o n s h i p between  g e o g r a p h i c d i s t a n c e and g e n e t i c d i s t a n c e , o v e r a l l t h e r e appears t o be no c l e a r r e l a t i o n s h i p between these two p a r a m e t e r s . These o b s e r v a t i o n s a r e i n agreement w i t h those o f K a h l e r et al. (1980), who found t h a t isozyme v a r i a b i l i t y i n Avena barbata was d i s t r i b u t e d i n mosaic p a t t e r n s which were not r e l a t e d t o g e o g r a p h i c d i s t a n c e . I n P. brachystemon, p o p u l a t i o n 1 (Sumas Mountain) and p o p u l a t i o n 18 (Campbell R i v e r ) a r e s e p a r a t e d by t h e g r e a t e s t g e o g r a p h i c d i s t a n c e and a l s o had t h e l a r g e s t g e n e t i c d i s t a n c e (D = 0.3775). However, p o p u l a t i o n 18 was t h e most g e n e t i c a l l y d i s s i m i l a r o f a l l P. brachystemon p o p u l a t i o n s  (D = 0.2396). T h i s l a r g e D i s comparable  t o some i n t e r - s p e c i f i c comparisons w i t h i n o t h e r genera. I t r e f l e c t s  117.  T a b l e 28. Population  Summary o f G e n e t i c D i s t a n c e s D  i n P. brachystemon  Most s i m i l a r t o  Most d i s t a n t from  Population  Population  D  0.1020  18  0.3775  13  D  1  0.1683  13  0.1025  7(3)  0.0565  18  0.2016  9  0.1140  15(1)  0.0549  18  0.2446  10  0.1777  0.1408  18  0.2586  1 and 13  7(1)  0.1051  15(2)  0.0273  18  0.2016  7(3)  0.0981  7(1)  0.0521  18  0.1488  15(1)  0.1055  7(1)  0.0432  18  0.2193  15(2)  0.1222  7(1)  0.0273  18  0.2646  18  0.2396  7(3)  0.1488  1  0.3775  118.  T a b l e 29.  ipulation  Summary o f g e n e t i c d i s t a n c e s i n P. congesta D  Most s i m i l a r t o Population  d i s t a n t: from  Most D  Population  D  2(1)  0.0492  14(2)  0.0044  15(2)  0.1191  2(2)  0.0554  2(1)  0.0070  15(2)  0.1121  3  0.0429  0.0210  15(2)  0.0858  5  0.0565  4  0.0308  9  10 7(2)  0.0083  8  0.1243  11  0.0109  17  0.0553  0.0555  4  0.0144  7(2)  0.0850  10  0.0333  11  0.0078  8  0.0526  11  0.0307  10  0.0078  8  0.0561  7(2)  0.0592  5  0.0083  8  0.1059  8  0.0679  3  0.0283  5  0.1243  14(1)  0.0477  14(2)  0.0076  15(2)  0.0925  14(2)  0.0457  2(1)  0.0044  15(2)  0.0970  15(1)  0.0633  7(2)  0.0199  2(2)  0.1080  15(2)  0.0768  5  0.0272  17  0.1418  17  0.0651  2(1)  0.0328  15(2)  0.1418  0.19,  GENETIC 1.00 0  0.952  0.928  0.904  1  1  1  1  1  15(2)  '  7(3)  (I)  0.976  7(1),  15(1)  IDENTITY  1  1  13 9  1  10  18  F i g u r e 44. A dendrograph d e p i c t i n g t h e g e n e t i c r e l a t i o n s h i p s among the sampled P. bvaahystemon p o p u l a t i o n s ( p o p u l a t i o n 16 e x c l u d e d ) : c o r r e l a t i o n s a r e based on N e i ' s Index o f G e n e t i c I d e n t i t y c a l c u l a t e d the a l l e l e f r e q u e n c i e s a t f i f t e e n l o c i .  from  120.  GENETIC 1 . 0 0 0  I  I D E N T I T Y (I) 0 . 9 7 0  —i  0 . 9 3 9  1  1 0 11  8  1 7  2 ( 2 )  1 4 ( 1 )  1 4 ( 2 ) 2 ( 1 )  1 5 ( 2 )  1 5 ( 1 )  7 ( 2 ) 5  F i g u r e 45. A .dendrograph d e p i c t i n g t h e g e n e t i c r e l a t i o n s h i p s among the sampled P. congesta p o p u l a t i o n s : c o r r e l a t i o n s a r e based on Nei's Index o f G e n e t i c I d e n t i t y c a l c u l a t e d from t h e a l l e l e frequencies a t twelve l o c i .  121.  the o b s e r v a t i o n t h a t t h i s p o p u l a t i o n was monomorphic f o r o r m a i n t a i n e d at r e l a t i v e l y h i g h f r e q u e n c y ,  a l l e l e s which- were g e n e r a l l y e i t h e r 89  r a r e o r absent i n o t h e r P. brachystemon p o p u l a t i o n s MDH-1  113  (Malahat  , PGI-3  3 3 r  and .PGM-3 ).  Sympatric p o p u l a t i o n s  115  D r i v e ) and 1 5 ( 1 ) : 15(3)  (i.e. EST-1  ,  7(1):7(3)  (Nanoose H i l l ) had r e l a t i v e l y low  g e n e t i c d i s t a n c e s : 0.0521 and.0.0489 r e s p e c t i v e l y .  However,  p o p u l a t i o n s 15(1) and 15(2) were more s i m i l a r t o p o p u l a t i o n 7 ( 1 ) , and 7(1) was more s i m i l a r t o 1 5 ( 2 ) , than they were t o t h e i r respective sympatric populations. c o l l e c t e d approximately  P o p u l a t i o n s 7(1) and 7(3) were  30 "m.apart ( a l t h o u g h i n s u c c e s s i v e y e a r s ) .  T h e i r a l l e l e f r e q u e n c i e s were compared u s i n g a t - t e s t Although  ( S p i e s s , 1977).  t h e s e two p o p u l a t i o n s were s e p a r a t e d by such a s m a l l  d i s t a n c e , they m a i n t a i n e d  the f o l l o w i n g a l l e l e s a t s i g n i f i c a n t l y  d i f f e r e n t frequencies: EST-1 , E S T - 1 8 9  and P G M - 1  106  (P<0.01); and E S T - 1  1 0 0  1 1 4  , LAP-1 , LAP-1  , PGI-2  9 5  8 1  and P G I - 2  In a d d i t i o n , these p o p u l a t i o n s had s i g n i f i c a n t l y heterozygosity values  100  ,-PGM-1  100  (PsO.05).  1 0 0  different  (P<0.01) a t EST-1, LAP-1, PGI-2 and PGM-1.  P. brachystemon p o p u l a t i o n s 15(1) and 15(2) were s e p a r a t e d by a d i s t a n c e o f not more than 100 m.(the exact d i s t a n c e i s unknown). W i t h i n these two p o p u l a t i o n s , t h e f o l l o w i n g a l l e l e s were at s i g n i f i c a n t l y d i f f e r e n t f r e q u e n c i e s  (P<0.01); E S T - 1  LAP-1  , PGM-1  1 0 0  , LAP-1  1 0 5  , PGI-3  3 3 r  , PGI-3  1 0 0 n  100  1 0 0  observed , EST-1  and P G M - 1  106  .  These p o p u l a t i o n s a l s o had s i g n i f i c a n t l y d i f f e r e n t h e t e r o z y g o s i t y v a l u e s a t LAP-1, PGI-3 and PGM-1  (P<0.01); and EST-1  (P<0.05). In  c o n t r a s t , P. congesta p o p u l a t i o n s 15(1) and 15(2) were compared  1 1 4  ,  122.  and s i g n i f i c a n t l y d i f f e r e n t f r e q u e n c i e s only LAP-1 , LAP-1 9 1  9 5  and L A P - 1  1 0 0  .  (P£0.01) were d e t e c t e d f o r  These d a t a suggest  that the l e v e l  o f g e n e t i c d i f f e r e n t i a t i o n between p o p u l a t i o n s o f P. bvachystemon:. i s not n e c e s s a r i l y a s s o c i a t e d w i t h the geographic separates  d i s t a n c e which  them. When p o p u l a t i o n s o f P. congesta were compared, t h e  correspondence between geographic  d i s t a n c e and g e n e t i c d i s t a n c e  appeared b e t t e r than t h a t found i n P. bvachystemon.  However,  t h e r e were s t i l l n o t a b l e e x c e p t i o n s t o any p a t t e r n .  F i g u r e 45  d e p i c t s a major dichotomy w i t h i n P. congesta. segregated  Populations  i n t o two unequal sub-groups w i t h p o p u l a t i o n s 15(1)  and 15(2) (Nanoose H i l l ) ,  7(2) (Malahat  D r i v e ) and 5 ( M i l l  Hill  Park) d i f f e r e n t i a t e d from t h e o t h e r e l e v e n p o p u l a t i o n s . T h i s 74 dichotomy was generated  by t h e r e l a t i v e f r e q u e n c y  o f a l l e l e EST-2  and t h e r e was no d i s c e r n i b l e p a t t e r n t o i t s d i s t r i b u t i o n  (Figure 35).  In p o p u l a t i o n s 1 5 ( 1 ) , 1 5 ( 2 ) , 7(2) and 5, i t was d e t e c t e d a t f r e q u e n c i e s >0.75 w h i l e i n t h e o t h e r e l e v e n p o p u l a t i o n s i t was never found w i t h a frequency>0.50.  W i t h i n t h e s m a l l sub-group, p o p u l a t i o n s 15(1)  and 15(2) were much more s i m i l a r t o p o p u l a t i o n s 7(2) and 5 r e s p e c t i v e l y , than they were t o each o t h e r . WA)  P o p u l a t i o n s 2(1) and 2(2) ( A n a c o r t e s ,  o c c u r on a s e r p e n t i n e o u t c r o p  (Kruckeberg,  these p o p u l a t i o n s were q u i t e s i m i l a r  1969).  Although  (D = 0.0070), p o p u l a t i o n 2(1)  was more s i m i l a r t o p o p u l a t i o n 14(2) ( J a c k ' s P o i n t : D--= 0.0044) than i t was t o p o p u l a t i o n 2 ( 2 ) .  P o p u l a t i o n 10 ( P r o s p e c t Lake) and  11 ( V i a d u c t Ave) were c o l l e c t e d . a p p r o x i m a t e l y one km a p a r t  ~ "  123.  and they were g e n e t i c a l l y more s i m i l a r t o each o t h e r than they were t o any o t h e r p o p u l a t i o n s . ( N i l e Creek) was  (D = 0.0078)  However, p o p u l a t i o n  most s i m i l a r t o p o p u l a t i o n 2(1)  (Anacortes,  17 WA)  a l t h o u g h t h e s e p o p u l a t i o n s are s e p a r a t e d by the g r e a t e s t , d i s t a n c e . 1  The  g e n e r a l l a c k o f correspondence between  d i s t a n c e and g e n e t i c d i s t a n c e suggests  geographic  t h a t gene f l o w i s not a major  f a c t o r i n f l u e n c i n g the macro-geographic d i s t r i b u t i o n o f a l l e l e s i n Pleotritis.  Regardless  o f the g e o g r a p h i c  distances involved,  P. oongesta p o p u l a t i o n s were, i n g e n e r a l , g e n e t i c a l l y more s i m i l a r t o each o t h e r than were P. braohystemon p o p u l a t i o n s ; D = 0.0520 and D = 0.1370 r e s p e c t i v e l y . T h i s i n d i c a t e s e i t h e r t h a t gene f l o w between p o p u l a t i o n s i s m o r e . p r e v a l e n t i n the o u t c r o s s e r and/or t h a t as a r e s u l t o f r e c o m b i n a t i o n o f v a r i a b i l i t y due i n the s e l f e r .  w i t h i n p o p u l a t i o n s , the l o s s  t o g e n e t i c d r i f t i s l e s s o f a f a c t o r than i t i s  S i n c e seeds o f b o t h s p e c i e s s h o u l d be e q u a l l y  d i s p e r s i b l e and t h e r e i s no evidence  t o suggest t h a t p o l l e n v e c t o r s  i n P. oongesta are e f f e c t i v e over l a r g e d i s t a n c e s , s t o c h a s t i c f o r c e s are p r o b a b l y more i m p o r t a n t ' t h a n  gene f l o w i n d e t e r m i n i n g  allele  d i s t r i b u t i o n s and. the r e l a t i v e l e v e l s o f g e n e t i c d i f f e r e n t i a t i o n observed  i n these taxa.  However, Lewontin (1974) c a u t i o n s t h a t  even s m a l l m i g r a t i o n r a t e s (i.:e. one i n d i v i d u a l i n a thousand per g e n e r a t i o n ) j a r e s u f f i c i e n t t o p r e v e n t d i f f e r e n t i a t i o n between p o p u l a t i o n s o f moderate s i z e .  124.  5.4.3 Taxonomic. and evolutionary  relationships  A l t h o u g h Morey; (1959, 1962) d i d n o t g i v e P. brachystemon s p e c i f i c r a n k , r e c o g n i z i n g i t as P. congesta s s p . brachystemon (F...§..M.) Morey, t h e isozyme d a t a support'  t h e r e c o g n i t i o n o f two  d i s t i n c t s p e c i e s . Standard g e n e t i c d i s t a n c e was averaged  over a l l  i n t e r - s p e c i f i c p a i r w i s e combinations o f p o p u l a t i o n s (Table 30) so t h a t t h e g e n e t i c d i v e r g e n c e between t h e s e two t a x a c o u l d be compared t o o t h e r s p e c i e s p a i r s o f known taxonomic rank.  These d a t a  were used a l s otooc o n s t r u c t ' a dendrograph (McCammon, 1968) which i s p r e s e n t e d i n F i g u r e 46.  G o t t l i e b (1977a) surveyed isozyme d a t a on  twenty-eight p l a n t species i n order t o evaluate the a p p l i c a t i o n s o f e l e c t r o p h o r e t i c d a t a i n p l a n t s y s t e m a t i c s . Reviewing  available  d a t a f o r t h i r t e e n p a i r s o f r e l a t e d s p e c i e s , he c a l c u l a t e d an average g e n e t i c i d e n t i t y f o r c o n g e n e r i c s p e c i e s , I = 0.67 + 0.07 ( e q u i v a l e n t t o D = 0.40)..This  demonstrates  that i n general,  congeneric species are s i g n i f i c a n t l y m o r e ' d i f f e r e n t i a t e d than s p e c i f i c p o p u l a t i o n s (I = 0 . 9 5 + 0.02).  intra-  Based on a sample o f t w e l v e  l o c i , t h e g e n e t i c i d e n t i t y o f P. brachystemon and P. congesta was I = 0.7311 (D = 0.3132).  This l e v e l o f d i f f e r e n t i a t i o n i s i n  e x c e l l e n t agreement w i t h G o t t l i e b ' s v a l u e f o r c o n g e n e r i c s p e c i e s . However, t h e e v o l u t i o n a r y r e l a t i o n s h i p o f t h e two s p e c i e s i s n o t so c l e a r .  G o t t l i e b had d a t a on t h r e e s p e c i e s p a i r s which were  known t o be r e l a t e d as p r o g e n i t o r and d e r i v a t i v e w i t h t h e d e r i v a t i v e b e i n g o f r e l a t i v e l y r e c e n t o r i g i n : Stephanomeria exiqua subsp. and S. malheurensis ( I = 0.94); Clarkia  biloba  and C.  lingulata  coronaria  125.  GENETIC 1 . 0 0 0  I  c  o  IDENTITY  0 . 9 7 0  0 . 9 3 9  1  1  0 . 9 0 9  1  (I)  0 . 8 7 8  1  1  10  E  <u  18  > u 03 l_ .O Q_-  13 7(3)  15(2)  15(1) 7(1)  F i g u r e 46. A dendrograph d e p i c t i n g t h e g e n e t i c r e l a t i o n s h i p s among the sampled Vlectvitis p o p u l a t i o n s : c o r r e l a t i o n s a r e based on N e i ' s Index o f G e n e t i c I d e n t i t y c a l c u l a t e d from t h e a l l e l e f r e q u e n c i e s at t w e l v e l o c i .  126.  (I = 0.88), and Guara longiflora  and G. demareei ( I = 0.99).  T h i s h i g h average g e n e t i c i d e n t i t y was i n t e r p r e t e d as e v i d e n c e that s h o r t l y a f t e r t h e i r o r i g i n , d e r i v a t i v e species are l i m i t e d g e n e t i c v e r s i o n s of" the p r o g e n i t o r .  I n o t h e r words, r e p r o d u c t i v e  i s o l a t i o n had e v o l v e d w i t h o u t a major g e n e t i c r e o r g a n i z a t i o n . P. braohystemon has been c o n s i d e r e d t o be a d e r i v a t i v e o f P. oongesta.  Y e t , t h e i r g e n e t i c i d e n t i t y was much lower  than  those c a l c u l a t e d f o r t h e t h r e e s p e c i e s p a i r s known t o be r e l a t e d as p r o g e n i t o r and d e r i v a t i v e .  When t h e gene p o o l s o f each s p e c i e s were  compared ( s e c t i o n 4.4),, 42% (14/33) o f t h e a l l e l e s d e t e c t e d i n P. oongesta were n o t s c o r e d i n P. braohystemon.  Only 17% (4/23)  o f t h e s e l f e r ' s a l l e l e s were n o t a l s o found i n t h e o u t c r o s s e r . Nineteen  a l l e l e s were p r e s e n t  i n b o t h s p e c i e s b u t seven o f these were  detected with s i g n i f i c a n t l y d i f f e r e n t frequencies Based on t h e i r l e v e l o f allozyme  (Table 1 7 ) .  differentiation, i t i shighly  u n l i k e l y t h a t P.- braohystemon i s a r e c e n t d e r i v a t i v e o f P. oongesta. The  i n a b i l i t y t o h y b r i d i z e P. oongesta and P. braohystemon (Carey and  Ganders, p e r s o n a l communication) a l s o i n d i c a t e s c o n s i d e r a b l e However, t h e m a j o r i t y o f t h e s e l f e r ' s a l l o z y m e s of the v a r i a t i o n detected i n the outcrosser.  divergence.  (83%) was a sub-set  The average g e n e t i c  d i s t a n c e (D=0.3132) between t h e two s p e c i e s was s m a l l e r t h a n t h e i n t r a - s p e c i f i c genetic distance populations-1  (D=0.3775) b e t w e e n ? , braohystemon  (Sumas Mountain) and 18 (Campbell R i v e r ) .  Therefore,  i t i s p o s s i b l e that the s e l f e r . i s a d e r i v a t i v e o f the outcrosser but t h a t enough t i m e has e l a p s e d s i n c e d i v e r g e n c e  f o r a considerable  127.  amount o f d i f f e r e n t i a t i o n t o have o c c u r r e d .  These o b s e r v a t i o n s  based, however, on the study o f a r e g i o n where b o t h s p e c i e s i n v a d e d s i n c e the r e t r e a t o f P l e i s t o c e n e g l a c i e r s . P. brachystemon  evolved  from P. congesta  o f the g l a c i a l boundary. represent  The  If  , i t p r o b a b l y d i d so s o u t h  species  more s i m i l a r f u r t h e r south: however, t h i s may  species.  are g e n e t i c a l l y  not n e c e s s a r i l y be  I n c o n c l u s i o n , t h e isozyme d a t a c o n f i r m e d the  o f P. brachystemon  not  g e n e t i c v a r i a t i o n o f the two  C o n s e q u e n t l y , i t i s p o s s i b l e t h a t the two  situation.  have  genotypes t h a t moved n o r t h may  a random sample.of the  are  as a d i s t i n c t s p e c i e s . However, i t i s  the  recognition impossible  t o make a d e f i n i t i v e statement on the e v o l u t i o n a r y r e l a t i o n s h i p o f t h e s e two  taxa without sampling populations  i n C a l i f o r n i a and  also  s t u d y i n g o t h e r s p e c i e s i n the genus.  5.4.4  Maintenance  A controversy  has  of observed  isozyme  polymorphisms  developed w i t h i n p o p u l a t i o n  genetics  because o f e f f o r t s t o e x p l a i n the maintenance o f observed enzyme polymorphisms. The  neutralists (neo-classicists) believe  that  most o f the e l e c t r o p h o r e t i c v a r i a t i o n , which i s o b s e r v e d , i s s e l e c t i v e l y n e u t r a l and m a i n t a i n e d by a b a l a n c e between m u t a t i o n r a t e and g e n e t i c d r i f t .  In c o n t r a s t , the s e l e c t i o n i s t s (neo-  D a r w i n i a n s ) b e l i e v e t h a t observed p r o t e i n polymorphisms are maintained. Ayala  A thorough d i s c u s s i o n o f t h e s e i s s u e s can be found i n  (1974), Lewontin (1974), Johnson (1973, 1975,  (1975).  selectively  1976)  and  Powell  128.  G e n e t i c d i s t a n c e s were compared between P.'brachystemon and P. congesta a t s y m p a t r i c and non-sympatric  s i t e s t o determine i f  s y m p a t r i c p o p u l a t i o n s were g e n e t i c a l l y more s i m i l a r than o t h e r i n t e r s p e c i f i c comparisons.  The purpose o f t h i s a n a l y s i s was t o t e s t f o r  the p o s s i b l e s e l e c t i v e maintenance o f isozyme polymorphisms. The r a t i o n a l e f o r t h i s c a p p r o a c h was t h a t i f both t a x a share t h e same a l l e l e s and t h e observed  a l l o z y m e polymorphisms a r e i n f l u e n c e d by  s e l e c t i o n , s i m i l a r environmental  regimes a t t h e s y m p a t r i c s i t e s  should  f a v o r t h e same a l l e l e s i n b o t h s p e c i e s . T h i s would be r e f l e c t e d by t h e i r g r e a t e r g e n e t i c s i m i l a r i t y . G e n e t i c d i s t a n c e s between s y m p a t r i c p o p u l a t i o n s a r e l i s t e d i n i t a l i c s i n T a b l e 30. The mean s t a n d a r d g e n e t i c d i s t a n c e f o r s y m p a t r i c comparisons was D = 0.3393 w h i l e i t was D = 0.3120 f o r n o n - s y m p a t r i c  comparisons.  d a t a do n o t s u p p o r t t h e assumption t h a t t h e observed are s e l e c t i v e l y m a i n t a i n e d .  Therefore,  these  isozyme polymorphisms  I t s h o u l d be n o t e d , however, t h a t P. congesta  and P. brachystemon may have d i f f e r e n t i a t e d s u f f i c i e n t l y t h a t t h i s comparison i s i n a p p r o p r i a t e . The e x i s t e n c e o f m u l t i - l o c u s a l l e l e complexes have been demonstrated i n s l e n d e r w i l d oats (e.g. C l e g g and A l l a r d , 1972; Hamrick and A l l a r d , 1972; A l l a r d et al., 1972;  Hamrick and Holden, 1979; K a h l e r et al., 1980) and b a r l e y (Clegg  et al., 1972).  These a u t h o r s p r e s e n t evidence and argue t h a t s e l e c t i o n  has s t r u c t u r e d t h e g e n e t i c r e s o u r c e s o f t h e s e s p e c i e s i n t o " h i g h l y i n t e r a c t i n g , co-adapted gene complexes."  I f f i t n e s s i s determined  by t h e c o o r d i n a t e d i n t e r a c t i o n o f l a r g e numbers o f l o c i ,  then  a l l e l e s which a r e s e l e c t i v e a t a few marker l o c i i n one s p e c i e s o f Plectritis  may n o t n e c e s s a r i l y be e q u a l l y advantageous i n t h e o t h e r .  T a b l e 30.  I n t e r - s p e c i f i c comparisons o f s t a n d a r d g e n e t i c d i s t a n c e (D) in  Pleotritis P. braohystemon p o p u l a t i o n s  1  13  9  10  2(1)  0.,3242  0.3060  0.1960  0. 2991  2(2)  0. 3393  0.2960  0.1868  3  0. 2858  0.3249  5  0.4524  4  7(3)  15(1)  15(2)  18  0. 2882  0 .2417  0 .2693  0.2854  0.2868  0. 2817  0. 2791  0 .2378  0 .2587  0.2881  0.2836  0.2552  0. 2662  0. 3585  012524  0 .3161  0.3700  0.3669  0.3983  0.2758  0. 3843  0. 3836  0 .3461  0 .3590  0.4121  0.4057  0. 2735  0.2595  0.1824  0. 2098  0. 2827  0 .2408  0 .2417  0.3111  0.3029  9  0. 2896  0.2943  0.2121  0. 2335  0. 3017  0 .2664  0 .2730  0.3402  0.3027  10  0. 3386  0.3452  0.2588  0. 2921  0. 3694  0 .2998  0 .3277  0.3862  0.3834  11  0. 3140  0.3330  0.2409  0. 2944  0. 3473  0 .2801  0 .3118  0.3594  0.3584  7(2)  0. 4412  0.4022  0.3003  0. 3853  0.3840 0. 3192 0 .3636  0.4020  0.4023  8  0. 2406  0.2846  0.2483  0. 1873  0. 3536  0 .2816  0 .2912  0.3718  0.3663  14(1)  0. 3200  0.2975  0.2046  0. 2724  0. 2997  0 .2388  0 .2703  0.3089  0.3089  14(2)  0. 2903  0.2763  0.1825  0. 2608  0. 2748  0 .2251  0 .2478  0.2892  0.2845  15(1)  0. 3677  0.4379  0.3427  0. 3915  0. 4562  0 .3676  0.4197 0.4588  0.4618  15(2)  0. 3990  0.3232  0.2452  0. 2678  0. 3540  0 .3560  0 .3041  0.4087 0.3998  17  0. 2695  0.3407  0.2699  0. 2586  0. 3625  0 .2878  0.3281  0.3740  7(1)  Comparisons between s y m p a t r i c p o p u l a t i o n s a r e i n i t a l i c s D (P.b. :Pv.o.) = 0.3132 I (P.b.:P.o.) = 0.7311  D ( s y m p a t r i c ) = 0.3393 I ( s y m p a t r i c ) = 0.7123  0.3385  130.  A second approach t o t h i s q u e s t i o n was t o compare the o b s e r v e d v a r i a n c e o f i n t e r - l o c u s h e t e r o z y g o s i t y (h) t o a t h e o r e t i c a l v a r i a n c e w h i c h assumes t h a t n e u t r a l m u t a t i o n s and g e n e t i c random d r i f t are balanced  ( N e i and.Roychoudhury, 1974). The t h e o r e t i c a l  v a r i a n c e i s g i v e n by Var  (h) = — :  —  (l+e) (2+6)(3+9) 2  where 9 = 4Nu7 mutation  N i s t h e e f f e c t i v e p o p u l a t i o n . , s i z e and y  i s the  r a t e p e r l o c u s p e r : g e n e r a t i o n . The v a l u e o f 6 may be  e s t i m a t e d by H / ( l - H ) ; i n . w h i c h H i s t h e e s t i m a t e o f average h e t e r o z y g o s i t y , as t h e e x p e c t a t i o n o f H i s 9/(1+9) . The e s t i m a t e s o f 9 , t h e o b s e r v e d v a r i a n c e o f h e t e r o z y g o s i t y and t h e t h e o r e t i c a l variance o f heterozygosity:are presented P. oongesta  i n Tables  f o r B. braohystemon  and  31 and 32 r e s p e c t i v e l y . The expected and  o b s e r v e d v a l u e s a r e g e n e r a l l y i n e x c e l l e n t agreement f o r P. braohystemon  and r e a s o n a b l e  agreement f o r P.  oongesta.  U n f o r t u n a t e l y , i t i s n o t p o s s i b l e t o assess t h e s i g n i f i c a n c e o f departures  from t h e t h e o r e t i c a l v a l u e . I n a d d i t i o n , i t cannot  be d e t e r m i n e d whether t h e d i f f e r e n c e i n t h e l e v e l o f agreement between v a r i a n c e s i n P. braohystemon i m p l i c a t i o n i n regard  and P. oongesta  has any  t o t h e r e l a t i v e r o l e s o f s e l e c t i o n and  d r i f t i n m a i n t a i n i n g o b s e r v e d polymorphisms w i t h i n t h e two s p e c i e s . However, i n g e n e r a l i t would appear t h a t t h e l e v e l s o f isozyme v a r i a t i o n , p a r t i c u l a r l y i n P. braohystemon, e x p e c t a t i o n s o f a s t o c h a s t i c model.  a r e i n agreement w i t h t h e  T h i s does n o t e l i m i n a t e t h e  p o s s i b i l i t y o f s e l e c t i o n because c e r t a i n t y p e s o f s e l e c t i o n and  131.  Table 31. A comparison between the observed and the t h e o r e t i c a l i n t e r - l o c u s variances o f h e t e r o z y g o s i t y (h) i n populations o f P. brachystemon  1 8  Population  Observed variance  2  T h e o r e t i c a l variance  1  0.056  0.022  0.016  13  0.066  0.022  0.018  9  0.032  0.006  0.010  10  0.006  0.001  0.002  7(1)  0.041  0.017  0.012  7(3)  0.164  0.045  0.035  15(1)  0.128  0.033  0.030  15(2)  0.063  0.024  0.018  16  0.014  0.001  0.004  18  0.075  0.034  0.020  3  8 = 4Np, where N = e f f e c t i v e population s i z e and u = mutation r a t e per locus per generation, 8 estimated by H/(l-H) s i n c e the expectation o f H = (8/(1+8) r  2  Observed i n t e r - l o c u s variance o f h e t e r o z y g o s i t y : V(h)= S (h, -H) / ( r - l ) th l k  where h^= estimate o f h e t e r o z y g o s i t y at the k  l o c i scored T h e o r e t i c a l i n t e r - l o c u s variance o f h e t e r o z y g o s i t y :  Var(H)=  28 (l+9) (2+6) (3+8) 2  =  locus and r = the number o f  132.  Table 32.  A comparison between the observed and the t h e o r e t i c a l i n t e r - l o c u s variance o f h e t e r o z y g o s i t y (h) i n populations o f  P. oongesta 6  Population 2(1) 2(2) 3 5 4 9 10 11 7(2) 8 14(1) 14(2) 15(1) 15(2) 17  '  Observed variance  Theoretical  0. 234  0.073  0.,043  0.268  0. 080  0..045  0. 325  0.,068  0.,048  0.274  0-066  0..045  ' 0.342  0.,078  0..049  0. 347  0.,073  0,.049  0.371  0.086  0..049  0. 352  0..081  0,.049  0. 350  0..061  0,.049  0.,272  0,.065  0,.045  0.,232  0,.065  0,.042  0.,221  0,.069  0..042  0.,306  0,.061  0 .047  0..230  0,.049  0 .042  0..295  0 .070  0 .047  variance  133.  v a r y i n g m u t a t i o n r a t e s may produce t h e same p a t t e r n s  ( L i , 1978).  In a d d i t i o n , t h e r e a r e a number o f p l a n t s t u d i e s which have demonstrated micro-habitat  s e l e c t i o n (Antonovics,  1968; Hamrick and A l l a r d , 1972;  M i t t e n et al., 1977; Hamrick and Holden, 1979; K a h l e r et al. the s a m p l i n g s t r a t e g i e s used i n t h e p r e s e n t  maintained  1980) and  s t u d y would n o t r e s o l v e  v a r i a t i o n patterns a t t h i s l e v e l . Considering iation  3  w i t h i n l o c a l populations  the quantity o f varand t h e v u l n e r a b i l i t y  o f i n d i v i d u a l s , p a r t i c u l a r l y s e e d l i n g s , s e l e c t i o n may be most important  operating at the level of micro-site differences.  5.5 Summary: the genetic Outcrossing o f P. brachystemon  consequences  of contrasting  breeding  r a t e s were c a l c u l a t e d f o r n i n e  and f i f t e e n p o p u l a t i o n s  of  systems  populations  P. congesta.  The  d i f f e r e n c e between t h e mean o u t c r o s s i n g r a t e s i n t h e two s p e c i e s was highly significant  (P^O.01).  These two s p e c i e s a r e i d e a l f o r  comparison because: ( i ) t h e y a r e c l o s e l y r e l a t e d and have a l a r g e p r o p o r t i o n o f t h e i r a l l e l e s i n common, ( i i ) t h e m u l t i - l o c u s o r g a n i z a t i o n o f t h e enzyme systems s t u d i e d i s homologous and ( i i i ) they have s i m i l a r l i f e - c y c l e s t r a t e g i e s and h a b i t a t r e q u i r e m e n t s . I t i s not uncommon, p a r t i c u l a r l y on s o u t h e r n Vancouver I s l a n d , t o f i n d t h e two s p e c i e s growing s y m p a t r i c a l l y . A number o f g e n e t i c parameters were measured w i t h i n each t a x o n t o assess that breeding  the e f f e c t  system has on p o p u l a t i o n s t r u c t u r e . The r e s u l t s a r e  summarized i n T a b l e 33. As measured by a l l p a r a m e t e r s , t h e s e l f e r maintained  s i g n i f i c a n t l y less v a r i a t i o n within local  than t h e o u t c r o s s e r a l t h o u g h  t h e i r detected  populations  levels of total variation  134.  Table 33.  Summary o f genetic d i f f e r e n c e s between P. brachystemon and  P. congesta P. brachystemon P. congesta  Parameter  Outcrossing  frequency  t  0.02410.008  0.70210.048  Percentage o f l o c i polymorphic per population  20.7613.49  53.3311.78  8.31'  Average number o f a l l e l e s / l o c u s per population  1.2510.04  1.98+0.05  11.41'  Average number o f a l l e l e s / p o l y m o r p h i c locus per population  2.2210.10  2.8410.08  4.84'  Expected % o f heterozygous l o c i / individual  5.8911.33  22.64+0.79  10.83'  Observed % o f heterozygous l o c i / individual  0.4510.13  17.7410.81  21.09'  0.166+0.021  0.26610.083  0.99  Gene d i v e r s i t y w i t h i n populations (H )  0.06010.021  0.266+0.071  2.24'  Proportion o f i n t e r - p o p u l a t i o n gene d i f f e r e n t i a t i o n (G<.,j.)  0.63910.053  0.15010.039  7.41'  Mean standard genetic distance between population (D)  0.137+0.083  0.05210.034  0.94  Mean g e n e t i c ' i d e n t i t y between populations (I)  0.872  0.949  T o t a l gene d i v e r s i t y  (Rj.)  13.84'  s  * (P 0.05) ** (P 0.01)  135.  (Ftp were not. s i g n i f i c a n t l y d i f f e r e n t . When t h e i n f l u e n c e o f s i t e v a r i a t i o n was c o n t r o l l e d by comparing o n l y s y m p a t r i c p o p u l a t i o n s o f the two s p e c i e s , a l l g e n e t i c d i f f e r e n c e s remained s i g n i f i c a n t . The reduced l e v e l o f v a r i a t i o n w i t h i n p o p u l a t i o n s o f t h e s e l f e r was r e f l e c t e d i n t h e F - s t a t i s t i c s and i n t h e a n a l y s i s o f gene d i v e r s i t y . On t h e average, P. congesta p o p u l a t i o n s  contained  85% o f t h e s p e c i e s ' v a r i a t i o n w h i l e o n l y 36% o f t h e v a r i a t i o n d e t e c t e d i n P. brachystemon was m a i n t a i n e d w i t h i n l o c a l p o p u l a t i o n s . In t h e s e l f e r , t h e reduced l e v e l o f g e n e t i c v a r i a t i o n w i t h i n p o p u l a t i o n s was accompanied by i n c r e a s e d p o p u l a t i o n a l d i f f e r e n t i a t i o n . T h i s i s g r a p h i c a l l y d e p i c t e d i n F i g u r e 46 which shows t h e r e d u c e d g e n e t i c i d e n t i t y among p o p u l a t i o n s o f P. brachystemon.  P r e l i m i n a r y d a t a suggest t h a t d i f f e r e n t i a t i o n ,  p a r t i c u l a r l y i n t h e s e l f e r , can o c c u r over s h o r t d i s t a n c e s . However, whether t h i s r e p r e s e n t s m i c r o - g e o g r a p h i c  d i f f e r e n t i a t i o n or d r i f t  i n small r e p r o d u c t i v i t y i s o l a t e d populations i s problematic. In t h i s t a x o n , a l l e l e f r e q u e n c i e s and s i n g l e l o c u s h e t e r o z y g o s i t i e s f l u c t u a t e d w i d e l y and d i f f e r e n t a l l e l e s were o f t e n f i x e d i n a d j a c e n t p o p u l a t i o n s . I n P. congesta, a l l e l e f r e q u e n c i e s and h e t e r o z y g o s i t i e s a l s o f l u c t u a t e d among p o p u l a t i o n s b u t n o t w i t h t h e a m p l i t u d e i n P. brachystemon.  observed  There appears t o be no r e l a t i o n s h i p between  g e n e t i c d i s t a n c e and g e o g r a p h i c  distance.  T h i s i m p l i e s t h a t gene  f l o w between p o p u l a t i o n s does n o t s i g n i f i c a n t l y i n f l u e n c e a l l e l e f r e q u e n c i e s . No evidence was found t o suggest t h a t t h e observed isozyme polymorphisms a r e s e l e c t i v e l y m a i n t a i n e d .  However, c o - o r d i n a t e d  gene complexes and m i c r o - h a b i t a t s e l e c t i o n would p r o b a b l y n o t have  136.  been d e t e c t e d . I t i s concluded  t h a t t h e observed  p o p u l a t i o n s t r u c t u r e o f P. .bvachystemon  d i f f e r e n c e s i n the  and P. oongesta  the r e s u l t o f t h e i r c o n t r a s t i n g b r e e d i n g systems.  i s primarily  Autogamy  restricts  gene f l o w between p o p u l a t i o n s and r e c o m b i n a t i o n w i t h i n p o p u l a t i o n s . Inbreeding s t r o n g l y i n f l u e n c e s the l e v e l s of v a r i a t i o n  maintained  w i t h i n p o p u l a t i o n s by l o w e r i n g t h e e f f e c t i v e p o p u l a t i o n s i z e . increases the p o t e n t i a l that s t o c h a s t i c forces w i l l affect a l l e l e frequencies w i t h i n populations.  This  significantly  Although  small  m i g r a t i o n r a t e s a r e s u f f i c i e n t t o p r e v e n t d i f f e r e n t i a t i o n between populations  (Lewontin,  i n P. oongesta  1974), t h e r e i s no e v i d e n c e t h a t p o l l e n f l o w  i s e f f e c t i v e over l a r g e d i s t a n c e s .  r e l a t i v e amounts o f r e c o m b i n a t i o n are p r o b a b l y t h e most i m p o r t a n t  and d r i f t w i t h i n l o c a l  oongesta.  populations  f a c t o r s i n f l u e n c i n g the c o n t r a s t i n g  l e v e l s and o r g a n i z a t i o n o f g e n e t i c v a r i a t i o n i n P. and P.  Therefore, the  braohystemon  137.  BIBLIOGRAPHY A l l a r d , R.W., B a b b e l , G.R., C l e g g , M.T., and K a h l e r , A.L. 1972. Evidence f o r c o a d a p t a t i o n i n Avena barbata. Proc. Nat. Acad. Sci. USA 69:3043-3048. A l l a r d , R.W., and J a i n , S.K. 1962. 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Studies in genetics V I . Univ. o f Texas p u b l . 7103. P. 49-90. S e l a n d e r , R. K. , Yang, S.Y., L e w o n t i n , R . C , and Johnson, W.E. 1970. G e n e t i c v a r i a t i o n i n t h e h o r s e s h o e c r a b (Limulus polyphemus), a p h y l o g e n e t i c " r e l i c " . Evolution 24:402-414. Shaw, C.R., and P r a s a d , R. 1970. 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 enzymes a c o m p i l a t i o n o f r e c i p i e s . Biochem. Genet. 4:297-320. S i c i l i a n o , M.J., and Shaw, C.R. 1976. S e p a r a t i o n and v i s u a l i z a t i o n o f enzymes on g e l s . P. 185-209, in I. Smith ( e d . ) . Chromatographic and electrophoretic techniques. I n t e r s c i e n c e . New York, N.Y. S o k a l , R.R., and Sneath, P.H.A. 1963. Principals Freeman. San F r a n c i s c o , CA.  of numerical taxonomy.  S o l b r i g , . 0 . T . 1972. Breeding system and g e n e t i c v a r i a t i o n i n Leavenworthia. Evolution 26:155-160. S o l b r i g , 0.T., and R o l l i n s , R.C. 1977. The e v o l u t i o n o f autogamy i n s p e c i e s o f t h e mustard genus Leavenworthia. Evolution 31:265-281.  144.  S p i e s s , E.B. 1977. Genes in populations. S t e b b i n s , G.L. 1950. Variation P r e s s . New York, N.Y.  J . W i l e y . New Y o r k , N.Y.  and evolution  in plants.  Columbia Univ.  . 1957. S e l f f e r t i l i z a t i o n and p o p u l a t i o n v a r i a b i l i t y i n t h e h i g h e r p l a n t s . Amer. Eat. 91:337-354. T o r r e s , A.M., Diedenhofen, U., Bergh, B.O., and K n i g h t R.J. 1978a. Enzyme polymorphisms as g e n e t i c markers i n t h e avocado. Amer. J. Bot. 65(2):134-139. T o r r e s , A.M., S o o s t , R.K., and Diedenhofen, U. 1978b. Leaf isozymes as g e n e t i c markers i n Citrus. Amer. J. Bot. 65(8):869-881. W a l l , J.R., and W h i t a k e r , T.W. 1971. G e n e t i c c o n t r o l o f l e u c i n e aminop e p t i d a s e and e s t e r a s e isozymes i n t h e i n t e r s p e c i f i c c r o s s Cucurbita equadorensis X C. maxima. Biochem. Genet. 5:223-229. Workman, P.L. 1969. The a n a l y s i s o f s i m p l e g e n e t i c polymorphisms. Human Biol. 41:97-114. W r i g h t , S. 1965. The i n t e r p r e t a t i o n o f p o p u l a t i o n s t r u c t u r e by F - s t a t i s t i c s w i t h s p e c i a l r e g a r d s t o systems o f m a t i n g . Evolution 19:395-420. Yeh, F.C., and L a y t o n , C. 1979. The o r g a n i z a t i o n o f g e n e t i c v a r i a b i l i t y i n c e n t r a l and m a r g i n a l p o p u l a t i o n s o f l o d g e p o l e p i n e Pinus contorta spp. latifolia. Can. J. Genet. Cytol. 21:487-503. Zouros, E. 1976. H y b r i d m o l e c u l e s and t h e s u p e r i o r i t y o f t h e h e t e r o z y g o t e . Nature 262:227-229.  145.  APPENDIX A. POPULATION LOCATIONS Sample designation  Location  L o c a t i o n and comments  Sumas Mountain  l a r g e , open s o u t h - f a c i n g s l o p e o f Sumas Mtn. ( e a s t o f A b b o t s f o r d , B.C), v i s i b l e from Trans-Canada Hwy.  2(1)  Anacortes,  WA  open s l o p e o v e r l o o k i n g Puget Sound, Washington Park (Anacortes C i t y P a r k ) : F i d a l g o Head, F i d a l g o I s l a n d ; on u l t r a m a f i c s o i l s d e r i v e d from p e r i o d o t i t e (Kruckeberg, 1969).  2(2)  Anacortes,  WA  l a r g e open meadow, Washington Park: ultramafic soils  13  W i l l i a m Head  l a r g e p o p u l a t i o n growing among G a r r y oak and a r b u t u s , M e t c h o s i n Rd. .6 km SW o f Pearson C o l l e g e Dr. M e t c h o s i n ,  B.C.  John Dean Park  p l a n t s were c o l l e c t e d on a s m a l l ledge and a l o n g a 15 m t r a n s e c t a t t h e base o f a c l e a r i n g l o c a t e d below a DND r a d a r i n s t a l l a t i o n , C e n t r a l S a a n i c h , B.C.  Mill Hill  open s o u t h - f a c i n g s l o p e ; p l a n t s a t t h i s s i t e bloom 2-4 wks. e a r l i e r than o t h e r c o l l e c t e d p o p u l a t i o n s , near V i c t o r i a , B.C.  Park  T h e t i s Lake Park  open meadow w i t h s c a t t e r e d G a r r y oak and a r b u t u s , near V i c t o r i a , B.C.  Francis  s y m p a t r i c p o p u l a t i o n , P..o. i s more abundant t h a n P.b.; D o u g l a s - f i r woodland, a much s h a d i e r s i t e t h a n most; S a a n i c h , B.C.  Park  10  P r o s p e c t Lake Rd.  s y m p a t r i c p o p u l a t i o n , P.a. i s more w i d e l y d i s t r i b u t e d t h a n P.b.; an open and somewhat d i s t u r b e d s i t e , e v i d e n c e of a b u r n ; Gaultheria shaUon and Cytisus soopavius are abundant; P r o s p e c t Lake Rd. .5 km N o f P r o s p e c t Lake G o l f C l u b . S a a n i c h , B.C.  11  V i a d u c t Avenue  s m a l l p o p u l a t i o n i n a d e p r e s s i o n between two r o c k o u t c r o p s ; p l a n t s were r o b u s t and d e n s i t y was h i g h , i n t e r s e c t i o n o f P r o s p e c t Lake Rd. and V i a d u c t Ave., S a a n i c h , B.C.  146. APPENDIX A. Sample designation  Location  POPULATION LOCATIONS L o c a t i o n and comments  7(1)  Malahat D r i v e  s y m p a t r i c p o p u l a t i o n , an exposed, SEf a c i n g r o c k y k n o l l beneath t h r e e Hydro t r a n s m i s s i o n towers o v e r l o o k i n g S a a n i c h I n l e t ; p u l l o f f l o c a t e d 4 km N o f " e n t e r i n g Malahat; Dr." s i g n on TransCanada Hwy..7(1)- P.b. c o l l e c t e d a l o n g a 15 m t r a n s e c t r u n n i n g down the. s l o p e b e g i n n i n g approx. 10 m N o f t r a n s m i s s i o n tower #1.  7(2)  Malahat D r i v e  P.a. - l o c a t e d beneath tower #2  7(3)  Malahat D r i v e  P.b. - NE s l o p e o f k n o l l , t o p o f k n o l l w i t h a few P.O.  Crofton Exit  S E - f a c i n g ledge approx. 5 m above Hwy. s i t e was q u i t damp a t time o f c o l l e c t i o n and h e r b . c o v e r was l u s h ; p l a n t s were c o l l e c t e d a l o n g a 30 m t r a n s e c t r u n n i n g NW from t h e ledge t o t h e back o f t h e c l e a r i n g ; Trans-Canada Hw. 1.7 km south o f C r o f t o n E x i t ( S a l t s p r i n g Island ferry).  14(1)  Jack's  Point  open s i t e b i s e c t e d by a g r a v e l r o a d , P.c. v e r y dense; s i t e has been d e s t r o y e d by c o n s t r u c t i o n o f Duke P o i n t I n d u s t r i a l Complex, Nanaimo, B.C.  14(2)  Jack's  Point  open s i t e a l o n g g r a v e l r o a d .3 km from A ( l ) ; s i t e a l s o d e s t r o y e d by construction.  15(1), 15(2)  Nanoose  16  L i t t l e Qualicum Falls  Hill  transmission  s y m p a t r i c s i t e w i t h P.b. b e i n g t h e most abundant; l a r g e S - f a c i n g s l o p e w i t h G a r r y oak and a r b u t u s , b o t h c o l l e c t i o n s from semi-shaded s i t e s beneath Quevous gavvyana. s i t e i s v i s i b l e from T r a n s Canada Hw. a t Nanoose Bay, n o r t h o f Nanaimo. p l a n t s were l o c a t e d on a s m a l l open ledge o v e r l o o k i n g t h e lower f a l l s ; L i t t l e Qualicum F a l l s Prov. Park.  147.  APPENDIX A. POPULATION LOCATIONS Sample designation  Location  L o c a t i o n and comments  17  N i l e Creek  p l a n t s c o l l e c t e d approx. 50 m from beach i n a g r a s s y a r e a o f s t a b i l i z e d sand among s c a t t e r e d Pseudotsuga menziesii. Access t o s i t e i s a g r a v e l r o a d o f f the Trans-Canada Hwy. a l o n g the n o r t h bank o f N i l e Creek, j u s t s o u t h o f Bowser, B.C.  18  E l k F a l l s Park  p l a n t s were c o l l e c t e d on an exposed g r a s s y ledge below the dam; E l k F a l l s P r o v . P a r k , Campbell R i v e r , B.C..  i  APPENDIX  B.  ENZYME SYSTEMS WHICH WERE NOT ADEQUATELY RESOLVED WITH AVAILABLE TECHNIQUES  ENZYME  RUNNING BUFFER  1. Acid phosphatease APH E.C. 3.1.3.2 2. Alcohol dehydrogenase AMI E.C. 1.1.1.1  I-VI  STAIN REFERENCE Roose 8 G o t t l i e b  (1976)  COMMENTS Adequate s t a i n i n g , but r e s o l u t i o n was poor.  I,II,IV,V  Scandalios (1969) G o t t l i e b (1973b)  No A c t i v i t y No A c t i v i t y  1,11  Scandalios (1969)  No A c t i v i t y  Delorenzo 8 Ruddle (1970)  With b u f f e r V I , and t o a l e s s e r degreee I, r e s o l u t i o n was sometimes very good but s t a i n i n g was u n r e l i a b l e . AAT appears to be a 2 or 3 , locus system with the slowest locus showing d i m e r i c i n heritance.  Shaw 6 Prasad (1970) S l c i l i a n o 8 Shaw (1976)  L i t t l e o r No A c t i v i t y No A c t i v i t y  6. Pructose-1, 6-diphosphatase I.II.IV-IX FDP E.C. 3.1.3.11  Allendorf,  Very l i g h t s t a i n i n g with b u f f e r s , I I , VI 8 VII I f 10 nig. NADP added to g e l , however staining i s unreliable.  7. Glutamate dehydrogenase GDII E.C. 1.4.1.3  Roose 8 G o t t l i e b (1976) A l l e n d o r f , et a l . (1977)  3. A l k a l i n e phosphatase ALPII E.C. 3.1.3.1 4. Aspartate aminotransferase ATT (=GOT) E.C. 2.6.1.1  S. Catalase CAT E.C. 1.11.1.6  I-VII  1,11,IV,VI  I,II,IV,V  et a l . (1977)  S t a i n i n g u n r e l i a b l e , sometimes r e s o l v e d a s i n g l e monomorphic band with b u f f e r I. There i s some question as to whether t h i s i s GDII as a s i n g l e band was sometimes resolved on other gels without the a d d i t i o n of s u b s t r a t e .  APPENDIX B.  (Continued)  ENZYME  RUNNING BUFFER  STAIN REFERENCE  COMMENTS  8. Glucose-6-phosphate dehydrogenase G6P E.C. 1.1.1.49  • 1,11 IV-IX  Roose 6 G o t t l i e b (1976) A l l e n d o r f , e t a l . (1977)  S t a i n i n g very d i f f u s e , was unable to r e s o l v e .  9. Peroxidase PER E.C. 1.11.1.7  I,II,IV,V,VI  Brown 6 A l l a r d (1969) G o t t l i e b (1973a) S i c i l i a n o 5 Shaw (1976)  Had success with G o t t l i e b ' s s t a i n and running b u f f e r s I 6 VI, however, s t a i n i n g was i n c o n s i s t e n t so system was not used. PER appears t o be a m u l t i l o c u s system with both anodal cathodal v a r i a n t s  10. S o r b i t o l dehydrogenase SDH E.C. 1.1.1.14  I,II V-IX  A l l e n d o r f , e t a l . (1977)  S i n g l e monomorphic band sometimes r e s o l v e d with Systems I 6 I I . However t h i s band s t a i n s without the a d d i t i o n o f s u b s t r a t e and i s t h e r e f o r e o f unknown function.  11. Succinate dehydrogenase SUCDH E.C. 1.3.99.1  1,11 V-IX  A l l e n d o r f , e t a l . (1977)  Results  12. Xanthine \ dehydrogenase XDH E.C. 1.2.1.37  I,II V-IX  A l l e n d o r f , e t a l . (1977)  Results i d e n t i c a l to SDH 6 SUCDH.  i d e n t i c a l t o SDH.  REFERENCE  RUNNING BUFFERS I Discontinuous L i - B o r a t e / T r i s - C i t r a t e pH 8.0 II Continous T r i s - C i t r a t e pH 7.0 III 0.3 M Borate  pH 8.0  IV Poulik V Ilistidine-Citrate  VII T r i s - M a l e a t e  IX Continuous  S i c i l i a n o S Shaw (1976) Shaw 6 Prasad  (1970)  Selander, e t a l .  VI Tris-Versene-Borate pH 8.0  VIII Continuous  Scandalios (1969), Selander, e t a l . (1971)  pH 7.4  (1971)  G o t t l i e b (1973a) S i c i l i a n o 5 Shaw (1976) Selander, e t a l . (1971)  T r i s - C i t r a t e I pH 6.3  Selander, e t a l . (1971)  T r i s - C i t r a t e II pH 8.0  Selander, e t a l . (1971)  151.  APPENDIX C.  H =  H ,  GENE AND GENOTYPE FREQUENCIES WITHIN POPULATIONS  e x p e c t e d p e r c e n t a g e o f h e t e r o z y g o u s l o c i / i n d i v i d u a l (see T a b l e s 22 and 23) =  o b s e r v e d p e r c e n t a g e o f heterozygous l o c i / i n d i v i d u a l (see T a b l e s 22 and 23)  n =  number o f i n d i v i d u a l s  (F) =  number o f f a m i l i e s  genotyped  r e p r e s e n t e d by genotyped  individuals.  152.  APPENDIX C . l .  Plectritis  brachystemon p o p u l a t i o n s  153. P o p u l a t i o n #77T1  Locus  Allele  'EST-I  89 100 114 h h  EST-2  100  LAP-1  85 91 100 h h  MDH-1  100  MDH-2  Siunas Mtn.  Frequency  .02 .53 .45 .516 .046 .  n  •(F)  130  (130)  19  (19)  19  (19)  1.00  19  (19)  100  1.00  19  (19)  PGI-1  100  1.00  131  (131)  PGI-2  100  1.00  131  (131)  PGI-3  lOOn  1.00  131  (131)  PGM-1  106  1.00  112  (112)  PGM-2  100  1.00  131  (131)  PGM-3  115  1.00  131  (131)  IDH-1  100  1.00  19  (19)  ME-1  100  1.00  19  (19)  6PG  100  1.00  19  (19)  SOD  100  1.00  19  (19)  0  0  1.00 .05 .84 .11 .280 .000  H=.0S31  Genotype  H = 0031 obs.  # Individuals  89/89 100/100 114/114 100/114  2 66 56 6  85/85 91/91 100/100  1 16 2  154. P o p u l a t i o n 77-13  Locus  Allele  EST-1  89 100 h h  EST-2  100  LAP-1  91 100 h h  MDH-1  100  MDH-2  W i l l i a m Head  Frequency .80 .20 . .320 .029  n  (F)  70  (70)  30  (30)  16  (16)  1.00  16  (16)  100  1.00  16  (16)  PGI-1  100  1.00  73  (73)  PGI-2  100  1.00  73  (73)  PGI-3  lOOn  1.00  73  (73)  PGM-1  100 106 h  48  (48)  PGM-2  100  1.00  73  (73)  PGM-3  115  1.00  73  (73)  IDH-1  100  1.00  30  (30)  ME-1  100  1.00  30  (30)  6PG-1  100  1.00  30  (30)  SOD  100  1.00  30  (30)  0  o  1.00 .06 .94 .113 .000  .52 .48 .499 .042  H=.0621  H =.0047 obs.  Genotype  JIndividi  89/89 100/100 89/100  55 13 2  91/91 100/100  1 IS  100/100 106/106 100/106  24 22 2  155.  Population 77-9  Locus  Allele  EST-1  EST-2  89 100 h h o 100  LAP-1  Francis Park  Frequency  n  .03 .97 .058 .023 .  43  (43)  1.00  14  (11)  100  1.00  14  (11)  MDH-1  100  1.00  14  (11)  MDH-2  100  1.00  14  (11)  PGI-1  100  1.00  S6  (43) .  PGI-2  94 100 h * h  .01 .99 .020 .017  56  0  ,  (F)  H=.0309  Genotype  H =.0042 obs  #Individi  89/89 100/100 89/100  . 1 41 1  (43)  100/100 94/100  55 1  PGI-3  33 lOOn h h  .19 .81 .308 .000  43  (43)  33/33 lOOn/lOOn  8 35  PGM-1  100 106 h h  .97 .03 .058 .000  40  (40)  100/100 106/106  39 1  PGM-2  100  43  (43)  PGM-3  115 182 h h  43  (43)  115/115 115/182  42 1  IDH-1  100  1.00  14  (11)  6PG  100  1.00  14  (U)  ME-1  100  1.00  14  (11)  SOD  100  1.00  14  (11)  0  0  0  1.00 .99 .01 .020 .023  156. Population 77-10  Locus  Allele  EST-1  Prospect Lake  Frequency  n  (F)  100  1.00  8  (8)  EST-2  100  1.00  21  (19)  LAP-1  100 105 "h ' h  22  (19)  MDH-1  113  1.00.  22  (19)  MDH-2  100  1.00  22  (19)  PGI-1  100  1.00  22  (19)  PGI-2  100  1.00  22  (19)  PGI-3  lOOn  1.00  22  (19)  PGM-1  106  i.oo  • 22  (19)  PGM-2  100  1.00  22  (19)  PGM-3  115  1.00  22  (19)"  IDH-1  100  1.00  22  (19)  ME-1  100  1.00  22  (19)  6PG  100  1.00  22  (19)  SOD  100  1.00  22  (19)  0  .95 .05 .095. .000  H=.0063  Genotype  100/100 ,105/105  H , =.0000 obs  #Individuals  21 1  157. Population  Locus  Allele  76-7(1)  Malahat Dr.  Frequency  n  (F)  H=.0393  ^=.0000  Genotype  'Individuals  EST-1  114  1.00  20  (20)  EST-2  100 \  1.00  20  (20)  LAP-1  95 100 h  .05 .95 .095 .000  20  (20)  95/95 100/100  1 19  MDH-1  100 113 h h  .55 .45 .495 .000  20  (20)  100/100 113/113  11 9  MDH-2  100  1.00  20  (20)  PGI-1  100  1.00  20  (20)  PGI-2  100  1.00  20  (20)  PGI-3  lOOn  1.00  20  (20)  PGM-1  100  1.00  20  (20)  PGM-2  100  1.00  20  (20)  PGM-3  115  1.00  20  (20)  IDH-1  100  1.00  20  (20)  ME-1  100  1.00  20  (20)  6PG  100  1.00  20  (20)  SOD  100  1.00  20  (20)  0  158.  Population 77-7(3)  Locus EST-1  Allele 89 100 114 h h  0  EST-2  100  LAP-1  95 100 h h  o  Malahat Dr.  Frequency  n  (F)  .56 . 12 .32 .570 .000  25  (24)  1.00  H-.140S  Genotype  H  ohs = .0000  'Individuals  89/89 100/100 114/114  14 3  8  25  (24)  .60 .40 .480 .000  25  (24)  95/9S 100/100  IS 10  .50 *.S0 .500 .000  24  (23)  100/100 113/113  12 12  81/81 100/100  3 22  MDH-1  100 113 h h  MDH-2  100  1.00  24  (23)  PGI-1  100  1.00  25  (24)  PGI-2  81 100 h h  25  (24)  PGI-3  lOOn  25  (24)  PGM-1  100 106 h h  .84 .16 .269 .000  25  (24)  100/100 106/106  21 4  PGM-2  100 lOOn h h  .96 .04 .077 .000  25  (24)  100/100 " lOOn/lOOn  24 1  PGM-3  11S  1.00  25  (24)  IDH-1  100  1.00  25  (24)  ME-1  100  1.00  2S  (24)  6PG  100  1.00  25  (24)  SOD  100  1.00  25  (24)  0  0  0  0  .12 .88 .211 .000 1.00  159. Population 76-15(1)  Locus  Alllele  EST-1  89 100 114 h : h  o  Nanoose H i l l  Frequency .06 ;22 • .72. .430 .111  n 9  •(F) (9)  EST-2  100  1.00  13.  (13)  LAP-1  100  1.00  13  (13)  MDH-1  100 . 113 h h  9  (9)  MDH-2  100  1.00  9  (9)  PGI-1  100-  1.00  13  (13)  PGI-2  42 100 h  .15 .85 .2S5 .000  13  PGI-3  33 lOOn h  .62 .38 .471 .000 .77 .23 .354 .000  0  h  o  .89 .11 .196 .000  Genotype 100/100 114/114 89/114  H , =.0074 obs  #Indiyidi  •  "  2 6 1  100/100 113/113  8 1  (13)  42/42 100/100  2 11  13  (13)  33/33 lOOn/lOOn  13  (13)  100/100 106/106  PGM-1  100 106 h h  PGM-2  100  1.00  13  (13)  PGM-3  US  1.00  13  (13)  IDH-1  100  1:00  13  (13)  ME-1  100  1.00  13  (13)  6PG  100  1.00  13  (13)  SOD  100  1.00  .13  (13)  o  H=.1137  8 5  10 3  160.  Population  Locus  Allele  77-15(2)  Nanoose H i l l  Frequency  n  (F) (2)  EST-1  114  . 1.00  7  EST-2  100  1.00  7  LAP-1  100 105. 112 h h  MDH-1  100  MDH-2  .S3 .39 .08 .561 .000  36  (32)  1.00  37  (32)  100  1.00  37  (32)  PGI-1  100  1.00  37  (32)  PGI-2  42 100 h  35  (30)  0  h  o  .16 .84 .269 .029  PGI-3  lOOn  1.00  37  (32)  PGM-1  100  1.00  37  (32)  PGM-2  100  1.00  37  (32)  PGM-3  115 182 h h  37  (32)  0  .97 .03 .0S8 .054  H=.0S92  Genotype  .  IDH-1  100  1.00  37  (32)  ME-1  100  1.00 ^  37  (32)  6PG  100  1.00  37  (32)  SOD  100  1.00  37  (32)  -  H =.005S obs  "Individuals  100/100 10S/10S 112/112  19 14 3  42/42 100/100 42/100  S 29 1.  115/115 . 115/182  35 2  161.  Population  77-16  L i t t l e Qualiciun Falls  H=.0136  H . =.0130 OOS  Locus  Allele  EST-1  Frequency  n  Genotype  "Individuals  Not Scored  EST-2  100  1.00  33  (33)  LAP-1  100  . 1.00  33  (33)  MDH-1  113  1.00 ,  33  (33)  MDH-2  100  1.00  33  (33)  PGI-1  100  1.00  33  (33)  PGI-2  94 100 h  33  (33)  (33)  h  o  .05 .95 .095 .091  PGI-3  lOOn  1.00  33  PGM-1  100-  1.00  33 . (33)  PGM-2  . 100  1.00  33  (33)  PGM-3  lis  33  (33)  IDH-1  182 h h o 100  1.00  33  (33)  ME-1  100  1.00  33  (33)  6PG  100  1.00  33  (33)  SOD  100  1.00  33  (33)  .95 .05 .095 .091  100/100 94/100  30 3  115/115 115/182  30 3  162. Population 77-18  Locus EST-1  Allele 89,  EST-2  100  LAP-1  85 100 112 h h  MDH-1  113  MDH-2  Elk Falls  Frequency  n  (Fl  1.00  74  (41)  1.00  1 2  (6)  9  (6)  1.00  9  (6)  100  1.00  9  (6)  PGI-1  100  1.00  74  (41)  PGI-2  100  1.00  74  (41)  PGI-3  33 lOOn h h  74  (41)  PGM-1  100  1.00  74  (41)  PGM-2  100  1.00  74  (41)  PGM-3  182  1.00  74  (41)  IDH-1 '  100  1.00  14  (6)  ME-1  ioo '•:  . 1.00  14  (6)  6PG  100  1.00 -  14  (6)  SOD  100  1.00  14  (6)  0  0  .17 .61 .22 .551 .111  .46 .54 .497 .000  H=.0699  Genotype  85/85 100/100 112/112 85/100  33/33 lOOn/lOOn  H . =.0074 obs  'Individuals  1 5 2 1  34 40  163.  APPENDIX C.2.  Pleotritis  oongesta  populations  164. P o p u l a t i o n 76-2(1)  Locus  Allele  EST-1  100  EST-2  74 100  -  LAP-1  h h 80 85 91 95 100 105 112  h h MDH-1  °  Q  113 125 139  h h  Anacortes  -.  H=.1897  Frequency  n  (F)  1.00  12  (12)  .06 .94 .113 .118  17  (17)  100/100 74/100  .04 .27 .23 .04 .15 .23 .04 .790 .615  13  (13)  85/85 91/91 100/100 105/105 80/105 85/91 85/95 85/100 91/100 105/112  .03 .32 .65 .474 .471  17  (17)  Genotype  H.  SIndividi  • . 15 2  I 1 1 1 1 1 1 1  125/12S 139/139 113/139 125/139  7 1 7  2  0  MDH-2  100  PGM-1  100 106  h h  1.00  17  (17)  .97 .03 .058 .059  18  (18)  100/100 100/106  17 1  .18 .79 .03 .343 .294  17  (17)  81/81 100/100 . 81/100 100/118  1 11 4 1  .47 .53 .498 .353  17  (17)  100/100 135/135 100/135  5 6 6  0  PGM-2  81 100 .118  h h PGM-3  °  100 135 '•  h h  o  IDH-1  100 •  1.00  18  (18)  ME-1  100  1.00  .18  (18)  6PG  100 "  1.00  18  (18)  SOD  100 '.  1.00  . 18  (18)  ;  =.1592  165. Population 76-2(2)  Locus  Allele  Frequency  Anacortes  H=.2113  H =.1428 obs t  n  (F)  21  (20)  .07 .93 .130 .143  21  . (20)  100/100 74/100  18 3  Genotype  'Individi  EST-1  100  EST-2  74 100 h h  LAP-1  80 85 91 95 100 105 h h Q  .13 .26 .09 , .07 .28 .17 .796 .478  23  (22)  80/80 85/85 91/91 95/95 100/100 105/105 80/100 8S/91 85/100 85/105 91/100 95/105 100/105  2 3 1 1 3 2 2 1 3 2 1 1 1  MDH-1  113 125 139 h h  .05 .57 .38 .528 .300  20  (19)  125/125 139/139 113/125 113/139 125/139  9 5 1 1 4  MDH-2  100  20 .  (19)  PGM-1  100 106 h h  .94 .06 .113 .042  24  (23)  100/100 106/106 100/106  22 1 1  PGM-2  81 100 118 h h o  .23 .67 .10 .488 .375  24  (23)  81/81 100/100 118/118 81/100 81/118 100/118  2 12 1 6 1 2  PGM-3  77 100 135 h h  .02 .63 .35 .480 .375  24  (23)  100/100 135/135 77/100 100/135  11 4 1 8  IDH-1  100  1.00  24  (23)  ME-1  100  1.00  24  (23)  6PG  100  1.00  24  (23)  SOD  100  1.00  24  (23)  0  1.00  0  0  0  1.00  166.  Population 76-3  John Dean Park  H=.2454  H ' =.2046 obs. •  Locus  Allele  Frequency  n  (F)  Genotype  EST-1  89 100 h h  .01 .99 .020 .009  53  (53)  100/100 100/89  52 1  EST-2  74 100 126 h h  .27 .69 .04 .450 .415  53  (53)  74/74 100/100 74/100 74/126 100/126  5 26 18 1 3  ,LAP-1  80 " 85 91 95 100 h h Q  .04 .03 .23 .57 .13 .603 .392  51  (51).  91/91 95/9S 100/100 80/95 8S/9I 95/95 91/9S 91/100 95/100  9 20 2 4 .1 2 4 1 8  MDH-1  113 125 139 h  i01 .31 .68 .441 .431  51  (51)  125/125 139/139 113/125 125/139  5 24 1 21  51  (SI)  0  0  h  o  # Individuals  MDH-2  100 • ;  PGM-1  94 100 106 117 h h  .01 .49 .38 .12 .601 .604  53  (S3)  100/100 106/106 94/106 100/106 100/117 106/117  14 7 1 18 6 7  PGM-2  81 100 h h  .25 .75 .375 .321  53  (53)  81/81 100/100 81/100  5 31 17  PGM-3  100 135 h h  .35 .65 .455 .283  53  100/100 135/135 100/135  11 27 15  IDH-1  100  1.00  S3  (53)  ME-1  100  1.00  S3  (53)  6PG  100  1.00  S3  (S3)  SOD  100  1.00  53  (53)  0  0  0  1.00  (53)  1 6 7 .  P o p u l a t i o n 76-5  Locus  Allele  Mill Hill  Frequency  n  (F)  1.00  12  (3) (3)  EST-1  100  EST-2  74 . 100 h h  .81 .19 .308 .231  13  LAP-1  85 91 95 100 h  .12 .21 .21 .46 .686 .583  12  .33 .67 .442 .667  12  (3)  12  (3)  0  o  h  Park  . (3)  H-.2150  Genotype  H  "Individi  74/74 100/100 74/100  9 1 3  91/91 95/95 100/100 85/100 91/100 95/100  1 2 2 3 3 1  139/139 125/139  4 8  MDH-1  125 139 h h  MDH-2  100  PGM-1  100 106 h h  .92 .08 .147 .154  13  (3)  100/100 100/106  11 2  PGM-2  81 100 h h  .54 .46 .497 .683  12  (3)  81/81 100/100 81/100  3 2 7  PGM-3  100 135 h h  .SO .50 .500 .455 •  11  (3)  100/100 135/135 100/135  3 3 5  IDH-1  100  1.00  13  (3)  ME-1  100  1.00  13  (3)  6PG  100  1.00  13  (3)  SOD  100  1.00  !3.  (3)  0  0  0  0  1.00  168. Population  76-4  Thetis Lake Park  H=.2S49  H  •  Locus  Allele  Frequency  n  1.00  15  (10)  (F)  Genotype  #Individ  EST-1  100  EST-2  74 100 h h  .37 .63 .466 .467  15  (10)  74/74 100/100 74/100  LAP-1  80 8S 91 95 100 105 h h  .03 .10 .20 .14 .50 .03 .662 .467  15 . (10)  91/91 95/9S 100/100 80/91 85/91 85/95 85/100 91/100 9S/10S  113 125 139 h h  .03 .50 .47 .528 .'.467  15  12S/12S 139/139 113/125 125/139  6  1.00  15  (10)  0  0  MDH-1  0  (10)  2 6 7  MDH-2  . 100  PGM-1  100 106 h h  .63 .37.466 .200  15  (10)  '100/100 106/106 100/106  8 4 3  PGM-2  81 100 h h  .20 .80 .320 . 133  IS  (10)  81/81 100/100 81/100  2 11 2  PGM-3  77 100 135 h h  .16 .47 .37 .617 .400  15  (10)  77/77 100/100 13S/13S 77/100 100/13S  1 4 4 3 3  IDH-1  100  1.00  IS  (10)  ME-1  100  1.00  IS.  (10)  6PG  100  1.00  15  (10)  SOD  100  1.00  15  (10)  0  0  0  169. Population 77-9  Locus  Allele  Francis Park  Frequency  (F)  H=.2S75  Genotype  H . =.1634 obs  "Individuals  EST-1  89 100 h h  .02 .98 .039 .011  88  (29)  89/89 100/100 89/100  1 86 1  EST-2  74 100 h h  .32 .68 .43S .341  41  (12)  74/74 100/100 74/100  6 21 14  LAP-1  80 85 91 100 h  .03 .07 .45 .45 .589 .267  30  (12)  85/85 91/91 100/100 80/85 80/91 85/91 91/100  1 10 11 1 1 1 5  .22 .51 .27 .619 .367  30  (12)  113/113 125/125 139/139 113/125 125/139  6 10 3 1 10  30  (12)  0  0  o  h  MDH-1  113 125 139 h h  MDH-2  100  PGM-1  100 106 117 h  .63 .23 .14 .531 .288  66  (25)  100/100 106/106 117/117 100 /106 106/117  34 6 7 15 4  PGM-2  81 100 118 h h  /37 .60 .03 .502 .403  72  (25)  81/81 100/100 81/100 100/118  14 29 25 4  PGM-3  100 135 h  .75 .25 .375 .282  71  (25)  100/100 135/135 100/135  43 8 20  0  0  h  o  1.00  IDH-1  100  1.00  44  (12)  ME-1  100  1.00  44  (12)  6PG  100  1.00  44  (12)  SOD .  100  1.00  44  (12)  170. Population 77-10  Prospect Lake  H=.2704  H . =.2005 OOS  Locus  Allele  EST-1  100  EST-2  74 100 h h  LAP-1  Frequency  n  (F)  Genotype  fflndividi  42  (13)  .49 .51 .500 .380  50  (20)  74/74 100/100 74/100  IS 16 19  80 8S 91 95 100 105 h h  .02 .16 .18 .29 .30 .05 .765 .375  48  (18)  8S/8S' 91/91 95/95 100/100 80/91 85/95 85/100 91/105 95/100 95/105  4 7 8 11 2 4 3 1 4 4  MDH-1  125 139 h  .36 .64 .461 .500  36  (11)  125/125 139/139 125/139  4 14 18  MDH-2  100  36  (ID  PGM-1  100 106 117 h  .62 .35 .03 .492 .291  55  (20)  100/100 106/106 100/106 100/117  26 13 13 3  1.00 :  0  0  h  o  1.00  PGM-2  81 100 118 h h  .SI .46 .03 .527 .560  50  (19)  81/81 100/100 81/100 81/118 100/118  12 10 25 2 1  PGM-3  100 135 h h  .49 .51 .500 .300  50  (19)  100/100 135/135 100/135  17 18 15  IDH-1  100  1.00  50  ME-1  100  1.00  50  6PG  100  1.00  50  SOD  100  1.00  50  0  0  171. Population 77-11  Locus  Allele  EST-1  100  EST-2  74 100 h h o 80 85 91 95 100 105 h h  LAP-1  MDH-1  MDH-2 PGM-1  125 139 h h 0 100  Viaduct Ave.  Frequency  1.00  n  (F)  Qbs  'Individuals  (20)  .45 .55 .495 .436  39  (20)  74/74 100/100 74/100  9 13 17  .08 .10 .33 .24 .22 .03 .768 .486  37  (18)  85/85 91/91 95/95 100/100 10S/105 80/91 80/95 80/100 85/91 85/100 91/95 91/100 95/100  1 6 7 4 1 4 1 1 3 2 2 4 1  .53 .47 .498 .385  39  (20)  125/125 139/139 125/139  13 11 15  39  (20)  .72 .28 .403 .410  39  (20)  100/100 106/106 100/106  20 3 16  .35 .60 .05 .515 .513  39  (20)  81/81 100/100 81/100 81/118 100/118  5 14 16 1 3  .33 .67 .442 .359  39  (20)  100/100 135/135 100/135  6 19 14  . 1.00  39  (20)  1.00  IDH-1 ME-1  100  1.00  39  (20)  6PG  100  1.00  39  (20)  SOD  100  1.00  39  (20)'  PGM-3  H =.21S8  39  100 106 h h 0 81 100 118 h h o 100 13S h h 0 100  PGM-2  Genotype  H=.2601  172. Population  Locus EST-1  Allele 89 100 114 h h  0  76-7(2)  Malahat Dr.  Frequency .03 ' . 89 .08 .201 .193  n  .  (F)  H=.2S92  Genotype  H . =.1896 obs  "Individuals  57  (19)  100/100 114/114 89/100 100/114  45 1 4 7  EST-2."  74 100 126 h h  .76 .16 .08 .390 .385 .  65  (19)  74/74 126/126 74/100 74/126 100/126  38 2 18 5 2  LAP-1  80 85 91 95 100 h h  .24 .13 .02 .38 .24 .723 .477  65  (19)  80/80 85/85 95/95 100/100 80/95 80/100 85/95 85/100 91/95 95/100  11 4 12 7 6 3 6 3 2 11  MDH-1  113 125 139 h h  .02 .34 .64 .474 .269  67  (19)  113/113 125/125 139/139 125/139  1 14 34 18  MDH-2  87 100 h h  .05 .95 .095 .075  67  (19)  87/87 100/100 87/100  1 61 5  PGM-1  100 106 h h  .83 .17 .282 .185  65  (19)  100/100 106/106 100/106  48 5 12  PGM-2  81 100 h h  .43 .57 .490 .338  68  (19)  81/81 100/100 81/100  18 27 23  PGM-3  .35 .6S .455 .353  68  (19)  100/100 13S/13S 100/135  12 32 24  IDH-1  100 135 h h o 100  1.00  68  (19)  ME-1  100  1.00  68  (19)  6PG  100  1.00  68  (19)  SOD  100  1.00  68  (19)  0  0  0  0  0  0  173. Population 76-8  Crofton Exit  *  Locus  Allele  EST-1  100  EST-2  74 100 126 h h 0 80 85 91 95 100 10S h h  LAP-1  MDH-1  MDH-2 PGM-1  PGM-2  113 125 139 h h 0 100 100 106 117 h h 0 81 100 h h  PGM-3  o  H=.2138  H . =.1823 /the  Frequency  n  CF)  1.00  32  (17)  .05 .86 .09 .250 • 219  32  (17) .  100/100 126/126 74/100 100/126  24 1 3 4  .27 .OS .11 .16 .33 .08 .770 .531  32  (17)  80/80 85/85 91/91 95/95 100/100 10S/10S 80/91 80/95 80/100 85/95 91/9S 95/100 100/105  2 1 2 3 5 2 2 2 9 1 1 1 1  .OS .18 .77 .372 .281  32  (17)  125/125 139/139 113/125 113/139 125/139  2 21 1 2 6  32  (17) .  .25 .73 .02 .404 .375  32  (17)  100/100 106/106 100/106 100/117  2 .18 11 1  .19 .81 .309 .313  32  (17)  81/81 100/100 81/100  1 21 10  .36 .64 .461 .469  32  (17)  100/100 135/135 100/135  4 13 15  1.00  IDH-1  100 135 h h 0 100  1.00  32  (17)  ME-1  100  1.00  32  (17)  6PG  100  1.00  32  (17)  SOD  100  1.00  32  (17)  Genotype  #Individui  174.  Population 14(1) Locus  Allele  EST-1  100  EST-2  74 100 126 h h 0 80 8S 91 95 100 105 h h 0  LAP-1  MDH-1  125 139 h  MDH-2  100  PGM-1  IDH-1  100 106 h h 0 81 100 h h o 49 100 13 S h h o 100  ME-1  Jack's Point  H=.188S  H . =.1521 obs.  n  CF)  30  (30)  .15 .78 .07 .364 .310  29  (29).  74/74 100/100 126/126 74/100 100/126  1 18 1 7 2  .12 .20 .07 .26 .25 .10 .801 .467  30  (30)  80/80 8S/8S 91/91 • 9S/95 100/100 105/105 80/100 85/91 85/95 85/100 8S/10S 91/95 9S/100  2 .. 2 1 6 3 2 3 1 1 4 2 . 1 2  125/125 139/139 125/139  1 21 7  Frequency 1.00  .16. .84 .269 .241  Genotype  # Individuals  29  (29)  29  (29)  .80 .20 .320 .200  30  (30)  100/100 106/106 100/106  21 3 6  . .04 .96 .077 .036  28  (28)  100/100 81/100  26 2  .02 .28 .70 .431 .571  28  (28)  135/135 49/100 100/135  12 1 15  i.oo  30  (30)  100  1.00  30  (30)  6PG  100  1.00  30  (30)  SOD  100  1.00  30  (30)  PGM-2  PGM-3  1.00  175.  Population 14 (2) Locus  Allele  EST-1  Jack's Point Frequency  n  CP)  100  1.00 .  27  (27)  EST-2  74 100 h h  .11 .89 .196 .152  33  C33)  LAP-1  80 8S 91 95 100 105 h h o. 125 139 h  .04 .22 .26 .17 .29 .02 .769 .276  29  .35 .65 .455 .367  0  MDH-1  h  o  MDH-2  100  PGM-1  100 106 h h  o  1.00 .85 .15 .255 .000  Genotype  # Indiv:  74/74 100/100 74/100  1 27 5  (29)  80/80 85/85 91/91 95/95 100/100 85/91 85/100 100/10S  1 3 7 5 S 1 6 1  30  (30)  125/125 139/139 125/139  5 14 11  30  (30)  27  (27)  100/100 106/106  23 4  100/100 135/135 100/135  8 10 IS  PGM-2  100  1.00  33  (33)  PGM-3  100 135 h h  .47 .53 .498 • .455  33  (33)  IDH-1  100  1.00  33  (33)  ME-1  100  1.00  33  (33)  6PG  100  1.00  33  (33)  SOD  100  1.00  33  (33)  0  H= .1811 H . =. obs.  176. Population  Locus  EST-1  76-15(1)  Allele  100 114 h h  Nanoose H i l l  Frequency  .99 .01 .020 .021  0 "  n  (F)  H=.2342  Genotype  H , =.1919 obs  'Individi;  47  (19)  100/100 100/114  46 1  .81 .13 .06 .323 .333  42  (18)  74/74 100/100 74/100 74/126  27 1 9 5  -  EST-2  74 100 126 h h  LAP-1  80 85 91 95 100 h  .17 .01 .51 .29 .02 .626 .396  48  (19)  80/80 91/91 9S/9S 80/91 80/9S 85/91 91/95 91/100  1 18 10 8 6 1 2 2  MDH-1  125 139 h  .29 .71 .412 .385  39  (16)  125/125 139/139 125/139  4 20 15  MDH-2  87 100 h h  .01 .99 .020 .026  39  (16)  100/100 87/100  38 1  PGM-1  100 106 h h o 81 100 ' 118 h h  .69 .31 .428 .333  48  (19)  100/100 106/106 100/106  25 6 16  .36 .62 .02 .486 .426  47  (19)  81/81 100/100 81/100 100/118  8 19 18 2  .45 .55 .495 .383  47  (19)  100/100 135/135 100/135  12 17 18  0  0  PGM-2  0 PGM-3  100 135 h h  IDH-1  100  1.00  48  (19)  ME-1  100  1.00  48  (19)  6PG  100  1.00  48  (19)  SOD  100  1.00  48  (19)  0  177. Population 77-•15(2)  Locus  Allele  Nanoose H i l l  Frequency  n  (F)  1.00  . 7  (3)  H=.1868  Genotype  H . =. obs  'Individuals  EST-1  . 100  EST-2  74 100  .83 .17 .282 .333  6  (3)  74/74 74/100  4 2  91 100  .05 .95 .095 .100  10  (8)  100/100 91/100  9 1  125 139  .43 .57 .490 .571  14  (10)  125/125 139/139 125/139  2 4 8  14  (10)  .58 .42 .487 .231  13  (9)  100/100 106/106 100/106  6 4 3  81 100  .29 .71 .412 .286  14  (10)  81/81 100/100 81/100  2 8 4  100 135  .39 .61 .476 .357  14  (10)  100/100 135/135 100/135  3 6 5  h " V LAP-1  h V  MDH-1  h h  0  MDH-2  100  PGM-1  100 106  h o  h  PGM-2  h h0  PGM-3  h h0  1.00  IDH-1  100  1.00  14  (10)  ME-1  100  1.00  14  (10)  6PG  100  1.00  14  (10)  SOD  100  1.00  14  (10)  178. . N i l e Creek  P o p u l a t i o n 76-17  Locus  EST-1  EST-2  LAP-1.  MDH-1  Allele  100 114 h h  0 74 100 126 h h • 0 80 85 91 95 100 105 h h  113 125 139 h h  0  0  Frequency  n  •CF)  (24)  100/100 100/114  35 1  .04 ' .94 .02 .114 .114  35  (23)  100/100 74/100 100/126  31 3 1  .07 .47 .39 .03. .01 .03 .620 .278  36  (24)  80/80 85/85 . 91/91 95/95 105/105 80/85 80/91 8S/91 85/100  1 13 10 1 1' 1 2 6 1  .21 .19 .60 .560 .514  35  (23)  113/113 125/125 139/139 113/125 113/139 125/139  3 1 13 2 7 9  35  (23)  .57 .43 .490 .441  34  (22)  100/100 106/106 100/106  12 7 15  .46 .51 .03 .527 .559  34  (22)  81/81 100/100 81/100 100/118  7 8 17 2  .28 .72 .403 .441  34  (22)  100/100 135/135 100/135  2 17 15  1.00  PGM-1  100 106 h h  PGM-2  81 100 118 h h  PGM-3  100 135 h h  IDH-1  100  1.00  36  (24)  ME-1  100  1.00  36  (24)  6PG  100  1.00  36  (24)  SOD  100  1.00  36  (24)  0  #Individu  36  100  0  Genotype  ob  .99 .01 .020 .028  MDH-2  0  H=.2278  

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