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Genetic differentiation of Hawaiian Bidens Helenurm, Kaius 1983

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GENETIC DIFFERENTIATION OF HAWAIIAN BIDENS by KAIUS HELENURM B.Sc, University Of Toronto, 1979 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Botany Department We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1983 © Kaius Helenurm, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Is'trr ^AJY  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 3E-6 (3/81) i i Abstract Adaptive radiat ion is the evolut ionary divergence of a group of organisms from a common ancestor to exp lo i t d i f f e ren t eco log ica l n iches. Bidens has undergone extensive adaptive rad iat ion on the Hawaiian Is lands. The 19 Hawaiian species exhibi t much more morphological and eco log ica l d i f f e r en t i a t i on than the cont inenta l members of the genus. However, the Hawaiian taxa are chromosomally homogeneous and reta in the capacity to interbreed in a l l poss ib le combinations. Thus the morphological and eco log ica l d i f f e r e n t i a t i o n in Hawaiian Bidens has been atta ined without the existence of reproductive i so l a t i ng mechanisms. Although some hybrid populat ions are known, hybr id iza t ion usual ly does not occur in nature because the species are found in d i f f e ren t hab i ta ts . Prel iminary genetic studies have suggested that some of the morphological d i f fe rences between taxa may be cont ro l l ed by very few genes. Genetic d i f f e r e n t i a t i o n may therefore not extend to other parts of the genome, such as to s t ruc tura l genes for enzymes. Most plant groups that have been studied e l ec t rophore t i ca l l y show a co r re l a t ion between morphological d i f f e r en t i a t i on and genetic d i f f e r e n t i a t i o n , but opposing pred ic t ions can be made about the extent of divergence at isozyme l o c i in Hawaiian Bidens. The morphological and eco log ica l data suggest that the taxa are highly d i f f e ren t i a t ed gene t i ca l l y , but the chromosomal s i m i l a r i t y of species, the genetic studies of morphological characters and the absence of genet i ca l l y cont ro l l ed i so l a t i ng mechanisms suggest that genetic d i f ferences among the taxa may be l imi ted to only a small port ion of the genome and may not include isozyme l o c i . Populations of the Hawaiian taxa of Bidens were compared at 23 l o c i con t ro l l i ng 9 enzyme systems. In genera l , populat ions are more polymorphic than populations of most other plant species that have been studied e l e c t rophore t i c a l l y . L i t t l e genetic d i f f e r e n t i a t i o n has occurred among taxa in sp i te of the high leve ls of genetic v a r i a b i l i t y , however. Genetic i den t i t i e s ca lcu la ted for pa i rs of populations show that populations of the same taxon are genet i ca l l y more s imi la r than populations belonging to d i f f e ren t taxa, but a l l values are h igh. The genetic d i f f e r e n t i a t i o n that has occurred among the taxa of Hawaiian Bidens is comparable to the genetic d i f ferences among populations of cont inenta l spec ies . Moreover, there i s no co r re l a t ion between the isozyme data and morphological data. No groups of taxa are evident in the genetic data although morphological groups ex i s t . Genetic d i f f e r en t i a t i on at isozyme l o c i has not occurred at the same rate as the acqu i s i t i on of adaptive morphological and eco log ica l characters in Hawaiian Bidens. Adaptive rad iat ion therefore does not require genetic change throughout the genome and may be l im i ted to the genes con t ro l l i ng morphological and eco log ica l characters . iv Table of Contents Abstract i i L i s t of Tables v L i s t of Figures v i Acknowledgement v i i I. INTRODUCTION 1 1.1 Adaptive Radiation And The Hawaiian Islands 1 1.2. Hawaiian Bidens 4 1.3 Genetic D i f f e r e n t i a t i o n Of Plant And Animal Populat ions 7 1.4 Electrophoresis And Isozymes 10 11 . MATERIALS AND METHODS 13 2.1 Population Samples 13 2.2 Growth Of Plant Material 20 2.3 Electrophoresis 20 2.4 Enzymes Studied 24 III . INHERITANCE OF ISOZYMES 28 3.1 Defi n i t i o n s And Nomenclature 28 3.2 Types Of Evidence 30 3.3 Monomorphic Enzymes 33 3.4 Polymorphic Enzyme Systems 33 3.4.1 Phosphoglucose Isomerase 40 3.4.2 Phosphoglucomutase 42 3.4.3 Malate Dehydrogenase ...44 3.4.4 Leucine Aminopeptidase 47 3.4.5 Diaphorase 47 3.5 American Taxa 47 IV. VARIABILITY WITHIN POPULATIONS 51 4.1 Hawaiian Populations 51 4.2 Hawaiian Taxa 53 4.3 American Species 55 V. DIFFERENTIATION OF POPULATIONS 56 5.1 Genetic Identities 56 5.2 Gene Diversity 64 5.3 P r i n c i p a l Component Analysis 67 5.4 Cluster Analysis 75 VI. DISCUSSION 77 BIBLIOGRAPHY 89 APPENDIX A - COMPLETE SCIENTIFIC NAMES OF BIDENS TAXA STUDIED 99 APPENDIX B - ALLELE FREQUENCIES IN BIDENS POPULATIONS STUDIED 100 APPENDIX C - ALLELE FREQUENCIES IN BIDENS TAXA STUDIED ..104 V L i s t of Tables 1. Co l l ec t ions of Hawaiian taxa studied 14 2. Co l l ec t ions of American species 19 3. Running condit ions used for enzyme systems 21 4. Composition of buffer so lut ions 22 5. Staining so lut ions employed for enzyme systems 25 6. Enzyme substructure, genetic contro l and number of l o c i scored 29 7. Genetic bas is of e lec t rophoret ic var iants 35 8. Genetic measures of v a r i a b i l i t y in 22 Hawaiian Bidens populations 52 9. Genetic measures of v a r i a b i l i t y in 15 Hawaiian Bidens taxa 54 10. Genetic ident i t y and distance values for populations of Hawaiian and American Bidens taxa 58 11. Genetic i d en t i t i e s for taxonomic comparisons of 22 Hawaiian Bidens populations 61 12. Genetic i d en t i t i e s for intertaxon geographic comparisons of 22 Hawaiian Bidens populations 61 13. Genetic iden t i t y and distance values for Hawaiian and American Bidens taxa 63 14. Gene d i ve r s i t y at 14 polymorphic l o c i in 22 populations of Hawaiian Bidens 66 15. Gene d i v e r s i t y for a l l l o c i in Hawaiian Bidens 66 16. D i s t r ibu t ion of Bidens taxa on the Hawaiian is lands ..81 v i L i s t of Figures 1. Map of the major (Windward) Hawaiian Islands 3 2. Locations of populations surveyed on Kauai and Oahu ..16 3. Locations of populations surveyed on Maui and Molokai 17 4. Locations of populations surveyed on Hawaii and Lanai 18 5. A l l e l e s of the PGI l o c i 41 6. Genotypes of common band PGI band patterns 41 7. Photograph of PGI band patterns 41 8. A l l e l e s of the PGM l o c i 43 9. Genotypes of common PGM band patterns 43 10. Photograph of PGM band patterns 43 11. A l l e l e s of the MDH l o c i 45 12. Genotypes of common MDH band patterns 45 13. Photograph of MDH band patterns 45 14. A l l e l e s of the LAP l o c i 48 15. Genotypes of common LAP band patterns 48 16. Photograph of LAP band patterns 48 17. A l l e l e s of the DIA l o c i 49 18. Genotypes of common DIA band patterns 49 19. Ordination of 22 Hawaiian Bidens populations on the f i r s t and second PCA axes 68 20. Ordination of 22 Hawaiian Bidens populations on the f i r s t and t h i r d PCA axes 69 21. Ordination of 22 Hawaiian Bidens populations on the second and t h i r d PCA axes 70 22. Ordination of 15 Hawaiian Bidens on the f i r s t and second PCA axes 72 23. Ordination of 22 Hawaiian Bidens populations and 4 populations of American taxa on the f i r s t and second PCA axes 73 24. Ordination of 15 Hawaiian and 4 American Bidens taxa on the f i r s t and second PCA axes 74 25. Dendrogram of 22 Hawaiian Bidens populations 75 A c k n o w l e d g e m e n t I am v e r y g r a t e f u l t o F r e d R. G a n d e r s , my s u p e r v i s o r , f o r m o r a l , i n t e l l e c t u a l , f i n a n c i a l and c r i t i c a l s u p p o r t , a n d t o Ken C a r e y a n d K e r m i t R i t l a n d f o r s u p p l y i n g me w i t h c o m p u t e r p r o g r a m s and h e l p i n g i n i n n u m e r a b l e l i t t l e ways. I w o u l d l i k e t o t h a n k my c o m m i t t e e members, B. Bohm and A . J . F . G r i f f i t h s f o r c r i t i c a l comments, and B. Bohm, G. B r a d f i e l d , E. Camm, C. L a y t o n , J . Maze, a n d W. N i c h o l l s f o r h e l p w i t h v a r i o u s a s p e c t s o f t h i s s t u d y . The U n i v e r s i t y o f B r i t i s h C o l u m b i a s u p p o r t e d me p a r t i a l l y w i t h a g r a d u a t e f e l l o w s h i p . 1 I. INTRODUCTION 1.1 Adaptive Radiation And The Hawaiian Islands Adaptive rad ia t ion is the evolut ion from a common ancestor of many species adapted to a var ie ty of eco log ica l niches. The evolut ionary divergence of a group of organisms to exp lo i t d i f f e ren t environments is a s i gn i f i c an t evolut ionary event, and occurred within the angiosperms in the Cretaceous, and within mammals in the Paleogene. The endemic mammals of South America and the marsupials of Aus t ra l i a are a lso examples of th i s phenomenon. Evidence for adaptive rad ia t ion on a smaller scale can be seen on oceanic i s lands . Oceanic is lands usual ly have unbalanced or disharmonic biotas in which genera and larger taxonomic categor ies are under-represented re l a t i ve to cont inenta l biotas (Car lqu is t , 1974). The number of species in each genus, however, is often r e l a t i v e l y la rge , and a high proport ion of species may be endemic. This is i l l u s t r a t e d by the many species of Darwin's f inches in the Galapagos Islands (Lack, 1947) and of honeycreepers in the Hawaiian Islands (Car lqu is t , 1970) in conjunction with the small number of genera of land birds found on these two groups of i s lands . Most oceanic is lands are created through volcanic a c t i v i t y and have never been connected to cont inenta l areas by land br idges. It is not su rp r i s ing , therefore, that the genera found on them are 2 few in number and are usually groups with adaptations for long-distance dispersal and establishment (Carlquist, 1974). Once populations of early colonizers have been established, opportunities for speciation may be greater than on older mainland areas because of the a v a i l a b i l i t y of habitats not yet occupied by other species. Rapid cladogenetic evolution within a small number of genera would occur i n i t i a l l y . As colonization by d i f f e r e n t groups of organisms continues, new ecological opportunities decrease and patterns of speciation may resemble those found on continents. The Hawaiian Islands are geologically young and are p a r t i c u l a r l y r i c h in habitats. The whole archipelago i s 3500 kilometres long, but the major islands (Figure 1) are clustered at the southeastern end. A l l the islands have been formed by the movement of the P a c i f i c plate over a fixed hot spot in the earth's mantle from which magma rises p e r i o d i c a l l y to form shield volcanoes (Dalrymple et a l . , 1973). As the plate moves in a northwesterly d i r e c t i o n , new islands are continually being formed and older ones are eroding to sea l e v e l and below. Although the chain i t s e l f i s tens of mi l l i o n s of years old, the major islands range in age from 5.6 to 3.8 m i l l i o n years for Kauai to less than 1.0 m i l l i o n years for Hawaii (Stearns, 1966). Volcanic a c t i v i t y s t i l l occurs on Hawaii, the youngest and highest isl a n d . A variety of habitats i s available, -ranging in temperature from below freezing to more than 30°C, in humidity from nearly 0 to 100%, and in median annual r a i n f a l l from over 11.8m to less K a u a i F i g u r e 1. Map o f t he m a j o r (Windward) H a w a i i a n I s l a n d s . 4 than 0.25m (Carlquist, 1970; T a l i a f e r r o , 1959). The archipelago is r i c h in examples of adaptive radiation; in addition to the honeycreepers there are the 650-700 endemic species of Drosophila that comprise about one-third of the entire genus (Johnson et a l . , 1975), and many examples in flowering plants, including Lipochaeta (Gardner, 1976), Bidens, Cyrtandra, Euphorbia, Palea, Scaevola, seven genera of l o b e l i o i d s (Carlquist, 1970), and three genera of the silversword a l l i a n c e (Carr and Kyhos, 1981) . 1.2 Hawaiian Bidens Bidens is a worldwide genus with centres of d i v e r s i t y in A f r i c a and the New World. Each of these areas has about 100 species of the t o t a l of approximately 280 in the genus (Sherff, 1937). Eurasia and the Caribbean have only about 5 species each but Polynesia has 66; a large number for such a small portion of the world's land area. Although the treatment of Sherff is in need of revision, the number of species he named probably r e f l e c t s the amount of morphological and ecological d i v e r s i t y found in a given region. Forty-three of his Polynesian species are Hawaiian and the other 23 are found in southeastern Polynesia. They comprise 2 of the 14 sections in the genus (one consisting solely of B. cosmoides on Kauai) and their closest a f f i n i t i e s are with American species. Because the southeast Polynesian species share s i m i l a r i t i e s with Hawaiian species but 5 exhib i t much less v a r i a b i l i t y than is found in the Hawaiian Islands ( G i l l e t t , 1972b, 1973), i t is l i k e l y that they are derived from Hawaiian taxa and that the o r i g i na l co lon iza t ion of Polynesia from the Americas occurred in Hawaii. S i m i l a r i t i e s shared by the Polynesian species and the r a r i t y of d i spersa l events to is lands so remote from any continent (evidence of which is the i r disharmonic biotas) suggest that there was perhaps only one introduct ion of the genus to the is lands (Ganders and Nagata, in prep.) although Fosberg (1948) and G i l l e t t (1975) bel ieved there were two. The Hawaiian taxa are extremely var iable morphologica l ly . Although only a few characters , such as the h e l i c o i d achene, are unique to the is lands ( G i l l e t t , 1975), no other region of the world contains as much d i v e r s i t y . The species d i f f e r in leaf shape (from simple to compound to highly d i ssec ted ) , flower head s ize and number, achene s ize and shape (from f l a t and s t ra ight to highly co i led ) and presence and type of d i spersa l mechanism (awns of various lengths and shapes, pubescence, and presence or absence of wings). They vary in growth form from small trees over 2m t a l l with a woody trunk to t a l l shrubs to erect and prostrate herbaceous forms. Di f ferences between species in a l l these characters are maintained under standard growing cond i t ions , which demonstrates that they are under strong genetic con t ro l . Eco log ica l d i f f e r e n t i a t i o n has occurred as we l l , since d i f f e ren t species are found in environments as disparate as coasta l sand dunes, montane bogs, a r i d cinder cones, open grassy areas and ra in fo res ts . 6 On the other hand, a l l species examined are chromosomally sim i l a r , with a d i p l o i d number of 72 (Skottsberg, 1953; G i l l e t t and Lim, 1970). Since the base number for Bidens i s 12, a l l Hawaiian species are hexaploid. Moreover, a l l i n t e r s p e c i f i c crosses attempted between Hawaiian species result in f e r t i l e hybrids ( G i l l e t t , 1972a, 1972b, 1973; G i l l e t t and Lim, 1970). Attempted crosses involving B. cosmoides were reported to be unsuccessful by G i l l e t t (1975) but B. cosmoides has been intercrossed successfully with other species by Ganders (pers. comm.). Crosses between Hawaiian and Marquesan species also produce viable although s t e r i l e progeny, but the Polynesian taxa w i l l not hybridize with B. p i l o s a , an American species often compared with them ( G i l l e t t , 1972b, 1973). Natural hybridization has been documented in nature between at least three pairs of species that are parapatric in d i s t r i b u t i o n (Degener, 1946; Mensch and G i l l e t t , 1972; Sherff, 1937; Ganders, pers. comm.), but in general taxa do not interbreed because they occur in d i f f e r e n t habitats. Experimental crosses between taxa and subsequent s e l f -f e r t i l i z a t i o n of progeny have revealed that many of the morphological differences between species may be controlled by a r e l a t i v e l y small number of genes. Leaf shape, awn length and stem pigmentation a l l seem to be under polygenic control because of the continuum of variation observed in the F z generation, but parental forms appear in the F z generation and minimum estimates of the number of genes involved using the Sinnott et a l . (1950) formula gave a value of one for the l a t t e r two characters. 7 Intraplant var ia t ion prevented quant i ta t ive ana lys is of the genetic basis of leaf form (G i l l e t t and Lim, 1970; Mensch and G i l l e t t , 1972). The information presented above leads to opposing pred ic t ions about the genetic d i f f e r e n t i a t i o n of Bidens on the Hawaiian Is lands. The extent of morphological and eco log ica l d i f f e r e n t i a t i o n suggests that the taxa are highly d i f f e ren t i a t ed gene t i c a l l y . However, the chromosomal s i m i l a r i t y of spec ies , absence of genetic i so l a t i ng mechanisms, and the p o s s i b i l i t y that many morphological characters may be cont ro l l ed by very few genes suggests that genetic d i f fe rences among the taxa may be l im i ted to a small port ion of the genome and may not r e f l ec t ove ra l l genetic d i f f e r e n t i a t i o n . The taxa may be d i s t i n c t only in ce r t a in morphological and eco log i ca l characters of adaptive s ign i f i cance commonly used by taxonomists. 1.3 Genetic D i f f e r en t i a t i on Of Plant And Animal Populat ions. Research during the f i r s t years of the ana lys is of isozyme va r i a t ion in natural populations suggested two genera l izat ions regarding the genetic re la t ionsh ips of populat ions. F i r s t , there was a co r re l a t i on between genetic s i m i l a r i t y of populat ions and taxonomic rank: c lose l y re lated taxa were more s imi la r genet i ca l l y than more d i s t an t l y re lated ones. This was evident at the subspeci f ic l eve l in the Drosophila w i l l i s t o n i group (Ayala et a l . , 1974) and in various vertebrate species as 8 well as at the leve l of species and genera in various animal groups (Avise, 1976). Got t l i eb (1977) found the same cor re l a t ion in a survey of studies involv ing higher p lants , with d i f f e ren t species being on average only about two-thirds as s imi la r gene t i ca l l y as conspec i f i c populat ions. S im i l a r i t y values much higher than the average were obtained in comparisons of species pa i rs that d i f f e r ed in very few morphological characters . Although taxonomic re l a t ionsh ip of organisms usual ly r e f l e c t s the degree of morphological s i m i l a r i t y between them, species may be named on the basis of very few morphological d i f ferences i f a r e l i ab l e d i scont inu i t y in va r ia t ion ex is ts and the species are genet i ca l l y i so l a t ed . In the genera C l a r k i a , Gaura, and Stephanomeria, the species pa i rs examined e l ec t rophore t i ca l l y are reproduct ively i so la ted and the morphological d i f ferences between them are very smal l , cons is t ing of only one or two characters (Got t l i eb , 1977). Thus, a second genera l izat ion was that there seemed to be a co r re l a t ion between the degree of morphological and genetic s i m i l a r i t y . This co r re l a t ion was a lso apparent in animals. When species within the genera Drosophi la , Lepomis, and Peromyscus were arranged according to genetic s i m i l a r i t y , the resu l t ing c l a s s i f i c a t i o n was very s imi la r to that produced on the basis of morphological c r i t e r i a alone (Avise, 1974). Subsequent studies of animals revealed numerous exceptions to both cor re la t ions (Avise e t . a l . , 1975; Kornf ie ld and Koehn, 1975; Carson et . a l . , 1975; Nixon and Tay lor , 1977; Mitton and Koehn, 1975) so that neither now seems tenable. In p lants , 9 however, they have generally been upheld, although there i s no apparent co r r e l a t i o n between morphological and genetic differences among species of Capsicum (Jensen et a l . , 1979), morphologically d i s t i n c t species of S u l l i v a n t i a cannot be distinguished by their isozymes ( S o l t i s , 1982), and morphological d i f f e r e n t i a t i o n i s exceeded by genetic d i f f e r e n t i a t i o n in subspecies of Coreopsis cyclocarpa (Crawford and Bayer, 1981). The plant groups in which genetic and morphological differences are generally well-correlated or in which genetic s i m i l a r i t y i s related to taxonomic rank include Coreopsis (Crawford and Bayer, 1981; Crawford and Smith, 1982), Sabatia (Bell and Lester, 1978), Solanum (Whalen, 1979), Capsicum (McLeod et a l . , 1979), Machaeranthera (Arnold and Jackson, 1978, 1979), Typha (Mashburn et a l . , 1978; Sharitz et a l . , 1980), Brassica (Yodava et a l . , 1979), Chenopodium (Crawford,1979; Crawford and Wilson, 1979), Phlox (Levin, 1978), Plantago (van Dijk and van Delden, 1981), Hordeum jubatum (Shumaker and Babbel, 1980), Desmodium nudiflorum (Schaal and Smith, 1980), and Chondri11a juncea (Burdon et a l . , 1980). The co r r e l a t i o n i s also present at the subpopulation l e v e l in populations of Veronica peregrina (Keeler, 1977). It is not known, however, whether the correlation of morphological and genetic d i f f e r e n t i a t i o n in plants holds on the scale of a recent and extensive adaptive radiation. Gottlieb (1976) concluded that the speciation process in many annual d i p l o i d plants occurs prior to the ac q u i s i t i o n of d i s t i n c t adaptations and is largely fortuitous, based on studies of Cla r k i a , Gaura, and 10 Stephanomer ia• Species in these genera are reproduct ively i so la ted because of chromosomal d i f f e rences , but divergence at isozyme l o c i has not yet occurred. However, Hawaiian Bidens are perennial and d i f f e r from the annuals because they exhibi t extensive morphological and eco log ica l d i f fe rences among taxa and lack reproductive i so l a t i ng mechanisms. It is therefore of considerable . interest to see whether e l ec t rophore t i ca l l y detectable genetic d i f f e r e n t i a t i o n has occurred among the Hawaiian taxa of Bidens. 1.4 E lectrophores is And Isozymes E lectrophores is e ssen t i a l l y cons is ts of subject ing plant or animal extracts to an e l e c t r i c current and separating molecules as they migrate through a gel or other medium on the basis of e l e c t ros t a t i c charge, s ize and shape. In starch gel e lect rophores is of enzymes, the molecules are separated mainly on the basis of d i f ferences in net charge determined by the number of charged amino ac ids they conta in . The pos i t ions of the enzymes on the gel are i d en t i f i ed using histochemical s ta in ing techniques (Hunter and Markert, 1957). The sta in ing methods couple the enzyme-substrate reaction with a react ion producing insoluble pigment v i s i b l e in the ge l , thus marking the pos i t ions to which the enzymes migrated. Mobi l i t y d i f ferences of enzymes indicate d i f ferences in the a l l e l e s producing them, but i den t i ca l migration rates are not necessar i ly evidence of i den t i c a l a l l e l e s . Approximately 30% of nucleot ide base 11 subst i tu t ions do not a f fec t amino ac id sequences of proteins (Selander, 1976), and many amino ac id subst i tu t ions do not a f fec t the net e l e c t ros t a t i c charge of enzymes because only 4 of the 20 common amino acids are charged in the pH ranges usual ly employed in e lec t rophores i s . Other d i f fe rences can be demonstrated in a group of enzymes migrating to the same pos i t ion on a ge l , such as d i f fe rences in heat s t a b i l i t y (Bernstein et a l . , 1973). King and Wilson (1975) estimated that only 27% of point mutations are e l ec t rophore t i ca l l y detectable . A further l im i t a t i on of e lectrophores is is that only s t ruc tura l genes, which make up less than 10% of the eukaryotic genome, can be surveyed (Selander, 1976). In sp i te of these l im i t a t i ons , gel e lect rophores is represents a major advance in surveying populations gene t i ca l l y . Genetic ana lys is of populations using only those genes in fer red from morphological data is inherently biased because only polymorphic genes producing var iant phenotypes can be i d e n t i f i e d . Examination of gene products permits considerat ion of monomorphic l o c i as we l l , since the technique depends simply on production of funct ional enzymes rather than upon the production of d i f f e ren t ones. Polymorphism at a locus i s in ferred from the number, pos i t ion and pattern of bands instead of being a prerequis i te for i d e n t i f i c a t i o n of a locus . Morphological characters may be determined not only by a large number of genes but by the environment as we l l , which makes genetic ana lys is d i f f i c u l t . Furthermore, simultaneous examination of many morphological characters is complicated by 12 the p l e io t rop i c e f fec ts of some genes. A d i rec t correspondence ex is ts between enzyme l o c i and the polypeptides they produce, al lowing unambiguous in terpretat ion of phenotypes. Their genetic contro l is general ly well-documented, moreover, in contrast to the sca rc i t y of genetic information regarding morphological characters . It is not known whether isozyme l o c i are the unbiased sample of the genome that they were o r i g i n a l l y hoped to be. Even i f extrapolat ion to the ent i re genome is not poss ib le , these genes are a part of the genome not prev iously access ib le to population b io log i s t s and conclusions derived from the i r study are of as much value as those based on any other subset of l o c i . The large number of genes made ava i lab le for invest igat ion by e lectrophores is and the quant i ta t ive estimates of a l l e l e frequencies derived for each locus allow precise descr ip t ion and comparison of populat ions, advantages not shared by other techniques. 13 II. MATERIALS AND METHODS 2.1 Population Samples Plants of the endemic Hawaiian taxa were co l l ec ted e i ther as seeds or cutt ings from natural populat ions. Table 1 l i s t s population l o c a l i t i e s , sample s izes and the type of mater ia l co l l e c t ed . Figures 2-4 show the locat ions of the populations in the Hawaiian Is lands. Table 2 gives s imi lar information for the American species, and also indicates the i r geographical di str ibut ion . Seed samples of populations comprise the majority of mater ia l examined in t h i s study, but, unfortunately , de ta i l ed information about many of the seed co l l e c t i ons is l a ck ing . Several co l l e c to r s provided seed samples, and often the seeds representing a taxon in a cer ta in l o c a l i t y were a bulk co l l e c t i on without ind ica t ion of the number of plants from which they were gathered. It is conceivable that a large seed, sample could be the progeny of a s ingle s e l f - f e r t i l i z e d i nd i v i dua l , and therefore perhaps not representat ive of the populat ion. It is impossible to know the extent of sampling bias in these cases. The larger co l l e c t i ons of cut t ings or seed fami l ies were co l l ec ted e i ther from a l l ind iv idua ls in a population or from a random sample, although sampling bias may be involved in l o c a l i t i e s at higher e levat ions where the steep te r ra in can make plants d i f f i c u l t to f ind or almost impossible to reach. TABLE 1. COLLECTIONS OF HAWAIIAN TAXA STUDIED POPULATION SAMPLE TYPE OF TAXON ACRONYM NUMBER SIZE MATERIAL 1 LOCALITY B . asymmetr i ca AS YM B4 27 S(5),P(3) Manoa Cl i f f s Trail, Oahu B90 1 1 S,P(9) Aiea Ridge, Oahu B . cerv i cata CERV B8 13 S(>2) Nualolo Valley, Kauai B83 1 P Makaha Ridge, Kauai B87 38 S(>4) Ohik'ilolo Ridge, Oahu B88 13 SO) Ohikilolo Ridge, Oahu B . forbesi1 ssp. forbes i i FORB F B12 9 S(>7),P( 1 ) Haena Dry Cave, Kauai B13 2 P Hanalei Bay, Kauai B14 25 S(7),P(2) Lumahai Beach, Kauai B74 2 P Na Pali Coast, Kauai B . hawa i ens i s HAWA B48 10 SO),P(3) Kehena, Hawa i i B50 52 S(7),P(6) Ka i mu, Hawa i i B231 137 S(>8) . Ka1apana, Hawa i i B . mau i ens i s MAUI B 10 16 S(>6) Waiehu, Maui . B27 3 S Zoo, Maui B126 4 S Ukumehame, Maui B129 3 P Awalua Gulch, Lanai B . merizies i i ssp. f i1i formi s MENZ F B109 48 S(>7) Nohonaohae, Hawaii B130 57 S Kipuka Kalawamauna, Hawaii B2 18 100 S(36) Puu Ahumoa, Hawaii B219 61 S(30) Puu Koko, Hawaii B224 7 S Puu Kana1opakanui, Hawaii B238 14 S Puu Waawaa, Hawaii B . micrantha ssp. micrantha MICR M B24 3 .p Iao Valley, Maui B25 2 p Kahoma Ditch Trail, Maui B78 21 S(4) Wa i1uku, Mau i B80 2 s Zoo, Maui B133 12 s Honokowai Ditch Trail, Mau TABLE 1 cont TAXON ACRONYM POPULATION NUMBER SAMPLE SIZE TYPE OF MATERIAL 1 LOCALITY B. micrantha ssp. ctenophy11 a B. m i crantha ssp. ka1ea1 aha B. mo 1 oka i ens i s B. popu1 i f o 1 i a B. sandv i cens i s ssp. sandv i cens i s B. sandv i cens i s ssp. confusa B. torta MICR C MICR K MOLO POPU SAND S SAND C TORT B. wiebkei WIEB B 149 80 S(>10) B9 1 1 S B197 22 S(>2) B1 1 2 P • B72 62 S(5) B42 18 P B1 15 1 P B35 9 P B44 2 1 S(5) B1 1 1 2 S(2) B1 12 8 S(12) ' B1 16 5 S(2) B200 79 S(>9),P(33) B33 2 P B34 21 S(3) B15 79 S(2) B37 64 S(1),P(60) B89 19 S(4) B1 10 3 S B213 1 10 S(>8) ,P(8) B215 5 S B257 49 S(6) B259 35 S(>5) Kona, Hawa i1 Kahikinui, Maui Kapalaoa Cabin, Mau i Diamond Head, Oahu Hoolehua, Molokai Kahana Valley, Kaaawa, Oahu Oahu Nuuanu Pali, Oahu Lanipo Trai1, Oahu Kalepa Summit Wailua, Kauai Haiku Valley, Waahi1 a Ridge Kaua i Oahu Oahu Waimea Canyon, Kauai Puu Ka Pele, Kauai Pahole Gulch, Oahu Palikea Trai1, Oahu Ohiki1olo R1dge, Oahu Kawai-Iki Ditch Trail, Oahu Mount Kaala, Oahu Kolekole Pass, Oahu Waianae Kai, Oahu Halawaiki Gulch, Molokai cn •S refers to seeds; the number in brackets is the number of plants from which they were collected. P refers to plants and the number following is the sample size. g u r e 2 . L o c a t i o n s o f p o p u l a t i o n s s u r v e y e d pn K a u a i and Oahu. 17 MOLOKAI B91 F i g u r e 3. L o c a t i o n s of p o p u l a t i o n s M o l o k a i . s u r v e y e d on Maui and F i g u r e 4. L o c a t i o n s of p o p u l a t i o n s L a n a i . s u r v e y e d on H a w a i i and TABLE 2. COLLECTIONS OF AMERICAN SPECIES SAMPLE MATERIAL TAXON SIZE COLLECTED' LOCALITY DISTRIBUTI ON * B. aropli ss i ma 4C Jer i cho Lake, Vancouver, British Columbi a Reputedly endemic to Vancouver Island but also found in the lower mainland of B.C. B . cynap i fo1i a 10 Hanauma Bay, Oahu Native to the V/est Indes and continental tropical America. An introduced weed in Hawaii fi r s t collected in 1929 on the island of Hawaii.3 B. frondosa 17 Spanaway Lake, Pierce County, Wash i ngton Newfoundland and Nova Scotia to Washington, and south to Louisiana, Virginia, and Ca1i forn i a. i. tr i part i ta 10 Jericho Lake, Vancouver, British Columbia A nearly cosmopolitan north temperate weed native to eastern North America and Europe. •P refers to wild collected plants and.S to seeds 'Hitchcock et al. (1955). 3 Degener (1946 ) . 20 On the other hand, some of the smaller co l l e c t i ons of cut t ings or seed fami l ies may in fact be representat ive because the populations were so smal l . As an extreme example, the only population of Bidens molokaiensis on Oahu consisted of seven mature plants and seven seedlings in 1979. A sample of two cu t t ings , which were homozygous and iden t i ca l at a l l isozyme l o c i s tudied, probably provides a completely representat ive sample of the taxon on th is i s l and . 2.2 Growth Of Plant Mater ia l Seeds were planted in vermicu l i te and grown in growth chambers with 14 hours l i gh t at 25°C and 10 hours dark at 15°C, and transplanted into s o i l about three weeks a f te r emergence. Cutt ings were dipped in rooting hormone and maintained under mist u n t i l well rooted. They were then planted in s o i l and grown in greenhouses at the Univers i ty of B r i t i s h Columbia. 2.3 E lectrophores is Hor izonta l starch gel e lectrophores is was used to assay isoenzymes. The methods used general ly fol low Layton (1980), with some modif icat ions of the gel rec ipes , composition of buffer so lut ions and running condi t ions (Tables 3 and 4). Most of the study was done using E lec t ros tarch Lot #307 but Lot #392 TABLE 3. RUNNING CONDITIONS USED FOR ENZYME SYSTEMS EXTRACTION GEL ELECTRODE CURRENT ENZYME BUFFER COMPOSITION1 BUFFER OR VOLTAGE (%w/v) •Malate dehydrogenase gel buffer A MDH E.C.1.1.1.37 A 12.5% starch A 350V 20% sucrose Phosphoglucose isomerase PGI E.C.5.3. 1.9 Phosphoglucomutase PGM E . C . 2 . 7.5. 1 Ma 1 i c enzyme ME E . C. 1 . 1. 1 .40 gel buffer B 12.5% starch 10% sucrose 350V /S—Gl ucos i dase GLU E.C.3 . 2 . 1 . Hexose aminidase HA E.C.3 . 2 . 1 . x dehydrogenase xDH 2130gel buffer C 12.5% starch 10% sucrose 350V D i aphorase DIA E.C. 1.6.4.3 Leucine aminopeptidase LAP E.C.3.4. 1 . 1 gel buffer D 12.5% starch 2 10% sucrose 75mA 'Gels were made the morning of a run with Lot #307, but the evening before with lot #392. !14.3% starch used with Lot #392. 22 TABLE 4. COMPOSITION OF BUFFER SOLUTIONS. BUFFER PH COMPOSITION' REFERENCE * Extraction buffer A 7 . 5 13.5mM Tris Yeh S O'Malley 4 . 3mM Citric acid monohydrate (1980) 0.75mM NAD 0.65mM NADP 1mM Ascorbic acid 1 mM EDTA 0. 1% BSA (w/v) 14mM 2-mercaptoethanol B 7 .8 100mM Tris Roose & Gott1ieb 10mM KC1 (1978) 1 .OmM MgCl 100mM Ascorbic acid 14mM 2-mercaptoethanol Electrode buffer A 6 . 1 40mM Citric acid monohydrate Clayton & Tretiak adjust pH with N-(3-aminopropy1)- ( 1972) morpho1i ne B 8 . 1 60mM L i th i um-hydrox i de R i dgeway et a 1. 30mM Boric acid (1970) C 8 .6 100mM NaOH Mi t ton et a 1. 30mM Boric acid ( 1977) D 8 .0 50mM Tris Shaw & Prasad 100mM Boric acid (1970) 18mM EDTA Gel buffer A G . 1 1:19 dilution of electrode Clayton & Tretiak buffer A (1972) B 8 . 5 30mM Tris R i dgeway et a 1 . 0. 5mM Citric acid,monohydrate (1970) 0.06mM Lithium hydroxide 0. 3mM Boric acid C 8 . 1 15mM Tris Mi t ton et a 1 . 3mM Citric acid anhydrous ( 1977) D 8 .0 1 : 9 dilution of electrode Shaw 8. Prasad buffer D (1970) 1pH of solutions adjusted 'Some buffer solutions are with si i i NaOH or HC1 unless otherwise noted, ghtly modified from these references. 23 was a lso used, which required modified methods of preparation to achieve s imi lar resu l ts (Table 3). The addi t ion of sucrose to the gels improved resolut ion of the bands. With Lot #307 starch was cooked, degassed, poured into 150x200*10mm p lex ig lass gel molds, wrapped in p l a s t i c and stored in the re f r ige ra to r the morning of a run. About 8mg of young leaf t issue was ground on ice (to i nh ib i t enzyme a c t i v i t y ) with one drop of extract ion buffer and 4mg polyv iny lpolypyrro l idone in spot p l a tes . The homogenate was absorbed onto 9x5mm wicks cut from Whatman 3MM chromatographic paper, and the wicks inserted into s lo ts cut 30mm from the end of the ge l . Migrat ion of enzymes was monitored with d i lu te red food colour ing absorbed onto wicks at e i ther end of the group of samples. Results were standardized by always inc luding one of three plants of known genotype in the run. E lect rophores is was performed in a re f r ige ra to r maintained at 0-4°C to avoid overheating which d i s to r t s migration and denatures enzymes. Gels were subjected to e l e c t r i c current for about four hours, using J-Cloths to complete the c i r c u i t between the ends of the gel and the electrode trays containing an electrode and buffer so lu t i on . A l l but one of the isozymes (a PGI-5 variant) migrated anodally in the condit ions employed ( i . e . , toward the pos i t i ve e lec t rode ) . Gels were s l i c ed with Gibson .008 p la in s tee l ba l l end gui tar s t r ing guided by 1.5mm thick p lex ig lass s t r i p s placed on e i ther side of the g e l . Top and bottom s l i c e s were discarded and the rest sta ined for appropriate enzymes using histochemical 24 sta in ing methods l i s t e d in Table 5. (Enzyme abbreviat ions are l i s t e d in Table 3.) In most cases, gels were bathed in 60 mis of s ta in so lu t i on , but with GLU and HA 5 mis of s ta in so lut ion were poured on top of the g e l . Most of the sta ins depended on react ions producing insoluble products v i s i b l e in natural l i g h t , but GLU and HA bands were only v i s i b l e under long-wave UV l i gh t and had to be scored before the bands d i f fused over the surface of the g e l . In attempting to s ta in for ADH (alcohol dehydrogenase) the appropriate bands rare ly appeared. Instead, a much more slowly developing band appeared overnight for each i nd i v i dua l , sometimes even in the absence of ethanol (but not in the absence of a sample). This mystery enzyme was accordingly termed xDH. After the sta in ing react ion was complete, PGI, PGM and MDH gels were f ixed in a 1:1 glycerine-water so lut ion ( S i c i l i ano and Shaw, 1976) and the rest in a 1:4:5 acet ic acid-methanol-water so lut ion (Al lendorf et a l . , 1977) to preserve r eso lu t i on . 2.4 Enzymes Studied The choice of enzyme systems used to estimate genetic v a r i a b i l i t y a f f ec t s the resu l ts obtained. Enzyme polymorphism has been shown to be corre la ted among enzymes of the g l y co l y t i c-Krebs cycle (Sing and Brewer, 1971). Johnson (1974) has suggested pos i t i ve re la t ionsh ips between enzyme polymorphism and regulatory character , and between the v a r i a b i l i t y of enzymes and the v a r i a b i l i t y of the i r substrates . Harr is et a l . (1977) 25 TABLE 5. STAINING SOLUTIONS EMPLOYED FOR ENZYME SYSTEMS. ENZYME STAIN RECIPE1 REFERENCE' MOH 200mM Tris-HCl, pH 8.0 S i c i1i ano & Shaw 80mM DL-ma1 i c ac i d ( 1976) 0.6mM NAD 0. 2mM MTT 0. 2mM NBT 0.25mM PMS PGI 1 30mM Tris-HCl, pH 8.0 Roose & Gott1ieb 5mM MgCl ( 1976) 0. 4U/ml Glucose-6-phosphate dehydrogenase 0.75mM D-fructose-6-phosphate 0. 2mM NADP 0. 2mM MTT 0.25mM PMS PGM 130mM Tri s-HCl , pH 8.0 Roose & Gottlieb 5mM MgCl ( 1976) 5 . 5mM -D-glucose-1-phosphate (Na salt) 0.004mM -D-glucose-1-phosphate (K salt) 0.4U/ml Glucose-6-phosphate dehydrogenase 0.2mM NADP 0 . 3mM MTT 0.25mM PMS ME 200mM Tris-HCl , pH 8 .0 S i c i1i ano S Shaw 5mM MgCl (1976) 25mM L-malie acid 0. 2mM NADP 0. 3mM MTT 0.25mM PMS GLU 50mM Citrate-phosphate, pH 4.0 Yeh & Layton 6mM 4-methy1 umbel 1i fery1-fi - (1979) D-glucos i de HA 25mM Citrate-phosphate, pH 4.0 S i c i1i ano & Shaw 5mM 4-methyl umbel 1iferyl-N-acetyl- (1976) P-D-glucosaminide xDH 200mM Trils-HCl , pH 8.5 S i c i1i ano & Shaw 4% 95% ethanol (1976) 0. 6mM NAD 0. 2mM MTT 0. 2mM NBT 0.25mM PMS : DIA 200mM Tris-HCl, pH 8.5 Yeh & 0'Mai ley 0.06mM 2,6-dichlorophenol-i ndophenol (1980) 0.06mM NADH 0. 2mM MTT LAP presoak ! solution Brewer S Sing 500mM Boric acid ( 1970) 5mM MgCl stain solution, pH 5.2 20mM Tris-HCl 20mM Ma 1 i c ac i d 0.65mM L-leucyl -^-naphthy1 amide HC1 0.0006% Fast black K salt (w/v) All gel slices were incubated at 40*C except for GLU and HA stains (room temperature) and the presoak step for LAP (one hour in refrigerator). 'Most of the recipes used are modifications of these references. 26 found that enzymes composed of more subunits have fewer v a r i a n t s i n humans, and Ward (1977) found a s i m i l a r c o r r e l a t i o n between quaternary s t r u c t u r e and polymorphism of enzymes in many v e r t e b r a t e and i n v e r t e b r a t e s p e c i e s . There may a l s o be a p o s i t i v e c o r r e l a t i o n between v a r i a b i l i t y and subunit molecular weight (Koehn and Eanes, 1977; but see Johnson, 1977, f o r an o p p o s i t e view) . S i c i l i a n o and Shaw (1976) p o i n t e d out that out of the approximately 1000 enzymes which have been i d e n t i f i e d , h i s t o c h e m i c a l s t a i n i n g techniques are a v a i l a b l e f o r fewer than 50. Twenty-seven of these were attempted f o r Bidens but enzyme a c t i v i t y was not recovered f o r a l l of them. Furthermore, adequate r e s o l u t i o n of bands was not achieved f o r many enzyme systems d e s p i t e the v a r i o u s types and combinations of e x t r a c t i o n , g e l and e l e c t r o d e b u f f e r s and g e l composition t e s t e d . F i n a l l y , chemical d i f f e r e n c e s among the Hawaiian taxa r e s u l t e d i n a f u r t h e r r e d u c t i o n of usable systems because of the i n a b i l i t y of any one method to achieve good r e s o l u t i o n i n a l l p o p u l a t i o n s . Using d i f f e r e n t techniques f o r d i f f e r e n t taxa would have made i t d i f f i c u l t to e s t a b l i s h homology of bands. T h i s problem e l i m i n a t e d the use of a c i d phosphatase, glutamate o x a l o a c e t a t e transaminase, and c e r t a i n regions of MDH and DIA. The nine enzyme systems f i n a l l y used to compare the Hawaiian p o p u l a t i o n s of Bidens were thus chosen not with due c o n s i d e r a t i o n of s t r u c t u r a l and f u n c t i o n a l c o n s t r a i n t s , but p u r e l y on the b a s i s of what systems worked. As with a l l e l e c t r o p h o r e t i c s t u d i e s , i t i s hard to know how 27 r e p r e s e n t a t i v e t h i s s u b s e t o f enzymes i s o f s t r u c t u r a l g e n e s o r o f t h e genome i n g e n e r a l . The number o f enzymes s t u d i e d i s a b o u t a v e r a g e f o r p l a n t p o p u l a t i o n s t u d i e s b u t t h e number o f l o c i i s w e l l a b o v e a v e r a g e . F o r t u n a t e l y , most o f t h e s e l o c i (17 o f 24) c o d e f o r enzymes e x a m i n e d i n o v e r 6 5% o f p r e v i o u s s t u d i e s o f p l a n t p o p u l a t i o n s ( G o t t l i e b , 1 9 8 1 ) , so t h a t c o m p a r i s o n s w i t h o t h e r p l a n t s a r e p o s s i b l e . 28 I I I . INHERITANCE OF ISOZYMES 3.1 D e f i n i t i o n s And Nomenclature The terms allozyme and isozyme were presumably conceived to d i s t i n g u i s h between enzymes that are a l l e l i c and those that are not. Thus, f o r enzymes c o n s i s t i n g of one p o l y p e p t i d e (monomeric enzymes) the products of a heterozygous l o c u s would be allozymes while products of d i f f e r e n t l o c i would be c a l l e d isozymes. M u l t i m e r i c enzymes pose a problem, however, because i t i s a s s o c i a t i o n s of gene products r a t h e r than the gene products themselves that are being compared. An enzyme composed of two u n l i k e subunits coded by d i f f e r e n t l o c i (an i n t e r l o c u s heterodimer) i s both a l l e l i c and n o n - a l l e l i c to an enzyme composed of i d e n t i c a l subunits coded by an a l l e l e at one of the l o c i . The convention i s to c a l l these isozymes ( G o t t l i e b , 1981) but t h i s makes allozymes very r a r e indeed when s e v e r a l genes are i n v o l v e d . Because even the monomeric enzymes that show polymorphism i n Bidens are c o n t r o l l e d by more than one l o c u s , I w i l l c o l l e c t i v e l y c a l l the m u l t i p l e forms of an enzyme system isozymes, i g n o r i n g the small subsets of molecules which c o u l d be d i s t i n g u i s h e d as allozymes. Table 6 l i s t s f o r each enzyme the s u b s t r u c t u r e , number of l o c i a p p a r e n t l y c o n t r o l l i n g i t and the number of l o c i scored -for t h i s study. The nomenclatural convention used i n the f o l l o w i n g s e c t i o n s i s to l a b e l l o c i n u m e r i c a l l y with the most a n o d a l l y TABLE G. ENZYME SUBSTRUCTURE, GENETIC CONTROL AND NUMBER OF LOCI SCORED. ENZYME SUBSTRUCTURE NUMBER OF LOCI EXPRESSED NUMBER OF LOCI SCORED MDH PGI PGM ME GLU HA xDH DIA LAP d i mer i c d i mer i c monomer i c tetrameri c monomer i c monomer i c >8 6 5 5 4 4 >2 . 1 >2 1 >2 1 1 1 >4 2 2 2 30 migrating locus for each enzyme system as "1" and the a l l e l e s alphabetically with the most anodally migrating a l l e l e at each locus as "a", with the rest proceeding in sequence according to position on the g e l . 3.2 Types Of Evidence Many l i n e s of evidence can be used to infer the genetic control of enzyme systems. The most fundamental of these is genetic analysis, the crossing of individuals with d i f f e r e n t band patterns and examining segregation in the progeny. The number of l o c i involved and the number of a l l e l e s at each can be deduced from a large number of such crosses. This i s a time-consuming procedure in Bidens, in which f l o r e t s are small, d i f f i c u l t to emasculate, and only produce one achene each. Although the ultimate test of the genetic hypothesis can only be of t h i s nature, there are fortunately easier ways to arrive at the actual inferences. The most useful of these i s knowing the substructure of the enzyme. A l l e l e s code for polypeptides which may be functional as monomeric molecules (e.g., LAP and PGM) or may need to bind to other polypeptides to function. Such multimeric enzymes are often dimeric (e.g., MDH and PGI), but tetramers such as catalase (Scandalios, 1965), hexamers such as glutamate dehydrogenase (Goldin and Frieden, 1971), and others also e x i s t . While i t is the composition of the a l l e l e product that a f f e c t s 31 e l e c t r o p h o r e t i c m o b i l i t y and thus the p o s i t i o n of bands on the g e l , i t i s the s u b s t r u c t u r e of the enzyme which determines the number of bands and t h e i r r e l a t i v e i n t e n s i t y . A heterozygote at one l o c u s w i l l produce two bands f o r a monomeric enzyme but three f o r a dim e r i c one: the a d d i t i o n a l band rep r e s e n t s the a s s o c i a t i o n of u n l i k e subunits (produced by the d i f f e r e n t a l l e l e s ) and has a m o b i l i t y intermediate to that of the molecules formed by the a s s o c i a t i o n of l i k e s ubunits (coded by the same a l l e l e ) . Because of the presumably random a s s o c i a t i o n of s u b u n i t s , molecules c o n s i s t i n g of u n l i k e subunits w i l l be twice as common as e i t h e r of the homodimers, r e s u l t i n g i n a 1:2:1 r a t i o of band i n t e n s i t i e s . When more than one lo c u s i s i n v o l v e d p a t t e r n s are more complex, but a b r i e f examination of band p a t t e r n s produced by i n d i v i d u a l s i n a p o p u l a t i o n s t i l l a f f o r d s a c l u e to the gen e t i c c o n t r o l of the enzyme system. The l i t e r a t u r e concerning enzymes and t h e i r use i n p o p u l a t i o n s t u d i e s i s a l s o h e l p f u l i n e l u c i d a t i n g the i n h e r i t a n c e of isozymes. In a d d i t i o n to general agreement i n su b s t r u c t u r e f o r a given enzyme i n d i f f e r e n t higher p l a n t s p e c i e s , there are a l s o s i m i l a r i t i e s i n the number of l o c i c oding f o r each enzyme at a given p l o i d y l e v e l ( G o t t l i e b , 1982). Because Hawaiian Bidens are h e x a p l o i d ( G i l l e t t and Lim, 1970), one would expect more l o c i than usual to be c o n t r o l l i n g each enzyme system. A n a l y s i s of h a p l o i d t i s s u e can s i m p l i f y band p a t t e r n s because of the presence of only one a l l e l e per l o c u s . T h i s i s e s p e c i a l l y v a l u a b l e in c o n i f e r s where megagametophytes are l a r g e 32 enough to be studied i n d i v i d u a l l y . In angiosperms an extract of pollen produced by a plant can be run instead. This is less useful because of the lumping of many haploid genotypes, but heteromeric enzymes w i l l not be formed through the association of subunits produced by d i f f e r e n t a l l e l e s of the same locus. A comparison of pollen and leaf tissue patterns can reveal which bands are composed of such heteromers, and can enable assignment of a l l e l e s to certain l o c i . This technique did not prove very useful with Bidens because of the small number of plants in flower, f a i n t staining of enzymes from pollen samples, and differences in the l o c i expressed in pollen and leaf tissue. Only PGI, PGM and MDH showed any a c t i v i t y at a l l in pollen samples, and in MDH an additional locus not active in leaves complicated the analysis. Because many genes code for each of these enzymes, only the pollen and leaf comparisons of double heterozygotes would have yielded information, and these were not encountered in the plants sampled. PGI, PGM and MDH isozymes are compartmentalized within the c e l l (Gottlieb, 1982). Although a l l are produced by nuclear genes, the products of some l o c i are found in p l a s t i d s and others in the cytoplasm. Furthermore, there i s no interaction between the polypeptides of p l a s t i d and cytoplasmic enzymes so that heterodimers are not formed between the subunits produced by the d i f f e r e n t l o c i . Because the p l a s t i d forms of PGI and PGM migrate to a di f f e r e n t region of the gel, genetic analysis simply treats each region separately. The compartmentalization of MDH in Bidens may not be refl e c t e d in separation of regions, 33 so that comparison of p a t t e r n s produced by c h l o r o p l a s t f r a c t i o n s and u n f r a c t i o n a t e d l e a f samples might h e l p e l u c i d a t e the g e n e t i c c o n t r o l . The r e s u l t s were ambiguous, however, so the attempt to look at c h l o r o p l a s t isozymes was dropped. 3.3 Monomorphic Enzymes S t a i n i n g f o r ME, xDH, GLU and HA r e s u l t e d i n an i d e n t i c a l , s i n g l e band f o r a l l i n d i v i d u a l s sampled from s e e d l i n g s and c u t t i n g s . The simplest i n t e r p r e t a t i o n of t h i s i s that each enzyme i s c o n t r o l l e d by one homozygous l o c u s , with a l l p l a n t s s h a r i n g the same a l l e l e . Adult p l a n t s o c c a s i o n a l l y had other bands for ME, GLU and HA, suggesting that more than one l o c u s codes f o r these systems although only one i s expressed i n younger p l a n t s . 3.4 Polymorphic Enzyme Systems Se v e r a l of the polymorphic enzymes had v a r i a n t s which migrated to two d i s t i n c t regions of the g e l . The zones of a c t i v i t y d i d not show any c o r r e l a t e d v a r i a t i o n and were t h e r e f o r e t r e a t e d independently with r e s p e c t to g e n e t i c hypotheses, each zone being c o n s i d e r e d to be c o n t r o l l e d by d i f f e r e n t l o c i . Because of small v a r i a t i o n among runs i n the m o b i l i t y of 34 bands, a l l e l e s were a c c e p t e d a s b e i n g d i f f e r e n t o n l y i f t h e r e was a l a r g e and c o n s i s t e n t m o b i l i t y d i f f e r e n c e between bands o r i f h e t e r o z y g o t e s were f o u n d f o r a l l e l e s c o d i n g f o r p o l y p e p t i d e s w i t h s m a l l m o b i l i t y d i f f e r e n c e s . N u l l a l l e l e s were i n v o k e d i n p r e f e r e n c e t o i n f e r r i n g s e v e r a l i d e n t i c a l uncommon a l l e l e s f o r d i f f e r e n t l o c i . The a s s u m p t i o n t h a t a g i v e n a l l e l e w ould be f o u n d a t one l o c u s o n l y w i t h o u t p r o o f t o t h e c o n t r a r y seems r e a s o n a b l e b e c a u s e of t h e s m a l l p r o b a b i l i t y of i n d e p e n d e n t m u t a t i o n s a t two l o c i h a v i n g e x a c t l y t h e same p h e n o t y p i c e f f e c t . The most common a l l e l e a t two PGI l o c i was i n f a c t s h a r e d , b u t t h e r e i s s t r o n g e v i d e n c e f o r t h i s . i n t h e band i n t e n s i t y p a t t e r n s . T h i s i s n o t t o o s u r p r i s i n g f o r a common a l l e l e g i v e n t h e p o l y p l o i d s t a t u s of H a w a i i a n B i d e n s i n w h i c h l o c i a r e p r e s u m a b l y d u p l i c a t e d . When more t h a n one l o c u s c o n t r o l l e d a r e g i o n of a c t i v i t y , a l l e l e s were a s s i g n e d t o them by f i n d i n g a g e n o t y p e homozygous f o r t h e common a l l e l e a t one l o c u s and h e t e r o z y g o u s a t t h e o t h e r . F i n d i n g s u c h h e t e r o z y g o t e s f o r e a c h a l l e l e p e r m i t t e d a s s i g n m e n t of a l l a l l e l e s t o l o c i . E v i d e n c e f o r t h e g e n e t i c h y p o t h e s e s f o r a l l p o l y m o r p h i c l o c i i s g i v e n i n T a b l e 7. A l t h o u g h sample s i z e s a r e s m a l l , t h e d a t a g e n e r a l l y c o n f o r m t o M e n d e l i a n e x p e c t a t i o n s . C r o s s e s i n v o l v i n g n u l l a l l e l e s show a p r o n o u n c e d l a c k of homozygous n u l l p r o g e n y , however. T h i s i s n o t u n e x p e c t e d b e c a u s e g e n o t y p e s u n a b l e t o p r o d u c e f u n c t i o n a l enzymes would n o t be l i k e l y t o s u r v i v e l o n g enough t o be 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 . 35 TABLE 7. GENETIC ANALYSIS OF ELECTROPHORETIC VARIANTS. PARENTAL OFFSPRING PHENOTYPES LOCUS PHENOTYPES bb be cc bd ed dd de ee dg gg an bn nn PGI-4 dd X dd ' cd X cd dg X dg PGI-5 cc X cc cd X cd 21 10 4 7 9 8 10 10 24 26 2 16 4 16 10 12 2 1 3 3 6 10 10 4 7 10 9 10 9 10 16 2 26 1 1 12 10 24 4 1 1 1 1 PGM- 1 bb X bb cc X cc bd X bd bn X bn 6 4 10 10 20 16 24 6 10 9 12 2 2 1 3 36 TABLE 7 c o n t . LOCUS PARENTAL PHENOTYPES ab bb OFFSPRING PHENOTYPES be ad bd cd PGM-2 bb X bb be X be bd X bd 4 10 7 9 10 1 1 20, 1G 24 6 10 4 16 12 1 6 PGM-3 ab X ab bb X bb bn X bn 1 2 21 8 6 7 9 1 1 20 16 24 6 10 4 6 10 16 12 12 1 6' PGM-4 aa X aa an X an 21 4 10 7 7 9 10 1 1 20 16 24 6 10 4 10 16 1 2 9 12 1 bb X bb 6 37 TABLE 7 cont. LOCUS PHENOTYPES OFFSPRING PHENOTYPES MDH-2 ad X ad dd X dd MDH-3 aa X aa 10 10 ab bb ac be cc ad bd cd dd an bn 12 10 7 15 ab X ab 2 4 ' 6 14 • 1 7 1 2 * 2 1 3 71 3 61 4 61 7 11' 5 19' bb X bb 9 3 8 10 1G 4 6 8 8 5 2 12 • 3 9 10 5 4 7 10 10 9 8 16 18 24 4 6 10 15 10 10 'Heterozygous ab and homozygous bb progeny were Impossible to distinguish, so they are summed under the bb column. 3 8 TABLE 7 cont. PARENTAL LOCUS PHENOTYPES OFFSPRING PHENOTYPES ab bb be ad bd ed dd bn MDH-5 aa X aa ac X ac ad X ad 10 10 10 24 15 4 6 4 3 9 10 10 8 16 18 5 3 5 2 3 14 2 5 4 MDH-6 aa X aa ab X ab 12 3 7 10 4 7 10 9 8 20 8 16 18 4 6 24 15 10 10 10 5 LAP- 1 bd X bd cd X cd dd X dd 1 4 9 10 12 7 10 16 18 4 39 TABLE 7 cont. PARENTAL OFFSPRING PHENOTYPES LOCUS PHENOTYPES — aa ab bb ac be cc ad bd cd dd an bn LAP-2 bb X bb 7 10 12 8 10 16 18 4 be X be -4 5 bn X bn 5 2 3 5 2 2 DIA-1 aa X aa 6 16 1 1 10 18 9 2 1 10 ab X ab 3 5 2 5 5 4 61 2 7 i 2 9' 4 41 bb X bb 4 7 6 DIA-2 aa X aa 16 1 1 ' 10 7 18 6 11 9 10 10 2 1 6 9 10 8 4 'Heterozygous ab and homozygous bb progeny were dif f i c u l t to distinguish, so they are summed under the bb column. 40 3.4.1 Phosphoglucose Isomerase PGI isozymes migrated to two regions of the gel (Figures 5-7). The anodal region was monomorphic while the slower zone was highly polymorphic. Two l o c i probably contro l the lower region since any pattern observed ( including both band pos i t ions and in tens i t i e s ) could be explained with four a l l e l e s . A to ta l of 14 a l l e l e s was invoked for these two l o c i , inc luding a nu l l for each. The l o c i share the i r most common a l l e l e so that a s ingle heavy band was frequently seen in th i s region. It is un l ike ly that a th i rd gene nearly always sharing the same a l l e l e is involved despite the taxa being hexaploid because d i f f e ren t band in tens i ty patterns would be expected, and because the occurrence of a genotype lacking that a l l e l e would be vanishingly small although several were actua l l y observed. Determining which a l l e l e s belonged to which locus was a l i t t l e more d i f f i c u l t because of the shared a l l e l e , but genotypes possessing two copies of an uncommon a l l e l e were assumed to be homozygous at one locus, al lowing unambiguous assignment of the other a l l e l e s to the second locus. A l l a l l e l e s were assigned to the l o c i by f inding genotypes homozygous for an uncommon a l l e l e at one locus and heterozygous at the other. The anodal region consisted of three evenly spaced bands of which the slowest was much l igh te r than the other two. Although two l o c i f ixed for d i f f e ren t a l l e l e s would produce a three-banded pat tern , the slowest band would not be l i gh tes t unless d i f f e r e n t i a l s ta in ing was involved. Because band in tens i ty seems well corre la ted with a l l e l e dosage in PGI a three locus 4 1 P G I - 1 P G I - 2 P G I - 3 PGI-4 PG I-5 F i g u r e 5 . A l l e l e s l o c i . o f t h e P G I laa laa laa laa 2aa 2aa 2aa 2aa 3aa 3aa 3aa 3aa 4dd 4dd 4cd 4cd 5dd 5cc 5cc 5cd F i g u r e 6 . G e n o t y p e s o f c o m m o n P G I b a n d p a t t e r n s . F i g u r e 7 . P h o t o g r a p h o f P G I b a n d p a t t e r n s . S a m p l e s 1 , 3 a n d 4 a r e 1 a a 2 a a 3 a a 4 c c 5 c c , s a m p l e s 2 , 1 1 , 1 2 a n d 1 3 a r e 1 a a 2 a a 3 a a 4 d d 5 c c , s a m p l e s 5 , 6 a n d 1 0 a r e 1 a a 2 a a 3 a a 4 d f 5 c c , a n d s a m p l e s 7 , 8 a n d 9 a r e 1 a a 2 a a 3 a a 4 f f 5 c c . 42 explanation i s preferable. Two l o c i fixed for one a l l e l e and a t h i r d for the other would produce a 4:4:1 r a t i o of band i n t e n s i t i e s which f i t s the observed pattern. PGI behaved as a dimeric enzyme in Bidens. This has also been reported in Festuca (Adams and A l l a r d , 1977), wheat (Hart, 1979), Lolium (Nielsen, 1980), Clarkia (Weeden and Gottlieb, 1979), Gaura (Gottlieb and P i l z , 1977), Citrus (Torres et a l . 1978), pitch pine (Guries and Ledig, 1978), ponderosa pine (Mitton et a l . , 1979), Douglas f i r (El Kassaby et a l . 1982) and P l e c t r i t i s (Layton, 1980). 3.4.2 Phosphoglucomutase Two independent zones of a c t i v i t y also appeared when gels were stained for PGM. Both were polymorphic and both best explained with two l o c i (Figures 8-10). The l o c i c o n t r o l l i n g the anodal region each had 5 a l l e l e s and the others had 3 and 4 a l l e l e s . A l l l o c i had a n u l l a l l e l e . PGM behaved l i k e a monomeric enzyme, with heterozygotes at one locus having two bands instead of one. This substructure has also been reported in. Citrus (Torres et a l . , 1978), ponderosa pine (Mitton et a l . , 1979), Douglas f i r (El Kassaby, 1982) and P l e c t r i t i s (Layton, 1980). 4 3 a-b-c-d- a-b-c-d-a-b-PGM-1 PGM-2 PGM-3 PGM-4 Ibb lbb lab 2bb 2bb 2b c 3bb 3bb 3ab 4aa 4ab 4aa F i g u r e 8 . A l l e l e s o f t h e P G M l o c i . F i g u r e 9 . G e n o t y p e s o f c o m m o n P G M b a n d p a t t e r n s . F i g u r e 1 0 . P h o t o g r a p h o f P G M b a n d p a t t e r n s . S a m p l e 1 i s 3 a b 4 a a , s a m p l e s 2 a n d 3 a r e 3 b b 4 a b , s a m p l e s 4 , 7 , 8 a n d 9 a r e 3 b b 4 a a , a n d s a m p l e s 5 a n d 6 a r e 3 b b 4 b b . T h e f a s t e r z o n e i s o v e r s t a i n e d a n d n o t d e c i p h e r a b l e . 44 3.4.3 Malate Dehydrogenase MDH behaved l i ke a dimeric molecule, had the most complex patterns and was cont ro l l ed by more l o c i than any other enzyme system (Figures 11-13). The slowest region on the gels was not scored because of poor reso lut ion in many taxa. The fastest band was iden t i ca l in a l l ind iv idua ls and probably cont ro l l ed by one homozygous locus. The cent ra l area was va r i ab le , having from 4 to 10 bands, but cer ta in sets of bands behaved independently of each other. The most common patterns are shown in F i g . 12. The zone l abe l l ed "C" appears to be cont ro l l ed by two l o c i since variant 3-banded patterns have e i ther l i gh t rheavy : l i gh t or heavyrmediumrlight band i n t ens i t i e s reminiscent of PGI and impossible to obtain with just one locus . Zone "B" is probably cont ro l l ed by three l o c i with the s implest , heavy:heavy: l ight pattern representing two l o c i f ixed for one a l l e l e and the th i rd for another. A t h i r d , intermediate a l l e l e at one of the faster l o c i would create the common 5-banded pattern and other faster a l l e l e s would cause the heavy:medium:light and l i gh t rheavy : l i gh t patterns at the anodal end of th is zone. As before, two l o c i are needed to explain these in tens i ty patterns at the top, and the t h i rd locus is necessary to account for the 3- or 5-banded patterns in the lower half of th i s zone. One problem was ass igning genotypes to the 5-banded pat tern . The 3- or 5-banded patterns were often f ixed for a populat ion, in which case homozygosity for the relevant genes was in fe r red . In populations var iab le at th is locus , however, 45 F i g u r e 13. Photograph of MDH band p a t t e r n s . Samples 1 and 9 are i n d e c i p h e r a b l e , samples 2, 3 and 5 are 2dd3aa4aa5ad6aa, samples 4 and 8 are 2dd3aa4aa5dd6aa, and samples 6 and 7 are 2dd3aa4aa5aa6aa. The most anodal band i s not i n c l u d e d i n the photograph. 46 i t was sometimes impossible to d i s t i ngu i sh between heterozygotes and homozygotes for the 5-banded pat tern . This is an unusual s i tua t ion in e lec t rophores i s , where a l l e l e dosage is usual ly revealed in simple re la t ionsh ips of band i n t e n s i t i e s , but in th i s instance patterns were so complex and expected in tens i ty re la t ionsh ips s u f f i c i e n t l y s imi la r that unambiguous in terpre ta t ion was not poss ib le . One poss ib le so lut ion to th i s sort of s i tua t ion is to ca lcu la te F, Wright 's coe f f i c i en t of inbreeding (which is theo re t i ca l l y the same for a l l l o c i ) for a few other l o c i , and use th is estimate to ca lcu la te the proportion of heterozygotes and homozygotes in the dominant 5-banded phenotype. The F values ca lcu la ted for other polymorphic l o c i turned out to be so var iable that using an average seemed quite a rb i t r a r y . Instead, the simpler Hardy-Weinberg model was used. The resu l t ing gene frequencies for populations var iab le at th is locus may not be s t r i c t l y accurate, but they s t i l l a f ford a basis for comparison of populat ions. MDH has a lso been reported to be dimeric in maize (Goodman et a l . , 1979), Acetabular ia (Serov et a l . , 1979), Eucalyptus (Brown et a l . , 1975), P l e c t r i t i s (Layton, 1980), p i t ch pine (Guries and Ledig, 1978), ponderosa pine (O'Malley et a l . , 1979), Douglas f i r (El Kassaby, 1982) and lodgepole pine (Yeh and Layton, 1979) . 47 3.4.4 Leucine Aminopeptidase LAP gels had from one to four bands and seemed to be cont ro l l ed by two l o c i , each with f ive act ive a l l e l e s and one nu l l (Figures 14-16). One of the a l l e l e s could not be unambiguously assigned to a locus so the choice was made a r b i t r a r i l y , but the e f fec t of th i s is minor since the frequency of the a l l e l e was only .01. It behaved as a monomer, as has been reported for Picea abies (Lundkvist, 1974), Pinus  s y l v e s t r i s (Rudin, 1977), Pisum sativum (Scandalios and E s p i r i t u , 1969) and Phaseolus (Wall, 1968). 3.4.5 Diaphorase Diaphorase isozymes a lso formed complex patterns but the slowest bands seemed independent of var ia t ion elsewhere. Two l o c i , one with four and the other with two a l l e l e s , were invoked to explain the patterns (Figures 17-18). Diaphorase behaved l i k e a monomeric enzyme with heterozygotes having two bands. 3.5 American Taxa The American taxa had very d i f f e ren t banding patterns for many enzyme systems. There is no sa t i s fac tory way to es tab l i sh gene homologies between the Hawaiian and American taxa by genetic ana lys is because the two groups w i l l not hybr id ize . 48 a • b-c d b-c • LAP-1 LAP-2 ldd 2bb ldd 2bc led 2bb F i g u r e 14. A l l e l e s of the LAP l o c i . F i g u r e 15. Genotypes of common LAP band p a t t e r n s . Figure 16. Photograph of LAP band p a t t e r n s . Samples 1-7 and 10 are 1dd2bb, and samples 8 and 9 are 1dd2bc. d D I A - 1 D I A - 2 l a a l b b l a b 2aa 2aa 2aa F i g u r e 17. A l l e l e s o f t h e DIA l o c i . F i g u r e 18. DIA G e n o t y p e s of common band p a t t e r n s . 50 Inferences from band pos i t ions are the best method ava i lab le and th is was f a c i l i t a t e d by the absence of v a r i a b i l i t y within populations of the American taxa and by the i r bands being at e i ther i den t i ca l pos i t ions to those in the Hawaiian species or in r ad i c a l l y d i f f e r en t pos i t i ons . A l l populations of American species were monomorphic, so a l l e l e frequencies of 1.00 were assigned ei ther for a l l e l e s shared with Hawaiian plants or for a l l e l e s unique to the American species. The American taxa appear to have fewer l o c i than the Hawaiian taxa for some enzyme systems, presumably because they are not a l l hexaploid. Bidens  frondosa is t e t r ap lo id and populations of B_^  t r i p a r t i t a are e i ther t e t r ap lo id or hexaploid (Fedorov, 1974). Chromosome counts for B^  amplissima and cynap i fo l i a have not been reported. To compare taxa of d i f f e ren t p lo idy l e ve l s , missing l o c i were treated as being f ixed for nu l l a l l e l e s . 51 IV. VARIABILITY WITHIN POPULATIONS 4.1 Hawaiian Populations Sample s izes for many of the l o c a l i t i e s l i s t e d in Table 1 are too small for reasonable estimates of v a r i a b i l i t y . Theore t i ca l l y , a sample of 30 ind iv idua ls provides a 0.95 probab i l i t y of detect ing an a l l e l e present in the population at a frequency of 0.05. Because of the small s ize of many natural populat ions, however, samples of over 20 are treated as large enough to be representat ive. Bidens p o p u l i f o l i a is included despite a sample of only 18 cutt ings because they represent most of the ind iv idua ls encountered at the s i t e . Table 8 l i s t s several genetic measures of v a r i a b i l i t y for 22 populat ions. Two values are given for percent polymorphic l o c i , number of a l l e l e s per polymorphic locus and number of a l l e l e s per locus : they were ca lcu la ted using a l l e l e s present at a minimum frequency of e i ther 0.05 or 0.01. The values obtained using the 0.01 c r i t e r i o n may be more representat ive of large populat ions, but the others may provide a better basis for comparison of small and large samples. The polymorphic index value is i den t i ca l to the mean or expected heterozygosity used by some authors and represents the heterozygote frequency (averaged over a l l l o c i ) in a population conforming to Hardy-Weinberg assumptions. A l t e rna t i v e l y , i t can be considered as the proport ion of l o c i at which the average ind iv idua l is 5 2 TABLE 8. GENETIC MEASURES OF VARIABILITY IN 22 HAWAIIAN BIDENS POPULATIONS. NUMBER OF NUMBER OF TAXON AND % LOCI ALLELES PER ALLELES PER POLYMORPHIC POPULATION n POLYMORPHIC POLYMORPHIC LOCUS' INDEX LOCUS' AS YM B4 27 30 4 (30 4) 3 57 (3 14) -1 48 ( 1 35) 0 1 18 CERV B87 38 34 8 ( 17 4) 3 25 (4 75) 1 52 ( 1 22) 0 073 FORB F B 14 25 34 8 (26 1 ) 3 25 (3 67) 1 52 ( 1 35) 0 102 HAWA B50 52 34 8 (26 1 ) 3 88 (4 00) 1 74 ( 1 43) 0 135 B23 1 137 30 4 (26 1 ) 3 86 (3 83) 1 56 ( 1 39) 0 099 MENZ F B109 48 47 8 (43 5) 2 91 (2 30) 1 78 ( 1 39) 0 129 B130 57 47 8 (39 1 ) 2 91 (2 67) 1 78 ( 1 43) 0 148 B2 18 100 43 5 (30 4) 3 30 (3 43) 1 83 ( 1 43) 0 1 15 B2 19 G 1 34 8 (30 4) 2 88 ( 3 14) 1 39 ( 1 35) 0 103 MICR M B7S 21 43 5 (39 1 ) 2 70 (2 78) 1 56 ( 1 48) 0 125 MICR C B149 80 52 2 (39 1 ) 2 67 (2 56) 1 78 ( 1 39) 0 136 MICR K B197 22 43 5 (43 5) 2 70 ( 2 60) 1 56 ( 1 52) 0 144 MOLO B72 62 30 4 ( 17 4) 3 00 (4 25) 1 30 ( 1 13) 0 043 POPU B42 18 30 4 (26 1 ) 3 00 (3 33) 1 30 ( 1 26 ) 0 084 SAND S B44 2 1 26 1 ( 17 4) 3 33 (4 50) 1 26 ( 1 17) 0 058 B200 79 47 8 ( 34 8) 3 00 ( 2 62) 1 83 ( 1 30) 0 09 1 SAND C B34 21 47 8 (43 5) 2 45 (2 50) 1 56 ( 1 48) 0 1 16 TORT B15 79 34 8 (26 1 ) 2 88 (3 33) 1 39 ( 1 26) 0 074 B37 64 39 1 (34 8) 3 67 (3 00) 1 83 ( 1 43) 0 082 B2 13 1 10 43 5 (34 8) 3 50 (2 75) 1 9 1 ( 1 35 ) 0 103 B257 49 47 8 (39 1 ) 3 55 (3 00) 2 09 ( 1 57) 0 123 WIEB B259 35 39 1 (30 4 ) 2 89 (3 00) 1 52 ( 1 30) 0 079 MEAN 39 4 ( 3 1 6 ) 3 14 (3 23 ) 1 6 1 ( 1 36) 0 104 SD 7.69 (8.29) « 0 40 (0 68 ) 0 22 (0 11) 0 028 1 Values are given for 1 oc i at which at wh i ch the . most common a 1 1 e 1 e ha frequency of <0.99 or <0.95 (in brackets). 'Values are given for alleles present at a frequency of 20.01 or >0.05 (in brackets ) . 53 heterozygous. It provides a usefu l re la t i ve measure of v a r i a b i l i t y regardless of the actual proportion of heterozygotes in natural populat ions. A l l of the Bidens populations f a l l well within the range of v a r i a b i l i t y found for plant populat ions described in a recent review (Got t l i eb , 1981). The average values for percent polymorphic l o c i , number of a l l e l e s per polymorphic locus and polymorphic index are much higher than the averages ca lcu la ted for 28 se l f i ng species (4.4, 2.26 and .001, respect ive ly ) and s imi la r to or somewhat higher than for 21 outcrossing species (37, 2.9 and .086, r espec t i ve l y ) . Populations of Hawaiian Bidens are therefore not except iona l , but tend to have higher l eve l s of v a r i a b i l i t y than most species examined to date. Of the 23 l o c i surveyed in populations of Hawaiian Bidens, 14 (60.9%) are polymorphic. Seventy-one a l l e l e s were found in a l l , for an average of 3.09 a l l e l e s per locus and 4.43 a l l e l e s per polymorphic locus for the group as a whole. 4.2 Hawaiian Taxa In order to include information from plants at a l l l o c a l i t i e s , measures of v a r i a b i l i t y were also ca lcu la ted for a l l taxa in which the number of plants sampled was over 20 (again with the exception of B_;_ p o p u l i f o l i a , which f e l l just short of th i s c r i t e r i o n ) . These are l i s t e d in Table 9 along with means and standard deviat ions for the 15 taxa. Bidens mauiensis is 54 TABLE 9. GENETIC MEASURES OF VARIABILITY IN 15 HAWAIIAN BIDENS TAXA. NUMBER OF NUMBER OF % LOCI ALLELES PER ALLELES PER POLYMORPHIC SPECIES n POLYMORPHIC POLYMORPHIC LOCUS' INDEX LOCUS' AS YM 38 39 1 (34.8) 3 22 (3 25) 1 65 ( 1 52) 0 142 CERV 65 47 8 (39.1) 3 09 ( 2 67) 1 87 ( 1 43) • 0 120 FORB F 38 47 8 (34 .8) 2 64 (2 88) 1 65 ( 1 39) 0 1 10 HAWA 199 39 1 (21.7) 3 44 (4 40) 1 74 ( 1 35 ) 0 1 16 MAUI 26 56 5 (52.2) 2 46 (2 50) 1 78 ( 1 70) 0 172 MENZ F 287 52 2 (39 . 1 ) 3 33 (2 67) 2 13 ( 1 43) 0 140 MICR M 40 52 2 (43.5) 2 67 (2 50) 1 78 ( 1 48) 0 137 MICR C 80 52 2 (39. 1 ) 2 67 (2 56) 1 78 ( 1 39) 0 136 MICR K 23 43 5 (39. 1 ) 3 00 (2 89) 1 70 ( 1 52) 0 150 MOLO 64 30 4 (13.0) 3 00 (5 67) 1 30 ( 1 13) 0 042 POPU 19 30 4 (26. 1) 3 00 (3 33) 1 30 ( 1 26) O 082 SAND S 124 52 2 (39. 1 ) 3 00 (2 44 ) 1 96 ( 1 35) 0 099 SAND C 23 47 8 (43.5) 2 64 ( 2 40) 1 65 ( 1 43) 0 1 17 TORT 329 60 9 (39.1) 3 14 (2 56) 2 30 ( 1 39) 0 12 1 WIEB 35 39 . 1 (30.4) 2 89 (3 00) 1 52 ( 1 30) 0 079 MEAN 46 . 1 (35.6) 2 95 (3 05) 1 74 ( 1 40) 0 1 18 SD 8.99 (9.62) 0 28 (0 89) 0 27 (0 13) 0 032 1 Values are given for loci at which at which the most common allele has a frequency of <0.99 or <0.95 (in brackets). 'Values are given for alleles present at a frequency of >0.01 or >0.05 (in brackets ) . 55 i n c l u d e d i n t h i s a n a l y s i s a l t h o u g h i t was e x c l u d e d f r o m t h e l a s t one b e c a u s e o f s m a l l s a m p l e s i z e s f r o m e a c h p o p u l a t i o n . I n g e n e r a l , v a l u e s a r e h i g h e r f o r t a x a t h a n f o r p o p u l a t i o n s , s h o w i n g t h a t some d i f f e r e n t i a t i o n o f p o p u l a t i o n s w i t h i n s p e c i e s h a s o c c u r r e d . The o n l y m e asure o f v a r i a b i l i t y t h a t i s c o n s i s t e n t l y l o w e r f o r t a x a i s t h e number o f a l l e l e s p e r p o l y m o r p h i c l o c u s ; t h i s o c c u r s b e c a u s e t h e r e i s a r e l a t i v e l y g r e a t e r d i f f e r e n c e i n t h e number o f p o l y m o r p h i c l o c i i n t a x a t h a n i n t h e t o t a l number of a l l e l e s f o u n d i n t a x a . 4.3 A m e r i c a n S p e c i e s I n c o n t r a s t t o H a w a i i a n t a x a , t h e weedy A m e r i c a n s p e c i e s showed no v a r i a b i l i t y i n any p o p u l a t i o n . No p o l y m o r p h i c l o c i e x i s t i n any o f t h e p o p u l a t i o n s , r e s u l t i n g i n t h e minimum v a l u e o f 1 a l l e l e p e r l o c u s a n d a p o l y m o r p h i c i n d e x o f 0. F o r t h e A m e r i c a n t a x a a s a g r o u p , 33 a l l e l e s e x i s t a t t h e n o m i n a l 23 l o c i f o r an a v e r a g e 1.43 a l l e l e s p e r l o c u s a n d 2.25 a l l e l e s p e r p o l y m o r p h i c l o c u s . 56 V. DIFFERENTIATION OF POPULATIONS 5.1 Genetic Ident i t i es Many indices of genetic s i m i l a r i t y have been proposed for comparison of populations using a l l e l e frequency data (Sanghvi, 1953; Cava l l i -S forza and Edwards, 1967; Balakrishnan and Sanghvi, 1968; Hedrick, 1971; Rogers, 1972; Ne i , 1972). Ne i ' s genetic ident i t y and genetic distance measures are preferable to the others , however, because they are the only ones that have a b i o l o g i c a l basis as opposed to being simply abstract measures: Ne i ' s genetic distance (1972) estimates the average number of codon d i f ferences per locus that are detectable using e lec t rophores i s . An even more compell ing reason to use Ne i ' s indices is that they are used to estimate s i m i l a r i t y of populations in most other plant populations s tud ies , f a c i l i t a t i n g d i rec t comparison of r e su l t s . The formula for Ne i ' s genetic ident i t y i s : •j -J XY  where J^y ,Jx and J y are the means of Sx-,y; , ^.x,2- and ^>y* over a l l l o c i , and x s and y; are the frequencies of the i-th a l l e l e in the two populations being compared. Genetic ident i ty can range from 0 to 1 and is a f fected by both the presence and the 57 frequency of a l l e l e s . The value for a pa i r of populations is 1 i f the populations not only share a l l of the i r a l l e l e s but a lso have them at i den t i ca l f requencies. The value is 0 i f the populations share no a l l e l e s at the l o c i s tudied. Ne i ' s genetic distance is defined as: D = -ln I which approximates 1-1 at high values of I. Go t t l i eb (1977) found average genetic i d en t i t i e s for conspec i f i c populations in 22 flowering plant species to be 0.95±0.02. Thirteen comparisons of congeneric plant species had a mean genetic ident i ty of 0.67±0.07. In a more recent review consider ing only the studies in which 11 or more l o c i were surveyed (Got t l i eb , 1981), the mean I for conspec i f i c populations i s 0.975 for -inbreeding species and 0.956 for outbreeding spec ies , with standard errors of 0.01 and 0.11 respect i ve ly . Table 10 l i s t s the genetic ident i ty and distance values for a l l pairwise comparisons of Bidens populat ions. The values for comparisons involv ing only Hawaiian populations ( inc luding both int ra- and intertaxon comparisons) range from 0.872 to 0.996 with a mean of 0.949, while a l l comparisons of Hawaiian and American taxa range from 0.510 to 0.727 with a mean of 0.603 and a standard deviat ion of 0.054. The s i m i l a r i t i e s between a l l of the Hawaiian populations are what would be expected of i n t r a spec i f i c comparisons even though 14 taxa are involved, while the i r re l a t ionsh ip to populations of American TABLE 10. GENETIC IDENTITY AND DISTANCE VALUES' FOR POPULATIONS OF HAWAIIAN AND AMERICAN BIDENS TAXA POPULATION ASYM CERV FORB F HAWA HAWA MENZ F MENZ F MENZ F MENZ F MICR M MICR C MICR K MOLO B4 B87 B14 B231 B50 B109 B130 B218 B219 B78 B149 B197 B72 ASYM B4 O .909 0 .919 0 . 964 0 .949 0 .913 0 . 904 0 .912 0 .908 0 .944 0 .907 0 .920 0 .923 CERV B87 0 .096 0 . 933 0 .946 0 . 936 0 .965 0 . 962 0 .940 0 .960 0 . 959 0 .894 0 . 947 0 .909 FORB F B 14 0 .085 0 .070 0 . 950 0 . 925 0 . 942 0 . 924 0 . 928 0 . 937 0 .962 0 . 893 0 . 953 0 .906 HAWA B23 1 0 .037 0 .055 0 .051 0 .985 0 .937 0 .918 0 .910 0 .924 0 .973 0 .883 0 .943 0 .901 HAWA 650 0 .052 0 .066 0 .078 <3 .015 0 .919 0 .915 0 . 903 0 .916 0 .949 0 . 872 0 .931 0 . 875 MENZ F B109 0 .091 0 .036 0 .060 0 .065 0 .085 0 .977 0 .971 0 . 978 0 .968 0 . 943 0 .961 0 .954 MENZ F B 1 30 0 . 101 0 .038 0 .079 0 .086 0 .089 0 .024 0 . 989 0 .987 0 . 958 0 . 955 0 .944 0 .949 MENZ F B218 0 .093 0 .062 0 .075 0 .094 0 . 102 0 .029 0 .01 1 0 .991 0 . 958 0 . 972 0 .944 0 .974 MENZ F B2 19 0 .097 0 .04 1 0 .065 0 .079 0 .088 0 .022 0 .013 0 .009 0 .966 0 . 965 0 . 947 0 .959 MICR M B78 0 .057 0 .04 1 0 .038 0 .028 0 .052 0 .032 0 .042 0. . 043 0. .035 0 . 932 0 . 960 0. . 947 MICR C B149 0 .098 0 .112 0 .113 0 . 124 0 . 137 0 .059 0 .046 0. .028 0. .035 0 .070 0 .919 0. , 952 MICR K B 197 0. .084 0. .055 0. .048 0 .059 0. .072 0. .039 0. .057 0. 058 0. .054 0. .040 0 .085 0. .918 MOLO B72 0. .080 0. .096 0. . 098 0 . 104 0. . 134 0. .047 0. .053 0. 026 0. .042 0. .054 0. .050 0. .086 POPU B42s 0, ;063 0. .062 0. .053 0. .053 0. 086 0. .044 0. .05 1 0. 037 0. 04 2 0. .019 0. .070 0. .068 0. 025 SAND S B44 0. .090 0. 094 0. .079 0. .084 0. 121 0. 046 0. 056 0. 038 0. 049 0, .029 0. ,055 0. 081 0. 024 SAND S B200 0. 084 0. 053 0. 059 0. 072 0. 098 0. 020 0. 031 0. 016 0. 019 0. 030 0. .052 0. 048 0. 017 SAND C B34 0. 086 0. 07G 0. 076 0. 083 0. 1 12 0. 043 0. 046 0. 029 0. 037 0. 036 0. 062 0. 068 0. 022 TORT B37 0. 089 0. 030 0. 048 0. 059 0. 080 0. 01 1 0. 033 0. 033 0. 025 0. 036 0. 073 0. 026 0. 055 TORT B15 0. 1 1 1 0. 043 0. 061 0. 079 0. 102 0. 025 0. 04'3 0. 040 0. 033 0. 049 0. 087 0. 036 0. 06 1 TORT B213 0. 056 0. 026 0. 027 0. 017 0. 038 0. 030 0. 048 0. 054 0. 041 0. 01 1 0. 093 0. 028 0. 075 TORT B2 15 0. 054 0. 027 0. 033 0. 021 0. 048 0. 029 0. 052 0. 058 0. 044 0. 015 0. 094 0. 028 0. 072 WI EB B260 0. 090 0. 044 0. 050 0. 069 0. 09 1 0. 019 0. 030 0. 019 0. 020 0. 029 0. 06 1 0. 038 0. 030 AMPL 0. 518 0. 527 0. 519 0. 539 0. 489 0. 546 0. 460 0. 455 0. 476 0. 486 0. 478 0. 510 0. 508 TRIP 0. 518 0. 527 0. 519 0. 539 0. 489 0. 546 0. 460 0. 455 0. 476 0. 486 0. 478 0. 510 0. 508 FRON • 0. 435 0. 403 0. 476 0. 464 0. 407 0. 402 0. 324 0. 319 0. 34 1 0. 402 0. 345 0. 405 0. 375 CYNA 0. 640 0. 649 0. 64 1 0. 663 0. 610 0. 67 1 0. 580 0. 573 0. 595 0. 606 0. 598 0. 633 0. 626 'Genetic identity values are in the upper region of the table, and genetic distances in the lower region. TABLE 10. cont POPULATION POPU SAND S SAND S SAND C TORT TORT TORT TORT WIEB AMPL TRIP FRON CYNA B42 B44 B200 B34 B37 B15 B213 B257 B260 ASYM B4 0 . 939 0 .914 0 .920 0 .917 0 .915 0 . 895 0 .946 0 .947 0 .914 0 . 596 0 . 596 0 . 647 0 . 503 CERV B87 0 .939 0 . 9 10 0 .949 0 . 926 0 . 970 0 .958 0 . 974 0 . 973 0 . 957 0 . 590 0 . 590 0 .668 0 . 500 FORB F BM 0 . 949 0 . 924 0 . 943 0 k927 0 .953 0 .941 0 . 973 0 .967 0 . 952 0 . 595 0 . 595 0 . 621 0 .503 HAWA B23 1 0 . 948 0 .919 0 . 930 0 *920 0 .943 0 .924 0 .983 0 .979 0 .933 0 . 583 0 . 583 0 .629- 0 . 491 HAWA B50 0 ..9 18 0 . 886 0 .906 0 .894 0 .923 0 .903 0 .963 0 .953 0 .913 0 .613 0 .613 0 .665 0 . 520 MENZ F B109 0 .957 0 .955 0 . 980 0 .958 0 .990 0 .975 0 .971 0 .972 0 .981 0 . 579 o . 579 0 .669 0 .486 MENZ F B130 0 . 950 0 . 946 0 . 970 0 . 955 0 . 968 0 . 958 0 . 953 0 .950 0 .970 0 .631 0 .631 0 . 723 0 . 537 MENZ F B2 18 0. . 964 0 . 963 0 . 984 0 .971 0 . 968 0. . 960 0 .947 0 .943 0 .981 0. .634 0 . 634 0 . 727 0 . 542 MENZ F B2 19 0. . 959 0 .952 0 . 98 1 0 .964 0 . 975 0. . 967 0 .960 0 .957 0 .981 0. .621 0. .621 0, .711 0. . 529 MICR M B78 0. . 98 1 0 . 972 0. . 970 0 . 964 0. . 964 0. .952 0 . 989 0, . 985 0. .971 0. .615 0, ,6 15 0, . 669 0. . 522 MICR C B149 0. .933 0 . 946 0. . 949 0 . 940 0. .930 0. .917 0. .912 0. .911 0. .941 0. .620 0. .620 0, . 708 0. . 527 MICR K B197 0. 935 0 . 922 0. .953 0 .934 0. . 974 0. 965 0. .972 0. .973 0, ,962 0. 601 0. ,601 0, .667 0. . 507 MOLO B72 0. 975 0. .977 0. 983 0 .979 0. 946 0. 941 0. .928 0. 930 0. 971 0. 602 0. 602 0. .687 0. .513 POPU B42 0. . 986 0. 979 0 . 980 0. 952 0. 944 0. .967 0. 964 O. .972 0. 619 0. 619 0. 675 0. 528 SAND S B44 0. 014 0. 975 0 .980 0. 941 0. 934 0. 945 0. 942 0. 966 0. 601 0. 601 0. 669 0. 512 SAND S 8200 0. 021 0. 025 0 .983 0. 981 0. 975 0. 965 0. 965 0. 992 0. 606 0. 606 0. 693 0. 515 SAND C B34 0. 020 0. 020 0. 017 0. 955 0. 962 0. 950 0. 951 0. 977 0. 608 0. 608 0. 685 0. 515 TORT B37 0. 049 0. 061 0. 020 0. .046 0. 990 0. 980 0. 981 0. 987 0. 580 0. 580 0. 669 0. 490 TORT B15 0. 058 0. 069 0. 025 0. 039 0. 010 0. 967 0. 973 O. 983 0. 576 0. 576 0. 667 0. 486 TORT B213 0. 034 0. 057 0. 036 0. 05 1 0. 02 1 0. 034 0. 996 0. 972 0. 598 o. 598 0. 656 0. 506 TORT B257 0. 036 0. 060 0. 035 0. 050 0. 019 0. 028 0. 004 0. 970 0. 589 0. 589 0. 650 0. 496 WIEB B2G0 0. 028 0. 035 0. 008 0. 024 0. 013 0. 018 0. 028 0. 030 0. 598 0. 598 0. 683 0. 508 AMPL 0. 479 0. 508 0. 500 0. 498 0. 544 0. 551 0. 515 0. 530 0. 514 0. 957 0. 826 0. 739 TRIP ' 0. 479 0. 508 0. 500 , 0. 498 . 0. 544 0. 551 0. 515 0. 530 0. 514 0. 044 0. 870 0. 739 FRON 0. 393 0. 403 0. 367 0. 378 0. 402 0. 405 0. 421 0. 431 0. 381 0. 191 0. 140 0. 652 CYNA 0. 638 0. 670 o; 663 0. 663 0. 714 0. 72 1 0. 68 1 0. 702 0. 678 0. 302 0. 302 o. 427 60 taxa conform to expected values for i n t e r spec i f i c comparisons. Despite the high mean and small range of genetic i d e n t i t i e s , the comparisons of Hawaiian Bidens populat ions can be analyzed according to taxonomic r e l a t i onsh ip . Table 11 l i s t s the mean and associated values for intrataxon, in te rsubspec i f i c and i n t e r spec i f i c comparisons. A one-way ana lys is of variance shows these "treatments" to have a s i gn i f i c an t e f fec t on sample means (F=12.61, P<0.001, d. f .=2l using the number of populations minus one as the minimum estimate of degrees of freedom). Comparison of sample means using a 1-tailed t-test with the error mean sum of squares for variance (Sokal and Rohlf, 1969) gives a s i gn i f i c an t value of 2.12 for intrataxon vs. in te rsubspec i f i c comparisons (minimum d.f.=16-1=15) but a non-s i gn i f i c an t value of 0.601 for in te rsubspec i f i c vs . i n t e r spec i f i c comparisons (df=21, P<0.28). The genetic i d en t i t i e s of populations of the same taxon are thus s i g n i f i c a n t l y higher than those of populations from d i f f e ren t taxa, but populat ions of d i f f e ren t subspecies of one species are no more s imi lar than populations of d i f f e ren t spec ies . The genetic ident i ty values can a lso be analyzed according to the extent of geographic separation of populations to determine i f the i r a f f i n i t i e s are re la ted to the distance between them. The treatments for th i s ANOVA are comparisons of populations from the same i s l and , from adjacent is lands and from d is tant i s l ands . A l l intrataxon comparisons were excluded because populations of the same taxon had already been shown to be s i g n i f i c a n t l y more s imi la r than populations of d i f f e ren t 61 TABLE 11. GENETIC IDENTITIES FOR TAXONOMIC COMPARISONS OF 22 HAWAIIAN BIDENS POPULATIONS. COMPARISON n HIGH LOW MEAN SD INTRATAXON 14 0. .996 0. .967 0. .9814 0 .0085 INTERSUBSPECIES 5 0. .983 0. .919 0, .9548 0 .0285 INTERSPECIES 212 0. .992 0. .872 0. 9472 0 .0254 TABLE 12. GENETIC IDENTITIES FOR INTERTAXON GEOGRAPHIC COMPARISONS OF 22 HAWAIIAN BIDENS POPULATIONS. COMPARISON n HIGH LOW MEAN SD INTRA ISLAND 50 0 . 986 0 . 872 0 .9417 . 0 .0272 ADJACENT ISLANDS 82 0 . 992 0 . 875 0 .9532 0. .0236 DISTANT ISLANDS 85 0 . 990 0 . 886 O . 9452 O. 0251 62 taxa. Intrataxon comparisons would have biased the analys is because populations of the same taxon are usual ly found on the same is land or on adjacent i s l ands . Maui, Molokai and Lanai were treated as one is land because of the i r proximity and the existence of land bridges between them during the Pleistocene (Stearns, 1966). Table 12 l i s t s the range, mean and standard deviat ion for each "treatment". A one-way ANOVA again gives a s i gn i f i c an t value (F=3.84, P<0.05, df=2,21). A p r i o r i 2-tai led t-tests based on the ANOVA show a s i gn i f i c an t d i f ference between genetic i den t i t i e s of comparisons between populations on the same is land and on adjacent is lands (t=2.56, df=21, P<0.05) and between genetic i den t i t i e s of populations from adjacent and distant is lands (t=2.08, df=21, P=0.05). Su rp r i s ing l y , the greatest s i m i l a r i t i e s are found between populations of d i f f e ren t taxa on adjacent i s l ands , with populations of d i f f e ren t taxa on the same is land being least s im i l a r . Table 13 l i s t s genetic i d en t i t i e s and distances for Bidens taxa. A l l ind iv idua ls sampled are included in th i s ana l y s i s . The overa l l resu l ts are s imi la r to those obtained from comparisons of ind iv idua l populat ions, but th i s ana lys is includes B. mauiensis. 63 TABLE 13. GENETIC IDENTITY AND DISTANCE VALUES' FOR HAWAIIAN AND AMERICAN BIDENS TAXA. TAXON ASYM CERV FORB F HAWA MAUI . MENZ F MICR M MICR C MICR K MOLO AS YM 0 . 9 4 0 0 . 9 4 3 0 . 9 5 7 0 . 9 5 5 0 . 9 4 3 0 . 9 5 7 0 . 9 3 2 0 . 931 0 . 9 5 5 CERV 0 . 0 6 2 0 . . 9 6 7 0 . 9 6 9 0 . 9 5 2 0 . 9 6 6 0 . 9 7 9 0 . 9 0 5 0 . 964 0 . 9 1 8 FORB F 0. 0 5 8 0 . . 0 3 4 0 , . 96 1 0. 9 5 7 0 . 9 5 7 0 . , 9 7 9 0 . 9 1 2 0 . 9 6 3 0 . 931 HAWA 0. 0 4 4 0 . .031 0 . . 0 4 0 0 . 9 5 8 0 . 931 0 . . 9 7 6 0 . 8 8 2 0 . 9 4 3 0 . 8 9 7 MAUI 0. 0 4 6 0 . 0 5 0 0 . . 0 4 4 0 . 0 4 3 0 . 9 3 7 0 . , 9 7 3 0 . 8 9 8 0 . 9 2 9 0 . 9 3 0 MENZ F 0. 0 5 9 0 . . 0 3 5 0 . . 0 4 4 0 . . 0 7 2 0 . 0 6 5 0 . . 9 6 4 0 . 9 6 7 0 . , 9 5 6 0 . 9 6 8 MICR M 0. 04 4 0 . . 02 1 0 . .021 0 . 0 2 4 0 . 0 2 7 0 . 0 3 6 0 . 924 0 . 9 7 0 0 . 9 3 7 MICR C 0. 0 7 0 0 . . 0 9 9 0 . . 0 9 2 0 . . 125 0 . 108 0 . 0 3 4 • 0 . , 0 7 9 0 . 9 1 9 0 . 9 5 2 MICR K 0. 0 7 1 0 . . 0 3 6 0 . . 0 3 8 0 . . 0 5 9 0 . 0 7 4 0 . 0 4 5 0 . . 0 3 0 0 . 0 8 4 0 . . 9 1 8 MOLO 0. . 0 4 6 0 . . 0 8 6 0 . . 0 7 2 0 . . 109 0 . 0 7 2 0 . 0 3 2 0 . , 0 6 5 0 . 0 4 9 0 . , 0 8 6 POPU 0. 0 3 9 0 . . 0 4 6 0 . .031 0 . . 0 5 9 0 . 0 3 1 . 0 . 0 3 5 0 . , 0 2 8 0 . 0 7 1 0 . , 0 6 7 0 , . 0 2 5 SAND S 0. 0 4 7 0 . 0 4 1 0 . . 0 3 2 0 . . 0 6 7 0 . 0 4 4 0 . 0 1 6 0 . . 0 2 6 0 , , 0 5 0 0 , . 0 4 7 0 , , 0 1 6 SAND C 0 . 0 5 1 0 . 0 5 3 0 . . 0 4 8 ' 0 . 0 8 2 0 , . 0 4 2 0 . . 0 2 6 0 . 0 4 2 0 . 06 1 0 , . 0 6 1 0 , . 02 1 TORT 0. . 0 4 7 0 . 0 0 9 0 . . 0 1 9 0 . . 0 2 7 0 . . 0 3 9 0 . . 0 2 9 0 .01 1 0 , . 0 8 3 ' 0 , ,021 0 , . 0 6 5 WIEB 0. . 0 6 6 0 . 0 3 5 0 . 0 3 6 0 . 0 7 3 0 . . 0 6 8 0 . . 0 1 7 0 . 0 3 9 0 . . 0 6 4 0 . 0 3 8 0 . 0 3 4 AMPL 0. . 5 0 0 0 . 511 0 . 494 0 . 5 2 5 0 . . 5 1 7 0 . . 4 7 0 0 . 5 0 9 0 . 4 7 8 0 . 5 0 9 0 . 5 0 6 TRIP 0 . 5 0 0 0 . . 511 0 . 4 9 4 0 . 5 2 5 0 . . 5 1 7 0 . . 4 7 0 0 . 5 0 9 0 , . 4 7 8 0 . 5 0 9 0 . 506 FRON 0. . 4 0 7 0 . 4 0 3 0 . 4 3 9 0 . 4 4 8 0 . . 4 4 6 0 . . 3 3 6 0 . 4 2 6 0 , . 3 4 5 0 . 4 0 6 0 . 374 CYNA 0 . 6 6 8 0 . 6 7 8 0 . 6 5 8 0 . 6 9 5 0 . . 69 1 0 . . 6 3 3 0 . 6 7 9 0 , . 64 1 0 . 6 8 0 0 . 6 6 6 'Genetic identity values are in the upper region of the table, and genetic distances in the lower region. TABLE 13. cont. TAXON POPU SAND S SAND C TORT WIEB AMPL TRIP FRON CYNA ASYM 0 . 96 1 0 . 9 5 4 0 . 9 5 0 0 . 954 0 . 9 3 6 0 . 6 0 7 0 . 6 0 7 0 . 6 6 6 0 . 5 1 3 CERV 0 . 9 5 5 0 . 9 6 0 0 , 9 4 8 0 . 9 9 1 0 . 9 6 5 0 . 6 0 0 0 . 6 0 0 0 . 6 6 8 0 . 5 0 7 FORB F 0 . 9 6 9 0 . 9 6 8 0 . 9 5 3 0 . 981 0 . 9 6 5 0 . 6 1 0 0 . 6 1 0 0 . 6 4 5 0 . 5 1 8 HAWA 0 . 94 2 0 . 9 3 5 0 . 92 1 0 . 9 7 3 0 . 9 3 0 0 . 591 0 . 591 0 . 6 3 9 0 . 4 9 9 MAUI 0 . 9 6 9 0 . 9 5 7 0 . 9 5 9 0 . 9 6 2 0 . 9 3 5 0 . 596 . 0 . 596 0 . 6 4 0 0 . 501 MENZ F 0 , 9 6 6 0 . 9 8 4 0 . 974 0 . 9 7 2 0 . 9 8 3 0 . 6 2 5 0 . 6 2 5 0 . 7 15 0 . 53 1 MICR M 0 9 7 2 0 . 9 7 4 0 . 9 5 9 0 . , 9 8 9 0 . 9 6 2 0 . 601 0 . .601 0 . 6 5 3 0 . 5 0 7 MICR C 0 , 9 3 2 0 . 951 0 . , 94 1 0 , , 9 2 0 0 . 9 3 8 0 . 6 2 0 0 . 6 2 0 0 . 7 0 8 0 . . 5 2 7 MICR K 0 , 9 3 5 0 . , 9 5 4 0 , , 94 1, 0 . , 9 8 0 0 . , 9 6 3 0., 601 0 . 601 0 . 6 6 6 0. . 5 0 6 MOLO o 9 7 6 0 , , 9 8 4 0 , , 9 7 9 0 . . 9 3 7 0 , 9 6 6 0 , 6 0 3 0 . . 6 0 3 0 . 6 8 8 0 . . 5 1 4 POPU 0 , , 9 8 8 0 , . 9 8 1 0 , . 9 6 6 0 . . 9 6 6 0 , .6 19 0 . .6 19 0 . . 6 7 5 0 . . 5 2 9 SAND S 0 . 0 1 2 0 . 9 9 0 0 , . 9 7 5 0 . , 9 8 7 0 , , 6 0 9 0 . 6 0 9 0 . . 6 8 6 0 . . 5 1 7 SAND C 0 . 0 2 0 0 . 0 1 0 0 . 9 6 4 . o . . 9 7 6 0 , . 6 0 5 0 . . 6 0 5 0 . . 6 8 4 0 . 5 1 2 TORT 0 . 0 3 5 0 , , 0 2 5 0 , . 0 3 7 0 , , 9 7 8 . 0 , , 596 0 . . 596 •o. . 6 6 2 0 . 5 0 3 WIEB 0 . 0 3 4 0 , 0 1 3 0 . 0 2 4 0 . 0 2 2 0 . . 5 9 7 0 . . 5 9 7 0 . . 6 8 3 0 . 5 0 6 AMPL 0 . 4 7 9 0 , 4 9 6 0 . 5 0 3 0 . 5 1 8 0 , .5 16 0 . . 9 5 7 0 . . 8 2 6 0 . 7 3 9 TRIP 0 . 4 7 9 0 , 4 9 6 0 . 5 0 3 0 . 5 1 8 0 , 5 1 6 0 . 0 4 4 - 0 . . 8 7 0 0 . 7 3 9 FRON o . 394 0 . 3 7 6 0 . 3 8 0 0 . 4 1 2 0 . 38 1 0 . 1 9 1 0 . 140 0 . 6 5 2 CYNA 0 . 6 3 8 0 . 6 5 9 0 . 6 6 9 0 . 6 8 8 0 . 68 1 0 . 3 0 2 0 . 3 0 2 0 . 4 2 7 64 5.2 Gene D ivers i t y The organizat ion of genetic v a r i a b i l i t y within a group of organisms can be invest igated using gene d i ve r s i t y ana lys is (Nei, 1975). This method reveals the subdiv is ion of genetic v a r i a b i l i t y in Hawaiian Bidens as a whole instead of simply making pairwise comparisons of s i m i l a r i t y . Total gene d i ve r s i t y is subdivided into components us ing: H T =H^  +D,T , where H-r is the to ta l gene d i v e r s i t y , H s is the average gene d i v e r s i t y within populations and D, T is the average gene d i v e r s i t y among populat ions. Values of H T and H s are obtained independently for each locus and then averaged for a l l l o c i . H T is ca l cu la ted as H =1-2x : i , where lc-, is the mean frequency of the i-th a l l e l e over a l l populat ions. H 5 i s ca lcu la ted for each population by H* = 1-£x,* , (which i s equivalent to expected heterozygosity in a population using the Hardy-Weinberg model and is i den t i ca l to the polymorphic index), and is then averaged for a l l populat ions. D S T i s obtained by subtract ion. If each population maintains the amount of genetic v a r i a b i l i t y found in the group as a whole, then H^ and H 5 w i l l 65 be iden t i ca l and D 6 T w i l l equal 0. If populations d i f f e r in a l l e l e frequencies (whether because of mutation, gene flow, random d r i f t , se lec t ion or meiotic d r i ve ) , H s w i l l be smaller than HT.. The re la t i ve extent of d i f f e r e n t i a t i o n among populations is given by G-ST =D ^/H-,-, where G 5 T i s the coe f f i c i en t of gene d i f f e r en t i a t i on and can vary from 0 to 1. A l t e rna t i ve l y , D^/Hj is used by some authors ( e .g . , Brown, 1979). Table 14 presents gene d i ve r s i t y values separately for the 14 polymorphic l o c i in populations of Hawaiian Bidens. Considerable v a r i a b i l i t y among l o c i is evident even without inc luding the monomorphic l o c i , a l l of which have values of 0 for each category. No c lear pattern seems to ex i s t , and the conclusion is that many l o c i should be sampled in order not to bias the resu l ts of a study of genetic v a r i a b i l i t y . Table 15 gives the resu l t s of gene d i ve r s i t y ana lys is at a l l l o c i for a l l populat ions, populations within a taxon and for subspecies within a species . The amount of d i f f e r e n t i a t i o n among populat ions, as measured by G S T , is greatest when a l l populations are considered, i s lower for d i f f e ren t subspecies within a spec ies , and is least for populations belonging to the same taxon. This pattern supports the observation that genetic i den t i t i e s are highest when populations of the same taxon are compared. When only polymorphic l o c i are considered, D^-r/H5 averages 0.114 for populations of one taxon and is 0.419 for a l l 66 TABLE 14. GENE DIVERSITY AT 14 POLYMORPHIC LOCI IN 22 POPULATIONS OF HAWAIIAN BIDENS. LOCUS H3 G '•5T1 /H, PGI-4 0. 4089 0. 3086 0. 1003 0. 25 0. 32 PGI-5 0. 2943 0. 2428 0. 0515 0. 18 0. 21 PGM- 1 0. 2745 0. 1810 0. 0935 0. 34 0. 52 PGM-2 0. 0988 0. 0867 0. 0121 0. 12 0. 14 PGM-3 0. 2101 0. 1727 0. 0374 0. 18 0. 22 PGM-4 0. 3487 0. 2948 0. .0540 0. 15 0. 18 LAP- 1 0. 1467 0. . 1233 0. .0234 0. 16 0. 19 LAP-2 0. . 3478 0. . 2508 0 .0970 0. 28 0. 39 DIA- 1 0. . 5320 0 . 2877 0 . 2443 0. 46 0. 85 DIA-2 0 .0667 • 0 .0435 0 .0232 0. 35 0. 53 MDH- 2 0 .0612 0 .0500 0 .0112 0. 18 0. 22 MDH-3 0 .4505 0 .2310 0 .2195 0. 49 0. 95 MDH-5 0 . 1306 0 .1012 0 .0293 0. 22 0. 29 MDH-6 0 .0072 0 .0068 0 .0004 0. 06 0. 06 , Q V r = I W H T TABLE 15. GENE DIVERSITY FOR ALL LOCI IN HAWAIIAN BIDENS. CATEGORY ' S T D^/H S A 11 popu1 at i ons 22 0. 147 O. 104 0. .043 0, . 295 0 .4 19 Populations of a taxon HAWA 2 0. 125 0. 118 0. .007 0. 056 0 .059 MENZ F 4 0. 1 36 0. 1 24 0. .012 0. .088 0 .097 SAND S 2 0 . 086 0 . 074 0 .012 0 . 140 0. . 162 TORT 4 0. 108 0. .095 0 .013 0. . 120 0 . 137 MEAN 0. .114 0. . 103 0 .011 . 0. . 101 0 .114 Subspec i es MICR 3 0. .17 1 0. . 135 0 .036 0. .211 0 . 267 'G4T=D^r/Fif 67 populat ions. Brown (1979) ca lcu la ted averages of 0.17 and 1.18 for 20 outbreeding and 13 inbreeding species , respec t i ve l y . A l l of the values for Hawaiian Bidens are low consider ing that species with presumably mixed mating systems are involved. Again, th is is in agreement with the unusually high genetic i den t i t i e s found among a l l populations of th is group. 5.3 P r inc ipa l Component Analys is The re la t ionsh ips of Bidens populations and taxa can be i l l u s t r a t e d using p r i n c i pa l component analys is (PCA). This technique ordinates the groups being studied in a space of fewer dimensions than the number of var iab les measured by f inding cor re la t ions between the va r i ab les . Unlike c lus te r ing techniques which force the groups into a h ierarch ic c l a s s i f i c a t i o n , PCA permits overlapping c lus te rs and is preferable in s i tuat ions where obvious c lus te rs are absent (Sokal, 1974). The axes of var ia t ion ca lcu la ted in the analys is are l inear combinations of the o r i g i na l var iables and account for the largest poss ib le proport ion of v a r i a t i on . Each successive axis thus accounts for a progress ive ly smaller amount of the to ta l v a r i a b i l i t y present, and each may be made up of components of a l l of the o r i g i na l va r i ab l es . Figures 19-21 are p lots of the 22 Hawaiian Bidens populations on the f i r s t and second, f i r s t and t h i r d , and second and th i rd PCA axes, respect i ve ly , of analyses where a l l e l e 68 • El4 AXIS 1 (13.4% variance) F i g u r e 19. O r d i n a t i o n o f 22 H a w a i i a n B i d e n s p o p u l a t i o n s on t h e f i r s t a n d s e c o n d PCA a x e s . 69 j i 1 — i — 1 AXIS 1 (13.4% v a r i a n c e ) F i g u r e 2 0 . O r d i n a t i o n o f 22 H a w a i i a n B i d e n s p o p u l a t i o n s on t h e f i r s t a n d t h i r d PCA a x e s . 70 . a) o G ca •H > 0 0 CO H 6 B t ^ 6.14 AXIS 2 (11.6% v a r i a n c e ) F i g u r e 2 1 . O r d i n a t i o n o f 22 H a w a i i a n B i d e n s p o p u l a t i o n s on t h e s e c o n d and t h i r d PCA a x e s . 71 frequencies are the var iab les descr ib ing each populat ion. The most notable conclusion from the ana l ys i s , aside from the fact that only one population seems cons is tent ly d is junct from the res t , is that the f i r s t axis accounts for only 13.4% of the variance present, and the f i r s t seven account for a mere 64.3%. The i n a b i l i t y of th i s method to f ind an axis expla in ing a larger proport ion of the to ta l v a r i a b i l i t y demonstrates the hyperspherical nature of the data set and emphasizes the absence of pattern in the var ia t ion of the populat ions. Figure 22 is an ordinat ion of 15 Hawaiian Bidens taxa on the f i r s t two PCA axes and shows essen t i a l l y the same th ing, with no taxon appearing d i s juc t from the res t . When American taxa are included (Figures 23 and 24) two non-overlapping c lus te rs appear; one for the American and one for the Hawaiian p lants . The f i r s t seven axes s t i l l account for only 63.2% (when populations were used) and 69.0% (when taxa were used) of the to ta l var iance, however. The coe f f i c i en t s descr ib ing the contr ibut ion of var iab les to each axis are also low. Their absolute values for the f i r s t ax is of the ana lys is of 22 Hawaiian populat ions, for example, range from 0.008 to only 0.257 and no s ingle a l l e l e has a predominant e f f e c t . 72 CJ C •H M > CN CO AXIS 1 (14.4% variance) F i g u re 22. O r d i n a t i o n of 15 H a w a i i a n B i d e n s t a x a on t h e f i r s t and s e c o n d PCA a x e s . 73 6 "PP^tiNj » C Y M K j L_ i : —i 1 » 1 1-AXIS 1 (22.4% v a r i a n c e ) F i g u r e 23. O r d i n a t i o n o f 22 H a w a i i a n B i d e n s p o p u l a t i o n s a n d 4 p o p u l a t i o n s o f A m e r i c a n t a x a on t h e f i r s t and s e c o n d PCA a x e s . 74 0j >~X~WU • 1? fro 1-4 0 M V c _ ^ - FA 1 AXIS 1 (24.5% variance) F i g u r e 24. O r d i n a t i o n of 15 Hawaiian and 4 American Bidens taxa on the f i r s t and second PCA axes. 75 5.4 Cluster Analys is The Hawaiian populations were a lso subjected to a l inkage c lus te r ing technique producing a dendrogram. An unweighted pair-group method using ar i thmetic averages (Sneath and Sokal , 1973) resu l ts in the dendrogram of Figure 25.. Although dendrograms produce c lear arrangements of populations belonging to we l l-d i f fe rent ia ted species in P l e c t r i t i s (Layton, 1980) and Limnanthes (R i t land, pers. comm.), the resul ts with Hawaiian Bidens are less sa t i s f ac to r y . Populations belonging to a s ingle species or subspecies seem to be separated as often as not and the groups formed by the ana lys is are therefore probably a r t i f a c t s of the method rather than a r e f l e c t i on of any actual h i e r a r ch i ca l re l a t ionsh ip ex i s t ing among them. The fact that most c lus te rs are formed simply by the addi t ion of one population a lso suggests that structure i s being forced upon the data rather than being revealed. The analys is demonstrates yet again the c lose s i m i l a r i t i e s of the Hawaiian p lan ts . 76 _ ASYM B4 - HAWA B231 - HAWA B50 - CERV B87 - MICR M B78 - TORT B213 - TORT B257 - MICR K B197 - MENZ F B109 TORT B37 - TORT B15 - SAND S B200 " WIEB B260 " MENZ F B130 ~ MENZ F B218 " MENZ F B219 " MOLO B72 - POPU B42 -SAND S B44 -SAND C B34 FORB F B14 " MICR C B149 0.08 • 0 .06 0.04 0.02 0.0 GENETIC DISTANCE F i g u r e 25. D e n d r o g r a m o f 22 H a w a i i a n B i d e n s p o p u l a t i o n s . 77 VI. DISCUSSION Most enzyme systems or subsets of an enzyme system showing independent va r ia t ion in Hawaiian Bidens appear to be cont ro l l ed by two rather than three l o c i . This i s unexpected in a plant known to be hexaploid. Gene dupl icat ion events can occur through unequal crossing-over (Ohno, 1970), by crosses between ind iv idua ls with d i f f e r e n t , p a r t i a l l y over lapping, rec iproca l t rans locat ions (Burnham, 1962), or by t ranspos i t ion (Dover, 1982), but these are mechanisms a f fec t ing only one or a few l o c i . A dupl icated PGI locus in C la rk ia (Gott l ieb and Weeden, 1979) and dupl icated MDH and ADH l o c i in maize (Goodman et a l . , 1980; Schwartz and Endo, 1966) are examples of these phenomena. The gene dup l i ca t ion observed in Bidens, however, is a resul t of genome dupl i ca t ion a f f ec t ing a l l l o c i . Since enzyme systems assayed with natural substrates have s imi lar numbers of l o c i governing the i r production in d i p l o i d f lowering plants (Got t l i eb , 1982), three times as many l o c i should be i den t i f i ed in a hexaploid. Nearly a l l homoeologous l o c i are expressed in hexaploid Tr i t icum species (Hart and Langston, 1977) although the dupl icated genes have diverged in structure and funct ion. The genetic contro l of a l l enzyme systems studied in t e t rap lo id Tragopogon species can be accounted for by the genes inher i ted from the i r d i p l o i d progeni tors , and even the r e l a t i v e a c t i v i t i e s of gene products at ADH l o c i in the d ip lo ids are retained in the te t rap lo ids (Roose and Go t t l i eb , 1976). The loss of dupl icate gene expression in Hawaiian Bidens has presumably occurred by the f i xa t ion of nu l l a l l e l e s at 78 cer ta in l o c i . The s i l enc ing of a locus is f a c i l i t a t e d in po lyp lo ids by the presence of other l o c i capable of f u l f i l l i n g the biochemical requirements of organisms. Fe r r i s and Whitt (1980) found that t e t rap lo id catostomid f ishes reta in dupl icated gene expression at only 47% of the i r isozyme l o c i . They suggest that the rate of formation and f i xa t i on of nu l l a l l e l e s may be cha rac te r i s t i c of some enzyme systems because they found s ingly expressed l o c i to be more polymorphic than dupl icate l o c i . A large number of a l l e l e s would be expected to ar ise at a locus at which mutations eas i l y a l t e r the enzyme produced: many of these would specify molecules d i f f e r i n g only in e lect rophoret ic mob i l i t y , but some would speci fy molecules s u f f i c i e n t l y d i f f e ren t that enzyme a c t i v i t y is no longer apparent (nul l a l l e l e s ) . Because nu l l a l l e l e s seem to ex is t at low frequencies at several isozyme l o c i studied in Hawaiian Bidens, i t is not un l ike ly that they have ar isen and become f ixed at other l o c i as we l l . The r e l a t i v e l y high leve ls of genetic polymorphism within populations of Hawaiian Bidens compared with other higher plants are in agreement with observations of hexaploid T r i t i cum, in which extensive d i f f e r e n t i a t i o n among genomes has occurred (Jaaska, 1969; Hart and Langston, 1977). The unexpectedly low leve ls of d i f f e r e n t i a t i o n of the Hawaiian populations at isozyme l o c i cannot therefore be a t t r ibuted to lack of v a r i a b i l i t y , as i t can in the genet i ca l l y depauperate Su l l i v an t i a species ( So l t i s , 1982). Although a large number of a l l e l e s ex is t at many of the l o c i , very l i t t l e corre la ted d i f f e r e n t i a t i o n is 79 observed at the isozyme l o c i as a whole. The genetic i d en t i t i e s show that d i f f e r e n t i a t i o n among the Hawaiian taxa is no greater than the degree of d i f f e r e n t i a t i o n that has been documented for populations of the same taxon in cont inenta l species. Fourteen d i f f e ren t c l a s s i f i c a t i o n s would resu l t i f a taxonomy were erected on the basis of each polymorphic locus, and the populations could be lumped into one species on the basis of the high genetic ident i ty values i f a l l l o c i were considered together. Patterns ex is t in the genetic ident i t y data despite the general ly high values obtained for a l l poss ib le comparisons of Hawaiian Bidens populat ions. The s i g n i f i c a n t l y d i f f e ren t genetic i d en t i t i e s of intrataxon and intertaxon comparisons show that populations of the same species are more s imi la r to each other than to populations of other spec ies . The gene d i ve r s i t y ana lys is shows greatest d i f f e r e n t i a t i o n among populat ions when a l l Hawaiian populations are considered and least d i f f e r e n t i a t i o n when only the populations belonging to one taxon are considered, corroborat ing the existence of a taxonomic pat tern. The PCA and dendrogram analyses f a i l to reveal groups of populations corre la ted with taxonomic c l a s s i f i c a t i o n based on morphology, so no cor re l a t ion between morphological characters and isozyme characters ex is ts above the l e ve l of populations of the same taxon. On morphological grounds, Bidens molokaiensis and B. mauiensis are very s im i l a r , and B. fo rbes i i and B. cerv ica ta are c lose l y re la ted and s imi la r to B. sandvicensis . Bidens micrantha, B. menz ies i i , and B. tor ta form a th i rd 80 morphological group, while both B. hawaiensis and B. p o p u l i f o l i a are r e l a t i v e l y d i s t i n c t from a l l other taxa. None of these re la t ionsh ips are evident in isozyme data. Although one population or another seems to be separated from the others in a given PCA plot or in the dendrogram, they vary from ana lys is to ana l y s i s , and the genetic distances in the dendrogram and the proportion of var ia t ion dealt with by the PCA are much too small for any conclusion except that there is no evidence of well-marked d i f f e r en t i a t i on in isozymes. Another pattern in the genetic i den t i t i e s is geographical . Most taxa are e i ther endemic to one is land or are found on adjacent i s lands , suggesting that i n te r i s l and co lon izat ions are f a i r l y rare (Table 16). In conjuct ion with the fact that subspecies of one species are usual ly found on d i f fe rent i s l ands , th is implies that divergence and spec iat ion events often occur a f ter d ispersa l to an adjacent i s l and . Comparison of populations using genetic ident i ty values supports th is hypothesis. If intrataxon comparisons are removed from the ana l y s i s , the genetic i den t i t i e s for populations found on adjacent is lands are s i g n i f i c a n t l y higher than for comparisons invo lv ing populations on the same is land or on d is tant i s lands . Moreover, even the genetic i den t i t i e s for comparisons of populat ions from distant is lands exceed those for populations on the same i s l and . Thus the most c lose l y re lated taxa occur on adjacent i s lands . This suggests that island-hopping may often resu l t in only s l i gh t d i f f e r e n t i a t i o n of populat ions. If populat ions diverge s u f f i c i e n t l y to be named d i f f e ren t taxa, but TABLE 16. DISTRIBUTION OF BIDENS TAXA ON THE HAWAIIAN ISLANDS TAXON NIIHAU KAUAI OAHU MOLOKAI LANAI MAUI HAWAII B i dens amp 1ectens B i dens asynime t r i ca B i dens campy i otheca campy1otheca B i dens campy 1otheca pentamera B i dens campy 1otheca wai ho iens i s B i dens cervica ta B i dens conjuncta B i dens cosmo i des Bidens forbesii forbesii B i dens forbes i i kahili ens i s  B i dens hawa i ens i s B i dens hi 11ebrand iana hillebrand i ana B i dens hillebrand i ana po1ycepha1 a B i dens macrocarpa B i dens mau i ens i s Bidens menziesii menziesii B i dens menz i es i i f i 1 i form i s Bidens micrantha micrantha B i dens m i crantha  B idens micrantha  B i dens mo 1 oka 1 ens i s  B i dens popu1 i fo 1 i a  B i dens sandv i cens i s  B i dens sandv i cens i s confusa  B i dens torta • B i dens va1 i da Bidens wiebkei ctenophy11 a ka1ea1 aha sandvi cens i s Co 82 not enough to change a l l e l e frequencies at isozyme l o c i apprec iably , then intertaxon genetic i den t i t i e s would be expected to be highest for comparisons of populations on d i f f e ren t i s l ands . Examination of polyacetylenes has revealed that Hawaiian Bidens produce a l imi ted array of these compounds compared to cont inenta l spec ies . Most of the taxa surveyed for isozymes e i ther have no polyacetylenes in the i r leaves or produce minute amounts, although the roots of the d i f f e ren t taxa do have one common polyacetylene (Marchant, pers. comm.). The polyacetylene data are in agreement with the isozyme data in showing no cor re l a t ion with taxonomic or morphological divergence of the taxa, but isozymes are much more var iable than the polyacety lenes. Comparisons of Hawaiian Bidens with American taxa y i e lds genetic distance values expected for i n t e r spec i f i c comparisons, and p r i nc ipa l components ana lys is separates the two groups without any over lap. The pattern of genetic d i f f e r en t i a t i on of the Hawaiian populations can therefore be considered d i s t i n c t i v e to the is lands and not cha rac te r i s t i c of the genus. Although Bidens amplissima and B. t r i p a r t i t a have a high genetic i den t i t y , they are a lso very s imi lar morphological ly and are probably very c lose l y re l a ted . Aside from th is instance, a l l comparisons of Hawaiian with American taxa or comparisons of American taxa with each other give genetic ident i t y values much lower than for comparisons of Hawaiian populations among themselves. 83 Patterns of d i f f e r en t i a t i on analogous to that in Hawaiian Bidens are found in other groups of unrelated organisms that have undergone recent adaptive r ad i a t i on . Both the si lversword a l l i ance and Lipochaeta on Hawaii exhib i t patterns of speciat ion s imi la r to that found in Hawaiian Bidens. The three genera of the s i lversword a l l i ance (Argyroxiphium, Dubautia, and Wilkesia) exhib i t great morphological and eco log ica l d i v e r s i t y , yet reta in the capacity to interbreed (Carr and Kyhos, 1981). I n te rspec i f i c and even intergener ic hybrids occur commonly in nature. However, there is much cy to log i ca l d i v e r s i t y as we l l , inc luding both rec iproca l t rans locat ions and aneuploidy. The two Hawaiian sections of Lipochaeta have also undergone adaptive rad ia t ion in a number of morphological characters (Gardner, 1976; Rabakonandrianina, 1980). Both in t rasec t iona l and in te r sec t iona l hybrids can be obtained, although in te rsec t iona l hybrids have abnormal meiosis because of the d i f f e ren t p lo idy leve ls of the two sect ions . In t rasect iona l hybrids are f u l l y f e r t i l e . As in Bidens, l i t t l e natural hybr id iza t ion occurs between species because of eco log ica l d i f ferences between them and the r a r i t y of habitat jux tapos i t ion . Speciat ion appears to have occurred on separate is lands in the t e t r ap lo id sect ion but on the same is land within the d i p l o i d section (Gardner, 1976). Unfortunately , no data on isozymes are ava i lab le for these genera. Isozyme data are ava i lab le for Tetramolopium, however, another genus that has undergone adaptive rad iat ion in Hawaii. As in Hawaiian Bidens, a l l species are s imi la r at the l o c i studied (Crawford, pers. comm.). 84 Hawaiian Drosophila have been extensively studied morphologica l ly , behavioura l ly , cy togenet ica l l y and e l e c t ropho re t i c a l l y . There i s a contrast between patterns of speciat ion in Hawaii and on the mainland in th i s genus. Continental Drosophila evolut ion involves speciat ion in which inversion d i f ferences between species prevent successful interbreeding, and d i f f e ren t species do not show marked morphological and behavioural d i f ferences (Ayala et a l . , 1974). In Hawaii, on the other hand, there are great morphological and behavioural d i f ferences with very l i t t l e concomitant cytogenetic d i f f e r e n t i a t i o n (Carson et a l . , 1970). Isozyme studies on cont inenta l species have revealed genetic d i f f e r e n t i a t i o n in isozymes with genetic i den t i t i e s conforming to the taxonomic re la t ionsh ips of populations and to the degree of morphological divergence observed (Ayala et a l . , 1974; Av ise , 1976). Hawaiian taxa, however, are in general very s imi la r to each other at isozyme l o c i although leve ls of polymorphism within populations are s imi lar to those found in cont inenta l populations (Carson and Johnson, 1975; Carson et a l . , 1975; Johnson et a l . , 1975; Carson and Kaneshiro, 1976; Sene and Carson, 1977; Craddock and Johnson, 1979). As in Hawaiian Bidens, absence of genetic v a r i a b i l i t y cannot explain the lack of isozyme d i f f e r e n t i a t i o n among d i f f e ren t taxa. Unlike Bidens, the Hawaiian Drosophila species are reproduct ively i so la ted from each other because of behavioural mechanisms. This may not represent a more profound genetic d i f ference between species than the reproductive i so l a t i on of Bidens species in d i f f e ren t habitats although i t is 85 probably a more r igorous mechanism of i s o l a t i o n . The extent of morphological d i v e r s i t y re la t i ve to mainland spec ies , the low leve l of cytogenetic d i f f e r e n t i a t i o n , and the absence of genetic d i f f e r e n t i a t i o n in s t ruc tura l genes seem to be common to adaptive rad iat ion on oceanic i s l ands . The example of i s land speciat ion most s imi la r to Bidens is found in Partula (Johnson, 1977; Murray and Clarke , 1980). Partula is a genus of land sna i l s cons is t ing of 9 taxa on the i s land of Moorea in French Polynes ia . The group is highly polymorphic morphologica l ly , yet is homogeneous chromosomally. The taxa are capable of interbreeding, although not in a l l poss ib le combinations. Sympatric species remain d i s t i n c t from each other poss ib ly through behavioural d i f f e rences . No taxon is reproduct ively i so la ted from a l l others, however, so i t is t heo re t i c a l l y poss ib le for an a l l e l e to be passed from any one taxon to any other. The polymorphic species are mainly outbreeding but the sna i l s are capable of s e l f - f e r t i l i z a t i o n . A l l the Moorean taxa are probably descendants of a s ingle introduct ion of the genus to the i s l and , and co lon iza t ion of other nearby is lands occurred from Moorea. The isozyme data a lso show high polymorphism within populations but, in contrast with morphological data, l i t t l e d i f f e r e n t i a t i o n among populations has occurred. A l l the Moorean taxa could be considered one species i f a taxonomy of the group were based only on isozyme data. A mean genetic ident i t y of 0.91 was obtained for conspec i f i c populations with a range of 0.89 to 0.95, and the mean for i n t e r spec i f i c comparisons was i d e n t i c a l : 86 0.91 with a range of 0.84 to 0.98. F i n a l l y , when comparisons of Moorean and Tahi t ian taxa are made, i t is evident that the most c lose l y re lated species occur on d i f f e ren t i s l ands . It seems l i k e l y that co lon iza t ion of other is lands may lead to spec ia t ion , but with r e l a t i v e l y l i t t l e i n i t i a l d i f f e r e n t i a t i o n . Species showing more divergence may have been i so la ted from each other for a longer per iod . Although speciat ion could occur as a consequence of co lon iza t ion of a d i f f e ren t part of the same is land as we l l , the degree of genetic i so l a t i on conferred by i n t e r i s l and co lon izat ions may make th is a more common method of spec ia t ion . It is evident that i s land genera in which adaptive rad ia t ion has occurred share many s i m i l a r i t i e s in patterns of d i f f e r e n t i a t i o n and speciat ion regardless of whether they are plants or animals. Great morphological d i v e r s i t y , a mechanism preventing interbreeding between populations (whether behavioural ar e co log i ca l ) , l i t t l e chromosomal evo lu t ion , and l i t t l e divergence at isozyme l o c i d i s t ingu i sh these s i tuat ions from patterns of cont inenta l evo lu t ion . The s i m i l a r i t i e s of Polynesian land s n a i l s , f r u i t f l i e s , and beggar's t i cks in patterns of evolut ionary d i v e r s i f i c a t i o n are much greater than the i r s i m i l a r i t i e s with the i r cont inenta l r e l a t i v e s . The underlying mechanisms that have caused the patterns may be d i f f e r en t in these groups, however. In Hawaiian Drosophi la , the rapid rates of speciat ion have been a t t r ibuted to founder e f f ec t s which r ad i c a l l y increase inbreeding and cause gametic d i sequ i l ib r ium in only a small subset of the genetic v a r i a b i l i t y 87 found in the a n c e s t r a l p o p u l a t i o n (Mayr, 1954, 1955). T h i s may a l t e r s e l e c t i v e values of genes and favour the a l l e l e s which are compatible i n homozygous form with c e r t a i n a l l e l e s at other, l o c i . Carson (1970, 1975) l i m i t e d t h i s argument to only the " c l o s e d " p o r t i o n of the genome (not i n c l u d i n g genes coding f o r isozymes) a f t e r the e l e c t r o p h o r e t i c s i m i l a r i t y of most Hawaiian s p e c i e s was d i s c o v e r e d . Recent t h e o r i e s i n c o r p o r a t e sexual s e l e c t i o n arguments ( S p i e t h , 1974) with founder e f f e c t models to e x p l a i n the e x t e n s i v e morphological and b e h a v i o u r a l d i v e r s i t y of D r o s o p h i l a on Hawaii (Templeton, 1980a, 1980b). E x p l a n a t i o n s r e l y i n g on e f f e c t s of i n b r e e d i n g and sexual s e l e c t i o n are not c o n v i n c i n g f o r Bidens s p e c i e s , i n which i n b r e e d i n g i s common and sexual s e l e c t i o n , i f present at a l l , i s c o n f i n e d to gynodioecy i n some taxa. They may not even apply to P a r t u l a completely because i n b r e e d i n g i s not uncommon in s n a i l s , e i t h e r . The morphological and e c o l o g i c a l d i f f e r e n t i a t i o n i n Bidens c o n s i s t s of c h a r a c t e r s of more d i r e c t adaptive value to the p h y s i c a l environment than the d i f f e r e n t i a t i o n of D r o s o p h i l a which i s r e l a t e d to sexual s e l e c t i o n . Although some u n i f i e d e x p l a n a t i o n may yet account f o r a l l i n s t a n c e s of i s l a n d s p e c i a t i o n , i t seems more l i k e l y that d i f f e r e n t e v o l u t i o n a r y mechanisms have produced a s i m i l a r p a t t e r n of s p e c i a t i o n . Hawaiian Bidens are h i g h l y d i f f e r e n t i a t e d m o r p h o l o g i c a l l y and e c o l o g i c a l l y , but they are chromosomally homogeneous and r e t a i n the c a p a c i t y to i n t e r b r e e d . Although the d i f f e r e n c e s between taxa are g e n e t i c a l l y c o n t r o l l e d , the genetic divergence does not extend to s t r u c t u r a l gene l o c i . P o p u l a t i o n s are more 88 v a r i a b l e t h a n most p l a n t p o p u l a t i o n s , b u t l i t t l e g e n e t i c d i f f e r e n t i a t i o n h a s o c c u r r e d i n t h i s p o r t i o n o f t h e genome. P o p u l a t i o n s t h a t a r e v e r y s i m i l a r m o r p h o l o g i c a l l y a n d a r e c l a s s i f i e d i n t h e same t a x o n a r e a l s o v e r y s i m i l a r g e n e t i c a l l y , b u t t h e c o r r e l a t i o n d o e s n o t e x t e n d t o i n t e r t a x o n c o m p a r i s o n s . P o p u l a t i o n s o f d i f f e r e n t s p e c i e s a r e a s s i m i l a r a s p o p u l a t i o n s b e l o n g i n g t o d i f f e r e n t s u b s p e c i e s , a n d no g r o u p s o f s p e c i e s a r e e v i d e n t i n t h e i s o z y m e d a t a a l t h o u g h m o r p h o l o g i c a l g r o u p s e x i s t . 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Hawa i i an Taxa B. asymmetrica ( L e v i . ) S h e r f f B. e e r y i c a t a S h e r f f B. f o r b e s i i S h e r f f ssp. f o r b e s i i B. hawaiens i s (Gray) S h e r f f B. mau i ens i s (Gray) S h e r f f B. m e n z i e s i i (Gray) S h e r f f ssp. f i 1 i formi s ( S h e r f f ) Ganders and Nagata B. m i c r a n t h a Gaud. ssp. m i c r a n t h a B. m i c r a n t h a Gaud. ssp. ctenophyl1 a ( S h e r f f ) Nagata and Ganders B. m i c r a n t h a Gaud. ssp. k a l e a l aha Nagata and Ganders B. m o l 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 B. s a n d v i c e n s i s L e s s . ssp. s a n d v i c e n s i s B. sandv i cens i s L e s s . ssp. c o n f u s a Nagata and Ganders B. t o r t a S h e r f f B. w i ebke i S h e r f f American Taxa B. ampl i ss i ma Greene B. c y n a p i f o l i a H.B.K. B. f r o n d o s a L. A P P E N D I X B . A L L E L E F R E Q U E N C I E S I N B I D E N S P O P U L A T I O N S S T U D I E D A L L E L E ASYM ' CERV FORB F HAWA HAWA MENZ F MENZ F MENZ F MENZ F MICR M MICR C MICR K MOLO B4 B 8 7 B 1 4 B231 B 5 0 B 1 0 9 B 1 3 0 B 2 1 8 B 2 1 9 B 7 8 B 1 4 9 B 1 9 7 B 7 2 P G I - 1 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 1 . 0 0 1 . 0 0 P G I - 2 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 1 . 0 0 1 . 0 0 P G I - 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 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 P G I - 3 b 0 . 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 0 . , 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G I - 4 a 0 . 17 0 . 0 0 0 . 0 0 0 , 0 0 0 . 0 9 0 . 0 0 0 . 01 0 . 0 5 0 . , 0 0 0 . 0 0 0 . , 0 8 0 . 0 0 0 . 0 0 P G I - 4 b 0 . 3 3 0 . 0 3 0 . 0 0 0 . , 0 0 0 . , 0 0 0 . 0 0 0 . 01 0 . 0 0 0 , , 0 0 0 . 0 0 0 . , 0 0 0 . 0 0 0 . 0 0 P G I - 4 c 0 . 0 2 0 . 82 0 . 0 2 0 . , 12 0 . 0 7 0 . 19 0 . 34 0 . 0 5 0 . , 17 0 . 24 0 . 0 3 0 . 2 0 0 . 0 0 P G I - 4 d 0 . 48 0 . 15 0 . 94 0 . , 4 9 0 . ,31 0 . 8 0 0 . 64 0 . 86 0 . , 8 3 0 . 76 0 . , 9 0 0 . 8 0 1. 0 0 P G I - 4 e 0 . 0 0 0 . 0 0 0 . . 0 4 0 . 0 2 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 I - 4 f 0 . . 0 0 0 . . 0 0 0 . 0 0 0 . . 0 0 0 . ,01 0 . 01 0 . 0 0 0 . 04 0 . , 0 0 0 . 0 0 0 . , 0 0 0 . 0 0 0 . 0 0 P G I - 4 g P G I - 4 k 0 . . 0 0 0 . , 0 0 0 . , 0 0 0 . , 36 0 , .51 0 . 0 0 0 . 0 0 0 . 0 0 0 , , 0 0 0 . 0 0 0 . . 0 0 0 . 0 0 0 . . 0 0 0 , 0 0 0 . , 0 0 0 . 0 0 0 . . 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 I - 4 1 0 . . 0 0 0 . , 0 0 0 . , 0 0 0 . , 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 , , 0 0 0 . 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 P G I - 4 n 0 , . 0 0 0 . , 0 0 0 . , 0 0 0 , , 0 0 0 , , 0 0 0 . . 0 0 0 . 0 0 0 , , 0 0 0 , . 0 0 0 . 0 0 0 . . 0 0 0 . 0 0 0 . . 0 0 P G I - 5 a 0 . . 0 0 0 . ,01 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 I - 5 b 0 . . 0 0 0 . ,01 0 . . 0 0 0 , . 0 0 0 , ,01 0 , . 0 0 0 . 0 0 0 . , 0 0 0 . . 0 0 0 . 0 0 0 , .01 0 . 0 0 0 . . 0 0 P G I - 5 c . 0 . .81 0 . , 6 0 1. . 0 0 0 . . 74 0 . , 4 5 0 . . 7 5 0 . 54 0 . , 78 0 . ,61 0 . 76 0 . . 8 5 0 . 9 5 1. . 0 0 P G I - 5 d 0 . . 0 0 0 , , 26 0 . . 0 0 0 . . 25 0 , , 5 2 0 . . 24 0 . ,41 0 . , 2 0 0 , . 3 9 0 . , 19 0 . , i 1 0 . 0 0 0 . . 0 0 P G I - 5 e 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 . .01 0 . 0 0 0 . , 0 0 P G I - 5 i 0 . . 0 0 0 . . 0 0 0 . . 0 0 . 0 , . 0 0 0 , , 0 0 0 . . 0 0 0 . 0 0 0 . , 0 0 0 . . 0 0 0 . 0 0 . 0 . , 0 0 . 0 . 0 0 0 . . 0 0 P G I - 5 J .0. . 0 0 0 , . 0 0 0 . . 0 0 0 , . 0 0 0 , , 0 0 0 . , 0 0 0 . 0 0 0 . , 0 0 0 , . 0 0 0 . 0 0 . 0 . . 0 0 0 . 0 0 0 . , 0 0 P G I - 5 k 0 . , 0 0 0 . , 0 0 0 , . 0 0 0 , 0 0 0 . 0 0 0 . , 0 0 0 . , 0 0 0 . . 0 0 0 . 0 0 0 . , 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 P G I - 5 n 0 . . 19 0 . 12 0 . . 0 0 0 ,01 0 .01 0 . ,01 0 . , 04 0 . , 0 2 0 , . 0 0 0 . 0 5 0 .01 0 , . 0 5 0 . , 0 0 PGM - 1 a 0 . . 0 0 0 , , 0 0 0 . . 0 0 0 . . 0 0 0 , 0 0 0 . . 0 0 0 . , 0 6 0 . . 0 2 0 . . 0 0 0 . , 0 0 0 . . 0 0 0 . . 0 0 0 . . 0 0 P G M - 1b 0 . 77 0 . 96 0 . . 78 1, . 0 0 0 , . 9 6 o. , 9 4 0 . , 47 0 , . 5 0 0 . . 53 0 . , 9 0 0 .21 0 . . 9 5 0 . , 9 8 P G M - 1c 0 . 0 0 0 . . 0 0 0 . . 0 0 0 . 0 0 0 , 0 0 0 . 0 0 0 . , 4 3 0 , . 26 0 . 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 P G M - 1 d 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 , 0 4 0 . . 0 4 0 , . 18 0 . 39 0 . . 10 0 . 79 0 , . 0 5 0 , , 0 2 P G M - 1 g 0 . 0 0 0 . 0 0 0 . . 0 0 0 . 0 0 0 , . 0 0 0 . . 0 0 0 . , 0 0 0 , . 0 0 0 . 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 P G M - 1 h 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 0 . . 0 0 0 . 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 P G M - 1 n ' 0 . 23 0 . 0 4 0 . 22 0 . 0 0 0 . 0 4 0 . 0 2 0 , . 0 0 0 . 0 3 0 . 0 8 0 . . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 a 0 . 0 0 0 . 0 0 0 . 0 4 0 . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 0 .01 0 . 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 b 1 . 0 0 1 . 0 0 0 . 9 6 0 . 8 9 0 . 6 8 0 . 9 6 1 . 0 0 0 . 9 8 0 . 9 8 0 . . 8 5 0 . 9 8 1 . 0 0 1 . 0 0 PGM-2C 0 . 0 0 0 . 0 0 0 . 0 0 0 . 10 0 .31 . 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 d 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 2 0 . 0 0 0 . 0 0 P G M - 2 f 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 g 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 h 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 n 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 .01 0 . 0 4 0 . 0 0 0 .01 0 . 0 2 0 . 15 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 3 a 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 28 0 . 18 0 . 0 9 0 . 0 0 0 . 18 0 . 4 9 0 . 22 0 . 0 0 P G M - 3 b . 1 . 0 0 0 . 9 9 0 . 96 0 . 9 9 1 . 0 0 0 . 7 2 0 . 8 0 0 .91 0 . 9 3 0 . 8 2 0 .51 0 . 78 •0 . 9 7 P G M - 3 n 0 . 0 0 0 .01 0 . 0 4 0 .01 0 . 0 0 0 . 0 0 0 .01 0 . 0 0 0 . 0 7 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 3 P G M - 4 a 0 . 58 0 . 8 8 0 . 74 0 . 76 0 . 36 0 . 9 2 0 . 6 8 0 . 6 3 0 . 8 1 0 . 9 0 0 .61 0 . 5 0 0 . 9 3 o o ALLELE ASYM CERV FORB F HAWA HAWA' MENZ F MENZ F MENZ F MENZ F MICR M MICR C MICR K MOLO B4 B87 B-14 B231 B50 B109 B130 B218 B219 B78 B149 B197 B72 PGM-4D 0. 00 PGM-4C 0. 00 PGM-4n 0. 42 LAP- 1a 0. 00 LAP-ID 0. 00 LAP- 1c 0. 00 LAP-1d 1 . 00 LAP - 1 e 0. 00 LAP- 1n 0. 00 LAP-2a 0. 00 LAP-2D 0. 11 LAP-2C 0. 00 LAP-2d 0. 85 LAP-2e 0. ,00 LAP-2n 0. ,04 DIA- 1a 0, , 11 DIA-1b 0. ,87 DIA-1C 0. ,02 DIA-1d 0. ,00 DIA- 1e 0. ,00 DI A-2a 1 . ,00 DIA-2b 0. ,00 DIA-2C 0. .00 MDH- 1a 1 . .00 MDH-2a 0, ,00 MDH-2b 0, ,00 MDH-2C 0. .00 MDH-2d 1 .00 MDH-3a 0 . 34 MDH-3b 0 .66 MDH-4a 1 . .00 MDH-5a 1 .00 MDH-5b 0 .00 MDH-5C 0 .00 MDH-5d 0 .00 MDH-5e 0 .00 MDH-6a 1 .00 MDH-Gb 0 .00 xDH-1 a 1 .00 ME -1a 1 .00 ME - 1b 0 .00 HA -1a 1 .00 HA - 1b 0 .00 GLU- 1a 1 .00 0. 00 0. 13 0. 07 0. 00 0. 00 0. 00 0. 12 0. 13 0. 17 0. 00 0. 00 0. 00 0. ,00 0. 00 0. 00 0. 00 0. 00 0. 00 1, ,00 1. 00 0. ,95 0, ,00 0. 00 0. ,05 0, ,00 0. 00 0. ,00 0. .00 0. 00 0. , 16 1. ,00 1. 00 0456 0, ,00 0. ,00 0, ,00 0, .00 0. 00 0, , 24 0, .00 0, ,00 0. .00 0, .00 0. ,00 0, ,04 0, . 76 . 0, 07 0, .00 0, , 24 0. . 37 1, ,00 0, ,00 0. .07 0, .00 0 .00 0. . 50 0 .00 0 ,00 o. .00 0, .00 0. . 97 0, . 50 1, .00 0 .03 0, . 50 0, ,00 0 .00 0, .00 0, .00 1, .00 1, ,00 1, ,ob 0 .00 0, . 12 0, .00 0 .00 0, ,00 0, .00 0 .00 0, ,00 0 .00 1 .00 0, , 88 1, .00 0 .00 0 .00 0 .00 1 .00 1, .00 1 .00 .1 .00 1 .00 1 .00 0 .96 1 .00 1 .00 0 .00 0 .00 0 .00 0 .04 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 0. 13 0. 00 0. 01 0. 00 0. 00 0. 00 0. 51 0. 08 0. 30 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 06 0. 00 0. 96 0. 90 0. 95 0. 04 0. 03 0. ,00 0. 00 0. 01 0. 05 0. 15 0. 04 0. ,00 0. 60 0. 74 0, ,96 0. 00 0. 00 0. ,00 0. ,24 0. 00 0. .00 0. 00 0. ,03 0. ,00 0. ,01 0. 19 0. .04 0. , 12 0. ,92 ' 0. .95 0; 88 0, ,08 0. ,05 0, ,00 0, ,00 0. .00 0, ,00 0. .00 0, .00 0. ,00 0, ,00 0. .00 1. .00 1, .00 1, ,00 o, ,00 0, ,00 0, .00 0. .00 0, .00 0. .00 1. ,00 1, ,00 1, ,00 0, ,00 0, .00 0, .00 0, ,00 o. . 32 0, . 12 0 ,00 0. .00 0, .00 1, .00 0, . 68 0 .88 0, ,00 0, .21 0 .44 1, .00 0, . 79 0 . 56 1, .00 1 .00 1 .00 1, .00 1 .00 0 .96 0, ,00 0 .00 0 .00 0, ,00 0, ,00 0 .04 0, .00 0 .00 0 .00 0, .00 0 .00 0 .00 1, .00 1 .00 1 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 1, .00 1 .00 1 .00 0, .00 0 .00 : 0 .00 1, .00 1 .00 1 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 . 0. .07 0. 00 0. 00 0, ,00 0. 00 0. 00 0. 30 0. 19 0. 10 0, ,00 0. 00 0. 00 0, ,00 0. 00 0. 00 0, ,00 0. 00 0. 00 1, ,00 1. 00 0. 98 0, .00 0. ,00 o. ,00 0, .00 0. ,00 0. ,02 0, .00 0, ,00 0, ,02 0, .92 1. ,00 0, 92 0, .00 0. ,00 0, ,00 0 .06 0. ,00 0, ,00 0 .02 0. ,00 0. ,00 0 .00 0. .00 0. .05 1 .00 . 0. .95 0, . 16 0 .00 0. .05 0. . 58 0 .00 0, ,00 0. . 26 0 .00 0, .00 0, .00 0 .00 0, ,00 0, .00 1 .00 1, ,00 1, .00 0 .00 0, .00 0, .00 0 .00 0 .00 0, .00 1 .00 1 .00 1 .00 0 .00 0, .00 0 .00 0 .02 0, .00 0 .00 0 .00 0 .00 0 .00 0 .98 1 .00 1 .00 0 .63 0 . 4 1 0 . 37 0 . 37 0 . 59 0 .63 1 .00 1 .00 1 .00 0 . 98 1 .00 1 .00 0 .00 0 .00 0 .00 0 .02 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 0. 00 0. 00 0. 00 0. 01 0. 00 0. 00 0. 39 0. 50 0. 07 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 07 0. 00 0. 92 0. 60 1. 00 0. 00 0. 33 0. 00 0. 08 0. 00 0. 00 0. 00 0. ,29 0. ,00 0. 61 0, ,71 0. 56 0. 00 0. .00 0. 00 0. 37 0, ,00 0, ,44 0. 00 0, ,00 0. ,00 0. 02 0, .00 0, ,00 0. 89 0 • 41 0, ,92 0. , 1 1 0, ,02 0, .08 0. 00 0, . 39 0, ,00 0. ,00 0, . 18 0, ,00 0. 00 0, ,00 0. .00 0. ,99 1, .00 1, ,00 0, ,01 0. .00 0, ,00 0, ,00 0. .00 0, .00 1, ,00 1. .00 1. .00 0. .00 0, .00 0, .00 0. .02 0, .00 0, .00 0, .00 0 .00 0, .00 0, .98 1 .00 1. .00 0, . 69 0 .00 0, . 97 0 .31 1 .00 0 .03 1, .00 1 .00 1 .00 1 .00 0 . 86 0 .97 0, .00 0 .00 0 .00 0 .00 0 . 14 0 .00 0 .00 0 .00 0 .03 0 .00 0 .00 0 .00 1 .00 0 .93 1 .00 0 .00 0 .07 0 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0 .00 0 .00 . 0 .00 1 .00 1 .00 1 .00 0 .00 0 .00. 0 .00 1 .00 1 .00 1 .00 A L L E L E POPU SAND S SAND S SAND C TORT TORT . TORT TORT WIEB AMPL T R I P FRON CYNA B 4 2 B 4 4 B 2 0 0 B34 B 3 7 B 1 5 B 2 1 3 B 2 5 7 B 2 6 0 P G I - 1 a 1 . 0 0 1. 0 0 1. 0 0 P G I - 2 a 1 . 0 0 1. 0 0 1. 0 0 P G I - 3 a 1 . 0 0 1. 0 0 1. 0 0 P G I - 3 b 0 . 0 0 0 . 0 0 0 . 0 0 P G I - 4 a 0 . 0 0 0 . 0 0 0 . 0 0 P G I - 4 b 0 . 0 0 0 . 0 0 0 . . 0 3 P G I - 4 C 0 . 22 0 . 0 0 0 . 0 6 P G I - 4 d 0 . 78 1. . 0 0 0 . , 9 0 P G I - 4 e 0 . 0 0 0 . 0 0 0 . , 0 0 P G I - 4 f 0 . 0 0 0 . 0 0 0 . ,01 P G I - 4 g 0 . 0 0 0 , , 0 0 0 . , 0 0 P G I - 4 k 0 . 0 0 0 . , 0 0 0 . . 0 0 P G I - 4 1 0 . 0 0 0 . , 0 0 . 0 . . 0 0 P G I - 4 n 0 . 0 0 0 . , 0 0 0 . . 0 0 P G I - 5 a 0 . 0 0 0 . , 0 0 0 , . 0 0 P G I - 5 b 0 . . 0 0 0 . . 0 0 0 . . 0 0 PGI-5C 1 . . 0 0 1. . 0 0 0 . . 9 9 P G I - 5 d 0 . . 0 0 0 , , 0 0 0 . . 0 0 P G I - 5 e . 0 . . 0 0 0 . . 0 0 0 , .01 P G I - 5 i 0 , . 0 0 0 , , 0 0 0 . 0 0 P G I - 5 J 0 , , 0 0 0 . . 0 0 0 . , 0 0 P G I - 5 k O. . 0 0 0 , . 0 0 0 . 0 0 P G I - 5 n 0 . , 0 0 0 . . 0 0 0 . 0 0 P G M - 1 a 0 . . 0 0 0 . 0 0 0 . 0 0 P G M - 1 b 1 . . 0 0 0 . 9 8 0 . 8 6 P G M - 1 c 0 , 0 0 0 . 0 0 0 . 0 0 P G M - 1 d 0 . 0 0 0 . 0 0 0 .01 P G M - 1 g 0 . 0 0 0 . 0 0 . 0 . 0 0 P G M - 1 h 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 1 n 0 . 0 0 0 . 0 2 ' 0 . 13 P G M - 2 a 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 b 1 . 0 0 1 . 0 0 0 . 86 P G M - 2 C 0 . 0 0 0 . 0 0 0 . 14 P G M - 2 d 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 f 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 g 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 h 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 n 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 3 a 0 . 0 0 0 .41 0 .01 P G M - 3 b 0 . 9 8 0 . 5 9 0 . 9 6 P G M - 3 n 0 . 0 2 0 . 0 0 0 . 0 3 P G M - 4 a 0 . 9 3 1 . 0 0 0 . 9 7 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 1. , 0 0 1. . 0 0 1. 0 0 1. 0 0 0 . 0 0 0 . , 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 0 . , 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 0 . , 04 0 . , 0 0 0 . 0 2 0 . 04 0 . , 07 0 . , 12 0 . . 11 0 . 27 0 . 31 0 . , 9 3 0 , , 8 0 0 . . 8 8 0 . 6 9 0 . 6 5 0 . . 0 0 0 . , 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 0 . , 0 5 0 . , 0 0 0 . 0 2 0 . 0 0 0 , * 0 . , 0 0 0 . . 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 , 0 0 0 . , 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 . . 0 0 0 . . 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 0 . . 0 0 0 . ,01 0 . 01 0 . 0 0 0 . . 0 0 0 . . 0 0 0 . . 0 0 0 . 0 0 0 , 01 0 . , 0 0 0 . . 0 2 0 . . 13 0 . , 0 3 0 . 04 0 , . 9 3 0 , .91 0 . , 87 0 . 85 0 . 88 0 , . 0 5 0 . . 0 6 0 , . 0 0 0 . 11 0 . , 0 6 0 , . 0 2 0 , . 0 0 0 . . 0 0 0 . , 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 0 . , 0 0 0 , , 0 0 0 . 0 0 0 . 0 0 0 , 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 .01 0 . 0 0 0 . . 0 2 0 . ,01 0 . 0 0 0 . 0 0 . 0 . 0 0 0 . . 0 0 0 . . 0 0 0 . 8 8 1, . 0 0 1 . 0 0 1. , 0 0 0 . . 9 8 0 . 0 0 0 , . 0 0 0 . 0 0 0 , . 0 0 0 .01 0 . 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 0 , . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 , 0 0 0 . 0 0 0 . 12 0 . 0 0 0 . 0 0 0 . 0 0 0 .01 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 9 8 1 . 0 0 1 . 0 0 0 . 9 0 0 .93 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 6 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 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 2 0 . 0 0 0 . 0 0 0 . 0 4 0 . 0 7 0 . 0 0 0 . 0 9 0 .01 0 . 0 4 0 . 0 2 0 . 9 0 0 . 8 8 0 . 9 9 0 . 9 3 0 . 9 0 0 . 10 0 . 0 4 0 . 0 0 0 . 0 4 0 . 0 8 0 . 9 0 0 . 8 6 0 . 9 0 0 . 8 0 0 . 9 1 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 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1. 0 0 1. 0 0 1. 0 0 1. 0 0 0 . 0 0 0 . 0 0 0 . 0 0 . 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 8 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 9 2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 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 1. 0 0 1. 0 0 1. 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1. 0 0 0 . 0 0 0 . 0 0 0 . 0 0 . 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 0 . . 0 0 0 . , 0 0 0 , 0 0 0 . . 0 0 0 . 9 8 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 2 0 , . 0 0 0 . , 0 0 0 . . 0 0 . 0 , . 0 0 0 . . 0 0 1, . 0 0 1. , 0 0 0 . . 0 0 0 , . 0 0 0. !oo 0 . . 0 0 0 . , 0 0 0 . . 0 0 1, . 0 0 0 . , 0 0 0 , . 0 0 0 . , 0 0 1. . 0 0 0 , . 0 0 0 . , 0 0 0 . . 0 0 0 . , 0 0 0 . . 0 0 0 . . 0 0 0 . . 0 0 0 . 0 0 0 . , 0 0 0 . . 0 0 0 , . 0 0 0 , . 98 0 , . 0 0 0 . . 0 0 0 , . 0 0 0 . , 0 0 0 . . 0 0 0 . . 0 0 0 . 0 0 0 , . 0 0 0 . 0 0 0 , . 0 0 0 . 0 0 0 , . 0 0 0 , . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 1, . 0 0 1, . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 0 : . 0 2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 o'. . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 9 2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 8 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 1 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 1 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 9 3 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 0 . 0 7 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 7 2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 A L L E L E POPU SAND S SAND S SAND C TORT TORT TORT TORT WIEB AMPL T R I P FRON CYNA B 4 2 B 4 4 B 2 0 0 B 3 4 B 3 7 B 1 5 B 2 1 3 B 2 5 7 B 2 6 0 P G M - 4 D 0 . . 0 0 PGM-4C 0 . . 0 0 P G M - 4 n 0 . .07 L A P - 1 a 0 . . 0 0 L A P - 1b 0 . . 0 0 L A P - 1c 0 . . 0 0 L A P - 1 d 1 , . 0 0 L A P - 1 e 0 . . 0 0 L A P - 1 n 0 . . 0 0 L A P - 2 a 0 . . 0 0 L A P - 2 b 0 . .81 L A P - 2 C 0 . . 0 0 L A P - 2 d 0 . . 0 0 L A P - 2 e 0 , , 19 L A P - 2 n 0 . . 0 0 D I A - 1a 0 . . 4 0 D I A - 1 b 0 . 6 0 D I A - 1c 0 . . 0 0 D I A - 1d 0 . . 0 0 DI A - 1 e 0 . . 0 0 D I A - 2 a 0 . 83 D I A - 2 b 0 . . 17 D I A - 2 C 0 . . 0 0 MDH-1 a 1 . 0 0 M D H - 2 a 0 . . 0 0 M D H - 2 b 0 . 0 0 M D H - 2 c 0 . 0 0 M D H - 2 d 1 . 0 0 M D H - 3 a 0 .77 MDH-.3b 0 . 23 M D H - 4 a 1 . 0 0 M D H - 5 a 1 . 0 0 M D H - 5 b 0 . 0 0 MDH-5C 0 . 0 0 M D H - 5 d 0 . 0 0 M D H - 5 e 0 . 0 0 M D H - 6 a 1 . 0 0 M D H - 6 b 0 . 0 0 x D H - 1 a 1 . 0 0 ME. - 1a 1 . 0 0 ME - 1 b 0 . 0 0 HA - 1 a 1 . 0 0 HA - 1b 0 . 0 0 G L U - 1a 1 . 0 0 0 . 0 0 0 . .01 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 , , 0 2 0 . 10 0 . 0 0 0 . , 0 2 0 , 0 0 0 . 0 0 0 , , 0 8 0 . 0 0 0 . 0 0 0 , , 0 0 0 , , 0 0 1. . 0 0 0 , . 9 0 0 . , 9 4 0 , 0 0 0 , , 0 0 0 . . 0 0 0 . , 0 0 0 . , 0 0 0 . , 0 6 0 . , 0 0 0 , , 17 0 , . 0 0 0 . , 9 3 0 , , 82 0 , ,81 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 7 0 . 0 2 0 . . 19 0 . . 5 0 0 , . 9 0 0 , . 6 8 0 . , 5 0 0 . 10 0 . , 32 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 1. , 0 0 1, . 0 0 1, , 0 0 0 . , 0 0 0 , . 0 0 0 . , 0 0 0 . , 0 0 0 , . 0 0 0 , , 0 0 1. , 0 0 ' 1, . 0 0 1, , 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 1. . 0 0 1 . 0 0 1, , 0 0 0 . , 9 3 0 . 5 5 , 0 , , 79 0 . . 0 7 0 , . 4 5 0 , .21 1. . 0 0 1 . 0 0 1, , 0 0 0 . , 9 8 0 . 9 5 0 , . 4 8 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 2 0 . 0 4 0 . 38 0 . . 0 0 0 . 0 0 0 , . 0 0 0 . 0 0 0 .01 0 . 14 1 . 0 0 1 . 0 0 1 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1, . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 1 . 0 0 1, . 0 0 0 . 0 0 . 0 . 0 0 0 , . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 0 . , 0 0 0 . , 0 0 0 . 0 2 0 . 0 0 0 , , 0 0 0 . 0 0 0 . , 14 0 . , 10 0 . 18 0 . , 0 0 0 . , 0 0 0 . 0 0 0 , , 0 0 0 , , 0 0 0 . 0 0 0 , . 10 0 , ,31 0 . 0 0 0 . . 8 3 0 , , 6 9 0 . 94 0 . . 0 2 0 . , 0 0 0 . 0 0 0 , . 0 6 0 . 0 0 0 , 0 6 0 , . 0 3 0 . . 0 0 0 . 0 6 0 . . 86 1. , 0 0 0 . , 9 0 0 . . 0 0 0 . . 0 0 0 . , 0 0 0 . . 0 2 0 . . 0 0 0 , . 0 0 0 , . 0 4 0 . . 0 0 0 . , 0 0 0 . . 0 6 0 , . 0 0 0 . , 0 4 0 , . 9 6 1, . 0 0 0 . ,31 0 , . 0 4 0 , . 0 0 0 . , 6 0 0 , , 0 0 0 , . 0 0 0 . 07 0 , , 0 0 0 , , 0 0 0 . 0 3 0 . , 0 0 0 , . 0 0 0 . , 0 0 1. , 0 0 1, . 0 0 0 . 97 0 , , 0 0 0 , . 0 0 0 . , 0 3 0 . , 0 0 0 , . 0 0 0 . 0 0 1, , 0 0 1, . 0 0 1, , 0 0 0 , , 0 0 0 , . 0 0 0 . , 0 0 0 , . 0 0 0 , . 0 0 0 , , 0 0 0 , , 0 9 0 , . 0 0 0 . , 0 0 0 .91 1 . 0 0 1, , 0 0 0 , . 0 0 0 , . 0 7 0 . , 0 0 1. . 0 0 0 . 9 3 1. . 0 0 1. . 0 0 1 . 0 0 1. . 0 0 0 . . 95 0 .61 0 . . 9 6 0 , . 0 0 0 . 0 0 0 . . 0 0 0 . 0 5 0 . 39 0 , . 0 4 0 , . 0 0 0 . 0 0 0 . . 0 0 0 , . 0 0 0 . 0 0 0 , , 0 0 1, . 0 0 0 . 9 9 1, . 0 0 0 , . 0 0 0 .01 0 , . 0 0 1 . 0 0 1 . 0 0 1, . 0 0 1, . 0 0 1 . 0 0 1, . 0 0 0 , . 0 0 0 . 0 0 0 . , 0 0 1, . 0 0 1 . 0 0 1, . 0 0 0 , . 0 0 0 . 0 0 0 . . 0 0 1, . 0 0 1 . 0 0 1, . 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 0 . , 0 0 0 . 27 0 , , 0 0 0 . 0 9 0 . 0 2 1. , 0 0 0 . 0 0 0 . , 0 0 0 , 0 0 0 . 0 5 0 . , 0 0 0 , , 0 0 0 . 19 0 . , 0 0 0 . . 0 0 0 . 74 1. . 0 0 0 , . 0 0 0 . 0 0 0 . , 0 0 0 , . 0 0 0 . , 0 2 0 , . 0 0 1, , 0 0 0 . , 0 2 0 , . 0 0 0 , 0 0 0 . , 75 1. . 0 0 1, , 0 0 0 . , 0 6 0 , . 0 0 0 , . 0 0 0 . , 0 8 0 . . 0 0 0 , . 0 0 0 , , 0 2 0 , , 0 0 0 , . 0 0 0 . , 0 6 0 , , 0 0 o. . 0 0 0 . , 34 0 . , 8 8 0 , . 0 0 0 . , 5 3 0 , , 0 5 0 , . 0 0 0 . 0 9 0 , , 0 7 0 , . 0 0 0 . 0 3 0 . . 0 0 0 , , 0 0 0 . 0 0 0 , , 0 0 1, . 0 0 0 . , 9 8 1, . 0 0 0 , . 0 0 0 . , 0 2 0 . , 0 0 0 . 0 0 0 . 0 0 0 , . 0 0 1, . 0 0 1. , 0 0 1. . 0 0 1, . 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 1. . 0 0 1 . 0 0 1 . 0 0 0 . . 0 0 0 , . 3 7 1 . 0 0 1, , 0 0 0 , . 6 3 0 . 0 0 1, . 0 0 1 . 0 0 1 . 0 0 0 , ,91 0 . 9 0 1 . 0 0 0 , . 0 0 0 . 0 2 0 . 0 0 0 , . 0 9 0 . 0 8 0 . 0 0 0 . . 0 0 0 , . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1, , 0 0 1 . 0 0 1 . 0 0 0 , , 0 0 0 . 0 0 0 . 0 0 1, . 0 0 1 . 0 0 . 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 0 , , 0 0 0 . 0 0 0 . 0 0 1, . 0 0 1 . 0 0 1 . 0 0 0 , 0 0 0 . 0 0 0 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 0 . , 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 0 . , 0 0 0 . oo 1. , 0 0 1. 0 0 1. 0 0 0 , , 0 0 0 . 0 0 0 , 0 0 0 , , 0 0 0 . , 0 0 0 . , 0 0 0 , . 0 0 0 . , 0 0 0 , , 0 0 0 , , 0 0 0 . , 0 0 0 , , 0 0 0 . . 0 0 0 , , 0 0 0 . . 0 0 1. . 0 0 1, , 0 0 1, . 0 0 0 . . 0 0 0 . , 0 0 0 , . 0 0 1, . 0 0 1. , 0 0 1, . 0 0 0 , . 0 0 0 . , 0 0 0 , . 0 0 0 . 0 0 0 . . 0 0 0 . . 0 0 0 , , 0 0 0 . . 0 0 0 , . 0 0 0 , . 0 0 0 , . 0 0 0 . . 0 0 0 , 0 0 1, . 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 1, . 0 0 0 , , 0 0 1, . 0 0 0 . . 0 0 1, , 0 0 0 . . 0 0 0 , . 0 0 0 , , 0 0 0 . , 0 0 1, . 0 0 0 , , 0 0 1, , 0 0 1 . 0 0 1. . 0 0 1, . 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 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1, . 0 0 1, . 0 0 0 . 0 0 0 . 0 0 0 , . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1, . 0 0 1 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 , . 0 0 0 , . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 04 APPENDIX C. A L L E L E FREQUENCIES IN BIDENS TAXA STUDIED. A L L E L E ASYM CERV FORB F HAWA MAUI MENZ F MICR M MICR C MICR K MOLO P G I - 1 a 1 . 0 0 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 P G I - 2 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 P G I - 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 1. 0 0 1. 0 0 P G I - 3 b 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 I - 4 a 0 . 1 2 0 . 0 0 0 . 0 0 0 . 0 2 0 . 0 4 0 . 0 2 0 . 0 0 0 . 0 8 0 . 0 0 0 . 0 0 P G I - 4 D 0 . 2 4 0 . 0 2 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 I - 4 C 0 . 0 9 0 . 5 9 0 . 0 3 0 . 1 0 0 . 19 0 . 1 8 0 . 1 9 0 . 0 3 0 . 2 0 0 . 0 0 P G I - 4 d 0 . 55 0 . 3 8 0 . 9 4 0 . 44 0 . 7 0 . 0 . 7 7 0 . 8 0 0 . 9 0 0 . 8 0 1. 0 0 P G I - 4 e 0 . 0 0 0 . 0 0 0 . 0 3 0 . 0 3 0 . 0 0 0 . qp 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G I - 4 f 0 . 0 0 0 . 0 2 0 . 0 0 0 . 0 0 0 . 0 7 0 . 0 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G I - 4 g 0 . OO 0 . 0 0 0 . 0 0 0 . 4 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G I - 4 k 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 PGI-41 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 I - 4 n 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 P G I - 5 a 0 . 0 0 0 . 0 1 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 I - 5 b 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 P G I - 5 C 0 . 8 7 0 . 6 9 0 . 9 5 0 . 6 6 0 . 7 8 0 . . 6 9 0 . 7 3 0 . 8 5 0 . 9 6 1. . 0 0 P G I - 5 d 0 . . 0 0 0 . 2 0 0 . 0 5 0 . . 3 1 0 . 17 0 . , 3 0 0 . 2 4 0 . 11 0 . . 0 0 0 . . 0 0 P G I - 5 e 0 . . 0 0 0 . 0 0 0 . . 0 0 o. . 0 0 0 . 0 0 0 . . 0 0 0 . . 0 0 0 . 0 1 0 . . 0 0 0 . 0 0 P G I - 5 i 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 I - 5 J 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 I - 5 k 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 I - 5 n 0 . 1 3 0 . . 0 8 0 . . 0 0 0 . . 0 2 0 . . 0 6 0 . 0 1 0 . . 0 3 0 . . 0 1 0 . 0 4 0 . 0 0 PGM-1 a 0 . 0 0 0 . 0 0 0 . 0 0 0 . . 0 0 0 . . 0 0 0 . 0 2 0 . 0 0 0 . . 0 0 0 . 0 0 0 . 0 0 PGM-1b 0 . 7 8 0 . 9 5 0 . 8 9 o . 9 9 0 . 9 8 0 . 6 0 0 . 9 1 0 . 2 1 o . 9 5 0 . 9 8 PGM- 1c O . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 • 19 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 PGM- 1 d 0 . 0 6 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 16 0 . 0 9 0 . 7 9 0 . 0 5 0 . 0 2 PGM- 1 g 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 PGM-1h 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 PGM- 1 n 0 . 1 6 0 . 0 5 0 . 11 0 . 0 1 0 . 0 2 0 . 0 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 a 0 . 0 0 0 . 0 2 0 . 0 3 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 b 1 . 0 0 . 0 . 9 0 0 . 9 7 0 . 8 4 0 . 7 3 0 . 9 8 0 . 9 1 0 . 9 8 1 . 0 0 1 . 0 0 P G M - 2 C 0 . 0 0 0 . 0 0 0 . 0 0 * 0 . 1 5 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 2 d 0 . 0 0 0 . 0 9 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 2 0 . 0 0 0 . 0 0 P G M - 2 f . 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 g 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 h 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 n 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 2 7 0 . 0 2 0 . 0 9 0 . 0 0 0 . 0 0 0 . 0 0 P G M - 3 a 0 . 0 0 0 . 0 5 0 . 0 0 0 . 0 0 0 . 0 6 0 . 1 3 0 . 18 0 . 4 9 0 . 2 6 0 . 0 0 PGM-3b 1 . 0 0 0 . 9 0 0 . 9 1 0 . 9 9 0 . 8 5 0 . 8 5 0 . 8 2 0 . 5 1 0 . 7 4 0 . 9 7 P G M - 3 n 0 . 0 0 0 . 0 5 0 . 0 9 0 . 0 2 0 . 1 0 0 . 0 2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 3 P G M - 4 a 0 . 6 9 0 . 8 2 0 . 7 0 0 . 6 4 0 . 9 0 0 . 7 3 0 . 9 1 0 . 6 1 0 . 5 2 0 . 9 3 P G M - 4 b 0 . 0 0 0 . 0 0 0 . 0 9 0 . 1 2 0 . 0 0 0 . 0 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 PGM-4C 0 . 0 0 0 .OO 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 0 0 . 0 0 P G M - 4 n 0 . 3 1 o . 18 o . 2 1 o . 2 4 0 . 1 0 o . 2 4 0 . 0 8 0 . 3 9 0 . 4 8 0 . 0 7 105 ALLELE ASYM CERV FORB F HAWA MAUI MENZ F MICR M MICR C MICR K MOLO LAP-1 a 0. 00 0. 00 0. 00 0. 00 LAP- 1b 0. 02 0. 04 0. 00 0. 00 LAP- 1C 0. 00 0. 00 0. 00 0. 00 LAP- 1 d 0. 98 0. 90 1. 00 0. 96 LAP-1e 0. 00 0. 04 0. 00 0. 04 LAP-1n 0. 00 0. 02 0. 00 0. 00 LAP-2a 0. 00 0. 00 0. 00 0. 15 LAP-2b 0. 20 0. 96 1. 00 0. 56 LAP-2C O. 00 0. 00 0. 00 0. 00 LAP-2d 0. 77 o. 00 0. 00 0. 25 LAP-2e 0. 00 0. 00 0. 00 0. 00 LAP-2n 0. 04 0. 04 0. 00 0. 04 DIA-1a 0. 26 0. 50 0. 13 0. 03 DIA- 1b 0. 65 0. 50 0. 50 0. 97 DIA-lc 0. 09 0. 00 0. 04 0. 00 DI A-1d 0. 00 0. 00 0. . 33 0. .00 DlA- 1e O. .OO 0. OO o. . oo o. .OO DIA-2a 1 , ,00 0, .97 0 .63 1. .00 DIA-2b 0. .00 0, .03 0 . 37 0 .00 DlA-2c o .00 0 .00 0 .00 0 .00 MDH-1 a 1 .00 1 .00 1 .00 1 .00 MDH-2a 0 .00 0 .00 0 .03 0 .00 MDH-2b 0 .00 0 .00 . 0 .00 0 .00 MDH-2c 0 .00 0 .00 0 .00 0 .00 MDH-2d 1 .00 1 .00 0 .97 1 .00 MDH-3a 0 . 54 0 .00 0 . 16 0 .00 MDH-3b 0 .46 1 .00 0 . 84 1 .00 MDH-4a 1 .00 1 .00 1 .00 1 .00 MDH-5a 0 . 88 0 . 89 0 . 98 1 .00 MDH-5b 0 .00 0 .00 0 .00 0 .00 MDH-5C 0 .03 0 . 1 1 0 .00 « 0 .00 MDH-5d 0 .00 0 .00 0 .02 0 .00 MDH-5e o .09 0 .OO 0 .00 0 .00 MDH-6a 1 .00 1 .00 1 .00 1 .00 MDH-6C 0 .00 0 .00 0 .OO 0 .00 xDH- 1 a •1 .00 1 .00 1 .00 1 .00 ME -1a 1 .00 1 .00 1 .00 1 .00 ME -1b 0 .00 0 .00 0 .00 0 .00 HA - 1a 1 .00 1 .00 1 .00 1 .00 HA -1b 0 .00 0 .00 0 .00 .0 .00 GLU- 1 a 1 .00 1 .00 1 .00 1 .00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 01 0. 11 0. 00 0. 07 0. 00 0. 93 0. 96 0. 84 0. 92 0. 57 1. 00 0. 07 0. 01 0. 03 0. 00 0. 32 0. 00 0. 00 0. 01 0. 01 0. 08 0. 04 0. 00 0. 00 0. 02 0. 24 0. 00 0. 27 0. 00 0. 52 0. 89 0. 73 0. 61 0. 73 0. 58 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 02 0. 00 0. 37 0. 00 0. 42 0. 00 0. 01 0. 00 0. 00 0. 00 0. 00 0. 48 0. 06 0. 03 0. 02 . 0. 00 0. 00 0. 05 0. 92 0. 14 0. 89 0. 39 0. 92 0. 87 0. 08 0. 62 0. 1 1 0. 07 0. 08 0. 08 0. 00 0. 24 0. 00 0. 37 0. 00 0. .00 0, .00 0. 00 0. .00 0. 17 0. ,00 0. ,oo •0 OO 0. OO 0. .oo 0. .00 0. .00 0 .87 1 .00 0. .97 0. . 99 1. .00 1 .00 0 . 13 0 .00 0. .03 0 .01 0 .00 0 .00 0 .00 0 .00 0. .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .08 0 .00 0 .02 0 .00 0 .00 0 .09 0 .00 0 .00 0 .00 0 .00 0 .00 0 .91 0 .92 1 .00 0 .98 1 .00 1 .00 0 . 50 0 .45 0 .21 0 .69 0 .00 0 .97 0 . 50 0 . 55 0 . 79 0 . 3 1 1 .00 0 .03 1 .00 1 .00 1 .00 . 1 .00 1 .00 1 .00 0 .61 0 .96 0 . 98 1 .00 0 .82 0 . 98 0 . 39 0 .03 0 .00 0 .00 0 .02 0 .00 0 .00 0 .01 0 .02 0 .00 0 . 14 0 .00 0 .00 0 .00 0 .00 o .00 0 .00 0 .02 0 .00 0 .00 0 .00 0 .00 0 .02 0 .00 1 .00 1 .00 1 .00 1 .00 0 .93 1 .00 0 .00 0 .00 0 .00 0 .00 0 .07 0 .oo 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .OO 1 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 06 A L L E L E P O P U S A N D S S A N D C T O R T W I E B A M P L T R I P F R O N C Y N A PGI-1a 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 PGI-2a 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 1 .00 PGI-3a 1 .00 1 .00 1 .00 1 .00 1 .00 0 .00 0 .00 0 .00 0 .00 PGI-3b 0 .00 0 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 1 .00 PGI-4a 0 .00 0 .00 0 .00 0 .00 0 .00 o .00 0 .00 0 .00 0 .00 PGI-4D 0 .00 0 .01 0 .00 0 .02 0 .00 0 .00 0 .00 0 .00 0 .00 PGI-4C 0 .21 0 .05 0 .07 0 . 24 0 . 10 0 .00 0 .00 0 .00 0 .00 PGI-4d 0 . 79 0 .94 0 .91 0 . 72 0 .90 0 .00 0 .00 0 .00 0 .00 PGI-4e 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 PGI-4f 0 .00 0 .01 0 .02 0 .02 0 .00 0 .00 0 .00 0 .00 0 .00 PGI-4g 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 . 0 .00 0 .00 0 .00 PGI-4k 0 .00 0 .00 0 .00 0 .00 0 .00 1 .00 1 .00 1 .00 0 .00 PGI-41 O .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 , o o 1 .00 PGI-4n 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 PGI-5a 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 PGI-5b 0 .00 0 .00 0 .00 0 .04 0 .00 0 .00 0 .00 0 .00 0 .00 PGI-5C 1 .00 0 .99 0 .93 0 .87 0 .99 0 .00 0 .00 0 .00 0 .00 PGI-5d 0 .00 0 .00 0 .04 0 .07 0 .00 0 .00 0 .00 0 .00 0 .00 PGI-5e 0 .00 0 .01 0 .02 0 .00 0 .01 0 .00 0 .00 0 .00 0 .00 PGI-5i 0 .00 . 0 .00 0 .00 0 .00 0 .00 1 .00 1 .00 0 .00 0 .00 PGI-5J 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 0 .00 1 .00 PGI-5k 0 . o o 0 .00 0 . o o 0 .00 0, .00 0 .00 0 .00 1 .00 0 .00 PGI-5n 0 .00 0 .00 0 .00 0 .02 0, .00 0, .00 0 .00 0 .00 0 .00 PGM-1 a 0 .00 0. .00 0. .00 o . O O 0. .00 0, ,00 0 .00 o .00 0 .00 PGM-1b 1. .00 0. .90 0. . 89 0. . 97 0. .97 0. .00 0 .00 0. ,00 0, .00 PGM- 1c 0. .00 0. .00 0. .00 0. ,00 0, ,00 0. .00 0. .00 0, .00 0, .00 PGM-1d 0, . o o 0. .01 0. .00 0. ,00 0. ,00 0, .00 0. .00 0. .00 0, .00 PGM-1g 0. .00 0. .00 0. .00 0. ,00 0. 00 1. .00 1. 00 1. ,00 . 0, .00 PGM-1h o . . o o 0. .00 0. .00 0. .00 0. ,00 0. .00 0. .00 o . ,00 1. .00 PGM-1n 0. .00 0. , 10 0. . 11 0. 02 0. 03 0. .00 0. ,00 0. .00 0. ,00 PGM-2a 0. .00 0. 00 0. .00 0. 00 0. 00 0. 00 0. ,00 0. 00 0. 00 PGM-2b 1. 00 0. 91 0, ,98 0. 94 0. 90 0. 00 0. 00 0. 00 0. 00 PGM-2C 0. 00 0. 08 0. 00 0. 03 0. 10 0. 00 0. ,00 0. 00 0. 00 PGM-2d 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 PGM-2f 0. 00 0. 00 0. 00 0. 00 0. 00 1. 00 0. 00 0. 00 0. 00 PGM-2g 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 1. 00 1. 00 0. 00 PGM-2h 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 1. 00 PGM-2n 0. 00 0. 01 0. 02 0. 03 0. 00 0. 00 0. 00 0. 00 0. 00 PGM-3a 0. o o 0. 10 0. 00 0. 04 0. 00 0. 00 0. 00 0. 00 0. 00 PGM-3b 0. 98 0. 86 0. 91 0. 93 0. 94 1. 00 1. 00 1. 00 1. 00 PGM-3n o . 02 0. 04 o . 09 0. 03 0. 06 0. 00 0. 00 0. 00 0. 00 PGM-4a 0. 93 0. 96 0. 91 0. 81 0. 64 0. 00 0. 00 0. 00 0. 00 PGM-4b 0. 00 0. 01 0. 00 0. 01 0. 00 0. 00 0. 00 0. 00 0. 00 PGM-4C 0. 00 0. 00 0. 00 0. 00 0. 34 0. 00 0. 00 0. 00 0. o o PGM-4n p. 07 0. 03 0. 09 0. 18 0. 01 1. 00 1. 00 1. 00 1. 00 1 07 A L L E L E POPU SAND S SAND C TORT WIEB AMPL T R I P FRON CYNA L A P - 1 a 0 . 0 0 0 . 01 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 L A P - 1b 0 . 0 0 0 . 0 7 0 . 0 0 0 . 01 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 L A P - 1c 0 . 0 0 0 . 01 0 . 0 0 0 . 11 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 L A P - 1d 1 . 0 0 0 . 9 0 0 . 9 5 0 . 8 3 1. 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 L A P - 1 e 0 . 0 0 0 . 0 0 0 . 0 0 0 . 01 0 . 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 0 . 0 0 L A P - 1n 0 . 0 0 0 . 0 0 0 . 0 5 0 . 0 5 0 . 0 0 1. 0 0 1. 0 0 1. 0 0 1. 0 0 L A P - 2 a 0 . 0 0 0 . 10 0 . 0 2 0 . 04 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 L A P - 2 b 0 . . 8 2 0 . 87 0 . 8 0 0 . 81 1. 0 0 1. 0 0 1. 0 0 1. 0 0 1. 0 0 L A P - 2 C 0 . . 0 0 0 . 0 0 0 . 0 0 0 . 01 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 L A P - 2 d 0 . , 0 0 0 . 0 0 0 . 0 0 0 . 0 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 L A P - 2 e 0 . . 18 0 . , 0 0 0 . 0 0 0 . 0 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 L A P - 2 n 0 . . 0 0 0 . , 0 4 0 . 18 0 . 0 8 0 . 0 0 0 . 0 0 0 . 0 0 0 . , 0 0 0 . , 0 0 D I A - 1a 0 , . 38 0 . ,71 0 . 7 0 0 . 46 0 . 9 0 0 . 0 0 0 . 0 0 1, , 0 0 0 . , 0 0 D I A - 1 b 0 . . 6 2 0 , , 29 0 . 3 0 0 . 46 0 . , 0 4 0 . , 0 0 0 . 0 0 0 . . 0 0 0 . , 0 0 D I A - l c 0 . . 0 0 0 . . 0 0 0 . , 0 0 0 , , 0 6 0 . 0 6 0 . 0 0 0 . , 0 0 0 , . 0 0 0 , , 0 0 D I A - l d 0 . . 0 0 0 , , 0 0 0 . 0 0 0 . , 0 2 0 . , 0 0 0 , , 0 0 0 . , 0 0 0 . 0 0 0 , . 0 0 D I A - 1 e 0 . 0 0 0 , . 0 0 0 . , 0 0 0 . , 0 0 0 . , 0 0 1, , 0 0 1. , 0 0 0 . 0 0 1 . 0 0 D I A - 2 a •o. . 8 4 0 , . 98 1, , 0 0 0 . . 9 8 1. , 0 0 0 . . 0 0 0 , . 0 0 1 . 0 0 0 . 0 0 D I A - 2 b 0 . 16 0 . 0 2 0 . , 0 0 0 , , 0 2 0 , . 0 0 0 , . 0 0 0 , , 0 0 0 . 0 0 0 . 0 0 D I A - 2 C 0 . . 0 0 0 . 0 0 0 . , 0 0 0 , . 0 0 0 . 0 0 1, . 0 0 1, . 0 0 0 . 0 0 ' 1 . 0 0 MDH-1 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 M D H - 2 a 0 . 0 0 0 . 0 0 0 , , 0 0 o. . 0 0 0 , . 0 0 0 , . 0 0 0 , . 0 0 0 . 0 0 0 . 0 0 M D H - 2 b 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 MDH-2C 0 . 0 0 0 . 0 0 0 , . 0 0 0 .01 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 M D H - 2 d 1 . 0 0 1 . 0 0 1 . 0 0 0 . 9 9 1 . 0 0 1 . 0 0 1 . 0 0 1 . Oo 1 . 0 0 M D H - 3 a 0 . 7 8 0 . 6 0 0 . 72 0 .01 0 . 32 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 M D H - 3 b 0 . 22 0 . 4 0 0 . 28 0 . 9 9 0 . 6 8 0 . 0 0 0 . 0 0 . 0 . 0 0 0 .oo M D H - 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 M D H - 5 a 1 . 0 0 0 . 9 4 0 . 5 0 0 . 8 8 0 . 9 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 M D H - 5 b 0 . 0 0 0 . 0 0 0 .bo • 0 . 0 0 0 .01 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 MDH-5C 0 . 0 0 0 . 0 4 0 . 36 o . 12 0 . 0 9 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 M D H - 5 d 0 . 0 0 0 . 0 0 0 . 14 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 M D H - 5 e 0 . 0 0 o . 0 2 0 .OO o . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 . 0 . 0 0 M D H - 6 a 1 . 0 0 1 . 0 0 1 . 0 0 0 . 9 9 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 M D H - G c 0 . 0 0 0 . 0 0 0 . 0 0 0 .01 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 x D H - 1 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 ME - 1 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 0 . 0 0 ME - 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 1 . 0 0 HA - 1a 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 1 . 0 0 0 . 0 0 HA - 1b 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 o . 0 0 0 . 0 0 1 . 0 0 G L U - 1 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 

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