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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., Vanderbilt University, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1980 © Charles Robert Layton, 1980 In presenting t h i s thesis i n p a r t i a l f u l f i l l m e n t of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v ailable f o r reference and study. I further agree that permission f o r extensive copying of t h i s thesis for s c h o l a r l y purposes may be granted by the Head of my department or his representative. It i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. The Uni v e r s i t y of B r i t i s h Columbia Vancouver, Canada V6T 1W5 ABSTRACT This study describes the consequences of contrasting breeding systems on l e v e l s and organization of isozyme v a r i a t i o n i n two c l o s e l y r e l a t e d taxa, Pleotvitis oongesta (Lindl.) D.C. and P. bvaahystemon F. £ M. (Valerianaceae). Both taxa are s e l f -compatible, herbaceous winter annuals which occur along the P a c i f i c coast from B r i t i s h Columbia to C a l i f o r n i a . 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 f i f t e e n l o c i i d e n t i f i e d i n Pleotvitis. Genetic models were proposed to explain the observed v a r i a t i o n . These models describe the multi-locus organization, sub:-unit structure and a l l e l i c v a r i a t i o n within each system. The models were tested with crosses and segregation analyses. When progeny classes were s u f f i c i e n t l y large, segregations were analyzed using the chi-square s t a t i s t i c . Although P. oongesta and P. bvaahystemon d i f f e r e d i n t h e i r a l l e l i c complements, the data suggest that the multi-locus organization of the enzyme systems surveyed;are i d e n t i c a l i n both species. Outcrossing rates were calculated f o r nine populations of P. hvaohystemon and f i f t e e n populations of P. oongesta. Their mean outcrossing rates were 2.4± 0.8% and 70.2± 4.8%, 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 highly s i g n i f i c a n t (P<0.01). A number of genetic parameters were measured within each taxon to assess the e f f e c t that breeding system has on population structure. As measured by a l l parameters, the s e l f e r maintains s i g n i f i c a n t l y less v a r i a t i o n within populations than the outcrosser although t h e i r detected l e v e l s of 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 d i f f e r e n t . These two species are i d e a l f or comparison because: (i) they are c l o s e l y r e l a t e d and have a large proportion of t h e i r a l l e l e s i n common, ( i i ) the multi-locus organization of the enzyme systems studied 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 rategies and habitat requirements. It i s not uncommon, p a r t i c u l a r l y on Vancouver Island, to f i n d the two species growing sympatrically (i.e. i n mixed populations). When the influence of 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 only sympatric populations of the two species, a l l genetic differences remained s i g n i f i c a n t . The reduced l e v e l of v a r i a t i o n within populations of the s e l f e r was r e f l e c t e d i n the F- s t a t i s t i c s and i n the analysis of gene d i v e r s i t y . On the average, P. oongesta populations contained 85% of the v a r i a t i o n detected within the species. Only 36% of the v a r i a t i o n detected i n P. braohystemon was maintained within l o c a l populations. In the s e l f e r , the reduced l e v e l of g e n e t i c " v a r i a t i o n within populations was accompanied by increased populational d i f f e r e n t i a t i o n . Preliminary data suggest that 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 the s e l f e r , can occur over short distances. However, whether t h i s represents micro-geographic d i f f e r e n t i a t i o n or d r i f t i n small reproductivelylxsolated populations i s problematic. In t h i s taxon, a l l e l e frequencies and single locus heterozygosities fluctuated widely and d i f f e r e n t a l l e l e s were often f i x e d i n adjacent populations. In P. oongesta, a l l e l e frequencies and heterozygosities also fluctuated among populations but not with the amplitude observed i n P. brachystemon. There appears to be no r e l a t i o n s h i p between genetic distance and geographic distance. This implies that gene flow between populations does not s i g n i f i c a n t l y influence a l l e l e frequencies. No evidence was.found to suggest that the observed isozyme polymorphisms are s e l e c t i v e l y maintained. However, co-ordinated gene complexes and micro-habitat s e l e c t i o n would probably not have been detected. It i s concluded that the observed differences i n the population structure of P. .bvachystemon and P. oongesta i s p r i m a r i l y the r e s u l t of t h e i r contrasting breeding systems. i v . TABLE OF CONTENTS CHAPTER ABSTRACT ' LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENTS 1.0 INTRODUCTION 1.1 Statement of the problem 1.2 Breeding system, and population structure 1.3 Morphological v a r i a t i o n in selfers 1.4 Isozyme v a r i a t i o n in selfers 1.5 Breeding system and genetic variability 2.0 MATERIALS AND METHODS 2.1 C o l l e c t i o n sites and sampling strategy 2.2 Potential sources of bias in the collections 19. 2.2.1 Sample size 19. 2.2.2 Phenology 19. 2.2.3 Seed wing . 1 9 . 2.2.4 Wahlund effect 20. 2.3 Methods of planting and growth regime 20. 2.4 Electrophoretic techniques 21. 3.0 INHERITANCE 29. 3.1 Isozyme nomenclature 29. 3.2 Esterase 31. 3.3 Leucine amine-peptidase 33. 3.4 Malate dehydrogenase 37. v. PAGE i i . v i i i . x. x i i i . 1. 1. 4. 6. 9. 10. 16. 16. CHAPTER,:: PAGE 3.5 Phosphoglucose isomerase 40. 3.6 Phosphoglucomuta.se 50. 3.7 Monomorphic enzyme systems 58. 3.8 Summary 60. 4.0 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 86. 5.1 Analysis of the breeding system 86. 5.2 Genetic variability within populations 94. 5.3 Analysis of gene diversity 102. 5.4 Comparisons of genetic identity and genetic distance HO. 5.4.1 Levels of inter-population differentiation 115. 5.4.2 Comparisons between genetic distance and 116. geographic distance 5.4.3 Taxonomic and evolutionary relationships 124. 5.4.4 Maintenance of observed isozyme polymorphisms 127. 5.5 Summary: the genetic consequences of contrasting 133. breeding systems v i . CHAPTER PAGE BIBLIOGRAPHY 137. APPENDIX A Population locations 1 4 5 -B Enzyme systems which were not adequately 148. resolved with a v a i l a b l e techniques C Gene and genotype frequencies within populations 151. C.l P. bvachystemon populations 152. ' C.2 Pteotritis oongesta populations 163.-v i i . LIST OF TABLES TABLE TITLE PAGE 1 C o l l e c t i o n s i t e s 17. 2 Running buffers 24. 3 Running buffer used, number of l o c i res.olved and 25. sta i n i n g references f o r enzyme systems studied i n P. brachystemon and P. congesta 4 Summary of enzyme s t a i n i n g procedures 27. 5 Stock solutions 28. 6 Analysis of allozyme v a r i a t i o n at EST-1 34. 7 Analysis of allozyme v a r i a t i o n at EST-2 35. 8 Analysis of allozyme v a r i a t i o n at LAP-1 38. 9 Analysis of allozyme v a r i a t i o n at MDH-1 43. 10 Analysis of allozyme v a r i a t i o n at PGI-2 51. 11 Analysis of allozyme v a r i a t i o n at PGI-3 51. 12 Analysis of allozyme v a r i a t i o n at PGM-1 55. 13 Analysis of allozyme v a r i a t i o n at PGM-2 56. 14 Analysis of allozyme var i at ion-rat PGM-3 57. 15 A l l e l e frequencies and heterozygosity values f o r 63. eight polymorphic l o c i i n P. brachystemon 16 A l l e l e frequencies and heterozygosity .values for 66. eight polymorphic l o c i i n P. congesta 17 Comparison of mean a l l e l e frequencies with 72. heterozygosity values for polymorphic l o c i i n P. brachystemon and P. congesta 18 Wright's f i x a t i o n index f o r seven polymorphic 88. l o c i i n P. brachystemon v i i i . TABLE TITLE PAGE 19 Wright's f i x a t i o n index f o r eight polymorphic 89. l o c i i n P. oongesta 20 Estimates o f ou t c r o s s i n g frequency f o r ten 92. populations of P. braohystemon 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 93. populations of P. oongesta 22 Summary of various genetic parameters f o r ten 95. populations of P. braohystemon 23 Summary of various genetic parameters f o r 96. f i f t e e n populations o f P. oongesta 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 106. d i f f e r e n t i a t i o n f o r f i f t e e n l o c i i n P. braohystemon 25 A n a l y s i s of gene d i v e r s i t y and degree o f .:*. . 107. 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 -26 Genetic i d e n t i t i e s and standard genetic 113. distances among populations of P. braohystemon 27 Genetic i d e n t i t i e s and standard genetic 114. distances among populations of P. oongesta 28 Summary of genetic distance i n P. braohystemon 117. 29 Summary of genetic distance i n P. oongesta 118. 30 I n t e r - s p e c i f i c comparison of genetic distance 129. i n Pleotritis 31 A comparison between the observed and the 131. t h e o r e t i c a l i n t e r - l o c u s variances of he t e r o z y g o s i t y i n populations of P. braohystemon 32 A comparison between the observed and the 132. t h e o r e t i c a l i n t e r - l o c u s variance of het e r o z y g o s i t y i n populations of P. oongesta 33 Summary of genetic d i f f e r e n c e s between 134. P. braohystemon and P. oongesta i x . LIST OF FIGURES FIGURE TITLE PAGE 1 Flowers of Vtectritis congesta and P. brachystemon 3. i l l u s t r a t i n g differences i n s i z e 2 A map showing the l o c a t i o n of sampled Plectritis 18. populations 3 The r e l a t i v e migration of allozymes detected at EST-1 32. 4 The r e l a t i v e migration of allozymes detected at EST-2 32. 5 Allozyme v a r i a t i o n at EST-1 32. 6 The r e l a t i v e migration of allozymes detected at LAP-1 36. 7 Allozyme v a r i a t i o n at LAP-1 i n P. congesta 36. 8 The r e l a t i v e migration of allozymes detected at MDH-1 41. 9 The r e l a t i v e migration of allozymes detected at MDH-2 41. 10 Allozyme v a r i a t i o n at MDH-1 42. 11 Allozyme v a r i a t i o n at MDH-1 42. 12 Segregation of allozyme v a r i a t i o n at PGI-2 45. and PGI-3 i n P. congesta 13 Ah:.allelic i n t e r p r e t a t i o n of the allozyme v a r i a t i o n 45. pictured i n Figure 12 14 A sample of the allozyme v a r i a t i o n detected at 46. PGI-2 and PGI-3 i n P. congesta 15 A sample o f the allozyme v a r i a t i o n detected at 46. PGI-2 and PGI-3 i n P. congesta 16 The r e l a t i v e migration of allozymes detected at PGI-2 48. 17 The r e l a t i v e migration of allozymes detected at PGI-3 48. x. FIGURE TITLE PAGE 18 Allozyme v a r i a t i o n at PGI-2 i n P. bvachystemon 48. 19 Allozyme v a r i a t i o n at PGI-2 i n P. bvachystemon 49. 20 A comparison of isozyme v a r i a t i o n i n PGI between 49. P. oongesta and P. bvachystemon 21 The r e l a t i v e migration of allozymes detected at PGM-1 53. 22 The r e l a t i v e migration of allozymes detected at PGM-2 53. 23 The r e l a t i v e migration of allozymes detected at PGM-3 53. 24 Allozyme v a r i a t i o n i n PGM i n P. oongesta 54. 25 A comparison of isozyme v a r i a t i o n i n PGM between 54. 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 of EST-1 a l l e l e s among populations 70. of P. bvachystemon 29 The d i s t r i b u t i o n of MDH-1 a l l e l e s among populations 70. of P. bvachystemon 30 The d i s t r i b u t i o n of LAP-1 a l l e l e s among populations 71. of P. bvachystemon 31 The d i s t r i b u t i o n of PGM-1 a l l e l e s among populations 71. of P. bvachystemon 32 The d i s t r i b u t i o n of LAP-1 a l l e l e s among populations 76. of P. oongesta 33 The d i s t r i b u t i o n of MDH-1 a l l e l e s among populations 76. of P. oongesta 34 The d i s t r i b u t i o n of PGM-3 a l l e l e s among populations 77. of P. oongesta 35 The d i s t r i b u t i o n of EST-2 a l l e l e s among populations 77. of?P. oongesta x i . F I G U R E ; TITLE PAGE 36 The d i s t r i b u t i o n of PGM-2 a l l e l e s among populations 78. of P. congesta 37 The d i s t r i b u t i o n of PGM-1 a l l e l e s among populations 78. of P. congesta 38 The d i s t r i b u t i o n of PGI-3 a l l e l e s among populations 80. of P.. brachystemon 39 The d i s t r i b u t i o n of PGM-3 a l l e l e s among populations 80. of P. brachystemon 40 The d i s t r i b u t i o n of PGI-2 a l l e l e s among populations 81. of P. brachystemon 41 The d i s t r i b u t i o n of EST-1 a l l e l e s among populations 81. of P. congesta .42 The r e l a t i o n s h i p between the outcrossing rate and 100. the expected heterozygosity among populations of P. brachystemon .43 The r e l a t i o n s h i p between the outcrossing rate and 100. the expected heterozygosity among populations of P. congesta 44 A dendrograph depicting the genetic r e l a t i o n s h i p s 119. among the sampled P. brachystemon populations 45 A dendrograph depicting the genetic r e l a t i o n s h i p s 120. :among the sampled P. congesta populations 46 A dendrograph depicting the genetic r e l a t i o n s h i p s 125. among the sampled P l e c t r i t i s populations x i i . ACKNOWLEDGEMENTS I would l i k e to express my gratitude to a l l those who have helped me with t h i s study and have made i t , i n retrospect, a p o s i t i v e experience. Dr. Fred Ganders, my advisor, introduced me to the plants and to many of the concepts from which the thesis evolved. Without his advice and t o t a l support, t h i s research would not have been conducted. I would l i k e to thank Dr. Tony G r i f f i t h s f o r h i s i n t e r e s t i n r e a l plants, h i s encouragement throughout the study and his c r i t i c a l evaluation of the genetic sections of th i s t h e s i s . A s p e c i a l thanks to Dr. Wilf S c h o f i e l d who was drafted, and agreed to p a r t i c i p a t e i n th i s p r oject, because of his open mind, patience and good humor. Dr. Francis Yeh advised and provided technical assistance with the data analysis. I am gr a t e f u l f or h i s suggestions regarding the discussions of population structure. In addition, I would l i k e to thank: Ken Carey for h i s advice on f i n d i n g , growing and handling Pteotvitis; Sue Krepp Denny, who i n i t i a l l y l e t me muddle about her lab and J e f f Archer and Richard O'Brien who assis t e d with the f i e l d work. My deepest gratitude belongs to Diane Layton whose patience, encouragement and assistance throughout the study made the process easier. 1. 1.0 INTRODUCTION 1.1 Statement of the problem This study describes the consequences of. contrasting breeding systems on l e v e l s and organization of isozyme v a r i a t i o n i n two 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 oongesta (Lindl.) D.C. and P l e o t r i t i s braohystemon F. § M. (Valerianaceae). Both taxa are self-compatible, herbaceous winter annuals which occur along the P a c i f i c coast from B r i t i s h Columbia to C a l i f o r n i a . P. braohystemon has 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 Charlotte Islands southward into southern C a l i f o r n i a . These two taxa are morphologically very s i m i l a r ; the only s i g n i f i c a n t differences are i n t h e i r f l o r a l characters. P. oongesta has r.elatively.large flowers. The c o r o l l a s average 6-10 mm. i n length, are bright pink and bear a nectariferous spur. They are strongly fragrant and are a c t i v e l y v i s i t e d by a number of p o l l i n a t o r s . Although self-compatible, P. oongesta i s strongly protandrous. However, since numerous flowers on the inflorescence may be open simultaneously, geitonogamous p o l l i n a t i o n s are p o s s i b l e . In contrast, the flowers of P. braohystemon are quite small. They are white to pale pink and average only 2-4 mm i n length. Corollas may be spurred, but i n some populations the spur i s reduced to a mere swelling. The flowers are not markedly protandrous. The stigma i s i n close proximity to the anthers at anthesis. The flowers of P. braohystemon have only a very f a i n t fragrance and p o l l i n a t o r s were not observed v i s i t i n g the flowers. This r a r i t y of insect v i s i t s has been reported e a r l i e r by Dempster (1958), Morey (1962) and Ganders 2. et al. (1977b). Figure 1 i l l u s t r a t e s the si z e difference between flowers of the two taxa. The differences i n f l o r a l characters strongly suggest contrasting breeding s t r a t e g i e s . This was investigated and confirmed by Ganders et al. (1977a, 1977b) who u t i l i z e d a seed wing dimorphism, present i n both taxa, to estimate outcrossing rates. The inheritance of the seed wing i s c o n t r o l l e d by a simple one gene, two a l l e l e system with the winged a l l e l e expressing dominance (Ganders et al., 1977a). Employing the progeny te s t method of Harding (1970), they found P. congesta to be p r i m a r i l y outcrossed; averaging 70% outcrossing over sevenrpopulations (Ganders et al., 1977a; Carey and Ganders, i n press). In contrast, P. brachystemon was found to be almost 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 , averaging only 2% outcrossing (Ganders et al., 1977b). These two taxa are c l o s e l y r e l a t e d ; P. brachystemon may be a d e r i v a t i v e of P. congesta. Stebbins (1957) argues that s e l f e r s are probably always derived from outcrossers. Many examples of the evolution of autogamous taxa from outcrossers have been demonstrated. Moore and Lewis (1965) presented c y t o l o g i c a l evidence that normally outcrossing, pink flowered, Clarkia xantiana has given r i s e to two d i s t i n c t small flowered autogamous populations; one with pink and the other with white flowers. Antonovics (1968) investigated the evolution of s e l f - f e r t i l i t y i n heavy metal tolerant populations of Agrostis tenuis and' Anthoxanthum odoratum from normally outcrossing non-tolerant populations. Arroyo (1973) concluded that autogamous Limnanthes floccosa has evolved from the outcrosser, L. alba (see also Ornduff and Crovello, 1968). 3. Figure 1. Flowers of (A) Weotritis oongesta and (B) P. braohystemon i l l u s t r a t i n g differences i n s i z e and i n the degree of separation of the stigma and anthers i n the two species. 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 receptive. 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 rategies and habitat preferences. In f a c t , sympatric {i.e. mixed) populations are not uncommon, at least on southern Vancouver Island. Babbel and Selander (1974) e l e c t r o p h o r e t i c a l l y surveyed two p a i r s of edaphically r e s t r i c t e d and widespreadcplant species and concluded that l e v e l s of genetic v a r i a b i l i t y are influenced by e c o l o g i c a l amplitude. Recently, Hamrick et al. (1979) reviewed a l l g e n e t i c a l l y interpretable, higher-plantallozyme l i t e r a t u r e published p r i o r to June, 1978. Using m u l t i v a r i a t e techniques, they attempted to correlate 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 with levels of allozyme v a r i a t i o n among 100 plant taxa. The l i f e - h i s t o r y 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 genetic variabilitycamong the studied taxa were: mating system, p o l l i n a t i o n mechanism, generation length, chromosome number, fecundity, geographic range and successional status. Since P. oongesta and P. braohystemon are c l o s e l y r e l a t e d {i.e. possibly as progenitor and 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 to study the genetic consequences of contrasting breeding s t r a t e g i e s while c o n t r o l l i n g f o r genetic background and other variables which also influence the genetic structure of populations. 1.2 Breeding system and population structure Populations of a predominantly outcrossed taxon are generally considered to consist of a large number of biotypes, each of which may be represented by only a s i n g l e i n d i v i d u a l . The l e v e l of v a r i a t i o n . maintained within the population gene p'ool depends on the e f f e c t i v e population s i z e (a function of the s i z e of the breeding population 5. and the outcrossing: r a t e ) , mutation rate, gene flow and the modes and s e v e r i t y of s e l e c t i o n . In contrast, the t r a d i t i o n a l concept of autogamous populations i s one of a number of d i s c r e t e homozygous biotypes i s o l a t e d from each other by breeding habit (DuRietz, 1930 and Stebbins, 1950, 1957). Each of these types may occupy a s l i g h t l y d i f f e r e n t adaptive peak and be represented -by a few or thousands of i n d i v i d u a l s . Successful biotypes are t r a d i t i o n a l l y considered to maintain themselves as homozygous l i n e s f o r p o t e n t i a l l y large numbersiof generations. Besides these reproductively i s o l a t e d sympatric l i n e s , the populations may also consist of numerous hybrids which are the r e s u l t of occasional crosses between these predominant inbred biotypes. The occurrence of these intermittent 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 of great evolutionary a c t i v i t y - w i t h i n the population (Stebbins, 1950, 1957). The proportion of the population composed of these i n t e r - r a c i a l hybrids w i l l depend on the l e v e l of outcrossing i n the population and the modes and s e v e r i t y of s e l e c t i o n . The F^ hybrids w i l l be h i g h l y heterozygous. These hybrids may or may not be more vigorous than ei t h e r of the parental types. I f they survive and s e l f , the generation w i l l show a wide range of segregation. Successive generationsr.wiil" continue to produce new segregants, i n diminishing numbers f o r seven to eight generations. The great majority of these types w i l l be less f i t than e i t h e r of the o r i g i n a l parents. But, should a new biotype with superior f i t n e s s be synthesized, i t can be the progenitor of a new l i n e . As Stebbins v i s u a l i z e s autogamous populations, the predominant biotypes are almost t o t a l l y homozygous and t h e i r i n t e r - r a c i a l hybrids are ephemeral. This model predicts that the vast majority of the v a r i a t i o n within the population w i l l be d i s t r i b u t e d between inbred l i n e s '.Ci'.e. f a m i l i e s ) . Consequently, the l e v e l of v a r i a t i o n maintained within the population w i l l be determined by the number of inbred l i n e s . This w i l l depend on the e f f e c t i v e population s i z e , mutation rate, gene flow, habitat d i v e r s i t y and modes and se v e r i t y of s e l e c t i o n . I f out-crossing rates are low and hybrids are ephemeral,.the two most important factors i n f l u e n c i n g the levels of v a r i a t i o n within populations should be habitat d i v e r s i t y and modes and s e v e r i t y of s e l e c t i o n . Baker (1959) predicts that under severe s e l e c t i o n only one biotype, or a very few, w i l l e x i s t and autogamous populations w i l l be uniform, i f present at a l l . 1.3 Morphological v a r i a t i o n in selfers A number of studies have investigated predominantly s e l f -p o l l i n a t e d taxa to determine i f the t r a d i t i o n a l population model, as proposed by Stebbins and others, agrees with quantitative phenotypic data and observations of morphological gene markers. J a i n and A l l a r d (1960) studied v a r i a t i o n at nine morphological gene l o c i within experimental barley populations. They concluded that the v a r i a b i l i t y maintained within these autogamous populations was not associated e n t i r e l y with sympatric homozygous l i n e s , and that le v e l s of outcrossing lower than those frequently observed i n s e l f e r s , i f accompanied by s e l e c t i o n for heterozygotes, could maintain considerable amounts of v a r i a b i l i t y . S i milar conclusions were reached when these same barley populations were analyzed f o r quantitative genetic characters (A l l a r d and J a i n , 1962). A l l a r d and Workman (1963) discovered that, i n lima beans, wide seasonal f l u c t u a t i o n s i n f i t n e s s were associated with c e r t a i n morphological markers. However, mean s e l e c t i v e values i n d i c a t 7. that heterozygotes l e f t an average of 20% - 30% more progeny than e i t h e r homozygote. Continuing t h i s work, Harding, A l l a r d and Smeltzer (1966) found that s e l e c t i o n f or heterozygotes was frequency-dependent. When heterozygotes were numerous, as i n early generations following an i n t e r -r a c i a l h y b r i d i z a t i o n , they had l i t t l e or no s e l e c t i v e value. Consequently, they were r a p i d l y reduced i n number by inbreeding. This process slowed and an equilibrium was reached when heterozygotes comprised approximately 7% of the experimental population. 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 values increased and at very low frequencies heterozygotes produced approximately three times as many progeny as homozygotes. When heterozygotes were a r t i f i c i a l l y reduced to a frequency of 2%, the 7% equilibrium value was restored within a si n g l e generation and t h i s was maintained i n the successive generation. Working with Avena fatua, Imam and A l l a r d (1965) discovered that f a m i l i e s , derived from s i n g l e plants c o l l e c t e d from natural populations, were highly heterogeneous for most measured phenotypic characters. In addition, f a m i l i e s derived from:the same s i t e showed a wide range of means for various characters. They concluded that populations of t h i s predominantly s e l f - p o l l i n a t i n g taxon consist of large numbers, of biotypes and that many i f not a l l i n d i v i d u a l s i n natural populations are heterozygous at numerous l o c i . Kannenberg and A l l a r d (1967) studied quantitative v a r i a t i o n i n eleven morphological characters within taxa of the Festuoa microstachys complex. This complex consists of eight species which frequently occur i n mixed populations and 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 . Since chasmogamous flowers are rare, Kannenberg and A l l a r d estimated that i t was u n l i k e l y that even one wild plant i n 1000 would be an F 7 hybrid and i t was probable 8. that the rate of outcrossing 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 large proportion of the t o t a l genetic variance i n a l l measured characters was at t r i b u t e d to within-population d i f f e r e n c e s . Species within sites.and families within species accounted f o r 10% and 30%, res p e c t i v e l y , of the t o t a l phenotypic variance i n t h e i r experimental p l o t s . Furthermore, when the means of fami l i e s within s i t e s were compared f or each of the eleven measurement characters, i t was found that there were few families at any one s i t e which did not d i f f e r , s i g n i f i c a n t l y with respect to at least one measurement character. In other words, any sing l e s i t e contained plants of many d i f f e r e n t genotypes. They then compared data on three characters to measurements c o l l e c t e d for two other species which had contrasting breeding systems; an obligate outcrosser Lolium multiflovvm and Avena fatua i n which outcrossing varies between 1% and 10%. Despite the differences: i n outcrossing rates, there were no consistent differences i n the lev e l s of within-population v a r i a t i o n among these three taxa. These studies of morphological v a r i a t i o n i n s e l f e r s are s i g n i f i c a n t because they c o n s i s t e n t l y found that the levels of v a r i a t i o n within populations was higher than expected: These r e s u l t s have led several investigators (e.g. Jain and A l l a r d , 1960; A l l a r d and J a i n , 1962; Imam and A l l a r d , 1965; Kannenberg and A l l a r d , 1967; S o l b r i g , 1972; Clegg and A l l a r d , 1972; Hamrick and A l l a r d , 1972; A l l a r d et dl. , 1972; Arroyo, 1975; So l b r i g and R o l l i n s , 1977; Levin, 1978; Keeler, 1978 and Hamrick and Holden, 1979).to question whether populations of autogamous taxa are as g e n e t i c a l l y uniform as t r a d i t i o n a l l y believed. 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 highly inbred populations where i n t e r - r a c i a l crosses are rare, bursts of segregation may be considerably more per s i s t a n t than the c l a s s i c a l model p r e d i c t s . A l l a r d and J a i n (1962) suggest, given heterosis, that populations i n which outcrossing i s common (they do not speci f y how common) might consist l a r g e l y of heterozygotes associated with numerous hybrid swarms overlapping i n time. Such populations would be highly v a r i a b l e and would not d i f f e r s i g n i f i c a n t l y from populations of predominantly outcrossed taxa. 1.4 Isozyme v a r i a t i o n in selfers More recent studies have u t i l i z e d gel electrophoresis i n order to investigate the d i s t r i b u t i o n of genetic v a r i a b i l i t y within autogamous taxa and to evaluate the ef f e c t s of breeding system.;on lev e l s and organization of genetic v a r i a b i l i t y . In Avena barbata, Clegg and A l l a r d (1972) found that a l l e l e frequencies, at f i v e enzyme loci.'.and two l o c i governing morphological t r a i t s , were d i s t r i b u t e d i n • s i g n i f i c a n t l y non-random, macro-geographic patterns which were c l o s e l y associated with the environment. A l l a r d et al. (1972) found a large proportion of t h i s v a r i a t i o n to be associated with two co-adapted a l l e l e complexes. Populations which occupied x e r i c s i t e s were monomorphic f o r a s p e c i f i c combination of a l l e l e s at f i v e enzyme l o c i . Populations at mesic s i t e s were monomorphic f o r ah alternate combination of allozymes while populations i n intermediate habitats were usually polymorphic at a l l f i v e l o c i . Therefore, they concluded that e p i s t a t i c i n t e r a c t i o n s among a l l e l e s were important i n the maintenance of genetic v a r i a b i l i t y i n A.'..barbata. In addition, Hamrick and A l l a r d (1972) and Hamrick and Holden (1979) have found that these co-adapted a l l e l e complexes i n 10. A. barbata are associated with micro-geographical v a r i a t i o n within populations. Kahler et al. (1980) studied isozyme v a r i a t i o n i n A. barbata i n I s r a e l . They found that: each population contained many isozyme phenotypes, populations were d i f f e r e n t i a t e d by t h e i r phenotypic arrays, 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 patterns, and particular:.phenotypes were correlated with c e r t a i n temperature and moisture-related v a r i a b l e s . Crawford and Wilson (1977) analyzed 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 populations of Chenopodivm fremontii. Most populations were monomorphic and the frequency of observed heterozygotes was extremely low (i.e. heterozygosity was detected i n only t h i r t e e n plants, each one, heterozygous at a s i n g l e locus). Keeler (1978) studied f i v e enzyme systems i n the hexaploid Veronica peregrina. No genetic analyses were attempted because of the p o l y p l o i d inheritance. However, through progeny testing, i n d i v i d u a l plants were found to be highly v a r i a b l e . It was concluded that hexaploid inheritance allowed the maintenance of i n d i v i d u a l v a r i a t i o n despite almost complete s e l f - p o l l i n a t i o n . Babbel and Wain (1977) estimated outcrossing rates and analyzed the population structure of the t e t r a p l o i d weed Hordeum jubatum. Four allozyme l o c i were surveyed and the outcrossing rates within two populations were calculated to be 1% and 3%. The observed heterozygosity at i n d i v i d u a l l o c i was low. However, a majority of the f a m i l i e s surveyed were fi x e d heterozygotes at two l o c i as a r e s u l t of polyploidy. 1.5 Breeding system and genetic variability A r e l a t i v e l y large number of published studies have examined lev e l s of genetic v a r i a t i o n i n either s e l f e r s or outcrossers. However, only a few investigations have attempted to describe the e f f e c t s of d i f f e r e n t breeding systems on the organization of genetic v a r i a t i o n i n r e l a t e d species. S o l b r i g (1972) compared three self-incompatible and four self-compatible species of Leavenwovthia for lev e l s and organization of enzyme v a r i a b i l i t y . S o l b r i g did not attempt any inheritance studies with the enzyme systems surveyed. Consequently, observed v a r i a t i o n could not be interpreted with respect to a l l e l e frequencies and number of l o c i surveyed. However, based on band frequencies, he concluded that the self-compatible species showed less within-family and within-population v a r i a t i o n than did the s e l f -incompatible species. S o l b r i g and R o l l i n s (1977) studied the evolution of autogamy i n Leavenwovthia (see also Lloyd, 1965) . They analyzed both enzyme data (i.e. just i n terms of band presence or absence and segregating versus non-segregating families) and morphological data on f r u i t characters. Generally, wider ranges of v a r i a t i o n were found i n the self-incompatible taxa. However, i n one self-compatible species, within-population v a r i a b i l i t y i n f r u i t characters was greater than that found i n two of the self-incompatible species. They concluded that the breeding system was the si n g l e most important factor determining population structure. Levin (1977) analyzed allozyme v a r i a t i o n at twenty l o c i i n two self-incompatible and one self-compatible species of Phlox. The self-compatible species P. euspidata s e l f s automatically i n e i t h e r the f i e l d or greenhouse and sets a f u l l compliment of seed. Using the maximum l i k e l i h o o d estimator of Brown and A l l a r d (1970), an outcrossing rate was calculated f o r a " t y p i c a l " population of t h i s species and found to be t = 0.22. When lev e l s of allozyme v a r i a t i o n were compared, P. euspidata had less t o t a l and within-population v a r i a b i l i t y 12. than e i t h e r of the self-incompatible species. Rick et al. (1977) studied genetic v a r i a t i o n at 11 enzyme and two morphological l o c i i n Lyoopevsioon pimpinellifolium. This i s a self-compatible species i n which outcrossing (i.e. measured as the proportion of c r o s s - p o l l i n a t i o n ) varied between zero and 40%. In t h i s taxon a l l measures of population v a r i a b i l i t y (i.e. heterozygosity and mean number of a l l e l e s per locus) were correlated p o s i t i v e l y (P<0.01) with f l o r a l characters (i.e. anther length and stigma exsertion) and proportion of cross-p o l l i n a t i o n (i.e. estimated by the proportion of non-parental a l l e l e s appearing i n progenies). In contrast, Arroyo (1975) detected no c l e a r 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 degree of autogamy among populations of the s e l f e r Linmanthes floooosa. She also concluded that although the s e l f e r L. floooosa has s l i g h t l y less within-population v a r i a t i o n than the outcrosser L. alba, the two species have s i m i l a r genetic structures. However, these conclusions should be accepted cautiously since no inheritance studies were conducted. Consequently, the number of loci•-surveyed was not determined and enzyme v a r i a b i l i t y could not be interpreted i n terms of a l l e l i c variation. In addition, J a i n (1978) c i t e s unpublished isozyme data which show that the s e l f e r L. floooosa i s much less v a r i a b l e within populations than the outcrosser L. alba. Investigations of the e f f e c t s of breeding system on the 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 has re s u l t e d i n some c o n f l i c t i n g conclusions. Arroyo (1975) detected no c l e a r r e l a t i o n s h i p between outcrossing rate and l e v e l of v a r i a t i o n i n two Linmanthes species. A l l a r d et al. (1975) reported that Avena barbata i s g e n e t i c a l l y less v a r i a b l e than A. fatua although t h e i r o u t c r o s s i n g r a t e s are 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 of allozyme 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 populations of Limnanth.es alba. In c o n t r a s t , Levin (1978), 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 d i f f e r e n c e s i n l e v e l s of v a r i a t i o n w i t h i n s e l f - c o m p a t i b l e versus s e l f - i n c o m p a t i b l e species of Phlox and Leavenwovthia, r e s p e c t i v e l y . Furthermore, i n s t u d i e s o f Leavenwovthia species ( S o l b r i g and R o l l i n s , 1977) and Lyoopevsioon p i m p i n e l l i f o l i u m (Rick e t al. 3 1977) i t was concluded that the breeding system was the s i n g l e most important f a c t o r i n f l u e n c i n g p o p u l a t i o n s t r u c t u r e . J a i n (1976) emphasizes that comparisons o f the genetic s t r u c t u r e between r e l a t e d species 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 nature and an e v a l u a t i o n of the s i g n i f i c a n c e of parameters used to describe the genetic v a r i a t i o n . In a comparison of se 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 species of 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 only one po p u l a t i o n o f the s e l f - c o m p a t i b l e species P. euspidata. Although he considered t h i s p o p u l a t i o n to be " t y p i c a l " , i t may not describe adequately the breeding system of t h i s s p e c i es. Outcrossing r a t e s among populations of s e l f - c o m p a t i b l e taxa o f t e n vary over wide l i m i t s ; e.g. zero t o 40% i n Lyoopevsioon p i m p i n e l l i folium (Rick et al., 1977); 48%-80% i n Pleetvitis oongesta (Ganders et al., 1977a) and 43%-97% i n Limnanthes alba ( J a i n , 1978). Outcrossing frequencies were not c a l c u l a t e d f o r the Leavenwovthia species ( 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 the breeding systems i n Limnanthes (Arroyo, 1975) were "estimated" by t h e i r degree of protandry and the a b i l i t y to set seed i n a p o l l i n a t o r - f r e e environment. In addition, no inheritance studies of enzyme v a r i a t i o n were conducted i n e i t h e r Leavenworthia or Limnanthes. Consequently, 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 could not be interpreted with respect to the number of l o c i scored and a l l e l e frequencies within populations. Therefore, most of the studies which have attempted to c o r r e l a t e breeding system with population structure i n r e l a t e d taxa have f a i l e d to e i t h e r : (i) quantify the mating system(s), ( i i ) i s o l a t e the e f f e c t s of the breeding system from other factors which influence population structure (e.g. genetic"background and 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 ) and/or ( i i i ) describe the organization of 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. a l l e l e s at l o c i ) . In an e f f o r t to c l a r i f y some of these issues, the present study analyzes the consequences of contrasting breeding strategies on the l e v e l s and organization of isozyme v a r i a t i o n i n P l e c t r i t i s brachystemon and P. congesta. Populations, sampling methods and the electrophoretic techniques which were adapted f o r P l e c t r - i t i s are described i n Chapter 2. In Chapter 3, the i n t e r p r e t a t i o n of enzyme mobility patterns i s discussed. Genetic models which explain the observed v a r i a t i o n are proposed and these are tested using the chi-square s t a t i s t i c . A l l e l i c v a r i a t i o n at f i f t e e n l o c i i s i d e n t i f i e d . The geographic d i s t r i b u t i o n of a l l e l e s among populations and the r e l a t i v e contributions of various l o c i to the genetic d i v e r s i t y (Nei, 1975) of each species are described i n Chapter 4. In Chapter 5, the analyses of the isozyme data are discussed. Both the outcrossing frequencies, calculated f o r each population, and the mean outcrossing rates f o r each taxon are compared. The l e v e l of v a r i a b i l i t y maintained within each population 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 qua n t i f i e d . Differences between the two species are interpreted with reference to t h e i r breeding s t r a t e g i e s . In addition to the e f f e c t s of breeding system on population genetic structure, a number of a d d i t i o n a l problems are addressed. Comparisons of genetic distance are used to make inferences regarding the l e v e l s of gene flow between populations and the taxonomic r e l a t i o n s h i p of P. oongesta and P. braohystemon. L a s t l y , the factors which influence the maintenance of the observed isozyme polymorphisms are discussed. 16. 2.0 MATERIALS AND METHODS 2.1 Collection sites and sampling strategy Isozyme v a r i a t i o n was analyzed i n ten c o l l e c t i o n s of Plectritis bvachystemon and f i f t e e n c o l l e c t i o n of P. congesta. Within each l o c a l population, seeds were c o l l e c t e d from i n d i v i d u a l s i n a randomized fashion. Seeds from each plant were kept separate and enzyme v a r i a t i o n was scored i n the progeny grown from these seeds. An e f f o r t was made to c o l l e c t seeds from an unbiased sample of ind i v i d u a l s that represented the genetic v a r i a b i l i t y present i n each l o c a l population. In the present study, a l o c a l population i s defined as a group of in d i v i d u a l s of the same taxon growing within a s p e c i f i e d area:: a topodeme i n the terminology of Gilmour and Gregor (1939). When P. bvachystemon and P. congesta grow intermixed or i n close proximity, t h i s i s defined as a sympatric population. The term population does not ne c e s s a r i l y imply that these aggregations of ind i v i d u a l s are panmictic units (i.e. gamodemes). Table 1 l i s t s c o l l e c t i o n s i t e s , species present, number of ind i v i d u a l s sampled and date of c o l l e c t i o n . The lo c a t i o n :of populations i s presented i n Appendix A. Sites are l i s t e d i n the order i n which they appear i n a l l tables. In order to i l l u s t r a t e possible macro-geographic patterns within each taxon, mainland populations are l i s t e d f i r s t , then Vancouver Island populations beginning with the V i c t o r i a area and moving north. Figure 2 shows the loc a t i o n of a l l sampled s i t e s . 17. Table 1 . Collection sites • Sample designation Locality Species Number of plants collected Date collected 1 Sumas Mountain P.b. 300 16 June 1977 2(1) Anacortes, Washington P.O. 36 17 June 1976 2(2) 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 Thetis Lake Park P.O. 47 21 June 1976 9 Francis 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 Exit 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 Nile Creek P.O. 61 24 June 1976 18 Elk Falls Park P.b.. 50 12 June 1977 18. Figure 2. The l o c a t i o n of sampled Tlectritis populations: • designate P . brachystemon populations, • designate P . congesta populations and • i d e n t i f y s i t e s where both species were c o l l e c t e d . 19. 2.2 Potential sources of bias in the collections 2.2.1 Sample size Given random mating, a sample s i z e of t h i r t y i n d i v i d u a l s per population i s s u f f i c i e n t to be 95% confident of detecting a l l e l e s within populations which are present at frequencies >0.05. It i s d i f f i c u l t to determine what constitutes an adequate, and comparable, sample i n P l e c t r i t i s because these two taxa have dramatically d i f f e r e n t outcrossing rates and neither species experiences complete random mating. However, t h i s sample s i z e compares favorably with most studies of isozyme v a r i a t i o n i n plant populations, regardless of the breeding system. Therefore, an attempt was made to sample at least 30 i n d i v i d u a l s per population. 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 large to achieve t h i s level of sampling, poor germination, combined with technical problems, produced v a r i a t i o n i n sample sizes among populations and between l o c i within populations. 2.2.2 Phenology Populations were sampled only once i n a given year, consequently, only those plants with mature seed at the time of c o l l e c t i o n were included i n the sample. 2.2.3 Seed wing As seeds mature i t i s possible that there are differences i n the retention of winged versus wingless seed within inflorescences. In populations polymorphic f o r the seed wing, t h i s would systematically bias frequencies of the winged and wingless a l l e l e s i n the sample. 20. Whether t h i s would bias a l l e l e frequencies at the enzyme l o c i surveyed i s not known since no linkage analysis was conducted. 2.2.4 Wahlund Effect.-, an apparent deficiency in heterozygotes as the result of sampling stratified populations At many sites,, plantes were d i s t r i b u t e d over a wide area that encompassed a number of dis c r e t e micro-habitats..As a r e s u l t of automgamy, v i c i n i s m and possible micro-site s e l e c t i o n , i t i s possible that topodemes may represent aggregates of sub-populations (i.e. neighborhoods), each with unique a l l e l e frequencies. Several workers have demonstrated that populations d i f f e r e n t i a t e over small distances (Antonovics, 1968; Antonovics and Bradshaw, 1970; Hamrick and A l l a r d , 1972; Hamrick and Holden, 1979). Bradshaw (1972) states that e f f e c t i v e population s i z e should be measured i n meters not kilometers. It was assumed, for the purposes of the present study, that a l l populations of each taxon were homogeneous and were comparable genetic u n i t s . However, lumping samples from subdivided populations biases, c alculated a l l e l e frequencies and produces over-estimates of expected heterozygosity (assuming Hardy-Weinberg equilibrium). 2.3 Methods of planting and growth regime Enzyme v a r i a t i o n was surveyed i n the progeny grown from the c o l l e c t e d seeds. A l l plants were grown under uniform conditions i n order to eliminate possible genotype - environment interactions which might modify isozyme phenotypes. Plants were grown i n a standard p o t t i n g s o i l mixture i n nursery f l a t s (254 mm. x 508 mm.). A l l f l a t s were given an i n i t i a l f e r t i l i z i n g (10 g. H i - S o l 20-20-20/1800 ml. water), allowed to d r a i n f o r 24 h r s . and then planted. Seeds were planted 5 mm. to 10 mm. deep, 200 seeds per f l a t . Wooden semi-micro g r i d locus markers (i.e. t o o t h p i c k s ) were placed next to each seed to i d e n t i f y i n d i v i d u a l s . Planted f l a t s were placed i n a growth chamber set on a "long day-cool" regime: 16 h r s . of l i g h t at 12°C and 8 h r s . of darkness at 7°C w i t h 50% r e l a t i v e humidity. This growth regime was chosen as i t promoted germination and prolonged the b a s a l r o s e t t e stage o f development. Seeds germinated w i t h i n ten days to two weeks and developed four true leaves three to four weeks a f t e r germination. S i n g l e b a s a l r o s e t t e leaves served as the enzyme source m a t e r i a l . 2.4 Electrophoretic techniques Gels were run h o r i z o n t a l l y and were prepared u s i n g E l e c t r o s t a r c h (Otto H i l l e r l o t #307) at a 12.5% w/v c o n c e n t r a t i o n . Sucrose (10% w/v) was added to the gels to improve the r e s o l u t i o n of c e r t a i n enzymes and to toughen gels f o r ease of handling. Gels were prepared the afternoon preceding a run. Starch was cooked, degassed and poured i n t o p l e x i g l a s s g e l molds (150 mm. x 200 mm. x 6 mm.). These were allowed to c o o l to room temperature, wrapped i n p l a s t i c and stored overnight at 6°C. Samples were prepared j u s t p r i o r to use as enzyme a c t i v i t y diminished upon f r e e z i n g and thawing. Leaves were removed from., p l a n t s and placed 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 placed on i c e to 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 or two b a s a l leaves were ground with two drops of 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 with 0.5% 2-mercapto-ethanol) and an equal amount of i n s o l u b l e PVP ( i . e . p o l y v i n y l -p o l y p y r r o l i d o n e ) . The crude homogenate was then absorbed onto f i l t e r paper wicks (4 mm. x 10 mm., Whatman 3 MM paper) and these were i n s e r t e d i n t o s l o t s cut 20 mm. from one end of the s t a r c h g e l . Runs were standardized by monitoring the mi g r a t i o n of a t r a c k i n g dye ( d i l u t e red food c o l o r i n g ) which was absorbed onto wicks and placed i n the outer sample s l o t s of each g e l . Genotype standards ] (i. e. i d e n t i f i e d homozygotes) were run,, when needed, to check the m o b i l i t y of isozyme v a r i a n t s . Once the samples were loaded, a l l but 10 mm at e i t h e r end of the gels were covered with p l a s t i c to prevent d e s i c c a t i o n during the run. Gels were then placed on the appropriate e l e c t r o d e t r a y s with the o r i g i n ( i . e . samples) at the cathode. Electrode wicks (Handi-wipes) were placed on the unwrapped ends of the g e l and current 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 appropriate 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 large glass f r o n t r e f r i g e r a t o r that was maintained at 0°-4°C. This prevented overheating which d i s t o r t s the 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 electrophoretic buffer 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 uffer, pH 8.0 (adapted from Scandalios, 1969); II Continuous T r i s - c i t r a t e b u ffer, pH 7.0 ( S i c i l i a n o and Shaw, 1976). The composition of these buffers i s given i n Table 2. Electrophoresis was conducted at the following voltages: I 350 V. (~60mA) u n t i l tracking dye migrated 50 mm., approximately 4 hrs.; II 250 V. (~60mA) u n t i l tracking dye migrated 110 mm., approximately 4.5-5.5 hrs. Upon completion of the run, gels were removed, sample wicks p u l l e d and the gels trimmed and s l i c e d f o r s t a i n i n g . P l e x i g l a s s s t r i p s 1.5 mm. thick were used as s l i c i n g guides. Monofilament f i s h i n g l i n e (2 l b . test) was used to s l i c e the gel. With a weight on top to prevent buckling, s l i c i n g s t r i p s were placed on either side of the ge l . The f i s h i n g l i n e was then held against the s t r i p s and drawn through the gel. Once a s l i c e had been made, another p a i r of s t r i p s were placed on eit h e r side of the gel and the procedure repeated. It was possible to obtain s i x s l i c e s from a g e l . The top s l i c e was discarded. The others, as needed, were placed i n the appropriate enzyme st a i n i n g solutions and incubated at 37°C u n t i l the banding patterns could be scored. Table 3 l i s t s the enzymes scored, the number of l o c i resolved, the appropriate running buffer and the sta i n i n g reference. The number following the 24. Table 2 . Running Buffers I. DISCONTINUOUS Li-BORATE/TRIS-CITRATE BUFFER pH 8.0 BUFFER A: .03 M LiOH-.19M Boric Acid 1.20 g Lithium hydroxide (monohydrate) 11.89 g Boric Acid H 20 to 1 l i t e r , adjust pH to 8.0 BUFFER B: .05 M T r i s - .008 M C i t r i c Acid 6.2 g T r i s 1.6 g C i t r i c acid (monohydrate) H 20 to 1 l i t e r , adjust pH to 8.0 ELECTRODE: BUFFER A GEL : 1:9 Mixture of Buffers A § B REFERENCE: adapted from Scandalios, J.G. (1969) and Selander, et a l . (1971). II. CONTINUOUS TRIS-CITRATE BUFFER pH 7.0 ELECTRODE: .13 M T r i s - .043 M C i t r i c Acid 16.35 g T r i s 9.04 g C i t r i c acid (monohydrate) H 20 to 1 l i t e r , adjust pH to 7.0 GEL : .013 M T r i s - .0043 M C i t r i c Acid 1:10 d i l u t i o n of Electrode Buffer (modified from reference) REFERENCE: adapted from S i c i l i a n o , M.J. § C.R. Shaw, (1976) Table 3 . Running Buffer Used, Number of Loci Resolved and Staining References for Enzyme Systems Studied i n P. bvaahystemon and P. oongesta. ENZYME # LOCI SCORED RUNNING BUFFER STAIN REFERENCE2 ESTERASE EST E.C. 3.1.1.1 ISOCITRATE DEHYROGENASE IDH E.C. 1.1. 1.42 LEUCINE AMINO PEPTIDASE LAP E.C. 3.4.11.1 MALATE DEHYDROGENASE MDH E.C. 1.1.1.37 MALIC ENZYME ME E.C. 1.1.1.40 PHOSPHOGLUCOSE ISOMERASE3 PGI E.C. 5.3.1.9 PHOSPHOGLUCOMUTASE PGM E.C. 2.7.5.1 6-PHOSPHOGLUCONIC DEHYDROGENASE 6PG E.C. 1.1.1.44 SUPEROXIDE DISMUTASE SOD E.C. 1.15.1.1 II II I or II II Gottlieb (1973a) Allendorf, et a l . (1977) Roose 6 Gottlieb (1976) S i c i l i a n o § Shaw (1976) Roose 5 Gottlieb (1976) Gottlieb (1973ad Roose 5 Gottlieb (1976) Allendorf, et a l . (1977) Running Buffers: I Discontinuous Li-Borate/Tris-Citrate Buffer ph 8.0 II Continuous Tris-Citrate Buffer pll 7.0 Most of the staining techniques used are modification of the or i g i n a l reference Due to problems with interpretation, this enzyme was not scored for P. congesta. 26. enzyme a b b r e v i a t i o n i s i t s Enzyme Commission code number ( F l o r k i n and S t o t z , 1973). Table 4 gives the s t a i n i n g procedures and Table 5 provides a l i s t of stock s o l u t i o n s . In a d d i t i o n , Appendix B l i s t s enzyme assays and running b u f f e r s which were attempted but proved to be unsuccessful with Plectritis. Gels were scored immediately 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 best. When s t a i n i n g was complete, gels were f i x e d i n e i t h e r a 1:4:5 a c e t i c acid-methanol-water s o l u t i o n ( A l l e n d o r f et al., 1977) or a 1:1 glycerine-water s o l u t i o n ( S i c i l i a n o and Shaw, 1976). The f i r s t f i x a t i o n mixture toughened and preserved gels as semi-permanent records b e t t e r than the l a t t e r . However, the glycerine-water f i x b e t t e r preserved the r e s o l u t i o n of c e r t a i n enzymes (i.e. MDH, PGI and PGM) and proved s u p e r i o r f o r gels that were to be photographed l a t e r . TABLE 4 . Summary of Enzyme Staining Procedures. ENZYME STAIN BUFFER1 NAD NADP/MgCl22 NBT/PMS3 MTT/PMS3 OTHER COMPONENTS 1. EST 2 60 mg a-napthyl acetate 30 mg g-napthyl acetate 100 mg Fast Blue RR 2. IDH 1 / / 200 mg DL - i s o c i t r i c acid 3. LAP 3 30 mg l-leucyl-g-napthylamide (dissolved in 5 ml N, N-dimethyl formamide) 25 mg. Black-K-salt added after 30 min, incubation. 4. MDH 1 25 mg / 5 ml IM Na-L-Malate 5. ME 4 / / 140 mg L-Malic Acid 6. PGI 1 / / 2 ml .018 M Fructose-6-phosphate 20 units G6PDH 7. PGM 1 / / 75 m g Na 2 a-D-Glucose-1-phosphate 3 ml .00017 M K 2 a-D-Glucose-1-phosphate 20 units G6PDH 8- 6PG 1 / / 20 mg 6-Phosphogluconic Acid (Naj Salt) 9- SOD Develops in light on gels stained for ME, PGI, PGM. ; 1 Stain Buffers: 1. .2M Tris-HCl pH 8.0 i 2 10 mg NADP + 1 ml 0.5 M MgCl ! 2. .IM K-P04 pH 6.4 3. . IM Tris-Maleate pH 6.5 3 10 mg NBT or, MTT + 5 mg PMS 4. .IM Tris-HCl pH 8.6 " ' Table 5 . Stock Solutions SOLUTION CONG. STORAGE ADDITIONAL STAINING BUFFERS 1. Tris-HCl pH 8.0 2. K-P0 4 pH 6.4 3. Tris-Maleate pH 6.5 4. Tris-HCl pH 8.6 DYES, COFACTORS, ENZYMES § SUBSTRATES Na-L-Malate MgCl 2 G6PDH (Glucose-6-phosphate dehydrogenase) NAD NADP NBT MTT PMS ,2M , IM ,1M ,1M IM .5M 10'units/ml, 10 mg/ml 10 mg/ml 10 mg/ml 10 mg/ml 5 mg/ml R R R R RT R R R R* R* R* 12.15g Na 2C0 3H 20 i n 50 ml. H 2 P Keep Cool, add 13.4g L-Malic a c i d H 20 to 100 ml. Dissolve i n .005M c i t r a t e pH 7.5 Dissolve i n .005M c i t r a t e pH 7.5 Dissolve i n .005M c i t r a t e pH 7.5 R: re f r i d g e r a t e RT: room temperature *: protect from l i g h t 29. 3.0 INHERITANCE 3.1 Isozyme nomenatatuve F i f t e e n l o c i , coding for nine enzymes, were i d e n t i f i e d i n Pteotritis. These enzymes are l i s t e d i n Table 3. Interpretations of enzyme mobility patterns on gels are presented and genetic models to explain the observed v a r i a t i o n are proposed. Two terms, isozyme (isoenzyme) and allozyme need to be defined. Isozymes are multiple molecular forms of enzymes. Allozyme designates isozymes which are coded for by a l l e l e s at a s i n g l e locus. The isozyme nomenclature adopted for P l e a t r i t i s was modified from Allendorf and Utter (1979). . An enzyme locus i s i d e n t i f i e d by the enzyme's abbreviation, plus a hyphenated numeral i f the system i s c o n t r o l l e d by multiple l o c i . A l l isozymes surveyed migrated toward the anode. The locus coding for the most anodally migrating proteins was designated one, the next as two and so on. Allozymes, and t h e i r corresponding a l l e l e s , are i d e n t i f i e d by a superscript according to t h e i r r e l a t i v e electrophoretic mobility. One a l l e l e , generally the most common, was a r b i t r a r i l y designated 100. Other allozymes were assigned numerical values according to t h e i r migration r e l a t i v e to the 100 form. For example, the a l l e l e at the most anodal esterase (EST) locus, which codes for the allozyme migrating 114 14% further than the common form was designated EST-1 . A l l e l e s coding for allozymes with no sta i n i n g a c t i v i t y (i.e. n u l l s ) were designated as lOOn (e.g. PGI-3'''^^n) ,i while allozymes with reduced st a i n i n g a c i t i v i t y are i d e n t i f i e d by an r following the mobility designation. 30. As a prelude to the inheritance studies, an i n i t i a l population survey was conducted to sample the extent of v a r i a t i o n present i n both species. Whenever v a r i a t i o n existed, enzyme systems were analyzed i n P. braohystemon and P. congesta separately, to insure that the mode of inheritance was i d e n t i c a l i n each species. Heterozygotes were t e n t a t i v e l y i d e n t i f i e d and s e l f e d . Segregation r a t i o s were then compared to the expected 1:2:1 r a t i o using chi-square analysis whenever progeny classes were s u f f i c i e n t l y large to v a l i d l y apply t h i s s t a t i s t i c . Heterozygotes i n P. bvachystemon were extremely rare and at some l o c i were never detected. Therefore, to v e r i f y a l l e l i c v a r i ants, crosses 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 of the f l o r a l structure and timing of anthesis i n P. bvachystemon, flowers had to be teased open with forceps and emasculated p r i o r to crossing. Plectvitis produces achenes and the flowers within an inflorescence mature separately over a period of a few weeks. In consequence, i t i s not an i d e a l organism for inheritance studies. Successful p o l l i n a t i o n s produced a s i n g l e seed and p o l l e n contamination was often a problem. As a r e s u l t , progeny classes i n the inheritance studies were generally small. I t i s assumed that the small sample sizes are responsible for the segregation r a t i o s which deviate from the expected. Presumed homozygotes were also s e l f e d and the progeny analyzed to confirm genetic i n t e r p r e t a t i o n s . Without exception, a l l such s e l f s produced progeny expressing the parental genotype. Each enzyme system surveyed i s described with respect to 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 (i.e. quaternary) 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 . Polymorphic enzymes are discussed f i r s t f ollowed by the monomorphic systems. 3.2 Esterase (EST) E.C. 3.1.1.1 Two major zones of a c t i v i t y appeared on gels s t a i n e d f o r EST. Various numbers of l i g h t l y s t a i n i n g bands were a l s o present but t h e i r i n h e r i t a n c e could not be determined. The major zones of s t a i n i n g were i n t e r p r e t e d to be separate l o c i . These were designated EST-1 and EST-2. Three a l l e l e s were detected at each locus (Figure 3 and Figure 4). the three EST-1 a l l e l e s were detected i n both species. However, i n c o n t r a s t to the high l e v e l of h e t e r o z y g o s i t y i n P. brachystemon, v a r i a t i o n was r a r e l y detected at t h i s locus i n P. congesta (Table 17). Three a l l e l e s were i d e n t i f i e d at EST-2 i n P. congesta w h i l e a l l populations of P. brachystemon were monomorphic f o r EST-2^^ (Table 17) . Heterozygotes at 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. This was i n t e r p r e t e d as evidence that EST i n Plectritis i s a monomeric p r o t e i n . This observation agrees w i t h the sub-unit s t r u c t u r e of EST reported i n other t a x a : Cucurbita- spp. (Wall and Whitaker, 1971), oats (Clegg and A l l a r d , 1973), b a r l e y (Kahler and A l l a r d , 1970), Norway spruce ( B a r t e l s , 1971), Scots pine (Rudin and Rasmuson, 1973), Stephanomeria ( G o t t l i e b , 1973a), Gaura ( G o t t l i e b and P i l z , 1976) and Drosophila subobscura (Loukas et al., 1979). Figure 5 i s a photograph of two gels s t a i n e d f o r EST. 32. EST-2 EST-1 Figure 3. The relative migration of allozymes detected at EST-1. Figure 4. The relative migration of allozymes detected at EST-2. EST-1 EST-2 EST-1 EST-2 1 15 28 Figure 5. Allozyme variation at EST-1; gel 1 - individuals 1-14 are the progeny from a selfed 1,100 homozygote, individuals 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 result of accidental s e l f s ) ; gel 2- individuals 1-15 demonstrate 1:2:1 segregation in the progeny of a selfed 100/114 heterozygote, individuals 16-29 are the progeny of a selfed llOO homozygote. EST-2 did not photograph well but i t is uniformly monomorphic in both gels. 33. It demonstrates the three a l l e l e s detected at EST-1 and i l l u s t r a t e s the method used to i n t e r p r e t isozyme patterns. Unfortunately, EST-2 did not photograph well but i t i s uniformly monomorphic i n these gels. EST-1 was not scored i n P. braohystemon population 16 ( L i t t l e Qualicum F a l l s ) . Staining 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 of spraying the plants with Malathion to combat an.aphid i n f e s t a t i o n . However, the i n s e c t i c i d e did not prevent the.expression of allozymes at EST-2. This observation suggests that the isozymes coded by these two l o c i represent d i f f e r e n t f u n c t i o n a l classes of EST. Segregation analyses are presented i n Table 6 f o r EST-1 and Table 7 f o r EST-2. 3-3 ' Leucine' aminopeptidase (LAP) E.C. 3.4.11.1 One locus was resolved on gels stained f o r LAP. In addition, other l i g h t l y s t a i n i n g bands were sometimes observed. This was a highly polymorphic enzyme i n both species (Table 17). Seven a l l e l e s were scored i n P. oongesta and a l l but one of these 80 (LAP-1 ) were detected i n P. braohystemon (Figure 6). Heterozygotes were i d e n t i f i e d by t h e i r two-banded pattern. This agrees with the monomeric structure of LAP i n other organisms: corn (Beckman et al., 1964)., Cuourbita spp. (Wall and Whitaker, 1971), knobcone pine (Conkle, 1971), Norway spruce (Lundkvist, 1974), Scots, pine (Rudin, 1977), avocado (Torres et al., 1978a.) and Drosophila subobsoura (Loukas et al., 1979). 34. Table 6. Analysis of allozyme variation at EST-1 P. brachystemon: Selfed heterozygotes 2 Individual Genotype Observed allozyme segregation in progeny x (2) A 1/A 1 A 1/A 2 A 2/A 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: Crosses Individual Genotype A1 /' A1 A1^ A2 A2/'A2 77-l-13a x 77-1-1 89/89 x 100/100 0 11 0 77-18-186 x 77-1-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: selfed homozygotes Individual Genotype Number of 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 ! 1 4 77-9-64b 100/100 8 77-9-65a 100/100 9 77-9-84a 100/100 13 * sample size i n s u f f i c i e n t for a v a l i d X test of significance ** a l l progeny expressed the maternal genotype 35. Table 7. Analysis of allozyme variation at EST-2 P. congesta. : Selfed heterozygotes Individual Genotype Observed allozyme segregation 2 in progeny V A i V A 2 A 2/A 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 * E 15 31 19 0.631 P. congesta: Selfed homozygotes Individual 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 size i n s u f f i c i e n t for a v a l i d x test of significance ** a l l progeny expressed the maternal genotype 36. L A P - 1 o , — — ; -jlOO -j112 _jl05 ^95 ^1 .85 .80 1 13 26 Figure 7. Allozyme v a r i a t i o n at LAP-1 i n P. congesta: genotypes -80/95, 95/95, bla n k , 95/100, blank, 95/100, 85/85, 100/100, 95/100 80/80, 91/91, 95/95, 100/100, 95/95, 80/80, 95/95, 80/100, blank, ' 80/91, 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 a sample of the v a r i a t i o n present i n P l e c t r i t i s . This gel was t y p i c a l of the genotype arrays detected i n populations of P. congesta. The r e s u l t s of crosses and segregation analyses are presented i n Table 8. 3.4 Malate dehydrogenase (MDH) E.C.I.1.1.37 There were three zones of a c t i v i t y on gels stained for MDH. These were a r b i t r a r i l y designated as zones A, B and C with A being the most anodal zone (Figures 10 and 11). As was found i n maize (Longo and Scandalios, 1969), and p i t c h pine (Guries and Ledig, 1978), these three s t a i n i n g zones may represent isozymes associated 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 (i.e. cytoplasm and mitochondria). The zone closest to the o r i g i n , C, was a poorly resolved band which may or may not represent a c t i v i t y at a s i n g l e locus. Zone B was composed of f i v e to seven well defined bands. Most commonly, i n d i v i d u a l s possessed f i v e bands. The middle band stained darkest, the outer two bands were much l i g h t e r and the other bands were intermediate i n both p o s i t i o n and s t a i n i n g . V a r i a t i o n was observed i n the number, the p o s i t i o n and the. r e l a t i v e s t a i n i n g i n t e n s i t i e s of bands. The banding patterns suggest that there are at least three l o c i coding f o r MDH isozymes which migrate to t h i s zone. The r e l a t i v e s t a i n i n g of t h i s s e r i e s of bands implies dosage ef f e c t s caused by the formation of hybrid multimeric isozymes which have overlapping m o b i l i t i e s . MDH has been found to be dimeric i n other 38. Table 8. Analysis of allozyme variation at LAP-1 P. braohystemon: Crosses Individuals Genotypes Observed allozyme segregation in progeny A 1/A 1 A 1/A 2 A 2/A 2 77-l-13a x 77-18-25C 91/91x100/100 0 12 0 P. oongesta: Selfed heterozygotes 2 Individual Genotype Observed allozyme segregation in progeny X(-2) A x/A x A x/A 2 A 2/A 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: Selfed homozygotes Individual Genotype Number of 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 size i n s u f f i c i e n t for a v a l i d x test of significance ** a l l progeny expressed the maternal genotype 39. organisms: horseshoe crab (Selander et al., 1970), Eucalyptis (Brown et al., 1975), p i t c h pine (Guries and Ledig, 1978), ponderosa pine (O'Malley et al., 1979), Douglas f i r (El-Kassaby, unpublished) and lodgepole pine (Layton and Yeh, unpublished). Unfortunately, more work i s needed to determine the inheritance of v a r i a t i o n i n zone B, consequently, these l o c i were not included i n the analysis. The v a r i a t i o n observed i n zone A can be described by the existence of two l o c i designated MDH-1 and MDH-2. Va r i a t i o n at MDH-1 was common i n both species while at MDH-2, no v a r i a t i o n was detected i n P. bvachystemon and v a r i a t i o n was extremely rare i n P. congesta. The presence of bands of intermediate mobility between allozymes with d i f f e r e n t .migrations ind i c a t e that the active enzymes coded by MDH-1 and MDH-2 are dimers (i.e. composed of two sub-units). Isozymes of multimeric enzymes are formed by the random assoc i a t i o n of pro t e i n sub-units. An i n d i v i d u a l , heterozygous f o r a l l e l e s A and B at a locus which codes f o r a dimeric enzyme, synthesizes three allozymes AA,.AB and BB i n a 1:2:1 r a t i o . E l e c t r o p h o r e t i c a l l y , heterodimers (hybrid dimeric enzymes) can be recognized because they have m o b i l i t i e s intermediate between the homodimeric allozymes and they s t a i n darker, t h e o r e t i c a l l y e x h i b i t i n g twice the a c t i v i t y of the homodimers. The i n t e r p r e t a t i o n of v a r i a t i o n at MDH-1 and MDH-2 was complicated. The observed s t a i n i n g patterns were consistant with a model which predicts that isozyme sub-units s p e c i f i e d by both l o c i associate with each other forming both i n t r a - and inter-locus heterodimers. The formation of inter-locus heterodimers of MDH has been observed i n ponderosa pine (O'Malley, et al., 1979), lodgepole pine (Layton and Yeh, unpublished) and Douglas f i r (El-Kassaby, unpublished). 40. Figure 8 and Figure 9 de p i c t the r e l a t i v e m i g r a t i o n of allozymes scored at MDH-1 and MDH-2 r e s p e c t i v e l y . MDH-1 1^ and MDH-l 1 1^ 113 125 139 were detected i n P. braohystemon while MDH-1 , MDH-1 and MDH-1 were found i n P. congesta. MDH-1"^ and M D H - h a v e i d e n t i c a l m o b i l i t i e s . The existence of MDH-2*^ was deduced from the presence of i n t e r - l o c u s heterodimers and confirmed with the discov e r y of the 87 rare MDH-2 v a r i a n t . Figure 10 and Figure 11 are photographs of gels s t a i n e d f o r MDH. The bands v i s i b l e i n zone A of Figure 10 are the i n t e r - l o c u s heterodimers formed between enzyme sub-units coded f o r by MDH-1 and MDH-2. Heterodimers g e n e r a l l y s t a i n twice as d a r k l y as homodimers and o f t e n , the MDH-1 homodimeric allozymes were not v i s i b l e . However, MDH-1 homodimers are v i s i b l e i n Figure 11. Data from crosses and segregation analyses f o r MDH-1 are presented i n Table 9. 3.5 Phosphoglucose isomerase (PGI) E.C.5.3.1.9 There were two zones of a c t i v i t y on gels 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 the most anodal zone (A) appeared to be c o n t r o l l e d by a s i n g l e locus which was designed PGI-1. A two locus model s u f f i c i e n t l y explained the v a r i a t i o n observed i n zone B and the corresponding l o c i were designated PGI-2 and PGI-3 (Figure 12). The m u l t i l o c u s o r g a n i z a t i o n of PGI i n Pleetritis i s i d e n t i c a l to that reported by G o t t l i e b (1977b) f o r the more advanced s e c t i o n of the genus .Clarkva. 4 1 . Z o n e - A Z o n e - B M_DH-1 •—— • B — ' _ _ • • — • tarn -j139 ^125 -j113 M D H - 2 1 0 0 Figure 8. The r e l a t i v e migration of allozymes detected at MDH-1. The v a r i a t i o n i n Zone A i s determined by two loci,. MDH-1 and MDH-2. MDH-1 1 0 0 and MDH-2l°° have i d e n t i c a l m o b i l i t i e s . The hollow bars represent the p o s i t i o n of heterodimers (hybrid dimeric isozymes). In MDH, these bands are interpreted to be in t e r - l o c u s heterodimers, formed by the random association of pro t e i n sub-units coded f o r by a l l e l e s at 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 of l o c i and was not interpreted i n the present study. M D H - 1 1 0 0 M D H - 2 8 7 Figure 9. The r e l a t i v e migration of allozymes detected at MDH-2. 42. 1 13 2 7 Figure 10. Allozyme v a r i a t i o n at 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 Figure 11. Allozyme v a r i a t i o n at MDH-1; a l l i n d i v i d u a l s are 100/100 homozygotes at MDH-2; note v a r i a t i o n i n zone B f o r plants 11 and 28. 43. Table 9. Analysis of allozyme variation at MDH-1 P. braohystemon: Crosses Individuals Genotypes Observed allozyme segregation i n progeny A'1/A1 A 1/A 2 A 2/A 2 77-l-13a x 77-18-25c 100/100x113/113 0 77 - 1 - l l l a x 77-18-18b 100/100x113/113 0 77-1-llOc x 77-18-19C 100/100x113/113 0 12 0 8 0 10 0 P. braohystemon: Selfed homozygotes Individual Genotype Number of progeny** 77-1-26C 100/100 6 77-l-73a 100/100 14 P. oongesta: Selfed heterozygotes Individual Genotype Observed allozyme segregation i n progeny x(2) P V A i V A 2 A 2/A 2 76-4-5 125/139 4 9 6 0.474 0.75-0.90 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 * Z 25 41 21 0.655 0.50-0.75 77-9-84a 113/125 1 1 1 * P. oongesta: Selfed homozygotes Individual Genotype Number of progeny** 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 size i n s u f f i c i e n t for a v a l i d X 2 test of significance ** a l l progeny expressed the maternal genotype 44. In P. congesta, an occasional plant appeared to be double banded at PGI-1. This suggests that allozymes at t h i s locus are either functional as monomers or that unlike sub-units do not dimerize. However, t h i s could not be confirmed as mobility differences between variants were small, r e s o l u t i o n was not always adequate and the appropriate segregation 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 patterns which were d i f f i c u l t to i n t e r p r e t . A two locus model, with i n t r a - l o c u s and i n t e r - l o c u s heterodimers, predicted a large percentage of the s t a i n i n g patterns observed i n segregation analyses (Figure 13). This model agrees, with the dimeric structure of PGI'which has been i n f e r r e d fromiisozyme studies i n other organisms: rabbits (Noltmanri, 1964), f i s h (Avise and K i t t o , 1973), Gaura (Gottlieb and P i l z , 1976),:Clarkia (Gottlieb, 1977b), Citrus (Torres et al., 1978b),. p i t c h pine (Guries and Ledig, 1978) and ponderosa pine (Mitten et al., 1979) . As more populations were surveyed, the number of a l l e l e s proposed to explain the observed v a r i a t i o n became unwieldy. Minor v a r i a t i o n s i n mobility and/or a c t i v i t y , inconsistancy i n the formation of heterodimers (also reported for AAT i n Citrus by Torres et al., 1978b) and the presence of n u l l s at both l o c i made i t impossible to confidently genotype i n d i v i d u a l s . Figures 14 and 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 i n P. congesta. 45. 1 2 3 4 5 6 7 8 9 10 11 12 1 3 14 Figure 12. Segregation of alloyzme variation at PGI-2 and PGI-3 in P. congesta: this is the Fj progeny of a selfed plant, heterozygous at PGI-2 and PGI-3 (individual 12 is P. brachystemon). Figure 13. An a l l e l i c interpretation of the allozyme variation pictured in Figure 12 (hollow bars represent the position of heterodimers). 46. p & r log-1 ••••• • • • • • f . r # U * t - f c - * 1 15 23 26 27 Figure 14. A sample of the allozyme variation detected at PGI-2 and PGI-3 i n P. oongesta (individuals 15, 23, 26 and 27 are P. braohystemon). A l - t » 15 Figure 15. A sample of the allozyme variation detected at PGI-2 and PGI-3 i n P. oongesta (individual 15 i s P. braohystemon). 47. I t was determined that more i n h e r i t a n c e work was r e q u i r e d to 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 allozymes. Consequently, t h i s system was not scored i n P. congesta. However, P. brachystemon proved to be much le s s polymorphic than P. congesta and the proposed i n h e r i t a n c e model explained the observed v a r i a t i o n . Therefore, PGI was included i n the p o p u l a t i o n surveys of t h i s s p e c ies. No v a r i a t i o n was detected at 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 species was not found i n P. congesta. This locus was used throughout the study as one of 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 species. P. brachystemon can be d i f f e r e n t i a t e d from P. congesta i n Figures 12, 14, 15, 19 and 20 by the presence of t h i s s l o w l y m i g r a t i n g allozyme. Figures 16 and 17 d e p i c t the v a r i a t i o n observed at 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 at PGI-2 and 100 94 two a l l e l e s were found at PGI-3. PGI-2 and PGI-2 are . 94 p i c t u r e d i n Figure 18. PGI-2 was extremely r a r e . I t was detected i n the heterozygous s t a t e i n only three 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. Since heterozygotes i n P. brachystemon were r a r e and these same i n d i v i d u a l s appeared to be heterozygous at PGM-3, these 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 designated as a PGI-2 allozyme because of 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 to c o n f i rm t h i s . Figure 19 shows PGI-2 i n both the homozygous and heterozygous s t a t e . This allozyme i s of i n t e r e s t because i t was unique.to Nanoose H i l l and y e t , i n populations 15(1) and 15(2), i t s frequency was 0.15. 4 8 . P G I - 2 2ioo 2 9 4 2 8 1 •>42 P G M P G I - 2 1 0 0 -Figure 1 6 . The relative migration of allozymes detected at PGI-2 in P. braohystemon. Figure 1 7 . The relative migration of allozymes detected at PGI-3 in P. braohystemon: the hollow bar represents an inter-locus heterodimer formed between PGI-2l°° a n ( j PGI-333r. ?CrV U 7 - 2 )100 , 3 3 r 8 9 15 Figure 1 8 . Allozyme variation at PGI-2 in P. braohystemon: individuals 8 and 9 are 94 / 1 0 0 heterozygotes, a l l other plants are homozygous for PGI-2 1 0 0; individual 1 5 is homozygous for PGI-3 3 3 r (the low mobility band is a 2 1 0 0/3 3 inter-locus heterodimer), a l l other plants are PGI-3 lOOn homozygotes, 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 variation at PGI-2 in P. brachystemon: individuals 7 and 9-13 are homozygous for PGI-2l°0; individuals 14 and 15 are homozygous for PGI-2 4 2; plant 8 is 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 for PGI-3 1 0 0 n except individual 4 which has the PGI-333r allozyme. PGrl * \ 0 4 1 14 20 23 27 Figure 20. A comparison of isozyme variation in 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 Fi progeny of a cross between plants homozygous for PGI-3l00n and ?GI-2>3ZT, the progeny are a l l heterozygotes except for 20, 23 and 27 which are the result of accidental s e l f s . 50. was o r i g i n a l l y assumed to have normal s t a i n i n g properties but a mobility i d e n t i c a l to PGI-2*^. This migration distance was designated as r e l a t i v e m o b i l i t y 100. A slowly migrating and f a i n t l y 33r s t a i n i n g allozyme, PGI-3 was l a t e r detected. The r e l a t i v e m o b i l i t y 33r . of the conspicuous band associated with PGI-3 [%7.e. intermediate between PGI-3"^ r and PGI-2^^), combined with the absence of any heterodimer associated with PGI-3^^ n, supported the assumption that PGI-S'''^11 and PGI-2^^ had i d e n t i c a l m o b i l i t i e s . However, with the 42 100 detection of PGI-2 homozygotes which show no s t a i n i n g at the PGI-3 p o s i t i o n (Figure 19), i t was possible to i d e n t i f y PGI-3''"^^11 as a n u l l a l l e l e and not a normally s t a i n i n g variant with a mobility i d e n t i c a l to PGI-2^^. N u l l a l l e l e s are not uncommon i n electrophoretic surveys and t h e i r frequencies tend to be underestimated because they are i generally not recognized i n heterozygotes. 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 at a locus to be a n u l l . The r e s u l t s of crosses and segregation analyses f o r PGI-2 and PGI-3 i n P. braohystemon are presented i n Table 10 and Table 11. 3.6 Phosphogluoomutase (PGM) E.C. 2.7.5.1 Gels stained f o r PGM displayed two zones of a c t i v i t y (A and B). Individuals had eit h e r one or two bands i n the most anodal zone (A) and two to four bands i n the slowly migrating zone (Figure 24). Bands i n zone A segregated as a l l e l e s at a single locus and was designated PGM-1. V a r i a t i o n i n zone B was determined to be co n t r o l l e d by two l o c i , PGM-2 and PGM-3. 51. Table 10. Analysis of allozyme variation at PGI-2 P. brachystemon: Selfed 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. Analysis of allozyme variation at PGI-3 P. brachystemon: Crosses Individuals Genotypes Observed allozyme segregation i n progeny A 1/A 1 A x/A 2 A 2/A 2 77-l-13a x 77-18-25c lOOn/lOOn x 33r/33r 0 12 0 P. brachystemon: Selfed homozygotes Individual Genotype Number of progeny** 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 expressed the maternal genotype Heterozygptes were i d e n t i f i e d by the presence of two bands which were as s o c i a t e d w i t h a s i n g l e locus. This suggests that PGM i n Plectritis i s a monomeric enzyme which agrees with the sub-unit s t r u c t u r e observed f o r PGM i n other organisms: horseshoe crab (Selander et al., 1970), Citrus (Torres et al.,1978b), ponderosa pine (Mitten et al., 1979), Drosophila subobsoura (Loukas et al., 1979) and Douglas f i r (El-Kassaby, unpublished). Four a l l e l e s were detected at PGM-1 and PGM-2. The i r r e l a t i v e ' m i g r a t i o n s are presented i n Figures 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 at PGM-3 (Figure 23). A l l four PGM-1 a l l e l e s were detected i n P. congesta but only two, PGM-1 1 0^ and PGM-1 °° were found i n P. brachystemon. At PGM-2, three a l l e l e s were found i n P.. congesta while P. brachystemon populations were e s s e n t i a l l y monomorphic f o r PGM-2 1 0 0. The only v a r i a n t a l l e l e detected 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 of p a r t i c u l a r i n t e r e s t because the two species were completely d i f f e r e n t i a t e d at t h i s locus. Two a l l e l e s , 115 182 PGM-3 and PGM-3 were detected i n P. brachystemon while four a l l e l e s , PGM-3 1 0 0, PGM-3 1 3 5, PGM-3 7 7 and PGM-3 4 9, were observed i n P. congesta. This was the only locus analyzed i n the study i n which the two species had no a l l e l e s i n common. Figures 24 and 25 i l l u s t r a t e the v a r i a t i o n observed i n PGM. The r e s u l t s of crosses and segregation analyses i n both species are presented f o r PGM-1 i n Table 12, PGM-2 i n Table 13.and PGM-3 i n Table 14. 53. P G M - 1 P G M - 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 n o t shown). PGM-3 P brachystemon P congesta P G M - 1 P G M - 2 311s 2<82 2 1 0 0 3 1 3 5 277 3 4 9 Figure 23. The r e l a t i v e migration of allozymes detected at PGM-3. 54. ,106 — ^ 0 0 2118 2 100 __> 3 135 — • 2 81 —* 3 100 pertA U 8 - Z • Figure 24. Allozyme variation in PGM in P. congesta. Z o n B 1 15 21 24 28 Figure 25. A comparison of isozyme variation in PGM between P. conge and P. brachystemon: plants 1-20, except 15, are P. congesta; plants 21-29 are P. brachystemon, the Fj progeny of a cross between plants homozygous for different alleles, 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 result of accidental selfs. 55. Table 12. Analysis of allozyme variation at PGM-1 P. brachystemon: Crosses Individuals Genotypes Observed V A i segregation of allozymes in progeny V A2 A 2 / A 2 77-l-13a x 77- 18-25 c 100/100 x 106/106 0 12 0 77-1-llla 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: Selfed homozygotes Individual Genotype Number of progeny** 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. congesta: Selfed heterozygotes Individual Genotype Observed segregation of allozymes i n progeny x 2 P V A i A :/A 2 A 2/A 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 * E 28 43 16 3.322 0.10-0.05 P. congesta: Selfed homozygotes Individual Genotype 77-9-45b 100/100 77-9-60c 100/100 77-9-64a 100/100 77-9-75b 100/100 77-9-65a 106/106 * sample size i n s u f f i c i e n t for a v a l i d x" test of significance ** a l l progeny expressed the maternal genotype Number of progeny** 11 14 13 10 9 56. Table 13. Analysis of allozyme variation at PGM-2 P. braohystemon : Selfed homozygotes Individual Genotype Number of 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: Selfed heterozygotes Individual Genotype Observed segregation of allozymes in progeny x .2 (2) V A i A 1/A 2 A 2/A 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 1. 100 P. oongesta: Selfed homozygotes Individual Genotype Number of progeny** 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 size i n s u f f i c i e n t for a v a l i d x " test of significance ** a l l progeny expressed the maternal genotype 57. Table 14. Analysis of allozyme variation at PGM-3 P. brachystemon: Crosses Individuals Genotypes Observed segregation of allozymes i n progeny A 1/A 1 A :/A 2 A 2/A 2 77-l-13a x 77-18-25c 115/115 x 182/182 0 12 0 77-1-llla x 77-18-18b 115/115 x 182/182 0 . 8 0 77-1-llOc x 77-18-19c 0 10 0 P. brachystemon: Selfed homozygotes Individual Genotype Number of progeny** 77-l-14c 115/115 15 77-l-26c 115/115 6 77-l-73a 115/115 14 77-9-llc 115/115 7 Individual P. congesta: Selfed heterozygotes Genotype Observed segregation of allozymes in progeny V A i A :/A 2 A 2/A 2 l(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 I 24 39 16 1.633 0.25-0.50 P. congesta: Selfed homozygotes Individual Genotype Number of 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 size i n s u f f i c i e n t for a v a l i d x" test of significa: ** a l l progeny expressed the maternal genotype 58. 3.7 Monomorphic enzyme systems Four enzymes were surveyed and the l o c i r e s o l v e d were found to be monomorphic f o r the same allozyme i n both species of Plectritis. These enzymes were i s o c i t r a t e dehydrogenase (IDH: E.C. 1.1.1.42), malic enzyme (ME:E.C. 1.1.1.40), 6-phosphogluconic dehydrogenase (6PG:E.C. 1.1.1.44) and superoxide dismutase (S0D:E.C.1.15.1.1). Gels s t a i n e d f o r IDH di s p l a y e d two areas of a c t i v i t y , a s i n g l e monomorphic band and a more slo 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 the monomorphic band was scored as the slower zone was not re s o l v e d . IDH has been shown to be dimeric i n horseshoe crabs (Selander et al., 1970), Drosophila subobscura (Loukas et al., 1979), ponderosa pine (O'Malley et al., 1979) and Douglas f i r (El-Kassaby, unpublished). Gels 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 plus one or two l i g h t l y s t a i n i n g and more slo w l y m i g r a t i n g bands (Figure 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 but segregations could not be i n t e r p r e t e d according to simple Mendelian i n h e r i t a n c e . Consequently, only ME-1 was scored. ME appears to be a t e t r a m e r i c enzyme i n Drosophila subobscura (Loukas et al., 1979). A gel s t a i n e d f o r 6PG i s p i c t u r e d i n Figure 27. A s i n g l e monomorphic band was observed i n a l l populations screened. 6PG has a dimeric s t r u c t u r e i n humans (Zouros, 1976), Drosophila subobsaura (Loukas et al., 1979) and Douglas f i r (El-Kassaby, unpublished). 5 9 . Figure 26. Allozyme activity in ME: note the variation in the number of lightly staining bands. Figure 27. Allozyme activity in 6PG. 60. SOD, which i s not i l l u s t r a t e d , appeared as a si n g l e mono-morphic white band on gels stained with tetrazolium dye. This enzyme i s often reported i n the l i t e r a t u r e as tetrazolium oxidase (TO) because, i n v i t r o , i t prevents the oxidation of tetrazolium dyes to blue formazan. L i p p i t t and F r i d o v i c h (1973), demonstrated that TO was a c t u a l l y SOD. Superoxide dismutase (or TO) has been shown to be a dimeric enzyme i n the following organisms: humans (Beckman, 1973); dogs (Brewer, 1967); potatoes (Oelschlegal § Stahmann, 1971); two species of Drosophila (Richmond and Powell, 1970; Ayala et aZ.j.1972); houseflies (McDonald et at., 1975) and Douglas f i r (El-Kassaby, unpublished). 3.8 Summary Nine enzyme systems were surveyed and f i f t e e n l o c i i d e n t i f i e d i n Vteotritis. Genetic models were proposed to explain the observed v a r i a t i o n . These models described the multi-locus organization, sub-unit structure and a l l e l i c v a r i a t i o n within each system. The models were tested with crosses and segregation analyses. When progeny classes were s u f f i c i e n t l y large, segregations were analyzed using the chi-square s t a t i s t i c . A model was proposed that explained observed segregations at PGI-2 and PGI-3 i n P . congesta. However, the l e v e l of polymorphism, inconsistancy i n the formation of heterodimers and the presence of n u l l s prevented a l l e l i c v a r i a t i o n at these l o c i 'from being confidently scored. Although P. congesta and P. braohystemon d i f f e r e d i n t h e i r a l l e l i c complements, the data 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 of the systems are i d e n t i c a l i n both sp e c i e s . U l t i m a t e l y , f i f t e e n l o c i were scored i n P. brachystemon and twelve l o c i i n P. congesta. The isozyme v a r i a t i o n detected w i t h i n each p o p u l a t i o n i s presented i n Appendix C. The l e v e l of polymorphism at each locus and the d i s t r i b u t i o n of a l l e l e s among populations i s described i n Chapter 4. 62. 4.0 VARIATION AMONG POPULATIONS A l l e l e frequencies, expected heterozygosity (h) and observed heterozygosity (h ) were calculated f o r each locus and are presented i n Table 15 for P. brachystemon and Table 16 f o r P. congesta. The expected proportion of heterozygotes at each locus was calculated using the following formula; 2 h = 1-Ex (Nei, 1975) i where x^ i s the frequency of the i a l l e l e at the locus i n question. This i s equivalent to the expected frequency of heterozygotes i f the population i s i n Hardy-Weinberg equilibrium. C a l c u l a t i n g expected heterozygosity, using the mean a l l e l e frequencies f o r each species, yielded the following sequence of l o c i ranked according to t h e i r t o t a l gene d i v e r s i t y ( i . e . H^, Nei, 1973): P. brachystemon - 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 (0.000, MDH-2 (0.000), ME-1 (0.000), SOD (0.000), 6PG (0.000). In order to emphasize differences i n the r e l a t i v e l e v e l s of v a r i a t i o n at homologous l o c i and f o r convenience of discussion, l o c i surveyed i n the two taxa are considered under the following three a r b i t r a r y categories: Table 15. A l l e l e Frequencies and Heterozygosity Values* for Eight Polymorphic Loci in PZectritis brachystemon. Locus A l l e l e 1 13 9 10 7(1) 7(3) 15(1) 15(2) 16 18 114 .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 0 .046 .029 .023 .000 .000 .000 . I l l .000 .000 112 .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 h 0 .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 .00 .55 .50 .89 1.00 .00 .00 h .000 .000 .000 .000 .495 .500 . 196 .000 .000 .000 h 0 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 Table 15. (Continued) Locus A l l e l e 1 13 9 10 7(1) 100 1 . 0 0 1 . 0 0 . 9 9 1 . 0 0 1 . 0 0 94 . 0 0 . 0 0 . 0 1 ; o o . 0 0 81 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 42 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 h . 0 0 0 . 0 0 0 . 0 2 0 . 0 0 0 . 0 0 0 h 0 . 0 0 0 . 0 0 0 .017 . 0 0 0 . 0 0 0 lOOn 1 . 0 0 1 . 0 0 . 8 1 1 . 0 0 1 . 0 0 33 . 0 0 . 0 0 .19 . 0 0 . 0 0 h . 0 0 0 . 0 0 0 . 3 0 8 . 0 0 0 . 0 0 0 h o . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 117 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 106 1 . 0 0 . 4 8 . 0 3 1 . 0 0 . 0 0 100 . 0 0 . 5 2 . 9 7 . 0 0 1 . 0 0 94 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 h • . 0 0 0 . 4 9 9 . 0 5 8 . 0 0 0 . 0 0 0 h 0 . 0 0 0 . 0 4 2 . 0 0 0 . 0 0 0 . 0 0 0 118 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 100 1. , 0 0 1. . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 81 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 lOOn . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 7 ( 3 ) 1 5 ( 1 ) 15(2) 16 18 . 8 8 . 8 5 . 8 4 . 9 5 1 . 0 0 . 0 0 . 0 0 . 0 0 . 0 5 . 0 0 . 1 2 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 15 . 1 6 . 0 0 . 0 0 .211 . 2 5 5 . 2 6 9 . 0 9 5 . 0 0 0 . 0 0 0 . 0 0 0 . 0 2 9 . 0 9 1 . 0 0 0 . 0 0 . 3 8 1 . 0 0 1 . 0 0 . 5 4 . 0 0 . 6 2 . 0 0 . 0 0 . 4 6 . 0 0 0 .471 . 0 0 0 . 0 0 0 . 4 9 7 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 1 6 . 2 3 . 0 0 . 0 0 . 0 0 . 8 4 . 7 7 1 . 0 0 1 . 0 0 1 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 2 6 9 . 3 5 4 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 9 6 1 . 0 0 1, . 0 0 1 . 0 0 1 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 4 . 0 0 . 0 0 . 0 0 . 0 0 Table 15 . (Continued) 1 13 9 10 7(1) 7(3) 15(1) 15(2) 16 18 h .000 .000 .000 .000 .000 .077 .000 .000 .000 .000 h 0 .000 .000 .000 .000 .000 .000 .000 .000 .000 .000 182 .00 .00 .01 .00 .00 .00 .00 .03 .05 • 1.00 135 .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 0 .000 .000 .023 .000 .000 .000 .000 .054 .091 .000 2 * h=l-Ex. 1 h = observed heterozygosity Table 16. A l l e l e Frequencies and Heterozygosity Values* for Eight Polymorphic Loci in Plectritis congesta. POPULATIONS Locus Alle l e 2(1) 2(2) 3 5 4 9 10 11 7(2) 8 14(1) 14(2) 15(1) 15(2) 114 .00 .00 .00 .00 .00 .00 .00 .00 .08 .00 .00 .00 .01 .00 .01 100 1.00 1.00 .99 1.00 1.00 .98 1.00 1.00 .89 1.00 1.00 1.00 .99 1.00 .99 89 .00 .00 .01 .00 .00 .02 .00 .00 .03 .00 .00 .00 .00 .00 .00 h .000 .000 .020 .000 .000 .039 .000 .000 .201 .000 .000 .000 .020 .000 .020 h 0 .000 .000 .009 .000 .000 .011 .000 .000 .193 .000 .000 .000 .021 .000 .028 126 .00 .00 .04 .00 .00 .00 .00 .00 .08' .09 .07 .00 .06 .00 .02 100 .94 .93 .69 . 19 .63 .68 .51 .55 .16 .86 .78 .89 .13 .17 .94 74 .06 .07 .27 .81 .37 .32 .49 .45 .76 .05 .15 .11 .81 .83 .04 h .113 .130 .450 .308 .466 .435 .500 .495 .390 .250 .364 .196 .323 .282 .114 h 0 .118 .143 .415 .231 .467 .341 .380 .436 .385 .219 .310 .152 .333 .333 .114 112 .04 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 105 .23 .17 .00 .00 .03 .00 .05 .03 .00 .08 .10 .02 .00 .00 .03 100 .15 .28 . 13 .46 .50 .45 .30 .22 .24 .33 .25 .29 .02 .95 .01 95 .04 .07 .57 .21 .14 .00 . 29 .24 .38 .16 .26 .17 .29 .00 .03 91 .23 .09 .23 .21 .20 .45 .18 .33 .02 .11 .07 .26 .51 .05 . .39 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 h .790 .796 .603 .686 .662 .589 .765 .768 .723 .770 .801 .769 .626 .095 .620 li o .615 .478 .392 .583 .467 .267 .375 .486 .477 .531 .467 .276 .396 .100 .278 * h = l-jx? h = observed heterozygosity Table 16 . (Continued) POPULATIONS Locus A l l e l e 2(1) 2(2) 3 5 4 9 10 11 7(2) 8 14(1) 14(2) 15(1) 15(2) 17 139 .65 .38 .68 .67 .47 .27 .64 .47 .64 .77 .84 .65 .71 .57 .60 125 .32 .57 .31 .33 .50 .51 .36 .53 .34 .18 .16 .35 .29 .43 .19 113 .03 .05 .01 .00 .03 .22 .00 .00 .02 .05 .00 .00 .00 .00 .21 100 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 h .474 .528 .441 .442 .528 .619 .461 .498 .474 .372 .269 .455 .412 .490 .560 h 0 .471 .300 .431 .667 .467 .367 .500 .385 .269 .281 .241 .367 .385 .571 .514 100 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 .95 1.00 1.00 1.00 .99 1.00 1.00 87 .00 .00 .00 .00 .00 .00 .00 .00 .05 .00 .00 .00 .01 .00 .00 h .000 .000 .000 .000 .000 .000 .000 .000 .095 .000 .000 .000 .020 .000 .000 h o .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 h 0 .059 .042 .604 .154 .200 .288 .291 .410 . 185. .375 .200 .000 .333 .231 .441 Table 16. (Continued) POPULATIONS Locus Alle l e 2(1) 2(2) 3 5 4 9 10 11 7(2) 8 14(1) 14(2) 15(1) 15(2) 17 118 .03 .10 .00 .00 .00 .03 .03 .05 .00 .00 .00 .00 .02 .00 .03 100 .79 .67 .75 .46 .80 .60 .46 .60 .57 .81 .96 1.00 .62 .71 .51 81 .18 .23 .25 .54 .20 .37 .51 .35 .43 .19 .04 .00 .36 .29 .46 lOOn .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 h .343 .488 .375 .497 .320 .502 .527 .515 .490 .309 .077 .000 .486 .412 .527 h .294 .375 .321 .583 .133 .403 .560 .513 .338 .313 .036 .000 .426 .286 .559 o 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 .72 115 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 100 .47 .63 .35 .50 .47 .75 .49 .33 .35 .36 .28 .47 ..45 .39 .28 77 .00 .02 .00 .00 .16 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 49 .00 .00 .00 .00 .00 .00 .00 .00 .00 .00 .02 .00 .00 .00 .00 h .498 .480 .455 .500 .617 .375 .500 .442 .455 .461 .431 .498 .495 .476 .403 h .353 .375 .283 .455 .400 .282 .300 .359 .353 .469 .571 .455 .383 .357 .441 ON OO o 69. H T> 0.400, 0.400>H T2 0.050 and H_<0.050. 4.1 Loci polymorphic with E^y 0.400 4.1.1 P. brachystemon - Four l o c i ; EST-1 (0.659), MDH-1 (0.484), LAP-1 (0.422) and PGM-1 (0.412) are considered within t h i s category. A l l e l e frequencies at these l o c i are p l o t t e d i n Figures 28, 29, 30 and 31. At each of these l o c i , no single a l l e l e i s predominant i n a l l populations. However, LAP-1 1 0 0 and PGM-1 1 0 0 occur with mean frequencies of 0.75 and 0.71 r e s p e c t i v e l y (Table 17). None of the a l l e l e s detected at EST-1, LAP-1 and PGM-1 are unique to P. brachystemon although E S T - 1 1 1 4 , E S T - 1 1 0 0 , LAP-1 1 0 0 and LAP-1 8 5 have s i g n i f i c a n t l y d i f f e r e n t frequencies i n the two species (Table 17). At these three l o c i , a l l e l e frequencies and heterozygosity values . o f l u c t u a t e over wide ranges with .no d i s c e r n i b l e pattern i n - • the d i s t r i b u t i o n of a l l e l e s . On the average, 1.89 EST-1 a l l e l e s , 2.00 LAP-1 a l l e l e s and 1.40 PGM-1 a l l e l e s were detected within 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 locus proved to be taxonomically diagnostic. Two a l l e l e s , MDH-1 1 0 0 and MDH-1 1 1 3 were detected with mean frequencies of 0.59 and 0.41 res p e c t i v e l y (Table 17). MDH-1 1 0 0 was 113 unique to P. brachystemon while MDH-1 was a low frequency a l l e l e , i n P. congesta. An average of 1.30 a l l e l e s were found within each population. The d i s t r i b u t i o n of these two a l l e l e s (Figure 29) some-what suggests c l i n a l v a r i a t i o n . MDH-1 1 0 0 was f i x e d at Sumas Mtn. and two of the three southern Vancouver Island populations. The Malahat Drive and one of the Nanoose H i l l populations were polymorphic while 70... EST-1 Population Figure 28. The d i s t r i b u t i o n of EST-1 a l l e l e s among populations of P. braohystemon. M D H - 1 >> u c <b 3 <u u u. 0) .60 J Population Figure 29. The d i s t r i b u t i o n of MDH-1 a l l e l e s among populations of P. braohystemon. • .71, L A P - 1 1 13 9 10 7(1) 7(3) 15(1) 15(2) 16 18 Population Figure 30. The d i s t r i b u t i o n of LAP-1 a l l e l e s among populations of P. brachystemon. P G M - 1 BO J cr ii i_ LL JD . 4 0 . tt) < . 20 J 1 13 9 10 7(1) 7(3) 15(1) 15(2) 16 18 Population Figure 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 populations of P. brachystemon. 72. Table 17. Comparison of Mean A l l e l e Frequencies with Heterozygosity Values for Polymorphic Loci i n p. braohystemon and P. oongesta. Locus A l l e l e P. braohystemon P. oongesta t EST.! 114 .39+.143 .01+.005 2.66* 100 .34±.134 .99+.007 -4.85*** 89 .27+.133 .00+.002 2.03 "T .659 .020 .422 .792 .484 .509 EST-2 126 - .03+.009 -3.33*** 100 1.00±.000 .60+.079 5.06*** 74 - .37+.079 -4.68*** Hj. -000 .502 LAP-1 112 .03+.022 .00+.003 1.36 105 .04+.039 .05±.018 - .23 100 .75±.100 .30+.059 3.88*** 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*** MDH-1 139 - . .60+.038 -15.79*** 125 - .36±.033 -10.91*** 113 .41+.142 .04±.019 2.59* 100 .591.142 - 4.15*** MDH-2 100 1.001.000 1.001.003 .00 87 - .001.003 .00 Hj, -000 .000 PGI-2 100 .951.021 NOT SCORED 94 .011.005 81 .011.012 42 .031.021 .096 73. Table 17. (Continued) Locus A l l e l e P. brachystemon P. congesta PGI-3 PGM-1 PGM-2 PGM-3 lOOn 33r *T 117 106 100 94 «T 118 100 81 lOOn "T 182 135 115 100 77 49 "T .874.072 .13±.072 .226 .29±.128 .71+.128 .412 1.00±.004 .00+.004 .000 .11+.099 .89+.099 .196 NOT SCORED .02+.012 .28+.047 .70+.050 .00+.001 .431 .02+.007 .69±.043 .29+.041 .439 .55±.036 .44+.033 .01±.011 .00+.001 .504 -1.67 .07 .07 .00 -2.86** 7.21*** -7.07*** .00 1.11 -15.28*** 8.99*** -13.33*** - .91 .00 »T .166±.058 .266±.083 *P <.05 **P <.01 ***P <.005 74. M O r l - l * ^ replaced MDH-1*^ at L i t t l e Qualicum F a l l s and Campbell River,, However, there were notable exceptions to the patterns: populat 113 10 (Prospect Lake) was fi x e d f o r MDH-1 while 15(2) (Nanoose H i l l ) was monomorphic f o r MDH-1^^. In add i t i o n , two plants c o l l e c t e d at 113 Cape Perpetua, Oregon, were homozygous f o r MDH-1 . Unfortunately, based on the present data, i t i s impossible to determine i f there i s a se l e c t i v e basis f o r t h i s pattern or i f i t i s coincidental and the r e s u l t of s t o c h a s t i c forces. 4.1.2 P. congesta - Six 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 t h i s category. A l l e l e frequencies f o r these l o c i are p l o t t e d i n Figures 32 through 37,, No sing l e a l l e l e was predominant i n a l l populations at any of these l o c i . However, PGM-2^^ was the most common a l l e l e i n a l l but two populations and PGM-1"^^ occurred with the highest frequency i n fourteen of the f i f t e e n populations (Table 16) on Seven a l l e l e s were detected at LAP-1 (Figure 32). LAP-1 , with a mean frequency of 0.09, was the only a l l e l e not detected i n P. bvachystemon (Table 17). L A P - l " ^ and LAP-1^* were detected i n a l l populations surveyed. On the average, 5.33 a l l e l e s were maintained within each population. Three a l l e l e s were detected at MDH-1 i n 139 P. congesta (Figure 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 populations maintained 2.53 a l l e l e s . A l l populations 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 detected i n eight of the f i f t e e n populations surveyed. 75. PGM-3 was a taxonomically diagnostic locus (Figure 34). Four a l l e l e s were i d e n t i f i e d at t h i s locus, none of which was detected i n P. braohystemon (Table 17). Two a l l e l e s , PGM-3 1 3 5 and PGM-3100, were found i n a l l populations while the other two a l l e l e s were either absent or occurred at low frequency. An average of 2.20 a l l e l e s was detected within each population. Three a l l e l e s were scored at the EST^2 locus (Figure 35). EST-2 7 4 and E S T - 2 1 2 6 were not detected i n P. braohystemon. The most common a l l e l e , E ST-2 1 0 0, was detected i n both taxa but at s i g n i f i c a n t l y d i f f e r e n t frequencies (Table 17). A l l populations were polymorphic f o r E S T - 2 1 0 0 and EST-2 7 4 while E S T - 2 1 2 6 was detected i n only s i x populations and always with a frequency of less than 0.10. On the average, 2.40 a l l e l e s were maintained within l o c a l populations. Three PGM-2 a l l e l e s were detected i n P. oongesta (Figure 36) Two of these a l l e l e s were not found i n P. braohystemon, while the most common a l l e l e , PGM-2 1 0 0, was detected i n both species but at s i g n i f i c a n t l y d i f f e r e n t frequencies (Table 17). A l l populations except 14(2) (Jack's Point) were polymorphic f o r PGM-2 1 0 0 and PGM-281. 118 PGM-2 was a rare a l l e l e . It was detected i n only seven populations and with the exception of population 2(2) (Anacortes, WA ), i t was never present with a frequency greater than 0.05. The average number of a l l e l e s detected per population was 2.40. Four a l l e l e s were scored at PGM-1 (Figure 37). A l l e l e s PGM-1 1 0 0 and PGM-1 1 0 6 were common, occurring i n a l l sampled populations. These a l l e l e s were detected also i n P. braohystemon with almost 117 i d e n t i c a l frequencies (Table 17). The two rare a l l e l e s , PGM-1 94 and PGM-1 , were not found i n P. braohystemon. On the average, L A P - 1 76.. 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 •Figure 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 populations of 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 Figure 33. The d i s t r i b u t i o n of MDH-1 a l l e l e s among populations of P. oongesta. 77. P G M-3 1.00 _ § .60 CT 1) <_ u. a; .40 < _ v -V—f T 2(1) 2(2) 3 ~T 7 T T' T — - T T T T-10 11 7(2) 8 14(1) 14(2) 1511) 16(2) 17 Population 135 -100 • 77 v 49 • Figure 34. The d i s t r i b u t i o n of PGM-3 a l l e l e s among populations of 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 Figure 35. The d i s t r i b u t i o n of EST-2 a l l e l e s "among populations of P. congesta. 78. P G M-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 Figure 36. The d i s t r i b u t i o n of PGM-2 a l l e l e s among populations of P. oongesta. P G M - 1 Figure 37. The d i s t r i b u t i o n of PGM-1 a l l e l e s among populations of P. oongesta. 79. 2.33 a l l e l e s were detected within l o c a l populations. 4.2 Loci -polymorphic with 0.400>HT>.0.050 4.2.1 P. brachystemon - Three l o c i ; PGI-3 (0.226), PGM-3 (0.196) and PGI-2 (0.096) were found to have th i s l e v e l of t o t a l gene d i v e r s i t y . At these l o c i , one a l l e l e was generally predominant i n a l l populations. A l l e l e frequencies f o r PGI-3 are pl o t t e d i n Figure 38. 33r PGI-3 was found i n only 3 populations: 9 (Francis Park), 15(1) (Nanoose H i l l ) and 18 (Campbell River). However, i n those populations i t was detected at frequencies of 0.19, 0.62 and 0.46 re s p e c t i v e l y (Table 15). This locus averaged 1.30 a l l e l e s per population. The two a l l e l e s detected at PGM-3 were unique to P. brachystemon and were therefore diagnostic of the species. Figure 39 describes the d i s t r i b u t i o n of these a l l e l e s . PGM-3 1 1 5 was eith e r f i x e d or occurred with a frequency20.95 i n nine of the ten populations. 182 surveyed. Population 18 (Campbell River) was monomorphic for PGM-3 Since no other populations from as f a r north as Campbell River were.-rsurveyed, i t was impossible to determine i f t h i s pattern represents, a l l e l e replace-ment along a l a t i t u d i n a l gradient. However, t h i s does not appear to be a simple c l i n e because the two plants c o l l e c t e d at Cape Perpetua,. Oregon, 182 were also found to be homozygous f o r PGM-3 . Unfortunately, without further study, one can only speculate whether., there i s a s e l e c t i v e basis f o r t h i s polymorphism or i f i t i s merely the r e s u l t of founder e f f e c t or d r i f t i n a small population. Four a l l e l e s were scored at PGI-2 (Figure 40). PGI-2 1 0 0 was the most common a l l e l e i n a l l populations while the other three 80, PGI -3 1 13 9 10 7(1) 7(3) 13(1) 16(2) 16 18 Population Figure 38. The d i s t r i b u t i o n of PGI-3 a l l e l e s among populations of P. braohystemon. P G M - 3 1 0 0 _ .80 .60 J U c <D rj 0) L U. ^ .40 4) 7(1) 7(3) 15(1) Population Figure 39. The d i s t r i b u t i o n of PGM-3 a l l e l e s among populations of P. braohystemon. 8 1 . PGI-2 1 13 9 10 7(1) 7(3) 15(1) 15(2) 16 18 Population •Figure 40. The d i s t r i b u t i o n of PGI-2 a l l e l e s among populations of P. brachystemon. EST-1 . 8 0 J .40 J 114 -1 0 0 « 8 9 " -i 1—-••—»-—i 14(1) 14(2) 15(1) 15(2) 17 —1 7(2) Population Figure 41. The d i s t r i b u t i o n of EST-1 a l l e l e s among populations of P. congesta. 82. a l l e l e s were rare or l o c a l l y endemic. On the average, 1.50 a l l e l e s were maintained within l o c a l populations. 4.2.2,P. oongesta - None of the l o c i surveyed had t h i s l e v e l of gene d i v e r s i t y . 4.3 S l i g h t l y polymorphic or monomorphic with H^,< 0.050 4.3.1 P. braohystemon - Eight l o c i were monomorphic or nearly so: PGM-2, EST-2, MDH-2, IDH-1, ME-1, PGI-1, SOD and 6PG. No v a r i a t i o n was detected at any of these l o c i except PGM-2. A si n g l e plant i n population 7(3) (Malahat Dr.) was homozygous f o r a n u l l a l l e l e . Although 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 the most common a l l e l e s at the homologous l o c i i n P. oongesta. A l l populations surveyed of both species, were fi x e d f o r the same a l l e l e s 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 while four 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 frequencies at EST-1 are p l o t t e d i n Figure 41. This locus was of p a r t i c u l a r i n t e r e s t because both taxa share the same three a l l e l e s . However, EST-1 was the most va r i a b l e locus i n P. braohystemon (Hj, = 0.659) while i t was one of the least v a r i a b l e l o c i i n P. oongesta (Hp = 0.020). On the average, populations maintained 1.40 a l l e l e s . 87 At MDH-2, a rare a l l e l e , MDH-2 was detected i n populations 7(2) (Malahat Drive) and 15(1) (Nanoose H i l l ) with frequencies of 83. 0.05 and 0.01 r e s p e c t i v e l y (Table 16). MDH-21UU and the a l l e l e s f i x e d at IDH-1, ME-1, SOD and 6PG were also f i x e d i n P. brachystemon. 4.4 Summary Table 17 compares the mean a l l e l e frequencies and t o t a l gene d i v e r s i t y values (H^ ,) for a l l polymorphic l o c i surveyed. IDH-1, ME-1, SOD and 6PG were also surveyed but were found to be monomorphic i n both species. In addition, no v a r i a t i o n was detected at PGI-1 i n P. brachystemon although i t was polymorphic i n the other species. Unfortunately, PGI could not be scored consistently i n P. congesta and was therefore excluded from.the study. In P. congesta, t h i r t y - t h r e e a l l e l e s were detected at the twelve l o c i surveyed, an average of 2.75 a l l e l e s per locus. In P. brachystemon, t h i r t y a l l e l e s were detected at f i f t e e n l o c i , an average of 2.00 a l l e l e s per locus. When only the twelve l o c i scored i n both species are compared, twenty-three a l l e l e s were detected i n P. brachystemon; an average of 1.92 a l l e l e s per locus. P. congesta had approximately 30% more a l l e l e s than P. brachystemon at the twelve l o c i surveyed. Fourteen of i t s t h i r t y -three a l l e l e s , 42%, were not shared with P. brachystemon. These unique a l l e l e s can be defined as common, rare or endemic. Common a l l e l e s were those detected i n at least f i v e populations or with a mean frequency >:0.05. Rare a l l e l e s were those which were found i n less than f i v e populations and had a mean frequency < 0.05. Endemics were a l l e l e s detected at low frequency i n a single population. Nine isozymes 84. ( EST-2 1 2 6, EST-2 7 4, LAP-1 8 0, MDH-1 1 3 9, MDH-1 1 2 5, PGM-2 1 1 8, PGM-281, PGM-3 1 3 5 and PGM-3 1 0 0), 27% of the a l l e l e s scored i n P. oongesta, 87 117 were common but unique to t h i s species. Three a l l e l e s (MDH-2 , PGM-1 , 77 94 49 and PGM-3 ), 9%, were rare and unique. Two a l l e l e s (PGM-1 and PGM-3 ), 6%, were endemics. In P. braohystemon, .four of i t s twenty-three a l l e l e s , 17%, 100 182 were not found i n P. congesta. Three of these (MDH-1 , PGM-3 and PGM-3 1 1 5) were common. The fourth, (PGM-2 1 0 0 n), was an endemic detected i n only a si n g l e plant. Nineteen a l l e l e s were present i n both species. However, seven of these, 37%, were detected at s i g n i f i c a n t l y d i f f e r e n t frequencies i n the two taxa (Table 17). Populations of the two species were d i f f e r e n t i a t e d by a l l e l e s and a l l e l e frequency differences at three l o c i (EST-2, MDH-1 and PGM-3). These l o c i were considered taxonomically diagnostic. The two species could also 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 addition, with the exception of a single p l a n t , PGM-2 was monomorphic i n a l l P. braohystemon populations and polymorphic i n a l l but one P. oongesta populations. None of the PGM-3 a l l e l e s were detected i n both species and a l l e l e s unique to each taxon were present at MDH-1, PGM-2 and EST-2. Generally, the most common a l l e l e i n P. congesta was ei t h e r f i x e d or also the most common a l l e l e i n P. braohystemon. However, t h i s was not the case at EST-1, MDH-1 and PGM-3. In P. braohystemon, a l l e l e frequencies.and si n g l e locus heterozygosities fluctuated widely:among populations. However, there were no discernable macro-geographic patterns to a l l e l e d i s t r i b u t i o n s , 85. except possibly MDH-1 and PGM-3. Populations maintained fewer a l l e l e s than i n P. congesta and d i f f e r e n t a l l e l e s were often, f i x e d i n populations. In contrast to P. brachystemon, a majority of the a l l e l e s detected i n P. congesta were maintained within each l o c a l population. A l l e l e frequencies and si n g l e locus heterozygosities fluctuated among populations but not with the amplitude found i n P. brachystemon. These observations suggest that i n P. brachystemon, the lev e l s of gene flow among populations and/or recombination within populations are lower than that which occurs i n P. congesta. 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 . Within each s e c t i o n , analyses and t h e i r r a t i o n a l e are explained, data presented and the r e s u l t s discussed. Section 5.1 describes the a n a l y s i s of the breeding system and the c a l c u l a t i o n of 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 l e v e l s of genetic 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 two species. S e c t i o n 5.3 analyzes 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 of v a r i a t i o n w i t h i n each taxon. Section 5.4 compares 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 estimates of genetic i d e n t i t y and genetic d i s t a n c e . This s e c t i o n has four sub-sections which u t i l i z e these i n d i c e s to make inferences r e l e v a n t to various taxonomic and e v o l u t i o n a r y questions. L a s t l y , s e c t i o n 5.5 summarizes the data presented i n the previous four sections;, iemphasizing d i f f e r e n c e s between the two species and d e s c r i b i n g the genetic consequences of 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 system The breeding system of any taxon can be .estimated using Wright's f i x a t i o n index, F (Wright, 1965). The f i x a t i o n index measures the extent to which observed genotypic d i s t r i b u t i o n s deviate ; ' 2 2 from Hardy-Weinberg proportions (i.e. p , 2pq, q ). F = 1- - 5 -* 2pqn ' where H i s the observed number of heterozygotes and 2pqn i s the expected number of heterozygotes assuming Hardy-Weinberg e q u i l i b r i u m . 87. Values of F range from -1 to +1. Negative values i n d i c a t e a percentage of heterozygotes i n excess of expectations and p o s i t i v e values represent a d e f i c i e n c y of heterozygotes. Wright's e q u i l i b r i u m law describes observed genotype d i s t r i b u t i o n s i n terms of a l l e l e frequencies and the f i x a t i o n index F: genotype - AA Aa aa 2 2 frequency - p +pqF 2pq(l-F) q +pqF. When F=0, the genotype d i s t r i b u t i o n i s described by a Hardy-Weinberg e q u i l i b r i u m . Deviations from Hardy-Weinberg proportions (F) are the r e s u l t of the j o i n t e f f e c t of 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 at a given locus: non-random mating, mutation, s e l e c t i o n , gene flow and genetic d r i f t . When inbreeding i s the only f a c t o r causing d e v i a t i o n s from expectations, 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 a n a l y s i s measures the p r o b a b i l i t y that u n i t i n g gametes, taken at 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 descent. This value i s defined as the 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 polymorphic locus w i t h i n each p o p u l a t i o n . These values are presented i n Table 18 f o r P. brachystemon and i n Table 19 f o r P. congesta.-. In a d d i t i o n , the s t a t i s t i c F. was c a l c u l a t e d f o r each locus. This i s value i s defined as the 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 to t h e i r own l o c a l p o p u l a t i o n . Large values of F^ s ( i . e . few heterozygotes) 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 large percentage of the u n i t i n g gametes are r e l a t e d 88. TABLE. 18. Wrights Fixation .Index (F) 1 for Seven Polymorphic Loci in P. brachystemon. Locus POPULATION EST-1 LAP-1 MDH-1 PGI-2 PGI-3 PGM-1 PGM-2 1 13 9 10 7(1) 7(3) 15(1) 15(2) 16 18 .911 .911 .599 1.000 .742 not scored 1.000 1.000 1.000 1.000 1.000 1.000 .798 1.000 1.000 1.000 1.000 1.000 .894 .917 1.000 1.000 1.000 1.000 1.000 1.000 1.000 F? = .865 .988 1.000 .949 1.000 .968 1.000 is H F = 1- : H = observed number of heterozygotes; 2pqn = expected number. 2pqn F. = weighted mean of F for a l l populations 89. TABLE 19. Wright's Fixation Index (F) 1 for Eight Polymorphic Loci in P. oongesta. POPULATION EST-1 EST-2 LAP-1 MDH-1 MDH-2 PGM-1 PGM-2 PGM-3 2(1) - -.041 .221 .007 - .042 .143 .291 2(2) - -.099 .399 .432 - .631 .232 .219 3 .057 .078 .350 .022 - -.005 .145 .378 5 .251 .ISO -.508 - -.047 -.174 .091 4 - -.001 .295 .116 - .571 .583 .352 9 .709 .215 .547 .408 - .458 .198 .249 10 - .240 .510 -.085 - .409 -.063 .400 11 - .119 .367 .228 - - .018 .004 .188 7(2) .040 .014 .340 .433 .215 .345 .310 .224 8 - .125 .310 .244 - .072 -.011 -.017 14(1) - .147 .417 .103 - .375 .072 -.326 14(2) - .227 .641 .194 - 1.000 - .083 15(1) -.064 -.032 .368 .067 -.282 .221 .124 .226 15(2) - -.182 -.053 -.166 - .526 .307 .250 17 -.389 -.003 .552 .082 - .100 -.060 -.095 F? = .180 .096' .398 .155 .032 .300 .128 .188 is 1 F = 1 - : H = observed number of heterozygotes; 2 pqn = expected number. 2pqn 2 F^ s = weighted mean of F for a l l populations 90. by descent. The d i f f e r e n c e s i n F^ s values f o r the two species are s t r i k i n g . At a l l polymorphic l o c i surveyed, except MDH-2 where v a r i a t i o n was r a r e , l o c a l populations of P. congesta show an average d e f i c i e n c y of heterozygotes between 10% (EST-2) and 40% (LAP-1). In c o n t r a s t , l o c a l populations of P. brachystemon had an average d e f i c i e n c y of heterozygotes between 86% (EST-1) and 100% (MDH-1, PGI-3 and PGM-2). This d i f f e r e n c e i n the observed l e v e l s of h e t e r o z y g o s i t y w i t h i n populations of the two species suggests that i n P. brachystemon, a much l a r g e r percentage of the u n i t i n g gametes are r e l a t e d . When populations are at e q u i l i b r i u m and non-random mating i s the only 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 of genotypes at a l o c u s , the f i x a t i o n index (F) i s r e l a t e d to the o u t c r o s s i n g frequency (t) according to the r e l a t i o n s h i p F= — (Nei and Syakudo, 1958). 1+t Outcrossing r a t e s can be c a l c u l a t e d from F by transforming the above equation to t= (Nei and Syakudo, 1958). 1 + r This method assumes that populations are at 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 estimator of t ( J a i n , 1979). Since t i s a f u n c t i o n of F, s i n g l e locus estimates of 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 , besides breeding system, which a f f e c t genotype frequencies w i t h i n populations (Workman, 1969). However, breeding system should be the only f a c t o r which a f f e c t s a l l l o c i w i t h i n a p o p u l a t i o n e q u a l l y . Consequently, when F i s averaged over 91. a number of l o c i and t h i s F i s used to 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 absolute estimator, i s c e r t a i n l y a r e l a t i v e estimator of o u t c r o s s i n g among populations. The average net d e v i a t i o n from Hardy-Weinberg pr o p o r t i o n s (F) and the o u t c r o s s i n g r a t e (t) were c a l c u l a t e d f o r each pop u l a t i o n and are presented i n Table 20 f o r P. braohystemon and Table 21 f o r P. oongesta. Wright's f i x a t i o n index can be c a l c u l a t e d only f o r polymorphic l o c i . Consequently, the number of l o c i c o n t r i b u t i n g to the o u t c r o s s i n g estimates v a r i e s among popu l a t i o n s . An o u t c r o s s i n g frequency was not l i s t e d f o r P. braohystemon p o p u l a t i o n 16 ( L i t t l e Qualicum F a l l s ) because the only v a r i a t i o n scored i n the p o p u l a t i o n was detected i n two p l a n t s , heterozygous at two l o c i (PGI-2 and PGM-3). This apparent anomaly produced an o u t c r o s s i n g r a t e which was u n r e a l i s t i c a l l y high f o r t h i s species (t=0.988). The estimates of o u t c r o s s i n g f o r P. braohystemon were between 0 and 7.4% (Francis Park). The average (± standard e r r o r ) f o r the nine populations was 2.4 ± 0.8% o u t c r o s s i n g . In P. oongesta the estimates ranged from a low of 42% (Francis Park and Jack's Point -2 ) to a high of 100% ( M i l l H i l l Park). The average ou t c r o s s i n g frequency f o r the f i f t e e n populations was 70.2 ±4.8%. These estimates are i n e x c e l l e n t agreement with those of Ganders et al. (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 of known i n h e r i t a n c e to estimate o u t c r o s s i n g i n both taxa. Using t h i s polymorphism and the progeny t e s t method of Harding (1970), they found o u t c r o s s i n g 92. TABLE 20. Estimates of Outcrossing Frequency f o r Ten Populations of P. braohystemon. POPULATION F 1 f 2 . • 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 - -18 .978 .011 AVERAGE .024±.008 F = weighted mean of F f o r a l l polymorphic l o c i 2 1-P t = — -1+F 93. TABLE 21. Estimates of Outcrossing Frequency f o r F i f t e e n Populations of P. congesta. POPULATION ......F . t 2 2(1) .105 .801 2(2) .307 .530 3 .146 .745 5 -.037 1.077 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 AVERAGE: .702±.048 F = weighted mean of F f o r a l l polymorphic l o c i 2 t = l _ F 1+F 94. i n P. bvaahystemon to average 2% and ou t c r o s s i n g i n P. congesta to range from 48% to 80% with a mean of 70%. The d i f f e r e n c e between the mean ou t c r o s s i n g r a t e s f o r these two c l o s e l y r e l a t e d species was h i g h l y s i g n i f i c a n t (PiO.Ol) but not s u r p r i s i n g . The observed divergence i n breeding s t r a t e g i e s had been a n t i c i p a t e d on the ba s i s of f l o r a l morphology and f i e l d observations and confirmed the e a r l i e r o u t c r o s s i n g estimates i n these taxa. 5.2 Genetic vaviability within populations In order to evaluate the consequences of c o n t r a s t i n g breeding s t r a t e g i e s on the popul a t i o n s t r u c t u r e of the two taxa, f i v e genetic 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 to describe l e v e l s of genetic 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 of l o c i t hat are polymorphic , average number of a l l e l e s per loc u s , average number of a l l e l e s per polymorphic l o c u s , expected percentage of heterozygous l o c i per i n d i v i d u a l and the observed percentage of heterozygous l o c i per i n d i v i d u a l . These values 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 pop u l a t i o n then averaged over a l l populations (± standard e r r o r ) f o r P. bvachystemon and P. congesta i n Tables 22 and 23 r e s p e c t i v e l y . The p r o p o r t i o n of l o c i that are polymorphic per po p u l a t i o n v a r i e d i n P. bvachystemon between 6.67% (Prospect Lake) and 40% (Malahat Drive-2) with an average of 20.76 ± 3.49%. The range i n P. congesta was not as great. Population 14(2) (Jack's Point) 95. Table 22. Summary of Various Genetic Parameters for Ten Populations of Plectritis brachystemon. Average Percentage Number Loci A l l e l e s / Population Polymorphic 1 Locus Average Number A l l e l e s / Polymorphic Locus Expected Percentage of Heterozygous Loci/ Individual 2 (H) Observed Percentage of 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. ,40 163 14. .28 1. .14+. ,10 2. ,00±. ,00 1.361.92 1.301. 88 18 13. .33 1. .20±. 14 2, .501. .17 6.9914.77 .741. ,74 AVERAGE 20.76+3.49 1.251.04 2.221.10 5.89+1.33 .451.13 1 The frequency of the most common a l l e l e i s < .99. 2 tjT f ° r r l o c i ; h=l-Ex?) expressed as a percentage. 3 A l l estimates with this population based on a sample of 14 l o c i (EST-1 Excluded) 96. Table 23. Summary of Various Genetic Parameters for Fifteen Populations of Plectritis oongesta. Percentage Loci Population Polymorphic 1 Average Average Number Number A l l e l e s / A l l e l e s / Polymorphic Locus Locus Expected Percentage of Heterozygous Loci/ Individual 2 C H ) Observed Percentage of Heterozygous Loci/ Individual v obs. 2(1) 50 .00 2 .08± .50 3 .17+ .79 18 .97±7 .78 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 11 50. .00 1, .92+, .42 2, ,83±, .65 26, .01+8. .23 21. .58+6.61 7(2) 66. ,67 2, ,17±. .34 2, ,75±, .37 25, .92+7.12 18. ,9615.02 8 50. 00 2. .08±. .43 3. , 17±. 60 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 AVERAGE 53.33+ 1.78 1. 98+. OS 2. 84±. 08 22. 641.79 17. 741.81 iThe frequency of the most common a l l e l e is < 99. =lj f o r l o c i ; h = 1- x?) expressed as a percentage. 2(H= 97. with 41.67%, had the lowest percentage of l o c i that were polymorphic. Populations 7(2) (Malahat Drive) and 15(1) (Nanoose H i l l ) with 66.67%, had the highest percentage of l o c i that were polymorphic. The average f o r the f i f t e e n populations was 5.3.33 ± 1.78%. The d i f f e r e n c e between the means f o r the two species was h i g h l y s i g n i f i c a n t (P_0.01). The average number of a l l e l e s detected per locus i n P. brachystemon ranged between 1.07 (Prospect Lake) and 1.47 (Malahat Drive-3) with a mean of 1.25 t 0.04. The values i n P. congesta were c o n s i s t e n t l y higher with a low of 1.67 ( M i l l H i l l ) and a high of 2.17 (John Dean Park, Malahat Drive and N i l e 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 the mean observed i n P. brachystemon (P^O.01). When average number of a l l e l e s per polymorphic locus was c a l c u l a t e d , P. congesta was again 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 the 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 polymorphic locus. The corresponding values i n the outcrosser were from 2.00 (Nanoose H i l l - 2 ) to 3.17 (Anacortes-1, -2 and Crofton e x i t ) . .The mean f o r P. congesta. was 2.84 ±0.08, and again the d i f f e r e n c e between the two taxa was h i g h l y s i g n i f i c a n t (P<0.01). The expected percentage of heterozygous l o c i per i n d i v i d u a l (H) was c a l c u l a t e d from the observed a l l e l e frequencies w i t h i n each p o p u l a t i o n . This i s the best s i n g l e s t a t i s t i c , f o r d e s c r i b i n g l e v e l s 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 defined i n the next s e c t i o n . In P. brachystemon, H ranged between 0.63% (Pospect Lake) and 14.05% (Malahat Drive-3) with an average value of 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% (Jack's Point-2) and 27.04% (Prospect Lake) f o r an aver.age of 22.64 +_ 0.79%. This value was s i g n i f i c a n t l y higher than the mean c a l c u l a t e d f o r the s e l f e r (PiiO.Ol). Although the absolute value of h e t e r o z y g o s i t y was lower i n P. brachystemon, the r e l a t i v e range of values was much l a r g e r than that observed i n P. congesta. The magnitude of the f l u c t u a t i o n s among populations of the s e l f e r suggests heightened 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 . This was p a r t i c u l a r l y s t r i k i n g when P. brachystemon c o l l e c t i o n s from sympatric s i t e s 7(1):7(3) (Malahat Drive) and 15(1):15 (2) (Nanoose H i l l ) were compared. These sympatric 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 v a r i a t i o n (H) and yet they were separated by distances of only t h i r t y meters at Malahat Drive and no greater than one hundred meters at Nanoose H i l l (the exact d i s t a n c e i s unknown). The magnitude of these 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 to be a r e s u l t of the breeding system. Autogamy r e s t r i c t s recombination and gene flow v i a p o l l e n vectors 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 of founder e f f e c t and d r i f t i n determining p o p u l a t i o n s t r u c t u r e . The observed percentage of heterozygous l o c i per i n d i v i d u a l ( H Q ^ S ) was c a l c u l a t e d by d i r e c t count and the d i f f e r e n c e s between the two species 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 Drive-1 and -3) to 1.30% ( L i t t l e Qualicum F a l l s ) w i t h a mean of only 0.45 * 0.13%. In P. oongesta, H ^ v a r i e d between 10.42% (Jack's Point-2) and 22.28% ( M i l l H i l l ) w ith a mean of 17.74 - 0.81%. Outcrossing r a t e s and l e v e l s of expected h e t e r o z y g o s i t y (H) were compared among populations w i t h i n each species. This i s p l o t t e d i n Figure 42 f o r P. braohystemon and Figure 43 f o r P. oongesta. However, as was found i n Limnanthes alba ( J a i n , 1978), and Pinus oontorta (Yeh and Layton, 1979), there was no c l e a r r e l a t i o n s h i p between estimated o u t c r o s s i n g r a t e s and genetic v a r i a b i l i t y among populations of the same taxon. I n t u i t i v e l y , 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 there could be s e v e r a l reasons f o r the lack of c o r r e l a t i o n . Sample s i z e s w i t h i n populations may have been too small to detect d i f f e r e n c e s . J a i n (1978) genotyped 20-30 f a m i l i e s per p o p u l a t i o n , Yeh and Layton (1979) genotyped 15 f a m i l i e s per popu l a t i o n and from 15 to 60 p l a n t s were genotyped per popu l a t i o n of Pleotritis. Secondly, the range of ou t c r o s s i n g r a t e s among populations of the same species may be too small to be a c c u r a t e l y measured with the estimators used. Although the technique employed by J a i n (1978) does not assume that the populations are i n Hardy-Weinberg e q u i l i b r i u m , the method used to c a l c u l a t e o u t c r o s s i n g i n lodgepole pine (Yeh and Layton, 1979) and Pleotritis makes that assumption. Any d e v i a t i o n s between the observed and expected frequencies of heterozygotes are a t t r i b u t e d s o l e l y to the r a t e of ou t c r o s s i n g . .10.0,. 30.0-^ 25.0 o >> 20.04 ."t_ 'to O O) 15.0 >> N O f r 10-0 (D (D x : 5.0 .01 .02 .03 .04 — i — .05 .06 — i — .07 ~08~ .09 — i .10 outcrossing rate Figure 42. The r e l a t i o n s h i p between the ou 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 populations of* "P. brachystemon. .10 .20 .30 .40 .50 .60 .70 .80 .90 1.00 outcrossing rate Figure 4.3. The r e l a t i o n s h i p between the ou 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 e among populations of P. congesta. 101. This assumption allows the.combined i n f l u e n c e of other f a c t o r s such as mic 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 to minimize breeding system d i f f e r e n c e s . L a s t l y , the i n d i c a t o r used to measure l e v e l s of v a r i a t i o n among populations presents problems because estimates of he t e r o z y g o s i t y have a large standard e r r o r . This occurs because each locus does not co n t r i b u t e e q u a l l y to a species' h e t e r o z y g o s i t y . Some l o c i are monomorphic while others are h i g h l y polymorphic. Consequently, without a very large sample of l o c i , the S.E. ass o c i a t e d with the estimate 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 the l e v e l s of v a r i a b i l i t y between populations of the same taxon (Figures 42 and 43). Although no r e l a t i o n s h i p was detected between o u t c r o s s i n g r a t e s and l e v e l s of h e t e r o z y g o s i t y among populations of the same species, the data suggest that the breeding system i s a major determinant of popu l a t i o n s t r u c t u r e . The co n t r a s t i n the out c r o s s i n g r a t e s of these two taxa 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 of v a r i a t i o n maintained w i t h i n populations. As measured by a l l parameters, populations of P. congesta maintain s i g n i f i c a n t l y higher l e v e l s of genetic v a r i a b i l i t y than do populations of i t s autogamous r e l a t i v e , P. bvachystemon. When 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 only those populations of both taxa growing s y m p a t r i c a l l y , a l l detected d i f f e r e n c e s remained s i g n i f i c a n t . 102. 5.3 Analysis of gene diversity The l e v e l of 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 with the a n a l y s i s of gene d i v e r s i t y (Nei, 1973, 1975). This a n a l y s i s permits genetic v a r i a t i o n to 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 of po p u l a t i o n s t r u c t u r e (i.e. w i t h i n versus between l o c a l or sub-populations). This technique i s a m o d i f i c a t i o n of Wright's F - s t a t i s t i c s expanded to 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 the d e t e c t i o n of genotype frequencies because i t does not measure d e v i a t i o n s between observed and expected l e v e l s of he t e r o z y g o s i t y . The b a s i s of the a n a l y s i s i s the c a l c u l a t i o n of expected, or t h e o r e t i c a l , h e t e r o z y g o s i t y per locus (h): h = l - I x 2 , l where x^ i s the frequency of the i ^ a l l e l e at the locus i n question. This i s designed to measure the amount of genetic v a r i a t i o n at a locus w i t h i n a popu l a t i o n . 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 are synonymous. The 2 concepts of h e t e r o z y g o s i t y and homozygosity (j=Ex^) i n populations were o r i g i n a l l y developed with respect to random mating populations. I f a pop 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 the 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 are not met, as when mating i s non-random, h does not equal the frequency of heterozygotes. i n the pop u l a t i o n . In order to avoid confusion when studying populations with non-random mating systems, Nei (1973) proposed 103. the term "gene d i v e r s i t y " f o r the he 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 the 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 of a popu l a t i o n i s estimated by H = I h k / r k=l which i s the average of h over the r l o c i sampled. This can be i n t e r p r e t e d as the average p r o p o r t i o n of heterozygotes per locus or the p r o p o r t i o n of l o c i at which an i n d i v i d u a l can be expected to be heterozygous. The o r g a n i z a t i o n of gene d i v e r s i t y w i t h i n a taxon can be described by the r e l a t i o n s h i p H T = Hs + D s r 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 the gene d i v e r s i t y w i t h i n l o c a l populations (Hg) and that between l o c a l populations (Dg^). These values are c a l c u l a t e d f o r each locus over a l l populations sampled, then averaged over a l l l o c i to get grand estimate f o r the taxon s t u d i e d . ILj, estimates the t o t a l genie v a r i a t i o n sampled over a l l populations as i t i s a f u n c t i o n of mean a l l e l e frequencies; H T = 1 " ^ i -i s the mean frequency of the i 1 " * 1 a l l e l e over a l l sampled pop u l a t i o n s . This s t a t i s t i c was c a l c u l a t e d from the data i n Tabl 17. H i s an estimate of the average amount of genetic v a r i a t i o n maintained w i t h i n l o c a l p o pulations. I f n populations are surveyed, the average gene d i v e r s i t y w i t h i n populations at a 104. p a r t i c u l a r locus i s n h H„ = E k/ S k = 1 n where h^ i s the expected h e t e r o z y g o s i t y i n the k l o c a l p o p u l a t i o n . I f a l l populations are members of a s i n g l e large panmictic u n i t and there i s no s e l e c t i o n f o r l o c a l adaptation, 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 the e n t i r e range of the taxon and H w i l l equal H . However, i n nature t h i s does not occur. Natural populations tend to d i f f e r e n t i a t e over time i n t o sub-populations as a r e s u l t of mutation, s e l e c t i o n , random d r i f t and r e s t r i c t e d gene flow. Therefore, H i s a subset H^. The l e v e l of d i f f e r e n t i a t i o n between populations i s measured by D , the gene d i v e r s i t y between po p u l a t i o n s , and i s given by DST = HT " HS-The greater the l e v e l of s u b - d i v i s i o n w i t h i n the taxon, the l a r g e r the value of Dg^-The r e l a t i v e amount of genetic d i f f e r e n t i a t i o n between l o c a l populations (G ) can be estimated by O 1 G = °ST/ ST / H T ' This value v a r i e s from zero to one and i s termed the c o e f f i c i e n t of gene d i f f e r e n t i a t i o n . The sampling variance of Gg^ can be c a l c u l a t e d (Chakraborty, 1974), a standard e r r o r obtained and the s i g n i f i c a n c e of the genetic d i f f e r e n t i a t i o n assessed. 105. The analyses of gene d i v e r s i t y i n P. brachystemon and P. congesta are presented i n Tables 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 of 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 both species which i s r e f l e c t e d by .the magnitude of the standard e r r o r s o f the means. S i m i l a r r e s u l t s have been obtained i n most other organisms t^hat . have been e x t e n s i v e l y surveyed f o r e l e c t r o p h o r e t i c v a r i a b i l i t y . The standard e r r o r s are o f t e n one-f i f t h to t w o - f i f t h s as large as the mean h e t e r o z y g o s i t i e s (Lewontin, 1974). Since a l l l o c i do not c o n t r i b u t e e q u a l l y to a species average gene d i v e r s i t y , i t i s important to survey as large a number l o c i as p o s s i b l e i n order to r e a l i s t i c a l l y estimate t h i s parameter (Nei and Roychoudhury, 1974). The values of H^ , for" P. brachystemon were between zero and 0.659 with a mean over a l l l o c i of 0.166+0.058. In P. congesta, the values ranged between zero and 0.792 with a mean of 0.266+0.083. Although P. brachystemon g e n e r a l l y has l e s s t o t a l gene d i v e r s i t y at each locus than P. congesta, the p a t t e r n of h e t e r o z y g o s i t y among l o c i of each species i s s i m i l a r . However, the EST l o c i are notable exceptions. Both species share the same three a l l e l e s at EST-1 yet t h i s locus had the l a r g e s t H^ value of a l l l o c i surveyed i n P. brachystemon and one of the lowest values observed i n P. congesta In..contrast, EST-2 had three a l l e l e s and was extremely heterozygous i n P. congesta while i t was monomorphic i n P. brachystemon. Esterases are a large and heterogeneous c l a s s of enzymes which are 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 1 0 6 . Table 24 . Analysis of Gene Diversity and Degree of Di f f e r e n t i a t i o n f o r 15 Loci i n Pleotritis braohystemon. Locus Total Gene Diversity (Hp) Gene Diversity Within Populations ay Proportion of I n t e r p e l l a t i o n Gene Di f f e r e n t i a t i o n (G S T) 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 Loci: .166±.058 .060±.021 ,639±.053 107. Table.25. A n a l y s i s of Gene D i v e r s i t y and Degree of 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 D i v e r s i t y (H T) Gene D i v e r s i t y Within Populations CHS) Pr o p o r t i o n of In 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 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 L o c i : .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 of allozyme a c t i v i t y at 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 Malathion suggests that allozymes at EST-1 and EST-2 may represent 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 ' these polymorphisms are adaptive ;' (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 represent an e v o l u t i o n a r y divergence of adaptive s t r a t e g i e s i n the two 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 of 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 of genetic d r i f t i s p u r e l y s p e c u l a t i v e . The d i f f e r e n c e between the mean H^ values of the two species was compared usi n g a t - t e s t with 25 d.f. and found to 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 of the d i f f e r e n c e between H T averaged over 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 to the la r g e standard e r r o r of the means. However, t h i s comparison demonstrates that d e s p i t e i t s high l e v e l of inbreed i n g , P. brachystemon maintains a l e v e l of t o t a l v a r i a t i o n which i s somewhat reduced, but comparable to the predominantly outcrossed P. congesta. I t i s the 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 , H , which r e f l e c t s the c o n t r a s t i n g breeding systems. I n t e r - l o c u s v a r i a t i o n i n . Hg was again l a r g e . Values ranged from zero to 0.218 i n P. brachystemon and zero to 0.671 i n P. congesta. When averaged over a l l l o c i , the mean genetic d i v e r s i t y w i t h i n l o c a l populations 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 equal t o the mean expected h e t e r o z y g o s i t y (H) averaged over a l l p o p u l a t i o n s . Consequently, H q 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 of heterozygotes per locus or the p r o p o r t i o n of l o c i at which an i n d i v i d u a l can be expected to be heterozygous. On the average, P. oongesta was heterozygous at 23% of i t s l o c i while P. braohystemonwas heterozygous at only 6%. Despite the i n t e r - l o c u s v a r i a t i o n , the d i f f e r e n c e between the means was s i g n i f i c a n t (P<0.05). This i l l u s t r a t e s that, the l e v e l of po p u l a t i o n s u b - d i v i s i o n i n the s e l f e r i s much l a r g e r than the l e v e l i n the outcrosser. As the gene d i v e r s i t y w i t h i n populations (H ) decreases, the gene d i v e r s i t y between {i.e. d i f f e r e n t i a t i n g ) . p o p u l a t i o n s (Dg-jO i n c r e a s e s . A c c o r d i n g l y , 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 populations (Gg^) a l s o increases because i t i s described by the r e l a t i o n s h i p % r = DS.T/_ H T When G was averaged over a l l l o c i , the values f o r P. braohystemon and P. oongesta were 0.639+0.053 and 0.150+0.039 r e s p e c t i v e l y . The d i f f e r e n c e , between the means was h i g h l y s i g n i f i c a n t (P<0.01). In P. braohystemon, 64% of the t o t a l gene d i v e r s i t y could be a t t r i b u t e d to 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 . This compared to only 15% in.P. congesta:- In other words, populations of P. congesta maintain, on the average, 85% of the v a r i a t i o n detected over the sampled range of the species. In c o n t r a s t , s i n g l e populations of P. braohystemon maintain an average of only 36% of the s e l f e r ' s array o f detected, v a r i a t i o n . Although G i s a good measure of the r e l a t i v e amount O 1 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 , i t s value i s h i g h l y dependent on (Nei, 1975). When i s low, may overestimate the magnitude of 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 not found to be s i g n i f i c a n t l y d i f f e r e n t between the two taxa, another measure o f po 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 . Standard genetic distance., D (Nei, 1972) estimates the number of net codon d i f f e r e n c e s per locus between pop u l a t i o n s . I t i s independent of the gene d i v e r s i t y w i t h i n populations-and t h e r e f o r e can be used f o r comparing the degree of 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 genetic distance (D ± i n t e r - l o c u s S.E.) among populations of 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 . In other words, f o r every 100 l o c i , there were approximately 5.2 codon d i f f e r e n c e s between populations of P. congesta and 13.7 codon d i f f e r e n c e s between populations of P. brachystemon. Although the D between populations of the s e l f e r was more than two and a h a l f times greater than the corresponding value i n the o u t c r o s s e r , the .difference was n o n - s i g n i f i c a n t (P<"0.40). However, t h i s l ack of s i g n i f i c a n c e can be a t t r i b u t e d to the i n t e r -locus 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 . 5.4 Comparisons of genetic identity and genetic distance Nei's (1972, 1975) index of genetic i d e n t i t y (I) and standard genetic distance (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 combinations of pop u l a t i o n s . There were four o b j e c t i v e s i n t h i s a n a l y s i s : i . to assess the r e l a t i v e l e v e l s 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 each species and c o r r e l a t e t h i s with the breeding system; 111. i i . t o determine i f genetic distance r e f l e c t s geographic distance between pop u l a t i o n s ; i i i . to c a l c u l a t e the 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 the two taxa i n order to evaluate t h e i r taxonomic r e l a t i o n s i p . and i v . to determine i f sympatric populations are g e n e t i c a l l y more s i m i l a r than other i n t e r - s p e c i f i c combinations. The purpose of t h i s comparison was to 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 of the observed isozyme polymorphisms. Nei's genetic i d e n t i t y s t a t i s t i c compares populations f o r both the presence/absence of i d e n t i c a l a l l e l e s and t h e i r occurrence at s i m i l a r frequencies. This measure i s c a l c u l a t e d f o r each locus: ^'(P j x) 2)(£(P j y) 2) where P_.^  and are the frequencies of 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 the locus and then d i v i d e d by the geometric mean of i d e n t i c a l homozygote frequencies. Two populations which have i d e n t i c a l a l l e l e frequencies at a l o c u s , have a genetic i d e n t i t y of one. Their genetic 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 are considered, the genetic i d e n t i t y between two populations i s c a l c u l a t e d by 112. 1 /"IXEp? ) (EEp^ YL1' i j * j x i j i j y The numerator, I s , i s an index of genetic s i m i l a r i t y (Sokal and Sneath, 1963): LK I = T- E E P. . . P. . . s L i j - I J X ijy In both the numerator and denominator, P.. and P.. are the IJX i j y corresponding frequencies of the j ^ a l l e l e at the i ^ n locus i n populations X and Y. L i s the number of l o c i surveyed. Spiess (1977) presents a more d e t a i l e d d i s c u s s i o n of the theory and c a l c u l a t i o n of t h i s s t a t i s t i c . The standard genetic distance between populations X and Y i s defined as D = - l o g e I . This i s approximately equal to 1-1. This value estimates the accumulated number of 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 of gene s u b s t i t u t i o n s i s i d e n t i c a l f o r a l l l o c i . The genetic i d e n t i t i e s and standard genetic distances between populations of P. braohystemon and P. oongesta are presented i n Tables 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 a l l e l e frequencies from f i f t e e n l o c i i n P. braohystemon and twelve l o c i i n P. oongesta. Population 16 ( L i t t l e Qualicum F a l l s ) of P. braohystemon was excluded from a l l genetic distance c a l c u l a t i o n s because a l l e l e frequencies f o r EST-1 were not a v a i l a b l e . Table 26. Genetic i d e n t i t y (I) and standard genetic distance (D) between populations of P.'.^brachystemon 1 1 13 0. 1020 9 0. 1433 10 0. 1408 7(1) 0. 1663 7(3) 0. 1382 15(1) 0. 1404 15(2) 0. 1384 18 0. 3775 I pop. 0. 8451 D pop. 0. 1683 13 9 0.9030 0.8665 0.9422 0.0595 0.1408 0.1442 0.0929 0.0868 0.0565 0.0904 0.0735 0.0549 0.0930 0.0884 0.2016 0.2446 0.9026 0.8923 0.1025 0.1140 10 7(1) 0.8687 0.8468 0.8687 0.9113 0.8657 0.9167 0.8433 0.1704 0.1426 0.0521 0.1784 0.0432 0.2461 0.0273 0.2586 0.2016 0.8372 0.9002 0.1777 0.1051 7(3) 15(1) 0.8709 0.8690 0.9451 0.9291 0.9136 0.9466 0.8671 0.8366 0.9492 0.9577 0.9180 0.0856 0.0707 0.0489 0.1488 0.2193 0.9066 0.8999 0.0981 0.1055 15(2) 18 0.8708 0.6856 0.9112 0.8174 0.9154 0.7830 0.7818 0.7721 0.9731 0.8174 0.9317 0.8617 0.9523 0.8031 0.7675 0.2646 0.8850 0.7869 0.1222 0.2396 Estimates of I above the diagonal Estimates of D above the diagonal I = 0.8720 D = 0.1370 + 0.0825 Table 27. Genetic i d e n t i t y (I) and standard genetic distance (D) between populations of P. oongesta. 2(1) 2(2) 3 2(1) .9930 .9576 2(2) .0070 .9427 3 .0433 .0590 5 .0759 .0773 .0649 4 .0299 .0226 .0253 9 .0477 .0281 .0616 10 .0455 .0461 .0210 11 .0332 .0359 .0183 7(2) .0831 .0884 .0502 8 .0623 .0783 .0283 14(1) .0142 .0416 .0223 14(2) .0044 .0182 .0304 15(1) .0907 .1080 .•0474 15(2) .1191 .1121 .0858 17 '."0328 .0535 .0431 I pop-i. .9520 .9461 .9580 D pop. :.0492 .0554 .0429 5 4 9 10 .9269 .9705 .9534 .9555 .9256 .9777 .9723 .9549 .9372 .9750 .9403 .9792 .9623 .9464 .9859 .0384 .9857 .9849 .0551 .0144 .9667 .0142 .0152 .0339 .0255 .0109 .0321 .0078 .0083 .0484 .0850 .0226 .1243 .0384 .0814 .0526 .0768 .0307 .0812 .0451 .0799 .0170 .0470 .0477 .0200 .0520 .0733 .0274 .0272 .0319 .0707 .0430 .1033 .0553 :0663 .(0444 .9451 .9697 .9460 .9672 .0565 .0308 .0555 .0333 11 7(2) 8 14(1) .9673 .9203 .9396 .9859 .9647 .9154 .9247 .9593 .9819 .9510 .9721 .9779 .9748 .9917 .8831 .9261 .9892 .9528 .9623 .9698 .9684 .9185 .9218 .9220 .9922 .9777 .9488 .9559 .9741 .9454 .9648 .0262 .8995 .9389 .0561 ..1059 .9662 .0358 .0630 .0344 .0318 .0813 .0470 .0076 .0267 .0199 .1020 .0774 .0499 .0441 .0979 .0925 .0402 .1019 .0423 .0453 .9698 .9425 .9344 .9534 .0307 .0592 .0679 .0477 14(2) 15(1) 15(2) 17 .9956 .9133 .8877 .9677 .9820 .8976 .8940 .9479 .9701 .9537 .9178 .9578 .9232 .9802 .9732 .9019 .9831 .9493 .9686 .9462 .9541 .9293 .9317 .9359 .9534 .9730 .9579 .9566 .9687 .9737 .9513 .9606 .9219 .9803 .9569 .9031 .9541 ;?030 .9067 .9586 .9924 .9255 .9116 .9557 .9188 .9076 .9550 '.'0847 .9393 .9097 .0970 .0626 .8678 .0460 .0946 .1418 .9553 .9387 .9261 .9370 .•0457 .0633 .0767 .0651 Estimates of I above the diagonal I = 0.9493 Estimates of D above the diagonal D = 0.0520 ± 0.0344 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 taxa can.be compared b y . c a l c u l a t i n g mean genetic i d e n t i t i e s (I) from a l l p a i r w i s e combinations of populations. On the average, populations of 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 populations of P. conge,sta (t = 0.9493). Based on a sample of twenty-eight s p e c i e s , G o t t l i e b (1977a) estimated the mean i d e n t i t i e s of i n t r a - s p e c i f i c p l a n t populations to be 0.951.02. He t h e r e f o r e concluded that i n g e n e r a l , a sample of one or a few populations 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 , . While the value of I f o r P. congesta i s i n e x c e l l e n t agreement with G o t t l i e b ' s estimate, the c a l c u l a t e d value f o r P'. brachystemon/is much lower. In f a c t , i t i s lower than estimates of I between f i v e congeneric species p a i r s ( G o t t l i e b , 1977a) : Clarkia biloba and C. lingulata (1=0.88), Gaura longiflora and G. demareei (I = 0.99), Hymenopappus scabrosaeus and H. artemisiaefolius (I = 0.90), Oenothera strigosa and 0. biennis '(I = 0.97), and Stephanomeria exigua subsp. coronaria and S. malheurensis (I = 0.94). The observed d i f f e r e n c e s i n the mean genetic i d e n t i t i e s i n Plectritis r e f l e c t s the r e l a t i v e degree of p a r t i t i o n i n g i n the gene pool of each species. In order f o r I to be l a r g e , l o c a l populations must maintain a large p r o p o r t i o n of the taxon's array of v a r i a t i o n . On the average, 85% of 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 . In c o n t r a s t , P. brachystemon populations contained only 34% of the v a r i a t i o n of the species. Large f l u c t u a t i o n s '.. i n a l l e l e frequencies between populations were much more prevalent i n the s e l f e r than the outcrosser (Figures 27-40). The 116. d i s t r i b u t i o n of many a l l e l e s i n P.•brachystemon might best be described as a "random walk." This p a t t e r n 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 to be a consequence o f the breeding system. Autogamy r e s t r i c t s gene flow and lowers the e f f e c t i v e p o p u l a t i o n s i z e . This enhances the importance o f s t o c h a s t i c forces i n determining a l l e l e frequencies w i t h i n p o p u l a t i o n s . This i s not to say that founder e f f e c t and genetic d r i f t are not important w i t h i n predominantly o u t c r o s s i n g taxa, only that these forces become r e l a t i v e l y more important when gene flow i s reduced. 5.4.2 Comparisons between genetic distance and geographic distance Genetic distance estimates are summarized i n Table 28 f o r P. brachystemon and i n Table 29 f o r P. congesta. In a d d i t i o n , dendrographs (McCammon, 1968) were generated using the estimates of genetic i d e n t i t y and these are presented i n Figures.44 and 45 f o r P. brachystemon and P. congesta r e s p e c t i v e l y . Although c e r t a i n comparisons suggest a r e l a t i o n s h i p between geographic distance and genetic d i s t a n c e , o v e r a l l there appears to be no c l e a r r e l a t i o n s h i p between these two parameters. These observations are i n agreement with those of Kahler et al. (1980), who found that 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 patterns which were not r e l a t e d to geographic d i s t a n c e . In P. brachystemon, p o p u l a t i o n 1 (Sumas Mountain) and po p u l a t i o n 18 (Campbell River) are separated by the greatest geographic d i s t a n c e and a l s o had the l a r g e s t genetic distance (D = 0.3775). However, p o p u l a t i o n 18 was the 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 populations (D = 0.2396). This large D i s comparable to some i n t e r - s p e c i f i c comparisons w i t h i n other genera. I t r e f l e c t s 117. Table 28. Summary of Genetic Distances i n P. brachystemon Population D Most s i m i l a r to Most d i s t a n t from Population D Population D 1 0.1683 13 0.1020 18 0.3775 13 0.1025 7(3) 0.0565 18 0.2016 9 0.1140 15(1) 0.0549 18 0.2446 10 0.1777 1 and 13 0.1408 18 0.2586 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. Table 29. Summary of genetic distances i n P. congesta i p u l a t i o n D Most s i m i l a r to Most d i s t a n t : from Population 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 10 0.0210 15(2) 0.0858 5 0.0565 7(2) 0.0083 8 0.1243 4 0.0308 11 0.0109 17 0.0553 9 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, G E N E T I C I D E N T I T Y (I) 1.00 0 0 . 9 7 6 0 . 9 5 2 0 . 9 2 8 0 . 9 0 4 1 1 1 1 7(1), 1 15(2) ' 15(1) 1 7(3) 1 13 9 1 10 18 Figure 44. A dendrograph d e p i c t i n g the genetic r e l a t i o n s h i p s among the sampled P. bvaahystemon populations (population 16 excluded): c o r r e l a t i o n s are based on Nei's Index of Genetic I d e n t i t y c a l c u l a t e d from the a l l e l e frequencies at f i f t e e n l o c i . 120. G E N E T I C I D E N T I T Y (I) 1 . 0 0 0 I 1 0 1 1 8 1 7 0 . 9 7 0 — i 0 . 9 3 9 1 2 ( 2 ) 1 4 ( 1 ) 1 4 ( 2 ) 2 ( 1 ) 1 5 ( 2 ) 1 5 ( 1 ) 7 ( 2 ) 5 Figure 45. A .dendrograph d e p i c t i n g the genetic 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 are based on Nei's Index o f Genetic I d e n t i t y c a l c u l a t e d from the a l l e l e frequencies at twelve l o c i . 121. the observation that t h i s p o p u l a t i o n was monomorphic f o r or maintained at r e l a t i v e l y high frequency, 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 or absent i n other P. brachystemon populations (i.e. EST-1 , MDH-1 1 1 3, P G I - 3 3 3 r and .PGM-3 1 1 5). Sympatric populations 7(1):7(3) (Malahat Drive) and 15(1): 15(3) (Nanoose H i l l ) had r e l a t i v e l y low genetic 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, populations 15(1) and 15(2) were more s i m i l a r to p o p u l a t i o n 7 (1), and 7(1) was more s i m i l a r to 15(2), than they were to t h e i r r e s p e c t i v e sympatric populations. Populations 7(1) and 7(3) were c o l l e c t e d approximately 30 "m.apart (although i n successive y e a r s ) . Their a l l e l e frequencies were compared using a t - t e s t ( Spiess, 1977). Although these two populations were separated by such a small d i s t a n c e , they maintained the f o l l o w i n g a l l e l e s at 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 8 9, E S T - 1 1 1 4 , LAP-1 9 5, LAP-1 1 0 0,-PGM-1 1 0 0 and PGM-1 1 0 6 (P<0.01); and E S T - 1 1 0 0 , P G I - 2 8 1 and P G I - 2 1 0 0 (PsO.05). In a d d i t i o n , these populations 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 values (P<0.01) at EST-1, LAP-1, PGI-2 and PGM-1. P. brachystemon populations 15(1) and 15(2) were separated by a distance of not more than 100 m.(the exact distance i s unknown). Within these two p o p u l a t i o n s , the f o l l o w i n g a l l e l e s were observed at s i g n i f i c a n t l y d i f f e r e n t frequencies (P<0.01); E S T - 1 1 0 0 , E S T - 1 1 1 4 , L A P - 1 1 0 0 , L A P - 1 1 0 5 , P G I - 3 3 3 r , P G I - 3 1 0 0 n , PGM-1 1 0 0 and PGM-1 1 0 6. These populations 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 values at 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 populations 15(1) and 15(2) were compared 122. and s i g n i f i c a n t l y d i f f e r e n t frequencies (P£0.01) were detected f o r only LAP-1 9 1, LAP-1 9 5 and L A P - 1 1 0 0 . These data suggest that the l e v e l of genetic d i f f e r e n t i a t i o n between populations of 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 with the geographic d i s t a n c e which separates them. When populations of P. congesta were compared, the correspondence between geographic distance and genetic d i s t a n c e appeared b e t t e r than that found i n P. bvachystemon. However, there were s t i l l notable exceptions to any p a t t e r n . Figure 45 de p i c t s a major dichotomy w i t h i n P. congesta. Populations segregated i n t o two unequal sub-groups with populations 15(1) and 15(2) (Nanoose H i l l ) , 7(2) (Malahat Drive) and 5 ( M i l l H i l l Park) d i f f e r e n t i a t e d from the other eleven p o p u l a t i o n s . This 74 dichotomy was generated by the r e l a t i v e frequency of a l l e l e EST-2 and there was no d i s c e r n i b l e p a t t e r n to i t s d i s t r i b u t i o n (Figure 35). In populations 15(1), 15(2), 7(2) and 5, i t was detected at frequencies >0.75 while i n the other eleven populations i t was never found with a frequency>0.50. Within the small sub-group, populations 15(1) and 15(2) were much more s i m i l a r to populations 7(2) and 5 r e s p e c t i v e l y , than they were to each other. Populations 2(1) and 2(2) (Anacortes, WA) occur on a serpentine outcrop (Kruckeberg, 1969). Although these populations were q u i t e s i m i l a r (D = 0.0070), p o p u l a t i o n 2(1) was more s i m i l a r to po p u l a t i o n 14(2) (Jack's P o i n t : D--= 0.0044) than i t was to po p u l a t i o n 2(2). Population 10 (Prospect Lake) and 11 (Viaduct Ave) were col l e c t e d . a p p r o x i m a t e l y one km apart ~ " 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 other (D = 0.0078) than they were t o any other p o p u l a t i o n s . However, p o p u l a t i o n 17 ( N i l e Creek) was most s i m i l a r to p o p u l a t i o n 2(1) (Anacortes, WA) although these populations are separated by 1 the greatest, d i s t a n c e . The general lack of correspondence between geographic distance and genetic distance suggests that gene flow 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 of a l l e l e s i n Pleotritis. Regardless of the geographic distances i n v o l v e d , P. oongesta populations 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 to each other 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 . This i n d i c a t e s e i t h e r that gene flow between populations i s more.prevalent i n the outcrosser and/or that as a r e s u l t of recombination w i t h i n p o p u l a t i o n s , the l o s s of v a r i a b i l i t y due to genetic d r i f t i s l e s s of a f a c t o r than i t i s i n the s e l f e r . Since seeds of both species should be e q u a l l y d i s p e r s i b l e and there i s no evidence to suggest that p o l l e n vectors 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 forces are probably more important'than gene flow i n determining a l l e l e 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 genetic d i f f e r e n t i a t i o n observed i n these taxa. However, Lewontin (1974) cautions that even small 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 to prevent d i f f e r e n t i a t i o n between populations of moderate s i z e . 124. 5.4.3 Taxonomic. and evolutionary relationships Although Morey; (1959, 1962) d i d not give P. brachystemon s p e c i f i c rank, r e c o g n i z i n g i t as P. congesta ssp. brachystemon (F...§..M.) Morey, the isozyme data support' the r e c o g n i t i o n of two d i s t i n c t s p e c i es. Standard genetic distance 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 populations (Table 30) so that the genetic divergence between these two taxa could be compared to other species p a i r s of known taxonomic rank. These data were used a l s o too construct' a dendrograph (McCammon, 1968) which i s presented i n Figure 46. G o t t l i e b (1977a) surveyed isozyme data on twenty-eight p l a n t species i n order to evaluate the a p p l i c a t i o n s of e l e c t r o p h o r e t i c data i n p l a n t systematics. Reviewing a v a i l a b l e data f o r t h i r t e e n p a i r s of 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 genetic i d e n t i t y f o r congeneric s p e c i e s , I = 0.67 + 0.07 (equivalent to 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 i n t r a -s p e c i f i c populations (I =0.95 + 0.02). Based on a sample of twelve l o c i , the genetic i d e n t i t y of P. brachystemon and P. congesta was I = 0.7311 (D = 0.3132). This l e v e l of 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 with G o t t l i e b ' s value f o r congeneric species. However, 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 of the two species i s not so c l e a r . G o t t l i e b had data on three species p a i r s which were known to 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 the d e r i v a t i v e being of r e l a t i v e l y recent o r i g i n : Stephanomeria exiqua subsp. coronaria and S. malheurensis (I = 0.94); Clarkia biloba and C. lingulata 1 2 5 . G E N E T I C I D E N T I T Y (I) 1 . 0 0 0 0 . 9 7 0 0 . 9 3 9 0 . 9 0 9 0 . 8 7 8 I 1 1 1 1 1 c o E <u > u 03 l_ .O Q_-10 18 13 7(3) 15(2) 15(1) 7(1) Figure 46. A dendrograph d e p i c t i n g the genetic r e l a t i o n s h i p s among the sampled Vlectvitis populations: c o r r e l a t i o n s are based on Nei's Index of Genetic I d e n t i t y c a l c u l a t e d from the a l l e l e frequencies at twelve l o c i . 126. (I = 0.88), and Guara longiflora and G. demareei (I = 0.99). This high average genetic i d e n t i t y was i n t e r p r e t e d as evidence 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 genetic v e r s i o n s of" the p r o g e n i t o r . In other words, reproductive i s o l a t i o n had evolved without a major genetic r e o r g a n i z a t i o n . P. braohystemon has been considered to be a d e r i v a t i v e of P. oongesta. Yet, t h e i r genetic 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 the three species p a i r s known to 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 the gene pools of each species were compared ( s e c t i o n 4.4),, 42% (14/33) of the a l l e l e s detected i n P. oongesta were not scored i n P. braohystemon. Only 17% (4/23) of the s e l f e r ' s a l l e l e s were not a l s o found i n the out c r o s s e r . Nineteen a l l e l e s were present i n both species but seven of these were detected w i t h s i g n i f i c a n t l y d i f f e r e n t frequencies (Table 17). Based on t h e i r l e v e l o f allozyme d i f f e r e n t i a t i o n , i t i s h i g h l y u n l i k e l y that P.- braohystemon i s a recent d e r i v a t i v e of P. oongesta. The i n a b i l i t y to h y b r i d i z e P. oongesta and P. braohystemon (Carey and Ganders, personal communication) a l s o i n d i c a t e s considerable divergence. However, the m a j o r i t y of the s e l f e r ' s allozymes (83%) was a sub-set of the v a r i a t i o n detected i n the outcrosser. The average genetic d i s t a n c e (D=0.3132) between the two species was smaller than the i n t r a - s p e c i f i c g enetic distance (D=0.3775) between?, braohystemon populations-1 (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 of the outcrosser but that enough time has elapsed since divergence f o r a considerable 127. amount of d i f f e r e n t i a t i o n to have occurred. These observations are based, however, on the study of a r e g i o n where both species have invaded s i n c e the r e t r e a t of P l e i s t o c e n e g l a c i e r s . I f P. brachystemon evolved from P. congesta , i t probably d i d so south of the g l a c i a l boundary. The genotypes that moved north may not represent a random sample.of the genetic v a r i a t i o n of the two species. Consequently, i t i s p o s s i b l e that the two species are g e n e t i c a l l y more s i m i l a r f u r t h e r south: however, t h i s may not n e c e s s a r i l y be the s i t u a t i o n . In c o n c l u s i o n , the isozyme data confirmed the r e c o g n i t i o n of P. brachystemon as a d i s t i n c t s p e c i e s . However, i t i s impossible to 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 of these two t a x a without sampling populations i n C a l i f o r n i a and a l s o studying other species i n the genus. 5.4.4 Maintenance of observed isozyme polymorphisms A controversy has developed w i t h i n p o p u l a t i o n genetics because of e f f o r t s to e x p l a i n the maintenance of observed enzyme polymorphisms. The n e u t r a l i s t s ( n e o - c l a s s i c i s t s ) b e l i e v e that most of 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 observed, i s s e l e c t i v e l y n e u t r a l and maintained by a balance between mutation r a t e and genetic 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-Darwinians) b e l i e v e that observed p r o t e i n polymorphisms are s e l e c t i v e l y maintained. A thorough d i s c u s s i o n of these issues can be found i n Ayala (1974), Lewontin (1974), Johnson (1973, 1975, 1976) and Powell (1975). 128. Genetic distances were compared between P.'brachystemon and P. congesta at sympatric and non-sympatric s i t e s to determine i f sympatric populations were g e n e t i c a l l y more s i m i l a r than other i n t e r -s p e c i f i c comparisons. The purpose of t h i s a n a l y s i s was to 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 thiscapproach was that i f both taxa share the same a l l e l e s and the observed allozyme polymorphisms are 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 at the sympatric s i t e s should favor the same a l l e l e s i n both species. This would be r e f l e c t e d by t h e i r greater genetic s i m i l a r i t y . Genetic distances between sympatric populations are l i s t e d i n i t a l i c s i n Table 30. The mean standard genetic distance f o r sympatric comparisons was D = 0.3393 while i t was D = 0.3120 f o r non-sympatric comparisons. Therefore, these data do not support the assumption that the observed isozyme polymorphisms are s e l e c t i v e l y maintained. I t should be noted, however, that 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 that t h i s comparison i s i n a p p r o p r i a t e . The existence of m u l t i - l o c u s a l l e l e complexes have been demonstrated i n slender w i l d oats (e.g. Clegg 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; Kahler et al., 1980) and b a r l e y (Clegg et al., 1972). These authors present evidence and argue that s e l e c t i o n has s t r u c t u r e d the genetic resources o f these species 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 the coordinated i n t e r a c t i o n of l a r g e numbers of l o c i , then a l l e l e s which are s e l e c t i v e at a few marker l o c i i n one species of Plectritis may not n e c e s s a r i l y be e q u a l l y advantageous i n the other. Table 30. I n t e r - s p e c i f i c comparisons of standard genetic distance (D) i n Pleotritis P. braohystemon populations 1 2(1) 0. ,3242 2(2) 0. 3393 3 0. 2858 5 0.4524 4 0. 2735 9 0. 2896 10 0. 3386 11 0. 3140 7(2) 0. 4412 8 0. 2406 14(1) 0. 3200 14(2) 0. 2903 15(1) 0. 3677 15(2) 0. 3990 17 0. 2695 13 9 0.3060 0.1960 0.2960 0.1868 0.3249 0.2552 0.3983 0.2758 0.2595 0.1824 0.2943 0.2121 0.3452 0.2588 0.3330 0.2409 0.4022 0.3003 0.2846 0.2483 0.2975 0.2046 0.2763 0.1825 0.4379 0.3427 0.3232 0.2452 0.3407 0.2699 10 7(1) 0. 2991 0. 2882 0. 2817 0. 2791 0. 2662 0. 3585 0. 3843 0. 3836 0. 2098 0. 2827 0. 2335 0. 3017 0. 2921 0. 3694 0. 2944 0. 3473 0. 3853 0.3840 0. 1873 0. 3536 0. 2724 0. 2997 0. 2608 0. 2748 0. 3915 0. 4562 0. 2678 0. 3540 0. 2586 0. 3625 7(3) 15(1) 0 .2417 0 .2693 0 .2378 0 .2587 012524 0 .3161 0 .3461 0 .3590 0 .2408 0 .2417 0 .2664 0 .2730 0 .2998 0 .3277 0 .2801 0 .3118 0 . 3192 0 .3636 0 .2816 0 .2912 0 .2388 0 .2703 0 .2251 0 .2478 0 .3676 0.4197 0 .3560 0 .3041 0 .2878 0 .3281 15(2) 18 0.2854 0.2868 0.2881 0.2836 0.3700 0.3669 0.4121 0.4057 0.3111 0.3029 0.3402 0.3027 0.3862 0.3834 0.3594 0.3584 0.4020 0.4023 0.3718 0.3663 0.3089 0.3089 0.2892 0.2845 0.4588 0.4618 0.4087 0.3998 0.3740 0.3385 Comparisons between sympatric populations are i n i t a l i c s D (P.b. :Pv.o.) = 0.3132 D (sympatric) = 0.3393 I (P.b.:P.o.) = 0.7311 I (sympatric) = 0.7123 130. A second approach to t h i s question was to compare the observed variance 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) to a t h e o r e t i c a l variance which assumes that n e u t r a l mutations and genetic random d r i f t are balanced (Nei and.Roychoudhury, 1974). The t h e o r e t i c a l v ariance i s given by Var (h) = — : — (l+e) 2(2+6)(3+ 9 ) where 9= 4Nu7 N i s the e f f e c t i v e population.,size and y i s the mutation r a t e per locus per:generation. The value of 6 may be estimated by H/(l-H); in. which H i s the estimate of average h e t e r o z y g o s i t y , as the expectation of H i s 9/(1+9) . The estimates of 9 , the observed variance of h e t e r o z y g o s i t y and the t h e o r e t i c a l v ariance of het e r o z y g o s i t y : a r e presented f o r B. braohystemon and P. oongesta i n Tables 31 and 32 r e s p e c t i v e l y . The expected and observed values are 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 reasonable agreement f o r P. oongesta. U n f o r t u n a t e l y , i t i s not p o s s i b l e to assess the s i g n i f i c a n c e of departures from the t h e o r e t i c a l value. In a d d i t i o n , i t cannot be determined whether the d i f f e r e n c e i n the l e v e l of agreement between variances i n P. braohystemon and P. oongesta has any i m p l i c a t i o n i n regard to the r e l a t i v e r o l e s of s e l e c t i o n and d r i f t i n maintaining observed polymorphisms w i t h i n the two species. However, i n general i t would appear that the l e v e l s of 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, are i n agreement with the expectations of a s t o c h a s t i c model. This does not e l i m i n a t e the p o s s i b i l i t y of s e l e c t i o n because c e r t a i n types of s e l e c t i o n and 131. Table 31. A comparison between the observed and the theoretical inter-locus variances of heterozygosity (h) i n populations of P. brachystemon 1 2 3 Population 8 Observed variance Theoretical 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 8 = 4Np, where N = effective population size and u = mutation rate per locus per generation, 8 estimated by H/(l-H) since the expectation of H = (8/(1+8) r 2 Observed inter-locus variance of heterozygosity: V(h)= S (h, -H) / ( r - l ) th k = l where h^= estimate of heterozygosity at the k locus and r = the number of l o c i scored Theoretical inter-locus variance of heterozygosity: Var(H)= 28  (l+9) 2(2+6) (3+8) 132. Table 32. A comparison between the observed and the theoretical inter-locus variance of heterozygosity (h) in populations of P. oongesta Population 6 Observed variance Theoretical variance 2(1) 0. 234 0. 073 0. ,043 2(2) 0. 268 0. 080 0. .045 3 0. 325 0. ,068 0. ,048 5 0. 274 0-066 0. .045 4 ' 0. 342 0. ,078 0. .049 9 0. 347 0. ,073 0, .049 10 ' 0. 371 0.086 0. .049 11 0. 352 0. .081 0, .049 7(2) 0. 350 0. .061 0, .049 8 0. ,272 0, .065 0, .045 14(1) 0. ,232 0, .065 0, .042 14(2) 0. ,221 0, .069 0. .042 15(1) 0. ,306 0, .061 0 .047 15(2) 0. .230 0, .049 0 .042 17 0. .295 0 .070 0 .047 133. v a r y i n g mutation r a t e s may produce the same patterns ( L i , 1978). In a d d i t i o n , there are a number of 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; Mi t t e n et al., 1977; Hamrick and Holden, 1979; Kahler et al. 3 1980) and the sampling s t r a t e g i e s used i n the present study would not r e s o l v e v a r i a t i o n patterns at t h i s l e v e l . Considering the q u a n t i t y of var-i a t i o n maintained w i t h i n l o c a l populations and the v u l n e r a b i l i t y of 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 l e v e l of m i c r o - s i t e d i f f e r e n c e s . 5.5 Summary: the genetic consequences of contrasting breeding systems Outcrossing r a t e s were c a l c u l a t e d f o r nine populations of P. brachystemon and f i f t e e n populations of P. congesta. The d i f f e r e n c e between the mean ou t c r o s s i n g rates i n the two species was h i g h l y s i g n i f i c a n t (P^O.01). These two species 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 large p r o p o r t i o n of 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 ( 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 requirements. I t i s not uncommon, p a r t i c u l a r l y on southern Vancouver I s l a n d , to f i n d the two species growing s y m p a t r i c a l l y . A number o f genetic parameters were measured wit h i n each taxon to assess the e f f e c t that breeding system has on po 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 are summarized i n Table 33. As measured by a l l parameters, the s e l f e r maintained s i g n i f i c a n t l y l e s s v a r i a t i o n w i t h i n l o c a l populations than the outcrosser although t h e i r detected l e v e l s of t o t a l v a r i a t i o n 134. Table 33. Summary of genetic differences between P. brachystemon and P. congesta Parameter P. brachystemon P. congesta t Outcrossing frequency 0.02410.008 0.70210.048 13.84' Percentage of l o c i polymorphic 20.7613.49 53.3311.78 8.31' per population Average number of alleles/locus 1.2510.04 1.98+0.05 11.41' per population Average number of alleles/polymorphic 2.2210.10 2.8410.08 4.84' locus per population Expected % of heterozygous l o c i / 5.8911.33 22.64+0.79 10.83' individual Observed % of heterozygous l o c i / 0.4510.13 17.7410.81 21.09' individual Total gene diver s i t y (Rj.) 0.166+0.021 0.26610.083 0.99 Gene diver s i t y within populations 0.06010.021 0.266+0.071 2.24' (H s) Proportion of inter-population gene 0.63910.053 0.15010.039 7.41' di f f e r e n t i a t i o n (G<.,j.) Mean standard genetic distance 0.137+0.083 0.05210.034 0.94 between population (D) Mean genetic'identity between 0.872 0.949 populations (I) * (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 the i n f l u e n c e of 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 only sympatric populations of the two s p e c i e s , a l l genetic 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 populations of the s e l f e r was r e f l e c t e d i n the F - s t a t i s t i c s and i n the a n a l y s i s of gene d i v e r s i t y . On the average, P. congesta populations contained 85% of the spec i e s ' v a r i a t i o n w h i le only 36% of the v a r i a t i o n detected i n P. brachystemon was maintained w i t h i n l o c a l p o p u l a t i o n s . In the s e l f e r , the reduced l e v e l of genetic v a r i a t i o n w i t h i n populations was accompanied by increased 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 . This i s g r a p h i c a l l y depicted i n Figure 46 which shows the reduced genetic i d e n t i t y among populations of P. brachystemon. P r e l i m i n a r y data suggest that 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 the s e l f e r , can occur over short d i s t a n c e s . However, whether t h i s represents micro-geographic 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 taxon, a l l e l e frequencies 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 widely 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 adjacent populations. In P. congesta, a l l e l e frequencies 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 populations but not with the amplitude observed i n P. brachystemon. There appears to be no r e l a t i o n s h i p between genetic distance and geographic d i s t a n c e . This i m p l i e s that gene flow between populations does not 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 frequencies. No evidence was found to suggest that the observed isozyme polymorphisms are s e l e c t i v e l y maintained. However, co-ordinated gene complexes and micro-habitat s e l e c t i o n would probably not have 136. been detected. I t i s concluded that 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 i s p r i m a r i l y the r e s u l t of t h e i r c o n t r a s t i n g breeding systems. Autogamy r e s t r i c t s gene flow between populations and recombination 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 populations by lowering the e f f e c t i v e p o p u l a t i o n s i z e . This 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 s i g n i f i c a n t l y a f f e c t a l l e l e frequencies w i t h i n p o p u l a t i o n s . Although small m i g r a t i o n r a t e s are s u f f i c i e n t to prevent d i f f e r e n t i a t i o n between populations (Lewontin, 1974), there i s no evidence that p o l l e n flow i n P. oongesta i s e f f e c t i v e over large d i s t a n c e s . Therefore, the r e l a t i v e amounts of recombination and d r i f t w i t h i n l o c a l populations are probably the most important 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 of genetic v a r i a t i o n i n P. braohystemon and P. oongesta. 137. BIBLIOGRAPHY A l l a r d , R.W., Babbel, G.R., Clegg, M.T., and Kahler, A.L. 1972. Evidence f o r coadaptation 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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The o r g a n i z a t i o n of genetic v a r i a b i l i t y i n c e n t r a l and marginal populations of lodgepole pine Pinus contorta spp. latifolia. Can. J. Genet. Cytol. 21:487-503. Zouros, E. 1976. Hybrid molecules and the s u p e r i o r i t y of the heterozygote. Nature 262:227-229. 145. APPENDIX A. POPULATION LOCATIONS Sample des i g n a t i o n Location Location and comments 2(1) Sumas Mountain Anacortes, WA 2(2) Anacortes, WA l a r g e , open south-facing slope of Sumas Mtn. (east of Abbotsford, B.C), v i s i b l e from Trans-Canada Hwy. open slope o v e r l o o k i n g Puget Sound, Washington Park (Anacortes C i t y Park): 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 -mafic s o i l s d e rived from p e r i o d o t i t e (Kruckeberg, 1969). large open meadow, Washington Park: u l t r a m a f i c s o i l s 13 W i l l i a m Head John Dean Park M i l l H i l l Park 10 Thet i s Lake Park Fra n c i s Park Prospect Lake Rd. 11 Viaduct Avenue large p o p u l a t i o n growing among Garry oak and arbutus, Metchosin Rd. .6 km SW of Pearson College Dr. Metchosin, B.C. p l a n t s were c o l l e c t e d on a small ledge and along a 15 m t r a n s e c t at the base of a c l e a r i n g l o c a t e d below a DND radar i n s t a l l a t i o n , C e n t r a l Saanich, B.C. open south- f a c i n g slope; p l a n t s at t h i s s i t e bloom 2-4 wks. e a r l i e r than other 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. open meadow with s c a t t e r e d Garry oak and arbutus, near V i c t o r i a , B.C. sympatric p o p u l a t i o n , P..o. i s more abundant than P.b.; D o u g l a s - f i r woodland, a much shadier s i t e than most; Saanich, B.C. sympatric p o p u l a t i o n , P.a. i s more widely d i s t r i b u t e d than P.b.; an open and somewhat d i s t u r b e d s i t e , evidence of a burn; Gaultheria shaUon and Cytisus soopavius are abundant; Prospect Lake Rd. .5 km N o f Prospect Lake G o l f Club. Saanich, B.C. small p o p u l a t i o n i n a depression between two rock outcrops; p l a n t s were robust 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 of Prospect Lake Rd. and Viaduct Ave., Saanich, B.C. 146. APPENDIX A. POPULATION LOCATIONS Sample d e s i g n a t i o n Location Location and comments 7(1) Malahat Drive sympatric p o p u l a t i o n , an exposed, SE-f a c i n g rocky k n o l l beneath three Hydro transm i s s i o n towers overlo o k i n g Saanich I n l e t ; p u l l o f f l o c a t e d 4 km N of "ent e r i n g Malahat; Dr." s i g n on Trans-Canada Hwy..7(1)- P.b. c o l l e c t e d along a 15 m t r a n s e c t running down the. slope beginning approx. 10 m N of tr a n s m i s s i o n tower #1. 7(2) Malahat Drive P.a. - located beneath t r a n s m i s s i o n tower #2 7(3) Malahat Drive P.b. - NE slope of k n o l l , top of k n o l l with a few P.O. Crofton E x i t 14(1) Jack's Point SE-facing ledge approx. 5 m above Hwy. s i t e was q u i t damp at time of c o l l e c t i o n and herb.cover was l u s h ; p l a n t s were c o l l e c t e d along a 30 m t r a n s e c t running NW from the ledge to the back of the c l e a r i n g ; Trans-Canada Hw. 1.7 km south of Crofton E x i t ( S a l t s p r i n g I s l a n d f e r r y ) . open s i t e b i s e c t e d by a gravel road, P.c. very dense; s i t e has been destroyed by c o n s t r u c t i o n of Duke Point I n d u s t r i a l Complex, Nanaimo, B.C. 14(2) Jack's P o i n t open s i t e along g r a v e l road .3 km from A ( l ) ; s i t e a l s o destroyed by c o n s t r u c t i o n . 15(1), 15(2) Nanoose H i l l sympatric s i t e with P.b. being the most abundant; large S-facing slope with Garry oak and arbutus, both 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 Trans-Canada Hw. at Nanoose Bay, north of Nanaimo. 16 L i t t l e Qualicum F a l l s p l a n t s were l o c a t e d on a small open ledge o v e r l o o k i n g the 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 des i g n a t i o n Location Location 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 grassy area of 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 road o f f the Trans-Canada Hwy. along the north bank of N i l e Creek, j u s t south of Bowser, B.C. 18 Elk 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 grassy ledge below the dam; Elk F a l l s Prov. Park, Campbell R i v e r , B.C.. i APPENDIX B. ENZYME SYSTEMS WHICH WERE NOT ADEQUATELY RESOLVED WITH AVAILABLE TECHNIQUES ENZYME RUNNING BUFFER STAIN REFERENCE COMMENTS 1. Acid phosphatease APH E.C. 3.1.3.2 2. Alcohol dehydrogenase AMI E.C. 1.1.1.1 3. Alkaline phosphatase ALPII E.C. 3.1.3.1 I-VI Roose 8 Gottlieb (1976) I,II,IV,V Scandalios (1969) Gottlieb (1973b) 1,11 Scandalios (1969) Adequate staining, but resolution was poor. No A c t i v i t y No A c t i v i t y No A c t i v i t y 4. Aspartate aminotransferase I-VII ATT (=GOT) E.C. 2.6.1.1 Delorenzo 8 Ruddle (1970) S. Catalase CAT E.C. 1.11.1.6 1,11,IV,VI Shaw 6 Prasad (1970) S l c i l i a n o 8 Shaw (1976) 6. Pructose-1, 6-diphosphatase I.II.IV-IX Allendorf, et a l . (1977) FDP E.C. 3.1.3.11 7. Glutamate dehydrogenase I,II,IV,V Roose 8 Gottlieb (1976) GDII E.C. 1.4.1.3 Allendorf, et a l . (1977) With buffer VI, and to a lesser degreee I, resolution was sometimes very good but staining was unreliable. AAT appears to be a 2 or 3 , locus system with the slowest locus showing dimeric i n -heritance. L i t t l e or No A c t i v i t y No A c t i v i t y Very li g h t staining with buffers, I I, VI 8 VII If 10 nig. NADP added to gel, however staining i s unreliable. Staining unreliable, sometimes resolved a single monomorphic band with buffer I. There is some question as to whether this is GDII as a single band was sometimes resolved on other gels without the addition of substrate. APPENDIX B. (Continued) ENZYME RUNNING BUFFER STAIN REFERENCE COMMENTS 8. Glucose-6-phosphate • 1,11 IV-IX dehydrogenase G6P E.C. 1.1.1.49 9. Peroxidase PER E.C. 1.11.1.7 10. Sorbitol dehydrogenase SDH E.C. 1.1.1.14 11. Succinate dehydrogenase SUCDH E.C. 1.3.99.1 12. Xanthine \ dehydrogenase XDH E.C. 1.2.1.37 I,II,IV,V,VI I,II V-IX Roose 6 Gottlieb (1976) Allendorf, et a l . (1977) Brown 6 Al l a r d (1969) Gottlieb (1973a) S i c i l i a n o 5 Shaw (1976) Allendorf, et a l . (1977) 1,11 V-IX Allendorf, et a l . (1977) I,II V-IX Allendorf, et a l . (1977) Staining very d i f f u s e , was unable to resolve. Had success with Gottlieb's stain and running buffers I 6 VI, however, staining was inconsistent so system was not used. PER appears to be a multilocus system with both anodal cathodal variants Single monomorphic band sometimes resolved with Systems I 6 II. However this band stains without the addition of substrate and i s therefore of unknown function. Results i d e n t i c a l to SDH. Results i d e n t i c a l to SDH 6 SUCDH. RUNNING BUFFERS I Discontinuous Li-Borate/Tris-Citrate 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 VI Tris-Versene-Borate pH 8.0 VII Tris-Maleate pH 7.4 VIII Continuous T r i s - C i t r a t e I pH 6.3 IX Continuous T r i s - C i t r a t e II pH 8.0 R E F E R E N C E Scandalios (1969), Selander, et a l . (1971) S i c i l i a n o S Shaw (1976) Shaw 6 Prasad (1970) Selander, et a l . (1971) Gottlieb (1973a) S i c i l i a n o 5 Shaw (1976) Selander, et a l . (1971) Selander, et a l . (1971) Selander, et a l . (1971) 151. APPENDIX C. GENE AND GENOTYPE FREQUENCIES WITHIN POPULATIONS H = expected percentage o f heterozygous l o c i / i n d i v i d u a l (see Tables 22 and 23) H , = observed percentage of heterozygous l o c i / i n d i v i d u a l (see Tables 22 and 23) n = number of i n d i v i d u a l s genotyped (F) = number of f a m i l i e s represented by genotyped i n d i v i d u a l s . 152. APPENDIX C . l . Plectritis brachystemon populations 153. Population #77T1 Siunas Mtn. H=.0S31 H = 0031 obs. Genotype # Individuals Locus A l l e l e Frequency n •(F) ' E S T - I 89 100 114 h h 0 .02 .53 .45 .516 .046 . 130 (130) E S T-2 100 1.00 19 (19) LAP-1 85 91 100 h h 0 .05 .84 .11 .280 .000 19 (19) MDH-1 100 1.00 19 (19) MDH-2 100 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) 89/89 2 100/100 66 114/114 56 100/114 6 85/85 1 91/91 16 100/100 2 154. Population 77-13 William Head H=.0621 H =.0047 obs. Locus Allele Frequency n (F) Genotype JIndividi EST-1 89 100 h h 0 .80 .20 . .320 .029 70 (70) 89/89 100/100 89/100 55 13 2 EST-2 100 1.00 30 (30) LAP-1 91 100 h h o .06 .94 .113 .000 16 (16) 91/91 100/100 1 IS MDH-1 100 1.00 16 (16) MDH-2 100 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 .52 .48 .499 .042 48 (48) 100/100 106/106 100/106 24 22 2 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) 155. Population 77-9 Francis Park H=.0309 H =.0042 obs Locus Allele Frequency n (F) Genotype #Individi EST-1 89 100 h h o .03 .97 .058 .023 . 43 (43) 89/89 100/100 89/100 . 1 41 1 EST-2 100 1.00 14 (11) LAP-1 100 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 0 .01 .99 .020 , .017 56 (43) 100/100 94/100 55 1 PGI-3 33 lOOn h h 0 .19 .81 .308 .000 43 (43) 33/33 lOOn/lOOn 8 35 PGM-1 100 106 h h 0 .97 .03 .058 .000 40 (40) 100/100 106/106 39 1 PGM-2 100 1.00 43 (43) PGM-3 115 182 h h 0 .99 .01 .020 .023 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) 156. Population 77-10 Prospect Lake H=.0063 H , =.0000 obs Genotype #Individuals Locus Allele Frequency n (F) EST-1 100 1.00 8 (8) EST-2 100 1.00 21 (19) LAP-1 100 .95 22 (19) 105 .05 "h ' .095. h 0 .000 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) 100/100 21 ,105/105 1 157. Population 76-7(1) Malahat Dr. H=.0393 ^=.0000 Genotype 'Individuals Locus Allele Frequency n (F) EST-1 114 1.00 20 (20) EST-2 100 \ 1.00 20 (20) LAP-1 95 .05 20 (20) 100 .95 h .095 .000 MDH-1 100 .55 20 (20) 113 .45 h .495 h 0 .000 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) 95/95 1 100/100 19 100/100 11 113/113 9 158. Population 77-7(3) Malahat Dr. H-.140S H ohs = .0000 Locus Allele Frequency n (F) Genotype 'Individuals EST-1 89 100 114 h h 0 .56 . 12 .32 .570 .000 25 (24) 89/89 100/100 114/114 14 3 8 EST-2 100 1.00 25 (24) LAP-1 95 100 h ho .60 .40 .480 .000 25 (24) 95/9S 100/100 IS 10 MDH-1 100 113 h h 0 .50 *.S0 .500 .000 24 (23) 100/100 113/113 12 12 MDH-2 100 1.00 24 (23) PGI-1 100 1.00 25 (24) PGI-2 81 100 h h 0 .12 .88 .211 .000 25 (24) 81/81 100/100 3 22 PGI-3 lOOn 1.00 25 (24) PGM-1 100 106 h h 0 .84 .16 .269 .000 25 (24) 100/100 106/106 21 4 PGM-2 100 lOOn h h 0 .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) 159. Population 76-15(1) Nanoose H i l l H=.1137 H , =.0074 obs Locus All l e l e Frequency n •(F) Genotype #Indiyidi EST-1 89 100 114 h : h o .06 ;22 • .72. .430 .111 9 (9) 100/100 114/114 89/114 2 • " 6 1 EST-2 100 1.00 13. (13) LAP-1 100 1.00 13 (13) MDH-1 100 . 113 h h 0 .89 .11 .196 .000 9 (9) 100/100 113/113 8 1 MDH-2 100 1.00 9 (9) PGI-1 100- 1.00 13 (13) PGI-2 42 100 h .15 .85 .2S5 .000 13 (13) 42/42 100/100 2 11 PGI-3 33 lOOn h ho .62 .38 .471 .000 13 (13) 33/33 lOOn/lOOn 8 5 PGM-1 100 106 h h o .77 .23 .354 .000 13 (13) 100/100 106/106 10 3 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) 160. Population 77-15(2) Nanoose H i l l H=.0S92 H =.005S obs Locus Allele Frequency n (F) Genotype "Individuals EST-1 114 . 1.00 7 (2) EST-2 100 1.00 7 LAP-1 100 105. 112 h h 0 .S3 .39 .08 .561 .000 36 (32) 100/100 10S/10S 112/112 19 14 3 MDH-1 100 1.00 37 (32) MDH-2 100 1.00 37 (32) PGI-1 100 1.00 37 (32) PGI-2 42 100 h h o .16 .84 .269 .029 35 (30) 42/42 100/100 42/100 S 29 1. 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 0 .97 .03 .0S8 . .054 37 (32) 115/115 . 115/182 35 2 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) 161. Population 77-16 Little Qualiciun Falls H=.0136 H . =.0130 OOS Locus Allele Frequency n Genotype "Individuals EST-1 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 ho .05 .95 .095 .091 33 (33) 100/100 94/100 30 3 PGI-3 lOOn 1.00 33 (33) PGM-1 100- 1.00 33 . (33) PGM-2 . 100 1.00 33 (33) PGM-3 lis 182 h h o .95 .05 .095 .091 33 (33) 115/115 115/182 30 3 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) 162. Population 77-18 Elk Falls H=.0699 H . =.0074 obs Locus Allele Frequency n ( F l EST-1 89, 1.00 74 (41) EST-2 100 1.00 1 2 (6) LAP-1 85 .17 9 (6) 100 .61 112 .22 h .551 h 0 .111 MDH-1 113 1.00 9 (6) MDH-2 100 1.00 9 (6) PGI-1 100 1.00 74 (41) PGI-2 100 1.00 74 (41) PGI-3 33 lOOn h h 0 .46 .54 .497 .000 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) Genotype 'Individuals 85/85 1 100/100 5 112/112 2 85/100 1 33/33 34 lOOn/lOOn 40 163. APPENDIX C.2. Pleotritis oongesta populations 1 6 4 . Population 76-2(1) Anacortes -. H=.1897 H. =.1592 Locus A l l e l e Frequency n (F) Genotype SIndividi EST-1 100 1.00 12 (12) EST-2 74 .06 17 (17) 100/100 • . 15 100 .94 74/100 2 h .113 - h ° .118 LAP-1 80 .04 13 (13) 85/85 I 85 .27 91/91 1 91 .23 100/100 1 95 .04 105/105 100 .15 80/105 1 105 .23 85/91 112 .04 85/95 1 h .790 85/100 1 h Q .615 91/100 1 105/112 1 MDH-1 113 .03 17 (17) 125/12S 2 125 .32 139/139 7 139 .65 113/139 1 h .474 125/139 7 h 0 .471 MDH-2 100 1.00 17 (17) PGM-1 100 .97 18 (18) 100/100 17 106 .03 100/106 1 h .058 h 0 .059 PGM-2 81 .18 17 (17) 81/81 1 100 .79 100/100 11 .118 .03 . 81/100 4 h .343 100/118 1 h ° .294 PGM-3 100 .47 17 (17) 100/100 5 135 '• .53 135/135 6 h .498 100/135 6 h o .353 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) 165. Population 76-2(2) Anacortes H=.2113 H t =.1428 obs Locus Allele Frequency n (F) Genotype 'Individi EST-1 100 1.00 21 (20) EST-2 74 .07 21 . (20) 100/100 18 100 .93 74/100 3 h .130 h 0 .143 LAP-1 80 .13 23 (22) 80/80 2 85 .26 85/85 3 91 .09 , 91/91 1 95 .07 95/95 1 100 .28 100/100 3 105 .17 105/105 2 h .796 80/100 2 h Q .478 8S/91 1 85/100 3 85/105 2 91/100 1 95/105 1 100/105 1 MDH-1 113 .05 20 (19) 125/125 9 125 .57 139/139 5 139 .38 113/125 1 h .528 113/139 1 h 0 .300 125/139 4 MDH-2 100 1.00 20 . (19) PGM-1 100 .94 24 (23) 100/100 22 106 .06 106/106 1 h .113 100/106 1 h 0 .042 PGM-2 81 .23 24 (23) 81/81 2 100 .67 100/100 12 118 .10 118/118 1 h .488 81/100 6 h o .375 81/118 1 100/118 2 PGM-3 77 .02 24 (23) 100/100 11 100 .63 135/135 4 135 .35 - 77/100 1 h .480 100/135 8 h 0 .375 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) 166. Population 76-3 John Dean Park H=.2454 H ' =.2046 obs. • Locus Allele Frequency n (F) Genotype # Individuals EST-1 89 .01 53 (53) 100/100 52 100 .99 100/89 1 h .020 h 0 .009 EST-2 74 .27 53 (53) 74/74 5 100 .69 100/100 26 126 .04 74/100 18 h .450 74/126 1 h 0 .415 100/126 3 ,LAP-1 80 " .04 51 (51). 91/91 9 85 .03 95/9S 20 91 .23 100/100 2 95 .57 80/95 4 100 .13 8S/9I .1 h .603 95/95 2 h Q .392 91/9S 4 91/100 1 95/100 8 MDH-1 113 i01 51 (51) 125/125 5 125 .31 139/139 24 139 .68 113/125 1 h .441 125/139 21 ho .431 MDH-2 100 • ; 1.00 51 (SI) PGM-1 94 .01 53 (S3) 100/100 14 100 .49 106/106 7 106 .38 94/106 1 117 .12 100/106 18 h .601 100/117 6 h 0 .604 106/117 7 PGM-2 81 .25 53 (53) 81/81 5 100 .75 100/100 31 h .375 81/100 17 h .321 0 PGM-3 100 .35 53 (53) 100/100 11 135 .65 135/135 27 h .455 100/135 15 h .283 0 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) 1 6 7 . Population 76-5 M i l l H i l l Park H-.2150 H Locus A l l e l e Frequency n (F) Genotype "Individi EST-1 100 1.00 12 (3) EST-2 74 . 100 h h 0 .81 .19 .308 .231 13 (3) 74/74 100/100 74/100 9 1 3 LAP-1 85 91 95 100 h h o .12 .21 .21 .46 .686 .583 12 . (3) 91/91 95/95 100/100 85/100 91/100 95/100 1 2 2 3 3 1 MDH-1 125 139 h h 0 .33 .67 .442 .667 12 (3) 139/139 125/139 4 8 MDH-2 100 1.00 12 (3) PGM-1 100 106 h h 0 .92 .08 .147 .154 13 (3) 100/100 100/106 11 2 PGM-2 81 100 h h 0 .54 .46 .497 .683 12 (3) 81/81 100/100 81/100 3 2 7 PGM-3 100 135 h h 0 .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) 168. Population 76-4 Thetis Lake Park H=.2S49 H • Locus Allele Frequency n (F) Genotype #Individ EST-1 100 1.00 15 (10) EST-2 74 100 h h 0 .37 .63 .466 .467 15 (10) 74/74 100/100 74/100 2 6 7 LAP-1 80 8S 91 95 100 105 h h 0 .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 MDH-1 113 125 139 h h 0 .03 .50 .47 .528 .'.467 15 (10) 12S/12S 139/139 113/125 125/139 6 MDH-2 . 100 1.00 15 (10) PGM-1 100 106 h h 0 .63 .37-.466 .200 15 (10) '100/100 106/106 100/106 8 4 3 PGM-2 81 100 h h 0 .20 .80 .320 . 133 IS (10) 81/81 100/100 81/100 2 11 2 PGM-3 77 100 135 h h 0 .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) Population 77-9 169. Francis Park H=.2S75 H . =.1634 obs Locus Allele Frequency (F) Genotype "Individuals EST-1 89 .02 88 100 .98 h .039 h .011 0 EST-2 74 .32 41 100 .68 h .43S h 0 .341 LAP-1 80 .03 30 85 .07 91 .45 100 .45 h .589 ho .267 MDH-1 113 .22 30 125 .51 139 .27 h .619 h 0 .367 MDH-2 100 1.00 30 PGM-1 100 .63 66 106 .23 117 .14 h .531 .288 PGM-2 81 /37 72 100 .60 118 .03 h .502 h .403 0 PGM-3 100 .75 71 135 .25 h .375 ho .282 IDH-1 100 1.00 44 ME-1 100 1.00 44 6PG 100 1.00 44 SOD . 100 1.00 44 (29) (12) (12) (12) (12) (25) (25) (25) (12) (12) (12) (12) 89/89 100/100 89/100 74/74 100/100 74/100 85/85 91/91 100/100 80/85 80/91 85/91 91/100 113/113 125/125 139/139 113/125 125/139 100/100 106/106 117/117 100 /106 106/117 81/81 100/100 81/100 100/118 100/100 135/135 100/135 1 86 1 6 21 14 1 10 11 1 1 1 5 6 10 3 1 10 34 6 7 15 4 14 29 25 4 43 8 20 170. Population 77-10 Prospect Lake H=.2704 H . =.2005 OOS Locus Allele Frequency n (F) Genotype fflndividi EST-1 100 1.00 42 (13) EST-2 74 : .49 - 50 (20) 74/74 IS 100 .51 100/100 16 h .500 74/100 19 h 0 .380 LAP-1 80 .02 48 (18) 8S/8S' 4 8S .16 91/91 7 91 .18 95/95 8 95 .29 100/100 11 100 .30 80/91 2 105 .05 85/95 4 h .765 85/100 3 h .375 91/105 1 0 95/100 4 95/105 4 MDH-1 125 .36 36 (11) 125/125 4 139 .64 139/139 14 h .461 125/139 18 .500 MDH-2 100 1.00 36 (ID PGM-1 100 .62 55 (20) 100/100 26 106 .35 106/106 13 117 .03 100/106 13 h .492 100/117 3 h o .291 PGM-2 81 .SI 50 (19) 81/81 12 100 .46 100/100 10 118 .03 81/100 25 h .527 81/118 2 h 0 .560 100/118 1 PGM-3 100 .49 50 (19) 100/100 17 135 .51 135/135 18 h .500 100/135 15 h 0 .300 IDH-1 100 1.00 50 ME-1 100 1.00 50 6PG 100 1.00 50 SOD 100 1.00 50 171. Population 77-11 Viaduct Ave. H=.2601 HQbs=.21S8 Locus Allele Frequency n (F) Genotype 'Individuals EST-1 100 EST-2 74 100 h h o LAP-1 80 85 91 95 100 105 h h 1.00 .45 .55 .495 .436 .08 .10 .33 .24 .22 .03 .768 .486 39 39 37 (20) (20) (18) 74/74 100/100 74/100 9 13 17 85/85 1 91/91 6 95/95 7 100/100 4 10S/105 1 80/91 4 80/95 1 80/100 1 85/91 3 85/100 2 91/95 2 91/100 4 95/100 1 MDH-1 125 139 h h 0 .53 .47 .498 .385 39 (20) 125/125 139/139 125/139 13 11 15 MDH-2 100 1.00 39 (20) PGM-1 100 106 h h 0 .72 .28 .403 .410 39 (20) 100/100 106/106 100/106 20 3 16 PGM-2 81 100 118 h h o .35 .60 .05 .515 .513 39 (20) 81/81 100/100 81/100 81/118 100/118 5 14 16 1 3 PGM-3 100 13S h h 0 .33 .67 .442 .359 39 (20) 100/100 135/135 100/135 6 19 14 IDH-1 100 . 1.00 39 (20) ME-1 100 1.00 39 (20) 6PG 100 1.00 39 (20) SOD 100 1.00 39 (20)' 172. Population 76-7(2) Malahat Dr. H=.2S92 H . =.1896 obs Locus Allele Frequency n (F) EST-1 89 .03 57 (19) 100 ' . 89 . 114 .08 h .201 h .193 0 EST-2." 74 .76 65 (19) 100 .16 126 .08 h .390 h .385 . 0 LAP-1 80 .24 65 (19) 85 .13 91 .02 95 .38 100 .24 h .723 h 0 .477 MDH-1 113 .02 67 (19) 125 .34 139 .64 h .474 h 0 .269 MDH-2 87 .05 67 (19) 100 .95 h .095 h .075 0 PGM-1 100 .83 65 (19) 106 .17 h .282 h .185 0 PGM-2 81 .43 68 (19) 100 .57 h .490 h .338 0 PGM-3 100 .35 68 (19) 135 .6S h .455 h .353 o IDH-1 100 1.00 68 (19) ME-1 100 1.00 68 (19) 6PG 100 1.00 68 (19) SOD 100 1.00 68 (19) Genotype "Individuals 100/100 114/114 89/100 100/114 74/74 126/126 74/100 74/126 100/126 80/80 85/85 95/95 100/100 80/95 80/100 85/95 85/100 91/95 95/100 113/113 125/125 139/139 125/139 87/87 100/100 87/100 100/100 106/106 100/106 81/81 100/100 81/100 100/100 13S/13S 100/135 45 1 4 7 38 2 18 5 2 11 4 12 7 6 3 6 3 2 11 1 14 34 18 1 61 5 48 5 12 18 27 23 12 32 24 173. Population 76-8 Crofton Exit H=.2138 H . =.1823 * /the Locus Allele Frequency n CF) Genotype #Individui EST-1 100 1.00 32 (17) EST-2 74 .05 32 (17) . 100/100 24 100 .86 126/126 1 126 .09 74/100 3 h .250 100/126 4 h • 219 0 LAP-1 80 .27 32 (17) 80/80 2 85 .OS 85/85 1 91 .11 91/91 2 95 .16 95/95 3 100 .33 100/100 5 10S .08 10S/10S 2 h .770 80/91 2 h .531 80/95 2 80/100 9 85/95 1 91/9S 1 95/100 1 100/105 1 MDH-1 113 .OS 32 (17) 125/125 2 125 .18 139/139 21 139 .77 113/125 1 h .372 113/139 2 h 0 .281 125/139 6 MDH-2 100 1.00 32 (17) . PGM-1 100 .25 32 (17) 100/100 2 106 .73 106/106 .18 117 .02 100/106 11 h .404 100/117 1 h 0 .375 PGM-2 81 .19 32 (17) 81/81 1 100 .81 100/100 21 h .309 81/100 10 ho .313 PGM-3 100 .36 32 (17) 100/100 4 135 .64 135/135 13 h .461 100/135 15 h 0 .469 IDH-1 100 1.00 32 (17) ME-1 100 1.00 32 (17) 6PG 100 1.00 32 (17) SOD 100 1.00 32 (17) 174. Population 14(1) Jack's Point H=.188S H . =.1521 obs. Locus Allele Frequency n CF) Genotype # Individuals EST-1 100 1.00 30 (30) EST-2 74 .15 29 (29). 74/74 1 100 .78 100/100 18 126 .07 126/126 1 h .364 74/100 7 h 0 .310 100/126 2 LAP-1 80 .12 30 (30) 80/80 2 8S .20 8S/8S .. 2 91 .07 91/91 • 1 95 .26 9S/95 6 100 .25 100/100 3 105 .10 105/105 2 h .801 80/100 3 h .467 85/91 1 0 85/95 1 85/100 4 8S/10S 2 . 91/95 1 9S/100 2 MDH-1 125 .16. 29 (29) 125/125 1 139 .84 139/139 21 h .269 125/139 7 .241 MDH-2 100 1.00 29 (29) PGM-1 100 .80 30 (30) 100/100 21 106 .20 106/106 3 h .320 100/106 6 h 0 .200 PGM-2 81 . .04 28 (28) 100/100 26 100 .96 81/100 2 h .077 h o .036 PGM-3 49 .02 28 (28) 135/135 12 100 .28 49/100 1 13 S .70 100/135 15 h .431 h o .571 IDH-1 100 i.oo 30 (30) ME-1 100 1.00 30 (30) 6PG 100 1.00 30 (30) SOD 100 1.00 30 (30) 175. Population 14 (2) Jack's Point H= .1811 H . =. obs. Locus Allele Frequency n CP) Genotype # Indiv: EST-1 100 1.00 . 27 (27) EST-2 74 .11 33 C33) 74/74 1 100 .89 100/100 27 h .196 74/100 5 h 0 .152 LAP-1 80 .04 29 (29) 80/80 1 8S .22 85/85 3 91 .26 91/91 7 95 .17 95/95 5 100 .29 100/100 S 105 .02 85/91 1 h .769 85/100 6 h o . .276 100/10S 1 MDH-1 125 .35 30 (30) 125/125 5 139 .65 139/139 14 h .455 125/139 11 ho .367 MDH-2 100 1.00 30 (30) PGM-1 100 .85 27 (27) 100/100 23 106 .15 106/106 4 h .255 ho .000 PGM-2 100 1.00 33 (33) PGM-3 100 .47 33 (33) 100/100 8 135 .53 135/135 10 h .498 100/135 IS h 0 • .455 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) 176. Population 76-15(1) Nanoose H i l l H=.2342 H , =.1919 obs Locus A l l e l e Frequency n (F) Genotype 'Individi; EST-1 100 .99 47 (19) 100/100 46 114 .01 - 100/114 1 h .020 h .021 0 EST-2 74 " .81 42 (18) 74/74 27 100 .13 100/100 1 126 .06 74/100 9 h .323 74/126 5 h 0 .333 LAP-1 80 .17 48 (19) 80/80 1 85 .01 91/91 18 91 .51 9S/9S 10 95 .29 80/91 8 100 .02 80/9S 6 h .626 85/91 1 .396 91/95 2 91/100 2 MDH-1 125 .29 39 (16) 125/125 4 139 .71 139/139 20 h .412 125/139 15 .385 MDH-2 87 .01 39 (16) 100/100 38 100 .99 87/100 1 h .020 h 0 .026 PGM-1 100 .69 48 (19) 100/100 25 106 .31 106/106 6 h .428 100/106 16 h o .333 PGM-2 81 .36 47 (19) 81/81 8 100 ' .62 100/100 19 118 .02 81/100 18 h .486 100/118 2 h 0 .426 PGM-3 100 .45 47 (19) 100/100 12 135 .55 135/135 17 h .495 100/135 18 h 0 .383 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) 177. Population 77-•15(2) Nanoose Hi l l H=.1868 H . =. obs Locus Allele Frequency n (F) Genotype 'Individuals EST-1 . 100 1.00 . 7 (3) EST-2 74 100 h " V .83 .17 .282 .333 6 (3) 74/74 74/100 4 2 LAP-1 91 100 h V .05 .95 .095 .100 10 (8) 100/100 91/100 9 1 MDH-1 125 139 h h 0 .43 .57 .490 .571 14 (10) 125/125 139/139 125/139 2 4 8 MDH-2 100 1.00 14 (10) PGM-1 100 106 h ho .58 .42 .487 .231 13 (9) 100/100 106/106 100/106 6 4 3 PGM-2 81 100 h h 0 .29 .71 .412 .286 14 (10) 81/81 100/100 81/100 2 8 4 PGM-3 100 135 h h 0 .39 .61 .476 .357 14 (10) 100/100 135/135 100/135 3 6 5 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. Population 76-17 . Nile Creek H=.2278 ob Locus A l l e l e Frequency n •CF) Genotype #Individu EST-1 100 .99 36 (24) 100/100 35 114 .01 100/114 1 h .020 h 0 .028 EST-2 74 .04 35 (23) 100/100 31 100 ' .94 74/100 3 126 .02 100/126 1 h .114 h • 0 .114 LAP-1. 80 .07 36 (24) 80/80 1 85 .47 85/85 13 91 .39 . 91/91 10 95 .03. 95/95 1 100 .01 105/105 1' 105 .03 80/85 1 h .620 80/91 2 h .278 8S/91 6 0 85/100 1 MDH-1 113 .21 35 (23) 113/113 3 125 .19 125/125 1 139 .60 139/139 13 h .560 113/125 2 h .514 113/139 7 0 125/139 9 MDH-2 100 1.00 35 (23) PGM-1 100 .57 34 (22) 100/100 12 106 .43 106/106 7 h .490 100/106 15 h 0 .441 PGM-2 81 .46 34 (22) 81/81 7 100 .51 100/100 8 118 .03 81/100 17 h .527 100/118 2 h 0 .559 PGM-3 100 .28 34 (22) 100/100 2 135 .72 135/135 17 h .403 100/135 15 h 0 .441 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) 

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