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Mixed mating systems and gynodioecy in Hawaiian Bidens Sun, Mei 1986

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MIXED MATING SYSTEMS AND GYNODIOECY IN HAWAIIAN BIDENS MEI SUN B . S c , Nanjing U n i v e r s i t y , 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES Botany Department We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1986 DOCTOR OF PHILOSOPHY i n © MEI SUN, 1986 «0 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r re f e r e n c e and study. I f u r t h e r agree that p ermission f o r ex t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of BOTANY The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: OCTOBER 15, 1986 i i A b s t r a c t A l l s p e c i e s of Bidens endemic t o the Hawaiian I s l a n d s evolved from a s i n g l e common ancestor through adaptive r a d i a t i o n . M o r p h o l o g i c a l d i f f e r e n t i a t i o n among these s p e c i e s i n c l u d e s d i f f e r e n c e s i n f l o r a l f e a t u r e s which i n f l u e n c e t h e i r mating systems. Twelve taxa of Hawaiian Bidens were s t u d i e d to assess the extent of v a r i a t i o n i n f l o r a l f e a t u r e s . O u t c r o s s i n g r a t e s were estimated f o r 15 p o p u l a t i o n s of e i g h t s p e c i e s using allozyme gene markers i n prog e n i e s . A l l sp e c i e s s t u d i e d had inte r m e d i a t e o u t c r o s s i n g r a t e s ranging from 58.0%-72.8%. O u t c r o s s i n g r a t e was not c o r r e l a t e d with flower s i z e , degree of protandry or seed set i n the absence of p o l l i n a t o r s , but d i d appear to be c o r r e l a t e d with i n f l o r e s c e n c e type. Strong protandry e f f i c i e n t l y prevents s e l f - f e r t i l i z a t i o n w i t h i n f l o w e r s , but numerous simultaneously f l o w e r i n g heads promote geitonogamy, which b r i n g s about a s u b s t a n t i a l l e v e l of s e l f i n g . The i n t e r m e d i a t e r a t e s of s e l f - f e r t i l i z a t i o n i n a l l s p e c i e s s t u d i e d do not support the p r e d i c t i o n of a bimodal d i s t r i b u t i o n of o u t c r o s s i n g r a t e s by Lande and Schemske (1985). The amount and s t r u c t u r e of ge n e t i c v a r i a t i o n was i n v e s t i g a t e d using allozyme data f o r 21 p o p u l a t i o n s of ten s p e c i e s of Hawaiian Bidens. Genetic v a r i a t i o n w i t h i n p o p u l a t i o n s was low. Genetic i d e n t i t i e s among p o p u l a t i o n s was high, probably because of t h e i r common a n c e s t r y . For the three commonly polymorphic l o c i , Pgi-1, Pgi-2, and Skdh-3, there was a s u b s t a n t i a l amount of g e n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s . T h i s suggests that founder e f f e c t s as w e l l as the mating system i i i have i n f l u e n c e d the d i s t r i b u t i o n of g e n e t i c v a r i a t i o n . There was a s i g n i f i c a n t heterozygote d e f i c i e n c y at one or more allozyme l o c i i n most p o p u l a t i o n s i n comparison with the Hardy-Weinberg e x p e c t a t i o n s . The heterozygote d e f i c i e n c y was p r i m a r i l y caused by i n b r e e d i n g , but a " h e t e r o z y g o s i t y paradox" was a l s o d e t e c t e d i n these p o p u l a t i o n s , s u g g e s t i n g the e f f e c t of other e v o l u t i o n a r y f a c t o r s on g e n e t i c s t r u c t u r e of Bidens p o p u l a t i o n s . T h i r t e e n of the 27 taxa of Hawaiian Bidens are gynodioecious. A b o r t i o n of microsporogenesis i n females i s caused by an e a r l y abnormal v a c u o l a t i o n of t a p e t a l c e l l s which leads to p r e m e i o t i c degeneration of microspore mother c e l l s . M i c r osporogenesis was developmentally s i m i l a r i n a l l nine taxa s t u d i e d . Genetic s t u d i e s p r o v i d e d evidence that male s t e r i l i t y i s c o n t r o l l e d by two r e c e s s i v e nuclear genes, and these genes are a l l e l i c i n a l l gynodioecious s p e c i e s . Cytoplasmic f a c t o r s are a p p a r e n t l y a l s o i n v o l v e d . Both developmental and g e n e t i c data suggest that male s t e r i l i t y i n Hawaiian Bidens i s homologous. The e v o l u t i o n of gynodioecy most l i k e l y o c c u r r e d autochthonously, and a l l gynodioecious taxa are probably the products of a d a p t i v e r a d i a t i o n of one a n c e s t r a l gynodioecious s p e c i e s . In gynodioecious p o p u l a t i o n s , females must have some s e l e c t i v e advantage to c o u n t e r a c t the disadvantage caused by reducing the f i t n e s s of male gametes to z e r o . T h e o r e t i c a l s t u d i e s of the maintenance of gynodioecy hypothesize t h a t females are maintained because of t h e i r o u t c r o s s i n g advantage or i v r e p r o d u c t i v e s u p e r i o r i t y as seed p a r e n t s . There was a s i g n i f i c a n t c o r r e l a t i o n between s e l f i n g r a t e s of hermaphrodites and f r e q u e n c i e s of females i n e i g h t gynodioecious p o p u l a t i o n s of Hawaian Bidens. T h i s r e s u l t p r o v i d e s the f i r s t e m p i r i c a l evidence that supports the o u t c r o s s i n g h y p o t h e s i s . H e t e r o z y g o s i t y at allozyme l o c i was higher i n the progenies of females than i n the progenies of hermaphrodites. I conclude that females are maintained i n gynodioecious p o p u l a t i o n s because of t h e i r o b l i g a t e o u t c r o s s i n g , and the consequence of gynodioecy i s that i t r a i s e s the l e v e l s of o u t c r o s s i n g and h e t e r o z y g o s i t y i n s e l f - c o m p a t i b l e p o p u l a t i o n s . However, estimated s e l f i n g r a t e s of hermaphrodites cannot by themselves account f o r the observed high f r e q u e n c i e s of females i n these p o p u l a t i o n s . Females must have some a d d i t i o n a l s e l e c t i v e advantage. Measurement of s e v e r a l f i t n e s s components of the two sexes f a i l e d to r e v e a l s i g n i f i c a n t d i f f e r e n c e s i n most cases. Information on the r e l a t i v e f i t n e s s of the two sexes i n nature i s needed to understand the maintenance of females i n gynodioecious s p e c i e s of Hawaiian Bidens. V Table of Contents A b s t r a c t . . . i i L i s t of Tables v i i i L i s t of F i g u r e s . . . x i Acknowledgement . . x i i i Chapter I GENERAL INTRODUCTION 1 Chapter II ELECTROPHORESIS 8 2.1 I n t r o d u c t i o n .....8 2.2 M a t e r i a l s .. 9 2.3 E l e c t r o p h o r e t i c Procedures 17 2.3.1 Gel P r e p a r a t i o n 17 2.3.2 E x t r a c t i o n . 18 2.3.3 E l e c t r o p h o r e s i s ....18 2.3.4 Enzyme A c t i v i t y S t a i n i n g 19 2.3.5 Enzyme Systems Resolved 20 2.4 Genetic I n t e r p r e t a t i o n s Of Zymograms 20 2.4.1 Phosphoglucose Isomerase (Pgi) 22 2.4.2 Shikimate Dehydrogenase (Skdh) 24 2.4.3 Leucine Aminopeptidase (Lap) 27 2.4.4 Malate Dehydrogenase (Mdh) 27 2.4.5 Phosphoglucomutase (Pgm) 33 2.4.6 6-Phosphoglucanate Dehydrogenase (6-Pgdh) 34 2.4.7 M a l i c Enzyme (Me), Hexose Aminidase (Ha) And 0-G l u c o s i d a s e (Glu) 34 2.4.8 Diaphorase And Aconitase 34 2.5 Summary 34 Chapter III MIXED MATING SYSTEMS IN HAWAIIAN BIDENS 38 3.1 I n t r o d u c t i o n .38 3.2 M a t e r i a l s And Methods 41 3.2.1 F i e l d C o l l e c t i o n 41 3.2.2 Measuring F l o r a l F eatures And Reproductive Parameters 42 3.2.3 E s t i m a t i o n Of O u t c r o s s i n g Rates 43 3.3 R e s u l t s 44 3.3.1 F l o r a l Features And Reproductive B i o l o g y 44 3.3.2 O u t c r o s s i n g Rates 56 3.3.3 C o r r e l a t i o n Of F l o r a l Features And O u t c r o s s i n g Rates 60 3.4 D i s c u s s i o n ......60 3.4.1 F l o r a l Features 60 3.4.2 O u t c r o s s i n g Rates 63 3.4.3 R e l a t i o n s h i p Between F l o r a l Mechanisms And Ou t c r o s s i n g Rates 72 3.4.4 E v o l u t i o n a r y I n t e r p r e t a t i o n Of Mixed Mating Systems In Hawaiian Bidens 75 3.5 Summary 77 v i Chapter IV GENETIC VARIATION AND STRUCTURE, AND THE EFFECT OF MIXED MATING SYSTEMS .79 4.1 I n t r o d u c t i o n ...79 4.2 M a t e r i a l s And Methods 83 4.3 R e s u l t s 83 4.3.1 Genetic D i v e r s i t y 83 4.3.2 Po p u l a t i o n Genetic S t r u c t u r e 88 4.3.3 Genetic D i v e r s i t y , S t r u c t u r e And The Mating System 92 4.3.4 Genetic D i s t a n c e s 95 4.4 D i s c u s s i o n 101 4.4.1 Genetic V a r i a b i l i t y And The Mating System .......101 4.4.2 D i s t r i b u t i o n Of Genetic V a r i a t i o n And The Mating System .. . 1 04 4.4.3 Po p u l a t i o n Genetic S t r u c t u r e And The Mating System 106 4.4.4 Genetic S i m i l a r i t y Among P o p u l a t i o n s 108 4. 5 Summary 110 Chapter V MICROSPOROGENESIS IN MALE-STERILE AND HERMAPHRODITIC PLANTS OF NINE GYNODIOECIOUS TAXA ..112 5.1 I n t r o d u c t i o n 112 5.2 M a t e r i a l s And Methods 114 5.3 R e s u l t s 116 5.3.1 Microsporogenesis In Hermaphrodites 116 5.3.2 Breakdown Of Microsporogenesis In Male S t e r i l e s 121 5.4 D i s c u s s i o n 125 5. 5 Summary 132 Chapter VI GENETICS OF GYNODIOECY 134 6.1 I n t r o d u c t i o n 134 6.2 M a t e r i a l s And Methods 135 6.3 R e s u l t s 1 36 6.4 D i s c u s s i o n .151 6.4.1 Cytoplasmic Male S t e r i l i t y 151 6.4.2 Genie Male S t e r i l i t y 151 6.4.3 Genic-cytoplasmic Model ..154 6. 5 Summary 159 Chapter VII THE MAINTENANCE OF GYNODIOECY 161 7.1 I n t r o d u c t i o n 161 7.2 M a t e r i a l s And Methods . 166 , 7.2.1 E l e c t r o p h o r e s i s 166 7.2.2 Seed Weight And Germination 166 7.2.3 V e g e t a t i v e And Reproductive Measures 167 . 7.2.4 P o l l e n S t a i n a b i l i t y ..168 7.3 R e s u l t s 1 68 7.3.1 S e l f i n g Rates Of Hermaphrodites And H e t e r o z y g o s i t y 168 7.3.2 Measurements Of F i t n e s s Components 172 7.3.3 Test For P l e i o t r o p i c E f f e c t s Of M a l e - s t e r i l e Genes .176 7.4 D i s c u s s i o n .182 7.4.1 S e l f i n g Rates Of Hermaphrodites And H e t e r o z y g o s i t y 182 7.4.2 S e v e r a l F i t n e s s Components Of Females And Hermaphrodites 187 7.4.3 P l e i o t r o p i c E f f e c t Of Male S t e r i l i t y 190 7.4.4 E v o l u t i o n a r y S t a b i l i t y And Maintenance Of Gynodioecy 192 7 . 5 Summary 1 94 Chapter VIII GENERAL CONCLUSIONS ...197 8.1 Mixed Mating Systems And F l o r a l Mechanisms .......197 8.2 Genetic V a r i a t i o n , S t r u c t u r e , And The E f f e c t Of Mating Systems 1 98 8.3 Gynodioecy 200 8.4 Summary 203 BIBLIOGRAPHY 205 APPENDIX A - NATIVE HAWAIIAN TAXA OF BIDENS. TAXA KNOWN TO BE GYNODIOECIOUS ARE PRECEDED BY AN ASTERISK ..223 APPENDIX B - ALLOZYME LOCI AND ALLELE FREQUENCIES IN 21 POPULATIONS OF HAWAIIAN BIDENS 225 v i i i L i s t of Tables 2-1. Twenty-one p o p u l a t i o n s of Hawaiian Bidens s t u d i e d e l e c t r o p h o r e t i c a l l y 14 2-2. E l e c t r o p h o r e s i s B u f f e r s used f o r Bidens 17 2- 3. Enzyme systems and the number of l o c i commonly recorded 21 3- 1. Species s t u d i e d f o r o u t c r o s s i n g r a t e s , and t h e i r h a b i t a t s , growth forms and i n f l o r e s c e n c e types .....45 3-2. F l o r a l s i z e s of 10 taxa of Hawaiian Bidens 47 3-3. C o r r e l a t i o n matrix of o u t c r o s s i n g r a t e ( t ) , f l o r a l f e a t u r e s , and autoseed set i n 8 taxa of Hawaiian Bidens 48 3-4. Du r a t i o n of protandry, p o l l e n and stigma phase and developmental r a t e of a n t h e s i s i n 10 taxa of Hawaiian Bidens 49 3-5. Percent seedset i n the absence of p o l l i n a t o r s i n 12 taxa of Hawaiian Bidens 54 3-6. O u t c r o s s i n g r a t e s of hermaphrodites and females, f r e q u e n c i e s of females, and weighted mean o u t c r o s s i n g r a t e s f o r 8 gynodioecious p o p u l a t i o n s of Hawaiian Bidens 57 3-7. O u t c r o s s i n g r a t e s of gynodioecious p o p u l a t i o n s with unknown maternal sex phenotypes 58 3-8. O u t c r o s s i n g r a t e s i n two p o p u l a t i o n s of a non-gynodioecious s p e c i e s , B. mauiensis ..59 3-9. R e l a t i o n s h i p between i n f l o r e s c e n c e types, o u t c r o s s i n g r a t e s (t) of hermaphrodites, and mean female f r e q u e n c i e s i n p o p u l a t i o n s of Hawaiian Bidens ......61 3-10. Mean o u t c r o s s i n g r a t e s (t) estimated using g e n e t i c markers and t h e i r i n t e r p o p u l a t i o n a l ranges i n n a t u r a l p l a n t p o p u l a t i o n s 64 3- 11. Mean o u t c r o s s i n g r a t e s (t) of 8 sp e c i e s of Hawaiian Bidens, weighted means are given f o r gynodioecious s p e c i e s 69 4- 1. A l l e l e f r e q u e n c i e s at three polymorphic l o c i i n 21 p o p u l a t i o n s of Hawaiian Bidens 84 i x 4-2. Genetic v a r i a b i l i t y and o u t c r o s s i n g r a t e s (t) i n 21 p o p u l a t i o n s of Hawaiian Bidens ... 85 4-3. Genetic d i v e r s i t y i n 21 p o p u l a t i o n s of Hawaiian Bidens at three polymorphic l o c i , and at 25 l o c i with monomorphic l o c i i n c l u d e d 87 4-4. Wright's observed f i x a t i o n index at three commonly polymorphic l o c i i n 19 polymorphic p o p u l a t i o n s of Hawaiian Bidens 90 4-5. F - s t a t i s t i c s f o r three polymorphic l o c i i n 19 polymorphic p o p u l a t i o n s of Hawaiian Bidens 91 4-6. Average values of Wright's observed f i x a t i o n index (Fo), expected f i x a t i o n index ( F e ) , A F and s e l f i n g r a t e (s) i n 19 polymorphic p o p u l a t i o n s of Hawaiian Bidens .93 4- 7. Genetic i d e n t i t y and g e n e t i c d i s t a n c e between 21 p o p u l a t i o n s of Hawaiian Bidens 98 5- 1. Gynodioecious taxa and an experimental h y b r i d i n v e s t i g a t e d f o r microsporogenesis 115 6-1. Segregation of females and hermaphrodites i n four gynodioecious p o p u l a t i o n s r a i s e d i n a greenhouse ..137 6-2. Segregation i n the F1 generat i o n i n experimental c r o s s e s between females of gynodioecious s p e c i e s and hermaphrodites of non-gynodioecious s p e c i e s .138 6-3. Segregation i n the F1 progenies i n experimental c r o s s e s between gynodioecious s p e c i e s 141 6-4. Segregation i n the F2 progenies 143 6-5. Segregation i n the progenies of backcrosses 146 6-6. Segregation i n the progenies of t r i p l e c r o s s e s ....147 6- 7. Segregation i n the progenies of quadruple c r o s s e s .149 7- 1. S e l f i n g r a t e s of hermaphrodites and f r e q u e n c i e s of females i n e i g h t gynodioecious p o p u l a t i o n s of Hawaiian Bidens 1 69 7-2. Observed h e t e r o z y g o s i t y at three polymorphic l o c i i n the progenies of hermaphrodites and females i n 11 gynodioecious p o p u l a t i o n s of Hawaiian Bidens 171 7-3. Achene weight and percentage of germination i n the progenies of hermaphrodites and females i n nine X gynodioecious p o p u l a t i o n s 173 7-4. Leaf dry weight of the progenies of hermaphrodites and females i n two experimental p o p u l a t i o n s ..174 7-5. V e g e t a t i v e growth and s u r v i v o r s h i p i n the progenies of experimental c r o s s e s 175 7-6. V e g e t a t i v e c h a r a c t e r s of the progenies of hermaphrodites and females i n three gynodioecious p o p u l a t i o n s r a i s e d i n a greenhouse p r i o r to re p r o d u c t i o n 177 7-7. Reproductive f e a t u r e s of the progenies of females and hermaphrodites i n two gynodioecious p o p u l a t i o n s r a i s e d i n a greenhouse 178 7-8. V e g e t a t i v e and r e p r o d u c t i v e measurements of females and hermaphrodites i n three gynodioecious p o p u l a t i o n s r a i s e d i n a greenhouse 179 7-9. F l o r a l and r e p r o d u c t i v e f e a t u r e s of hermaphrodites and females segregated from an experimental c r o s s : MS FORB (12) X (MS HAWA (51 A) X H MAUI (10B)) 181 7-10. Reproductive f e a t u r e s of hermaphrodites and females i n the F2 progeny of an experimental c r o s s : MS FORB (12) X H MAUI (10) 183 7-11. O u t c r o s s i n g r a t e s a v a i l a b l e i n four gynodioecious p o p u l a t i o n s 185 x i L i s t of F i g u r e s 1-1. M a l e - s t e r i l e d i s c flower (MS) and hermaphroditic d i s c flower (H) i n a gynodioecious s p e c i e s of Hawaiian Bidens 6 2-1. L o c a t i o n s of p o p u l a t i o n s s t u d i e d e l e c t r o p h o r e t i c a l l y on the i s l a n d of Kauai ..10 2-2. L o c a t i o n s of p o p u l a t i o n s s t u d i e d e l e c t r o p h o r e t i c a l l y on the i s l a n d of Oahu 11 2-3. L o c a t i o n s of p o p u l a t i o n s s t u d i e d e l e c t r o p h o r e t i c a l l y on the i s l a n d of Maui 12 2-4. L o c a t i o n s of p o p u l a t i o n s s t u d i e d e l e c t r o p h o r e t i c a l l y on the i s l a n d of Hawaii 13 2-5. A l l e l e s of Pgi-1 and Pgi-2 23 2-6. E l e c t r o p h o r e t i c p a t t e r n s and g e n e t i c a n a l y s i s of Pgi at the c a t h o d a l region 25 2-7. Sample g e l s of Pgi-1 and Pgi-2 allozymes 26 2-8. A l l e l e s and commonly observed banding p a t t e r n s of Skdh-3 28 2-9. Two sample g e l s showing Skdh-3 29 2-10.Loci, a l l e l e s and commonly observed banding p a t t e r n s of Lap allozymes 30 2-11.Two sample g e l s of Lap allozymes 31 2-12.Loci, a l l e l e s and some banding p a t t e r n s of Mdh allozymes 32 2-13.Loci, a l l e l e s and commonly observed banding p a t t e r n s of Pgm allozymes 35 2-14.Loci, a l l e l e s and commonly observed banding p a t t e r n s of 6-Pgdh allozymes 36 4-1. Regression of observed f i x a t i o n index on s e l f i n g r a t e i n 15 p o p u l a t i o n s of Hawaiian Bidens 94 4-2. R e l a t i o n s h i p between o u t c r o s s i n g r a t e and / J F i n p o p u l a t i o n s of Hawaiian Bidens 96 4-3. Dendrogram based on g e n e t i c d i s t a n c e matrix f o r x i i p o p u l a t i o n s of Hawaiian Bidens .100 5-1 - 5-12. Microsporogenesis i n hermaphroditic p l a n t s from gynodioecious p o p u l a t i o n s of Bidens c e r v i c a t a 118 5- 13 - 5-21. Abnormal microsporogenesis i n m a l e - s t e r i l e p l a n t s from a gynodioecious p o p u l a t i o n of Bidens  c e r v i c a t a 122 6- 1. A l l e l i s m f o r male s t e r i l i t y i n nine gynodioecious s p e c i e s of Hawaiian Bidens demonstrated by i n t e r s p e c i f i c c r o s s e s ..150 7- 1. Regression of frequency of females on s e l f i n g r a t e of hermaphrodites i n e i g h t gynodioecious p o p u l a t i o n s of Hawaiian Bidens 170 x i i i Acknowledgement I am very g r a t e f u l to my s u p e r v i s o r , Dr. Fred R. Ganders, fo r i n t e l l e c t u a l and f i n a n c i a l support, and v a l u a b l e c r i t i c i s m and comments. I am a l s o g r a t e f u l to Drs. K. Cole, G. B r a d f i e l d , A.J.F. G r i f f i t h s and J . Myers, who served as my t h e s i s committee members, f o r t h e i r c r i t i c a l comments. I would l i k e to thank Kermit R i t l a n d f o r p r o v i d i n g and h e l p i n g with computer programs. I am indebted to Rob Scagel f o r encouragement and h e l p with many aspects of t h i s study. My a p p r e c i a t i o n i s extended to Bob Kantymir and Elena K l e i n f o r a s s i s t a n c e with t r a n s p o r t a t i o n and greenhouse experiments. I w i l l "always be g r a t e f u l to Dr. R.F. Scagel f o r being s u p p o r t i v e d u r i n g the course of my graduate study. My s i n c e r e thanks a l s o go to many other f a c u l t y members and graduate students of the Department of Botany f o r t h e i r encouragement, i n t e r e s t and f r i e n d s h i p . The s c h o l a r s h i p granted by the M i n i s t r y of Education, People's Republic of China, and a graduate f e l l o w s h i p from the U n i v e r s i t y of B r i t i s h Columbia are g r a t e f u l l y acknowledged. 1 I. GENERAL INTRODUCTION Three types of mating systems e x i s t i n p l a n t s p e c i e s , i . e . , o u t c r o s s i n g , mixed mating (mixture of both o u t c r o s s i n g and s e l f -f e r t i l i z a t i o n ) , and s e l f - f e r t i l i z a t i o n . O u t c r o s s i n g occurs i n sp e c i e s with mechanisms to prevent s e l f - f e r t i l i z a t i o n or promote o u t c r o s s i n g , such as s e l f - i n c o m p a t i b i l i t y , h e t e r o s t y l y , and dio e c y . S e l f - f e r t i l i z a t i o n i s common i n s e l f - c o m p a t i b l e s p e c i e s l a c k i n g s p a t i a l and temporal s e p a r a t i o n between mature stamens and stigma, such as autogamy w i t h i n buds before a n t h e s i s , or clei s t o g a m y . However, many s p e c i e s have f l o r a l mechanisms a l l o w i n g both s e l f - f e r t i l i z a t i o n and o u t c r o s s i n g , r e s u l t i n g i n mixed-mating systems. T h e o r e t i c a l and e m p i r i c a l s t u d i e s of the e v o l u t i o n of p l a n t mating systems and the e v o l u t i o n a r y r o l e s of mating systems have accumulated i n the l i t e r a t u r e of p o p u l a t i o n b i o l o g y f o r more than a ce n t u r y . Many t h e o r e t i c a l s t u d i e s p r e d i c t the r e l a t i v e e v o l u t i o n a r y s t a b i l i t y of mating systems on the b a s i s of c o s t s and b e n e f i t s a s s o c i a t e d with s e l f i n g and o u t c r o s s i n g (e.g., L l o y d , 1979; Schoen and L l o y d , 1984; Lande and Schemske, 1985; Uyenoyama, 1986). In the absence of in b r e e d i n g d e p r e s s i o n , genes that promote s e l f i n g without reducing p o l l e n d i s p e r s a l would have a two f o l d advantage which d e r i v e s from a dou b l i n g of p a r e n t - o f f s p r i n g r e l a t e d n e s s under u n i p a r e n t a l r e p r o d u c t i o n compared to u n r e l a t e d b i p a r e n t a l r e p r o d u c t i o n (Uyenoyama, 1986, and r e f e r e n c e s t h e r e i n ) . H i s t o r i c a l l y s e l f i n g s p e c i e s can maintain s e l f i n g by d e c r e a s i n g g e n e t i c loads i n the p o p u l a t i o n s , whereas a h i s t o r y of o u t c r o s s i n g can favor o u t c r o s s i n g by 2 s e l e c t i o n a g a i n s t homozygotes f o r d e l e t e r i o u s r e c e s s i v e genes (Lande and Schemske, 1985). Two views e x i s t on the e v o l u t i o n and maintenance of mixed mating systems. Mixed mating systems may represent t r a n s i t i o n a l s t a t e s and the d i r e c t i o n of e v o l u t i o n depends on the l e v e l of inb r e e d i n g d e p r e s s i o n i n the p o p u l a t i o n s (Lande and Schemske, 1985; Schemske and Lande, 1985), or mixed mating systems can represent balanced or optimal mating systems and be maintained by s e l e c t i o n ( J a i n , 1976; Waller i n Uyenoyama, 1986) or they can be e v o l u t i o n a r i l y s t a b l e under c e r t a i n b i o l o g i c a l l y tenable c o n d i t i o n s (Maynard Smith, 1978; L l o y d , 1979; H o l s i n g e r , 1986; Uyenoyama, 1986). E m p i r i c a l s t u d i e s of p l a n t mating systems i n about 60 s p e c i e s show a wide range of v a r i a t i o n i n o u t c r o s s i n g r a t e along the continuum from complete or predominant s e l f i n g to complete or predominant o u t c r o s s i n g (see Schemske and Lande, 1985, and r e f e r e n c e s t h e r e i n ) , and a wide v a r i a t i o n i n o u t c r o s s i n g r a t e a l s o occurs among p o p u l a t i o n s w i t h i n s p e c i e s (see Table 10 i n Chapter 3 f o r r e f e r e n c e s ) . Although some of the e m p i r i c a l s t u d i e s do g r e a t l y c o n t r i b u t e to our understanding of the d i s t r i b u t i o n of o u t c r o s s i n g r a t e s i n n a t u r a l p o p u l a t i o n s , the e m p i r i c a l data set does not represent a random sample of p l a n t s p e c i e s . Almost h a l f of the s p e c i e s s t u d i e d are annual weeds or cr o p p l a n t s i n the Gramineae and the Leguminosae or f o r e s t t r e e s i n the Pinaceae. Few i f any s t u d i e s have been conducted to estimate o u t c r o s s i n g r a t e s f o r s p e c i e s o c c u r r i n g on i s o l a t e d oceanic i s l a n d s (but see R i t l a n d and Ganders, 1985). The mating systems of i s l a n d s p e c i e s are a su b j e c t of .3 e v o l u t i o n a r y s i g n i f i c a n c e . P o l l i n a t o r f a i l u r e or p o p u l a t i o n b o t t l e n e c k s , which f r e q u e n t l y occur i n founder p o p u l a t i o n s of sp e c i e s c o l o n i z i n g remote i s l a n d s , can reduce genetic loads by purging the p o p u l a t i o n of r e c e s s i v e d e l e t e r i o u s a l l e l e s , and thus s e l f i n g can evolve i n a h i s t o r i c a l l y o u t c r o s s i n g s p e c i e s and be maintained by s e l e c t i o n f o r l o c a l a d a p t a t i o n (Stebbins, 1957; Antonovics, 1968; J a i n , 1976; L l o y d , 1979, Lande and Schemske, 1985). But many i s l a n d s p e c i es are probably s u b j e c t to s e l e c t i v e pressure f o r o u t c r o s s i n g to generate or maintain c e r t a i n l e v e l s of g e n e t i c v a r i a b i l i t y , as suggested by r e l a t i v e l y high p r o p o r t i o n s of d i c l i n o u s , dichogamous and wind p o l l i n a t e d s p e c i e s on the Hawaiian I s l a n d s . C a r l q u i s t (1974) r e p o r t e d 27.7% d i o e c i o u s , 5% monoecious, and 2.6% gynodioecious s p e c i e s i n the Hawaiian f l o r a , with the t o t a l d i c l i n o u s s p e c i e s of about 44%, dichogamous s p e c i e s of about 30% and wind p o l l i n a t e d s p e c i e s of about 20%. In other words, over 90% of the s p e c i e s i n the Hawaiian f l o r a have high o u t c r o s s i n g p o t e n t i a l s which are f a c i l i t a t e d by f l o r a l mechanisms. Although d i f f e r e n t views on the e v o l u t i o n of dioecy on oceanic i s l a n d s e x i s t (Baker, 1955, 1967; Baker and Cox, 1984; C a r l q u i s t , 1965, 1966, 1974; G i l m a r t i n , 1968; Godley, 1979; Bawa, 1980, 1982), autochthonous e v o l u t i o n of dioecism a f t e r l o n g - d i s t a n c e d i s p e r s a l cannot be r u l e d out ( C a r l q u i s t , 1974; Baker and Cox, 1984). H a b i t a t d i v e r s i t y , which i s common i n high i s l a n d s and e s p e c i a l l y i n the Hawaiian I s l a n d s , probably p l a y s an important r o l e i n promoting s e l e c t i o n f o r dioecy, as suggested by the c o r r e l a t i o n between the height of i s l a n d s and the percent 4 d i o e c i s m i n the study of Baker and Cox (1984). Q u a n t i t a t i v e l y c h a r a c t e r i z i n g the mating systems of i s l a n d s p e c i e s i s thus of p a r t i c u l a r importance i n understanding the e v o l u t i o n of p l a n t mating systems and the e v o l u t i o n a r y r o l e of mating systems i n determining the l e v e l and d i s t r i b u t i o n of g e n e t i c v a r i a t i o n i n i s l a n d p o p u l a t i o n s . Species of Bidens (Asteraceae) o c c u r r i n g on the Hawaiian I s l a n d s provide i d e a l m a t e r i a l s f o r c a r r y i n g out such s t u d i e s . The Hawaiian I s l a n d s a l l arose as submarine volcanoes i n the P a c i f i c Ocean, and most of them are over a m i l l i o n years o l d . They are the most i s o l a t e d i s l a n d s from any c o n t i n e n t . The e c o l o g i c a l d i v e r s i t y of the i s l a n d s i s e x t r a o r d i n a r y (see C a r l q u i s t , 1970). The r e l a t i v e a n t i q u i t y , extreme remoteness, and e c o l o g i c a l r i c h n e s s of the i s l a n d s may have s i g n i f i c a n t e f f e c t s on the e v o l u t i o n of b i o t a s e s t a b l i s h e d subsequent to l o n g - d i s t a n c e d i s p e r s a l . Species of Bidens endemic to Hawaii occur on a l l the 8 major i s l a n d s , with o l d e r and l a r g e r i s l a n d s having more endemic s p e c i e s than the younger or s m a l l e r ones. T h i s p a t t e r n of d i s t r i b u t i o n suggests a long e v o l u t i o n a r y h i s t o r y of Bidens on the Hawaiian I s l a n d s . M o r p h o l o g i c a l , chromosomal, bi o c h e m i c a l and g e n e t i c s t u d i e s of Hawaiian s p e c i e s of Bidens have p r o v i d e d some v a l u a b l e i n f o r m a t i o n on t h e i r o r i g i n , s p e c i a t i o n , adaptive r a d i a t i o n , p o p u l a t i o n g e n e t i c s i m i l a r i t y , and p o p u l a t i o n s u b s t r u c t u r e (e.g., S k o t t s b e r g , 1953; G i l l e t t and Lim, 1970; G i l l e t t , 1975; Ganders and Nagata, 1983, 1984, Marchant et a l . , 1984; Helenurm and Ganders, 1985; R i t l a n d and Ganders, 1985), which p r o v i d e s a background necessary f o r 5 the s t u d i e s of t h e i r mating systems and the e f f e c t s of the mating systems on ge n e t i c v a r i a t i o n and i t s d i s t r i b u t i o n i n Hawaiian Bidens. Of 19 sp e c i e s and 8 subspecies of Hawaiian Bidens, 9 sp e c i e s and 4 a d d i t i o n a l subspecies are gynodioecious ( G i l l e t t , 1975; Ganders, p e r s . comm.) Gynodioecy, the occurrence of hermaphrodite p l a n t s and m a l e - s t e r i l e p l a n t s w i t h i n a p o p u l a t i o n ( F i g . 1-1), i s t r a d i t i o n a l l y c o n s i d e r e d as a type of mating system to promote o u t c r o s s i n g . Despite many t h e o r e t i c a l and e m p i r i c a l s t u d i e s of gynodioecy, the g e n e t i c b a s i s of male s t e r i l i t y and the mechanism of maintenance of females i n p o p u l a t i o n s remain p r o b l e m a t i c . Although comparative s t u d i e s of microsporogenesis i n hermaphrodites and m a l e - s t e r i l e mutants have been conducted i n many cr o p s p e c i e s t o i n v e s t i g a t e the developmental e x p r e s s i o n of male s t e r i l i t y , developmental s t u d i e s have r a r e l y been c a r r i e d out f o r n a t u r a l gynodioecious s p e c i e s . A complete study of gynodioecy i n Hawaiian Bidens, i n c l u d i n g the three t o p i c s mentioned above, i s not only e s s e n t i a l f o r understanding the e v o l u t i o n of gynodioecy i n t h i s group of s p e c i e s , but a l s o badly needed to understand the e v o l u t i o n a r y s i g n i f i c a n c e of gynodioecy i n g e n e r a l . The o b j e c t i v e s of t h i s study are 1) to estimate o u t c r o s s i n g r a t e s i n n a t u r a l p o p u l a t i o n s of Hawaiian s p e c i e s of Bidens using allozyme markers, and to study the r e l a t i v e importance of f l o r a l mechanisms as ge n e t i c determinants of mating systems (Chapter 3); 2) to i n v e s t i g a t e the e f f e c t of mating systems on the l e v e l of g e n e t i c v a r i a t i o n and the d i s t r i b u t i o n of t h i s v a r i a t i o n i n 6 Figure 1-1. Male-sterile disc flower (MS) and hermaphroditic disc flower (H) in a gynodioecious species of Hawaiian Bidens. 7 p o p u l a t i o n s of Hawaiian Bidens (Chapter 4); 3) to study gynodioecy i n Hawaiian Bidens, i n c l u d i n g comparative s t u d i e s of microsporogenesis (Chapter 5), g e n e t i c s t u d i e s of the i n h e r i t a n c e of male s t e r i l i t y (Chapter 6), and s t u d i e s on the maintenance of females i n p o p u l a t i o n s (Chapter 7). H o r i z o n t a l s t a r c h g e l e l e c t r o p h o r e s i s has served as a b a s i c method f o r the r e s e a r c h presented i n Chapters 3, 4, and 7, thus d e t a i l e d d e s c r i p t i o n s of the e l e c t r o p h o r e t i c procedures and g e n e t i c i n t e r p r e t a t i o n of allozyme banding p a t t e r n s are given i n an i n d i v i d u a l chapter (Chapter 2). Chapter 8 presents the general c o n c l u s i o n s of t h i s study. Chapters 3-7 have been prepared f o r p u b l i c a t i o n . These chapters are presented i n the form of i n d i v i d u a l papers. 8 I I . ELECTROPHORESIS 2.1 I n t r o d u c t i o n E l e c t r o p h o r e s i s has made i t p o s s i b l e s i n c e the l a t e 1960's to i d e n t i f y l a r g e numbers of p a r t i c u l a r gene l o c i and to enumerate the l e v e l of g e n e t i c v a r i a t i o n i n n a t u r a l p o p u l a t i o n s . E l e c t r o p h o r e s i s exposes s i n g l e codominant gene markers that can be used f o r e s t i m a t i n g the o u t c r o s s i n g r a t e and the e f f e c t of mating systems on genotypic s t r u c t u r e and g e n e t i c v a r i a t i o n i n n a t u r a l p o p u l a t i o n s . P r e v i o u s l y such s t u d i e s were l i m i t e d because of d i f f i c u l t i e s i n f i n d i n g good polymorphic m o r p h o l o g i c a l marker genes and a l s o because the a l l e l e s at these gene l o c i commonly e x h i b i t dominant-recessive r e l a t i o n s h i p s . Although e l e c t r o p h o r e s i s has i t s l i m i t a t i o n s , as noted by H a r t l (1980) and G o t t l i e b (1981), e.g., r o u t i n e e l e c t r o p h o r e s i s d e t e c t s only those amino a c i d s u b s t i t u t i o n s that r e s u l t i n charge d i f f e r e n c e s i n the p r o t e i n s and thus amount of polymorphism may be underestimated, 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 s i s and the number of s t u d i e s i n p l a n t p o p u l a t i o n g e n e t i c s continue to m u l t i p l y . H o r i z o n t a l s t a r c h g e l e l e c t r o p h o r e s i s was used i n t h i s study. E l e c t r o p h o r e t i c procedures were b a s i c a l l y the same as d e s c r i b e d by Helenurm (1983). Some m o d i f i c a t i o n s were necessary i n order to improve allozyme r e s o l u t i o n and to achieve high e f f i c i e n c y . In t h i s chapter, I present 1) s i t e i nformation on 21 p o p u l a t i o n s of Hawaiian Bidens s t u d i e d e l e c t r o p h o r e t i c a l l y ; 2) e l e c t r o p h o r e t i c procedures used; 3) enzyme systems r e s o l v e d 9 and allozyme l o c i recorded; and 4) ge n e t i c i n t e r p r e t a t i o n of allozyme zymograms. 2.2 M a t e r i a l s Twenty-one p o p u l a t i o n s , r e p r e s e n t i n g 12 taxa, of Hawaiian Bidens d i s t r i b u t e d on four major i s l a n d s (Kauai, Oahu, Maui, and Hawaii) were s t u d i e d e l e c t r o p h o r e t i c a l l y . These p o p u l a t i o n s were chosen p u r e l y because of seed a v a i l a b i l i t y . The d i s t r i b u t i o n s of these p o p u l a t i o n s on the four i s l a n d s are given i n F i g . 2-1 - 2-4, and the l o c a l i t i e s , s i z e s , h a b i t a t d e s c r i p t i o n s and dates of c o l l e c t i o n s of these p o p u l a t i o n s are given i n Table 2-1. Seeds c o l l e c t e d from these p o p u l a t i o n s were germinated i n v e r m i c u l i t e and r a i s e d i n growth chambers at 25 C f o r 12 h day l e n g t h and at 17 C f o r 12 h i n dark. Seedlings were ready f o r e l e c t r o p h o r e s i s 2-3 weeks a f t e r g e r m i n a t i o n . Sample s i z e s of f a m i l i e s and i n d i v i d u a l s per f a m i l y are given i n the t a b l e s i n each r e l e v a n t chapter. 10 Figure 2-1. Locations of populations studied electrophoretically on the island of Kauai. AMPL 2 TORT 1 TORT 2 ISLAND of OAHU ELEVATION Contour I n t t r v i l M O O O SAND 1 Figure 2 - 2 . Locations of populations studied e l e c t r o p h o r e t i c a l l y on the i s l a n d of Oahu. Figure 2-3. Locations of populations studied electrophoretically on the island of Maui. 13 Figure 2-4. Locations of populations studied electrophoretically on the island of Hawaii. T a b l e 2-1. Twenty-one p o p u l a t i o n s of Hawaiian Bidens s t u d i e d e l e c t r o p h o r e t i c a l l y (see Appendix A f o r complete s c i e n t i f i c names of t h e s e s p e c i e s ) . S p e c i e s P o p u l a t i o n P o p u l a t i o n P o p u l a t i o n F l o w e r i n g H a b i t a t Date of acronym L o c a l i t y S i z e time D e s c r i p t i o n c o l l e c t i o n amplectens AMPL K e a l i a T r a i 1 , Waianae Mtns., Oahu >500 Feb.-May Elev.220-240m. Dry sunny ledges and c l i f f s , growing with lowland s h r u b s . dune, 1984 B. asymmetrica ASYM A i e a Loop T r a i 1 Koolau Mtns., Oahu <25 dan.-Apr. E1ev.300-450m. S l o p e s and r i d g e top exposed or under wet f o r e s t canopy. dune, 1984 B. c e r v i c a t a CERV Makaha Ridge, Kauai 100-500 Mar.-May Elev.between 750-1000m. Dry r i d g e or under t r e e s . dune, 1984 B. f o r b e s i i FORB F1 K a l a l a u t r a i l , s s p . f o r b e s i i Na P a l i Coast, Kauai >1000 Feb.-Apr. E1ev.50-150m. Wet exposed Feb., 1984 a few i n Oct. c o a s t a l c l i f f s w i t h s h r u b s . B. f o r b e s i i ssp. k a h i 1 i e n s i s FORB K Mt. K a h i l i , Kauai 100-500 May-dun. Ele v . a b o u t 850m. Slo p e and dune, 1984 r i d g e under wet f o r e s t s . B . hawaiejnsi s HAWA Hi 1ina P a l i Road, K i l a u e a , Hawai1 Feb.-Mar. El e v . a b o u t 950m. Open shrub Feb., 1984 v e g e t a t i o n on l a v a f l o w s . B. h i 1 l e b r a n d i a n a HILL E East of M a l l k o Bay, 100-500 May-dun. El e v . a b o u t 30m. windswept June, 1984 Table 2-1 cont. ssp. polycephala Makawao, Maul HILL W West of Mallko Bay, Makawao, Maul 100-500 B. maulens is MAUI 1 Walehu, Walluku, west Maui 100-500 MAUI 2 Manawalnul Gulch, east Maul <50 B. menziesi1 ssp. f i l l f o r m l s MENZ 1 West slope of Mauna Kea, Ahumoa, Hawaii >1000 MENZ 2 South slope of Mauna Kea, Puu Koohe, Hawaii >500 MENZ 3 West slope of Mauna Kea, 100-500 Nohona 0 Hae, Hawaii MENZ 4 Southwest slope of Mauna Kea, Puu Koko, Hawai1 50-100 a few 1n Feb. ocean c l i f f s on bare s o i l May-Jun. Elev.10-25m. On edge of windswept ocean c l i f f s on bare s o i l or with grasses. June, 1984 Feb.-dun. Elev.30-40m. dry 11thifled a few In Dec. sand dunes with sparse low shrubs and herbs. June, 1984 Feb.-Jun. a few In Dec. Elev.about 30m. Very dry, rocky area with sparse vegetatIon. June, 1984 Oct.-Nov. Elev.about 2000m. Dry, open forest on large cinder cone. Nov., 1984 Oct.-Nov. Elev.1700m. Dry open forest on large cinder cone. Nov.. 1984 Sept.-Nov. Elev.970m. Dry open cinder cone, recently burned. Nov., 1984 Sept.-Nov. Elev.2000m. Dry, low shrub vegetation with grasses, on c1nder cone. Nov. 1984 T a b l e 2-1 c o n t . S. sandvi cens i s s s p . s a n d v i c e n s i s SAND 1 Waahila Ridge, Manoa V a l l e y , Oahu 100-500 Feb.-Mar. a few i n Feb. Apr.-Jun. E1ev.350-400m. s t e e p g r a s s y June, 1984 s l o p e i n a mesic f o r e s t . SAND 2 Highway 580, a l o n g Wailua R i v e r , Kawai <50 Feb.-Mar. E1ev.50-100m. On rocky, s t e e p road c u t . June, 1984 SAND 3 Waimea Canyon, Kauai 100-500 Dec.-Jun. E l e v . a b o u t 360m. On s t e e p a few i n Oct. canyon s l o p e s w i t h s p a r s e shrubs and herbs. June, 1984 B. s a n d v i c e n s i s s s p . c o n f u s a SAND C I l i a u loop t r a i l , Waimea Canyon, Kauai >500 Dec.-Jun. a few i n Oct. E l e v . a b o u t 910m. Open June, 1984 v e g e t a t i o n on rim of canyon. B. s a n d v i c e n s i s X c o n f u s a * SAND X Waimea Canyon, Kauai >1000 Dec.-Jun. Elev.450-600m. Open shrubby, g r a s s y v e g e t a t i o n on the f l a t a r e a of the canyon rim. June, 1984 B. t o r t a TORT 1 Waianae Kai , Waianae Mtns., Oahu 100-500 Dec.-Mar. E1 ev. 500-700m. On open r i d g e D e c , 1983 or Jun. i n mesic f o r e s t s . TORT 2 P a l i k e a t r a i 1 Southern Waianae, Oahu 100-500 Dec.-Mar. Elev.800m. On open r i d g e or Jun. i n mesic f o r e s t s . Nov., 1981 *: T h i s p o p u l a t i o n i s i n t e r m e d i a t e between s a n d v i c e n s i s subsp. s a n d v 1 c e n s i s and subsp. c o n f u s a , or B_^  c e r v i c a t a . 17 2.3 E l e c t r o p h o r e t i c Procedures 2.3.1 G e l P r e p a r a t i o n E l e c t r o s t a r c h Lot #392 was g e n e r a l l y used f o r the m a t e r i a l s i n t h i s study. The c o n c e n t r a t i o n of s t a r c h used was 14%, with 10% sucrose added to improve r e s o l u t i o n . Two b u f f e r systems, A (Clayton and T r e t i a k , 1972) and B (Ridgway et a l . , 1970), were used i n i t i a l l y (Table 2-2). Table 2-2. E l e c t r o p h o r e s i s B u f f e r s used f o r Bidens B u f f e r system code E l e c t r o d e b u f f e r components pH Gel b u f f e r components pH 0.04M C i t r i c a c i d (anhydrous) a d j u s t pH with N-(3-aminopropyl)• morpholine 0.002M C i t r i c a c i d (anhydrous) 6.1 (made as 1:20 d i l u t i o n of e l e c t r o d e b u f f e r ) 6.1 B 0.03M b o r i c a c i d 0.06M l i t h i u m hydroxide 0.005M C i t r i c a c i d 8.1 (anhydrous) 0.03M T r i s ; p l u s 1% e l e c t r o d e b u f f e r 8.5 18 A f t e r minor m o d i f i c a t i o n s , b u f f e r system A was found to be g e n e r a l l y a p p l i c a b l e to 9 enzyme systems (Table 2-3), and i t was thus s e l e c t e d as a r o u t i n e b u f f e r system f o r e l e c t r o p h o r e t i c runs. S t a r c h was measured and mixed with g e l b u f f e r s o l u t i o n s , cooked i n a microwave oven to b o i l i n g , degassed, and poured i n t o p l e x i g l a s s molds. D i f f e r e n t p l e x i g l a s s molds were chosen depending on the t h i c k n e s s of g e l s d e s i r e d . U s u a l l y , each g e l was prepared f o r s t a i n i n g s i x enzyme systems. The g e l s were covered with p l a s t i c , kept at room temperature o v e r n i g h t , and cooled down to 2-4 C p r i o r to use. 2.3.2 E x t r a c t i o n Young l e a f t i s s u e s were macerated i n 2 drops of c o l d e x t r a c t i o n b u f f e r s o l u t i o n (Yeh and O'Malley, 1980) i n m i c r o t i t r a t i o n w e l l s . The i n d i v i d u a l s of each f a m i l y were p l a c e d i n adjacent w e l l s to f a c i l i t a t e r e c o r d i n g . An e l e c t r i c d r i l l motor with a p e s t l e attachment was used to homogenize the p l a n t t i s s u e . T h i r t y samples were ground up at a time, and the homogenates were each absorbed onto 12x3 mm Whatman #3 chromatography paper wicks. S a t u r a t e d wicks were p l a c e d s e q u e n t i a l l y i n each small t r a n s v e r s e s l o t near the edge of a r e c t a n g u l a r g e l . 2.3.3 E l e c t r o p h o r e s i s The g e l s loaded with samples were pl a c e d h o r i z o n t a l l y between two t r a y s c o n t a i n i n g e l e c t r o d e s and b u f f e r s o l u t i o n s (Table 2-2). E l e c t r o d e s were connected to each end of the g e l by J - c l o t h s soaked i n b u f f e r s o l u t i o n , and a c u r r e n t of 50mA at 19 about 350V was a p p l i e d a c r o s s the g e l . A l l enzymes r e s o l v e d i n t h i s study migrate from the c a t h o d a l end to the anodal end. The e n t i r e e l e c t r o p h o r e t i c run was conducted at about 4 C i n a r e f r i g e r a t o r , commonly f o r 4 hours. The m i g r a t i n g f r o n t was monitored using marker wicks s t a i n e d with red dye #2. The r a t e of m i g r a t i o n appeared to be a f f e c t e d by the t h i c k n e s s of a g e l , the c o n c e n t r a t i o n s of s t a r c h , g e l b u f f e r and e l e c t r o d e b u f f e r , and pH of the b u f f e r systems. A f t e r e l e c t r o p h o r e s i s , g e l s were each h o r i z o n t a l l y cut i n t o 6-7 s l i c e s with monofilament l i n e guided by 1.5mm t h i c k p l e x i g l a s s s t r i p s , and the top s l i c e of each g e l was always d i s c a r d e d . 2.3.4 Enzyme A c t i v i t y S t a i n i n g The s t a i n i n g b u f f e r s were s t o r e d i n a r e f r i g e r a t o r , and warmed to about 30 C p r i o r to s t a i n i n g . The d e s i r e d amount of s t a i n i n g b u f f e r and s t a i n i n g chemicals f o r each enzyme system f o r a known number of g e l s l i c e s was measured i n t o brown b o t t l e s to prevent d e n a t u r a t i o n of l i g h t - s e n s i t i v e c h e m i c a l s . The enzyme s p e c i f i c s t a i n i n g s o l u t i o n s were poured i n t o t r a y s , and g e l s l i c e s were immersed i n the s t a i n i n g s o l u t i o n s and incubated at about 35 C i n the dark u n t i l the c o l o r e d allozyme bands were w e l l developed. To stop the enzyme r e a c t i o n , the s t a i n i n g s o l u t i o n was d i s c a r d e d , and the g e l s l i c e s were r i n s e d with c o l d tap water. The sharpness of the allozyme bands was g e n e r a l l y s a t i s f a c t o r y . Only o c c a s i o n a l l y was the f i x a t i o n s o l u t i o n 5 water : 4 ethanol : 1 a c e t i c a c i d ( A l l e n d o r f et a l . , 1977), or 50% g l y c e r o l ( S i c i l i a n o and Shaw, 1976) used to preserve r e s o l u t i o n . The banding p a t t e r n s were recorded f i r s t and 20 genotypes were determined f o r each i n d i v i d u a l l a t e r . 2.3.5 Enzyme Systems Resolved T h i r t y - s i x enzyme systems were t e s t e d i n d i f f e r e n t b u f f e r systems, but only 11 were r e s o l v e d . Of those enzyme systems r e s o l v e d i n both A and B b u f f e r systems, Pgm showed d i f f e r e n t banding p a t t e r n s . Data were recorded based on b u f f e r system A fo r t h i s enzyme system f o r a l l p o p u l a t i o n s s t u d i e d . Of a t o t a l of 38 allozyme l o c i r e s o l v e d (Table 2-3), only P g i - 1 , Pgi-2 and Skdh-3 are commonly polymorphic. D e t a i l e d i n f o r m a t i o n on genetic i n t e r p r e t a t i o n of the banding p a t t e r n s are given f o r these three l o c i . Most l o c i of other enzyme systems were monomorphic, and a few polymorphic l o c i appeared to be c o n t r o l l e d by simple Mendelian f a c t o r s . Only b r i e f d e s c r i p t i o n s of these enzyme systems are presented. 2.4 Genetic I n t e r p r e t a t i o n s Of Zymograms The g e n e t i c b a s i s of the isozyme v a r i a t i o n was i n t e r p r e t e d in r e f e r e n c e to the r e s u l t s of c o n t r o l l e d c r o s s e s i n t h i s study and i n the study of Helenurm (1983), but p r i m a r i l y i n f e r r e d from the comparison of phenotypes found i n progeny a r r a y s from the 21 populat i o n s . T a b l e 2-3. Enzyme systems and the number of l o c i commonly r e c o r d e d f o r 21 p o p u l a t i o n s of Hawaiian B i d e n s . No. of l o c i Enzyme Code Acronym S t a i n i n g r e f e r e n c e s c o r e d Phosphoglucose isomerase E ,C .5 .3. 1 .9 Pgi Tanks l e y , 1980 5 Sh i k i m a t e dehydrogenase E .C . 1 . 1 . 1 .25 Skdh T a n k s l e y and R i c k , 1980 3 L e u c i n e aminopept1dase E ,C. 3 .4. 1 . 1 Lap V a l l e j o s , 1983 6 Ma l a t e dehydrogenase E ,C . 1 . 1 . 1 . 37 Mdh Brown e t a l . , 1978 7 Phosphoglucomutase E .C. 2 .7. 5 . 1 Pgm Ta n k s l e y , 1979 8 6-p-gluconate dehydrogenase E ,C. 1 . 1 . 1 .44 6-Pgdh V a l l e j o s , 1983 6 M a i i c enzyme E .C . 1 . . 1 . 1 .40 Me S i c i l i a n o and Shaw, 1976 1 Hexose aminidase E . ,C . 3. .2 . 1 .30 Ha S l c l l l a n o and Shaw, 1976 1 B-glucos1dase E , C. 3. 2 . 1 .21 Qlu Yeh and La y t o n , 1979 1 22 2.4.1 Phosphoqlucose Isomerase (Pgi) Pgi behaved as a d i m e r i c enzyme (heterozygotes having three isozyme bands) in Bidens, as has been r e p o r t e d f o r many other p l a n t s p e c i e s (see Helenurm, 1983 f o r r e f e r e n c e s t h e r e i n ) . Two independent regions of isozyme bands were r e s o l v e d f o r P g i . The banding p a t t e r n s at the c a t h o d a l region were v a r i a b l e , i n which i n d i v i d u a l s can each show 1, 3, 4, 5, 6 or 7 bands, whereas the anodal region was monomorphic, i n which a l l i n d i v i d u a l s e x h i b i t e d three evenly spaced bands i n a l l p o p u l a t i o n s s t u d i e d . Isozymes at t h i s region are commonly l o c a t e d i n c h l o r o p l a s t s , but a l s o c o n t r o l l e d by nuclear genes ( G o t t l i e b , p e r s . comm.). According to band i n t e n s i t i e s , which are c o r r e l a t e d with a l l e l e dosage, three l o c i were assig n e d to the anodal r e g i o n . T h i s i s i n agreement with Helenurm (1983). Allozymes at the c a t h o d a l r e g i o n are c o n t r o l l e d by two l o c i , designated Pgi-1 and Pgi-2. The p r o t e i n subunits produced by these two l o c i randomly u n i t e to form both i n t r a - and i n t e r - l o c u s dimers. Four a l l e l e s at Pgi-1 and three a l l e l e s at Pgi-2 were f r e q u e n t l y present i n p o p u l a t i o n s of Hawaiian Bidens i n t h i s study ( F i g . 2-5). I t was sometimes d i f f i c u l t to determine which a l l e l e belonged to which l o c u s due to a l l e l e o v e r l a p p i n g . I n d i v i d u a l banding p a t t e r n s on each g e l were always recorded f i r s t , but genotypes were as s i g n e d to i n d i v i d u a l s only a f t e r a l l banding p a t t e r n s i n a p o p u l a t i o n and genotypic s t r u c t u r e w i t h i n f a m i l i e s were ana l y z e d . Family genotype a n a l y s i s u s u a l l y allowed 23 a — Pgi-1 Pgj-2 F i g u r e 2-5. A l l e l e s of Pgi-1 and P g i - 2 . For each l o c u s , the a l l e l e s are d e s i g n a t e d by l e t t e r with a f o r that coding the most c a t h o d a l allozyme, b f o r the next and so f o r t h ( A l l other enzyme systems were a l s o d e s i g n a t e d f o l l o w i n g t h i s r u l e ) . The most common a l l e l e s a t the two l o c i , Pgi-1b and Pgi-2c, have the same m i g r a t i o n p o s i t i o n . The l e s s frequent a l l e l e s , Pgi-1a and Pgi-2b a l s o share the same p o s i t i o n . 24 unambiguous assignment of a l l e l e s to each l o c u s . D i f f e r e n t combinations of a l l e l e s at the two l o c i r e s u l t i n more than 23 banding p a t t e r n s i n the polymorphic r e g i o n . Some commonly recorded banding p a t t e r n s and t h e i r g e n e t i c i n t e r p r e t a t i o n s are given i n F i g . 2-6. D e s i g n a t i o n of a l l e l e s at t h i s region i n the present study p a r t i a l l y d i f f e r e d from Helenurm (1983). Some bands i n t e r p r e t e d as a l l e l e s by Helenurm were a c t u a l l y i n t r a - or i n t e r - l o c u s heterodimers, such as Pgi-4e i n Helenurm. S e v e r a l a l l e l e s were not found i n any p o p u l a t i o n s of t h i s study, such as a l l e l e s P g i -4b, Pgi-4g, Pgi-5a, Pgi-5b and Pgi-5e of Helenurm. A rare n u l l a l l e l e was observed i n t h i s r e g i o n , but i t appeared to be independent of Pgi-1 or Pgi-2. T h i s a l l e l e was recorded f o r some p o p u l a t i o n s , but excluded from the f i n a l data a n a l y s i s because of i t s u n c e r t a i n t y . Some sample g e l s of Pgi-1 and Pgi-2 i n d i f f e r e n t p o p u l a t i o n s of Hawaiian Bidens are presented i n F i g . 2-7. 2.4.2 Shikimate Dehydrogenase (Skdh) Skdh i s a monomeric enzyme i n Bidens, as has been r e p o r t e d f o r L y c o p e r s i c o n esculentum and Solanum p e n n e l l i i (Tanksley and Rick, 1980). Isozyme bands were present i n two r e g i o n s . The c a t h o d a l r e g i o n was monomorphic. Commonly the same two bands were present i n a l l i n d i v i d u a l s . Two l o c i were assigned to t h i s r e g i o n . The anodal r e g i o n was polymorphic, a p p a r e n t l y c o n t r o l l e d by one l o c u s . Four a l l e l e s at the locus were d e t e c t e d . The r e l a t i v e a l l e l e p o s i t i o n s , the commonly observed banding p a t t e r n s i d F i g u r e 2-6. E l e c t r o p h o r e t i c p a t t e r n s and g e n e t i c a n a l y s i s of Pgi at the c a t h o d a l r e g i o n . Two l o c i a r e a s s i g n e d to t h i s r e g i o n , Pgi-1 and Pgi - 2 . The number a t t a c h e d to each band i n d i c a t e s the r e l a t i v e i n t e n s i t i e s of the band i n the i n d i v i d u a l . Genotypes are g i v e n f o r i n d i v i d u a l banding p a t t e r n s w i t h numbers i n d i c a t i n g the a l l o z y m e l o c i and l e t t e r s i n d i c a t i n g the a l l e l e s a t the locus (Arrow i n d i c a t e s the m i g r a t i n g f r o n t ) . Genotypes of these i n d i v i d u a l s from l e f t to r i g h t a r e : 1) 1bb2cc; 2) 1bc2cc; 3) 1cc2cc; 4) 1bb2bb; 5) 1bb2bc; 6) 1bc2bc; 7) 1bb2ab or 1ab2ac; 8) 1ab2aa; 9) 1bb2aa; 10) 1cc2bc; 11) 1bc2bb or 1ac2bc; 12) 1cc2bb; 13) 1cc2ab; 14) 1bc2ab or 1ac2ac; 15) 1bc2aa; 16) 1cc2ac; 17) 1bc2ac; 18) 1ab2ab or 1aa2ac; 19) 1bb2ac; 20) 1bd2cc; 21) 1dd2cc; 22) 1bd2ac. 26 F i g u r e 2-7. Sample g e l s of Pgi-1 and Pgi-2 a l l o z y m e s . A. E x p e r i m e n t a l c r o s s e s between p l a n t s P1 (genotype 1bb2cc) and P2 (genotype 1bb2aa) g i v i n g F1 progeny of genotype 1bb2ac. B. A sample g e l of Pgi-1 and Pgi-2 i n a p o p u l a t i o n of B_^  sandv i cens i s. Genotypes of the numbered i n d i v i d u a l s a r e : 1. 1bb2cc; 2. 1cc2cc; 3. 1bc2bc; 4. 1bb2bc; 5. 1bc2cc; 6. 1bb2bb; 7. 1bb2ac. C. A sample g e l of Pgi-1 and Pgi-2 i n a p o p u l a t i o n of B_;_ m e n z i e s i i (MENZ 2). Genotypes of the numbered i n d i v i d u a l s a r e : 1) 1bb2bb; 2) 1bb2bc; 3) 1bb2aa. D. A sample g e l of Pg1-1 and Pgi-2 i n a p o p u l a t i o n of B. hawa1 ens i s (HAWA). Genotypes of the numbered i n d i v i d u a l s a r e : 1. 1bb2ac; 2. 1bc2cc; 3. 1bb2cc. 27 and assignment of genotypes are given i n F i g . 2-8. Two sample g e l s of Skdh-3 are presented i n F i g . 2-9. 2.4.3 Leucine Aminopeptidase (Lap) Lap behaved as a monomeric enzyme i n Bidens, as has been r e p o r t e d f o r other p l a n t s p e c i e s (see Helenurm, 1983 f o r r e f e r e n c e s t h e r e i n ) . Two independent regions of isozyme a c t i v i t i e s were d e t e c t e d . The c a t h o d a l region showed the same two monomorphic bands f o r a l l i n d i v i d u a l s . The anodal region showed 4 or 5 bands. The a l l e l e assignment and genotypes of some commonly recorded banding p a t t e r n s are shown i n F i g . 2-10. Two sample g e l s of Lap are shown i n F i g . 2-11. 2.4.4 Malate Dehydrogenase (Mdh) Mdh behaved as a d i m e r i c enzyme i n Bidens, as has been r e p o r t e d f o r many other p l a n t s p e c i e s (see Helenurm, 1983 f o r r e f e r e n c e s t h e r e i n ) . Three independent regions of enzyme a c t i v i t i e s were d e t e c t e d . The c a t h o d a l r e g i o n (Mdh-1 and Mdh-2 in F i g . 2-12) was polymorphic, 1 or 3 bands were commonly observed. Three bands commonly appeared i n the intermediate r e g i o n (Mdh-3, Mdh-4 and Mdh-5) but v a r i a t i o n i n number and r e l a t i v e i n t e n s i t y of bands was o c c a s i o n a l l y observed ( F i g . 2-12). Three l o c i were assig n e d to t h i s r e g i o n a c c o r d i n g to t h e i r r e l a t i v e band i n t e n s i t i e s . The a n a l y s i s of these banding p a t t e r n s i s given i n F i g . 2-12. The anodal region (Mdh-6 and Mdh-7) showed the same two bands f o r a l l i n d i v i d u a l s . Two l o c i were assig n e d to t h i s r e g i o n . The p a t t e r n s 5 and 7 shown i n F i g . 2-12 were o c c a s i o n a l l y observed. I suspect that these v a r i a n t s 28 F i g u r e 2-8. A l l e l e s and commonly observed banding p a t t e r n s of Skdh-3 at the anodal r e g i o n . The a l l e l e s are d e s i g n a t e d by l e t t e r with a f o r that coding the most c a t h o d a l allozyme, b f o r the next and so f o r t h . Genotypes of i n d i v i d u a l s from l e f t t o r i g h t a re: c c ; be; bb; ac; cd; and bd. 2 9 Figure 2-9. Two sample gels of Skdh-3. A. Experimental crosses between plants P1 (genotype cc) and P2 (genotype bb) giving F1 progeny of genotype be. B. A sample gel of Skdh-3 in a populations of B^  hawaiensis (HAWA). Genotypes of numbered individuals are: 1. cc; 2. be; 3. bb. 30 4b 4a 3a 2b lb - 2a l a A F i g u r e 2-10. L o c i , a l l e l e s and commonly observed banding p a t t e r n s of Lap at the anodal r e g i o n . The allozyme l o c i are d e s i g n a t e d by numbers and a l l e l e s at each locus are desig n a t e d by l e t t e r s (Arrow i n d i c a t e s the d i r e c t i o n of m i g r a t i o n ) . Genotypes of i n d i v i d u a l s from l e f t to r i g h t a r e : 1-3. 1bb2bb3aa4aa; 4. 1aa2aa3aa4aa; 5. 1bb2bb3aa4ab; 6. 1bb2bb3aa4bb. 31 Figure 2-11. Two sample gels of Lap allozymes. A. Experimental crosses between plants P1 (genotype 1bb2bb3aa4aa) and plant P2 (genotype 1aa2aa3aa4aa) giving F1 progeny of genotype 1ab2ab3aa4aa. B. A sample gel of Lap in a population of B^  menziesi i . Al l individuals are homozygous (genotype 1bb2bb3aa4aa). 32 s = = = = = = = | 4 a, 5a — U 3a • — -lb,2a — 1 a F i g u r e 2-12. L o c i , a l l e l e s and some banding p a t t e r n s of Mdh. The allozyme l o c i are d e s i g n a t e d by numbers and a l l e l e s of each l o c u s by l e t t e r s (Arrow i n d i c a t e s the d i r e c t i o n of m i g r a t i o n ) . Genotypes of i n d i v i d u a l s from l e f t to r i g h t a r e : 1, 2 and 6 a r e : 1bb2aa3aa4aa5aa6aa7aa; 3. 1ab2aa3aa4aa5aa6aa7aa; 4. 1aa2aa3aa4aa5aa6aa7aa. 33 were produced by a r t i f a c t s . F ast m i g r a t i o n of some breakdown products of isozymes c o u l d r e s u l t i n the banding p a t t e r n 5, and the f a i l u r e to produce an intermediate heterodimer band i n some i n d i v i d u a l s c o u l d r e s u l t i n the banding p a t t e r n 7. The present g e n e t i c i n t e r p r e t a t i o n s f o r t h i s enzyme system d i f f e r from those i n Helenurm (1983). Helenurm assig n e d 5 a l l e l e s (Mdh-5a, 5b, 5c, 5d, 5e) f o r one l o c u s and 2 a l l e l e s (Mdh-6a, 6b) f o r the other l o c u s at the c a t h o d a l r e g i o n , whereas i n t h i s study, only Mdh-5a, 5d ( e q u i v a l e n t to Mdh-1a, 1b i n t h i s study) and Mdh-6a ( e q u i v a l e n t to Mdh-2a i n t h i s study) were de t e c t e d i n t h i s r e g i o n . A t o t a l of 7 a l l e l e s at 3 l o c i were as s i g n e d to the intermediate r e g i o n i n Helenurm (1983), i n which 2 l o c i were i n t e r p r e t e d as polymorphic. Because the a l l e l e s Mdh-2a, 2b, 2c, and Mdh-3b in Helenurm were not observed i n t h i s study and the m a j o r i t y of p o p u l a t i o n s of t h i s study showed no v a r i a t i o n i n t h i s r e g i o n , only three monomorphic l o c i were as s i g n e d to t h i s r e g i o n . The random a s s o c i a t i o n of p r o t e i n s u b u n i t s coded by these l o c i can r e s u l t i n the commonly observed 4:4:1 r a t i o i n r e l a t i v e band i n t e n s i t y . 2.4.5 Phosphoglucomutase (Pqm) Pgm behaved as a monomeric enzyme i n Bidens, as has been shown i n other p l a n t s p e c i e s (see Helenurm, 1983 f o r r e f e r e n c e s t h e r e i n ) . The banding p a t t e r n s of Pgm r e s o l v e d i n the two b u f f e r systems A and B are e n t i r e l y d i f f e r e n t . The banding p a t t e r n s i n B u f f e r system B were the same as d e s c r i b e d by Helenurm (1983), but the banding p a t t e r n s r e s o l v e d i n B u f f e r system A were commonly more monomorphic than the ones i n B u f f e r B. B u f f e r 34 system A was used f o r a l l p o p u l a t i o n s of t h i s study. The banding p a t t e r n s r e s o l v e d i n B u f f e r A are given i n F i g . 2-13. 2.4.6 6-Phosphoglucanate Dehydrogenase (6-Pqdh) Commonly 5 or 6 bands were r e s o l v e d f o r t h i s enzyme system. Locus 1 and Locus 2 each had a rare a l l e l e . The enzyme system appears to be dimeric i n Bidens, as has been r e p o r t e d f o r humans, D r o s o p h i l a subobscura and Douglas f i r (see Layton, 1980 f o r re f e r e n c e s t h e r e i n ) . The banding p a t t e r n s commonly observed are given i n F i g . 2-14. 2.4.7 M a l i c Enzyme (Me), Hexose Aminidase (Ha) And ft - Gl u c o s i d a s e (Glu) These three enzyme systems were g e n e r a l l y monomorphic i n the p o p u l a t i o n s of Hawaiian Bidens s t u d i e d , A minimum one l o c u s was ass i g n e d to each of the three enzyme systems. 2.4.8 Diaphorase And A c o n i t a s e These two enzyme systems were c l e a r l y r e s o l v e d i n only a few p o p u l a t i o n s of Hawaiian Bidens. They were excluded from the f i n a l data a n a l y s i s . 2.5 Summary In summary, a t o t a l of 38 allozyme l o c i c o n t r o l l i n g 9 enzyme systems were r e s o l v e d i n p o p u l a t i o n s of Hawaiian Bidens. These l o c i a r e : Pgi-1, Pgi-2, Pgi-3, Pgi-4, Pgi-5; Skdh-1, Skdh-2, Skdh-3; Lap-1, Lap-2, Lap-3, Lap-4, Lap-5, Lap-6; Mdh-1, Mdh-2, Mdh-3, Mdh-4, Mdh-5, Mdh-6, Mdh-7; Pgm-1, Pgm-2, Pgm-3, Pgm-4, Pgm-5, Pgm-6, Pgm-7, Pgm-8; 6-Pgdh-1, 6-Pgdh-2, 6-Pgdh-3, 35 t 8 b, 7c 7 b, 8a 6a,7a 5a 4a 3a,2b 2a l b l a F i g u r e 2-13. L o c i , a l l e l e s and commonly observed banding p a t t e r n s of Pgm. The allozyme l o c i are desi g n a t e d by numbers and a l l e l e s of each lo c u s by l e t t e r s (Arrow i n d i c a t e s the d i r e c t i o n of m i g r a t i o n ) . Genotypes of i n d i v i d u a l s from l e f t to r i g h t a r e : 1. 1ab2aa3aa4aa5aa6aa7bb8bb; 2-7. 1bb2aa3aa4aa5aa6aa7bb8bb. Pgm-4 o c c a s i o n a l l y e x h i b i t e d a n u l l a l l e l e . 36 3a 2b lb, 2a — — la F i g u r e 2-14. L o c i , a l l e l e s and commonly observed banding p a t t e r n s of 6-pgdh. The allozyme l o c i are desi g n a t e d by numbers and a l l e l e s of each lo c u s by l e t t e r s (Arrow i n d i c a t e s the d i r e c t i o n of m i g r a t i o n ) . Genotypes of i n d i v i d u a l s from l e f t to r i g h t a r e : 1-6. 1bb2bb3aa4aa5aa6aa; 7. 1ab2bb3aa4aa5aa6aa. 37 6-Pgdh-4, 6-Pgdh-5, 6-Pgdh-6; Me-1; Ha-1; Glu-1. Of these l o c i , 5 l o c i were polymorphic at the 95% c r i t e r i o n ( Pgi-1, Pgi-2, Skdh-3, Pgm-8 and Lap-4), and 5 more l o c i were polymorphic at the 99% c r i t e r i o n (Pgm-1, Pgm-2, Pgm-7, 6-Pgdh-1 and 6-Pgdh-2). Only th r e e commonly polymorphic l o c i , Pgi-1, Pgi-2, and Skdh-3, were used as g e n e t i c markers to estimate o u t c r o s s i n g r a t e s i n 15 polymorphic p o p u l a t i o n s . These p o p u l a t i o n s are AMPL, CERV, FORB F1, HAWA, MAUI 1, MAUI 2, MENZ 1, MENZ 2, SAND 1, SAND 2, SAND 3, SAND C, SAND X, TORT 1, and TORT 2. The parameters of g e n e t i c v a r i a b i l i t y w i t h i n p o p u l a t i o n s were estimated based on a l l l o c i r e s o l v e d i n the p o p u l a t i o n , but only 25 commonly recorded l o c i were used f o r N e i - s t a t i s t i c a n a l y s i s . These l o c i and a l l e l e f r e q u e n c i e s are given i n Appendix B. 38 I I I . MIXED MATING SYSTEMS IN HAWAIIAN BIDENS 3.1 I n t r o d u c t i o n In a recent t h e o r e t i c a l study on the e v o l u t i o n of breeding systems, c o n s i d e r i n g the "cost of m e i o s i s " and in b r e e d i n g d e p r e s s i o n , Lande and Schemske (1985) p r e d i c t e d that only complete o u t c r o s s i n g or complete s e l f i n g can be e v o l u t i o n a r i l y s t a b l e . Intermediate s e l f i n g r a t e s , or mixed mating systems, are e v o l u t i o n a r i l y u n s t a b l e , and would evolve towards complete o u t c r o s s i n g or complete s e l f - f e r t i l i z a t i o n depending on whether the l e v e l of in b r e e d i n g depression i n the p o p u l a t i o n was over or below the t h r e s h o l d of 50%. Schemske and Lande (1985) surveyed e m p i r i c a l o b s e r v a t i o n s over the l a s t three decades and concluded that o u t c r o s s i n g r a t e s among 55 s p e c i e s s t u d i e d are bimodally d i s t r i b u t e d , corresponding to the two e v o l u t i o n a r i l y s t a b l e r e p r o d u c t i v e modes of predominant o u t c r o s s i n g and predominant s e l f i n g , as p r e d i c t e d by t h e i r model (Lande and Schemske, 1985). D i f f e r e n t i n t e r p r e t a t i o n s of the e m p i r i c a l data a r i s e i f d i f f e r e n t p o l l i n a t i o n syndromes are taken i n t o account (Aide, 1986), or the non-randomness of the sampled s p e c i e s i s c o n s i d e r e d . Moreover, q u a n t i t a t i v e s t u d i e s of mating systems of the s p e c i e s o c c u r r i n g on oceanic i s l a n d s are m i s s i n g from the e m p i r i c a l data s e t . The 19 sp e c i e s and 8 subspecies of Bidens endemic to the Hawaiian I s l a n d s e x h i b i t f a r more d i v e r s i t y i n morphology and ecology than the r e s t of the genus (about 200 sp e c i e s ) on f i v e c o n t i n e n t s . These s p e c i e s are b e l i e v e d to be the product of 39 a d a p t i v e r a d i a t i o n i n a s i n g l e a n c e s t r a l s p e c i e s f o l l o w i n g a long d i s t a n c e d i s p e r s a l event ( G i l l e t t and Lim, 1970; G i l l e t t , 1975; Ganders and Nagata, 1983b, 1984; Marchant et a l . , 1984; Helenurm and Ganders, 1985). A l l Hawaiian s p e c i e s of Bidens are herbaceous or woody p e r e n n i a l s with e i t h e r p r o s t r a t e or e r e c t h a b i t , and range from l e s s than 0.3 m to over 3 m i n h e i g h t . They occur i n h a b i t a t s that range from a r i d to s e m i a r i d l a v a flows, rocky, windy c o a s t a l s i t e s , open grassy r i d g e s , montane bogs, to dense r a i n f o r e s t s , with moisture g r a d i e n t s extending from 0.3 m to 7.0 m of annual r a i n f a l l , and through e l e v a t i o n s extending from sea l e v e l to over 2000 m ( G i l l e t t , 1975; Ganders and Nagata, 1984). Besides e x t e n s i v e d i f f e r e n c e s i n growth forms, branching p a t t e r n s , and l e a f and f r u i t c h a r a c t e r s , m o rphological d i v e r s i f i c a t i o n i n t h i s group of s p e c i e s a l s o occurs i n f l o r a l c h a r a c t e r s . There are d i f f e r e n c e s i n flower s i z e , i n c l u d i n g the diameters of flower heads ( c a p i t u l a ) and the numbers of ray and d i s c f l o w e r s ; i n degree of protandry ( p o l l e n r e l e a s e d before the stigmas are r e c e p t i v e ) ; artd i n the amount of seed set i n the absence of p o l l i n a t o r s . F l o r a l f e a t u r e s determine or a f f e c t p l a n t mating systems, as shown i n N i c o t i a n a r u s t i c a (Breese, 1959), T r i t i c u m spp. (Zohary and Imber, 1963), C l a r k i a spp. (Moore and Lewis, 1965; Vasek, 1965), Limnanthes spp. (Arroyo, 1973), Lupinus nanus (Harding et a l . , 1974), Impatiens c a p e n s i s (Waller, 1979), Ipomoea spp. (Ennos, 1981), G i l i a a c h i l l e i f o l i a (Schoen, 1982), Wahlenbergia albomaqinata ( L l o y d et a l . , 1982), and P l e c t r i t i s spp. (Layton and Ganders, 1985). In a d d i t i o n , 40 s p e c i e s of Hawaiian Bidens d i f f e r i n i n f l o r e s c e n c e type, and/or in the t o t a l number of flower heads borne on the p l a n t s . Environmental h e t e r o g e n e i t y and f l o r a l d i v e r s i f i c a t i o n suggest that Hawaiian s p e c i e s of Bidens might have evolved d i v e r g e n t breeding systems d u r i n g the process of adaptive r a d i a t i o n i n response to d i f f e r e n t s e l e c t i v e regimes in e c o l o g i c a l l y d i v e r s e habitats.. Adaptive r a d i a t i o n , and thus r a p i d s p e c i a t i o n , i n Hawaiian Bidens a l s o suggests frequent occurrences of founder events. S p e c i a t i o n and s u b s p e c i a t i o n i n Hawaiian Bidens have o f t e n o c c u r r e d a f t e r marginal p o p u l a t i o n s invaded new h a b i t a t s on the same i s l a n d s , or c o l o n i z e d d i f f e r e n t , u s u a l l y younger i s l a n d s (Ganders and Nagata, 1984). Repeated c o l o n i z a t i o n of new areas by s i n g l e or a few i n d i v i d u a l s , and s e l e c t i o n f o r l o c a l a d a p t a t i o n , t h e o r e t i c a l l y , promote the e v o l u t i o n of s e l f -f e r t i l i z a t i o n ( Stebbins, 1957; Antonovics, 1968; J a i n , 1976; Lloyd,1979; Lande and Schemske, 1985). Hawaiian s p e c i e s of Bidens are s e l f - c o m p a t i b l e ( G i l l e t t and Lim, 1970; R i t l a n d and Ganders, 1985; present study), as are most s p e c i e s i n the Hawaiian f l o r a (Stephens, 1964; R o e l o f s , 1979; Kores, 1979; Rabakonandrianina, 1980; Cory, 1984; Lowrey and Crawford, 1985). Regardless of s e l f - c o m p a t i b i l i t y , a l l Hawaiian s p e c i e s of Bidens are s t r o n g l y protandrous, and 13 of them are gynodioecious. There i s evidence that gynodioecy i n Hawaiian Bidens evolved a f t e r the long d i s t a n c e d i s p e r s a l (see Chapters 5-7). Protandry and gynodioecy suggest a high l e v e l of o u t c r o s s i n g i n these s p e c i e s . Mating systems of Hawaiian Bidens, and l i k e w i s e , of 41 many other Hawaiian s p e c i e s , t h e r e f o r e , must be a dynamic outcome of c o u n t e r a c t i v e e v o l u t i o n a r y f o r c e s . A q u a n t i t a t i v e study of the mating system i s necessary f o r a b e t t e r understanding of e v o l u t i o n of p l a n t s p e c i e s o c c u r r i n g on oceanic i s l a n d s . In t h i s chapter, I p r e s e n t : 1) measurements of f l o r a l f e a t u r e s f o r 12 taxa of Hawaiian Bidens; 2) estimates of o u t c r o s s i n g r a t e s of 15 p o p u l a t i o n s d i s t r i b u t e d on 4 major Hawaiian i s l a n d s over a wide range of h a b i t a t s , to determine i f o u t c r o s s i n g r a t e s are bimodally d i s t r i b u t e d w i t h i n t h i s group of s p e c i e s of the same genus; and 3) an a l y s e s of the r e l a t i o n s h i p between f l o r a l f e a t u r e s and o u t c r o s s i n g r a t e s i n t h i s group of s p e c i e s , to eva l u a t e the r e l a t i v e importance of f l o r a l mechanisms as g e n e t i c determinants of mating systems. 3.2 M a t e r i a l s And Methods 3.2.1 F i e l d C o l l e c t i o n Seeds (achenes) from 21 p o p u l a t i o n s of 12 taxa o c c u r r i n g on 4 major Hawaiian i s l a n d s (Kauai, Oahu, Maui and Hawaii) were c o l l e c t e d i n February, June, and November, 1984, and February 1985. D e t a i l e d s i t e i n f o r m a t i o n on i n d i v i d u a l p o p u l a t i o n s has been given i n Table 2-1 i n Chapter 2. Seeds c o l l e c t e d from each i n d i v i d u a l p l a n t , r e f e r r e d t o as f a m i l i e s , were kept separate. In l a r g e p o p u l a t i o n s (>500 p l a n t s ) , many p l a n t s (>100) d i s t r i b u t e d i n a broad area were sampled f o r seeds. In small p o p u l a t i o n s (<100 p l a n t s ) , every i n d i v i d u a l was searched f o r seeds. Maternal sex types were recorded f o r gynodioecious p o p u l a t i o n s i f the p o p u l a t i o n s were f l o w e r i n g and maternal sex 42 types were d i s t i n g u i s h a b l e . Seeds were a l s o c o l l e c t e d f o r those p o p u l a t i o n s , known to be gynodioecious, but past blooming season. These seeds were kept i n separate envelopes at room temperature u n t i l use. 3.2.2 Measuring F l o r a l Features And Reproductive Parameters The numbers of ray flowers and d i s k f l o w e r s , and diameter of the c a pitulum were measured f o r p l a n t s r a i s e d i n greenhouses. These p l a n t s were i n i t i a l l y c o l l e c t e d from w i l d p o p u l a t i o n s on the Hawaiian I s l a n d s as seeds or c u t t i n g s . The f l o r a l f e a t u r e s of Hawaiian s p e c i e s of Bidens are c o n s i s t e n t w i t h i n taxa and show l i t t l e d i f f e r e n c e between greenhouse grown p l a n t s and w i l d p l a n t s ( G i l l e t t and Lim, 1970). T h e r e f o r e , small sample s i z e s f o r these f l o r a l f e a t u r e s are s u f f i c i e n t . The developmental r a t e of a n t h e s i s of the d i s c flowers w i t h i n s i n g l e c a p i t u l a was measured f o r greenhouse p l a n t s by r e c o r d i n g t i m i n g d i f f e r e n c e s i n a n t h e s i s among d i s c f l o w e r s . I checked the tagged f l o w e r i n g c a p i t u l a once or twice a day to observe the way i n which flowers i n each c a p i t u l u m opened and the d u r a t i o n of a n t h e s i s f o r each i n d i v i d u a l flower and f o r each capitulum. The degree of protandry, represented by the time i n t e r v a l between p o l l e n p r e s e n t a t i o n and stigma r e c e p t i v i t y , was recorded f o r marginal d i s c flowers of the cap i t u l u m , s i n c e c e n t r a l d i s c flowers o f t e n vary i n degree of protandry depending on the s i z e of the capitulum. Seed set i n the absence of p o l l i n a t o r s was i n v e s t i g a t e d by p l a c i n g p l a n t s i n s i d e l a r g e i n s e c t - p r o o f cages i n greenhouses. The number of d i s c flowers of each randomly sampled flower head was counted at a n t h e s i s , and the t o t a l seeds on each of the caged p l a n t s were 43 counted three weeks l a t e r . The seedset per ovule of each of these caged p l a n t s was c a l c u l a t e d by d i v i d i n g the number of autoseeds ( i . e . , seeds produced by automatic s e l f - f e r t i l i z a t i o n ) by the t o t a l number of d i s c flowers sampled. The i n d i v i d u a l percentages of auto-seeds were averaged to o b t a i n a mean value f o r each s p e c i e s . A r t i f i c i a l p o l l i n a t i o n s were conducted to t e s t stigma r e c e p t i v i t y . S e l f - c o m p a t i b i l i t y was i n v e s t i g a t e d by rubbing f l o w e r i n g heads on the same p l a n t s together to b r i n g about s e l f - f e r t i l i z a t i o n . P o l l e n - o v u l e r a t i o s were i n v e s t i g a t e d f o r p l a n t s of B_;_ hawaiensis, B. m e n z i e s i i , B. m o l o k a i e n s i s and B. s a n d v i c e n s i s grown in greenhouses, with a sample s i z e of 10 flowers f o r each s p e c i e s . P o l l e n g r a i n s were a c e t o l y z e d and counted with a hemocytometer using the methods of Ganders (1974). For determining p o l l e n s t a i n a b i l i t y , more than.200 . f r e s h p o l l e n g r a i n s were c o l l e c t e d r e s p e c t i v e l y from p l a n t s of B.  s a n d v i c e n s i s subsp. confusa, B. f o r b e s i i subsp. f o r b e s i i , and F1 and F2 h y b r i d s of s e v e r a l other s p e c i e s r a i s e d i n greenhouses, and s t a i n e d with l a c t o p h e n o l blue o v e r n i g h t , and counted under a microscope. 3.2.3 E s t i m a t i o n Of O u t c r o s s i n g Rates H o r i z o n t a l s t a r c h g e l e l e c t r o p h o r e s i s was conducted f o r 21 n a t u r a l p o p u l a t i o n s of Hawaiian Bidens. D e t a i l e d e l e c t r o p h o r e t i c procedures and enzyme systems sampled have been given i n Chapter Chapter 2. Of 21 p o p u l a t i o n s s t u d i e d , both p o p u l a t i o n s of B.  h i l l e b r a n d i a n a were completely f i x e d at the 31 isozyme l o c i assayed. The p o p u l a t i o n s of B^ asymmetrica and IK f o r b e s i i subsp. k a h i l i e n s i s l a c k e d s u f f i c i e n t polymorphic l o c i to 44 estimate o u t c r o s s i n g r a t e ; and sample s i z e was too small to estimate o u t c r o s s i n g r a t e s i n two of the four p o p u l a t i o n s of B.  menziesi i subsp. f i l i f o r m i s . These p o p u l a t i o n s were thus excluded from o u t c r o s s i n g s t u d i e s . Of 38 isozyme l o c i sampled, onl y 3 l o c i , Pgi-1, Pgi-2 and Skdh-3, are g e n e r a l l y polymorphic i n most p o p u l a t i o n s . These three l o c i were used as g e n e t i c markers to estimate o u t c r o s s i n g r a t e s . In g e n e r a l , about 30 f a m i l i e s of 10 p l a n t s each were assayed e l e c t r o p h o r e t i c a l l y i n r e l a t i v e l y l a r g e p o p u l a t i o n s , and a l l a v a i l a b l e seed f a m i l i e s were assayed f o r small p o p u l a t i o n s , with v a r y i n g numbers of progeny depending on the a v a i l a b i l i t y of seeds. The m u l t i l o c u s mixed-mating model of R i t l a n d and J a i n (1981) was used to estimate average o u t c r o s s i n g r a t e (t) f o r the 15 p o p u l a t i o n s with s u f f i c i e n t polymorphic markers. 3.3 R e s u l t s 3.3.1 F l o r a l Features And Reproductive B i o l o g y I n f l o r e s c e n c e - Three types of i n f l o r e s c e n c e are present i n the Hawaiian s p e c i e s of Bidens : s o l i t a r y ; simple, or u s u a l l y compound cymes with a few to many c a p i t u l a ; and compact compound cymes with numerous c a p i t u l a (Table 3-1). Bidens mauiensis has s o l i t a r y i n f l o r e s c e n c e s , each having a long peduncle. The i n f l o r e s c e n c e s of B_;_ amplectens, B. c e r v i c a t a , B. f o r b e s i i , B.  hawaiensis, B. h i l l e b r a n d i a n a , B. s a n d v i c e n s i s T a b l e 3-1. S p e c i e s s t u d i e d f o r o u t c r o s s i n g r a t e s , and t h e i r h a b i t a t s , growth forms and I n f l o r e s c e n c e types S p e c i e s H a b i t a t Growth form Inf 1orescence B. amplectens B. c e r v i c a t a B. f o r b e s i i Dry, windward c l i f f s of mtns. on s e a c o a s t . Dry, under t r e e s & a l o n g r o a d c u t s , on r i d g e . Wet c o a s t a l c l i f f s . subsp. forbes11 Medium woody shrub. Medium t o t a l l woody shrub. Medium to t a l l woody shrub. Loose compound cyme, fewer heads. Loose compound cyme, many heads. Loose compound cyme, many heads. B. h a w a i e n s i s B. h i 1 l e b r a n d l a n a subsp. p o l y c e p h a l a  B. m a u i e n s i s B. m e n z i e s i i subsp. f i 1 i f o r m i s  B. sandv i cens i s subsp. c o n f u s a subsp. sandv i cens i s Dry, hot, exposed l a v a f l o w s . Sea c l i f f s , exposed to sea s p r a y . Dry, rocky or sandy h i l l s , near s e a c o a s t . Dry c i n d e r cones. Dry to wet canyon s1 opes. Mesic r i d g e s & r o a d c u t s . Medium t o t a l l woody shrub. Low, p r o s t r a t e to decumbent herb. Low, p r o s t r a t e to decumbent herb. T a l l woody shrub. Low, s l i g h t l y woody herb. Low, s l i g h t l y woody herb. Loose compound cyme, fewer heads. Loose compound cyme, fewer heads. S o l i t a r y . Compact compound cyme, many heads. Loose compound cyme, fewer heads. Loose compound cyme, fewer heads. 4> 46 and torta are loose compound cymes. Of these, IK amplectens and IK hawaiensis have fewer capitula on t h e i r inflorescences than the other species, but have larger flower s i z e . Bidens  menziesi i has large compact compound cymes with numerous flowering heads at anthesis. F l o r a l size - A l l Hawaiian species of Bidens have showy ray flowers. These ray flowers are bright-yellow, and s t e r i l e . The disc flowers are perfect in hermaphroditic species and in the hermaphrodites of gynodioecious species, but are completely male-s t e r i l e in female plants. The diameters of flowering heads (with disc and ray flowers included), although variable within species, are d i s c r e t e l y d i f f e r e n t among some species. The most variable component of f l o r a l size within a species i s the number of disc flowers, ranging from 4-12 in B_j_ menziesi i subsp. f i l i f ormis, and from 43-108 in IK_ amplectens. The diameters of flowering heads vary from about 15mm in IK menziesii subsp. f i l i f o r m i s to about 50-70mm in IK amplectens among the species studied (Table 3-2). The number of ray flowers varies from 3 to more than 11, but i s usually f i v e in those species with r e l a t i v e l y small flowering heads. The measures of f l o r a l size are p o s i t i v e l y correlated with each other (Table 3-3). Protandry and flowering phenology - Anthesis of disc flowers occurs over several days, proceeding c e n t r i p e t a l l y in the capitulum. The length of time from the f i r s t to the last disc flower to bloom i s commonly 3-4 days, ranging from 2-5 days, among these species (Table 3-4). A l l the disc flowers are strongly protandrous. Prior to anthesis, adjacent anther T a b l e 3-2. F l o r a l s i z e s of 10 taxa of Hawaiian Bidens. Taxon Sample No. of pi a n t s s i z e No. of c a p i tu1 a D i ameter of c a p i t u l u m (mm) No. of d i s c f l o w e r s per c a p i t u l u m (range) No. of ray f l o w e r s per c a p i t u l u m (range) B. amplectens 10 25 50-70 73±17(43-108) 8 B. c e r v i c a t a 3 10 18±2 (16-21 ) 5 (3-5) B. f o r b e s i i subsp. f o r b e s i i 1 1 €8 25-30 18±3 (13-24) 5 (3-6) B. ha w a i e n s i s 2 1 1 35-40 16±3 (10-20) 7 (5-8) B. h i 1 1 e b r a n d i a n a subsp. p o l y c e p h a l a 4 22 15 18±2 (11-22) 5 (5-6) B. ma u i e n s i s 17 17 38 25±4 ( 17-31 ) 6-1 1 B. menz i es11 subsp. f i l i f o r m Is 15 100 15 9£1 ( 4-12) 5 (4-6) B. s a n d v i c e n s I s subsp. c o n f u s a 83 399 25-40 14±2 ( 8-24) 5 (5-6) subsp. s a n d v i c e n s 1 s 40 1 16 21-25 15*3 (10-22) 5 (5-6) B. t o r t a 9 83 25 16±3 (10-22) 5 (4-8) Table 3-3. Correlation matrix of outcrossing rate (t), floral features, and autoseeds in 8 taxa of Hawaiian Bidens. 8 O F - 6 R» . 0 5 0 0 ' . 7 0 6 7 R» . 0 1 O O - . 8 3 4 3 VAR 1 ABLE if 1 t 1 . 0 0 0 0 2 N d l s c . 1 4 6 5 1 O O O O 3 Nray . 4 1 1 5 . 6 7 1 5 1 O O O O 4 . Diameter . 3 4 1 2 9 6 2 9 * * . 7 3 4 5 * 1 O O O O 5 .Pexpos. - . 0 7 3 1 . 3 0 5 0 - . 1 3 0 9 . 3 3 2 9 1 O O O O 6 Sexpos. . 1 G 0 7 4 0 1 2 . 0 3 2 5 . 4 2 3 6 9 1 5 7 * * 1 O O O O 7 Sphase(d) - . 1 6 4 7 - . 0 3 4 7 - . 0 8 2 8 . 0 7 5 3 5 9 1 9 4 1 5 3 1 oooo 8 . P r o t a n d r y - . 3 0 1 6 - . 4 5 8 8 - . 5 6 8 2 - . 4 5 7 5 - . 1 4 5 6 - . 2 6 9 2 . 4 5 6 1 1 . 0 O 0 0 9 .Pphase(h) - . 3 0 3 6 . 1 1 1 2 - . 3 3 5 4 . 1 2 6 0 ** . 8 6 4 8 . 7 7 8 2 * * . 7 8 0 3 . 3 4 0 7 1 O O O O 1 0 .Sphaseth) - . 0 3 6 1 . 1 1 3 4 - . 0 7 3 1 . 2 0 7 9 * . 7 9 9 9 . 7 1 4 2 * ** . 9 2 9 5 . 2 8 8 5 . 9 1 7 5 * * 1 O O O O 11 .Autoseeds . 1 8 0 O - . 1 8 0 7 - . 2 9 2 9 - . 1 7 3 2 . 1 9 1 8 . 1 4 3 0 - . 3 6 3 4 - . 3 7 3 7 - . 1 1 0 0 - . 2 3 2 2 1 O O O O 1 . 2 3 . 4 . 5 . 6 . 7 8 9 1 0 1 1 t N d l s c Nray Diameter Pexpos. Sexpos. Sphase(d) P r o t a n d r y Pphse(h) Sphase(h) Autoseeds * p < .05. **p < .01. // 2: No. of disc flowers per head; 3: No. of ray flowers per head; A: Diameter of flower heads; 5: Length of pollen exposing for all disc flowers; 6: Length of stigma exposing for a l l disc flowers; 7: Stigma phase in the disc flower; 8: Degree of protandry; 9: Pollen phase per head; 10: Stigma phase per head; 11: Seed set In the absence of pollinators. 49 T a b l e 3-4. D u r a t i o n of p r o t a n d r y , p o l l e n and stigma phase, and developmental r a t e of a n t h e s i s ( r e p r e s e n t e d by the l e n g t h of time i n which p o l l e n o r stigmas a r e exposed i n a l l d i s c f l o w e r s of one head) i n 10 taxa of Hawaiian B1 dens. A n t h e s i s i n the cap 1tulum P r o t a n d r y Expos i ng Expos i ng Pol 1 en St igma Taxon (days) pol1 en s t i gmas phase phase (days) (days) (days) (days) B. amplectens 3 4-5 3-4 6-7 7-8 B. ca m p y l o t h e c a 7 5 4 1 1 9 B. c e r v i c a t a 3.5-4 4 3 6-6.5 6.5-7.5 B. f o r b e s i i 4 3 2 6 6-6.5 subsp. f o r b e s i i B. h a w a i e n s i s 3 2 2 5 4 8. m a u i e n s i s 3-3.5 4 3 6-6.5 8-9 B. menz i e s 1 i 3 2 2 5 5 subsp. m e n z i e s i 1 B. s a n d v i c e n s 1 s subsp. c o n f u s a 4-5 3-4 3 7-8 7-8 subsp. s a n d v 1 c e n s I s 3-4 3-4 3 6-7 7 B. t o r t a 3 4 2-3.5 5-6.5 5-6.5 5 0 l o c u l e s dehisce to r e l e a s e p o l l e n g r a i n s i n s i d e the anther tube. P o l l e n g r a i n s are then c o l l e c t e d by the p a p i l l o s e s t y l e as i t c o n t i n u e s growing through the anther tube. P o l l e n g r a i n s i n a l l Hawaiian s p e c i e s of Bidens are spiny, which may f a c i l i t a t e the attachment of the p o l l e n mass to the c l o s e d s t y l e branches as the s t y l e extends out of the anther tube s h o r t l y a f t e r the d i s c flower opens. P o l l e n i s presented on the c l o s e d s t y l e branches 2-3mm above anthers commonly f o r 3-4 days, with an extreme of 7 days i n IK campylotheca (Table 3-4) before the two s t y l e branches d i v e r g e to expose the s t i g m a t i c s u r f a c e s . The e n t i r e p o l l e n phase i n a s i n g l e c a p i t u l u m i s u s u a l l y about 6-8 days, which equals the l e n g t h of protandry. i n a s i n g l e f l o r e t p l u s the l e n g t h of time r e q u i r e d f o r a n t h e s i s of a l l f l o r e t s i n the ca p i t u l u m . The c e n t r a l d i s c flowers g e n e r a l l y remain protandrous one day l e s s than those i n the margin of the capitulum. I t takes 1.5-2 days f o r s t y l e branches to diverge and recurve i n t o a c i r c l e , and they remain i n t h i s s t a t e f o r 2 or more days before the d i s c flower f a l l s o f f . Stigma r e c e p t i v i t y i n a s i n g l e d i s c flower i s 36-48 hours, which s t a r t s a f t e r s t y l e branches have d i v e r g e d i n t o a l i n e (about 180 degree), or beyond, as shown by a r t i f i c i a l p o l l i n a t i o n s performed on IK campylotheca, B.  hawaiensis, B. p o p u l i f o l i a , B. s a n d v i c e n s i s subsp. s a n d v i c e n s i s , using s a n d v i c e n s i s subsp. confusa as a common p o l l e n donor. The e n t i r e r e c e p t i v e stigma phase i n a s i n g l e c a p i t u l u m i s about 6-8 days i n most s p e c i e s , which equals the l e n g t h of the stigma phase f o r a s i n g l e f l o r e t p l u s the time of a n t h e s i s i n the capitulum minus 1 day. 51 Almost a l l Hawaiian s p e c i e s of Bidens have a prolonged f l o w e r i n g season, ranging from about 2 months i n JK f o r b e s i i subsp. k a h i l i e n s i s to about h a l f a year i n IK s a n d v i c e n s i s subsp. confusa. Some taxa have a peak f l o w e r i n g season f o r a few months, but c o n t a i n some f l o w e r i n g i n d i v i d u a l s a l l through the year. The main f l o w e r i n g season f o r most s p e c i e s tends to be from February to May, as i n IK amplectens, B. asymmetrica, B.  c e r v i c a t a , B. cosmoides, B. f o r b e s i i subsp. f o r b e s i i , B.  hawaiensis, B. h i l l e b r a n d i a n a , B. mauiensis, and B.  s a n d v i c e n s i s subsp. s a n d v i c e n s i s . The peak f l o w e r i n g season i s May-June i n IK f o r b e s i i subsp. k a h i l i e n s i s ; October-November i n IK menziesi i subsp. f i l i f o r m i s ; October-June i n B.  s a n d v i c e n s i s subsp. confusa; and December-March i n IK t o r t a . The dry season i s from July-September, d u r i n g which most p o p u l a t i o n s probably experience drought s t r e s s (Ganders, p e r s . comm.). P o l l i n a t i o n - P o l l i n a t o r s which have been observed to v i s i t Bidens i n n a t u r a l p o p u l a t i o n s are mainly honeybees (Apis  m e l l i f e r a ) , s y r p h i d f l i e s i n the fa m i l y Syrphidae, t a c h i n i d f l i e s i n the fa m i l y T a c h i n i d a e , and o c c a s i o n a l l y moths i n the fa m i l y P y r a l i d a e and some n a t i v e wasps. True bugs (Nysius spp.) and b e e t l e s have a l s o been observed on flowers of Bidens. These i n s e c t s feed on ovules and p o l l e n , but whether they a c t u a l l y b r i n g about p o l l i n a t i o n i s unknown (Ganders, p e r s . comm., and Guppy, p e r s . comm.). P o l l i n a t o r s v i s i t d i s c f l owers f o r nectar, and probably f o r p o l l e n as w e l l . Disc flowers s e c r e t e nectar from a t u b u l a r nectary at the base of the c o r o l l a tube. The 52 nectar produced o f t e n f i l l s the e n t i r e c o r o l l a tube of a d i s c flower i n e a r l y a n t h e s i s . The amount of nectar p r o d u c t i o n and sugar c o n c e n t r a t i o n were s i m i l a r i n the s p e c i e s of present study, 0.3 - 0.5 p i per d i s c flower, with an average sugar c o n c e n t r a t i o n of 62% (Ganders and Nagata, 1983). P o l l i n a t o r behavior was observed i n a greenhouse. Bees v i s i t i n g f l o w e r i n g heads i n s e r t e d t h e i r probosces i n t o the c o r o l l a tubes f o r n e c t a r , and v i s i t e d n e a r l y a l l d i s c flowers which were at e a r l y a n t h e s i s . The p o l l e n g r a i n s on s t y l e branches c o u l d be c a r r i e d on the heads of the bees, and then d e p o s i t e d on the exposed stigma s u r f a c e s of other open d i s c f l o w e r s . The p o l l i n a t o r s o f t e n v i s i t e d more than one f l o w e r i n g head on the same p l a n t before f l y i n g to other p l a n t s . Geitonogamy ( p o l l i n a t i o n of one flower by another flower on the same p l a n t ) c o u l d thus be brought about f r e q u e n t l y . 53 S e l f - c o m p a t i b i l i t y and seed-set i n the absence of  p o l l i n a t o r s - A l l experimental s e l f - p o l l i n a t i o n s produced abundant to f u l l complements of seeds. S e l f - i n c o m p a t i b i l i t y was not found i n any of the s p e c i e s s t u d i e d . A l l i n t e r s p e c i f i c experimental c r o s s e s produced f e r t i l e F1 and F2 progenies (see Chapter 6). In the absence of p o l l i n a t o r s , however, most s p e c i e s set no seeds (Table 3-5). The low seed s e t , about 1%, i n B.  mauiensis, B. s a n d v i c e n s i s subsp. s a n d v i c e n s i s and BL v a l i d a was probably due to a c c i d e n t a l s e l f - f e r t i l i z a t i o n , or geitonogamy brought about by p o l l e n f a l l i n g on the stigma of d i s c flowers of the same p l a n t . The higher percent of auto-seedset observed i n B. c e r v i c a t a , B. h i l l e b r a n d i a n a subsp. p o l y c e p h a l a , B.  m e n z i e s i i subsp. f i l i f o r m i s and B_^  t o r t a c o u l d not be accounted fo r by a c c i d e n t a l geitonogamy, but was more l i k e l y caused by a low frequency of n a t u r a l s e l f - f e r t i l i z a t i o n or autogamy. V a r i a t i o n s i n auto-seedset among p l a n t s w i t h i n these taxa were observed. In BL h i l l e b r a n d i a n a , of 4 p l a n t s caged, 2 i n d i v i d u a l s set no seeds, and the others set 7-8% seeds of the t o t a l number of o v u l e s borne on the heads. In B^ t o r t a , seed set was about 10% on average i n 3 p l a n t s , with one of the i n d i v i d u a l s s e t t i n g 26% seeds. T h i s i n d i v i d u a l p l a n t came from a d i f f e r e n t p o p u l a t i o n than the o t h e r s . I t i s not c l e a r whether the u n u s u a l l y high auto-seedset observed i n t h i s p l a n t i s c h a r a c t e r i s t i c of the e n t i r e p o p u l a t i o n , or j u s t of t h i s i n d i v i d u a l . Two of the caged p l a n t s of EL t o r t a set no seeds. The h i g h e s t auto-seedset observed i n IK hawaiensis, 24% on average, i s due to r e v e r s e d stigma r e c e p t i v i t y i n the flower 54 T a b l e 3-5 P e r c e n t s e e d s e t In the absence of p o l l i n a t o r s i n 12 taxa of Hawaiian B i dens . Sample s i z e T o t a l "A Taxon No. of No. of No. of No. of seed s e t p l a n t s c a p i t u l a d i s c f l o w e r s seeds (range) B. amp 1ectens 1 7 B. campy 1otheca 1 2 B . e e r y 1cata 2 32 B. f o r b e s i i subsp. f o r b e s i i 1 10 B. hawa i ens i s 2 10 B. h i l l e b r a n d i ana subsp. po1ycepha1 a 4 32 B. mau i ens i s 6 57 B . menz i es i t subsp. f i 1 i form i s 4 117 B. sandv i cens i s subsp. c o n f u s a 1 13 subsp. sandv i cens i s 7 138 B. t o r t a 5 112 B. va 1 i da 2 6 256 0 0 50 0 0 541 26 5 ( 1 - 6 ) 140 0 0 166 39 24 (16-33) 583 29 5 (0-9) 1117 16 1 (0-6) 800 4 1 5 (1-10) 199 0 0 2270 12 1 (0-2) 1643 162 10 (0-26) 140 2 1 (0-1) 55 heads, i . e . , the s t y l e branches of c e n t r a l d i s c flowers open before those of marginal d i s c f l owers, which r e s u l t s i n the weakest protandry i n the c e n t r a l d i s c flowers i n t h i s s p e c i e s . The g e n e r a l l y low a u t o - f e r t i l i z a t i o n i n Hawaiian Bidens was a l s o confirmed by o b s e r v a t i o n s of zero to low auto-seedset i n p l a n t s grown i n greenhouses dur i n g winter months, when p o l l i n a t o r a c t i v i t y i s r a r e . Agamospermy does not occur i n the Hawaiian s p e c i e s of Bidens. V e g e t a t i v e r e p r o d u c t i o n i n Hawaiian s p e c i e s of Bidens i s uncommon. V e g e t a t i v e r e p r o d u c t i o n was only observed i n p o p u l a t i o n s of B_^  cosmoides, and r a r e l y i n B_^  t o r t a . The lower branches on some p l a n t s can root at the nodes where they touch the s o i l s u r f a c e , as reported by G i l l e t t and Lim ( 1 9 7 0 ) . B.  cosmoides, which u s u a l l y grows under the wet f o r e s t canopy, can p o s s i b l y form l a r g e c l o n e s . P o l l e n - ovule r a t i o , p o l l e n s i z e and p o l l e n s t a i n a b i l i t y Each d i s c flower c o n t a i n s only one ovule and so can produce only one seed (achene) i n Bidens. The average number of p o l l e n g r a i n s i n the anthers of the i n d i v i d u a l d i s c flowers of a s p e c i e s thus equals the p o l l e n - ovule r a t i o of the s p e c i e s . The numbers of p o l l e n g r a i n s produced by d i s c flowers were counted f o r B.  hawaiensis, B. m e n z i e s i i subsp. f i l i f o r m i s , B. m o l o k a i e n s i s , and B_^  s a n d v i c e n s i s subsp. s a n d v i c e n s i s. The average value of the p o l l e n - o v u l e r a t i o i n these s p e c i e s was 3 3 8 2 ( ± 2 4 0 1 ) , and the average p o l l e n s i z e was 1 9 . 8 ( + 3 . 9 J u m . P o l l e n s t a i n a b i l i t y was g e n e r a l l y high, 9 8 % - 1 0 0 % , f o r a l l p l a n t s of any s p e c i e s examined. 56 3.3.2 O u t c r o s s i n g Rates O u t c r o s s i n g r a t e s were obtained f o r 15 p o p u l a t i o n s of 9 taxa, of which 8 taxa are gynodioecious. O u t c r o s s i n g r a t e s were estimated s e p a r a t e l y f o r the progenies of hermaphrodites and females i n 8 gynodioecious p o p u l a t i o n s of 5 taxa. In the other 5 gynodioecious p o p u l a t i o n s r e p r e s e n t i n g 4 taxa with unknown maternal sex types, o u t c r o s s i n g r a t e s , estimated from the mixed progenies of hermaphrodites and females, represent the average o u t c r o s s i n g r a t e s over sexes i n these p o p u l a t i o n s . O u t c r o s s i n g r a t e s i n gynodioecious p o p u l a t i o n s - O u t c r o s s i n g r a t e s estimated from the progeny of hermaphrodites i n 8 gynodioecious p o p u l a t i o n s range from 0.242 to 0.887, with an average of 0.562. The average o u t c r o s s i n g r a t e s estimated f o r the progeny of female p l a n t s was 0.862, ranging from 0.750 to 1.000. The mean o u t c r o s s i n g r a t e s weighted over sex range from 0.425 to 0.881, with an average of 0.639 (Table 3-6). The d i f f e r e n c e s i n sample s i z e s had no s i g n i f i c a n t e f f e c t s on o u t c r o s s i n g r a t e estimates i n these p o p u l a t i o n s . O u t c r o s s i n g r a t e s i n those gynodioecious p o p u l a t i o n s i n which i n f o r m a t i o n on maternal sex types were not a v a i l a b l e are given i n Table 3-7. The mean o u t c r o s s i n g r a t e i n these p o p u l a t i o n s was 0.651, ranging from 0.560 to 0.803. O u t c r o s s i n g r a t e s i n non-gynodioecious p o p u l a t i o n s Although two non-gynodioecious s p e c i e s , IK_ h i l l e b r a n d i a n a and B.  mauiensis, were s t u d i e d e l e c t r o p h o r e t i c a l l y , only IK mauiensis had s u f f i c i e n t polymorphic l o c i to estimate o u t c r o s s i n g r a t e s (Table 3-8). The two estimates of o u t c r o s s i n g Table 3-6. Outcrossing rates (t) of hermaphrodites and females, frequencies of females (%), and weighted mean outcrossing rates for 8 gynodioecious populations of Hawaiian Bidens. Hermaphrodltes Pop. No. of No. of families progeny t(s.e.) Females No. of No. of families progeny t(s.e.) Frequency Weighted of females mean (%) t CERV 28 344 .608(.078) 14 116 .833(.121) 25 .664 FORB F1 28 262 .506(.148) 19 201 .860(.169) 30 .612 MENZ 1 20 306 .450(.098) 20 176 .755(.155) 44 .584 MENZ 2 25 317 .474(.173) 19 117 1.000(.320) 34 .653 SAND C 24 375 .887(.115) 26 261 .835(.115) 11 .881 SAND 1 30 249 .877(.180) SAND 2 20 191 .242(.107) 11 157 .750(.201) 36 .425 SAND 16 133 .465(.160) 1 1 113 1.000(.160) 35 .652 Mean (s.e.) 23(8) 272(80) .564(.221) 17(5) 163(55) .862(.103) 28(12) .639(.135) U l Table 3-7. O u t c r o s s i n g r a t e s of gynodioecious p o p u l a t i o n s with unknown maternal sex phenotypes. No. of No. of Pop. f a m i l i e s progeny t ( s .e.) AMPL 33 339 .588( . 161 ) HAWA 25 422 .634( . 118) SAND 3 36 391 .803( .142) TORT 1 32 609 .670( .092) TORT 2 1 6 218 .560( .146) Mean(s.e.) 28(8) 396(142) .651 ( .095) Table 3-8. O u t c r o s s i n g r a t e s i n two p o p u l a t i o n s of a non-gynodioecious s p e c i e s , IK mauiensis. Pop. No. of f a m i l i e s No. of progeny t (s . e.) MAUI 1 February 48 463 .682( .094) June 28 243 .580( .125) MAUI 2 15 120 .731 ( .170) Mean(s.e.) 27(16) 237(165) .681 ( .071 ) 60 r a t e i n the p o p u l a t i o n o c c u r r i n g on west Maui were from seeds c o l l e c t e d i n February and June of the same f l o w e r i n g season. February i s e a r l y i n the f l o w e r i n g season, whereas June i s l a t e . The o u t c r o s s i n g r a t e estimated from seeds c o l l e c t e d i n June was lower than i n February, although the d i f f e r e n c e was not s i g n i f i c a n t . The small sample s i z e i n the p o p u l a t i o n o c c u r r i n g on east Maui was due to the small p o p u l a t i o n s i z e . The o u t c r o s s i n g r a t e i n t h i s p o p u l a t i o n was higher, although not s i g n i f i c a n t l y so. The mean o u t c r o s s i n g r a t e f o r t h i s s p e c i e s i s 0.681 . 3.3.3 C o r r e l a t i o n Of F l o r a l Features And O u t c r o s s i n g Rates Although s i g n i f i c a n t d i f f e r e n t i a t i o n occurs i n f l o r a l f e a t u r e s among these s p e c i e s of Hawaiian Bidens, there were no s i g n i f i c a n t c o r r e l a t i o n s between most f l o r a l f e a t u r e s and o u t c r o s s i n g r a t e s (Table 3-3). O u t c r o s s i n g r a t e s , however, appeared to be r e l a t e d to i n f l o r e s c e n c e types i n these s p e c i e s (Table 3-9). S i g n i f i c a n t c o r r e l a t i o n s were found between c e r t a i n f l o r a l f e a t u r e s , such as between the lengths of p o l l e n phase and stigma phase, and between the numbers of d i s c and ray flowers and the diameter of the c a p i t u l u m . 3.4 D i s c u s s i o n 3.4.1 F l o r a l Features Although v a r i a t i o n e x i s t s w i t h i n p l a n t s and taxa of Hawaiian Bidens i n the diameter of c a p i t u l a , number of ray flowers and number of d i s c f l owers, flower and capitulum s i z e s are h e r i t a b l e w i t h i n taxa, and vary s i g n i f i c a n t l y among taxa. T a b l e 3-9. R e l a t i o n s h i p between I n f l o r e s c e n c e types, o u t c r o s s i n g r a t e s ( t ) of h e r m a p h r o d i t e s , and mean female f r e q u e n c i e s i n p o p u l a t i o n s of Hawaiian B i dens. Type of 1 n f ) o r e s c e n c e s p e c l e s No. of popu1 a t f ons t Mean f r e q u e n c y of females {%) Sol 1tary B. m a u i e n s i s 2 .681 0 Compound cymes B. c e r v i c a t a B. f o r b e s i i subsp. f o r b e s i i B. s a n d v i c e n s i s 6 .600 24 Compact compound cymes B. menz i es i i subsp. mez i e s 1 i 2 . 462 39 62 The number of c a p i t u l a borne on the p l a n t s appears to be c o r r e l a t e d with i n f l o r e s c e n c e types, and/or flower s i z e . Bidens  amplectens and IK hawaiensis have the l a r g e s t flower s i z e i n t h i s group of taxa, but have fewer flower heads on the p l a n t s than other taxa of the same i n f l o r e s c e n c e type. On the other hand, IK m e n z i e s i i subsp. f i l i f o r m i s has the s m a l l e s t flower s i z e , but has the l a r g e s t number of flower heads on the p l a n t s . A l l s p e c i e s of Hawaiian Bidens are s t r o n g l y protandrous, and have a long p o l l e n phase and stigma phase i n i n d i v i d u a l flower heads, although c o n s i d e r a b l e v a r i a t i o n occurs among s p e c i e s . The d u r a t i o n of p o l l e n phase and stigma phase per flower head i s c o r r e l a t e d with the l e n g t h of time r e q u i r e d f o r a n t h e s i s of a l l d i s c flowers i n the head (Table 3 - 4 ) , and a f f e c t e d by degree of protandry and the number of d i s c flowers i n the head. Strong protandry i n h i b i t s autogamy, and prolonged p o l l e n phase and stigma phase i n c r e a s e chances of p o l l i n a t i o n as w e l l as p o l l e n d i s p e r s a l . The d i f f e r e n c e i n degree of protandry among d i s c flowers w i t h i n a s i n g l e flower head, about one day l e s s i n the c e n t r a l d i s c flowers than i n the marginal ones, may a l s o have some b i o l o g i c a l s i g n i f i c a n c e . P o l l i n a t o r s p r e f e r e n t i a l l y v i s i t f r e s h l y opened d i s c f l o w e r s . When most of d i s c flowers have passed the optimum nectar producing p e r i o d , the flower head i s l e s s a t t r a c t i v e to p o l l i n a t o r s . The s h o r t e n i n g of protandry i n the c e n t r a l d i s c flowers probably f a c i l i t a t e s the synchrony of the s t i g m a t i c phase of the e n t i r e flower head, and thus i n c r e a s e s the chance of p o l l i n a t i o n of c e n t r a l d i s c f l o w e r s . Having l a r g e f l o r a l d i s p l a y s , extended p e r i o d s of a n t h e s i s w i t h i n i n d i v i d u a l 63 c a p i t u l a , and an extended f l o w e r i n g season, a l l Hawaiian s p e c i e s of Bidens are s u c c e s s f u l i n a t t r a c t i n g p o l l i n a t o r s ( a l s o see Chapter 7). Although s e l f - c o m p a t i b l e , as shown by f u l l seedset i n a r t i f i c i a l s e l f - p o l l i n a t i o n s , the absence or low l e v e l of seedset i n the absence of p o l l i n a t o r s i n d i c a t e s that protandry i s strong enough to prevent s e l f - f e r t i l i z a t i o n of d i s c f l o w e r s , and a l s o i n d i c a t e s the absence of apomixis in Hawaiian s p e c i e s of Bidens. The low l e v e l of seedset i n the absence of p o l l i n a t o r s i n a few s p e c i e s (Table 3-5) may r e s u l t from o c c a s i o n a l breakdown of protandry i n some d i s c f l o w e r s . Although v a r i a b l e among the taxa examined, the p o l l e n - o v u l e r a t i o i n Hawaiian Bidens i s , on average, between the c a t e g o r i e s of xenogamous s p e c i e s and facultative.xenogamous s p e c i e s i n comparison with the data given by Cruden (1977). 3.4.2 O u t c r o s s i n g Rates O u t c r o s s i n g r a t e i s a key component of the mating system. A v a i l a b l e estimates of o u t c r o s s i n g r a t e i n many p l a n t s p e c i e s have shown that a s p e c i e s ' mating system i s not a constant (see Table 3-10). S p a t i a l and temporal v a r i a t i o n s occur both among po p u l a t i o n s w i t h i n s p e c i e s and among c l o s e l y r e l a t e d s p e c i e s . V a r i a t i o n i n o u t c r o s s i n g r a t e i s o f t e n due to d i f f e r e n c e s i n f l o r a l c h a r a c t e r s , p o l l i n a t o r behavior or abundance, p o p u l a t i o n d e n s i t y and probably a d d i t i o n a l g e n e t i c and environmental f a c t o r s . V a r i a t i o n i n o u t c r o s s i n g r a t e - O u t c r o s s i n g r a t e s of hermaphrodites i n 8 gynodioecious p o p u l a t i o n s vary from 64 Table 3-10. Mean outcrossing rates (t) estimated using genetic markers and their interpopulatlonal ranges in natural plant populations. No. of Range Taxon populations t of t Source Asteraceae 81 dens menziesi 1 subsp. fi1If o r m l s  B o r r i c h l a frutescens  Helianthus annuus  Seneclo vulgar 1 s Borag1naceae Amsinckia spectabl1 is Caryophyllaceae Spergularia media .52 .35-.63 RMtland and Ganders, 1985 1.13 Ant 1 finger, 1982 .76 .54-.91 E l l s t r a n d et a l . , 1978 .105 .028-.199 Marshall and Abbott, 1982 .30 .03-.53 Ganders et a l . , 1985 .12 .08-.15 Sterk and DIJkhulzen. 1972 S. mar i na Convo1vu1aceae Ipomoea hederacea I. purpurea Grami neae Avena barbata f atua .015 .01-.02 Sterk and DIJkhulzen, 1972 .07 Ennos, 1981 .70 Ennos, 19S1 .013 Hamrlck and A l l a r d , 1972 .045 .014-.075 Marshall and A l l a r d , 1970 .02 .01-.08 A l l a r d and Kahler, 1971 .020 A l l a r d et a l . , 1972 .06 .01-.12 Imam and A l l a r d , 1965 65 T a b l e 3-10 c o n t . .01 o- .04 A l l a r d and K a h l e r , 1971 A . s t e r 1 1 1 s .04 .00- .09 J a i n , 1975 Bromus mol1 Is 1 .096 .08- .11* Brown et a l . , 1974 Cynosurus c r i s t a t u s 1 .98 .93- 1 .02* Ennos, 1985 Elymus c a n a d e n s i s 15 .069 o- .25 Sanders and Hamrick, 1980 F e s t u c a m i c r o s t a c h y s .0001 o- .0001 Kannenberg and A l l a r d , 1 9 6 7 14 .005 0- .067 Adams and A l l a r d , 1982 Hordeum iubatum 2 .02 .01- .03 Babbel and Wain, 1977 H. spontaneum 26 .016 O- .096 Brown et a l . , 1978 H. v u l g a r e .04 .00- . 15 J a i n , 1975 L o l i u m m u l t i f l o r u m .98 .82 .90- 1 .OO A l l a r d and K a h l e r . 1971 Schemske and Lande, 1985 T r i t i c u m d i c o c c o i d e s 12 .03 Nevo et a l . , 1982 T. s p e l t o i d e s .85 Zohary and Imber, 1963 ib i a t a e Thymus v u l g a r i s 4 .68 .51- .90 V a l d e y r o n et al.,1977 ;gum i nosae Lu p i n u s a f f i n i s 2 . 35 . 13-.50 H a r d i n g e t a1., 1974 L. b i c o l o r 9 .041 .00- . 29 H a r d i n g e t a l . , 1974 L. nanus 21 .45 . o-1 .00 H a r d i n g et a l . , 1974 L. p a c h y l o b u s 1 .00 H a r d i n g et a l . , 1974 L. p o l y c a r p u s 2 .01 o- . 10 H a r d i n g et a l . , 1974 L. s u c c u l e n t u s 29 .46 . 138 -.971 H a r d i n g and Barnes, 1977 L. p i 1 o s u s 1 . 30 H o r o v i t z and Harding, 1983 Medicago polymorpha .082 .03- . 15 J a i n , 1975 S t y l o s a n t h e s s c a b r a 2 .018 .017 - .026 S t a c e , 1982 T r i f o l i u m s u b t e r r a n e a n 4 .0015 0- .0022 M a r s h a l l and Broue, 1973 T. h i r t u m .045 .01- .09 d a i n , 1975 66 T a b l e 3-10 c o n t . 21 L i mnanthaceae L imnanthes a l b a * * doug 1 as i i Myrtaceae E u c a l y p t u s d e l e g a t e n s i s E. o b i i q u a E. pauc i f 1 o r a Onagraceae C1 ark i a ex 11 i s C. t e m b l o r i e n s i s C, ungu i c u 1 a t a Oenothera organens i s Papavaraceae Papaver dub i um P i naceae P i c e a ab i es P i n u s e l 1iot11 P. ponderosa P. r a d l a t a P. s y 1 v e s t r 1 s  Pseudotsuga menzies11 .047 .01-. 10 J a i n and M a r t i n s , 1979 .78 .43-.97 J a i n , 1978 .80 R i t l a n d and J a i n , 1981 .76 .75-.76 K e s s e l i and J a i n , 1984 .77 .66-.85* Moran and Brown, 1980 .76 .64-.84 Brown e t a l . . 1975 .69 .62-.84 P h i l l i p s and Brown. 1977 .44 .43-.45 Vasek, 1964, 1967 .57 .38-.89 Vasek and Ha r d i n g , 1976 .55 .08-.83 Vasek and h a r d i n g , 1976 .96 Vasek, 1965 1.05 .74-1.27 L e v i n e t a l . , 1979 .25 .19-.29 Humphreys and Gale, 1974 .89 .87-93 L u n d q u i s t , 1979 .94 Schemske and Lande, 1985 . 96 Mi t t o n et a l . , 198 1 .96 Mi t t o n et a l . , 1977 .98 Schemske and Lande, 1985 .95 G u r i e s and L e d i g . 1982 .91 Schemske and Lande, 1985 .90 .86-.96 Shaw and A l l a r d , 1982 .90 E l - K a s s a b y e t a l . , 1981 Polemonlaceae 67 T a b l e 3-10 c o n t . G i 1 i a a c h i 1 l e i f o l i a 7 Phi ox c u s p i d a t a 1 P r o t e a c e a e B a n k s i a a t t e n u a t a 1 B. menz i es i i • 1 S c r o p h u l a r i aceae C o l l i ns i a heterophy11 a 3 C. s p a r s i f 1 o r a Va1er i anaceae P 1 e c t r i t i s brachystemom 9 P. c o n g e s t a 15 . 57 .22 1 . 10 1 .04 .93 . 10 .024 . 702 15-.96 .84-1.00 .01-.50 0-.074 .421-1.077 Schoen, 1982 L e v i n , 1978 S c o t t , 1980 S c o t t , 1980 Wei 1 and A l l a r d , 1964 A l l a r d and K a h l e r , 1971 Layton and Ganders, 1985 Layton and Ganders, 1985 Range of o u t c r o s s i n g r a t e s among s i t e s or s u b p o p u l a t i o n s w i t h i n p o p u l a t i o n s : O u t c r o s s i n g r a t e s of h e r m a p h r o d i t e s i n g y n o d i o e c i o u s p o p u l a t i o n s . 68 0.242-0.887, with a mean value of 0.564. However, v a r i a t i o n i n weighted mean o u t c r o s s i n g r a t e s f o r these p o p u l a t i o n s (ranging from 0.425-0.881, with a mean value of 0.639) i s l e s s e x t e n s i v e . The mean weighted o u t c r o s s i n g r a t e s f o r s p e c i e s show even l e s s v a r i a t i o n , ranging from 0.588-0.728, with an average value of 0.643 over s p e c i e s (Table 3-11). In IL s a n d v i c e n s i s , of which 5 p o p u l a t i o n s were s t u d i e d , o u t c r o s s i n g r a t e s , estimated from progeny of hermaphrodites, v a r i e d among p o p u l a t i o n s from 0.242-0.887, which covered the e n t i r e range of o u t c r o s s i n g r a t e s estimated f o r a l l the p o p u l a t i o n s of Hawaiian Bidens i n t h i s study. V a r i a t i o n i n o u t c r o s s i n g r a t e s i n p o p u l a t i o n s of B.  s a n d v i c e n s i s i s c o r r e l a t e d with female f r e q u e n c i e s (see Chapter 7), thus s e l e c t i v e f o r c e s , which are a c t i v e i n m a i n t a i n i n g gynodioecy i n t h i s s p e c i e s , probably account f o r most v a r i a t i o n in o u t c r o s s i n g r a t e s among the p o p u l a t i o n s w i t h i n t h i s s p e c i e s , as w e l l as among gynodioecious s p e c i e s of Hawaiian Bidens. V a r i a t i o n i n o u t c r o s s i n g r a t e s among p o p u l a t i o n s w i t h i n a s p e c i e s and between congeneric s p e c i e s has been d i s c u s s e d by s e v e r a l authors ( J a i n , 1975; Schoen, 1982; Hamrick, 1982; Schemske and Lande, 1985). High i n t e r p o p u l a t i o n a l v a r i a t i o n i n o u t c r o s s i n g r a t e s has been r e p o r t e d i n v i r t u a l l y a l l o u t c r o s s i n g s p e c i e s i n which more than 3 p o p u l a t i o n s have been s t u d i e d (see Table 3-10). The l a r g e s p a t i a l and/or temporal v a r i a t i o n i n o u t c r o s s i n g r a t e s i n these s p e c i e s are a s s o c i a t e d with environmental determinants of the breeding system, such as p l a n t or flower d e n s i t y ( A l l a r d and Kahler, 1971, A l l a r d et a l . , 1977). 69 Table 3-11. Mean o u t c r o s s i n g r a t e s ( t) of 8 s p e c i e s of Hawaiian Bidens. Weighted means are given f o r gynodioecious s p e c i e s . No. of Taxa p o p u l a t i o n s t Range of t B. c e r v i c a t a  B. amplectens  B. f o r b e s i i subsp. f o r b e s i i  B. hawaiensis  B. mauiensis  B. m e n z i e s i i subsp. f i l i f o r m i s  B. s a n d v i c e n s i s B. t o r t a 1 1 1 1 2 2 5 2 .664 .588 .612 .634 .681 .619 .728 .615 .631-.731 .584-.653 .425-.881 .560-.670 70 p o l l i n a t o r a c t i v i t y (Harding, i n Schemscke and Lande, 1985), or a s s o c i a t e d with g e n e t i c determinants of the breeding system, such as f l o r a l s t u c t u r e (Vasek, 1967; Vasek and Harding, 1976; Ganders et a l . , 1985), or protandry (Schoen, 1982). P o p u l a t i o n s w i t h i n a s p e c i e s of Hawaiian Bidens have the same or very s i m i l a r f l o r a l f e a t u r e s , s i m i l a r protandry, and g e n e r a l i z e d p o l l i n a t o r s . These f a c t o r s are l e s s l i k e l y to be r e s p o n s i b l e f o r the i n t e r p o p u l a t i o n a l v a r i a t i o n i n o u t c r o s s i n g r a t e i n Hawaiian Bidens. However, p o p u l a t i o n s of Hawaiian Bidens d i f f e r i n p l a n t d e n s i t y . O u t c r o s s i n g r a t e c o u l d be i n f l u e n c e d by p l a n t d e n s i t y through density-dependent p o l l i n a t o r behavior (Levin and K e r s t e r , 1.974). O u t c r o s s i n g r a t e s were n e g a t i v e l y c o r r e l a t e d with p l a n t d e n s i t y i n H e l i a n t h u s annuus ( E l l s t r a n d , et a l . , 1977), but c o r r e l a t i o n was not found i n G i l i a a c h i l l e i f o l i a (Schoen, 1982). Congeneric s p e c i e s can have d i v e r g e n t breeding systems (see Table 3-10 f o r r e f e r e n c e s ) . D i f f e r e n c e s i n o u t c r o s s i n g r a t e s i n c l o s e l y r e l a t e d s p e c i e s are o f t e n g e n e t i c a l l y c o n t r o l l e d through f l o r a l mechanisms. D i f f e r e n t i a t i o n i n f l o r a l s i z e , i n s e p a r a t i o n between stigma and anthers, and i n degree of protandry i n two s p e c i e s of P l e c t r i t i s r e s u l t s i n a c o n t r a s t i n g breeding system, one s p e c i e s being p r i m a r i l y s e l f - f e r t i l i z e d , ( t < 0.03), and another s p e c i e s being predominantly o u t c r o s s e d (t = 0.70) as r e p o r t e d by Ganders et a l . (1977) and Layton and Ganders (1985). The d i v e r g e n t o u t c r o s s i n g r a t e s i n two s p e c i e s of Ipomoea are a s s o c i a t e d with the d i f f e r e n c e i n anther-stigma s e p a r a t i o n (Ennos, 1981). F l o r a l mechanisms are a l s o r e s p o n s i b l e f o r the divergence i n the breeding system i n the A e q i l o p s - T r i t i c u m group 71 of s p e c i e s (Zohary and Imber, 1963). However, mating systems of p h y l o g e n e t i c a l l y r e l a t e d s p e c i e s are probably not a l l b i o l o g i c a l l y independent. S i m i l a r i t y i n f l o r a l c h a r a c t e r s i n the s p e c i e s of Hawaiian Bidens, i n g e n e r a l , g i v e s r i s e to the same mixed mating system, with l i t t l e v a r i a t i o n i n o u t c r o s s i n g r a t e s at the s p e c i e s l e v e l , r e g a r d l e s s of wide g e o g r a p h i c a l or e c o l o g i c a l i s o l a t i o n among these s p e c i e s . Sources of i n b r e e d i n g - Although s e l f - c o m p a t i b l e , strong protandry i n a l l s p e c i e s of Hawaiian Bidens e f f i c i e n t l y prevents automatic s e l f - f e r t i l i z a t i o n . However, s u b s t a n t i a l s e l f i n g occurs i n a l l s p e c i e s s t u d i e d , with s e l f i n g r a t e s of hermaphrodites ranging from 0.319-0.538. These r e l a t i v e l y high r e a l i z e d s e l f i n g r a t e s are probably p r i m a r i l y due to geitonogamy. O u t c r o s s i n g r a t e s may be i n f l u e n c e d by the number of f l o w e r i n g heads on the p l a n t s which i s a s s o c i a t e d with the d i f f e r e n c e i n i n f l o r e s c e n c e types (Table 3-9). In the i n f l o r e s c e n c e of B.  menziesi i , numerous synchronous f l o w e r i n g heads f a c i l i t a t e geitonogamy. P o l l i n a t o r behavior, which has o f t e n been shown to be i n f l u e n c e d by flower d e n s i t y , c o u l d r e g u l a t e the l e v e l of o u t c r o s s i n g by a l t e r i n g the r a t i o of v i s i t s made to the flowers on the same p l a n t to those made to the flowers on d i f f e r e n t p l a n t s . The h i g h flower d e n s i t y i n the i n f l o r e s c e n c e of B.  menziesi i may i n c r e a s e the p r o p o r t i o n of geitonogamy, whereas the few, s o l i t a r y i n f l o r e s c e n c e s of EL mauiensis may p o t e n t i a l l y promote o u t c r o s s i n g . The opposing e v o l u t i o n a r y c o n s t r a i n t s of massive f l o w e r i n g to a t t r a c t p o l l i n a t o r s and the r e s u l t i n g e x c e s s i v e s e l f - f e r t i l i z a t i o n due to geitonogamy are c o n f l i c t s 72 a l s o common to many t r o p i c a l t r e e s (Bawa, 1983; Koptur, 1984). P o p u l a t i o n s u b s t r u c t u r i n g , which can promote consanguineous mating, i s another source of in b r e e d i n g ( E l l s t r a n d and F o s t e r , 1983). Evidence that consanguineous matings c o n t r i b u t e to s e l f i n g has been shown f o r p o p u l a t i o n s of m e n z i e s i i subsp. f i l i f o r m i s ( R i t l a n d and Ganders, 1985). The r e l a t i v e c o n t r i b u t i o n to s e l f i n g by geitonogamy and consanguineous mating can be assessed through the o u t c r o s s i n g r a t e s of female p l a n t s i n the gynodioecious p o p u l a t i o n s of Hawaiian Bidens (Table 3-6). Since geitonogamy i s not p o s s i b l e f o r female p l a n t s , the apparent s e l f i n g r a t e s of female p l a n t s are most probably a t t r i b u t a b l e to mating to r e l a t i v e s . The apparent s e l f i n g r a t e s of females i n 7 gynodioecious p o p u l a t i o n s t h a t ranged from 0-0.25, with a mean value of 0.14, probably r e f l e c t the l e v e l of consanguineous mating i n the p o p u l a t i o n s of Hawaiian Bidens, although a c t u a l l e v e l s of mating to r e l a t i v e s would vary with the extent of p o p u l a t i o n s t r u c t u r i n g w i t h i n each i n d i v i d u a l p o p u l a t i o n . 3.4.3 R e l a t i o n s h i p Between F l o r a l Mechanisms And O u t c r o s s i n g  Rates Gynodioecy - The weighted mean o u t c r o s s i n g r a t e s vary with the r e l a t i v e p r o p o r t i o n s and r e a l i z e d o u t c r o s s i n g r a t e s of hermaphrodites and females i n gynodioecious p o p u l a t i o n s (Table 3-8). The r e a l i z e d o u t c r o s s i n g r a t e s of females i n 4 of 7 p o p u l a t i o n s are s i g n i f i c a n t l y d i f f e r e n t from the expected value of u n i t y under random mating, which i s l i k e l y due to p o p u l a t i o n s u b s t r u c t u r i n g . The mean o u t c r o s s i n g r a t e (0.462) of hermaphrodites of B^ m e n z i e s i i subsp. f i l i f o r m i s i n t h i s study 73 i s lower than the mean o u t c r o s s i n g r a t e (0.52) repo r t e d i n a pr e v i o u s study of the s p e c i e s ( R i t l a n d and Ganders, 1985), but the weighted mean f o r the s p e c i e s (0.619) i n the present study i s h i g h e r . In the present study, progenies of female p l a n t s were s e p a r a t e l y assayed f o r e s t i m a t i n g o u t c r o s s i n g r a t e s , i n order to be a b l e to assess the e f f e c t of male s t e r i l i t y i n i n c r e a s i n g the l e v e l of o u t c r o s s i n g i n gynodioecious p o p u l a t i o n s . In the pr e v i o u s study, the seed progenies c o l l e c t e d i n the p o p u l a t i o n s of the s p e c i e s were not d i s t i n g u i s h a b l e with respect to the maternal genotypes. The di s c r e p a n c y i n o u t c r o s s i n g estimates f o r B. m e n z i e s i i subsp. f i l i f o r m i s between t h i s study and the pr e v i o u s study c o u l d be due to temporal v a r i a t i o n i n e i t h e r f r e q u e n c i e s of females or o u t c r o s s i n g r a t e s of hermaphrodites or females i n p o p u l a t i o n s of t h i s s p e c i e s . -The weighted mean o u t c r o s s i n g r a t e (0.619) i n t h i s study i s about 16% higher than the mean o u t c r o s s i n g r a t e of hermaphrodites (0.462), i n d i c a t i n g t h a t male s t e r i l i t y s u b s t a n t i a l l y i n c r e a s e s the l e v e l of o u t c r o s s i n g i n t h i s s p e c i e s , probably p r i m a r i l y due to the hig h f r e q u e n c i e s of females i n the p o p u l a t i o n s . The average i n c r e a s e of mean o u t c r o s s i n g r a t e caused by male s t e r i l i t y i s 7.5% over 8 gynodioecious p o p u l a t i o n s (Table 3-6). The mean o u t c r o s s i n g r a t e (0.651) estimated from mixed progenies f o r the r e s t of the gynodioecious p o p u l a t i o n s with unknown maternal sex types i s i n c l o s e agreement with the weighted mean value of o u t c r o s s i n g r a t e i n the gynodioecious taxa with d i s t i n g u i s h a b l e maternal sex. The mean o u t c r o s s i n g r a t e (0.681) of a non-gynodioecious s p e c i e s , B.  mauiensis, i s a l s o i n c l o s e agreement with the mean o u t c r o s s i n g 74 r a t e of gynodioecious s p e c i e s . Gynodioecy has been t r a d i t i o n a l l y b e l i e v e d to be a mechanism that i n c r e a s e s o u t c r o s s i n g , and t h i s has been confirmed by t h i s study. But the e f f i c i e n c y of gynodioecy i n i n c r e a s i n g the l e v e l of o u t c r o s s i n g depends h e a v i l y on the c u r r e n t o u t c r o s s i n g r a t e of hermaphrodites, p o p u l a t i o n s u b s t r u c t u r e , and the frequency of females w i t h i n each p o p u l a t i o n , as a l s o suggested by the present study. The o u t c r o s s i n g r a t e s of hermaphrodites are h i g h l y c o r r e l a t e d with the f r e q u e n c i e s of females i n gynodioecious p o p u l a t i o n s of Hawaiian Bidens. The e v o l u t i o n a r y s i g n i f i c a n c e of t h i s c o r r e l a t i o n l i e s i n the maintenance of gynodioecy (see Chapter 7 fo r more d i s c u s s i o n ) . F l o r a l f e a t u r e s - Flower s i z e , although v a r y i n g s i g n i f i c a n t l y among Hawaiian s p e c i e s of Bidens, appears t o have no e f f e c t on o u t c r o s s i n g r a t e s i n these s p e c i e s . Flower s i z e i n Lupinus ( H o r o v i t z and Harding, 1972) and i n G i l i a a c h i l l e i f o l i a (Schoen, 1982) i s not c o r r e l a t e d with o u t c r o s s i n g r a t e . Degree of protandry and seedset i n the absence of p o l l i n a t o r s i n non-apomictic p l a n t s are more r e l i a b l e as p r e d i c t o r s of breeding systems as shown i n N i c o t i a n a r u s t i c a (Breese, 1959), C l a r k i a ssp. (Moore and Lewis, 1965; Vasek, 1965), Limnanthes spp. (Arroyo, 1973), Lupinus nanus (Harding et a l . , 1974), and G i l i a  a c h i l l e i f o l i a (Schoen, 1982). P o l l e n - o v u l e r a t i o serves as a very c o n s e r v a t i v e i n d i c a t o r of breeding system (Cruden, 1977). Strong protandry, extremely low auto-seedset, and r e l a t i v e l y high p o l l e n - o v u l e r a t i o s i n the Hawaiian s p e c i e s of Bidens a l l p r e d i c t a p r i m a r i l y o u t c r o s s i n g breeding system i n these s p e c i e s . 75 Although g e n e r a l l y upheld by t h i s study, the high l e v e l of s e l f i n g i n some s p e c i e s , such as IK m e n z i e s i i subsp. f i l i f o r m i s , i s unexpected, i f only these f a c t o r s are c o n s i d e r e d . I n f l o r e s c e n c e type, which i n d i c a t e s the number of simultaneously f l o w e r i n g heads on a p l a n t , i s a l s o an important determinant of the breeding system i n Hawaiian Bidens. Species such as B.  m e n z i e s i i may have s e v e r a l thousand flowers per p l a n t open at the same time. Geitonogamy r e s u l t s i n s u b s t a n t i a l s e l f i n g i n these s p e c i e s . The mixed mating system i n Hawaiian Bidens i s p r i m a r i l y g e n e t i c a l l y c o n t r o l l e d and probably i n f l u e n c e d by p l a n t d e n s i t y and p o l l i n a t o r behavior. In the s a l t marsh Composite, B o r r i c h i a f r u t e s c e n s , the percentage of p o l l i n a t o r f l i g h t s among flowers on d i f f e r e n t p l a n t s was about 91%, and on the same p l a n t s was 9%. The high o u t c r o s s i n g rate estimated f o r the sp e c i e s (1.13) confirmed the p r e d i c t i o n of a low l e v e l of geitonogamy ( A n t l f i n g e r , 1982). The stro n g protandrous chasmogamous flowers i n Impatiens c a p e n s i s f a i l to set f r u i t i n the absence of p o l l i n a t o r s , and p o l l i n a t o r movements among the flowers on the same p l a n t s b r i n g about a geitonogamy rate of 0.086 (Waller, 1980). 3.4.4 E v o l u t i o n a r y I n t e r p r e t a t i o n Of Mixed Mating Systems In  Hawaiian Bidens N e i t h e r h i g h l y s e l f e d nor very h i g h l y o u t c r o s s e d s p e c i e s have evolved i n Hawaiian Bidens. The int e r m e d i a t e o u t c r o s s i n g r a t e i n a l l s p e c i e s s t u d i e d does not support Schemske and Lande's bimodal p r e d i c t i o n . Mixed mating systems may represent t r a n s i e n t or nonadaptive s t a t e s (Schemske and Lande, 1985), but they can be 76 e v o l u t i o n a r i l y s t a b l e i f the e f f i c i e n c y of p o l l i n a t i o n depends on the s e l f i n g r a t e ( L l o y d , 1979; Schoen and L l o y d , 1984); the degree of i n b r e e d i n g d e p r e s s i o n v a r i e s from gener a t i o n to g e n e r a t i o n (Maynard Smith, 1978); or s e l f e d progeny are l e s s s u c c e s s f u l migrants than outbred progeny ( H o l s i n g e r , 1986). A recent study by Uyenoyama (1986) i n d i c a t e s that the e v o l u t i o n of an optimal mixed mating system i s p o s s i b l e i n p o p u l a t i o n s p r a c t i c i n g b i p a r e n t a l i n b r e e d i n g , because b i p a r e n t a l i n b r e e d i n g ( i . e . , mating to r e l a t i v e s ) i n t r o d u c e s frequency-dependent c o s t s and b e n e f i t s a s s o c i a t e d with s e l f i n g and o u t c r o s s i n g by i n c r e a s i n g r e l a t e d n e s s between parents and t h e i r b i p a r e n t a l l y d e r i v e d o f f s p r i n g and reducing the r e l a t i v e i n b r e e d i n g d e p r e s s i o n s u f f e r e d by u n i p a r e n t a l o f f s p r i n g . B i p a r e n t a l i n b r e e d i n g has been d e t e c t e d i n at l e a s t . 4 gynodioecious p o p u l a t i o n s of Hawaiian Bidens (Table 3-6). The o u t c r o s s i n g r a t e s of females are expected to be u n i t y i f b i p a r e n t a l i n b r e e d i n g does not occur i n these p o p u l a t i o n s . A s s o r t a t i v e movements of p o l l i n a t o r s among s i m i l a r f l o r a l morphs, p l a n t d e n s i t y and density-dependent p o l l i n a t o r behavior, and r e s t r i c t e d gene flow w i t h i n p o p u l a t i o n s may r e s u l t i n b i p a r e n t a l i n b r e e d i n g (Uyenoyama, 1986 and r e f e r e n c e s t h e r e i n ) . There are no d i f f e r e n c e s i n flower morphs among hermaphrodites w i t h i n p o p u l a t i o n s of Hawaiian Bidens. B i p a r e n t a l i n b r e e d i n g and d i f f e r e n c e s i n the l e v e l of b i p a r e n t a l i n b r e e d i n g i n Hawaiian Bidens are most l i k e l y due to p l a n t d e n s i t y and p o p u l a t i o n s t r u c t u r i n g . As has been d i s c u s s e d above, p o p u l a t i o n s u b s t r u c t u r e r e s u l t s i n mating among r e l a t i v e s i n p o p u l a t i o n s of IK m e n z i e s i i subsp. f i l i f o r m i s ( R i t l a n d and 77 Ganders, 1985). Regardless of the mechanism m a i n t a i n i n g the i n t e r m e d i a t e r a t e of s e l f - f e r t i l i z a t i o n , the l i m i t e d v a r i a t i o n i n o u t c r o s s i n g r a t e s among sp e c i e s of Hawaiian Bidens suggests that mixed mating systems are probably e v o l u t i o n a r i l y s t a b l e i n t h i s group of s p e c i e s . 3.5 Summary A l l Hawaiian s p e c i e s of Bidens are s t r o n g l y protandrous and almost h a l f of them are gynodioecious, i n d i c a t i n g a high o u t c r o s s i n g p o t e n t i a l of t h e i r mating systems. F l o r a l f e a t u r e s r e l a t e d to the mating systems and o u t c r o s s i n g r a t e s i n 15 p o p u l a t i o n s were q u a n t i t a t i v e l y s t u d i e d f o r 10 taxa of Hawaiian Bidens using allozyme gene markers and s t a t i s t i c a l methods. These s p e c i e s d i f f e r s i g n i f i c a n t l y i n flower s i z e , i n c l u d i n g the diameter of flower heads and numbers of d i s c flowers and ray flowers per head. A l l s p e c i e s e x h i b i t strong protandry, ranging from 2-7 days. A n t h e s i s proceeds c e n t r i p e t a l l y w i t h i n the flower head f o r 2-5 days i n these s p e c i e s , r e s u l t i n g i n a prolonged p o l l e n phase and stigma phase, ranging from 5-11 days and 4-9 days r e s p e c t i v e l y i n i n d i v i d u a l flower heads. A l l the s p e c i e s s t u d i e d i n greenhouses e x h i b i t e d low seedset i n the absence of p o l l i n a t o r s (ranging from 0-24%, mean: 4%), i n d i c a t i n g t h a t protandry e f f i c i e n t l y prevents s e l f - f e r t i l i z a t i o n . A wide range of o u t c r o s s i n g r a t e s occurs i n p o p u l a t i o n s of Hawaiian Bidens. O u t c r o s s i n g r a t e s i n hermaphrodites of 8 gynodioecious p o p u l a t i o n s ranged from 0.242-0.887, and i n females ranged from 0.75-1.00. The presence of females i n c r e a s e s the l e v e l of o u t c r o s s i n g of the p o p u l a t i o n as a whole. The 78 e f f i c i e n c y of gynodioecy as an o u t c r o s s i n g mechanism depends on the frequency of females and the l e v e l of o u t c r o s s i n g i n hermaphrodites i n the p o p u l a t i o n . The weighted mean o u t c r o s s i n g r a t e s i n these gynodioecious p o p u l a t i o n s ranged from 0.425-0.881, with an average of 0.639 (±0.135), which does not s i g n i f i c a n t l y d i f f e r from the average value of 0.651 (±0.095) i n another f i v e gynodioecious p o p u l a t i o n s with unknown maternal sexual forms. The non-gynodioecious s p e c i e s mauiensis has a mean o u t c r o s s i n g r a t e of 0.681(±0.071), not s i g n i f i c a n t l y d i f f e r e n t from the mean o u t c r o s s i n g r a t e s i n gynodioecious p o p u l a t i o n s . The s u b s t a n t i a l l e v e l s of s e l f - f e r t i l i z a t i o n , ranging from 0.272-0.412 i n the e i g h t s p e c i e s s t u d i e d might be due to geitonogamy ( i . e . , c r o s s p o l l i n a t i o n of flowers borne on the same i n d i v i d u a l ) . Numerous simultaneously f l o w e r i n g heads on the same p l a n t promote s e l f i n g . Another source of i n b r e e d i n g probably comes from consanguineous mating, which has been shown i n s u b s t r u c t u r e d p l a n t p o p u l a t i o n s . The intermediate r a t e s of o u t c r o s s i n g i n a l l s p e c i e s of Hawaiian Bidens i n t h i s study do not support the t h e o r e t i c a l p r e d i c t i o n of a bimodel d i s t r i b u t i o n of o u t c r o s s i n g r a t e s i n n a t u r a l p l a n t p o p u l a t i o n s by Lande and Schemske (1985). 79 IV. GENETIC VARIATION AND STRUCTURE, AND THE EFFECT OF MIXED MATING SYSTEMS 4.1 I n t r o d u c t i o n T h e o r e t i c a l l y , p l a n t s p e c i e s that r e g u l a r l y p r a c t i c e s e l f -f e r t i l i z a t i o n should have a low l e v e l of g e n e t i c v a r i a t i o n and h e t e r o z y g o s i t y w i t h i n p o p u l a t i o n s and a high l e v e l of g e n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s . Outbreeding s p e c i e s are p r e d i c t e d to have a high l e v e l of g e n e t i c v a r i a t i o n and h e t e r o z y g o s i t y w i t h i n p o p u l a t i o n s and a low l e v e l of g e n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s (Wright, 1921, 1969; Mather; 1943, D a r l i n g t o n and Mather, 1949; Stebbins, 1950, 1957; Baker, 1953; Grant, 1958; A l l a r d et a l . , 1968; J a i n , 1968; H a r t l , 1980). Numerous s t u d i e s have i n v e s t i g a t e d the r e l a t i o n s h i p between the breeding system, genetic v a r i a t i o n and s t r u c t u r e of p l a n t p o p u l a t i o n s . Most s t u d i e s based on allozyme surveys support the t h e o r e t i c a l e x p e c t a t i o n s ( S o l b r i g , 1972; G o t t l i e b 1975; L e v i n , 1977, 1978; Schaal, 1975; Brown and J a i n , 1979; Brown, 1979; Hamrick et a l . , 1979; Schoen, 1982; Layton and Ganders, 1984). There a l s o appears to be a general t r e n d that o u t c r o s s i n g s p e c i e s show l e s s h e t e r o z y g o s i t y and inbreeders show more w i t h i n p o p u l a t i o n s than expected at the known l e v e l of o u t c r o s s i n g of the p o p u l a t i o n s (Brown, 1979), d e s p i t e the c o n t r a r y e v o l u t i o n a r y p r e s s u r e on the mating system (Lande and Schemske, 1985). Brown (1979) r e f e r s to t h i s phenomenon as the " h e t e r o z y g o s i t y paradox". Most i n v e s t i g a t o r s have tended to e x p l a i n t h i s paradox by r e f e r r i n g to the e f f e c t s of other f o r c e s which mold g e n e t i c 80 s t r u c t u r e of p l a n t p o p u l a t i o n s , e.g., s e l e c t i o n , gene flow, g e o g r a p h i c a l or mic r o g e o g r a p h i c a l d i f f e r e n t i a t i o n (Brown, 1979 and r e f e r e n c e s t h e r e i n ) . Although much re s e a r c h has i n v e s t i g a t e d the e f f e c t of breeding systems on p l a n t p o p u l a t i o n s , the m a j o r i t y of p l a n t p o p u l a t i o n s that have been s t u d i e d occur on mainlands. P l a n t p o p u l a t i o n s on oceanic i s l a n d s have been r a r e l y s t u d i e d . The d i s t i n c t i v e c h a r a c t e r i s t i c s of the fauna and f l o r a of oceanic i s l a n d s have been reco g n i z e d s i n c e Darwin (1859) and Wallace (1880). Most s t u d i e s have concerned the l o n g - d i s t a n c e d i s p e r s a l and subsequent establ i s h m e n t , and adapt i v e r a d i a t i o n . But the maintenance and development of g e n e t i c v a r i a b i l i t y i n p l a n t p o p u l a t i o n s i n i t i a t e d from one or a few immigrants to oceanic i s l a n d s has r e c e i v e d l i t t l e a t t e n t i o n . According to C a r l q u i s t (1966, 1974), immigrant s p e c i e s must overcome the r e s t r i c t i o n of ge n e t i c m a t e r i a l s caused by the very small s i z e of the i n i t i a l p o p u l a t i o n f o r s u c c e s s f u l e s t a b l i s h m e n t . Whether the i n t r o d u c t i o n event from the c o n t i n e n t to the oceanic i s l a n d s i s by one seed or a s t a l k of propagales, the o r i g i n a l g e n e t i c v a r i a t i o n i s l i m i t e d and the i n i t i a l p o p u l a t i o n s i z e very s m a l l . The c i r c u l a t i o n of g e n e t i c v a r i a b i l i t y w i t h i n and/or among p o p u l a t i o n s i s thus of importance i n m a i n t a i n i n g a c e r t a i n amount of g e n e t i c v a r i a b i l i t y i n immigrant s p e c i e s . Besides the breeding system, many other e c o l o g i c a l f a c t o r s p l a y important r o l e s i n m a i n t a i n i n g and s t r u c t u r i n g g e n e t i c v a r i a t i o n i n n a t u r a l p o p u l a t i o n s , such as e c o l o g i c a l h e t e r o g e n e i t y , gene flow, p o p u l a t i o n s i z e , g e o g r a p h i c a l range, and l i f e c y c l e (Nevo, 1978; 81 Clarke,1979; Hamrick et a l . , 1979; L o v e l e s s and Hamrick,1984; McClenaghan and Beauchamp,1986). Species of Bidens endemic to the Hawaiian I s l a n d s range from herbaceous to woody p e r e n n i a l s , have mixed mating systems, occupy extremely d i v e r s e h a b i t a t s , maintain l o c a l i z e d g e o g r a p h i c a l d i s t r i b u t i o n s , commonly have small p o p u l a t i o n s i z e s , and most of the p o p u l a t i o n s were probably e s t a b l i s h e d from a l i m i t e d number of founders. These e c o l o g i c a l parameters have c o n t r a r y e f f e c t s on the g e n e t i c v a r i a t i o n and s t r u c t u r e of Hawaiian Bidens p o p u l a t i o n s . The mixed mating system of Hawaiian Bidens (see Chapter 3) should r e s u l t i n a s u b s t a n t i a l l e v e l of g e n e t i c v a r i a b i l i t y w i t h i n p o p u l a t i o n s and a low l e v e l of g e n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s (Loveless and Hamrick, 1984). The p e r e n n i a l l i f e c y c l e r e t a r d s l o s s of v a r i a t i o n w i t h i n p o p u l a t i o n s and reduces e f f e c t s of g e n e t i c d r i f t and thus slows ge n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s (see L o v e l e s s and Hamrick, 1984, and r e f e r e n c e s t h e r e i n ) . However, the endemic and l o c a l i z e d g e o g r a p h i c a l ranges, commonly small p o p u l a t i o n s i z e s , and founder e f f e c t s i n Hawaiian Bidens should r e s u l t i n depauperate g e n e t i c v a r i a b i l i t y w i t h i n p o p u l a t i o n s and high g e n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s due to d r i f t and i s o l a t i o n (see L o v e l e s s and Hamrick, 1984, and r e f e r e n c e s t h e r e i n ) . Environmental h e t e r o g e n e i t y p r o v i d e s d i v e r g e n t s e l e c t i v e p r e s s u r e s and thus adaptive d i f f e r e n t i a t i o n , as evidenced by the morphological v a r i a t i o n i n Hawaiian Bidens ( G i l l e t t and Lim, 1970; G i l l e t t , 1975; Ganders and Nagata, 1983a, 1983b). 82 N a t u r a l h y b r i d i z a t i o n , an i n d i c a t i o n of gene flow among c l o s e l y r e l a t e d s p e c i e s , i s a l s o a frequent phenomenon on oceanic i s l a n d s ( C a r l q u i s t , 1974). In the Hawaiian f l o r a , n a t u r a l h y b r i d i z a t i o n has been r e p o r t e d f o r G o l d i a , Hedyotis and Coprosma i n the Rubiaceae; Bidens, Daubautia, L i p o c h a e t a , Argyroxiphium i n the Asteraceae; Clermontia i n the Campanulaceae; Scaevola i n the Goodeniaceae; P i p t u r u s i n the U r t i c a c e a e ; V i o l a i n the V i o l a c e a e ; and Vaccinium i n the E r i c a c e a e ( S h e r f f , 1935, 1937; G i l l e t t , 1966, 1975; G i l l e t t and Lim, 1970; C a r l q u i s t , 1974). The l e v e l s of g e n e t i c v a r i a t i o n w i t h i n p o p u l a t i o n s and ge n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s of Hawaiian Bidens, t h e r e f o r e , must be the j o i n t r e s u l t of the mating system, s e l e c t i o n , g e n e t i c d r i f t and other e v o l u t i o n a r i l y important f o r c e s . S t u d i e s that c o r r e l a t e p o p u l a t i o n g e n e t i c v a r i a t i o n , s t r u c t u r e and d i f f e r e n t i a t i o n with the breeding system, i n concert with g e o g r a p h i c a l i s o l a t i o n and n a t u r a l h y b r i d i z a t i o n may h e l p to understand s p e c i a t i o n , a d a p t a t i o n , and adaptive r a d i a t i o n i n Hawaiian Bidens, and t h e r e f o r e the c h a r a c t e r i s t i c e v o l u t i o n a r y phenomena on oceanic i s l a n d s . The o b j e c t i v e s of the s t u d i e s r e p o r t e d i n t h i s chapter are 1) to measure g e n e t i c v a r i a t i o n at allozyme l o c i w i t h i n and among n a t u r a l p o p u l a t i o n s of Hawaiian Bidens and 2) to assess the r e l a t i v e r o l e s of the breeding system, g e n e t i c d r i f t and other f a c t o r s i n determining g e n e t i c v a r i a t i o n , s t r u c t u r e and d i f f e r e n t i a t i o n w i t h i n and among Hawaiian Bidens p o p u l a t i o n s . 83 4.2 M a t e r i a l s And Methods Twenty-one p o p u l a t i o n s of 12 taxa of Hawaiian Bidens were i n v e s t i g a t e d f o r t h e i r g e n e t i c v a r i a b i l i t y and p o p u l a t i o n s t r u c t u r e at allozyme l o c i u s i ng e l e c t r o p h o r e t i c methods (Table 4-1). The e f f e c t of mating systems on g e n e t i c v a r i a t i o n and s t r u c t u r e were e v a l u a t e d through c o r r e l a t i o n s t a t i s t i c a n a l y s i s f o r 15 p o p u l a t i o n s f o r which o u t c r o s s i n g r a t e s had been determined. D e t a i l e d i n f o r m a t i o n on these p o p u l a t i o n s , and d e s c r i p t i o n s of s t a r c h g e l e l e c t r o p h o r e s i s and ge n e t i c i n t e r p r e t a t i o n s of isozyme banding p a t t e r n s have been given i n Chapters 2 and 3. Sample s i z e s f o r the s t u d i e s presented i n t h i s chapter are given i n the t a b l e s . 4.3 R e s u l t s 4.3.1 Genetic D i v e r s i t y A l l e l e f r e q u e n c i e s f o r a l l l o c i s t u d i e d are given i n Appendix B. Co n s i d e r a b l e v a r i a t i o n i n a l l e l e f r e q u e n c i e s at three commonly polymorphic l o c i , Pgi-1, Pgi-2, and Skdh-3, was found among these p o p u l a t i o n s (Table 4-1). Of a t o t a l of 38 allozyme l o c i assayed, 5 l o c i (13.2%) are polymorphic at the 95% c r i t e r i o n , and 10 l o c i (26.3%) are polymorphic at the 99% c r i t e r i o n among these p o p u l a t i o n s . Table 4-2 presents g e n e t i c v a r i a t i o n parameters estimated f o r 21 p o p u l a t i o n s of Hawaiian Bidens. The expected p r o p o r t i o n of heterozygotes (He), a l s o c a l l e d the polymorphic index ( P I ) , i n i n d i v i d u a l p o p u l a t i o n s was 84 Table 4-1. A l l e l e f r e q u e n c i e s at three polymorphic l o c i i n 21 Hawaiian p o p u l a t i o n s of Bidens. Pgi-1 Pgi-2 Skdh-3 Pop. a b c a b c a b c d AMPL .19 .80 .01 0 .02 .98 0 0 .99 0 ASYM .12 .88 0 0 0 1 .00 0 0 1 .00 0 CERV .20 .72 .08 .06 .34 .60 .01 .22 .77 0 FORB F1 .02 .98 0 0 .05 .95 0 .19 .81 0 FORB K 0 1 .00 0 0 0 1 .00 0 .19 .81 0 HAWA .03 .97 0 .03 0 .97 0 .72 .27 .01 HILL E 0 1 .00 0 0 0 1 .00 0 1 .00 0 0 HILL W 0 1 .00 0 0 0 1 .00 0 1 .00 0 0 MAUI 1 .22 .77 .01 .08 .15 .77 0 0 1 .00 0 MAUI 2 .01 .99 0 .43 0 .57 0 .07 .81 .12 MENZ 1 .35 .65 0 .05 .13 .82 0 .01 .99 0 MENZ 2 .02 .98 0 0 .84 .16 0 0 1 .00 0 MENZ 3 .08 .91 .01 .03 .04 .93 0 .04 .96 0 MENZ 4 .09 .91 0 0 .47 .53 0 0 1 .00 0 SAND 1 .07 .92 .01 .05 .02 .93 0 0 1 .00 0 SAND 2 0 1 .00 0 0 0 1 .00 0 .30 .63 .07 SAND 3 .05 .92 0 .04 .05 .91 .03 .03 .93 .01 SAND C .02 .97 .01 .03 .34 .63 .01 .09 .90 0 SAND X .16 .78 .06 .10 .10 .80 0 .04 .96 0 TORT 1 .20 .80 0 .18 0 .82 0 0 1 .00 0 TORT 2 .05 .94 .01 .17 .02 .81 0 .17 .80 .03 85 Table 4-2. Genetic v a r i a b i l i t y and outcrossing rates (t) in 21 populations of Hawaiian B i dens. k1: Number of a l l e l e s per polymorphic locus; k2: Number of a l l e l e s per locus; PLP: % l o d polymorphic; Ho(p): Average observed heterozygosity at polymorphic l o c i ; H o ( a l l ) : Average observed heterozygosity at a l l l o c i ; PI(He): Polymorphic Index or expected heterozygosity (see Table 2-1 for population acronym). No. Popu1 at i on N loc i t k1 k2 PLP Ho(p) Ho(al1) PI (H HILL E 182 31 - - 1 .000 0 .0 .000 .000 .000 HILL W 180 31 - - 1 .000 0 .0 .000 .000 .000 ASYM 182 28 - 2 .000 1 .036 3 .6 .093 .003 .010 FORB K 300 29 - 2 .000 1 .068 6 .9 . 173 .012 .015 SAND 2 208 19 . 307 3 .000 1 . 105 5 . 3 . 333 .018 .027 SAND X 133 33 . 455 2 .667 1 . 152 9 . 1 . 256 .016 .031 MENZ 1 306 30 .450 2 . 330 1 . 143 16 . 7 . 191 .020 .036 MENZ 2 317 33 . 474 2 .OOO 1 .121 12 . 1 . 1 18 .008 .037 MENZ 3 69 27 - 2 .667 1 . 185 1 1 . 1 .096 .01 1 .016 MENZ 4 64 28 - 2 .000 1 . 143 14 . 3 . 289 .022 .033 FORB F 1 262 35 .506 2 . 143 1 . 229 20 .0 .061 .010 .024 TORT 2 218 16 .560 3 .000 1 . 375 18 . 8 . 162 .030 .051 AMPL 339 24 . 588 2 . 330 1 . 167 12 . 5 . 150 .013 .019 CERV 344 31 .608 2 .625 1 . 149 25 , 8 .300 .040 .081 HAWA 422 25 .634 2 . 500 1 . 240 16 . 0 . 121 .015 .025 TORT 1 G09 18 .670 2 . 500 1 . 167 1 1 . 1 .242 .027 .034 MAUI 1 463 29 .682 2 .500 1 . 207 13 . 8 . 335 .036 .039 MAUI 2 120 12 .731 2 . 333 1 .333 25 .0 .205 .051 .060 SAND 3 391 31 .803 2 .667 1 . 323 19 . 4 . 135 .015 .024 SAND 1 249 28 .877 3 .000 1 . 143 7. . 1 . 137 .010 .013 SAND C 375 30 .887 2 . 750 1 . 233 13 . 3 . 196 .020 .035 Mean 273 27 .615 2 .474 1 . 168 12. 2 . 189 .018 .029 (s.e.) (136) (6) ( . 17) ( .34) ( . 10) ( 7. 0) ( .08) ( .01) ( .02) 86 c a l c u l a t e d as He = 1 - 1 / n ^ 2 - P ± £ , where n i s the number of l o c i , and p i s the frequency of the i t h a l l e l e a t the" kth l o c u s . Observed h e t e r o z y g o s i t y was c a l c u l a t e d as mean percent of h e t e r o z y g o s i t y per i n d i v i d u a l over polymorphic l o c i , Ho(p), and over a l l l o c i , H o ( a l l ) , f o r each p o p u l a t i o n . Number of a l l e l e s per l o c u s was c a l c u l a t e d f o r polymorphic l o c i (k1) and f o r a l l l o c i (k2). The d i f f e r e n c e s i n sample s i z e among p o p u l a t i o n s have no s i g n i f i c a n t c o r r e l a t i v e e f f e c t on e s t i m a t i n g these g e n e t i c parameters. Parameters of gene d i v e r s i t y are given i n Table 4-3 f o r 21 p o p u l a t i o n s at 3 polymorphic l o c i , and 25 l o c i with monomorphic l o c i i n c l u d e d . T o t a l gene d i v e r s i t y , H T , was measured as H T = 1 - £ p ^ ( N e i , 1973, 1975), where p i s the mean frequency of the i t h of k a l l e l e s among these p o p u l a t i o n s . Hp i s a measure of the mean h e t e r o z y g o s i t y expected under random mating. T o t a l gene d i v e r s i t y may be p a r t i t i o n e d i n t o average gene d i v e r s i t y w i t h i n p o p u l a t i o n s (Hs) and between p o p u l a t i o n s ( D S T ), Hs = 1 - 1 / n Z . S l p ^ , and D S T = Bp - Hs, where p i s the frequency of the i t h a l l e l e at the kth l o c u s w i t h i n p o p u l a t i o n s . The a b s o l u t e measure of gene d i f f e r e n t i a t i o n , Dm, independent of gene d i v e r s i t y w i t h i n subpopulations, which serves as a measure of the minimum net gene d i f f e r e n c e s between p o p u l a t i o n s , i s Dm = nDST / ( n - 1 ) , (Nei, 1975), where n i s the number of p o p u l a t i o n s sampled. Both Dsr and Dm are a b s o l u t e measures of gene d i f f e r e n t i a t i o n , but Dm excludes comparisons of p o p u l a t i o n s with themselves. Dm i s u s e f u l f o r comparing the i n t e r p o p u l a t i o n a l gene d i v e r s i t y , Rsx, which i s c a l c u l a t e d as R S T = Dm/Hs (Nei, 1975). 87 Table 4-3. Gene d i v e r s i t y i n 21 p o p u l a t i o n s of Hawaiian Bidens at 3 polymorphic l o c i , and at 25 l o c i with monomorphic l o c i i n c l u d e d . Locus H T Hs D S T G S T Dm Rsr D S T / H S Pgi-1 0. 183 0. 163 0. 020 0. 109 0. 021 0 . 1 27 0. 123 Pgi-2 0. 312 0. 216 0. 097 0. 31 1 0. 102 0 .471 0. 449 Skdh-3 0. 335 0. 145 0. 190 0. 567 0. 199 1 .370 1 . 310 Mean 0. 277 0. 175 0. 1 02 0. 368 0. 1 07 0 .614 0. 583 25 l o c i 0. 045 0. 029 0. 016 0. 361 0. 017 0 .593 0. 552 88 A v e r a g i n g o v e r a l l l o c i a n d a l l s p e c i e s , t h e t o t a l g e n e d i v e r s i t y ( H T ) i n H a w a i i a n B i d e n s i s 0 . 0 4 5 , b u t m u c h h i g h e r w h e n o n l y t h r e e c o m m o n l y p o l y m o r p h i c l o c i , P g i - 1 , P g i - 2 , a n d S k d h - 3 , w e r e u s e d . F o r a n y o n e p o l y m o r p h i c l o c u s , e x c e p t S k d h - 3 , a l a r g e p r o p o r t i o n o f t h e t o t a l g e n e d i v e r s i t y w a s a t t r i b u t a b l e t o w i t h i n p o p u l a t i o n g e n e d i v e r s i t y ( H s ) . T h e m e a n w i t h i n p o p u l a t i o n g e n e d i v e r s i t y a c c o u n t s f o r 6 3 % o f t h e m e a n t o t a l g e n e d i v e r s i t y a t t h e t h r e e p o l y m o r p h i c l o c i . R s T a v e r a g e d o v e r a l l l o c i i s 0 . 6 1 4 , i n d i c a t i n g t h a t t h e r e i s a p p r o x i m a t e l y 60% a s m u c h v a r i a t i o n b e t w e e n p o p u l a t i o n s a s t h e r e i s w i t h i n p o p u l a t i o n s ( T a b l e 4 - 3 ) . 4 . 3 . 2 P o p u l a t i o n G e n e t i c S t r u c t u r e T h e o r g a n i z a t i o n o f g e n e t i c v a r i a t i o n w a s e x a m i n e d u s i n g F -s t a t i s t i c s i n 21 p o p u l a t i o n s o f H a w a i i a n B i d e n s . O b s e r v e d W r i g h t ' s f i x a t i o n i n d e x , F o , ( W r i g h t , 1 9 2 2 ; J a i n a n d W o r k m a n , 1 9 6 7 ) , w h i c h r e p r e s e n t s d e v i a t i o n o f h e t e r o z y g o t e f r e q u e n c i e s f r o m H a r d y - W e i n b e r g p r o p o r t i o n d u e t o t h e c o m b i n e d e f f e c t s o f f i n i t e p o p u l a t i o n s i z e , s e l e c t i o n , i n b r e e d i n g , a n d o t h e r f o r c e s s h a p i n g t h e g e n e t i c m a k e u p o f t h e p l a n t p o p u l a t i o n s , w a s e s t i m a t e d a s F o = 1 - H o / ( 1 - X P 2 ) , w h e r e Ho i s t h e o b s e r v e d 1 n u m b e r o f h e t e r o z y g o t e s , a n d 1 - "Z. P ? i s t h e e x p e c t e d n u m b e r . F o w a s c a l c u l a t e d f o r e a c h o f t h e t h r e e c o m m o n l y p o l y m o r p h i c l o c i , P g i - 1 , P g i - 2 , a n d S k d h - 3 , i n p o l y m o r p h i c p o p u l a t i o n s o f H a w a i i a n B i d e n s , a n d t h e v a r i a n c e o f F o w a s e s t i m a t e d u s i n g t h e e q u a t i o n < T 2 = ( 1 - F ) [ 2 p q ( 1 + F ) + F ( 2 - F ) ( 1 - 2 p ) 2 ] / 2 n p q , ( R a s m u s s e n , 1 9 6 4 ) , w h e r e p i s t h e f r e q u e n c y o f t h e p r e d o m i n a n t a l l e l e a t t h e l o c u s , a n d q i s t h e summed f r e q u e n c y o f t h e r e s t o f t h e a l l e l e s a t t h e l o c u s . T h e s i g n i f i c a n c e o f h e t e r o z y g o t e d e v i a t i o n f r o m H a r d y - W e i n b e r g 89 p r e d i c t i o n s at each l o c u s was t e s t e d by Chi-square goodness of f i t s t a t i s t i c s (Table 4-4). Most p o p u l a t i o n s show s i g n i f i c a n t heterozygote d e f i c i e n c y at one or more l o c i . The mean f i x a t i o n index over a l l p o p u l a t i o n s i s F i s , which rep r e s e n t s the average d e v i a t i o n of the p o p u l a t i o n s ' genotypic p r o p o r t i o n s from Hardy-Weinberg e q u i l i b r i u m . The extent of h e t e r o g e n e i t y i n a l l e l e f r e q u e n c i e s among p o p u l a t i o n s i s estimated by F S T , which i s measured as F S T = ( T / p ( 1 - p ) , whereC i s the v a r i a n c e of a l l e l e f r e q u e n c i e s among p o p u l a t i o n s , and p i s the mean a l l e l e frequency among p o p u l a t i o n s . The o v e r a l l f i x a t i o n index, F I T , which rep r e s e n t s the 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 the t o t a l p o p u l a t i o n , was estimated as F I T = F is + ( 1 - F i s ) F s T . Nei (1975) a s s e r t s t hat G S T i s e q u i v a l e n t to 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 , F S T , which r e p r e s e n t s r e l a t i v e gene d i f f e r e n t i a t i o n among p o p u l a t i o n s , and was estimated as G S T = D S T / H t D i f f e r e n c e s between estimates of Fsj and G S T are n e g l i g i b l e . Estimates of F I S , Fsx, and F I T f o r Pgi-1, Pgi-2, and Skdh-3 are given i n Table 4-5. The mean value of Fj.s i s 0.457, i n d i c a t i n g that the observed genotypic p r o p o r t i o n s d e v i a t e s i g n i f i c a n t l y from those expected at Hardy-Weinberg e q u i l i b r i u m . Chi-square t e s t s were performed f o r the values of F i x using the formula of L i and H o r v i t z (1953), x 2 = F 2 N ( k - l ) f o r k(k-1)/2 degree of freedom, where F i s the estimate of F I T f o r a p o p u l a t i o n sample, N, with k a l l e l e s . S i g n i f i c a n t d e f i c i e n c i e s of heterozygotes compared to panmixia e x p e c t a t i o n s were found at Table 4-4. Wright's observed fixation Index at three commonly polymorphic loci In 19 populations of Hawaiian Bidens. Pqi -1 Pfl1-2 Skdh -3 Popualtlon N Fo(s.e.) P N Fo(s e. ) P N Fo(s • e. ) P AMPL 339 . 1 17( .006) 339 . 129( .016) ASYM 182 .571( .014) ** CERV 343 .361( .006) ** 343 .324( .005) ** 318 .214( .007) * * FORB F1 262 .270( .022) 262 .474( .015) * 232 .205( .010) * FORB K 45 .492( .048) * HAWA 422 .481( .012) * 422 .017( .006) 282 .304( .007) ** MAUI 1 243 .294( .009) * * 243 .302( .009) ** 463 .048( .004) 463 . 126( .005) * MAUI 2 120 . 150( .058) 120 .349( .015) • * 1 12 . 147( .020) MENZ 1 306 . 1B9( .006) ** 306 . 349( .008) ** 243 .350( .035) MENZ 2 325 . 486( .019) 325 .210( .007) * MENZ 3 69 .416( .043) * 69 . 185( .046) 69 -.061( .017) MENZ 4 64 -.096( .005) 64 . 185( .030) SAND 1 249 . 132( .011) 249 -.051( .005) SAND 2 191 .343( .010) ** SANO 3 391 . 174( .007) • 391 .040( .006) 89 . 133( .033) SAND C 306 . 172( .014) 306 .227( .006) ** 309 .095( .008) SAND X 133 .206( .016) 133 .290( .017) • TORT 1 609 . 153( .004) * * 609 .278( .004) * * TORT 2 187 .475( .020) * 187 . 158( .012) 128 .457( .017) ** *: Chi-square test of genotype distribution * P<0.05; **: p<0.01. Table 4-5. F - s t a t i s t i c s f o r 3 polymorphic l o c i i n 19 p o p u l a t i o n s of Hawaiian Bidens. Locus F I S F I T F S T Pgi-1 0.399 0.472* 0.122 Pgi-2 0.383 0.576** 0.312 Skdh-3 0.588 0.836** 0.601 Mean 0.457 0.628 0.345 *: p<0.05; **: p<0.0l. 92 a l l polymorphic l o c i . Genie d i f f e r e n t i a t i o n , as measured by F s T , ranges from 0.122 f o r Pgi-1 to 0.601 f o r Skdh-3 with a mean over the three polymorphic l o c i of 0.345, i n d i c a t i n g s i g n i f i c a n t d i f f e r e n c e s i n gene f r e q u e n c i e s among p o p u l a t i o n s . The v a l u e s of G S T were i n c l o s e agreement with those of Fsx. 4.3.3 Genetic D i v e r s i t y , S t r u c t u r e And The Mating System The mean number of a l l e l e s per polymorphic lo c u s ( k l ) ranges from 2.00-3.00, mean number of a l l e l e s per l o c u s (k2) ranges from 1.00-1.375, percent of l o c i polymorphic (PLP) w i t h i n p o p u l a t i o n s ranges from 0-26%, mean observed h e t e r o z y g o s i t y per i n d i v i d u a l (Ho) ranges from 0-0.051, and o u t c r o s s i n g r a t e s range from 0.307-0.887 (Table 4-2). No s i g n i f i c a n t c o r r e l a t i o n was found between any of the parameters of g e n e t i c v a r i a t i o n i n Table 2 and o u t c r o s s i n g r a t e i n these p o p u l a t i o n s . However, the mean observed f i x a t i o n i n d i c e s are s i g n i f i c a n t l y c o r r e l a t e d with s e l f i n g r a t e s (Table 4-6) i n these p o p u l a t i o n s , as i n d i c a t e d i n F i g . 4-1 (r=0.78, p<0.0l). The p o s i t i v e c o r r e l a t i o n between observed f i x a t i o n i n d i c e s and s e l f i n g r a t e s i n these p o p u l a t i o n s i n d i c a t e s that the heterozygote d e f i c i e n c i e s i n p o p u l a t i o n s of Hawaiian Bidens are p r i m a r i l y due to i n b r e e d i n g . The i n b r e e d i n g c o e f f i c i e n t , i . e . , Wright's expected f i x a t i o n index ( F e ) , was c a l c u l a t e d f o r i n d i v i d u a l p o p u l a t i o n s using the formula Fe = ( l - t ) / ( l + t ) , where t i s the o u t c r o s s i n g r a t e of the p o p u l a t i o n . Fe values represent Hardy-Weinberg p r o p o r t i o n s a d j u s t e d f o r a known amount of s e l f - f e r t i l i z a t i o n . The d i f f e r e n c e between observed and expected val u e s of the f i x a t i o n index i s A F (Brown, 1979), which i s d e f i n e d by the 93 Table 4-6. Average values of Wright's observed f i x a t i o n index (Fo), expected f i x a t i o n index ( F e ) , ^ F and s e l f i n g r a t e (s) in 19 polymorphic p o p u l a t i o n s of Hawaiian Bidens. No. of Pop. l o c i Fo Fe A F S ASYM 1 0.571 — — — FORB K 2 0.322 - - -SAND 2 1 0.343 0.530 -0.187 0.693 SAND X 2 0.248 0.375 -0.127 0.545 MENZ 1 3 0.296 0.379 -0.083 0.550 MENZ 2 2 0.348 0.357 -0.009 0.526 MENZ 3 3 0. 180 - - -MENZ 4 2 0.045 - - -FORB F1 3 0.316 0.328 -0.012 0.494 TORT 2 3 0.363 0.282 0.081 0.440 AMPL 2 0. 123 0.259 -0. 136 0.412 CERV 3 0.300 0.244 0.056 0.392 HAWA 3 0.267 0.224 0.043 0.366 TORT 1 2 0.216 0. 198 0.018 0.330 MAUI 1 3 0. 1 53 0. 189 -0.036 0.318 MAUI 2 3 0.215 0. 1 55 0.060 0.269 SAND 3 3 0.116 0.109 0.007 0. 197 SAND 1 2 0.041 0.066 -0.025 0. 123 SAND C 3 0.161 0.060 0.101 0.113 Mean 0.243 0.250 -0.017 0.385 0.5 o.o-| 1 1 1 1 1 , 1 1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Selfing rate in population (s) F i g u r e 4 - 1 . R e g r e s s i o n o f o b s e r v e d f i x a t i o n i n d e x on s e l f i n g r a t e i n p o p u l a t i o n s ^ of H a w a i i a n B i d e n s . 95 formula A F = Fo - Fe. P o s i t i v e A F values i n d i c a t e a d e f i c i t of heterozygotes compared with e x p e c t a t i o n s under Wright's n e u t r a l i n b r e e d i n g law. A negative A F value i n d i c a t e s that the p o p u l a t i o n c o n t a i n s more heterozygotes than expected. The mean A F v a l u e , averged over polymorphic l o c i f o r i n d i v i d u a l p o p u l a t i o n s , was c a l c u l a t e d f o r the 15 p o p u l a t i o n s with o u t c r o s s i n g r a t e s a v a i l a b l e (Table 4-6). The o v e r a l l mean A F v a l u e , averaged over 15 p o p u l a t i o n s , i s -0.017. The p o p u l a t i o n s of Hawaiian Bidens are, i n g e n e r a l , only s l i g h t l y more heterozygous than expected. There i s a t r e n d that the p o p u l a t i o n s with lower o u t c r o s s i n g r a t e s (<0.60) tend to have negative A F values (with a mean value of -0.068), whereas the p o p u l a t i o n s with higher o u t c r o s s i n g r a t e s (>0.60) tend to have p o s i t i v e A F values (with a mean value of 0.028). The c o r r e l a t i o n between o u t c r o s s i n g r a t e and A F value i s shown i n F i g . 4-2 (r=0.622, p<0.0l). 4.3.4 Genetic D i s t a n c e s In s t u d i e s of gene d i f f e r e n c e s between p o p u l a t i o n s , the concept of genetic d i s t a n c e was developed to u t i l i z e e l e c t r o p h o r e t i c data as a measure of the accumulated number of d e t e c t a b l e gene s u b s t i t u t i o n s per loc u s (Nei, 1972). Nei's g e n e t i c d i s t a n c e r e p r e s e n t s the amount of between p o p u l a t i o n d i f f e r e n c e i n a l l e l e frequency summed over a l l l o c i , given as D = - l o g I, where I i s the normalized i d e n t i t y of genes between p o p u l a t i o n s of X and Y, d e f i n e d as I = Jvv/J J J . Where J v , J AI X Y A Y and are the a r i t h m e t i c means of j x, j y , and j r e s p e c t i v e l y , over a l l l o c i , i n c l u d i n g both polymorphic and monomorphic l o c i 0.3-1 0.4 0.6 0.8 Outcrossing rate (t) Figure 4-2. Correlation between outcrossing rate (t) and A, F i n 15 populations of Hawaiian Bidens (r = 0. 622, p4 0.01). ON 97 ( J X = Z x2 , J Y = Z y2 , j ^ =21 x^y. ; x and y are the f r e q u e n c i e s of the i t h a l l e l e s i n p o p u l a t i o n X and Y r e s p e c t i v e l y (Nei, 1 9 7 4 ) ) . A computing program, GDID, s u p p l i e d by Kermit R i t l a n d , was used to c a l c u l a t e g e n e t i c d i s t a n c e and g e n e t i c i d e n t i t y between p o p u l a t i o n s of Hawaiian Bidens. The estimates of I and D f o r these p o p u l a t i o n s are given i n Table 4-7. The v a l u e s of g e n e t i c i d e n t i t y range from 0 . 9 2 6 - 1 . 0 0 0 among 2 1 0 p a i r w i s e comparisons i n 21 p o p u l a t i o n s of Hawaiian Bidens, with a mean value of 0 . 9 8 2 ( i o . 0 1 8 ) . The lowestvalues of I were found between p o p u l a t i o n s of B^ h i l l e b r a n d i a n a subsp. p o l y c e p h a l a and the other taxa. Bidens h i l l e b r a n d i a n a subsp. p o l y c e p h a l a i s completely monomorphic f o r a l l l o c i assayed and a rare a l l e l e was f i x e d at a commonly polymorphic l o c u s , S k d h - 3 . A dendrogram was produced based on the g e n e t i c d i s t a n c e matrix, using an unweighted p a i r -group method a c c o r d i n g to a r i t h m e t i c averages (Sneath and Sokal, 1 9 7 3 ) , which shows that the r e l a t i o n s h i p among the p o p u l a t i o n s of Hawaiian Bidens at the allozyme l e v e l does not agree with the taxonomic grouping p a t t e r n ( F i g 4 - 3 ) . P o p u l a t i o n s of the same taxa, such as B^ menziesi i subsp. f i l i f o r m i s , are o f t e n separated i n t o d i f f e r e n t sub-groups and p o p u l a t i o n s of d i f f e r e n t taxa i n t o the same sub-groups. N e i t h e r i s there a geographic p a t t e r n c o r r e l a t e d with g e n e t i c d i s t a n c e s among these p o p u l a t i o n s . P o p u l a t i o n s d i s t r i b u t e d on the same i s l a n d s or adjacent to each other are not grouped together i n F i g . 4 - 3 . Table 4-7. between Genetic identity (above the diagonal) and genetic distance (below the diagonal) 21 populations of Hawaiian Bidens. AT I ON AMPL ASYM CERV FORB F1 FORB K HAWA HILL E HILL W MAUI 1 MAUI 2 AMPL ASYM CERV FORB Fl FORB K HAWA HILL E HILL W MAUI 1 MAUI 2 MENZ 1 MENZ 2 MENZ 3 MENZ 4 SAND 1 SAND 2 SAND 3 SAND C SAND X TORT 1 TORT 2 0.000 0.01 1 0.004 0.005 0.032 0.053 0.053 0 002 0.018 0 003 0.04 1 0.001 0.013 0.001 0008 0.002 0.010 0 001 O 002 O 006 1 .000 0 012 0.003 0 002 0.029 0.050 0.050 0O03 0.017 0.004 0.039 0.000 0.013 0.000 0.009 0.001 0.007 0.002 0.003 0.005 0.989 O 988 0.008 0.011 0.021 0.036 O 036 0.006 0.019 0.006 0.017 O.OOB 0005 O 010 0.013 0.010 0.004 0.005 0.010 0.010 0.996 O 998 0.992 OOOO 0.014 0.028 0.028 0.006 0.014 0O07 0.030 0.001 0 011 0.002 0.002 0.001 0.005 0.003 0.006 0.002 0.995 0.998 0.989 1 .000 0.016 0.032 0.032 0007 0 015 0.010 0 04 1 0002 0.015 0.003 0002 0.002 0 007 0.004 O. 008 0 002 0 969 0 97 1 0.979 0.986 O 984 0.003 0O03 0.037 0.047 0O32 0.059 0.022 0.037 0 027 0O10 0 027 0.028 0.024 O 038 0O26 O 948 0.951 0.964 0.972 0.969 0.997 OOOO 0 060 0.072 0.053 0.077 0.042 0058 O 047 0.024 0 050 0.047 0.04 1 0.062 0O49 0.948 0.951 0.964 0.972 0.969 0.997 1 000 0.060 0.072 0 053 0O77 0.042 O 058 0.047 O 024 0.050 0.047 0.04 1 0.062 0.049 0.998 0 997 O 994 0.994 0.993 0.964 0.942 0 942 0.013 O.OOI O.030 0 002 0O07 O 002 0 015 0.003 0.004 O 000 O. 001 0005 0 982 O 984 0 981 0.986 0.985 0.954 O 930 O 930 O 987 0 023 0 046 0015 O 023 0012 0.019 0013 0.012 0 01 1 O.O10 O 006 00 Table 4-7. cont POPULATION MENZ t MENZ 2 MENZ 3 MENZ 4 SAND 2 SANO 2 SAND 3 SANO C SANO X TORT 1 TORT 2 AMPL 0 997 0 . 960 0 .999 0 987 0 999 0 .992 0 . 998 0 . 990 0 .999 0 998 0 .994 ASYM 0 .996 0 962 1 000 0 987 1 .000 0 991 0 999 O 993 0 998 0 997 0 995 CERV 0 994 0 983 0 .992 0 .995 0 990 0 987 0 .990 0 996 0 .995 0 990 0 990 FORB F 1 0 993 0 970 0 999 0 989 0 998 0 998 0 .999 0 995 0 997 0 .994 0 998 FORB K 0 990 0 960 0 998 0 .985 0 .997 0 998 0 998 0 .993 0 996 0 .992 0 998 HAWA 0 969 O 942 0 978 O 964 0 973 0 99 1 O .974 0 .972 O .977 O 963 0 975 HILL E O 948 0 926 0 959 0 943 0 954 0 976 0 951 0 954 0 960 O 940 0 952 HILL W 0 948 0 926 0 959 0 943 0 .954 0 976 O 95 1 0 954 0 960 0 940 0 952 MAUI 1 0 999 0 971 0 998 0 993 0 998 0 986 0 997 0 996 1 000 O 999 0 995 MAUI 2 0 977 0 955 0 985 0 977 0 988 0. 982 0 987 0 989 0 989 O 990 0 994 MENZ 1 0 972 0 996 0 992 o 995 0. 983 0 994 0 991 0 999 O. 997 0. 988 MENZ 2 0 029 0 970 0 993 0 967 0 951 0 965 0 985 0. 976 0. 960 0. 952 MENZ 3 0 004 0 030 0 992 1 OOO 0. 994 1 OOO 0 995 0 999 0 998 0. 997 MENZ 4 0 O08 0 007 0 008 0. 990 0. 978 O 990 O 998 0 994 O 987 0. 983 SAND 1 0 005 0 033 0 000 0 010 0. 993 1 OOO 0 994 0. 999 0. 998 0. 996 SAND 2 0 0)7 0 051 0 006 0 023 o 007 0. 992 O. 98 7 o 991 O. 989 0. 996 SAND 3 0 006 0 036 0 OOO 0 010 0. 000 0. 008 0. 995 0. 999 0. 997 0. 997 SAND C 0 009 0 015 0 005 0 002 0. 006 0. 013 0. 005 0 996 0. 990 0. 994 SAND X 0 OOI 0 024 0 001 0 006 0 001 0 009 0. OOI O 004 0. 999 0. 997 TORT 1 0 003 0. 04 1 0 002 0 013 0 002 0 01 1 0. 003 0. 001 0 OOI 0 996 TORT 2 0 012 0 049 0 003 0 017 0 004 0. 004 0 003 0. 006 0. 003 0 004 100 I 1 1 1 — 0 . 0 5 0 . 0 2 NEI GENETIC DISTANCE HflWfl H I L L E H I L L W AMPL 2 RSYM MENZ 3 SAND 1 SAND 3 MAUI 1 J L SAND X L T 0 R T 1 MENZ 1 r FORB F1 J ^ F Q R B K i— SAND 2 f t H : TORT CERV MENZ SAND MAUI MENZ 4 C 2 2 1 0 . 0 Figure 4-3. Dendrogram based on genetic distance matrix for 21 populations of Hawaiian Bidens. 101 4.4 D i s c u s s i o n 4.4.1 Genetic V a r i a b i l i t y And The Mating System Many s t u d i e s of enzyme l o c i have shown c o n s i d e r a b l e g e n e t i c v a r i a t i o n both w i t h i n and between p l a n t p o p u l a t i o n s ( G o t t l i e b , 1977; Brown, 1979; Hamrick et a l . , 1979). D i f f e r e n c e s i n amount of g e n e t i c v a r i a t i o n among p l a n t s p e c i e s are o f t e n c o r r e l a t e d with breeding systems, l o n g e v i t y , g e o g r a p h i c a l ranges, f e c u n d i t i e s , p o l l i n a t i o n mechanisms, chromosome number, and stage of s u c c e s s i o n (Hamrick et a l . , 1979). E m p i r i c a l s t u d i e s of 110 p l a n t s p e c i e s give mean values of PLP, k(=k2), PI, and Ho(=Ho(all)) of 36.8%, 1.69, 0.141, and 0.156 r e s p e c t i v e l y (Hamrick et a l . , 1979), with c o n s i d e r a b l e v a r i a t i o n among the s p e c i e s s t u d i e d . In comparing the values of PLP, k2, and PI i n the p o p u l a t i o n s of Hawaiian Bidens with the ranges of these measures of g e n e t i c v a r i a t i o n i n 110 s p e c i e s given by Hamrick et a l . ( l 9 7 9 ) , a l l the three parameters of g e n e t i c v a r i a t i o n i n p o p u l a t i o n s of Hawaiian Bidens are p o s i t i o n e d at the low extremes. The average observed h e t e r o z y g o s i t y (Ho) i s s i g n i f i c a n t l y l e s s than the expected (PI) (p<0.025), i n d i c a t i n g t h a t a c o n s i d e r a b l e heterozygote d e f i c i e n c y e x i s t s i n p o p u l a t i o n s of Hawaiian Bidens i n comparison with Hardy-Weinberg expectat i o n s . Although the o v e r a l l low l e v e l of g e n e t i c v a r i a t i o n (PLP=12.2%, H o ( a l l ) = 0 . 0 l 8 , and k2=1.17), might be unexpected c o n s i d e r i n g the mixed mating system and high chromosome number (72) i n a l l Hawaiian s p e c i e s of Bidens, i t i s not s u r p r i s i n g on 102 the b a s i s of small p o p u l a t i o n s i z e , g e n e t i c d r i f t and other consequences of founder events. One compensation f o r low g e n e t i c v a r i a b i l i t y might be " f i x e d h e t e r o z y g o s i t y " , the formation of h y b r i d d i m e r i c isozymes between two homozygous l o c i . F i x e d h e t e r o z y g o s i t y has been g e n e r a l l y observed f o r P g i , Mdh, and p o s s i b l y 6-Pgdh i n p o p u l a t i o n s of Hawaiian Bidens. F i x e d h e t e r o z y g o s i t y has been demonstrated f o r Pgi i n C l a r k i a ( G o t t l i e b , 1977) and Festuca (Adams and A l l a r d , 1977), and i n many bony f i s h e s (Avise and K i t t o , 1973). I t has been demonstrated for other enzyme systems i n T r i t i c u m , Zea, Stephanomeria, Hordeum and Tragopogon (see Lack and Kay, 1986; and r e f e r e n c e s c i t e d t h e r e i n ). Another compensating s t r a t e g y f o r the low l e v e l of g e n e t i c v a r i a b i l i t y at i n d i v i d u a l l o c i i n p o p u l a t i o n s of Hawaiian Bidens i s probably "gene d u p l i c a t i o n " . Most enzyme systems i n t h i s study show a d i v e r s i t y of homozygous isozyme bands, an e f f e c t of gene d u p l i c a t i o n . Gene d u p l i c a t i o n can occur i n p l a n t e v o l u t i o n both v i a p o l y p l o i d y and v i a r e p e t i t i o n s i n DNA sequences ( G o t t l i e b , 1977, 1981; Lack and Kay, 1986). Although the chromosomes d i s p l a y d i p l o i d p a i r i n g behavior ( S k o t t s b e r g , 1953), the h e x a p l o i d chromosome number of Hawaiian Bidens may account f o r the m u l t i p l e bands i n the homozygous c o n d i t i o n , which are g e n e r a l l y observed f o r Pgm (8 bands), 6-Pgdh (6 bands), Mdh (6 bands), Pgi (minimum 4 bands), Lap (6 bands), and Skdh (3 bands) (see Chapter 2 f o r d e t a i l e d i n f o r m a t i o n ) . The combined e f f e c t s of f i x e d h e t e r o z y g o s i t y and gene d u p l i c a t i o n c o u l d counterbalance the p a u c i t y of g e n e t i c v a r i a b i l i t y at i n d i v i d u a l l o c i i n p o p u l a t i o n s of Hawaiian Bidens. 103 T h e o r e t i c a l l y , predominantly o u t c r o s s i n g p o p u l a t i o n s can maintain higher l e v e l s of i n t r a - p o p u l a t i o n g e n e t i c v a r i a t i o n than predominantly i n b r e e d i n g p o p u l a t i o n s . Schoen (1982) has shown th a t the o u t c r o s s i n g r a t e s were s i g n i f i c a n t l y c o r r e l a t e d with the l e v e l of PLP, k, and Ho i n G i l i a a c h i l l e i f o l i a , although i n b r e e d i n g i t s e l f only reduces the l e v e l of Ho, not the l e v e l of PLP, or k. The lack of c o r r e l a t i o n between Ho and t i n p o p u l a t i o n s of Hawaiian Bidens suggests that other f o r c e s a f f e c t i n g the l e v e l of h e t e r o z y g o s i t y i n these p o p u l a t i o n s may have o v e r r i d d e n the e f f e c t of the mating system. The average observed h e t e r o z y g o s i t y at polymorphic l o c i f o r the 19 polymorphic p o p u l a t i o n s of Hawaiian Bidens i s 0.189 (+0.083) (Table 4-2). Brown (1979) r e p o r t e d an average observed h e t e r o z y g o s i t y of 0.345 w i t h i n p l a n t p o p u l a t i o n s f o r 18 o u t c r o s s i n g taxa, and 0.126 f o r 11 i n b r e e d i n g taxa at polymorphic allozyme l o c i . The average observed h e t e r o z y g o s i t y at polymorphic l o c i i n Hawaiian Bidens i s between the o u t c r o s s e r s and s e l f e r s , which i s i n agreement with the mixed mating system. T h e r e f o r e , the extremely low l e v e l of observed h e t e r o z y g o s i t y per i n d i v i d u a l when averaged over a l l l o c i f o r a l l p o p u l a t i o n s , 0.018, i s o b v i o u s l y due to the low l e v e l s of PLP and k (k1 and k2) i n p o p u l a t i o n s of Hawaiian Bidens. Complete monomorphism at a l l allozyme l o c i assayed was found i n both p o p u l a t i o n s of B.  h i l l e b r a n d i a n a subsp. p o l y c e p h a l a , and the commonly polymorphic l o c i , P gi-1, Pgi-2, Skdh-3 are f i x e d f o r e i t h e r the frequent a l l e l e s or r a r e a l l e l e s i n some p o p u l a t i o n s of B_^  amplectens, B.  asymmetrica, B. f o r b e s i i subsp. k a h i l i e n s i s , B. mauiensis, B. 104 menziesi i subsp. f i l i f o r m i s , B. s a n d v i c e n s i s subsp. s a n d v i c e n s i s , and EL t o r t a . And these l o c i have skewed a l l e l e f r e q u e n c i e s i n most polymorphic p o p u l a t i o n s (Table 4-1). Genetic d r i f t and d i f f e r e n t i a l s e l e c t i o n pressure i n combination with h i s t o r i c a l f a c t o r s , such as common anc e s t r y and the nature of founders, probably p l a y a major r o l e i n determining genetic v a r i a b i l i t y i n p o p u l a t i o n s of Hawaiian Bidens. There i s a s i g n i f i c a n t d i s c r e p a n c y between the l e v e l of g e n e t i c v a r i a t i o n found i n t h i s study and that r e p o r t e d i n a p r e v i o u s study by Helenurm and Ganders (1985). They reported PLP, k1, k2, and PI of 40.0%, 2.56, 1.64, and 0.102 r e s p e c t i v e l y (12.2%, 2.47, 1.17 and 0.029 r e s p e c t i v e l y i n t h i s s t u d y ) . T h i s i s probably due to the d i f f e r e n c e s i n p o p u l a t i o n sample s i z e and i n number of allozyme l o c i sampled. A r e l a t i v e l y small p o p u l a t i o n sample s i z e and small number of allozyme l o c i were assayed i n the p r e v i o u s study. Although most of enzyme systems assayed i n t h i s study were the same as i n the p r e v i o u s study, a d i f f e r e n t b u f f e r system was used i n t h i s study, which enabled more l o c i to be r e s o l v e d . Most of the newly r e s o l v e d l o c i were monomorphic. There have been arguments that a small number of very polymorphic l o c i have l e d to i n f l a t e d estimates of h e t e r o z y g o s i t y i n woody p l a n t s (Guries and L e d i g , 1982). 4.4.2 D i s t r i b u t i o n Of Genetic V a r i a t i o n And The Mating System Allozyme r e l a t i o n s h i p s are not c o r r e l a t e d with taxonomic r e l a t i o n s h i p s in Hawaiian Bidens (Helenurm and Ganders, 1985; F i g . 4-3). Furthermore, a l l Hawaiian s p e c i e s are i n t e r f e r t i l e . T h e r e f o r e , a l l the p o p u l a t i o n s i n the present study can be 105 t r e a t e d together f o r g e n e t i c d i v e r s i t y a n a l y s i s . The value of Hp i s i n f l u e n c e d by PLP, k, and the evenness of mean a l l e l e f r e q u e n c i e s (Loveless and Hamrick, 1984). The average Hx value i n the 21 p o p u l a t i o n s of Hawaiian Bidens i n the present study, 0.045, i s much lower than the mean H T value at polymorphic l o c i , 0.277 (Table 4-3), due to the low percentage of polymorphic l o c i i n these p o p u l a t i o n s . In r e v i e w i n g 163 s t u d i e s , L o v e l e s s and Hamrick (1984) c a l c u l a t e d the average val u e s of Hf at polymorphic l o c i f o r autogamous, mixed-mating, and predominantly outcrossed s p e c i e s , which are 0.291, 0.242, and 0.251 r e s p e c t i v e l y . There was no c o r r e l a t i o n between Hx values and breeding systems found i n these s t u d i e s , but the mean 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 (Hs) and among po p u l a t i o n s (Gsx) were c o r r e l a t e d with breeding systems. Low average val u e s of Hs (0.128) and high v a l u e s of Gsx (0.523) were found i n autogamous s p e c i e s , intermediate Hs (0.174) and Gsx (0.243) i n mixed-mating s p e c i e s , and high Hs (0.214) and low GSj (0.118) i n predominantly o u t c r o s s e d s p e c i e s , i n d i c a t i n g t h a t the breeding systems a f f e c t g e n e t i c d i v e r s i t y i n p l a n t p o p u l a t i o n s p r i m a r i l y i n the way i n which v a r i a t i o n at polymorphic l o c i i s p a r t i t i o n e d (see L o v e l e s s and Hamrick, 1984). The mean Hs value at three commonly polymorphic l o c i i n p o p u l a t i o n s of Hawaiian Bidens i s 0.175, i n c l o s e agreement with the mixed-mating system, but the mean Gsx v a l u e , 0.368, i s much higher than that r e p o r t e d f o r mixed-mating s p e c i e s , suggesting that a r e l a t i v e l y high l e v e l of g e n e t i c d i f f e r e n t i a t i o n among the p o p u l a t i o n s of Hawaiian Bidens has o c c u r r e d at these l o c i . 1 0 6 Probably genetic d r i f t and s e l e c t i o n f o r l o c a l a d a p t a t i o n are p l a y i n g more important r o l e s than the mating system i n b r i n g i n g about t h i s l e v e l of ge n e t i c d i f f e r e n t i a t i o n i n t h i s group of s p e c i e s . Endemic or narrowly d i s t r i b u t e d s p e c i e s are expected to have hig h Gst va l u e s , although t h i s was not confirmed by L o v e l e s s and Hamrick ( 1 9 8 4 ) . A l l the s p e c i e s of Bidens endemic to the Hawaiian I s l a n d s have very l o c a l i z e d d i s t r i b u t i o n s and most e x h i b i t g e o g r a p h i c a l and e c o l o g i c a l i s o l a t i o n (Ganders and Nagata, 1 9 8 4 ) . N a t u r a l h y b r i d i z a t i o n between g e o g r a p h i c a l l y i s o l a t e d s p e c i e s has become a r e l a t i v e l y r a r e occurrence, i n d i c a t i n g that g e o g r a p h i c a l i s o l a t i o n has e f f i c i e n t l y l i m i t e d gene flow among p o p u l a t i o n s of Hawaiian Bidens. Probably both reduced gene flow - and e x t e n s i v e e c o l o g i c a l d i v e r s i f i c a t i o n promote genetic d i f f e r e n t i a t i o n among p o p u l a t i o n s . D S T / H S emphasizes the p a r t i t i o n i n g of the ge n e t i c v a r i a t i o n d e t e c t e d at polymorphic l o c i . Brown ( 1 9 7 9 ) r e p o r t e d the valu e s of D S T / H S at polymorphic l o c i f o r 2 8 s p e c i e s , which averaged 0 . 1 7 f o r outbreeders and 1 . 1 8 f o r • i n b r e e d e r s . The value of D S T / H S i n Hawaiian Bidens of 0 . 5 8 3 i n d i c a t e s an intermediate p o r t i o n of the g e n e t i c v a r i a t i o n i s d i s t r i b u t e d among p o p u l a t i o n s . 4 . 4 . 3 P o p u l a t i o n Genetic S t r u c t u r e And The Mating System Observed f i x a t i o n i n d i c e s represent the observed frequency of heterozygotes compared with panmictic e x p e c t a t i o n s . The average value of Fo over 1 9 polymorphic p o p u l a t i o n s of Hawaiian Bidens (Table 4 - 6 ) i s 0 . 2 4 3 ( + 0 . 1 2 7 ) , i n d i c a t i n g a s i g n i f i c a n t h eterozygote d e f i c i e n c y . The c o r r e l a t i o n between the observed 1 07 f i x a t i o n i n d i c e s and s e l f i n g r a t e s i n these p o p u l a t i o n s suggests that the observed heterozygote d e f i c i t s are p r i m a r i l y due to i n b r e e d i n g ( F i g . 4-1). Adjusted f o r the known l e v e l of i n b r e e d i n g i n the p o p u l a t i o n , Wright's f i x a t i o n index i s the i n b r e e d i n g c o e f f i c i e n t ( i . e . , F e ) , which r e p r e s e n t s the p o r t i o n of the heterozygote d e f i c i t caused by i n b r e e d i n g . The d i f f e r e n c e between Fo and Fe, A F , r e p r e s e n t s the magnitude or s t r e n g t h of other f o r c e s i n shaping g e n e t i c s t r u c t u r e of the p o p u l a t i o n . The average value of Fo c a l c u l a t e d f o r 23 o u t c r o s s i n g s p e c i e s by Brown (1979) i s 0.16, and the average A F value f o r 7 i n b r e e d i n g s p e c i e s i s 0.08. The h e t e r o z y g o s i t y paradox i s a l s o d e t e c t e d i n Hawaiian Bidens. The p o p u l a t i o n s with low o u t c r o s s i n g r a t e s tend to have negative A F v a l u e s , i n d i c a t i n g heterozygote excesses; and the p o p u l a t i o n s with high o u t c r o s s i n g r a t e s tend to have p o s i t i v e A F v a l u e s , i n d i c a t i n g heterozygote d e f i c i e n c i e s , as shown i n F i g . 4-2. The average A F value f o r the 15 p o p u l a t i o n s which had o u t c r o s s i n g r a t e s estimated i s -0.017, suggesting that the observed heterozygote frequency, on average, i s c l o s e to that expected f o r the known l e v e l of o u t c r o s s i n g i n p o p u l a t i o n s of Hawaiian Bidens. However, the negative values of A F i n the p o p u l a t i o n s having higher s e l f i n g r a t e s (s>0.40), with a mean value of -0.068, d e v i a t e d more from 0 than those i n the p o p u l a t i o n s having lower s e l f i n g r a t e s (s<0.40), with a mean value of 0.028 (Table 4-5), suggesting that besides the mating system, s e l e c t i o n , g e o g r a p h i c a l and m i c r o g e o g r a p h i c a l d i f f e r e n t i a t i o n , and gene flow are probably a l s o i n v o l v e d with 108 the observed l e v e l of f i x a t i o n i n d i c e s i n p o p u l a t i o n s of Hawaiian Bidens. 4.4.4 Genetic S i m i l a r i t y Among Po p u l a t i o n s In a review, G o t t l i e b (1981) repo r t e d that the mean value of g e n e t i c i d e n t i t y f o r c o n s p e c i f i c p o p u l a t i o n s i s 0.956, and f o r congeneric p o p u l a t i o n s i s 0.67. Genetic i d e n t i t y among the p o p u l a t i o n s of Hawaiian Bidens i s 0.982 on average, even higher than gene t i c i d e n t i t y values found i n c o n s p e c i f i c p o p u l a t i o n s . T h i s i s a r e s u l t of the low l e v e l of g e n e t i c v a r i a b i l i t y i n Hawaiian Bidens, as supported by the f a c t t h a t much lower g e n e t i c i d e n t i t y v a l u e s were obtained when only polymorphic l o c i were used f o r N e i - s t a t i s t i c s (Sun, unpu b l i s h e d ) . A high g e n e t i c i d e n t i t y value (0.9989 ± 0.0002) was r e c e n t l y r e p o r t e d by McClenaghan and Beauchamp (1986) f o r p o p u l a t i o n s of C a l i f o r n i a fan palm, which d i s p l a y an extremely low g e n e t i c v a r i a b i l i t y w i t h i n p o p u l a t i o n s and low g e n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s . The authors a t t r i b u t e the high g e n e t i c s i m i l a r i t y among the i s o l a t e d p o p u l a t i o n s to t h e i r common a n c e s t r y . L i k e the C a l i f o r n i a fan palm p o p u l a t i o n s , a l l Hawaiian Bidens p o p u l a t i o n s probably evolved from the same a n c e s t r a l p o p u l a t i o n . The high g e n e t i c s i m i l a r i t y among p o p u l a t i o n s of Hawaiian Bidens i s l i k e l y due to common a n c e s t r y . Contrary to Helenurm and Ganders (1985), i t cannot be i n t e r p r e t e d as evidence of an absence of g e n e t i c d i f f e r e n t i a t i o n because the high Gs^ and F S T v a l u e s i n d i c a t e that s u b s t a n t i a l g e n e t i c d i f f e r e n t i a t i o n has oc c u r r e d among the p o p u l a t i o n s of Hawaiian Bidens at three commonly polymorphic l o c i . 109 S o l t i s (1982) found that taxa of S u l l i v a n t i a , which are m o r p h o l o g i c a l l y d i s t i n c t and possess d i s t i n c t i v e a r r a y s of f l a v o n o i d c o n s t i t u e n t s , cannot be d i s t i n g u i s h e d a l l o z y m i c a l l y . Examples of absence of c o r r e l a t i o n between a l l o z y m i c v a r i a b i l i t y and m o r p h o l o g i c a l , chemical and c y t o l o g i c a l v a r i a b i l i t y can a l s o be found i n Turner (1973), Larson and Highton (1978), and Jensen et a l . ( l 9 7 9 ) . In the present study, the d i s c r e p a n c y between taxonomic groups based p r i m a r i l y on morphological data and the allozyme p a t t e r n shown i n F i g . 4-3 may suggest that the l o c i c oding f o r morphological c h a r a c t e r s are independent of the l o c i c oding f o r allozymes. Although the morphological and e c o l o g i c a l d i f f e r e n c e s among Hawaiian s p e c i e s of Bidens suggest a high degree of g e n e t i c d i f f e r e n t i a t i o n among s p e c i e s , these d i f f e r e n c e s appear to be c o n t r o l l e d by a r a t h e r small number of genes, as r e v e a l e d by experimental c r o s s e s ( G i l l e t t and Lim, 1970; Mensch and G i l l e t t , 1972). T h i s s i t u a t i o n might be e x p l a i n e d by a d a p t a t i o n to environmental f a c t o r s . The genes which c o n t r o l m orphological and e c o l o g i c a l d i f f e r e n c e s i n Hawaiian Bidens are most l i k e l y to be those genes that p r o v i d e each s p e c i e s with the adaptive p r o p e r t i e s needed to meet the l o c a l h a b i t a t requirements. S p e c i a t i o n can occur with l i t t l e d i vergence at gene l o c i coding f o r allozymes ( G o t t l i e b , 1973, 1974; G o t t l i e b and P i l z , 1976; Rick et a l . , 1976). Adaptive r a d i a t i o n i n Hawaiian Bidens c e r t a i n l y has not r e q u i r e d a comparative degree of allozyme d i f f e r e n t i a t i o n . 110 4.5 Summary The e f f e c t of the mating system on g e n e t i c v a r i a t i o n and s t r u c t u r e i n 21 p o p u l a t i o n s of Hawaiian Bidens was i n v e s t i g a t e d u sing allozyme data and s t a t i s t i c a l methods. A l l p o p u l a t i o n s e x h i b i t e d low l e v e l s of g e n e t i c v a r i a b i l i t y . Percent polymorphic l o c i (PLP) ranged from 0-25.8%, with an average of 12.2% over a l l p o p u l a t i o n s . Observed l e v e l s of h e t e r o z y g o s i t y (Ho) at polymorphic l o c i ranged from 0.061-0.335, with an average of 0.189 over polymorphic p o p u l a t i o n s . However, the mean Ho v a l u e s were very low when c a l c u l a t e d based on a l l l o c i recorded, ranging from 0.000-0.051, with an average of 0.018 over a l l p o p u l a t i o n s . The parameters of g e n e t i c v a r i a b i l i t y are not c o r r e l a t e d with o u t c r o s s i n g r a t e s i n these p o p u l a t i o n s . The low l e v e l s of g e n e t i c v a r i a b i l i t y w i t h i n p o p u l a t i o n s are expected i n founder p o p u l a t i o n s , or p o p u l a t i o n s which have experienced severe b o t t l e n e c k s . Observed mean f i x a t i o n i n d i c e s at three polymorphic l o c i i n 15 p o p u l a t i o n s ranged from 0.041-0.571, with an average value of 0.243 over p o p u l a t i o n s . Chi-square t e s t s of genotypic f r e q u e n c i e s r e v e a l e d a s i g n i f i c a n t heterozygote d e f i c i e n c y i n comparision with Hardy-Weinberg e x p e c t a t i o n s at one or more l o c i i n most p o p u l a t i o n s . S e l f i n g r a t e s were found to be c o r r e l a t e d with the observed f i x a t i o n i n d i c e s i n 15 polymorphic p o p u l a t i o n s (r=0.78, p < 0.01), i n d i c a t i n g that the heterozygote d e f i c i e n c y was p r i m a r i l y caused by i n b r e e d i n g . A s i g n i f i c a n t c o r r e l a t i o n was a l s o found between o u t c r o s s i n g r a t e s and ^ F values i n these p o p u l a t i o n s (r=0.622, p<0.0l), which suggests t h a t , besides 111 mating systems, other e v o l u t i o n a r y f o r c e s (e.g., s e l e c t i o n , g e n e t i c d r i f t , e t c . ) might a l s o p a r t i c i p a t e i n shaping the ge n e t i c s t r u c t u r e of p o p u l a t i o n s of Hawaiian Bidens. A low l e v e l of t o t a l gene d i v e r s i t y (H^ = 0.045) was found i n the 21 p o p u l a t i o n s s t u d i e d . H T was much higher at the three commonly polymorphic l o c i , Pgi-1, Pgi-2, and Skdh-3, with a mean value of 0.277. The t o t a l gene d i v e r s i t y was p r i m a r i l y d i s t r i b u t e d w i t h i n p o p u l a t i o n s (HS=0.175), but a c o n s i d e r a b l e amount of gene d i v e r s i t y was d i s t r i b u t e d among p o p u l a t i o n s . High G S T value (0.368) and Fsr value (0.345) i n d i c a t e s a r e l a t i v e high l e v e l of ge n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s . Geographical i s o l a t i o n and commonly small p o p u l a t i o n s i z e s are c h a r a c t e r i s t i c of s p e c i e s of Bidens o c c u r i n g on the Hawaiian I s l a n d s . Genetic d r i f t probably p l a y s a major r o l e i n the d i s t r i b u t i o n of allozyme v a r i a t i o n i n these p o p u l a t i o n s . Genetic i d e n t i t y among 21 p o p u l a t i o n s of Hawaiian Bidens i s high, ranging from 0.926-1.000, with a mean value of 0.982. The high g e n e t i c s i m i l a r i t y among these p o p u l a t i o n s i s probably due to t h e i r common a n c e s t r y . 1 12 V. MICROSPOROGENESIS IN MALE-STERILE AND HERMAPHRODITIC PLANTS OF NINE GYNODIOECIOUS TAXA 5.1 I n t r o d u c t i o n A l l indigenous taxa of Bidens i n the Hawaiian I s l a n d s are b e l i e v e d to be the products of adaptive r a d i a t i o n from one a n c e s t r a l immigrant s p e c i e s (Ganders and Nagata, 1983b, 1984). These Bidens s p e c i e s e x h i b i t e x t e n s i v e morphological and e c o l o g i c a l d i v e r s i t y and have r e c e n t l y been c l a s s i f i e d i n 19 s p e c i e s and 8 subspecies (Ganders and Nagata, 1983a). Mensch and G i l l e t t (1972) f i r s t r e p o r t e d m a l e - s t e r i l e p l a n t s i n EL micrantha ssp. c t e n o p h y l l a (= IL c t e n o p h y l l a S h e r f f ) , and G i l l e t t (1975) r e p o r t e d gynodioecious p o p u l a t i o n s i n four other s p e c i e s : IL_ menziesi i , EL_ t o r t a (= IL_ f ulvescens S h e r f f ) , IL hawaiensis (= IL_ s k o t t s b e r g i i S h e r f f ) and B.  f o r b e s i i . Four more s p e c i e s and s e v e r a l subspecies are a l s o gynodioecious (Appendix A ) . Some p l a n t s i n gynodioecious p o p u l a t i o n s have hermaphroditic d i s c flowers while other p l a n t s have n e a r l y isomorphic, m a l e - s t e r i l e f l o w e r s . At a n t h e s i s , male-s t e r i l e p l a n t s can be re c o g n i z e d by t h e i r s l i g h t l y s maller flower heads, and s m a l l e r , shrunken, l i g h t c o l o r e d anthers l a c k i n g p o l l e n . There i s no morp h o l o g i c a l v a r i a t i o n i n the ex p r e s s i o n of male s t e r i l i t y among m a l e - s t e r i l e p l a n t s . The f r e q u e n c i e s of male s t e r i l e s vary i n d i f f e r e n t gynodioecious p o p u l a t i o n s , ranging from 9% to 44%. G i l l e t t (1975) suggested that male s t e r i l i t y i n |L hawaiensis (= IL s k o t t s b e r g i i ) was c o n t r o l l e d by a r e c e s s i v e gene. The r e s u l t s of experimental c r o s s e s show 113 d i g e n i c r e c e s s i v e c o n t r o l of male s t e r i l i t y i n s e v e r a l other gynodioecious taxa (Chapter 6). Comparative microsporogenesis has been e x h a u s t i v e l y s t u d i e d i n normal and cytoplasmic or g e n i c - c y t o p l a s m i c m a l e - s t e r i l e crop p l a n t s (see Edwardson, 1970; Laser and L e r s t e n , 1972; Horner and Rogers, 1974; Horner, 1977). A few s t u d i e s have d e a l t with genie male s t e r i l i t y i n crop p l a n t s (Rick, 1948; C h i l d e r s , 1952; Nakashima and Hosokawa, 1974; A l b e r t s e n and Palmer, 1979; Greyson, Walden and Cheng, 1980; Palmer et a l . , 1980; A l b e r t s e n and P h i l l i p s , 1981). On the other hand, microsporogenesis i n s p e c i e s e x h i b i t i n g g e n i c a l l y c o n t r o l l e d male s t e r i l i t y i n n a t u r a l gynodioecious p o p u l a t i o n s has been v i r t u a l l y u nstudied. D e t a i l e d s t u d i e s of normal anther d i f f e r e n t i a t i o n and microsporogenesis have been rep o r t e d f o r only a few Asteraceae (Davis, 1961, 1962a, 1962b, 1964, 1966; Nakashima and Hosokawa, 1974; Horner, 1977), and have not i n c l u d e d Bidens. I have i n v e s t i g a t e d anther d i f f e r e n t i a t i o n and microsporogenesis i n male s t e r i l e s and hermaphrodites of nine gynodioecious taxa of Bidens using l i g h t microscopy. T h i s study had three purposes: to compare normal anther development and microsporogenesis i n Bidens with other Asteraceae, to compare microsporogenesis i n male s t e r i l e s i n n a t u r a l l y gynodioecious s p e c i e s with m a l e - s t e r i l e mutants i n crop p l a n t s , and to see i f microsporogenesis i n male s t e r i l e s d i f f e r s among the gynodioecious s p e c i e s of Bidens. The r e s u l t s of t h i s study are r e l e v a n t to understanding the e v o l u t i o n of gynodioecy i n Hawaiian Bidens. 1 14 5.2 M a t e r i a l s And Methods Microsporogenesis was i n v e s t i g a t e d i n p l a n t s of nine gynodioecious taxa of Bidens n a t i v e to the Hawaiian I s l a n d s and one i n t e r s p e c i f i c h y b r i d (Table 5-1). P l a n t s were grown i n greenhouses from c u t t i n g s or seeds c o l l e c t e d from n a t u r a l p o p u l a t i o n s . The h y b r i d was produced e x p e r i m e n t a l l y . Flower buds at v a r i o u s developmental stages were c o l l e c t e d i n December, 1983, January, 1984 and January, 1986. Six to ten flower buds of each developmental stage were sampled from each taxon and the h y b r i d . Both JB-4 and p a r a f f i n s e c t i o n i n g were used f o r c e r v i c a t a , and p a r a f f i n s e c t i o n i n g was used f o r a l l the other m a t e r i a l s s t u d i e d . Samples f o r p a r a f f i n s e c t i o n i n g were f i x e d i n 2% FAA (2% f o r m a l i n , 4% g l a c i a l a c e t i c a c i d and 70% e t h a n o l ) , dehydrated i n t - b u t a n o l , embedded i n P a r a p l a s t , s e c t i o n e d using a Wetzlar r o t a r y microtome at 8-12 pm. The s e c t i o n s were s t a i n e d e i t h e r i n s a f r a n i n 0 - f a s t green or i n s a f r a n i n 0 - orange G - i r o n alum (Johansen, 1940). S a f r a n i n 0 - f a s t green gave b e t t e r c o n t r a s t although both s t a i n s were s a t i s f a c t o r y . Buds f o r JB-4 s e c t i o n i n g were cut i n h a l f and f i x e d with 2.5% g l u t a r a l d e h y d e i n 0.1M c a c o d y l a t e b u f f e r (pH 7.2) f o r 3 h at room temperature and washed with the same b u f f e r twice f o r another 30 min, and dehydrated i n a graded ethanol s e r i e s . The samples were then taken d i r e c t l y from 95% a l c o h o l to c a t a l y z e d S o l u t i o n A f o r i n f i l t r a t i o n f o r 3 days at room temperature i n g e n t l e r o t a t i o n . Three changes of c a t a l y z e d JB-4 s o l u t i o n were a p p l i e d d u r i n g i n f i l t r a t i o n . Embedding s o l u t i o n was c a t a l y z e d 115 Table 5-1. Gynodioecious taxa and an experimental h y b r i d i n v e s t i g a t e d f o r microsporogenesis Taxa Sexes i n v e s t i g a t e d * Native l o c a l i t y B. c e r v i c a t a B. f o r b e s i i ssp. f o r b e s i i MS & H MS O h i k i l o l o Ridge, Oahu Haena, Kauai ssp. k a h i l i e n s i s  B. hawaiensis  B. m e n z i e s i i ssp. menziesi i  B. micrantha ssp. micrantha  B. p o p u l i f o l i a  B. s a n d v i c e n s i s ssp. s a n d v i c e n s i s B. t o r t a  B. f o r b e s i i ssp. f o r b e s i i X B. t o r t a MS & H MS & H MS & H MS & H MS & H MS H MS & H Mt. K a h i l i , Kauai Kaimu, Hawaii Olowalu Gorge, Maui Kahoma D i t c h , Maui Kahana V a l l e y , Oahu Mauumae Ridge, Oahu Waahila Ridge, Oahu Waianae Mtns., Oahu MS Experimental h y b r i d * MS: m a l e - s t e r i l e ; H: hermaphrodite. 1 16 S o l u t i o n A / S o l u t i o n B i n a r a t i o of 25:1. The embedding procedures were conducted on i c e to r e t a r d p o l y m e r i z a t i o n . The s e c t i o n s were cut on dry g l a s s knives on a S o r v a l l microtome at 2-3 um and s t a i n e d with 0.05% t o l u i d i n e blue 0 i n benzoate b u f f e r at pH 4.4 (Yeung, 1984), and with 0.73% t o l u i d i n e blue 0 and 0.135% b a s i c f u c h s i n i n 30% ethanol (Burns and B r e t s c h n e i d e r , 1981). Both p a r a f f i n s e c t i o n s and JB-4 s e c t i o n s were photographed. The photographs i n t h i s chapter were taken with a C a r l Z e i s s photomicroscope and are JB-4 s e c t i o n s of B.  c e r v i c a t a . 5.3 R e s u l t s Anther development from the a r c h e s p o r i a l stage to m a t u r i t y was i n v e s t i g a t e d i n both m a l e - s t e r i l e s and hermaphrodites. The o b s e r v a t i o n s reported i n the present paper were based on s e v e r a l thousand c r o s s s e c t i o n s of more than 500 flower buds. Normal anther ontogeny i n the hermaphrodites and the a b o r t i o n of microsporogenesis in male s t e r i l e s were the same i n a l l taxa and the h y b r i d s t u d i e d . The d e s c r i p t i o n s of the r e s u l t s are d i v i d e d i n t o two p a r t s . Normal anther d i f f e r e n t i a t i o n and microsporogenesis in the hermaphrodites are d e s c r i b e d f i r s t and abnormal microsporogenesis i n the m a l e - s t e r i l e s second. 5.3.1 Microsporogenesis In Hermaphrodites The young anther i s a 4-lobed s t r u c t u r e . The archesporium d i v i d e s to form a p a r i e t a l l a y e r towards the o u t s i d e and a primary sporogenous c e l l i n s i d e . The primary p a r i e t a l c e l l s undergo p e r i c l i n a l d i v i s i o n s to give r i s e to a s i n g l e l a y e r e d 117 tapetum i n s i d e and a secondary p a r i e t a l l a y e r o u t s i d e . The secondary p a r i e t a l c e l l s undergo f u r t h e r p e r i c l i n a l d i v i s i o n s to g i v e r i s e to a s i n g l e c e l l t h i c k middle l a y e r i n s i d e and a s i n g l e l a y e r e d endothecium o u t s i d e . During the d i f f e r e n t i a t i o n of the anther w a l l the primary sporogenous c e l l s undergo one or two m i t o t i c d i v i s i o n s to form more sporogenous c e l l s i n most microsporangia or remain undivided and f u n c t i o n d i r e c t l y as the microspore mother c e l l (mmc) i n some microsporangia. Upon the completion of anther d i f f e r e n t i a t i o n , an epidermal l a y e r , an e n d o t h e c i a l l a y e r , a middle l a y e r and a t a p e t a l l a y e r c o n s t i t u t e the anther w a l l and there are 1-4 sporogenous c e l l s v i s i b l e i n the t r a n s v e r s e s e c t i o n of each l o c u l e ( F i g . 5-1, 5-2). The tapetum --The t a p e t a l c e l l s enlarge i n s i z e and s t a i n densely as soon as e a r l y anther d i f f e r e n t i a t i o n i s completed. Some t a p e t a l c e l l s reach the s i z e of the sporogenous c e l l s b e fore the onset of m e i o s i s . During t h i s p r e m e i o t i c enlargement the t a p e t a l c e l l s are c h a r a c t e r i z e d by a s i n g l e , l a r g e , deeply s t a i n i n g nucleus and dense cytoplasm. Spaces between t a p e t a l c e l l s and sporogenous c e l l s have appeared at the formation of mmcs ( F i g . 5-3). The tapetum appears detached from the anther w a l l though s t i l l remaining i n t a c t and p e r i p h e r a l ( F i g . 5-3 -5-7), and the middle l a y e r of the anther w a l l i s not apparent d u r i n g meiosis ( F i g . 5-3 - 5-7), although these may be a r t i f a c t s caused by shrinkage d u r i n g f i x a t i o n and embedding. Nuclear s i z e i n the t a p e t a l c e l l i n c r e a s e s and k a r y o k i n e s i s i s i n i t i a t e d p r i o r to l e p t o t e n e of m e i o s i s I and some t a p e t a l c e l l s become b i n u c l e a t e d u r i n g meiosis ( F i g . 5-7). T a p e t a l cytoplasm 118 119 F i g u r e 1-12. Micro s p o r o g e n e s i s i n hermaphrodites from the gynodioecious s p e c i e s IK c e r v i c a t a . A l l f i g u r e s are c r o s s - s e c t i o n s of a n t h e r s . 1. E a r l y stage of d i f f e r e n t i a t i o n of anther l o c u l e c o n s i s t i n g of epidermis, endothecium, middle l a y e r , tapetum and sporogenous t i s s u e . x550. 2. Sporogenous mass stage. T a p e t a l c e l l s enlarge and middle l a y e r has become h i g h l y v a c u o l a t e d . x550. 3. mmc stage. Tapetum s t a i n s more densely. Separation i s apparent between mmcs (arrow). Middle l a y e r has become more f l a t t e n e d . x550. 4. Metaphase of M e i o s i s I. T a p e t a l c e l l s undergo k a r y o k i n e s i s . Middle l a y e r b a r e l y d e t e c t a b l e . x500. 5. E a r l y t e t r a d stage. x550. 6. L a t e r t e t r a d stage. Tapetum remains dense. Microspores are surrounded by c a l l o s e . x550. 7. Same stage as 6. Arrow i n d i c a t e s b i n u c l e a t e t a p e t a l c e l l . x550. 8. E a r l y -v a c u o l a t e microspore stage. Released microspores from t e t r a d s are rounded and have t h i n exine w a l l s with s p i n e s . Tapetum has l o s t c e l l w a l l s and i s v a c u o l a t e but s t i l l p e r i p h e r a l . Middle l a y e r has disappeared. x550. 9. Mid-v a c u o l a t e microspore stage. Tapetum i n t r u d e s c e n t r i p e t a l l y to surround microspores. Microspores have become vacuolate and have more prominent exine and s p i n e s . x550. 10. L a t e - v a c u o l a t e microspore stage. Microspores surrounded by p l a s m o d i a l tapetum d i s p l a y two w a l l l a y e r s . x480. 11. Vacu o l a t e d p o l l e n stage. Plasmodial tapetum i s h i g h l y v a c u o l a t e and degenerate. x470. 12. Cross s e c t i o n of e n t i r e anther. Anther d e h i s c e s to r e l e a s e p o l l e n . x90. 120 becomes v a c u o l a t e d and c e l l w a l l s have disappeared by the time microspores are r e l e a s e d from the t e t r a d s ( F i g . 5-8). The protoplasm of the t a p e t a l c e l l s flows inward to surround the young va c u o l a t e d microspores ( F i g . 5-9 - 5-10). The plasmodial tapetum g r a d u a l l y becomes h i g h l y v a c u o l a t e d ( F i g . 5-11) and disa p p e a r s p r i o r to the engorged p o l l e n stage ( F i g . 5-12). The sporogenous t i s s u e - The sporogenous c e l l s are surrounded by the tapetum and are appressed to each other at the completion of e a r l y anther d i f f e r e n t i a t i o n ( F i g . 5-1, 5-2). These c e l l s are i n i t i a l l y c h a r a c t e r i z e d by densely s t a i n i n g n u c l e i and cytoplasm ( F i g . 5-1) and s t a i n l e s s densely a f t e r d i s t i n c t c e n t r a l sporogenous t i s s u e has formed ( F i g . 5-2). The o r i g i n a l sporogenous c e l l w a l l s disappear d u r i n g the formation of mmcs. The mmcs become s p h e r i c a l i n shape and separate from each other as w e l l as from the surrounding tapetum ( F i g . 5-3). M e i o s i s I begins when each nunc becomes more s p h e r i c a l i n shape and more separated ( F i g . 5-4). The t e t r a d s remain embedded i n c a l l o s e f o r about one to two days be f o r e microspores are r e l e a s e d . A w a l l i s formed around each microspore p r e c e d i n g i t s r e l e a s e from the t e t r a d . The microspores r a p i d l y become s p h e r i c a l a f t e r t h e i r r e l e a s e , and cytoplasm i s dense with a s i n g l e nucleus at the center at t h i s stage ( F i g . 5-8). Upon the p l a s m o d i a l e x t e n s i o n of the p a r i e t a l t a p e t a l c e l l s i n t o the l o c u l e , l a r g e vacuoles have formed i n the cytoplasm of each microspore and exine w a l l s become t h i c k e r ( F i g . 5-9). While remaining w i t h i n the pla s m o d i a l tapetum, microspores e n l a r g e i n s i z e and become h i g h l y v a c u o l a t e d ( F i g . 5-10), and then form 121 p o l l e n g r a i n s ( F i g . 5-11). The h i g h l y v a c u o l a t e p o l l e n g r a i n s g r a d u a l l y f i l l with r e s e r v e s . The anther w a l l — The middle l a y e r becomes va c u o l a t e soon a f t e r i t s d i f f e r e n t i a t i o n ( F i g . 5-2), and appears more f l a t t e n e d with f u r t h e r development of the anther w a l l ( F i g . 5-3). I t i s not apparent j u s t p r i o r t o the formation of the p l a s m o d i a l tapetum ( F i g . 5-8). The e n d o t h e c i a l c e l l s r a d i a l l y e n l a r g e d u r i n g l a t e v a c u o l a t e microspore and e a r l y v a c u o l a t e p o l l e n stages ( F i g . 5-11), and the t y p i c a l f i b r e bands are formed i n the t r a n s v e r s e and inner e n d o t h e c i a l c e l l w a l l s d u r i n g the maturation of p o l l e n g r a i n s . By a n t h e s i s , many s m a l l , dense p a r t i c l e s appear i n the epidermis, which may account f o r the dark c o l o r of anthers at a n t h e s i s . The s e p t a l c e l l s between adjacent anther l o c u l e s disappear and the two adjacent l o c u l e s become c o n f l u e n t a f t e r the formation of the p o l l e n g r a i n s . Dehiscence of the anther w a l l occurs p r i o r to f l o r e t opening to r e l e a s e p o l l e n g r a i n s ( F i g . 5-12), which are then c o l l e c t e d by c l o s e d , p a p i l l o s e s t y l e branches which emerge from the f l o r a l tube through the surrounding tube of a n t h e r s . Protandry i s t y p i c a l of a l l taxa of Hawaiian Bidens. 5.3.2 Breakdown Of Microsporogenesis In Male S t e r i l e s E a r l y anther d i f f e r e n t i a t i o n i n male s t e r i l e s appears normal, as d e s c r i b e d f o r the c o r r e s p o n d i n g stages i n hermaphrodites above. The tapetum - Many smal l vacuoles are apparent i n the cytoplasm of t a p e t a l c e l l s soon a f t e r e a r l y anther d i f f e r e n t i a t i o n ( F i g . 5-13). At a comparable developmental 122 123 F i g u r e 5-13 - 5-21. Abnormal microsporogenesis i n m a l e - s t e r i l e p l a n t s from the gynodioecious s p e c i e s JK c e r v i c a t a . A l l f i g u r e s are c r o s s - s e c t i o n s of p o r t i o n s of an t h e r s . 13. E a r l y stage of d i f f e r e n t i a t i o n of an anther l o c u l e c o n s i s t i n g of epidermis, endothecium, p a r t i a l l y formed middle l a y e r , tapetum, and sporogenous t i s s u e . Development i s normal up to t h i s stage, when small vacuoles appear i n t a p e t a l c e l l s . Sporogenous c e l l s appear normal. x625. 14. Large vacuoles are apparent i n t a p e t a l c e l l s . Middle l a y e r i s w e l l d i f f e r e n t i a t e d . x625. 15. F u r t h e r abnormal v a c u o l a t i o n of t a p e t a l c e l l s . Cytoplasm of sporogenous c e l l s shows s i g n s of v a c u o l a t i o n . x575. 16. T a p e t a l c e l l s are almost completely v a c u o l a t e with only a nucleus l e f t , and begin to enlarge r a d i a l l y . Many smal l vacuoles are v i s i b l e i n the sporogenous c e l l s . x575. 17. S l i g h t l y l a t e r stage. T a p e t a l c e l l s f u r t h e r e n l a r g e d . x575. 18. Sporogenous c e l l s are l e s s dense and n u c l e i are abnormal. x575. 19. T a p e t a l c e l l s f u r t h e r i n t r u d e i n t o l o c u l e . Sporogenous c e l l s begin to degenerate. x575. 20. Sporogenous c e l l s have d i s i n t e g r a t e d , and tapetum has become d i s o r g a n i z e d c o n c o m i t a n t l y . Middle l a y e r and endothecium i n t r u d e i n t o l o c u l e . x575. 21. Cross s e c t i o n of e n t i r e anther. Most c e l l s surrounding each l o c u l e have degenerated and l o c u l e s have disappeared. x120. 1 24 stage i n hermaphrodites, t a p e t a l cytoplasm i s denser (Fig.5-1, 5-2). These small vacuoles i n the tapetum a p p a r e n t l y fuse to produce fewer, l a r g e vacuoles ( F i g . 5-14) and the cytoplasm of each t a p e t a l c e l l g r a d u a l l y d i s a p p e a r s with only a nucleus v i s i b l e i n some c e l l s ( F i g . 5-15, 5-16). At a comparable stage i n hermaphrodites, the cytoplasm of t a p e t a l c e l l s s t a i n s more densely and t a p e t a l c e l l s r a d i a l l y e nlarge i n s i z e ( F i g . 5-2, 5-3). The h i g h l y v a c u o l a t e d t a p e t a l c e l l s p ress upon the sporogenous c e l l s ( F i g . 5-16, 5-17) at about the stage when normal t a p e t a l c e l l s undergo k a r y o k i n e s i s . The t a p e t a l c e l l s c ontinue to r a d i a l l y extend upon the d i s i n t e g r a t i n g sporogenous c e l l s ( F i g . 5-19) and both d i s o r g a n i z e and disappear ( F i g . 5-20). The sporogenous t i s s u e — The sporogenous c e l l s of male-s t e r i l e s appear as normal as those of hermaphrodites (Fig.. 5-13, 5-14) u n t i l the e a r l y abnormal v a c u o l a t i o n of the tapetum. These sporogenous c e l l s are l a r g e , have dense cytoplasm and l a r g e n u c l e i , as d e s c r i b e d f o r the hermaphrodites above. With v a c u o l a t i o n of the t a p e t a l c e l l s , the cytoplasm of the sporogenous c e l l s a l s o becomes l e s s dense and many small vacuoles appear ( F i g . 5-15 - 5-18), while normal sporogenous c e l l s i n the hermaphrodite are rounding up to become mmcs and undergoing meiosis ( F i g . 5-3 - 5-8). The sporogenous c e l l s degenerate when the t a p e t a l c e l l s have extended r a d i a l l y i n t o the l o c u l e ( F i g . 5-19, 5-20). The anther w a l l --There seems to be no abnormality i n the epidermis and the endothecium at the e a r l y stages ( F i g . 5-13 125 5-15). The l a y e r s of the anther w a l l become l e s s d i s t i n c t with the v a c u o l a t i o n of the tapetum. A l l the c e l l l a y e r s r a d i a l l y e n large toward the l o c u l e upon the degeneration of sporogenous c e l l s and most c e l l s become d i s o r g a n i z e d r e s u l t i n g i n the disappearence of the l o c u l e ( F i g . 5-21). At a n t h e s i s , the anther i s composed of a s i n g l e - l a y e r e d epidermis with a few remnant c e l l s of the endothecium and the c o n n e c t i v e t i s s u e . The remaining e n d o t h e c i a l c e l l s do not have the t y p i c a l f i b e r bands as shown i n the normal endothecium, and the epidermal c e l l s c o n t a i n only a few dense p a r t i c l e s , which may account f o r the much l i g h t e r c o l o r of m a l e - s t e r i l e anthers at a n t h e s i s . 5.4 D i s c u s s i o n Anther d i f f e r e n t i a t i o n and microsporogenesis are the same in hermaphrodites of a l l gynodioecious taxa of Hawaiian Bidens. A l l e x h i b i t an ephemeral middle l a y e r and a plasmodial tapetum c h a r a c t e r i s t i c of Asteraceae. I n v e s t i g a t i o n s of microsporogenesis i n other taxa of the Asteraceae (Davis, 1961, 1962a, 1962b, 1964; Nakashima and Hosokawa, 1974; Horner, 1977) have demonstrated a general s i m i l a r i t y i n the development of normal a n t h e r s . Among taxa p r e v i o u s l y s t u d i e d , the sunflower (Helianthus annuus) i s a c l o s e r e l a t i v e of Bidens. In comparing a l l the stages of microsporogenesis, as d e f i n e d by Horner (1977), development of the tapetum, mmcs, microspores and p o l l e n g r a i n s are the same i n the "normal l i n e " of sunflower and the hermaphrodite of Bidens (see Horner, 1977; Nakashima and Hosokawa, 1974). The middle l a y e r i n Bidens seems to disappear s l i g h t l y e a r l i e r than r e p o r t e d f o r the sunflower by Horner 126 (1977). The breakdown of microsporogenesis o c c u r r e d i n the same manner i n the m a l e - s t e r i l e p l a n t s of a l l gynodioecious taxa of Bidens i n v e s t i g a t e d , as w e l l as i n the i n t e r s p e c i f i c h y b r i d between B^ f o r b e s i i and B^ t o r t a . However, the timing of a b o r t i o n i n m a l e - s t e r i l e Bidens d i f f e r s from both g e n i c a l l y i n h e r i t e d male s t e r i l e s i n the sunflower (Nakashima and Hosokawa, 1974) and c y t o p l a s m i c male s t e r i l e s i n the sunflower (Horner, 1977). Male s t e r i l i t y i n Bidens i s c h a r a c t e r i z e d by a complete p r e m e i o t i c degeneration of the sporogenous c e l l s f o l l o w i n g the e a r l y , abnormal, complete v a c u o l a t i o n of the tapetum. In m a l e - s t e r i l e sunflowers with genie i n h e r i t a n c e (Nakashima and Hosokawa, 1974), the tapetum appeared to be normal u n t i l the l a t e t e t r a d stage or e a r l y v a c u o l a t e microspore stage. The tapetum remained p e r i p h e r a l , e nlarged, and maintained c e l l w a l l s at the time that the normal tapetum became p l a s m o d i a l , and d i s i n t e g r a t e d l a t e r . In c y t o p l a s m i c male-s t e r i l e sunflower (Horner, 1977), the f i r s t v i s i b l e evidence of abnormal development was enlargement of the t a p e t a l c e l l s at the e a r l y t e t r a d stage. The t a p e t a l c e l l w a l l s d i s o r g a n i z e d and the en l a r g e d t a p e t a l c e l l s became vac u o l a t e and degenerated at the l a t e t e t r a d stage. In both cases, the abnormal behavior of the tapetum was suggested to be the cause of a b o r t i o n . There are s e v e r a l f e a t u r e s of anther a b o r t i o n i n the male-s t e r i l e p l a n t s of Hawaiian Bidens that are worthy of comment. F i r s t , the g e n e t i c f a c t o r ( s ) which c o n t r o l the a b o r t i o n of microsporogenesis act with remarkable p r e c i s i o n and c o n s i s t e n c y . 127 No v a r i a t i o n was observed i n the t i m i n g , order and r a t e of the breakdown of microsporogenesis among m a l e - s t e r i l e s of a l l taxa s t u d i e d . T h i s p r e c i s e developmental e x p r e s s i o n of male s t e r i l i t y i s notable when compared with many other s p e c i e s where male s t e r i l i t y has been s t u d i e d c y t o l o g i c a l l y . Great v a r i a t i o n i n the breakdown of microsporogenesis has been r e p o r t e d f o r c y t o p l a s m i c male s t e r i l i t y . An extreme example i s shown i n m a l e - s t e r i l e wheat (Chauhan and Singh, 1966), where three types of p o l l e n a b o r t i o n occurred w i t h i n the same s p i k e l e t , although only one o c c u r r e d i n any s i n g l e f l o r e t . In type (a) a b o r t i o n , the tapetum d i s i n t e g r a t e d p r e m e i o t i c a l l y , which i s analogous to the case i n Bidens; i n type (b), the normal degeneration of the tapetum was delayed; and i n type (c) a t a p e t a l periplasmodium was formed. Another example of such v a r i a t i o n i s shown i n c y t o p l a s m i c m a l e - s t e r i l e sugar beets. Artschwager (1947) observed two types of degeneration i n the flowers of a s i n g l e i n f l o r e s c e n c e . One type of a b o r t i o n was a s s o c i a t e d with a t a p e t a l Plasmodium and the other was a s s o c i a t e d with a c e l l u l a r tapetum. However, the wide v a r i a t i o n r e p o r t e d i n some e a r l i e r s t u d i e s may be the r e s u l t of the f i x a t i o n or other p r e p a r a t i v e methods used. V a r i a b i l i t y i n the e x p r e s s i o n of male s t e r i l i t y i s a l s o r e p o r t e d f o r g e n i c a l l y c o n t r o l l e d cases. Rick (1948) found that microsporogenesis i n a r e c e s s i v e m a l e - s t e r i l e mutant i n tomato, ms7, c o u l d be d i s r u p t e d at any stage of m e i o s i s . The mode of a c t i o n of another r e c e s s i v e gene, ms5, i s l e s s p r e c i s e i n that 128 normal p o l l e n developed i n c e r t a i n microsporangia while microsporogenesis was i n t e r r u p t e d at v a r i o u s stages i n o t h e r s . In the case of completely m a l e - s t e r i l e Medicaqo s a t i v a , where male s t e r i l i t y was c o n t r o l l e d by a s i n g l e r e c e s s i v e gene, the degenerative changes which took p l a c e i n the anther showed great v a r i a b i l i t y i n r e s p e c t to ; t i m i n g of onset ( C h i l d e r s , 1952). There was no v a r i a t i o n , however, i n the e x p r e s s i o n of male s t e r i l i t y r e p o r t e d i n both genie and c y t o p l a s m i c m a l e - s t e r i l e sunflower (Nakashima and Hosokawa, 1974; Horner, 1977). Second, the p r e c o c i o u s v a c u o l a t i o n of the tapetum i n male-s t e r i l e Bidens appears to be the cause of the degeneration of the sporogenous c e l l s . U n t i l the time of abnormal v a c u o l a t i o n i n the t a p e t a l c e l l s , anther ontogeny proceeds normally. F u r t h e r d i g e s t i o n of the tapetum i s f o l l o w e d by a r a p i d , d i s i n t e g r a t i o n of sporogenous c e l l s , and the tapetum then degenerates c o n c o m i t a n t l y . Examples of male s t e r i l i t y a s s o c i a t e d with t a p e t a l a b n o r m a l i t i e s are numerous. The hypothesis that m a l f u n c t i o n i n g of the tapetum i s a primary cause f o r the i n i t i a t i o n of the a b o r t i v e process has been supported by experimental i n v e s t i g a t i o n s i n A l l i u m , Beta, Capsicum, C r e p i s , Cucumis, C u c u r b i t a , D a c t y l i s , Daucus, Ga1i um, Hordeum, Phaseolus, Rununculus, Sorghum, Tabernaemontana, T r i t i c u m and Zea (see V a s i l , 1967, Horner, 1977 and r e f e r e n c e s c i t e d t h e r e i n ) . Although the breakdown of microsporogenesis may occur at any developmental stage, p r e m e i o t i c degeneration of sporogenous c e l l s f o l l o w i n g the e a r l y v a c u o l a t i o n of the tapetum has not 129 been r e p o r t e d as f r e q u e n t l y as a b o r t i o n o c c u r r i n g at l a t e r developmental stages. Laser and L e r s t e n (1972) reviewed c y t o p l a s m i c male s t e r i l i t y i n 62 taxa, which had been i n v e s t i g a t e d a n a t o m i c a l l y and c y t o l o g i c a l l y . In no case d i d the complete breakdown of microsporogenesis take p l a c e between the sporogenous c e l l stage and the pr e m e i o t i c mmc stage, the f i r s t stage of microsporogenesis recognized by Laser and L e r s t e n (1972). Degeneration appeared at l a t e r stages i n a l l these t a x a . A l b e r t s e n and P h i l l i p s (1981) d e s c r i b e d microsporogenesis i n 13 n o n - a l l e l i c r e c e s s i v e m a l e - s t e r i l e mutants i n Zea mays, i n which the e a r l i e s t stage where a b o r t i o n occurred was durin g mmc meio s i s i n one of the mutants. S i m i l a r l y , i n nine male-s t e r i l e mutants of tomato (Rick 1948) the e a r l i e s t stage at which breakdown occ u r r e d was d u r i n g prophase I i n the mmcs. C h i l d e r s (1952) and Greyson et a l . (1980) observed the degeneration of mmcs p r i o r to meiosis i n Medicago s a t i v a and i n a male s t e r i l e mutant of Zea mays, ms9. However, i n both cases the tapetum p e r s i s t e d a f t e r the degeneration of the mmcs, i n c o n t r a s t to the concomitant degeneration of the tapetum i n Bidens. Singh and Rhodes (1961) rep o r t e d p r e m e i o t i c degeneration of sporogenous c e l l s i n C u c u r b i t a maxima, i n which male s t e r i l i t y was c o n t r o l l e d by a monogenic r e c e s s i v e . They observed that the sporogenous c e l l s developed v a c u o l e s and the n u c l e i became h y p e r t r o p h i e d before degenerating, and the tapetum s t a r t e d to degenerate a f t e r the mmcs had s t a r t e d degeneration, which i s s i m i l a r to the o b s e r v a t i o n s d e s c r i b e d here. The r o l e of the tapetum i n normal microsporogenesis i s 1 30 v i t a l ( E c h l i n , 1971). The c h i e f f u n c t i o n of the tapetum has long been suggested to be the prod u c t i o n and t r a n s p o r t of enzymes, hormones and n u t r i e n t s d u r i n g microsporogenesis. These m a t e r i a l s are t r a n s p o r t e d to the sporogenous c e l l s or mmcs dur i n g the p r e - c a l l o s e p e r i o d , when the t r a n s f e r of "meiotic s i g n a l s " i s v i t a l to the onset of meio s i s i n mmcs (see V a s i l 1967). In Bidens, the a c t i v e f u n c t i o n of normal t a p e t a l c e l l s p r i o r to meio s i s i s suggested by t h e i r dense cytoplasm , l a r g e n u c l e i and k a r y o k i n e s i s , while the malfunct i o n of the tapetum i n male s t e r i l e s i s suggested by the e a r l y v a c u o l a t i o n of cytoplasm and the absence of k a r y o k i n e s i s . E i t h e r the f a i l u r e to supply the sporogenous c e l l s with e s s e n t i a l m a t e r i a l s f o r DNA s y n t h e s i s or the f a i l u r e to send m e i o t i c s i g n a l s to the sporogenous c e l l s may r e t a r d the onset of meiosis i n m a l e - s t e r i l e sporangia. The complete d i g e s t i o n of cytoplasm i n the t a p e t a l c e l l s appears to r e s u l t i n the v a c u o l a t i o n of cytoplasm and the d i s i n t e g r a t i o n of n u c l e i i n sporogenous c e l l s . Developmental s i m i l a r i t y among the male s t e r i l e s i n Bidens suggests a common mechanism of male s t e r i l i t y i n a l l gynodioecious taxa of Bidens i n the Hawaiian I s l a n d s . T h i s a l s o suggests that the same gene(s) c o n t r o l male s t e r i l i t y i n a l l t a x a . I f male s t e r i l i t y were c o n t r o l l e d by d i f f e r e n t genes i n d i f f e r e n t s p e c i e s of Bidens, one might expect d i f f e r e n c e s i n the ontogeny of m a l e - s t e r i l e anthers (Rick, 1948; A l b e r t s e n and P h i l l i p s , 1981). Genetic s t u d i e s of the i n h e r i t a n c e of male s t e r i l i t y have proved that the nuclear genes which cause male s t e r i l i t y are a l l e l i c i n a l l gynodioecious s p e c i e s of Hawaiian 131 Bidens (Chapter 6). I t suggests that gynodioecy o r i g i n a t e d only once i n Hawaiian Bidens. T h i s mutational event undoubtedly o c c u r r e d a f t e r Bidens immigrated to the Hawaiian I s l a n d s because gynodioecy i s unknown i n any c o n t i n e n t a l s p e c i e s . The occurrence of gynodioecy i n 13 taxa of Hawaiian Bidens may be a r e s u l t of s p e c i a t i o n and adaptive r a d i a t i o n from the o r i g i n a l taxon i n which the m a l e - s t e r i l e mutation event occurred, or, i n some cases i t c o u l d be the r e s u l t of n a t u r a l h y b r i d i z a t i o n between gynodioecious and hermaphroditic taxa. H y b r i d i z a t i o n among a l l Hawaiian taxa i s p o s s i b l e , although n a t u r a l i n t e r s p e c i f i c h y b r i d s are r e l a t i v e l y uncommon (Ganders and Nagata, 1984). If the m a l e - s t e r i l e mutation was e s t a b l i s h e d i n an e a r l y a n c e s t r a l p o p u l a t i o n , the descendant taxa would be expected to be gynodioecious. I f male s t e r i l i t y evolved only once i n the ada p t i v e r a d i a t i o n of Bidens on the Hawaiian I s l a n d s , one might expect that the gynodioecious taxa would be more c l o s e l y r e l a t e d to each other than to the s t r i c t l y hermaphroditic taxa. C e r t a i n gynodioecious s p e c i e s are s i m i l a r m o r p h o l o g i c a l l y and appear to represent c l o s e l y r e l a t e d groups. Bidens m e n z i e s i i , B.  micrantha and B_^  t o r t a are s i m i l a r and share d e r i v e d c h a r a c t e r s such as glabrous achenes and numerous small flower heads. Bidens c e r v i c a t a , B. f o r b e s i i and p o s s i b l y B^ s a n d v i c e n s i s are c l o s e l y r e l a t e d and may be a s i s t e r group or a n c e s t r a l to the B.  m e n z i e s i i group. However, B_^  amplectens, B. hawaiensis and B.  p o p u l i f o l i a are m o r p h o l o g i c a l l y d i f f e r e n t from the other gynodioecious s p e c i e s as w e l l as being rather d i f f e r e n t from 132 each o t h e r . With p r e s e n t l y a v a i l a b l e data i t i s not p o s s i b l e to determine whether gynodioecy i n these three s p e c i e s r e p r e s e n t s p a r a l l e l e v o l u t i o n , a t r a i t i n h e r i t e d from a common ancestor, or whether i t has r e s u l t e d from i n t e r s p e c i f i c h y b r i d i z a t i o n . The r e s u l t s of the present study do suggest that gynodioecy i n a l l Hawaiian s p e c i e s of Bidens i s homologous. 5.5 Summary Microsporogenesis was i n v e s t i g a t e d i n hermaphroditic and m a l e - s t e r i l e p l a n t s i n nine gynodioecious taxa of Hawaiian Bidens. Normal microsporogenesis i n hermaphrodites and the onset of a b o r t i o n i n male s t e r i l e s were s i m i l a r i n a l l taxa and i n a h y b r i d between two gynodioecious s p e c i e s . The e a r l y abnormal v a c u o l a t i o n of t a p e t a l c e l l s i s the f i r s t v i s i b l e evidence l e a d i n g to p r e m e i o t i c a b o r t i o n of microsporogenesis i n male s t e r i l e s . The sporogenous c e l l s d i s i n t e g r a t e r a p i d l y a f t e r the v a c u o l a t i o n of the tapetum, r e s u l t i n g i n a shrunken, i n d e h i s c e n t anther which i s composed of only the epidermal l a y e r with some remnant c e l l s of the endothecium and the c o n n e c t i v e at a n t h e s i s . In hermaphrodites, the t a p e t a l c e l l s remain dense and undergo k a r y o k i n e s i s to become b i n u c l e a t e d u r i n g meiosis I. The tapetum becomes plasmodial a f t e r microspores are r e l e a s e d from t e t r a d s and g r a d u a l l y d i s a p p e a r s d u r i n g p o l l e n formation. The g e n e t i c f a c t o r ( s ) which cause the a b o r t i o n a c t with remarkable p r e c i s i o n and c o n s i s t a n c y i n a l l taxa i n v e s t i g a t e d . T h i s suggests t h a t gynodioecy i n a l l Hawaiian Bidens i s homologous and the establishment of male s t e r i l i t y i n Hawaiian Bidens o c c u r r e d o n l y once. The spread of the g e n e t i c m a l e - s t e r i l e 133 f a c t o r ( s ) may be the r e s u l t of adaptive r a d i a t i o n of the o r i g i n a l gynodioecious s p e c i e s or n a t u r a l i n t e r s p e c i f i c h y b r i d i z a t i o n . 134 VI.. GENETICS OF GYNODIOECY 6.1 I n t r o d u c t i o n The g e n e t i c b a s i s of many m a l e - s t e r i l e mutants i n crop p l a n t s has been i n v e s t i g a t e d , but not much i s known of the i n h e r i t a n c e of male s t e r i l i t y i n n a t u r a l gynodioecious s p e c i e s . The importance of st u d y i n g the i n h e r i t a n c e of male s t e r i l i t y i s , as has been s t a t e d by Charlesworth (1981), that " Despite having been s t u d i e d f o r a c o n s i d e r a b l e time, the problem of how females are maintained i n gynodioecious s p e c i e s remains unsolved. U n t i l the g e n e t i c b a s i s f o r male s t e r i l i t y i s known, i t seems u n l i k e l y that we w i l l understand how t h i s type of polymorphism i s maintained". To date, i n h e r i t a n c e of male s t e r i l i t y has been s t u d i e d f o r only about 12 n a t u r a l gynodioecious s p e c i e s (see Charlesworth, 1981; Van Damme, 1984, and r e f e r e n c e s t h e r e i n ) . The r e s u l t s of these s t u d i e s i n d i c a t e t hat the i n h e r i t a n c e of male s t e r i l i t y i n these s p e c i e s i s more complicated than simple s i n g l e - f a c t o r nuclear or cyt o p l a s m i c c o n t r o l . Cytoplasmic-genic mechanisms appear to be i n a c t i o n i n almost a l l of these spec i e s . For a f u r t h e r understanding of n a t u r a l l y o c c u r r i n g gynodioecy, the 13 gynodioecious taxa of Hawaiian Bidens provide e x c e l l e n t m a t e r i a l s f o r st u d y i n g the g e n e t i c b a s i s of male s t e r i l i t y . The developmental s t u d i e s of microsporogenesis r e p o r t e d i n Chapter 5 suggest that male s t e r i l i t y i s homologous in a l l gynodioecious s p e c i e s . T h i s study p r i m a r i l y aimed at i n v e s t i g a t i n g the mode of g e n e t i c c o n t r o l of male s t e r i l i t y , and 135 at t e s t i n g the hypothesis that male s t e r i l i t y i n d i f f e r e n t g ynodiecious taxa of Hawaiian Bidens i s homologous. 6.2 M a t e r i a l s And Methods Seeds and c u t t i n g s of Hawaiian s p e c i e s of Bidens were c o l l e c t e d and r a i s e d to f l o w e r i n g p l a n t s i n greenhouses. These p l a n t s i n c l u d e d hermaphrodites and females, and served as the p a r e n t a l generation f o r many experimental c r o s s e s . A l l the c r o s s e s were made by hand i n greenhouses. Flowers were p r o t e c t e d from p o l l i n a t o r contamination by p l a c i n g the p l a n t s i n t o i n s e c t - p r o o f growth chambers or cages. Most c r o s s e s made were between females and hermaphrodites by simply rubbing hermaphrodite flower heads with copious p o l l e n g r a i n s onto the female flower heads at the optimum r e c e p t i v e phase. S e l f i n g of hermaphrodites was performed by rubbing flower heads on the same p l a n t t o g e t h e r . In a few cases where hermaphrodite p l a n t s were used as maternal parents, the p o l l e n g r a i n s were blown away from the flower heads before the stigmas were r e c e p t i v e . Strong protandry i n the Hawaiian s p e c i e s of Bidens e f f i c i e n t l y prevents s e l f - f e r t i l i z a t i o n . No attempt was made to emasculate the flow e r s s i n c e a l l d i s c flowers are very s m a l l . Male s t e r i l i t y c o u l d be e a s i l y i d e n t i f i e d i n the F1 and F2 progeny, and i n the progenies of backcrosses and t r i p l e - c r o s s e s . M a l e - s t e r i l e p l a n t s were reco g n i z e d on the b a s i s of flower and anther morphology. M a l e - s t e r i l e flowers commonly have smaller flower s i z e and l i g h t e r anther c o l o r than hermaphroditic f l o w e r s . In a d d i t i o n , m a l e - s t e r i l e anthers are s m a l l , i n d e h i s c e n t , and s h r i v e l l e d i n appearence. In cases of 136 u n c e r t a i n t y , the flower heads were checked under a microscope f o r the presence or absence of p o l l e n g r a i n s . There i s no intermediate form of male s t e r i l i t y among flowers w i t h i n p l a n t s or among p l a n t s , so that phenotypes can be c l a s s i f i e d unambiguously. The i n h e r i t a n c e of male s t e r i l i t y i n a l l gynodioecious s p e c i e s was s t u d i e d using F1, F2, and backcross g e n e r a t i o n s . T r i p l e - c r o s s e s and quadruple-crosses were performed to t e s t a l l e l i s m among gynodioecious s p e c i e s . F u r t h e r d e t a i l s of the c r o s s i n g designs are presented i n Ta b l e s 6-6 and 6-7. The segr e g a t i o n r a t i o s of females and hermaphrodites were recorded from n a t u r a l l y p o l l i n a t e d hermaphrodites and females from p o p u l a t i o n s of f o r b e s i i subsp. f o r b e s i i , B. p o p u l i f o l i a , and EL s a n d v i c e n s i s subsp. s a n d v i c e n s i s . Seeds of these p o p u l a t i o n s were c o l l e c t e d i n the f i e l d , and grown i n a greenhouse. 6.3 R e s u l t s Segregations of hermaphrodites and females i n progeny grown from seeds c o l l e c t e d from four n a t u r a l p o p u l a t i o n s are given i n Table 6-1. Both female and hermaphroditic maternal p l a n t s i n n a t u r a l p o p u l a t i o n s of these gynodioecious s p e c i e s produced hermaphrodite and female progeny. Table 6-2 prese n t s the seg r e g a t i o n r a t i o s i n F1 progeny f o r 27 experimental c r o s s e s between females of a l l gynodioecious s p e c i e s of Hawaiian Bidens and hermaphrodites of non-gynodioecious s p e c i e s . The r e s u l t s are homogeneous. A l l F1 p l a n t s were hermaphrodites, i n d i c a t i n g Table 6-1. Segregation of females and hermaphrodites i n 4 gynodioecious p o p u l a t i o n s r a i s e d i n a greenhouse. Maternal NO. Of progeny Species sex type* H MS B. f o r b e s i i FORB F1 H 18 2 MS 13 3 FORB F2 H 29 8 MS 29 9 B. p o p u l i f o l i a H 18 2 B. s a n d v i c e n s i s H 28 5 MS 43 9 *: H: hermaphrodites; MS: male s t e r i l e s . Table 6-2. Segregation in the F1 generation in experimental crosses between females of gynodioecious species and hermaphrodites of non-gynodioecious species. A l l male-sterile plants have the same genotype: mmnn, and a l l hermaphrodites have the same genotype: MMNN. The empty ( ) designates that cytoplasm can be either type: (A) or (B). The N locus has no ef f e c t in (A) cytoplasm (Genotype assignments 1n a l l the tables of th i s chapter are based on the genlc-cytoplasmic model). No. of F1 progeny Experimental cross H MS MS ( )mmnn X H ( )MMNN B. ce r v l c a t a (8B) X B. campylotheca (114A) 4 0 X B. cosmoides (9) 8 0 X B. hi 1lebrandiana (68B) 2 0 B. forbes11 (12A) X B. B. cosmoides (9) 5 0 X B. hi 1lebrandiana (68B) 2 0 X B. mauiensis (10B) 6 0 X B. moloka lens is (11) 6 0 X B. valIda (131 A) 6 O B. hawaiensis (51A) X B. mauiensis (10A) 6 0 X B. moloka lens is (11 & 72A) 9 0 B. menziesi1 (30 & 84) X B. mau1 ens 1s 4 0 X B. campylotheca (114A) 4 0 139 Table 6-2 cont. X #179B 3 0 B. mlcrantha (133A) X B. campylotheca (195C & 114A) a 0 X B. valIda (131A) 5 0 B. p o p u l i f o l i a (42B) X B. cosmoldes (9) 16 0 X B. macrocarpa (23) 4 0 X B. mauiensis (129) 9 0 X B. moloka lens Is (72) 5 0 X AM79A 8 0 B. sandvicensis (7A & 112) X B. cosmoldes (9) 5 0 X /C179A 4 0 X B. campylotheca (114) 3 0 B. torta (18) X B. mauiensis (10A) 7 0 *58B X B. moloka lens is (72) 2 0 X B. maulensis (10A) 3 0 X B. valIda (131A) 3 O *: H: hermaphrodites; MS: male s t e r i l e s . H: 179A and 179B are F1 hybrids of B. valIda X B. molokaiens 1s. 58B 1s a natural hybrid of B. torta X B.amplectens. 140 that the hermaphrodite c o n d i t i o n i s dominant to the male-s t e r i l e . The c r o s s e s made between females and hermaphrodites of gynodioecious s p e c i e s g i v e e i t h e r a l l hermaphrodites or both hermaphrodites and females (Table 6-3). In the c r o s s e s which gave female progeny, the genotypes of the hermaphrodites used as p a t e r n a l parents are probably heterozygous f o r sex-determining genes, s i n c e female progeny were repe a t e d l y produced i n d i f f e r e n t c r o s s e s when the same hermaphrodite p l a n t s were used as p o l l e n donors. A s i m i l a r s e g r e g a t i o n p a t t e r n i s shown in w i t h i n - s p e c i e s c r o s s e s . If the p a t e r n a l hermaphrodite c o n t a i n s r e c e s s i v e male s t e r i l i t y genes, m a l e - s t e r i l e progeny w i l l be produced by female p l a n t s and s e g r e g a t i o n w i l l occur when heterozygous hermaphrodites are s e l f e d . T h i s was found i n B.  p o p u l i f o l i a p l a n t 42A (Table 6-3). The number of genes c o n t r o l l i n g male s t e r i l i t y was i n v e s t i g a t e d by s e l f i n g F1 hermaphrodites from the c r o s s e s between male s t e r i l e s and hermaphrodites of non-gynodioecious s p e c i e s shown in Table 6-2, and a l s o hermaphrodites of gynodioecious s p e c i e s . Table 6-4 g i v e s the s e g r e g a t i o n r a t i o s in the F2 g e n e r a t i o n . In IK_ hawaiensis, B. f o r b e s i i subsp. f o r b e s i i , and IK_ p o p u l i f o l i a , male s t e r i l i t y appears to be c o n t r o l l e d by a s i n g l e r e c e s s i v e gene, g i v i n g 3:1 r a t i o s i n the F2. However, i r r e g u l a r s e g r e g a t i o n s i n the F2 appeared i n B.  menziesi i subsp. f i l i f o r m i s and IK s a n d v i c e n s i s subsp. s a n d v i c e n s i s , the r a t i o s are c l o s e r to 15:1 than to 3:1, suggesting a d i g e n i c c o n t r o l of male s t e r i l i t y . If male s t e r i l i t y i s monogenic r e c e s s i v e , a 1:1 r a t i o should be obtained T a b l e 6-3. S e g r e g a t i o n i n the F l p r o g e n i e s i n e x p e r i m e n t a l c r o s s e s between g y n o d i o e c i o u s s p e c i e s . No. of F1 progeny E x p e r i m e n t a l c r o s s H MS MS X H ( )mmnn ( )M_NN. (B)mmNN N o n - s e g r e g a t i n g c r o s s e s : or ( )MM_ B. c e r v i c a t a (8B) X B . f o r b e s i l (13A) 3 0 X B . s a n d v i c e n s i s (43B) 6 0 B . f o r b e s i l (12A) X B . menz i es i1 (31) 10 0* X B . m i c r a n t h a (24 & 25) 3 0 X B . t o r t a (39A) 6 0 B . h a w a i e n s i s (51A) X B. f o r b e s i l (71) 2 0 B . m e n z i e s i i (30 & 84) X B . amplectens ( 1 ) 5 0 B . p o p u l i f o l i a (42B) X B . amplectens (1) 36 0 X B. c e r v i c a t a (8C) 21 0 X B . f o r b e s i l (71A) 5 0 X B. m i c r a n t h a (24C) 4 0 X B. menziesi1 ( 163A) 2 0 X B. s a n d v i c e n s i s (20A) 3 0 X 065A 15 O B . s a n d v i c e n s i s (7A) X B . amplectens ( 1 ) 8 O X B . c e r v i c a t a (8C) 13 0 X B. m i c r a n t h a (24C) 2 0 T a b l e 6-3 c o n t . X B . t o r t a 3 0 S e g r e g a t i n g c r o s s e s : MS ( )mmnn X H ( )Mmnn, (B)mmNn or ( )MmNn B. h a w a i e n s i s (5 1A) X B . asyrametrica (4) 1 1 X B. c e r v i c a t a (8D) 4 4 X B. menz i e s 1 i (31) 1 • 1* X B . p o p u l i f o l i a (42A) 1 1 1 X B . s a n d v i c e n s i s (112) 15 6 B. m e n z i e s i i (30 & 84) X B . s a n d v i c e n s i s (112) 4 2 B. p o p u l i f o l i a (42B) X B . asymmetrica (4) 3 3 X B . h a w a i e n s i s (52) 2 3 X B . menz i es i i (31) 0 3* B. s a n d v i c e n s i s (7A) X B . menz i es i i (31) 0 2* X B . asymmetrica (4) 4 2 W i t h i n s p e c i e s : B. p o p u l i f o l i a (42B) X B . p o p u l i f o l i a (42C, (A)MM .) 12 0 (A) mmnn X B . p o p u l i f o l i a (42A. (A)Mm—) 3 4 B. p o p u l i f o l i a (42C) s e l f 5 0 B. p o p u l i f o l i a (42A) s e l f 10 12 It: 65A i s a n a t u r a l h y b r i d of ET_ t o r t a X B^ amplectens . *: Genotype of H 31 must be he t e r o z y g o u s a c c o r d i n g to these r e s u l t s . Table 6-4 Segregation in F2 progenies. Parental cross No. of F2 progeny H MS X*(3:1) X'(15: 1 ) Crosses with non-gynodioecious species: MS 1) B. f o r b e s i i (12A) X B. mauiensis (10B) (A)mmnn (A)MMNN 2) B. hawaiensis (51A) X B. mauiensis (10A) (A)mmnn (A)MMNN 3) B. p o p u l i f o l i a (42B) X B. mauiensis (129A) (A)mmnn (A)MMNN 72 16 2.182 0.20-0.10 21.382 <0.005 32 12 0.121 0.80-0.70 33.188 <0.005 56 16 0.296 0.70-0.50 31.348 <0.005 4) B. menziesi1 (30) (B)mmnn 5) 6) B. torta (18) (B)mmnn B. mauiensis (A)MMNN B. mo1 oka 1ens 1s ( )MMNN B. mauiensis (10A) (A)MMNN 16 26 3.313 0.10-0.05 0.004 0.95 1.200 0.30-0.20 0.240 0.70-0.5 2.178 0.20-0.10 2.569 0.20-0.1 Table 6-4. cont Crosses with gynodioecious species: 7) B. menz i es11 (B )mmnn 8) 9) B. sandvicensis X B. hawaiensis (A) M_N_, M nn or mmN. X B. sandvicensis (B) M_N_ X B. torta ( )M_N_ Cross between hermaphrodites: H X H 10) B. mauiensis (27C) X B. cervicata (8D) (A)MMNN ( )Mm 0.000 >0.99 5.081 <0.05 5.170 <0.05 0.000 >0.99 7.200 <0.01 0.860 0.50-0. 1.545 0.30-0. 7.200 <0.01 145 i n the backcross; and i f d i g e n i c , a 3:1 r a t i o should be obt a i n e d . The r e s u l t s of the backcrosses made f o r B.  hawaiensis, B. p o p u l i f o l i a , and IK t o r t a are given i n Table 6-5. Regardless of the g e n e r a l l y small sample s i z e , the progeny r a t i o s are c l o s e to 1:1 i n hawaiensis and IK p o p u l i f o l i a , i n agreement with the 3:1 r a t i o i n the F2 f o r these two s p e c i e s , and the r a t i o i s c l o s e to 3:1 i n IK t o r t a , i n agreement with 15:1 r a t i o i n the F2 i n t h a t s p e c i e s . The a l l e l i s m of the m a l e - s t e r i l e genes i n these s p e c i e s was t e s t e d by 17 t r i p l e - c r o s s e s (Table 6-6) and 4 quadruple c r o s s e s (Table 6-7). In most of the c r o s s e s , female progeny were segregated, which i s i n d i c a t i v e of a l l e l i s m of m a l e - s t e r i l e genes i n the s p e c i e s i n v o l v e d r e g a r d l e s s of the se g r e g a t i o n r a t i o . The r a t i o s are c e r t a i n l y h e l p f u l i n de-termining the number of gene l o c i i n v o l v e d , which w i l l be d i s c u s s e d l a t e r . The a l l e l i c r e l a t i o n s h i p r e v e a l e d by s e g r e g a t i n g c r o s s e s i n Table 6-3 and by t r i p l e - c r o s s e s (Table 6-6) and quadruple-c r o s s e s (Table 6-7) can be summarized i n F i g . 6-1. The r a t i o s of t r i p l e c r o s s e s i n Table 6-6 are e i t h e r 1:1 or 3:1, which i s i n agreement with the backcross r a t i o s of monogenic and d i g e n i c r e c e s s i v e s r e s p e c t i v e l y . In c o n s i d e r i n g the a l l e l i c r e l a t i o n s h i p between female p a i r s i n these t r i p l e c r o s s e s , the r a t i o s p r o v i d e a d d i t i o n a l support f o r the monogenic or d i g e n i c r e c e s s i v e models. A l l quadruple-crosses gave 3:1 r a t i o s , i n d i c a t i n g d i g e n i c c o n t r o l of.male s t e r i l i t y . Table 6-5. Segregation in the progeny of backcrosses (see Appendix A for species acronym) Backcross No. of progeny H MS X (1:1) X 2(3:1) MS ( MS H ) 1) MS HAWA(51A) X (A)mmnn X (MS HAWA(51A) X H AMPL(1)) ( )M (MS HAWA(51A) X H MAUI(10A)) (A)MMNN (MS HAWA(51A) X H CERV(8D)) ( )Mm_ Pooled 1.286 0.30-0.20 0.692 0.50-0.30 8.04 <0.005 9.256 <0.005 2) MS P0PU(42B) X H (MS P0PU(42B) X H AMPL(O) (A)mmnn ( )M 12 10 0.183 0.70-0.50 57.709 <0.005 3) MS T0RT(18) X H (MS T0RT(18) X H MAUI(10A)) (B)mmnn (A)MMNN 19 9.783 <0.005 0.710 0.50-0.30 Table 6-6. Segregation in t r i p l e crosses (see Appendix A for species acronym). No. of progeny Tr i p l e cross H MS XZ(1:1) p X 2(3:1) p MS X ( MS X H ) 1) MS CERV(88) X (MS F0RB(12A) X H MAUI(10B)) 6 0 6.000 <0.05 2.000 0.20-0.10 (B)mmnn (A)mmnn (A)MMNN 2) X (MS MENZ(30) X H ASYM(4)) 7 2 2.778 0.10-0.05 0.037 0.90-0.80 (B)mmnn ( )MmN 3) MS F0RB(12A) X (MS HAWA(51A) X H MAUI(10A)) 25 18 1.140 0.30-0.20 6.519 <0.05 (A) mmnn (A)mmnn (A)MMNN 4) MS MENZ(163) X (MS P0PU(42B) X H MAUI(129)) 13 1 10.286 <0.005 2.381 0.20-0.10 (B) mmnn (A)mmnn (A)MMNN 5) X (MS F0RB(12A) X H MAUI(10B)) 8 5 0.692 0.50-0.30 1.256 0.30-0.25 (A) mmnn (A)MMNN 6) X (MS MENZ(30) X H ASYM(4)) 2 2 0.000 >0.99 1.333 0.25-0.20 (B) mmnn ( )MmN_ 7) MS MICR(24) X (MS P0PU(42B) X H AMPL(1)) 9 6 0.600 0.50-0.30 1.800 0.20-0.10 (B)mmnn (A)mmnn ( )M_N_ 8) X (MS P0PU(42B) X H HILL(68)) 7 0 7.000 <0.01 2.083 0.20-0.10 (A)mmnn ( )MMNN 9) X (MS HAWA(51A) X H MAUI(10A)) 2 3 0.200 0.70-0.50 3.267 0.10-0.05 (A)mmnn (A)MMNN 10) MS MICR(133) X (MS F0RB(12A) X H MAUI(IOB)) 18 4 8.909 <O.OQ5 0.545 0.50-0.30 e 6 - 6 c o n t . ( B ) m m n n 1 1 ) M S S A N D ( 1 1 3 ) ( B ) m m n n 1 2 ) 1 3 ) M S T O R T ( 1 9 ) ( B ) m m n n 1 4 ) 1 5 ) 1 6 ) M S T 0 R T ( 2 2 5 ) ( B ) m m n n 1 7 ) ( A ) m m n n ( M S F O R B ( 1 2 A ) ( A ) m m n n ( M S M E N Z ( 3 0 ) ( B ) m m n n ( M S P 0 P U ( 4 2 B ) ( A ) m m n n ( M S H A W A ( 5 1 A ) ( A ) m m n n ( M S P 0 P U ( 4 2 B ) ( A ) m m n n ( M S F O R B ( 1 2 A ) ( A ) m m n n ( M S M E N Z ( 3 0 ) ( B ) m m n n ( A ) M M N N X H M A U I ( 1 0 B ) ) ( A ) M M N N X H A S Y M ( 4 ) ) ( ) M m N — X H H I L L ( 6 8 ) ) ( ) M M N N X H M A U I ( 1 0 A ) ) ( A ) M M N N X H A M P L ( 1 ) ) ( ) M _ N _ X H M A U I ( 1 0 B ) ) ( A ) M M N N X H A S Y M ( 4 ) ) ( ) M m N _ 11 3 4 . 5 7 1 < 0 . 0 5 0 . 0 9 5 0 . 8 0 - 0 . 7 0 1 1 0 . 0 0 0 > 0 . 9 9 0 . 6 6 7 0 . 5 0 - 0 . 3 0 2 0 2 . 0 0 0 0 . 2 0 - 0 . 1 0 0 . 6 6 7 0 . 5 0 - 0 . 3 0 3 O 3 . 0 0 0 0 . 1 0 - 0 . 0 5 1 . 0 0 0 0 . 5 0 - 0 . 3 0 1 1 0 . 0 0 0 > 0 . 9 9 0 . 6 6 7 0 . 5 0 - 0 . 3 0 3 7 2 7 1 . 5 6 3 0 . 3 O - 0 . 2 O 1 0 . 0 8 3 < 0 . 0 0 5 3 4 2 6 1 . 0 6 7 0 . 5 0 - 0 . 3 0 1 0 . 7 5 6 < 0 . 0 0 5 Table 6-7. Segregation in the progeny of quadruple crosses (see Appendix A for species acronym) No. of progeny Quadruple cross H MS X l( 1:1) p X J(3:1) p MS ( MS X H ) X H ( MS X H ) 1) MS 186 (MS MICR X H HAWA) X H (MS FORB(12A) X H MAUI(10B)) 16 4 7 . 200 <0 .01 0 . 267 0 .70- 0 .50 (B)mmnn (B)mmnn (A)MmN_ (A)MmNn (A)mmnn (A)MMNN (A )Mmnn 2) X H (MS P0PU(42B) X H MAUI(129)) 19 5 8 . 167 <0 .005 0. 333 0. . 70-0. 50 (A)MmNn (A)mmnn (A )MMNN 3) MS 190 (MS MICR X H HAWA) X H (MS HAWA(51A) X H MAUI(10A)) 8 1 5 . 444 <0. ,05 0. 926 0. 50- 0. 30 (B)mmnn (B)mmnn (A)MmN_ (A)MmNn (A)mmnn (A)MMNN (A)Mmnn 4) X H (MS MENZ(30) X H MAUI ) 15 4 6 . 368 <0. 05 0. 158 0. 70- 0. 50 (B)mmnn (B)mmnn (A)MMNN •J>-B. h a w a i e n s i s B.asymmetrica B.populifolia Figure 6-1. Allelism for male sterility in nine gynodioecious species of Hawaiian Bidens demonstrated by interspecific crosses. o 151 6.4 D i s c u s s i o n Three general modes of i n h e r i t a n c e of male s t e r i l i t y have been proposed f o r m a l e - s t e r i l e mutants i n a g r i c u l t u r a l crops or n a t u r a l gynodioecious s p e c i e s , i . e . , c y t o p l a s m i c ; genic-c y t o p l a s m i c ; and genie c o n t r o l of male s t e r i l i t y . S e v e r a l models of genetic c o n t r o l of male s t e r i l i t y i n Hawaiian Bidens can be t e s t e d using the r e s u l t s of the experimental c r o s s e s . 6.4.1 Cytoplasmic Male S t e r i l i t y I f male s t e r i l i t y were s o l e l y c o n t r o l l e d by cytoplasm, progenies of females would a l l be females and progenies of hermaphrodite mothers would be a l l hermaphrodites. T h i s model can be r e a d i l y r u l e d out based on the r e s u l t s presented i n Tables 6-1, 6-2, and 6-3. To choose among the remaining models, the f i r s t step i s to determine whether n u c l e a r c o n t r o l of male s t e r i l i t y can account f o r a l l of the r e s u l t s . 6.4.2 Genie Male S t e r i l i t y There are s e v e r a l p o s s i b l e n u c l e a r gene models, depending on the dominance r e l a t i o n s h i p and the number of l o c i i n v o l v e d . 1. Male s t e r i l i t y dominant If male s t e r i l i t y i s caused by a s i n g l e dominant nuclear gene, females can have only the genotype Mm, while hermaphrodites can have only the genotype mm. The segregation r a t i o s (H:MS) should always be 1:1 i n the progenies of females, and 1:0 i n the progenies of maternal hermaphrodites. With two or more dominant l o c i , r e g a r d l e s s of gene i n t e r a c t i o n , progenies 152 of females should always c o n t a i n females. T h i s model can be r e a d i l y e l i m i n a t e d based on the data i n Tables 6-1, 6-2, and 6-3 (progenies of females that were a l l hermaphrodites). 2. Male s t e r i l i t y r e c e s s i v e One l o c u s model If male s t e r i l i t y i s caused by a s i n g l e r e c e s s i v e n u c l e a r gene, mm w i l l be the only genotype f o r females, and MM or Mm f o r hermaphrodites. T h i s model can account f o r a l l the r e s u l t s i n the F1 (Tables 6-2, 6-3), and the 3:1 F2 r a t i o s and 1:1 backcross r a t i o s obtained f o r f o r b e s i i , B. hawaiensis and B. p o p u l i f o l i a (Tables 6-4, 6-5), but cannot e x p l a i n the 15:1 F2 r a t i o s i n IK m e n z i e s i i and IK s a n d v i c e n s i s (Crosses 8, 9 i n Table 6-4), or the 3:1 r a t i o i n the backcross of IK_ t o r t a (Cros-s 3 i n Table 6-5). Nor can t h i s model account f o r the 3:1 r a t i o i n t r i p l e c r o s s e s (Crosses 1, 4, 8, 10, 11 i n Table 6-6) or the quadruple c r o s s e s (Table 6-7). Two l o c u s model If male s t e r i l i t y i s determined by two d u p l i c a t e r e c e s s i v e genes m and n, females w i l l have only the genotype, mmnn, while a l l other genotypes are hermaphrodites. F1 r a t i o s can be 1:0, 1:1, or 3:1 and F2 r a t i o s can be 15:1 or 3:1, depending on the genotypes of p a r e n t a l hermaphrodites. T h i s model can e x p l a i n a l l the r e s u l t s i n F1 progenies (Table 6-2, 6-3), but cannot e x p l a i n the F2 r a t i o s i n c r o s s e s 1, 2, 3 i n Table 6-4 i f a l l i n d i v i d u a l s of the hermaphroditic s p e c i e s IK mauiensis have the same genotype MMNN. If assuming that hermaphroditic s p e c i e s were f i x e d with the genotypes e i t h e r MM or NN, the 3:1 153 r a t i o s i n c r o s s e s 1, 2, 3 and 4 (Table 6-4) c o u l d be ex p l a i n e d . Under t h i s assumption, however, no c o n s i s t e n t genotype can be assi g n e d to the i n d i v i d u a l , H 10A, of IK mauiensis i n the c r o s s e s 2 and 6 (Table 6-4). In the F2 progenies of c r o s s 6, 26 H : 4 MS were segregated, the r a t i o f i t both 3:1 and 15:1 with c l o s e p r o b a b i l i t y . The backcross 3 (Table 6-5) gave a 3:1 r a t i o , i n agreement with the 15:1 r a t i o i n the F2. Thus the only genotype which can be assigne d to H 10A i s MMNN, whereas i t has to be MMnn or mmNN i n Cross 2. Nor can a c o n s i s t a n t genotype be assigned to H 10B in c o n s i d e r i n g the F2 r a t i o i n Cross 1 (Table 6-4) and d i s c o r d a n t r a t i o s i n t r i p l e c r o s s e s 1, 10, 11, 16 (Table 6-6) and in the quadruple c r o s s 1 (Table 6-7). Thus, t h i s model cannot s u f f i c i e n t l y e x p l a i n the data of t h i s study. Another two lo c u s model, i n which e i t h e r l o c u s causes male s t e r i l i t y when homozygous r e c e s s i v e , would give a 9:7 r a t i o i n the F2. None of the experimental c r o s s e s gave the 9:7 r a t i o i n the F2. T h i s model can be r e a d i l y r u l e d out. 3. Two locus e p i s t a t i c model T h i s model has been p r e v i o u s l y proposed f o r the gynodioecious s p e c i e s Origanum v u l g a r e (Lewis and Crowe, 1956) and C o r t a d e r i a spp. (Connor, 1973). In t h i s system, the dominant gene F causes male s t e r i l i t y and a dominant f e r t i l i t y -r e s t o r i n g gene H i s e p i s t a t i c to F, thus females can only have genotypes FFhh or Ffhh, whereas hermaphrodites can be any of the r e s t of the genotypes: FFHH, FFHh, FfHH, FfHh, ffHH, ffHh and f f h h . Crosses between females and hermaphrodites can give F1 154 r a t i o s 1 : 0 , 1 : 1 , 5 : 3 , 3 : 1 , o r 0 : 1 ; F 2 r a t i o s 3 : 1 , 1 3 : 3 , o r 1:0; a n d t h e b a c k c r o s s r a t i o s 1 : 1 , 3 : 1 , o r 5 : 3 . T h e d i a g n o s t i c r a t i o s u s e f u l i n d i s t i n g u i s h i n g t h i s m o d e l f r o m t h e o t h e r s a r e 0 : 1 i n F 1 , 1 3 : 3 a n d 1 : 0 i n F 2 , a n d 5 : 3 i n t h e b a c k c r o s s . F o r a s m a l l s a m p l e s i z e , t h e 5 : 3 r a t i o i s h a r d l y d i s t i n g u i s h a b l e f r o m a 1 :1 r a t i o . A l t h o u g h t h e 1 3 : 3 r a t i o h a s a h i g h e r p r o b a b i l i t y t h a n e i t h e r 3 : 1 o r 1 5 : 1 r a t i o i n c r o s s 1 a n d 6 ( T a b l e 6 - 4 ) , t h e 0 : 1 r a t i o i n F1 a n d 1 : 0 r a t i o i n F 2 w e r e n e v e r f o u n d f o r a n y c r o s s e s w i t h a s a m p l e s i z e n > 3 . C r o s s e s 3 ( T a b l e 6 - 5 ) , 6 ( T a b l e 6 - 4 ) a n d 1 , 4 , a n d 10 ( T a b l e 6 - 6 ) h a v e t o o many h e r m a p h r o d i t e s i n t h e p r o g e n i e s c o m p a r e d t o t h e e x p e c t e d 1 : 1 o r 5 : 3 r a t i o s . T h e s e r a t i o s a r e e x p e c t e d b e c a u s e t h e i n d i v i d u a l s o f B_j_ m a u i e n s i s i s m u s t b e h o m o z y g o u s d o m i n a n t a t t h e H l o c u s t o a l w a y s g i v e h e r m a p h r o d i t e p r o g e n i e s i n t h e F1 ( T a b l e 6 - 2 ) . S o t h i s m o d e l i s a l s o u n l i k e l y . 6 . 4 . 3 G e n i c - c y t o p l a s m i c M o d e l A l l e x p e r i m e n t a l c r o s s e s s u g g e s t t h a t e i t h e r o n e o r t w o r e c e s s i v e g e n e s c o n t r o l m a l e s t e r i l i t y . H o w e v e r , c r o s s e s o f t h e same h e r m a p h r o d i t e p l a n t t o f e m a l e s o f d i f f e r e n t s p e c i e s g a v e d i f f e r e n t r e s u l t s , s h o w i n g t h a t t h e r e m u s t b e g e n e t i c d i f f e r e n c e s a m o n g f e m a l e s a s w e l l . T h i s r e s u l t d o e s n o t a p p e a r t o b e e x p l a i n a b l e b y a n y s t r i c t l y n u c l e a r m o d e l f o r t h e i n h e r i t a n c e o f m a l e s t e r i l i t y . H o w e v e r , i f m a l e s t e r i l i t y i s c o n t r o l l e d b y i n t e r a c t i o n s b e t w e e n c y t o p l a s m a n d n u c l e a r g e n e s , t h e d i f f e r e n c e s b e t w e e n f e m a l e s c a n b e a c c o u n t e d f o r b y c y t o p l a s m i c d i f f e r e n c e s b e t w e e n s p e c i e s o r p o p u l a t i o n s . T h i s m o d e l p o s t u l a t e s t h a t t h e r e a r e 155 two types of cytoplasm (A) and (B), and two nucl e a r genes, m and n. In cytoplasm (A), homozygous r e c e s s i v e s at the m lo c u s are male s t e r i l e , r e g a r d l e s s of the genotype at the n l o c u s . In cytoplasm (B), both the m and n l o c i must be homozygous r e c e s s i v e f o r male s t e r i l i t y to be expressed. T h e r e f o r e , depending on the cytoplasm, male s t e r i l i t y can appear to be c o n t r o l l e d by e i t h e r one or two r e c e s s i v e n u c l e a r genes. Cytoplasm Female genotype A mmN_ or mmnn B mmnn With (A) cytoplasm, only the m lo c u s a f f e c t s male s t e r i l i t y , so that the F2 from a double heterozygote g i v e s a 3:1 phenotypic r a t i o 9 (A)M_N_ : 3 (A)mmN_ : 3 (A)M_nn : 1 (A)mmnn H MS H MS Whereas i n cytoplasm (B), both dominant f e r t i l i t y - r e s t o r i n g genes f u n c t i o n , g i v i n g a 15:1 r a t i o i n the F2. 9 (B)M_N_ : 3 (B)mmN_ : 3 (B)M_nn : 1 (B)mmnn H H H MS Th i s model r e q u i r e s no d i f f e r e n c e at m lo c u s i n the nuclear genotypes of females i n d i f f e r e n t gynodioecious s p e c i e s . T h i s i s c o n s i s t a n t with developmental data, which suggested a uniform 156 mechanism of male s t e r i l i t y i n Hawaiian Bidens. I t a l s o agrees with the a l l e l i c nature of male s t e r i l i t y genes among these s p e c i e s ( F i g . 6-1). Th i s model alone appears to be able to account f o r a l l data i n t h i s study. Genotypes were thus a s s i g n e d , based on t h i s model, to a l l i n d i v i d u a l s f o r a l l c r o s s e s presented i n the t a b l e s . Cytoplasmic male s t e r i l i t y appears to r e a d i l y e v o l v e , as suggested by numerous r e p o r t s on cyto p l a s m i c and g e n i c -c y t o p l a s m i c male s t e r i l i t y i n crop p l a n t s (see Edwardson, 1970; Laser and L e r s t e r n , 1972 f o r re v i e w s ) . However, simple c y t o p l a s m i c male s t e r i l i t y i s rare i n n a t u r a l l y o c c u r r i n g gynodioecious s p e c i e s . So i s simple nuclear genie male s t e r i l i t y , although genie m a l e - s t e r i l e mutants have a l s o been f r e q u e n t l y r e p o r t e d f o r crop p l a n t s ( J a i n , 1958). In almost a l l w e l l s t u d i e d s p e c i e s , gynodioecy appears to be c o n t r o l l e d by the i n t e r a c t i o n between cytoplasmic f a c t o r s and nuclear genes (see Ross, 1978; Charlesworth, 1981 f o r r e v i e w s ) . The i n h e r i t a n c e of male s t e r i l i t y i n Origanum vulgar e was f i r s t proposed as d i g e n i c and e p i s t a t i c as d i s c u s s e d above (Lewis and Crowe, 1956), but recent s t u d i e s of t h i s s p e c i e s by Kheyr-Pour (1980, 1981) re v e a l e d a complicated c y t o p l a s m i c - g e n i c system of male s t e r i l i t y . Ross (1969) proposed a d i g e n i c r e c e s s i v e mechanism of male s t e r i l i t y i n Plantago l a n c e o l a t a , however, two or more types of mechanisms were r e c e n t l y r e p o r t e d fo r t h i s s p e c i e s , each showing complicated g e n i c - c y t o p l a s m i c nature (Van Damme and Van Delden, 1982; Van Damme, 1983). The ba s i c g e n e t i c mechanism f o r male s t e r i l i t y i n Thymus v u l g a r i s i s 157 a l s o g e n i c - c y t o p l a s m i c (Valdeyron et a l . , 1977). Gynodioecy i n Limnanthes d o u g l a s i i was i n i t i a l l y i n t e r p r e t e d as monogenic dominant i n h e r i t a n c e (Baker, 1966; J a i n et a l . , 1978), but a l a t e r study of the s p e c i e s gave r e s u l t s s u g g e s t i n g that male s t e r i l i t y appeared to be g e n i c - c y t o p l a s m i c ( K e s s e l i and J a i n , 1984). C l e a r evidence f o r c y t o p l a s m i c i n h e r i t a n c e with nuclear r e s t o r e r genes i s shown in Nemophila m e n z i e s i i (Ganders, 1978). Lack of simple cytoplasmic or simple genie male s t e r i l i t y i n n a t u r a l gynodioecious s p e c i e s may r e s u l t from t h e i r e v o l u t i o n a r y i n s t a b i l i t y . Simple nuclear genie male s t e r i l i t y can r e a d i l y evolve to dioecy through gradual accumulation of completely l i n k e d r e c e s s i v e genes f o r male s t e r i l i t y and of dominant or nondominant genes f o r p a r t i a l female s t e r i l i t y (Ross and Weir, 1975; Ross, 1978). Mutations reducing female f e r t i l i t y of hermaphrodites appear to be a common occurrence (Ross, 1978; Charlesworth, 1981). Simple c y t o p l a s m i c male s t e r i l i t y i s r a r e because r e s t o r e r mutations can convert c y t o p l a s m i c i n h e r i t a n c e of male s t e r i l i t y i n t o c y t o p l a s m i c - g e n i c i n h e r i t a n c e (Charlesworth, 1981). In c o n t r a s t , g e n i c -c y t o p l a s m i c male s t e r i l i t y appears to be e v o l u t i o n a r i l y s t a b l e because l i n k a g e can h a r d l y occur between n u c l e a r p a r t i a l female s t e r i l i t y and c y t o p l a s m i c male s t e r i l i t y (Ross, 1978). A l l Hawaiian s p e c i e s of Bidens evolved from a s i n g l e a n c e s t r a l s p e c i e s a f t e r l o n g - d i s i s t a n c e d i s p e r s a l ( G i l l e t t , 1975; Ganders and Nagata, 1983,1984; Marchant et a l . , 1984; Helenurm and Ganders, 1975), but only about h a l f of them are gynodioecious. Gynodioecy i s not known i n s p e c i e s of Bidens 158 o u t s i d e the Hawaiian I s l a n d s (Ganders, u n p u b l i s h e d ) . Gynodioecy in Hawaiian Bidens, t h e r e f o r e , most l i k e l y evolved autochthonously. Although male s t e r i l i t y might be c o n t r o l l e d by d i f f e r e n t g e n e t i c mechanisms i n d i f f e r e n t gynodioecious s p e c i e s , the homogeneous phenotypic and developmental e x p r e s s i o n of male s t e r i l i t y suggests the same g e n e t i c mechanism (Chapter 5). I found evidence of a l l e l i c r e l a t i o n s h i p among male s t e r i l i t y genes i n d i f f e r e n t gynodioecious s p e c i e s ( F i g . 6-1). Thus I conclude that the male s t e r i l i t y mutation event most l i k e l y o c c u r r e d only once i n Hawaiian Bidens. M a l e - s t e r i l e mutants are at a s e l e c t i v e disadvantage because they t r a n s m i t genomes to the next g e n e r a t i o n only through o v u l e s , whereas hermaphrodites transmit genomes through both ovules and p o l l e n and through- the ovules of m a l e - s t e r i l e mutants. However, male s t e r i l i t y can evolve to gynodioecy under f a v o r a b l e c o n d i t i o n s (see Chapter 7 ) . The 13 c u r r e n t l y known gynodioecious taxa c o u l d be the products of a d a p t i v e r a d i a t i o n of one a n c e s t r a l gynodioecious s p e c i e s . N a t u r a l h y b r i d i z a t i o n c o u l d a l s o spread male s t e r i l i t y genes i n t o hermaphroditic p o p u l a t i o n s . I conclude that male s t e r i l i t y i n a l l gynodiecious s p e c i e s of Hawaiian Bidens i s c o n t r o l l e d by the same g e n e t i c mechanism. Among the p o s s i b l e mechanisms, g e n i c - c y t o p l a s m i c i n h e r i t a n c e i s the only one which e x p l a i n s the r e s u l t s of a l l of the experimental c r o s s e s . 159 6.5 Summary In h e r i t a n c e of male s t e r i l i t y i n a l l gynodioecious s p e c i e s of Hawaiian Bidens was i n v e s t i g a t e d by experimental c r o s s e s conducted i n greenhouses. Both m a l e - s t e r i l e i n d i v i d u a l s and hermaphrodites were segregated i n the progenies of open-p o l l i n a t e d female and hermaphroditic p l a n t s growing i n nature. Females of e i g h t gynodioecious s p e c i e s gave only hermaphrodite progenies when p o l l i n a t e d by hermaphrodites of seven hermaphroditic (non-gynodioecious) s p e c i e s . When females were p o l l i n a t e d by hermaphrodites of nine gynodioecious s p e c i e s and B. asymmetrica, some c r o s s e s produced progenies with both MS and H while the r e s t gave a l l H pr o g e n i e s . Segregation of both MS and H occu r r e d when females were p o l l i n a t e d by hermaphrodites which segregated MS progeny upon s e l f i n g . The r a t i o s (H/MS) of 1:0, 1:1, 3:1 were observed i n the F1 ge n e r a t i o n ; 3:1, 15:1, or 13:3 were observed i n F2 g e n e r a t i o n s ; and 1:1 and 3:1 i n backcrosses. T h i r t e e n i n t e r s p e c i f i c t r i p l e c r o s s e s and a l l quadruple c r o s s e s gave MS progenies, i n d i c a t i n g a d i r e c t l y a l l e l i c r e l a t i o n s h i p among male s t e r i l i t y genes i n a l l gynodioecious s p e c i e s . Based on these r e s u l t s , models of ge n e t i c i n h e r i t a n c e i n gynodioecious s p e c i e s of Hawaiian Bidens were compared. The model d i g e n i c - c y t o p l a s m i c appears to be the best choice among a l l the models d i s c u s s e d . T h i s model al l o w s females i n a l l gynodioecious s p e c i e s of Hawaiian Bidens to have the same nuc l e a r genotype, mmnn. Male s t e r i l i t y i s caused by two r e c e s s i v e genes, and f e r t i l i t y can be r e s t o r e d i n the presence 160 of a dominant gene M, or N. T h e r e f o r e , a 15:1 r a t i o i s expected i n the F2 i f the p a r e n t a l females have cytoplasm (B). However, i f the p a r e n t a l females have cytoplasm (A), i n which only the gene M f u n c t i o n s , the r a t i o i n the F2 i s 3:1. T h i s model appears to be able to e x p l a i n the observed d i s c r e p a n c y i n the s e g r e g a t i o n r a t i o s i n the F2 i n d i f f e r e n t gynodioecious s p e c i e s . Based on the r e s u l t s of t h i s study and the developmental s t u d i e s i n the previous chapter, the male s t e r i l i t y mutation event probably became e s t a b l i s h e d only once i n Hawaiian Bidens. The 13 present gynodioecious taxa might r e s u l t from adaptive r a d i a t i o n of the o r i g i n a l gynodioecious s p e c i e s , or from n a t u r a l h y b r i d i z a t i o n between gynodioecious taxa and hermaphroditic taxa. 161 V I I . THE MAINTENANCE OF GYNODIOECY 7.1 I n t r o d u c t i o n Developmental d e f e c t s which cause a b o r t i o n of microsporogenesis r e s u l t i n male s t e r i l i t y . M a l e - s t e r i l e mutants f r e q u e n t l y occur i n both n a t u r a l and c u l t i v a t e d p l a n t s p e c i e s , but n a t u r a l gynodioecious p o p u l a t i o n s are r e l a t i v e l y r a r e (Lewis, 1941; Rick, 1948; J a i n , 1959). T h i s i s because females are at a s e l e c t i v e disadvantage i n gynodioecious p o p u l a t i o n s . Hermaphrodites c o n t r i b u t e genes to the next gene r a t i o n through both p o l l e n and ovules, whereas females c o n t r i b u t e genes only through o v u l e s . T h i s i s e q u i v a l e n t to a stro n g s e l e c t i v e pressure a g a i n s t females (Lewis, 1941). I f females do not have some compensating s e l e c t i v e advantages, they w i l l not be ab l e to e s t a b l i s h i n o r i g i n a l l y hermaphroditic p o p u l a t i o n s or be maintained i n gynodioecious p o p u l a t i o n s . How females are maintained i n gynodioecious s p e c i e s has remained as an unsolved problem ever s i n c e Darwin (1877), d e s p i t e many t h e o r e t i c a l s t u d i e s (Lewis, 1941, 1942; J a i n , 1961; Ross and Shaw, 1971; Ho and Ross, 1973; Valdeyron et a l . , 1973; L l o y d , 1974, 1975, 1976, 1982; Charlesworth and Charlesworth, 1978a, 1978b; Charlesworth and Ganders, 1979; Charlesworth, 1981; Gregori u s et a l . , 1982; Ross and Gr e g o r i u s , 1985). T h e o r e t i c a l s t u d i e s of the maintenance of gynodioecy i n d i c a t e that requirements f o r the maintenance of females are d i f f e r e n t depending upon the mode of i n h e r i t a n c e of male s t e r i l i t y (Lewis, 1941; L l o y d , 1974, 1975; Charlesworth, 1981). 162 There e x i s t three types of ge n e t i c c o n t r o l of male s t e r i l i t y ; : c y t o p l a s m i c , genie, and c y t o p l a s m i c - g e n i c . Cytoplasmic male s t e r i l i t y does not s u f f e r much s e l e c t i v e disadvantage and females can be maintained i n p o p u l a t i o n s i f t h e i r f e r t i l i t y i s only s l i g h t l y above t h a t of the hermaphrodites (Lewis, 1941). However, both genie and c y t o p l a s m i c - g e n i c male s t e r i l i t y r e q u i r e l a r g e f i t n e s s d i f f e r e n c e s between females and hermaphrodites f o r females to be maintained (Lewis, 1941; L l o y d , 1974, 1975; Charlesworth and Charlesworth, 1978; Charlesworth and Ganders, 1979; Charlesworth, 1981). The model of Lewis (1941) f o r a single-gene c o n t r o l l e d male s t e r i l i t y has demonstrated that females must be more than twice as f e r t i l e i n seed f e c u n d i t y as hermaphrodites i f they are to e x i s t ( i . e . , the p r o p o r t i o n of females p > 0) i n a p o p u l a t i o n . Since Lewis (1941), t h e o r e t i c a l s t u d i e s of the maintenance of male s t e r i l i t y have been extended to v a r i o u s modes of i n h e r i t a n c e , with many f a c t o r s a c t i n g on the f i t n e s s of the sexual forms c o n s i d e r e d , and to s p e c i e s with p a r t i a l s e l f i n g i n the hermaphrodite i n v o l v i n g i n b r e e d i n g d e p r e s s i o n and overdominance ( J a i n , 1961; Ross and Shaw, 1971; Valdeyron et a l . , 1973; Ho and Ross, 1973, 1974; L l o y d , 1974a, 1974b, 1975, 1976; Ross and Weir, 1975; Charlesworth, 1981; Charlesworth and Charlesworth, 1978; Charlesworth and Ganders, 1979; Gregorius et a l . , 1982; Ross and G r e g o r i u s , 1985). L l o y d (1976) demonstrated that the r e l a t i v e s u r v i v a l and seed p r o d u c t i o n of the two sexes and the inbre e d i n g of hermaphrodites have i d e n t i c a l e f f e c t s on the e q u i l i b r i u m sex r a t i o i n a l l genie modes of i n h e r i t a n c e , i f 163 the d i f f e r e n t genotypes w i t h i n a sex do not d i f f e r i n average f i t n e s s . L l o y d ' s model i n d i c a t e s t hat a s t a b l e sexual dimorphism can be maintained by an o u t c r o s s i n g advantage of females. The o u t c r o s s i n g hypothesis of gynodioecy p o s t u l a t e s that the disadvantage of females can be c o u n t e r a c t e d because t h e i r progeny a l l r e s u l t from o u t c r o s s i n g , whereas some inbred progeny of hermaphrodites are l e s s f i t because of i n b r e e d i n g d e p r e s s i o n . Females can be maintained i n p o p u l a t i o n s i f sd > sd > 1/2, where s i s the s e l f i n g r a t e of hermaphrodites, and d i s the i n b r e e d i n g d e pression of progeny from s e l f i n g ( L l o y d , 1974, 1975; Ganders, 1978; Charlesworth and Charlesworth, 1978; Charlesworth and Ganders, 1979; Charlesworth, 1981). The frequency of females maintained i n p o p u l a t i o n s i n c r e a s e s as sd i n c r e a s e s ( L l o y d , 1975). Because the s e l f i n g r a t e of hermaphrodites and the degree of i n b r e e d i n g d e p r e s s i o n expressed i n t h e i r progeny are not b i o l o g i c a l l y independent (Valdeyron et a l . , 1973; Ganders, 1978), the c o n d i t i o n s r e q u i r e d f o r m a i n t a i n i n g females based on the mixed mating of hermaphrodites and reduced f i t n e s s of i n b r e d progeny i n L l o y d ' s model are as s t r i n g e n t as those r e q u i r e d i n Lewis' model based on r e l a t i v e f e r t i l i t y d i f f e r e n c e between the two sexual forms. However, the j o i n t advantages of females i n both o u t c r o s s i n g , and i n d i f f e r e n t i a l seed p r o d u c t i o n over hermaphrodites are probably s u f f i c i e n t f o r the i n i t i a l spread of m a l e - s t e r i l e gene(s) and the establishment of gynodioecy, i f the o v e r a l l f i t n e s s of females i s at l e a s t twice that of hermaphrodites (Ho and Ross, 1973; L l o y d , 1975; Ross, 1978). 164 T h i r t e e n of 27 taxa of Hawaiian Bidens are gynodioecious (Appendix A). The m a l e - s t e r i l e i n d i v i d u a l s of these taxa are homogeneous i n phenotypic e x p r e s s i o n w i t h i n p o p u l a t i o n s or taxa, and s i m i l a r among gynodioecious s p e c i e s except that they may d i f f e r i n flower s i z e . Females can be e a s i l y r e c o g n i z e d at a n t h e s i s by t h e i r s maller flower heads, and p a l e c o l o r e d , s h r i v e l e d , non-dehiscent and empty anthers. A comparative study of microsporogenesis i n male s t e r i l e s and hermaphrodites r e v e a l s that e a r l y abnormal v a c u o l a t i o n of the tapetum and subsequently degeneration of sporogenous c e l l s p r i o r to m e i o s i s r e s u l t s i n complete male s t e r i l i t y i n a l l 9 gynodioecious taxa s t u d i e d (Chapter 5). Male s t e r i l i t y i n a l l gynodioecious s p e c i e s of Hawaiian Bidens i s probably c o n t r o l l e d by r e c e s s i v e nuclear genes, and these genes are a l l e l i c (Chapter 6). Both developmental and g e n e t i c s t u d i e s i n d i c a t e a homologous o r i g i n of male s t e r i l i t y i n a l l gynodioecious Hawaiian s p e c i e s of Bidens. These gynodioecious s p e c i e s occur on a l l major Hawaiian I s l a n d s , suggesting a r e l a t i v e l y long e v o l u t i o n a r y h i s t o r y of gynodioecy i n Hawaiian Bidens. How females are maintained i n these s p e c i e s i s t h e r e f o r e of c o n s i d e r a b l e e v o l u t i o n a r y i n t e r e s t . If the o b l i g a t e o u t c r o s s i n g of females i s the major mechanism f o r m a i n t a i n i n g females i n n a t u r a l gynodioecious p o p u l a t i o n s , the f r e q u e n c i e s of females should be p o s i t i v e l y c o r r e l a t e d with s e l f i n g r a t e s of hermaphrodites i n p o p u l a t i o n s , and the progeny of females should be more heterozygous than those of the hermaphrodites. However, t h i s p r e d i c t i o n of the 165 o u t c r o s s i n g hypothesis i n Ll o y d ' s model has not been p r e v i o u s l y t e s t e d , so to t e s t i t i n n a t u r a l gynodioecious p o p u l a t i o n s of Hawaiian Bidens i s a major o b j e c t i v e of t h i s study. The hypo t h e s i s of d i f f e r e n t i a l seed p r o d u c t i o n between sexes i n Lewis' model p o s t u l a t e s that females can be maintained only i f they produce at l e a s t twice as many seeds as hermaphrodites. The high seed r e p r o d u c t i o n of females c o u l d be r e l a t e d to the m a l e - s t e r i l e form i t s e l f ( i . e . , the p l e i o t r o p i c e f f e c t of male s t e r i l i t y genes, or resource r e a l l o c a t i o n , see Charlesworth and Charlesworth, 1978), or r e l a t e d to the e f f e c t of allogamy ( i . e . , o b l i g a t e o u t c r o s s i n g ) . Because e s t i m a t i n g r e l a t i v e f i t n e s s of females, hermaphrodites, and t h e i r progenies i n n a t u r a l gynodioecious p o p u l a t i o n s i s d i f f i c u l t , an attempt has been made to measure some f i t n e s s components at d i f f e r e n t l i f e c y c l e stages of hermaphrodites and females i n s e v e r a l experimental p o p u l a t i o n s . In t h i s chapter I present 1) the str o n g p o s i t i v e c o r r e l a t i o n between s e l f i n g r a t e s of hermaphrodites and fr e q u e n c i e s of females i n 8 gynodioecious p o p u l a t i o n s of Hawaiian Bidens ; 2) the estimates of f i t n e s s components of progenies of females and hermaphrodites at s e v e r a l stages of t h e i r l i f e c y c l e s i n experimental gynodioecious p o p u l a t i o n s and in c o n t r o l l e d experimental c r o s s e s ; and 3) an i n v e s t i g a t i o n of the p l e i o t r o p i c e f f e c t of male s t e r i l i t y i n experimental gynodioecious p o p u l a t i o n s and i n the progenies of experimental c r o s s e s . 166 7.2 M a t e r i a l s And Methods 7.2.1 E l e c t r o p h o r e s i s E i g h t gynodioecious p o p u l a t i o n s of 5 Hawaiian taxa of Bidens were sampled i n Feburary, June and November, 1984. Frequency of female p l a n t s was estimated i n the f i e l d from counts of f l o w e r i n g p l a n t s . Seeds were c o l l e c t e d from i n d i v i d u a l hermaphrodites and females and grown i n growth chambers to provide progenies f o r e l e c t r o p h o r e s i s . P o p u l a t i o n s s t u d i e d and sample s i z e s are given i n the t a b l e s of t h i s c h a p t e r . Three commonly polymorphic l o c i , P gi-1, Pgi-2, and Skdh-3, were used f o r e s t i m a t i n g s e l f i n g r a t e s (s) and h e t e r o z y g o s i t y (Ho) i n these p o p u l a t i o n s . D e t a i l e d e l e c t r o p h o r e t i c methods and g e n e t i c i n t e r p r e t a t i o n of allozyme banding p a t t e r n s are d e s c r i b e d i n Chapter 2. The m u l t i l o c u s method of R i t l a n d and J a i n (1981) was used f o r e s t i m a t i n g s e l f i n g r a t e s of hermaphrodites i n these p o p u l a t i o n s . 7.2.2 Seed Weight And Germination Twenty to s i x t y seeds from each f a m i l y of females and hermaphrodites were weighed together on a microbalance f o r average seed weight. These seeds were sown s e p a r a t e l y by f a m i l i e s i n v e r m i c u l i t e i n a growth chamber to determine percent germination of seeds i n each f a m i l y . Because mature seeds from the p l a n t s r a i s e d i n greenhouses can germinate at any photoperiod at the high l a t i t u d e (49 N) i n Vancouver, I assume that the seeds of a l l Hawaiian s p e c i e s of Bidens are d a y - n e u t r a l i n germination requirments. Seeds were thus germinated i n a 167 constant 12-hour day l e n g t h at 25 C and 12-hour night l e n g t h at 17 C. S e e d l i n g s of each f a m i l y of females and hermaphrodites were counted 4 weeks a f t e r sowing, and the counts were repeated i n the f o l l o w i n g week to recover some l a t e germinated seedings. S e e d l i n g s were normally kept growing i n v e r m i c u l i t e f o r another week f o r root development and then were t r a n s p l a n t e d to i n d i v i d u a l s o i l pots i n greenhouses and r a i s e d t here to r e p r o d u c t i o n . 7.2.3 V e g e t a t i v e And Reproductive Measures Progenies of females and hermaphrodites were r a i s e d i n the same greenhouse c o n d i t i o n s . P l a n t height and diameter of the stem, l e a f dry weight and t o t a l l e a f p a i r s on the p l a n t were measured. The l e a f weight and number of l e a f p a i r s served as a measure of v e g e t a t i v e growth and development at e a r l y v e g e t a t i v e stages. Environmental l i m i t s to growth can be ignored at these stages. F r e s h l e a f p a i r s were oven d r i e d f o r 48 hours at 125 C before weighing. Reproductive f e a t u r e s were measured f o r females and hermaphrodites f o r greenhouse r a i s e d p l a n t s . T o t a l flower heads, i n c l u d i n g buds, f l o w e r i n g heads and seed heads, were counted f o r progenies of females and hermaphrodites at two developmental stages: e a r l y f l o w e r i n g and e a r l y f r u i t i n g . Most data presented i n t h i s chapter are pooled data from the two measurements. The numbers of f l o r e t s per head and seeds per head were counted from each i n d i v i d u a l p l a n t and averaged over p o p u l a t i o n s . The r e p r o d u c t i v e c h a r a c t e r s mentioned above were a l s o measured fo r females and hermaphrodites segregated i n the 168 progenies of two experimental c r o s s e s to i n v e s t i g a t e p l e i o t r o p i c e f f e c t s of male s t e r i l i t y genes. 7.2.4 P o l l e n S t a i n a b i l i t y P o l l e n g r a i n s were c o l l e c t e d from s e v e r a l randomly sampled p l a n t s i n progenies of females and hermaphrodites i n an experimental p o p u l a t i o n of s a n d v i c e n s i s ssp. confusa. P o l l e n was s t a i n e d with l a c t o p h e n o l blue o v e r n i g h t and at l e a s t 200 g r a i n s were counted per p l a n t f o r determining p o l l e n v i a b i l i t y . 7.3 R e s u l t s 7.3.1 S e l f i n g Rates Of Hermaphrodites And H e t e r o z y g o s i t y The f r e q u e n c i e s of females ranged from 9 - 44% i n the 8 gynodioecious p o p u l a t i o n s s t u d i e d . The s e l f i n g r a t e s of hermaphrodites i n these p o p u l a t i o n s ranged from 11.3 - 75.8% (Table 7-1). They were s i g n i f i c a n t l y c o r r e l a t e d with the f r e q u e n c i e s of females (r=0.91, p<0.0l), as shown in F i g . 7-1. D i f f e r e n c e s i n sample s i z e s of f a m i l i e s , i n d i v i d u a l s per f a m i l y , and number of l o c i have no s i g n i f i c a n t e f f e c t s on the estimates of s e l f i n g r a t e s . The l e v e l of h e t e r o z y g o s i t y at the allozyme l o c i P gi-1, Pgi-2, and Skdh-3 i n the progenies of m a l e - s t e r i l e p l a n t s was g e n e r a l l y higher than i n the progenies of hermaphrodites i n i n d i v i d u a l p o p u l a t i o n s , as expected of the females (Table 7-2). Average h e t e r o z y g o s i t y at these three l o c i was higher i n progeny of females i n e i g h t out of ten p o p u l a t i o n s . T a b l e 7-1. S e l f i n g r a t e s of hermaphrodites (%) and f r e q u e n c i e s of females (%) i n 8 g y n o d i o e c i o u s p o p u l a t i o n s of Hawaiian Bidens. S p e c i e s Sample s i z e % S e l f i n g i n P o p u l a t i o n progeny f a m i l i e s hermaphrodites ( s . e . ) % females i n p o p u l a t i o n [sample s i z e ] B. c e r v i c a t a CERV 344 28 39.2 (7.8) 25 [12] B. f o r b e s i i s s p . f o r b e s i i FORB F1 262 28 49.4 (15.6) 30 [325] B. m e n z i e s i i s s p . f 1 1 i formi s MENZ 1 306 20 55.0 (9.8) 44 [136] MENZ 2 317 25 52.6 (17.3) 34 [178] B. s a n d v i c e n s i s ssp. c o n f u s a SAND C 375 24 11.3 (11.5) 11 [54] B. s a n d v i c e n s i s s s p . s a n d v i c e n s i s SAND 1 SAND 2 249 30 191 20 12.3 (18.0) 75.8 (10.7) 9 [68] 36 [31] SAND X 133 16 53.5 (16.0) 35 [518] 60 y - .50x • 6.11 I l 1 1 1 ' 0 20 40 60 80 100 Selling In hermaphrodites (%) Figure 7-1. Regression of frequency of females on s e l f i n g rate of hermaphrodites i n eight gynodioecious populations of Hawaiian Bidens. 4^ o Table 7-2. Observed heterozygosity (Ho) at 3 polymorphic loci in the progenies of male s t e r i l e s and hermaphrodites in 11 gynodioecious populations of Hawaiian Bidens. Progeny of hermaphrod i tes Progeny of females Species Population Observed heterozygos i ty Observed heterozygos1ty N Pgi-1 Pgi-2 Skdh-3 Mean N Pgi-1 Pg1-2 Skdh-3 Mean B. cervic a t a CERV 344 . 265 . 351 . 283 . 300 1 18 . 356 .483 .461 .433 B. forbes1i ssp. forbes i 1 FORB F1 264 .027 .049 . 238 . 105 201 . 149 .080 . 209 . 146 ssp. kahi1iensis FORB K 30 .000 .000 .033 .01 1 90 .000 .000 . 156 .052 B. menz i es11 ssp. f i1 i formis MENZ 1 306 .369 . 190 .000 . 186 176 . 405 .329 .000 .245 MENZ 2 314 .000 .229 .OOO .076 1 17 .000 . 197 .000 .066 MENZ 3 69 . 101 . 101 .087 .096 68 .412 .426 .088 . 309 MENZ 4 64 . 172 .406 .000 . 193 80 .413 .463 .000 . 292 B. sandvicensis ssp. confusa SAND C 309 .048 . 379 . 162 . 196 265 .068 . 377 . 143 . 196 ssp. sandvicensis SAND 1 251 .131 . 143 .OOO .091 0 - - - -SAND 2 289 .003 .007 . 321 . 1 10 199 .000 .015 . 333 . 1 16 SAND X 133 .286 .233 - . 260 1 13 . 292 .354 - . 323 Mean 216 . 127 . 190 .112 . 148 130 .210 . 272 . 154 .218 172 Average h e t e r o z y g o s i t y f o r a l l p o p u l a t i o n s was 0.218 i n progeny of females and 0.148 i n progeny of hermaphrodites. 7.3.2 Measurements Of F i t n e s s Components Seed (achene) weight and percent seed germination i n 9 gynodioecious p o p u l a t i o n s showed no s i g n i f i c a n t d i f f e r e n c e s between the progenies of females and hermaphrodites, except i n one p o p u l a t i o n of IK s a n d v i c e n s i s , i n which seeds of females germinated b e t t e r (p<0.0l) than those of hermaphrodites (Table 7-3). Seed weight was not c o r r e l a t e d with germination percentage i n these p o p u l a t i o n s . D i f f e r e n c e s i n sample s i z e s of number of seeds and number of f a m i l i e s had no s i g n i f i c a n t e f f e c t s on measuring these two parameters. Leaf dry weight of progeny of females at an e a r l y v e g e t a t i v e stage was s i g n i f i c a n t l y higher (p<0.05) than that of hermaphrodites i n a p o p u l a t i o n of IK f o r b e s i i ssp. k a h i l i e n s i s , but not so i n a p o p u l a t i o n of IK_ f o r b e s i i ssp. f o r b e s i i (Table 7-4). Dry l e a f weight of progeny of females was a l s o higher than those of hermaphrodites i n experimental c r o s s e s (Table 7-5). The l e a f weight of the progeny i n a c r o s s between a female of B.  p o p u l i f o l i a and a hermaphrodite of IK cosmoides was a l s o s i g n i f i c a n t l y higher than those i n a w i t h i n p o p u l a t i o n " c r o s s " of IK_ p o p u l i f o l i a . The progeny of females a l s o s u r v i v e d b e t t e r than those of hermaphrodites i n the c o n t r o l l e d c r o s s e s . The d i f f e r e n t i a l r a t e s of s u r v i v o r s h i p , however, c o u l d be a r e s u l t of experimental e r r o r , s i n c e more than 80% of s e l f e d progeny of 173 Table 7-3. Seed (achene) weight and percentage of germination In the progenies of male s t e r i l e s and hermaphrodites In 9 gynodioecious populations. Maternal No. No. seed weight % Populat ion sexual form seeds fam i1i es (mg/achene) germi nat ion CERV H 700 14 .85( .21) .60( . 19) MS 675 14 .81( .09) • 52( .21 ) FORB F1 H 1025 30 . 98 ( . 18) .62( .22) MS 735 21 • 97( .20) .51( . 22) FORB F2 H 299 15 1 . 13( .27) -MS 296 15 1 . 10( .21) -FORB K H 368 7 1. 17( . 16) • 37( . 18) MS 389 7 1 . 28( . 20) . 36( . 10) MENZ 1 H 1314 20 1 .07( .22) .63( . 16) MS 1438 29 1 .05( . 19) • 71( . 16) MENZ 2 H 929 19 .87( . 12) • 25( . 12) MS 930 19 .85( . 12) • 31( .11) MENZ 3 H 180 9 .84( .09) .83( . 17) MS 180 9 • 74( . 13) • 76( . 18) MENZ 4 H 260 13 1 . 32( • 24) . 59( .25) MS 280 14 1 . 24( . 14) .74( .24) SAND X H 400 20 1 .04( .21) .43( . 22)** MS 400 20 1 .04( . 15) .65( . 16) Mean(s.e.) 600(389) 16(7) 1 .02( • 17) .51( . 21) **' t - t e s t : p<0.01 174 Table 7-4. Leaf dry weight of the progenies of male s t e r i l e s and hermaphrodites i n two gynodioecious p o p u l a t i o n s of B. f o r b e s i i r a i s e d in a greehouse. Maternal No. of Leaf dry weight Pop. sexual form progeny ( g / l e a f p a i r ) FORB F1 H 21 0.218(0.045) MS 19 0.210(0.106) FORB K H 23 0.238(0.078)* MS 25 0.284(0.061) * t - t e s t : p<0.05. T a b l e 7-5 V e g e t a t i v e growth and s u r v i v o r s h i p i n the p r o g e n i e s of e x p e r i m e n t a l c r o s s e s . Type of No. Leaf d r y weight Ig)** % c r o s s e s * progeny 3 r d p a i r 6 t h p a i r S u r v i v o r s H P0PU(42c) s e l f e d 12 .014(.012) .174(.061) 50 MS P0PU(42b) x H P0PU(42c) 21 .028(.007) .322(.114) 95 MS P0PU(42b) x H C0SM(9c) 10 .040(.011) - 100 tt: 42c i s a he r m a p h r o d i t e of B^ p o p u l i f o l i a , 42b i s a female p l a n t of the same s p e c i e s . 9c i s a h e r m a p h r o d i t i c p l a n t of cosmoides . **: t - t e s t p<0.01 f o r any two measurements. 176 a hermaphrodite of p o p u l i f o l i a r a i s e d i n the same greenhouse s u r v i v e d w e l l i n a l a t e r experiment. The d i f f e r e n c e i n v e g e t a t i v e growth between outbred progeny of females and those from s e l f p o l l i n a t i o n , as w e l l as h y b r i d v i g o r among the s p e c i e s of Hawaiian Bidens, was a l s o f r e q u e n t l y observed i n other experiments conducted f o r g e n e t i c s t u d i e s of gynodioecy i n greenhouses. V e g e t a t i v e growth, p l a n t h e i g h t , and diameter of stems measured at p r e - r e p r o d u c t i v e and e a r l y f l o w e r i n g stages f o r three experimental p o p u l a t i o n s are given i n Table 7-6. There were no s i g n i f i c a n t d i f f e r e n c e s between progenies of females and hermaphrodites i n these measurements. No d i f f e r e n c e s between progenies of females and hermaphrodites were found i n t o t a l flower heads per p l a n t , d i s c flowers per head or seeds per head (Table 7-7). Flower head s i z e was s i g n i f i c a n t l y l a r g e r (p<0.0l) i n the progeny of m a l e - s t e r i l e p l a n t s than i n those of hermaphrodites. 7.3.3 Test For P l e i o t r o p i c E f f e c t s Of M a l e - s t e r i l e Genes F l o r a l t r a i t s - The f l o r a l f e a t u r e s of hermaphrodites i n Hawaiian Bidens are c h a r a c t e r i s t i c of Asteraceae. The c a p i t u l u m (flower head) bears from f i v e to more than one hundred d i s c flowers ( f l o r e t s ) i n d i f f e r e n t s p e c i e s (see Chapter 3). The number of f l o r e t s per head i s s i m i l a r i n females and hermaphrodites w i t h i n each s p e c i e s (Table 7-8). The main morp h o l o g i c a l d i f f e r e n c e between females and hermaphrodites i s the diameter of flower heads. M a l e - s t e r i l e flower heads are s i g n i f i c a n t l y smaller than hermaphrodite heads r e g a r d l e s s of the 177 Table 7-6. V e g e t a t i v e c h a r a c t e r s of the progenies of females and hermaphrodites i n three greenhouse r a i s e d gynodioecious p o p u l a t i o n s p r i o r to r e p r o d u c t i o n . Pop. Maternal sexual type NO. Of progeny Pl a n t height cm (s.e.) Stem diameter mm (s.e.) FORB F1 H 20 58. 2(18.1) 3.7(0.3) MS 14 64.1(10.4) 3.9(0.4) FORB K H 17 63.0(20.0) 4.2(0.5) MS 32 70.0(19.0) 4.2(0.5) SAND C H 32 85.0(17.1) 3.0(0.3) MS 51 92.2(18.6) 3.0(0.4) Prob. N.S. N.S. T a b l e 7-7. R e p r o d u c t i v e f e a t u r e s of the p r o g e n i e s of females and he r m a p h r o d i t e s i n two g y n o d i o e c i o u s p o p u l a t i o n s r a i s e d i n a greenhouse. Head Heads F l o r e t s Seeds P o p u l a t i on Materna1 No. diameter per p l a n t per head per head seeds sexual form progeny mm ( s . e . ) ( s . e . ) ( s . e. )# ( s . e . )# per ovul FORB F1 H 20 16.8(3. 1 )** 1 16(68) 14(3) [24] 9(3) [ 131] .61 MS 14 20.5(0.7) 133(67) 16(3) [5] 10(2) [7] .60 SAND C H 32 26.2(6.0)** 67(52) 14(3) [117] 10(2) [378] .71 MS 51 30.3(4.6) 77(42) 14(2) [282] 11(3) [556] . 79 H: Number i n [ ] I n d i c a t e s the sample s i z e of f l o w e r heads. ** t - t e s t : p<0.01. Table 7-8. Vegetative and reproductive measurements of females and hermaphrodites in three gynodioecious populations raised in a greenhouse. Progeny Head F1orets PI ant Stem Heads Seeds Species & Materna1 No. diameter per head he i ght d1ameter per plant per head popu1 at i on sex sex pi ants mm C N ] cm mm (s.e.) [ N ] (s.e.) (s.e.) (s.e.) (s.e.) (s.e.) FORB F1 MS MS 3 - - 69(10) 4.3(0.2) 138(36) -H 13 21(1) 16(3) [5] 63(11) 3.8(0.5) 133(73) 10(2) [7] MS 2 13(0)** 12(2) [3] 62(7) 3.8(0.3) 140(153) 11(2) [15] H 18 18(0) 14(3) [21] 58(19) 3.8(0.3) 1 1 1 (66) 8(3) [115] POPU H MS 2 - 20(3) [5] 76(1) 2.9(0.2) 33( 1) 8(7) [11] H 18 - 19(4) [47] 66(19) 2.9(0.3) 41(20) 10(6) [55] SAND C MS MS 9 27(3)** 14(2) [48] 89(21) 3.0(0.3) 68(30) 12(2) [102] H 42 32(5) 14(2) [234] 93(18) 3.0(0.3) 81 (44) 11(3) [454] H MS 5 19(8)** 14(3) [18] 80(22) 3.2(0.3) 1 17(56)** 10(1) [107] H 27 28(4) 15(3) [99] 86(16) 3.0(0.3) 55(45) 10(3) [271] ** t - t e s t : p<0.01 180 maternal sexual form i n two gynodioecious p o p u l a t i o n s i n v e s t i g a t e d (Table 7-8), as w e l l as i n an experimental c r o s s (Table 7-9). The r e d u c t i o n i n female head s i z e i s caused by sma l l e r ray flower s i z e and d i s c flower s i z e . Anther s i z e of female flowers i s a l s o s i g n i f i c a n t l y smaller than that of hermaphrodite f l o w e r s . Stigma r e c e p t i v i t y - Normal c y l i n d r i c anthers of hermaphrodites are dark-brown or bl a c k , surrounding the c l o s e d two stigma branches when the d i s c flower j u s t opens. Anthers dehisce one-half day l a t e r and p o l l e n g r a i n s are presented on p r o t r u d i n g s t y l e branches. The two branches of the s t y l e remain c l o s e d f o r u s u a l l y three days a f t e r p o l l e n p r e s e n t a t i o n (see Chapter 3), then open to r e v e a l r e c e p t i v e stigmas. In c o n t r a s t , the two s t y l e branches of m a l e - s t e r i l e flowers have a l r e a d y opened at a n t h e s i s , or open to be r e c e p t i v e soon a f t e r the d i s c flower opens. T h e - r e c e p t i v i t y of the stigma l a s t s f o r about 2-3 days i n hermaphroditic f l o w e r s , but about 4-5 days i n male-s t e r i l e f l o wers i n most s p e c i e s . Ray flowers of both female p l a n t s and hermaphrodites are completely s t e r i l e , although m o r p h o l o g i c a l o v a r i e s are pr e s e n t . M a l e - s t e r i l e i n d i v i d u a l s flowered at about the same time as hermaphrodites i n a l l experimental p o p u l a t i o n s and c o n t r o l l e d c r o s s e s , and both sexual forms have a prolonged f l o w e r i n g season. V e g e t a t i v e and r e p r o d u c t i v e t r a i t s - There were no s i g n i f i c a n t d i f f e r e n c e s between sexes per se i n e i t h e r v e g e t a t i v e growth or r e p r o d u c t i v e output i n three gynodioecious p o p u l a t i o n s r a i s e d i n a greenhouse, except i n one case. T a b l e 7-9. F l o r a l and r e p r o d u c t i v e f e a t u r e s of male s t e r i l e s and hermaphrodites s e g r e g a t e d from an e x p e r i m e n t a l c r o s s : MS F0RB(12) X H [MS HAWA(51A) X H MAUI(10)]#. Head Ray f l o w e r Ray f l o w e r D i sc c o r o l 1 a Anther Sexua1 No. diameter 1ength width l e n g t h 1ength F 1 o r e t s Seeds Heads form p i a n t s mm mm mm mm - mm per head per head per p l a n t ( s . e . ) ( s . e . ) ( s . e . ) (s.e.) ( s . e . ) ( s . e . ) ( s . e . ) ( s . e . ) H 5 33.3(2.9) 17.0(1.0) 7.3(.6) 7.0(1.7) 1 .8(.4) 23(3) 15(1) 39(58) MS 5 23.3(2.9) 12.5(4.1) G.0(.5) 5.0(.5) 1.2(.2) 17(3) 15(5) 77(50) Prob. < .01 <.05 <.01 <.05 < .01 < .01 N.S. N.S. #: MS FORB(12) i s a male-s t e r i1e p i ant of B. f o r b e s i1 ssp. f o r b e s i i MS HAWA(51A) i s a ma 1 e - s t e r i1e p i ant of B. hawaiensis . H MAUI(10) 1s a h e r m a p h r o d i t i c p l a n t of B^ mauiensis . 182 Although having the same hermaphrodite maternal parent, female progeny produced twice as many flower heads per p l a n t as hermaphrodite progeny i n the p o p u l a t i o n SAND C of B. s a n d v i c e n s i s subsp. confusa (Table 7-8). No s i g n i f i c a n t d i f f e r e n c e s were d e t e c t e d i n r e p r o d u c t i v e output between female and hermaphrodite progenies i n two experimental c r o s s e s (Table 7-9, 7-10), although m a l e - s t e r i l e p l a n t s produced fewer d i s c flowers ( f l o r e t s ) per head than hermaphrodite p l a n t s i n one c r o s s (Table 7-9). When m a l e - s t e r i l e and hermaphroditic p l a n t s were p l a c e d i n i n s e c t - p r o o f cages, no females and few hermaphrodites set seeds. Some hermaphrodites of a few s p e c i e s set seeds at a low frequency (see Table 3-4 i n Chapter 3 f o r d a t a ) , probably r e s u l t i n g from a low l e v e l of s e l f - f e r t i l i z a t i o n . Agamospermy or apomixis was not found i n e i t h e r sexual form. P o l l e n s t a i n a b i l i t y was very high, 96-99%, i n a l l hermaphrodite p l a n t s t e s t e d . These p l a n t s were progeny of both females and hermaphrodites i n a greenhouse r a i s e d gynodioecious p o p u l a t i o n of IK s a n d v i c e n s i s ssp. confusa. 7.4 D i s c u s s i o n 7.4.1 S e l f i n g Rates Of Hermaphrodites And H e t e r o z y g o s i t y Although the maintenance of male s t e r i l i t y has been s t u d i e d e x p e r i m e n t a l l y i n a v a r i e t y of n a t u r a l gynodioecious s p e c i e s , most of these s t u d i e s emphasize measuring v a r i o u s f i t n e s s components at d i f f e r e n t stages of the l i f e c y c l e . Only a few s t u d i e s have given o u t c r o s s i n g r a t e s of hermaphrodites 183 Table 7-10. Reproductive f e a t u r e s of male s t e r i l e s and hermaphrodites i n the F2 progeny of an experimental c r o s s : MS POPU(12) X H MAUI(10). Heads I n f l o r e s c e n c e s Sexual No.of per p l a n t per p l a n t Heads per form p l a n t s (s.e.) (s.e.) i n f l o r e s c e n c e H MS 24 12 50(29) 56(35) 6(2) 6(3) 8 9 prob. N.S. N.S. N.S. 184 (Table 7-11). None of these experimental s t u d i e s t e s t e d the o u t c r o s s i n g h y p o t h e s i s of the t h e o r e t i c a l s t u d i e s , as represented by L l o y d ' s model. The d i s c o v e r y of the strong p o s i t i v e c o r r e l a t i o n between s e l f i n g r a t e s of hermaphrodites and the f r e q u e n c i e s of females i n n a t u r a l gynodioecious p o p u l a t i o n s of Hawaiian Bidens i n t h i s study p r o v i d e s the f i r s t e m p i r i c a l evidence which confirms that the o b l i g a t e o u t c r o s s i n g of females i s a major f a c t o r m a i n t a i n i n g females i n p o p u l a t i o n s . Gynodioecy has been t r a d i t i o n a l l y i n t e r p r e t e d as an o u t c r o s s i n g mechanism (Mather, 1940). The s e l e c t i v e advantage of gynodioecy i n Hawaiian Bidens might be that the progeny of females have g r e a t e r f i t n e s s because they r e s u l t e d from o u t c r o s s i n g and are more heterozygous. L e v e l s of h e t e r o z y g o s i t y have a l s o been r e p o r t e d f o r two gynodioecious p o p u l a t i o n s of Limnanthes d o u q l a s i i ( K e s s e l i and J a i n , 1984), i n which the observed h e t e r o z y g o s i t y i n the progeny of females was g e n e r a l l y higher than that i n the progeny of hermaphrodites, which agrees with the present study. The p o s s i b l e a d a p t i v e s i g n i f i c a n c e of gynodioecy as a mechanism to promote o u t c r o s s i n g has been d i s c u s s e d i n s e v e r a l t h e o r e t i c a l s t u d i e s (Lewis, 1941; J a i n , 1961; Valdeyron et a l . , 1973; Gouyon and Vernet, 1982; Ross and G r e g o r i u s , 1985). Male s t e r i l i t y may be p a r t i c u l a r l y important as a means of generating necessary h y b r i d i t y or m a i n t a i n i n g h y b r i d i t y optima i n predominantly s e l f - f e r t i l i z e d p o p u l a t i o n s ( J a i n , 1961), The study of Gouyon and Vernet (1982) i n v e s t i g a t e d the e f f e c t of gynodioecy on the genotypic s t r u c t u r e of a p o p u l a t i o n of T a b l e 7-11. O u t c r o s s i n g r a t e s a v a i l a b l e f o r 4 g y n o d i o e c i o u s s p e c i e s Spec ies No. of popu1 at 1ons t % fema1es Source C i r s i u m p a l u s t r e - .83 - C o r r e n s , 1916 ( I n Ross,1978). C o r t a d e r l a M c h a r d l 1 - .25 38 Connor. 1973. Thymus v u l g a r i s 4 .64 10-90 V a l d e y r o n e t a l . , 1977. .51 .65 .90 Limnanthes d o u g l a s l l 2 . 75 10-20 K e s s e l 1 8> J a i n , 1984. . 76 oo 186 Thymus v u l g a r i s . They found that m a l e - s t e r i l e i n d i v i d u a l s i n themselves are more heterozygous than hermaphrodites, as p r e d i c t e d by t h e i r model. Greater h e t e r o z y g o s i t y was a l s o found i n the progenies of females than i n those of hermaphrodites i n t h i s s p e c i e s . I f heterozygotes or new recombinant types are at a s e l e c t i v e advantage at some l o c i , females should be favored as w e l l as t h e i r p rogenies, and the i n c r e a s e i n female frequency i n c r e a s e s the h e t e r o z y g o s i t y of the whole p o p u l a t i o n . There i s evidence which suggests that females may be favored under c o m p e t i t i o n or i n d i s t u r b e d areas, probably because they promote g e n e t i c recombination (Dommee et a l . , 1978; Bonnenasion et a l . , 1979, i n Gouyon and Vernet, 1982). S e l f - i n c o m p a t i b i l i t y i s a more e f f i c i e n t o u t c r o s s i n g breeding system than gynodioecy. Gynodioecy c o u l d not evolve i n s e l f - i n c o m p a t i b l e s p e c i e s , such as Plantago l a n c e o l a t a (Baker, 1963; Ross, 1970) and H i r s c h f e l d i a incana ( H o r o v i t z and B e i l e s , 1980), i f the e v o l u t i o n of gynodioecy depends s o l e l y on i t s o u t c r o s s i n g consequences d i s c u s s e d above. Since a l l i n d i v i d u a l s outbreed i n s e l f - i n c o m p a t i b l e gynodioecious p o p u l a t i o n s , the l e v e l s of h e t e r o z y g o s i t y should be the same i n females and hermaphrodites, as supported by the experimental data i n Plantago l a n c e o l a t a (Krohne et a l . , 1980). T h e r e f o r e , the establishment of gynodioecy i n s e l f - i n c o m p a t i b l e s p e c i e s i s o b v i o u s l y due to other s e l e c t i v e advantages which are independent of o u t c r o s s i n g . I t has been argued that gynodioecy might be s e l e c t e d as a p o p u l a t i o n a l s t r a t e g y f o r i n c r e a s i n g r e p r o d u c t i v e output because a b o r t i o n of p o l l e n may r e s u l t i n a 187 r e a l l o c a t i o n of resource to seed p r o d u c t i o n w i t h i n the p l a n t (Darwin, 1877; H o r o v i t z and B e i l e s , 1980). The high f r e q u e n c i e s of females i n gynodioecious p o p u l a t i o n s of Hawaiian Bidens cannot be brought about by the r e l a t i v e o u t c r o s s i n g advantage of females a l o n e . According to L l o y d (1975) and Ganders (1978), the frequency of females (p) i n a gynodioecious p o p u l a t i o n i s determined by the formula, p = (2sd - l ) / ( 2 s d ) , assuming that females and hermaphrodites are equal i n e v e r y t h i n g except that the inbred progeny of hermaphrodites s u f f e r i n b r e e d i n g d e p r e s s i o n . The observed f r e q u e n c i e s of females are much higher than expected, even given the h i g h e s t d value of 1 ( i . e . , a l l inbred progeny having a f i t n e s s of z e r o ) , except i n the p o p u l a t i o n SAND 2 of B. s a n d v i c e n s i s subsp. s a n d v i c e n s i s . Obviously, females must have some a d d i t i o n a l s e l e c t i v e advantages. The observed l a r g e v a r i a t i o n i n the f r e q u e n c i e s of females i n n a t u r a l p o p u l a t i o n s of Hawaiian Bidens, however, i n d i c a t e s that f a c t o r s a c t i n g on the f i t n e s s of females are rather complicated and each.of them probably has v a r i a b l e s e l e c t i v e values depending on the environment (Assouad et a l , 1978). 7.4.2 S e v e r a l F i t n e s s Components Of Females And Hermaphrodites There were no s i g n i f i c a n t d i f f e r e n c e s i n seed weight and percent germination between the progenies of females and hermaphrodites i n gynodioecious p o p u l a t i o n s of Hawaiian Bidens i n v e s t i g a t e d , except i n p o p u l a t i o n SAND X, a p o p u l a t i o n showing i n t e r g r a d a t i o n between B^ s a n d v i c e n s i s ssp. s a n d v i c e n s i s and ssp. confusa, i n which the progeny of females germinated b e t t e r 188 (65%) than those of hermaphrodites (43%). Seed weight has been measured as a parameter of seed q u a l i t y f o r s e v e r a l gynodioecious p o p u l a t i o n s . In Plantago l a n c e o l a t a , i n which hermaphrodites are s e l f - i n c o m p a t i b l e , the seed weight of progeny of females r e l a t i v e to that of hermaphrodites i s 1.15 and 0.9 r e p e c t i v e l y i n the two m a l e - s t e r i l e phenotypes, MS1 and MS2 (Van Damme, 1984; Van Damme and Van Delden, 1984), but no d i f f e r e n c e s were found i n seed germination. In Geranium s y l v a t i c u m , the r a t i o of seed s i z e i s 0.9 (Vaarama and J a a s k e l a i n e n , 1967). Webb (1979, 1981) found no d i f f e r e n c e in G i n g i d i a montana and Scandia g e n i c u l a t a i n the average weight of seeds or mericarps. The r a t i o s of seed weight per p l a n t i n male s t e r i l e s r e l a t i v e to hermaphrodites ranged from 1.10-1.40 i n H i r s c h f e l d i a incana ( H o r o v i t z and B e i l e s , 1980). Females produce much heavier seeds than hermaphrodites i n C o r t a d e r i a s e l l o a n a , with a r a t i o of 1.47, and i n CL f u l v i d a , with a r a t i o of 1.40. A high r a t i o of germination of females to hermaphrodites, 7.7, was a l s o found i n C. s e l l o a n a (Connor, 1973). A 3.45 germination r a t i o was found i n C i r s i u m arvense ( L l o y d and M y a l l , 1976). There appears to be no ge n e r a l t r e n d i n seed weight and germination between the two sexual forms, and the d i f f e r e n c e s i n seed weight were not g e n e r a l l y found to be c o r r e l a t e d with d i f f e r e n c e s i n seed ger m i n a t i o n . T h i s may suggest that heavier seeds produced by females i n some gynodioecious s p e c i e s c o u l d represent an advantage i n the form of longer seed v i a b i l i t y or more vigorous s e e d l i n g s other than higher p r o p o r t i o n of germination. Inbreeding d e p r e s s i o n or h e t e r o s i s was found i n p o p u l a t i o n 189 FORB K of B_j_ f o r b e s i i subsp. k a h i l i e n s i s (Table 7-4), and i n the progeny of s e l f p o l l i n a t e d B^ p o p u l i f o l i a as shown in v e g e t a t i v e growth (Table 7-5). Leaves were twice as heavy (dry weight) i n the progeny of a female as i n the progeny of a s e l f e d hermaphrodite. However, no d i f f e r e n c e s were found i n p l a n t height or diameter (Table 7-6), nor i n seed set (Table 7-7) between the progenies of females and hermaphrodites i n three experimental gynodioecious p o p u l a t i o n s measured at l a t e r stages. T h i s i m p l i e s that i n b r e e d i n g d e p r e s s i o n , i f i t does e x i s t at these l i f e c y c l e stages i n gynodioecious p o p u l a t i o n s of Hawaiian Bidens, c e r t a i n l y was not expressed under greenhouse c o n d i t i o n s . F i e l d c o n d i t i o n s may accentuate v e g e t a t i v e and r e p r o d u c t i v e d i f f e r e n c e s between the two sexual forms, however (Assouad et a l 1978). Higher l e v e l s of h e t e r o z y g o s i t y i n the progeny of females may give them g r e a t e r homeostasis and thus they might be favoured i n d i f f i c u l t environmental conditons (Valdeyron et a l . , 1973; Dommee et a l . , 1978). On the other hand, lack of f i t n e s s d i f f e r e n c e at these stages might be due to l i m i t a t i o n under c u l t i v a t i n g c o n d i t i o n s , as a l l the p l a n t s i n these three experimental p o p u l a t i o n s were each r a i s e d i n i n d i v i d u a l s o i l pots from seeds c o l l e c t e d i n nature. Although there should be no environmental l i m i t a t i o n at the s e e d l i n g or e a r l y v e g e t a t i v e stage, the l i m i t e d s o i l pot space might u l t i m a t e l y a f f e c t the p o t e n t i a l growth r a t e or r e p r o d u c t i v e output i n these p o p u l a t i o n s . The r e p r o d u c t i v e c a p a c i t i e s of the two sexual forms have been s t u d i e d i n a number of n a t u r a l gynodioecious s p e c i e s . 190 Females have an advantage over hermaphrodites i n most cases (Darwin, 1877; Lewis and Crowe, 1956; Burrows, 1960; Connor, 1973; Young, 1972; L l o y d and M y a l l , 1976; Assouad e t a l . , 1978; Ho r o v i t z and B e i l e s , 1980; Krohno et a l . , 1980; P h i l i p p , 1980; Webb, 1981; Van Damme, 1984; Van Damme and Van Delden, 1984). L l o y d (1976) showed that the frequency of females i n gynodioecious s p e c i e s of 7 genera i n d i v e r s e f a m i l i e s can be ex p l a i n e d i n terms of h i s model: C = (1-2p)/[ 2 (1-p).], where C = r e l a t i v e ovule c o n t r i b u t i o n of hermaphrodites to females, and p = the e q u i l i b r i u m p r o p o r t i o n of females i n the p o p u l a t i o n . F r e q u e n t l y , however, not enough advantage was found i n females to account f o r the observed female f r e q u e n c i e s (e.g., Krohno et a l . , 1980; P h i l i p p , 1980; Webb, 1981; Uno, 1982; K e s s e l i and J a i n , 1984). 7.4.3 P l e i o t r o p i c E f f e c t Of Male S t e r i l i t y P l e i o t r o p i c e f f e c t s of male s t e r i l i t y genes were shown i n f l o r a l c h a r a c t e r s , such as smaller flower heads, e a r l i e r and longer stigma r e c e p t i v i t y , and reduced anthers. The r e d u c t i o n i n c o r o l l a s i z e of females apppears to be a general f e a t u r e a s s o c i a t e d with male s t e r i l i t y ( L l o y d and Webb, 1977). A s i m i l a r d i f f e r e n c e between the two sexual forms i n the le n g t h of r e c e p t i v i t y of stigmas has been r e p o r t e d i n Thymus v u l g a r i s (Assouad and Valdeyron, 1975). Although the small e r s i z e of m a l e - s t e r i l e flower heads may seem to be a disadvantage because they may be l e s s a t t r a c t i v e to p o l l i n a t o r s , high r a t e s of p o l l i n a t i o n of m a l e - s t e r i l e flowers were recorded i n n a t u r a l p o p u l a t i o n s of IK m e n z i e s i i subsp. f i l i f o r m i s and IK_ f o r b e s i i 191 ssp. f o r b e s i i (Ganders, p e r s . comm.). High e f f i c i e n c y of p o l l i n a t i o n of females i s suggested i n other n a t u r a l gynodioecious p o p u l a t i o n s of Bidens by t h e i r high percentage of seedset (pers. o b s e r v a t i o n ) . S i m i l a r o b s e r v a t i o n s have been r e p o r t e d f o r Thymus v u l g a r i s (Assouad et a l , 1978). The high p o l l i n a t i o n e f f i c i e n c y may a l s o be a t t r i b u t a b l e t o the " f l a g e f f e c t " of flowers (Arroyo and Raven, 1975), i . e . , numerous flowers or flower heads i n the i n f l o r e s c e n c e s a t t r a c t p o l l i n a t o r s i n s t e a d of i n d i v i d u a l f l o w e r s . T h i s i s probably the case i n Hawaiian s p e c i e s of Bidens. Most Hawaiian s p e c i e s have i n f l o r e s c e n c e s t hat are compound cymes with numerous si m u l t a n o u s l y f l o w e r i n g heads (see Chapter 3). The longer stigma r e c e p t i v i t y of m a l e - s t e r i l e flowers may a l s o i n c r e a s e the chance of p o l l i n a t i o n . High seed set on m a l e - s t e r i l e flower heads was commonly observed i n n a t u r a l gynodioecious p o p u l a t i o n s of Bidens, as w e l l as i n open p o l l i n a t e d m a l e - s t e r i l e i n d i v i d u a l s r a i s e d i n greenhouses. The present data from greenhouse grown gynodioecious p o p u l a t i o n s and from c o n t r o l l e d c r o s s e s • do not suggest the presence of p l e i o t r o p i c e f f e c t s of male s t e r i l i t y genes on v e g e t a t i v e growth or seed p r o d u c t i o n , except i n p o p u l a t i o n SAND C of B. s a n d v i c e n s i s subsp. confusa. The o b s e r v a t i o n of higher seed p r o d u c t i o n i n m a l e - s t e r i l e progeny of hermaphrodites, but not i n m a l e - s t e r i l e progeny of females, i n t h i s p o p u l a t i o n might be j u s t chance c o n s i d e r i n g the small sample s i z e of female progeny. Seed-set of females was observed to be s i g n i f i c a n t l y higher 192 than that of hermaphrodites i n three n a t u r a l gynodioecious p o p u l a t i o n s of Ek menziesi i ssp. f i l i f o r m i s , and one p o p u l a t i o n of EL_ s a n d v i c e n s i s , but no d i f f e r e n c e s were observed i n the numbers of flowers per head between the two sexual forms i n these p o p u l a t i o n s (Ganders, p e r s . comm.). The low seed-set of hermaphrodites i n one p o p u l a t i o n of B_j_ m e n z i e s i i ssp. f i l i f o r m i s was caused by the abnormal behavior of t h e i r s t y l e branches. The s t y l e branches of a l a r g e p r o p o r t i o n of d i s c flowers of hermaphrodites f a i l e d to open to expose stigma s u r f a c e s , but presented p o l l e n g r a i n s normally. T h i s o b s e r v a t i o n suggests that the g r e a t e r seed p r o d u c t i o n of females i n t h i s s p e c i e s i s caused by reduced female f e r t i l i t y i n hermaphrodites, not by the p l e i o t r o p i c e f f e c t s of male s t e r i l i t y genes. Females set more seeds than hermaphrodites i n many gynodioecious s p e c i e s (Darwin, 1877; Burrows, 1960; Assouad e t . a l . , 1978; Webb, 1979, 1981; P h i l i p p , 1980; Van Damme and Van Delden, 1984), but i t i s not c l e a r whether the d i f f e r e n t i a l seed p r o d u c t i o n r e s u l t s from p l e i o t r o p i c e f f e c t s of male s t e r i l i t y genes per se, or from resource r e a l l o c a t i o n , or from reduced female f e r t i l i t y of hermaphrodites. 7.4.4 E v o l u t i o n a r y S t a b i l i t y And Maintenance Of Gynodioecy Two types of gynodioecy have been d i s t i n g u i s h e d based on t h e i r e v o l u t i o n a r y s t a b i l i t y (Ross, 1978): s t a b l e gynodioecy (which does not evolve towards dioecy) and unstable gynodioecy (which evolves to d i o e c y ) . Normal female f u n c t i o n i n hermaphrodites c h a r a c t e r i z e s the s t a b l e type and reduced female 193 f u n c t i o n i n hermaphrodites c h a r a c t e r i z e s the u n s t a b l e type. I f female f u n c t i o n of hermaphrodites i s a r e l i a b l e c r i t e r i o n of the e v o l u t i o n a r y s t a b i l i t y of gynodioecy, gynodioecy i n B.  m e n z i e s i i subsp. f i l i f o r m i s i s probably u n s t a b l e because of the reduced female f u n c t i o n i n hermaphrodites d i s c u s s e d above. T r a n s i t i o n from hermaphroditism v i a gynodioecy i s among the f i v e e v o l u t i o n a r y pathways to dioecy d i s c u s s e d by Ross (1978, 1980, 1982). Some e x i s t i n g gynodioecious s p e c i e s on the Hawaiian I s l a n d s are probably a l s o at t r a n s i t i o n a l stages from hermaphroditism to dioecism, such as some s p e c i e s i n the genera C h a r p e n t i e r a and G o u l d i a ( C a r l q u i s t , 1974). Examples of t h i s pathway have been suggested i n Pimelea (Burrows, 1960), Fuchsia (Arroyo and Raven, 1975), C i r s i u m arvense ( L l o y d and M y a l l , 1976) and a p i o i d U m b e l l i f e r a e (Webb, 1979). The high f r e q u e n c i e s of females i n p o p u l a t i o n s of B.  m e n z i e s i i subsp. f i l i f o r m i s , 34-44%, cannot be e x p l a i n e d by the s e l f i n g r a t e s of hermaphrodites a l o n e . The reduced female f u n c t i o n of hermaphrodites, which c o n f e r s about a three f o l d advantage i n seed p r o d u c t i o n to females, t h e r e f o r e , must act as another major f a c t o r m a i n t a i n i n g females i n t h i s s p e c i e s . The maintenance and e v o l u t i o n a r y s t a b i l i t y of gynodioecy i n p o p u l a t i o n s of other s p e c i e s cannot be f u l l y e x p l a i n e d based on p r e s e n t l y a v a i l a b l e d a ta. Besides o u t c r o s s i n g , other s e l e c t i v e advantages females must have to maintain t h e i r c u r r e n t f r e q u e n c i e s are yet to be d i s c o v e r e d . F u r t h e r i n f o r m a t i o n on the r e l a t i v e f i t n e s s of females and hermaphrodites i n n a t u r a l h a b i t a t s i s needed. 194 7.5 Summary S e v e r a l f a c t o r s a f f e c t i n g the maintenance of gynodioecy i n p o p u l a t i o n s of Hawaiian Bidens were s t u d i e d . S e l f i n g r a t e s of hermaphrodites were h i g h l y c o r r e l a t e d with the f r e q u e n c i e s of females i n e i g h t gynodioecious p o p u l a t i o n s . T h i s d i s c o v e r y p r o v i d e s the f i r s t e m p i r i c a l evidence that supports the o u t c r o s s i n g hypothesis of the maintenance of gynodioecy. The l e v e l s of h e t e r o z y g o s i t y i n the progenies of females were g e n e r a l l y higher than i n the progenies of hermaphrodites at three polymorphic allozyme l o c i , Pgi-1, Pgi-2, and Skdh-3, i n d i c a t i n g t h at male s t e r i l i t y does a f f e c t the g e n e t i c s t r u c t u r e of gynodioecious p o p u l a t i o n s . Estimates of l e a f dry weight suggested the presence of i n b r e e d i n g d e p r e s s i o n or h e t e r o s i s at e a r l y stages of v e g e t a t i v e growth i n one gynodioecious p o p u l a t i o n of IK f o r b e s i i subsp. k a h i l i e n s i s (FORB K) and i n experimental p o l l i n a t i o n s of B.  p o p u l i f o l i a (POPU). However, no s i g n i f i c a n t d i f f e r e n c e s were found i n seed weight or germination between females and hermaphrodites i n nine open p o l l i n a t e d n a t u r a l gynodioecious p o p u l a t i o n s , except that seeds produced by females germinated b e t t e r than those of hermaphrodites i n a p o p u l a t i o n of B.  s a n d v i c e n s i s (SAND X). P l a n t height and diameter, and ovule ( = f l o r e t ) and seed p r o d u c t i o n were a l s o s i m i l a r i n the progenies of the two sexual forms as shown in three gynodioecious p o p u l a t i o n s r a i s e d i n a greenhouse. The p l e i o t r o p i c e f f e c t s of male s t e r i l i t y genes were de t e c t e d mainly i n f l o r a l c h a r a c t e r s . . M a l e - s t e r i l e flower heads 195 are s i g n i f i c a n t l y smaller than hermaphroditic ones. The r e d u c t i o n i n the diameter of flower heads i n females i s due to the s i g n i f i c a n t l y reduced ray flowers and d i s c f l o w e r s . Females have longer stigma r e c e p t i v i t y than hermaphrodites. P l e i o t r o p i c e f f e c t s of male s t e r i l i t y genes on seed p r o d u c t i o n was suggested i n only one greenhouse r a i s e d p o p u l a t i o n of s a n d v i c e n s i s subsp. confusa, i n which MS progeny of hermaphrodites had a higher seed y i e l d than t h e i r H progeny. Data i n t h i s study do not support the hypothesis of resource r e a l l o c a t i o n . No g e n e r a l c o n c l u s i o n s can be drawn about the e v o l u t i o n a r y s t a b i l i t y of gynodioecy i n Hawaiian Bidens u n t i l f u r t h e r i n f o r m a t i o n i s a v a i l a b l e on r e l a t i v e female f e r t i l i t y of hermaphrodites i n n a t u r a l p o p u l a t i o n s . I conclude that females are maintained i n n a t u r a l gynodioecious p o p u l a t i o n s of Hawaiian Bidens at l e a s t p a r t i a l l y due to t h e i r o b l i g a t e o u t c r o s s i n g , while the progenies of hermaphrodites e x h i b i t i n b r e e d i n g d e p r e s s i o n . The s e l f i n g r a t e s of hermaphrodites, however, are not s u f f i c i e n t by themselves to account f o r the observed f r e q u e n c i e s of females i n these p o p u l a t i o n s . F i t n e s s components at s e v e r a l l i f e c y c l e stages were i n v e s t i g a t e d . Females were found to have an advantage over hermaphrodites only i n seed p r o d u c t i o n or only i n e a r l y v e g e t a t i v e growth of t h e i r progenies i n two p o p u l a t i o n s . The observed f i t n e s s d i f f e r e n c e s between the two sexual forms i n t h i s study a l s o appeared to be i n s u f f i c i e n t f o r m a i n t a i n i n g females i n p o p u l a t i o n s . F i t n e s s d i f f e r e n c e s between the two sexual forms o c c u r r i n g i n n a t u r a l h a b i t a t s need to be 196 i n v e s t i g a t e d f o r a thorough understanding of the maintenance of gynodioecy i n Hawaiian Bidens. 197 V I I I . GENERAL CONCLUSIONS 8.1 Mixed Mating Systems And F l o r a l - Mechanisms A l l s p e c i e s of Hawaiian Bidens s t u d i e d e x h i b i t e d i n t e r m e d i a t e o u t c r o s s i n g r a t e s , ranging from 0.588-0.728. The mixed mating system i n Hawaiian Bidens i s l a r g e l y g e n e t i c a l l y determined. F l o r a l mechanisms, such as male s t e r i l i t y and protandry, promote o u t c r o s s i n g , but s e l f - c o m p a t i b i l i t y and geitonogamy r e s u l t i n s u b s t a n t i a l s e l f - f e r t i l i z a t i o n . Environmental f a c t o r s , such as p l a n t d e n s i t y , p o p u l a t i o n s t r u c t u r e and p o l l i n a t o r behavior may modify the mating system by r e g u l a t i n g the amount of geitonogamy and consangineous mating i n Hawaiian Bidens. B i p a r e n t a l i n b r e e d i n g , which t h e o r e t i c a l l y i n t r o d u c e s the p o s s i b i l i t y of an optimal mating system that i n c o r p o r a t e s both modes of r e p r o d u c t i o n ( b i p a r e n t a l and u n i p a r e n t a l r e p r o d u c t i o n ) , may serve to maintain the mixed mating systems i n Hawaiian Bidens. The l i m i t e d v a r i a t i o n i n o u t c r o s s i n g r a t e s among s p e c i e s of Hawaiian Bidens suggests that mixed mating systems i n t h i s group of s p e c i e s are p o s s i b l y e v o l u t i o n a r i l y s t a b l e . A wide range of i n t e r p o p u l a t i o n v a r i a t i o n i n o u t c r o s s i n g r a t e i s found i n IK s a n d v i c e n s i s , i n which 5 p o p u l a t i o n s were s t u d i e d . In viewing e m p i r i c a l s t u d i e s on p l a n t mating systems, i n t e r p o p u l a t i o n v a r i a t i o n i n o u t c r o s s i n g r a t e i s t y p i c a l of o u t c r o s s i n g s p e c i e s or s p e c i e s with mixed mating systems i n which more than three p o p u l a t i o n s have been s t u d i e d . V a r i a t i o n in o u t c r o s s i n g r a t e i s u s u a l l y caused by d i f f e r e n c e s among 198 p o p u l a t i o n s i n e i t h e r g e n e t i c or environmental determinants of the mating system. Environmental f a c t o r s are most l i k e l y r e s p o n s i b l e f o r i n t e r p o p u l a t i o n v a r i a t i o n observed w i t h i n s p e c i e s i n Hawaiian Bidens, s i n c e f l o r a l mechanisms are s i m i l a r w i t h i n each s p e c i e s . C o n s i d e r a b l e v a r i a t i o n i n f l o r a l f e a t u r e s occurs among sp e c i e s of Hawaiian Bidens, but d i f f e r e n c e s i n f l o r a l f e a t u r e s are, i n g e n e r a l , not c o r r e l a t e d with d i f f e r e n c e s i n o u t c r o s s i n g r a t e . I n f l o r e s c e n c e type, or the number of flower heads borne on p l a n t s , tends to be c o r r e l a t e d with o u t c r o s s i n g r a t e due to i t s e f f e c t on geitonogamy. The lack of c o r r e l a t i o n between f l o r a l f e a t u r e s and o u t c r o s s i n g r a t e s i n Hawaiian Bidens i s not s u r p r i s i n g . Flower s i z e has been shown to be a poor p r e d i c t o r of the mating system i n other e m p i r i c a l s t u d i e s (Schoen, 1982, and r e f e r e n c e s t h e r e i n ) . Protandry i s r e l i a b l e i n p r e d i c t i n g the mating system, but strong protandry i n a l l s p e c i e s of Hawaiian Bidens has s i m i l a r e f f i c i e n c y i n p r e v e n t i n g or reducing autogamy i n s p i t e of some degree of v a r i a t i o n i n timing among s p e c i e s . Seed set i n the absence of p o l l i n a t o r s i s g e n e r a l l y low i n Hawaiian Bidens as a consequence of g e n e r a l l y strong protandry. 8.2 Genetic V a r i a t i o n , S t r u c t u r e , And The E f f e c t Of Mating  Systems The l e v e l of g e n e t i c v a r i a t i o n at allozyme l o c i w i t h i n and among p o p u l a t i o n s of Hawaiian Bidens i s low compared to other s p e c i e s with mixed mating systems. The t o t a l gene d i v e r s i t y at polymorphic l o c i i s p r i m a r i l y d i s t r i b u t e d w i t h i n p o p u l a t i o n s , 199 but a s u b s t a n t i a l amount i s d i s t r i b u t e d among p o p u l a t i o n s , i n d i c a t i n g that a r e l a t i v e l y high degree of genetic d i f f e r e n t i a t i o n has o c c u r r e d among p o p u l a t i o n s of Bidens. The l e v e l of g e n e t i c v a r i a b i l i t y w i t h i n p o p u l a t i o n s i s not c o r r e l a t e d with o u t c r o s s i n g r a t e , which suggests that h i s t o r i c a l f a c t o r s and g e n e t i c d r i f t may p l a y more important r o l e s than the mating systems in g e n e r a t i n g the c u r r e n t l e v e l of g e n e t i c v a r i a t i o n w i t h i n p o p u l a t i o n s of Hawaiian Bidens. C o r r e l a t i o n between g e n e t i c 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 has r a r e l y been found w i t h i n s p e c i e s with mixed mating systems unless the p o p u l a t i o n s have d i f f e r e n t f l o r a l mechanisms (Ganders et a l . , 1985). S i g n i f i c a n t heterozygote d e f i c i e n c i e s i n comparison with panmictic e x p e c t a t i o n were found i n most polymorphic p o p u l a t i o n s of Hawaiian Bidens. D i f f e r e n c e s i n observed Wright's f i x a t i o n index are c o r r e l a t e d with d i f f e r e n c e s i n o u t c r o s s i n g r a t e among these p o p u l a t i o n s , i n d i c a t i n g that the d e v i a t i o n s of heterozygote f r e q u e n c i e s from Hardy-Weinberg p r e d i c t i o n s are p r i m a r i l y caused by i n b r e e d i n g . The d i f f e r e n c e s between the observed f i x a t i o n i n d i c e s and those expected on the b a s i s of known l e v e l s of o u t c r o s s i n g i n p o p u l a t i o n s of Hawaiian Bidens are, on average, c l o s e to zero, but there seems to be a trend that p o p u l a t i o n s with lower o u t c r o s s i n g r a t e s have negative A F v a l u e s , and p o p u l a t i o n s with higher o u t c r o s s i n g r a t e s p o s i t i v e v a l u e s , the so c a l l e d " h e t e r o z y g o s i t y paradox" of Brown (1979). ^ F v a l u e s i n the p o p u l a t i o n s with low o u t c r o s s i n g r a t e s d e v i a t e more from 0, suggesting the e f f e c t of other e v o l u t i o n a r y f o r c e s 200 i n molding the g e n e t i c s t r u c t u r e of p o p u l a t i o n s of Hawaiian Bidens. High g e n e t i c i d e n t i t i e s among p o p u l a t i o n s of Hawiian Bidens are a p p a r e n t l y due to t h e i r common a n c e s t r y and the low l e v e l of g e n e t i c v a r i a b i l i t y , and cannot be i n t e r p r e t e d as evidence f o r the absence of g e n e t i c d i f f e r e n t i a t i o n among p o p u l a t i o n s without c o n s i d e r i n g the hig h values of G S T and Fsr at the three commonly polymorphic l o c i . The h i e r a r c h i c a l r e l a t i o n s h i p among p o p u l a t i o n s of Hawaiian Bidens based on allozyme v a r i a t i o n does not resemble the taxonomic p a t t e r n based on morphological v a r i a t i o n . T h i s absence of c o r r e l a t i o n between allozyme d i v e r s i t y and morphological and bi o c h e m i c a l d i v e r s i t y has a l s o been found i n other p l a n t s p e c i e s . 8.3 Gynodioecy Gynodioecy as a mechanism to promote o u t c r o s s i n g has been confirmed i n t h i s study, but i t s e f f i c i e n c y depends on the c u r r e n t l e v e l of o u t c r o s s i n g i n hermaphrodites, the frequency of females, and the degree of p o p u l a t i o n s u b s t r u c t u r e . S t u d i e s on microsporogenesis i n nine gynodioecious taxa of Hawaiian Bidens r e v e a l e d homologous developmental e x p r e s s i o n of male s t e r i l i t y i n Hawaiian Bidens. Females are completely m a l e - s t e r i l e due to a p r e m e i o t i c a b o r t i o n of microsporogenesis. The g e n e t i c f a c t o r s which cause male s t e r i l i t y a c t with remarkable p r e c i s i o n and c o n s i s t e n c y i n a l l taxa i n v e s t i g a t e d , suggesting homologous g e n e t i c c o n t r o l of male s t e r i l i t y . Genetic s t u d i e s of gynodioecy c o n f i r m that male s t e r i l i t y i s c o n t r o l l e d by the same r e c e s s i v e nuclear genes i n a l l gynodioecious s p e c i e s of Hawaiian 201 Bidens. Apparently two gene l o c i , and probably c y t o p l a s m i c f a c t o r s a l s o , are i n v o l v e d . The a l l e l i c r e l a t i o n s h i p among male s t e r i l i t y genes p r o v i d e s f u r t h e r evidence that d i f f e r e n t gynodioecious s p e c i e s of Hawaiian Bidens have a common a n c e s t r y . Simple cytoplasmic or simple genie c o n t r o l of male s t e r i l i t y has been found t o be r a r e i n n a t u r a l gynodioecious s p e c i e s (Ross, 1978; Charlesworth, 1981). A strong p o s i t i v e c o r r e l a t i o n was found between female f r e q u e n c i e s and s e l f i n g r a t e s of hermaphrodites i n e i g h t gynodioecious p o p u l a t i o n s of Hawaiian Bidens, and o f f s p r i n g of females are more heterozygous than those of hermaphrodites. Maintenance of females i n gynodioecious p o p u l a t i o n s of Hawaiian Bidens i s at l e a s t p a r t l y due to t h e i r o b l i g a t e o u t c r o s s i n g . But the observed s e l f i n g r a t e s of hermaphrodites are i n s u f f i c i e n t by themselves to account f o r the frequency of females i n these p o p u l a t i o n s . Estimates of f i t n e s s components i n s e v e r a l experimental gynodioecious p o p u l a t i o n s e x h i b i t e d no s i g n i f i c a n t d i f f e r e n c e s i n v e g e t a t i v e v i g o r or r e p r o d u c t i v e c a p a c i t i e s between hermaphrodites and females and between t h e i r p r o g e n i e s . Females do not set more seeds than hermaphrodites i n most n a t u r a l gynodioecious p o p u l a t i o n s of Hawaiian Bidens, but seed f e c u n d i t y of hermaphrodites i s s i g n i f i c a n t l y lower i n some p o p u l a t i o n s of B_;_ menziesi i subsp. f i l i f o r m i s . The high female f r e q u e n c i e s i n p o p u l a t i o n s of t h i s s p e c i e s are due to the j o i n t advantages of females i n seed f e c u n d i t y and o b l i g a t e o u t c r o s s i n g of females. Developmental and g e n e t i c s t u d i e s p r o v i d e evidence that 202 g y n o d i o e c y o r i g i n a t e d o n l y o n c e i n H a w a i i a n B i d e n s , a n d t h e m a l e s t e r i l i t y m u t a t i o n e v e n t o c c u r r e d a f t e r B i d e n s i m m i g r a t e d t o t h e H a w a i i a n I s l a n d s . T h e c u r r e n t 13 g y n o d i o e c i o u s t a x a , r e p r e s e n t i n g n i n e s p e c i e s , may b e a r e s u l t o f a d a p t i v e r a d i a t i o n o f t h e a n c e s t r a l g y n o d i o e c i o u s p o p u l a t i o n , o r a r e s u l t o f n a t u r a l h y b r i d i z a t i o n b e t w e e n g y n o d i o e c i o u s s p e c i e s a n d h e r m a p h r o d i t i c s p e c i e s . T h e e v o l u t i o n a r y f a t e o f g y n o d i o e c y i n g y n o d i o e c i o u s p o p u l a t i o n s o f H a w a i i a n B i d e n s c o u l d b e d i f f e r e n t , a s s u g g e s t e d b y t h e d i f f e r e n c e s i n f e m a l e f r e q u e n c i e s , i n s e l f i n g r a t e s o f h e r m a p h r o d i t e s a n d i n f e m a l e f e r t i l i t y o f h e r m a p h r o d i t e s . F e m a l e s c o u l d b e g r a d u a l l y e l i m i n a t e d b e c a u s e t h e y l a c k s u f f i c i e n t a d v a n t a g e o v e r h e r m a p h r o d i t e s t o c o m p e n s a t e f o r t h e f a i l u r e o f m a l e f u n c t i o n , a s s u g g e s t e d b y l o w f r e q u e n c i e s o f f e m a l e s , h i g h o u t c r o s s i n g r a t e s a n d h i g h f e m a l e f e r t i l i t y o f h e r m a p h r o d i t e s , a n d t h u s s e c o n d a r y h e r m a p h r o d i t i s m c o u l d e v o l v e . On t h e o t h e r h a n d , g y n o d i o e c y a p p e a r s t o b e e v o l v i n g t o w a r d s d i o e c y , a s s u g g e s t e d b y h i g h f r e q u e n c i e s o f f e m a l e s , a n d d e c r e a s e d f e m a l e f u n c t i o n o f h e r m a p h r o d i t e s i n p o p u l a t i o n s o f B ^ m e n z i e s i i s u b s p . f i l i f o r m i s . H o w e v e r , g y n o d i o e c y c a n b e e v o l u t i o n a r i l y s t a b l e i f f e m a l e s h a v e s u f f i c i e n t a d v a n t a g e s w h i c h a r e n o t d e r i v e d f r o m d e c r e a s e d f e m a l e f e r t i l i t y o f h e r m a p h r o d i t e s , a s i s p r o b a b l y t h e c a s e i n m o s t o t h e r g y n o d i o e c i o u s p o p u l a t i o n s o f H a w a i i a n B i d e n s . 203 8.4 Summary In summary, I conclude t h a t : a. A l l s p e c i e s of Hawaiian Bidens s t u d i e d have intermediate r a t e s of s e l f - f e r t i l i z a t i o n . The mixed mating systems are p r i m a r i l y c o n t r o l l e d by a combination of f l o r a l mechanisms. Protandry and gynodioecy promote o u t c r o s s i n g , but geitonogamy r e s u l t s i n s e l f i n g . b. P o p u l a t i o n s of Hawaiian Bidens have g e n e r a l l y low l e v e l s of ge n e t i c v a r i a t i o n , and s u b s t a n t i a l g e n e t i c d i f f e r e n t i a t i o n has occurred among p o p u l a t i o n s , suggesting the e f f e c t of founder events. Most p o p u l a t i o n s are c l o s e to inbree d i n g e q u i l i b r i u m , but a " h e t e r o z y g o s i t y paradox" suggests the e f f e c t of other e v o l u t i o n a r y f a c t o r s . c. Male s t e r i l i t y i s due to p r e m e i o t i c degeneration of microspore mother c e l l s . The homogeneous developmental ex p r e s s i o n of male s t e r i l i t y suggests a common ge n e t i c mechanism. d. Male s t e r i l i t y i s g e n e t i c a l l y c o n t r o l l e d . Two r e c e s s i v e n u c l e a r genes and probably two types of cytoplasm are i n v o l v e d . The mode of i n h e r i t a n c e of male s t e r i l i t y appears to be g e n i c - c y t o p l a s m i c . 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Enzyme v a r i a t i o n s i n n a t u r a l p o p u l a t i o n s of d o u g l a s - f i r , Pseudotsuga m e n z i e s i i (Mirb.) Franco, from B r i t i s h Columbia. 1. Genetic v a r i a t i o n p a t t e r n s i n c o a s t a l p o p u l a t i o n s . S i l v a e Genetica 29:83-92. YOUNG, D. A. 1972. The r e p r o d u c t i v e b i o l o g y of Rhus i n t e g r i f o l i a and Rhus ovata (Anacardiaceae). E v o l u t i o n 26: 406-414. ZOHARY, D. AND D. IMBER. 1963. Genetic dimorphism i n f r u i t types i n A e q i l o p s s p e l t o i d e s . H e r e d i t y 18:223-231. 223 Appendix A. N a t i v e Hawaiian taxa of B idens. Taxa known to be g y n o d i o e c i o u s a r e preceded by an a s t e r i s k . Taxon S p e c i e s acronym D i s t r i b u t i o n * B^ amplectens S h e r f f B. asymmetr tea (H. Lev.) S h e r f f B. campy1otheca Schz. B i p . ssp . campy 1otheca  B. cam p y l o t h e c a s s p . pentamera ( S h e r f f ) Ganders & Nagata B. ca m p y l o t h e c a s s p . w a i h o i e n s i s S t . John * B_^  c e r v i c a t a S h e r f f B. c o n j u n c t a S h e r f f B. cosmoides (A. Gray) S h e r f f * B^ f o r b e s i 1 S h e r f f s s p . forbes11 * B^ f o r b e s i i s s p . k a h i 1 i e n s i s Ganders & Nagata * §_;_ hawa i ens i s A . Gray B. h i 1lebrandiana (Drake) Deg. ex S h e r f f s s p . h i l l e b r a n d i a n a  B. h i 1lebrandiana s s p . p o l y c e p h a l a Nagata & Ganders AMPL ASYM CERV FORB F FORB K HAWA HILL Oahu Oahu Hawaii, L a n a l , Oahu Maul Maul K a u a i , Oahu, N i i h a u Maul Kaua 1 Kaua 1 Kaua 1 Hawal1 Hawal1 Maul, Moloka 1 224 Appendix A c o n t . B. macrocapa (A. Gray) S h e r f f B. m a u i e n s i s (A. Gray) * B_^  menz 1 es i 1 (A. Gray) S h e r f f s s p . m e n z i e s i i * B_^  menz 1 es 1 1 s s p . f i l l f ormi s ( S h e r f f ) Ganders & Nagata * B^ m i c r a n t h a Gaud. s s p . m i c r a n t h a * B^ m i c r a n t h a s s p . c t e n o p h y l l a ( S h e r f f ) Nagata S Ganders B. m i c r a n t h a s s p . k a l e a l aha Nagata & Ganders B. mo 1 o k a i e n s i s ( H i l l e b r . ) S h e r f f * B^ p o p u l i f o l i a S h e r f f * Bj_ sandv i cens i s L e s s . s s p . s a n d v i c e n s i s * sandv i cens 1 s s s p . conf usa Nagata & Ganders * t o r t a S h e r f f B. v a l I d a S h e r f f B. w i ebke1 S h e r f f MAUI MENZ Oahu L a n a i , Maul Maul, Moloka 1 MICR Hawai1 Maul Hawat1 MOLO POPU L a n a i , Maul Moloka 1, Oahu Oahu SAND S SAND C TORT kaua i , Oahu Kaua i Oahu Kaua 1 Moloka 1 Appendix B. Allozyme lo c i and a l l e l e frequencies in 21 populations of Hawaiian Bidens (see Table 2-1 for population acronym). A l l e l e AMPL ASYM CERV FORB F1 FORB K HAWA HILL E HILL W MAUI 1 MAUI 2 Pgi-1a 0 .01 0 .00 0 .08 0 .00 0 .00 0 .00 0 .00 0 .00 0 .01 0 .00 Pg1-1b 0 .80 0 .88 0 .72 0 .98 1 .00 0 .97 1 .00 1 .oo 0 .77 0 .99 Pgi-1c 0 . 19 0 . 12 0 .20 0 .02 0 .00 0 .03 0 .00 0 .oo 0 .22 0 .01 Pg1-1d 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 Pgi-2a 0 .00 0 .00 0 .06 0 .00 0 .00 0 .03 0 .00 0 .oo 0 .08 0 ,43 Pgi-2b 0 .02 0 .00 0 . 34 0 .05 0 .00 0 .00 0 .00 0 .00 0 . 15 0 oo Pgi-2c 0 .98 1 .00 0 .60 0 .95 1 .00 0 .97 1 .00 1 .00 0 .77 0 .57 Pgi-3a 1 .00 1 .00 1 .00 1 .00 1 .00 1. .00 1 .00 1 .00 1 .00 1, ,00 Pgi-4a 1 .00 1 .00 1 .00 1 .00 1 .00 1, .00 1 .00 1 .00 1 .00 1 .00 Pgi-5a 1 .00 1 .00 1. .00 1 ,00 1 .00 1. ,00 1 .00 1 .00 1 .00 1, ,00 Skdh-3a 0 .00 0 .00 0. .01 0 ,00 0, ,00 0. ,00 0, .00 0, .00 0, .00 0. ,00 Skdh-3b 0. ,00 0, .00 0. 22 0. 19 0. , 19 0. 72 1 , .00 1. 00 0. ,00 o. 07 Skdh-3c 0. .99 1. .00 0. ,77 0. ,81 0, ,81 0. 27 0. .00 0. 00 1, ,00 0. .81 Skdh-3d 0. .00 0, .00 0. 00 0. 00 0. .00 0. 01 0, ,00 0. 00 0. 00 0. 12 Mdh-3a 1 . 00 1 .00 1 . 00 1 . 00 1. 00 1. ,00 1. 00 1. ,00 1. 00 Pgm-1a 1 . 00 1, .00 0. 98 0. 97 1 . ,00 1. 00 1, 00 1. ,00 1, ,00 1. .00 Pgm-1b 0. ,00 0. ,00 0. 02 0. 03 0. 00 0. 00 0. ,00 0. 00 0. .00 0. 00 Pgm-2a 0. 00 0. 00 0. 03 0. 01 0. 00 0. 00 0. 00 0. 00 0. 00 Pgm-2b 1. 00 1. ,00 0. 97 0. 99 1 . oo 1. 00 1. oo 1. oo 1. oo Pgm-3a 1. 00 1. 00 1. 00 1. 00 1 . 00 1. 00 1. 00 1. 00 1. 00 1. 00 A l l e l e MENZ 1 MENZ 2 MENZ 3 MENZ 4 SAND 1 SAND 2 SAND 3 SAND 3 SAND X TORT 1 TORT 2 P g i - 1 a 0 . 0 0 0 . 0 0 0 .01 0 . 0 0 0 .01 0 .oo 0 . 0 0 0 . 0 1 O . 0 6 0 . 0 0 0 . 0 1 P g i - l b 0 . 6 5 0 . 9 8 0 .91 0 .91 0 . 9 2 1 . 0 0 0 . 9 2 0 . 9 7 0 . 7 8 0 . 8 0 0 . 9 4 P g i - 1c 0 . 3 5 0 . 0 2 0 . 0 8 0 . 0 9 0 . 0 7 0 . 0 0 0 . 0 5 0 . 0 2 0 . 16 0 . 2 0 0 . 0 5 P g l - 1 d 0 . 0 0 0 . 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 3 0 . 0 0 0 . 0 0 0 . 0 0 0 .OO P g i - 2 a 0 . 0 5 0 . 0 0 0 . 0 3 0 . 0 0 0 . 0 5 0 . 0 0 0 . 0 4 0 . 0 3 0 . 10 0 . 18 0 . 17 P g i - 2 b 0 , . 13 0 . 8 4 0 , . 0 4 0 , . 4 7 0 . 0 2 0 . 0 0 0 , . 0 5 0 . 34 0 . . 10 0 . 0 0 0 . 0 2 P g i - 2 c 0 . 82 0 . 16 o. . 9 3 0 , . 5 3 0 . 9 3 1 .oo 0 . .91 o . 6 3 o. . 8 0 0 . 8 2 0 .81 P g i - 3 a 1 . 0 0 1 .OO 1. , 0 0 1, . 0 0 1 . 0 0 1 . 0 0 1. . 0 0 1. . 0 0 1. . 0 0 1. . 0 0 1 OO P g i - 4 a 1 0 0 1 . 0 0 1. , 0 0 1, . 0 0 1 . 0 0 1. . 0 0 1. . 0 0 1 . 0 0 1. . 0 0 1 . 0 0 1. , 0 0 P g i - 5 a 1 , . 0 0 1 . 0 0 1, , 0 0 1 . 0 0 1 . 0 0 1. . 0 0 1. . 0 0 1 . 0 0 1 . 0 0 1. . 0 0 1 . 0 0 S k d h - 3 a 0 . . 0 0 0 . 0 0 0 . . 0 0 0 . . 0 0 0 . . 0 0 0 . . 0 0 0 . 0 3 0 , . 01 0 . . 0 0 0 . , 0 0 0 . , 0 0 S k d h - 3 b 0 . .01 0 . . 0 0 0 . 04 0 . . 0 0 0 . 0 0 0 . . 3 0 0 . 0 3 0 . . 0 9 0 . 0 4 0 . , 0 0 0 . 17 S k d h - 3 c 0 . 9 9 1. . 0 0 0 . 9 6 1. 0 0 1. . 0 0 0 . 6 3 0 . 9 3 0 . 9 0 0 . 9 6 1. 0 0 0 . 8 0 S k d h - 3 d 0 0 0 0 . 0 0 0 . 0 0 0 , , 0 0 0 . . 0 0 0 . . 07 0 . 01 0 . . 0 0 o. 0 0 0 . . 0 0 0 . 0 3 M d h - 3 a 1, 0 0 1. . 0 0 1. 0 0 1. , 0 0 1. 0 0 1. 0 0 1. . 0 0 1. 0 0 P g m - 1 a 1. 0 0 1. . 0 0 1. 0 0 1. 0 0 1. . 0 0 1. 0 0 1. OO 1. . 0 0 1. 0 0 1. 0 0 1. OO P g m - 1b 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 P g m - 2 a 0 . 0 0 0 , , 0 0 0 . OO o. 0 0 0 . 0 0 0 . 0 0 o. OO o. OO 0 . 0 0 0 . 0 0 P g m - 2 b 1. OO 1. , 0 0 1. 0 0 1. 0 0 1. 0 0 1. 0 0 1. 0 0 1. OO 1. oo 1. OO P g m - 3 a 1. 0 0 1. , 0 0 1. OO 1. 0 0 1. oo 1. oo 1. OO 1. 0 0 1. 0 0 1. oo 1. 0 0 to ON A l l e l e AMPL ASYM CERV FORB Pgm-4a 1 .00 1 .00 1 .00 0 .99 Pgm-7a 0.00 0 .00 0 .02 0 .01 Pgm-7b 1 .00 1 .00 0 .95 0 .99 Pgm-7c 0.00 0 .00 0 .03 0 .00 pgm-8a 0.00 o .00 0 . 14 0 .00 Pgm-8b 1 .00 1 .00 0 .86 1 .00 Lap-1a 1 .00 1 .00 1 .00 Lap-2a 1 .00 1 .00 1 .00 Lap-3a 1 .00 1 .00 1 .00 Lap-4a 1 .00 0 .89 0 .99 Lap-4b 0.00 0. .00 0. . 11 0 .01 Pgd-1a* - 1. oo 1. .00 1. oo Pgd-1b - 0, .00 0. .00 0. 00 Pgd-2a - 1. .00 1. .00 1. 00 Pgd-2b - 0. .00 0. ,00 0. 00 Pgd-3a - 1. .00 1. ,00 1. 00 Pgd-4a - 1. .00 1. .00 1. 00 Pgd-5a - 1. 00 1. 00 1. 00 Ha-1a 1 .00 1. 00 1. 00 1. 00 Glu-1a 1 .00 1. 00 1. 00 Me-1a 1 .00 1. 00 1. 00 1. 00 FORB K HAWA HILL E HILL W MAUI 1 MAUI 1 . 00 1 . 00 1 . 00 O.00 O.00 O.OO 1 . 00 1 . 00 1 . 00 0.00 0.00 0.00 O.00 - 0.00 1.00 - 1.00 1 .00 1 .00 1.00 1.00 1.00 1.00 1 .00 1 .00 0.00 0.00 1 . OO 1 . 00 1 . oo 0. 00 0. 00 0. 00 1.00 0.98 1.00 0.00 0.02 0.00 1 .00 1 .00 1 .00 1.00 1.00 1.00 1.00 1.00 1.00 1 .00 1 .00 1 .00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0. 00 O . 00 0 . 00 1 . 00 1 .00 1 . 00 O.OO 0.00 o.oo 0. OO 0. 00 o. oo 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .OO 1.oo 0.OO O.00 1.00 1.00 0.00 0.00 1.00 1.00 1 .00 1 .00 1.00 1.00 -1.OO 1.00 1.00 1.00 1.00 A l l e l e MENZ 1 MENZ 2 MENZ 3 MENZ 4 SAND 1 SAND 2 SAND 3 SAND C SAND X TORT 1 TORT 2 Pgm-4a Pgm-7a Pgm-7b Pgm-7c pgm-8a Pgm-8b Lap-1a Lap-2a Lap-3a Lap-4a Lap-4b Pgd-1a* Pgd-1b Pgd-2a Pgd-2b Pgd-3a Pgd-4a Pgd-5a Ha-1a Glu-1a Me- 1a 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 1 .00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 O. 79 0.00 1 .00 0.00 0. 11 0.89 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 1 .00 0.00 1 .00 1 .00 1 .00 1 .oo 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 1 .00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 0.97 0.03 1 .00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 1 .00 0.00 1 .00 1 .oo 0.00 1 .00 0.00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 0.00 1 .00 0.00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 0.00 1 .00 1 .00 0.00 1 .00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 1 .00 0.00 1 .00 1 .00 1 .00 1 .oo 1 .00 1 .00 1 .00 0.00 1 .00 0.00 0.00 1 .00 1 .00 1 .00 1 .00 1 .00 0.00 1 .00 0.00 1 .00 0.00 0.00 1 .00 1 .00 1 .00 1 .00 1 .oo 1 .oo *: Pgd = 6-pgdh. NJ ho co 

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