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Genetic studies in populations of Collinsia parviflora Dougl. ex Lindl. (Scrophulariaceae) Krause, Gerda Rosa 1978

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Genetic studies in populations of Colli n s i a parviflora Dougl.ex Lindl . (Scrophulariaceae) by Gerda Rosa Krause Sc. (Hon.), University of Briti s h Columbia, 1975 A thesis submitted i n partial fulfillment of the requirements for the degree of Master of Science in The Faculty of Graduate Studies Department of Botany We accept this thesis as conforming to the required standard The University of Br i t i s h Columbia Gerda Rosa Krause, 1978 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at 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 , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 Date - i i -Abstract The Collinsia populations in southwestern B r i t i s h Columbia and northwestern Washington show considerable variation i n leaf and flower characters both within and between populations. One of the purposes of this study was to determine the genetic mechanisms con-t r o l l i n g some of these characters. Two leaf polymorphisms were studied. They were the presence or absence of purple anthocyanin spots on the surface of the leaves and the presence or absence of a silvery sheen, also on the surface of the leaves. Each was shown to be controlled by a single gene with two a l l e l e s . The spotted leaf character was dominant over the un-spotted leaf character and the silvery sheen was dominant over the normal green leaf character. Flower size was also studied and shown to be controlled by polygenic inheritance. Two mutant flower colours, white and magenta, are found in this region of study, in addition to the normal blue colour. The inher-itance of flower colour was not conclusively determined but the data indicate that two genes may be involved, one controlling the pro-duction of the magenta pigment from the colourless precursor and one controlling the production of the blue pigment from the magenta one. Another purpose of this work wa6 to determine the chromosome number of the Collinsias in this area. Both diploid (n=7) and tetraploid (n=l*f) counts have been reported. Six populations were studied and a l l were found to be tetraploid. - i i i -Finally, the cytological and genetic data were used in con-junction with morphological data to revise the taxonomy of the Collinsias in this region of study. Most authors divide them into two species, £. grandiflora Dougl, ex Lindl, and parviflora Dougl, ex Lindl, However this study indicates that they are only one highly variable species, C_, parvi f l o r a . - i v -T a b l e o f C o n t e n t s Page A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s v L i s t o f F i g u r e s a n d I l l u s t r a t i o n s v i i Acknowledgement i x C h a p t e r 1 I n t r o d u c t i o n a ) Taxonomy 1 b) C y t o g e n e t i c s 2 c ) G e n e t i c s 3 d) M a t e r i a l s and M e t h o d s 5 C h a p t e r 2 L e a f S p o t P o l y m o r p h i s m a) I n t r o d u c t i o n 10 b) M a t e r i a l s and M e t h o d s 13 c) R e s u l t s 15 d) D i s c u s s i o n 21 C h a p t e r 3 S i l v e r y L e a f P o l y m o r p h i s m a) I n t r o d u c t i o n 2 4 b) M a t e r i a l s a n d M e t h o d s 2If c) R e s u l t s 2 6 d) D i s c u s s i o n 33 C h a p t e r k F l o w e r C o l o u r M u t a n t s a) I n t r o d u c t i o n 3k fc) M a t e r i a l a n d M e t h o d s 38 c) R e s u l t s ifO d) D i s c u s s i o n k9 C h a p t e r 5 I n h e r i t a n c e o f F l o w e r S i z e a) I n t r o d u c t i o n 51 b) M a t e r i a l s and M e t h o d s 53 c) R e s u l t s 57 d) D i s c u s s i o n 73 C h a p t e r 6 Chromosome C o u n t s a) I n t r o d u c t i o n 77 fc) M a t e r i a l s and M e t h o d s 78 c) R e s u l t s 78 d) D i s c u s s i o n 79 C h a p t e r 7 Taxonomy a) I n t r o d u c t i o n 81 b) M a t e r i a l s and M e t h o d s 81 c) R e s u l t s 85 d) D i s c u s s i o n 90 B i b l i o g r a p h y V i t a - v -L i s t of Tables Page Table I Population numbers of the C o l l i n s i a populations used i n the crossing experiments • • • • • • • • • • • • • • • • 7 Table II Si and F^ progeny of the crosses i n v o l v i n g the spotted l e a f character • • • 16 Table I I I S 2 and Fp progeny of heterozygous spotted S^  and F^ plants • • • • • • • • • 18 Table IV Sg and Fg progeny of unspotted S^  and Fx plants • • • • • • • 19 Table V Sg and Fg progeny of homozygous spotted S, and F, plants • • • • • • • • • • • • • 22 Table VI S^ and F]_ progeny of the crosses i n v o l v i n g the s i l v e r y - l e a f character Table VII S 2 and F 2 progeny of the crosses i n v o l v i n g the s i l v e r y - l e a f character Table VIII F 2 progeny of the #11 green (9) x #25 s i l v e r y (d1) cross • • • • • • • Table IX S^ and So progeny of the populations used i n the crosses i n v o l v i n g flower colour 41 Table X F^ and Fp progeny r e s u l t i n g from crosses between blue-flowered and white-flowered plants • • • • • • • • • • • • • • • • • • 43 Table XI F 2 progeny of the #22 (?) x #9 blue (<3) cross that showed segregation f o r flower colour • • • • • • • • • • • • • • • • • • kk Table XII F^ and F 2 progeny of the crosses between blue-flowered plants and magenta-flowered plants • • • • • « • • • • • • • • • • • • 46 Table XIII Fi and F 2 progeny of the crosses between white-flowered magenta-flowered plants • • 48 Table XIV C o r o l l a length classes assigned to C, p a r v i f l o r a and C. g r a n d i f l o r a by various authors • • • • • • • • • • • • • • 32 Table XV Populations of C. b a r v i f l o r a used i n the study of flower s i z e v a r i a t i o n . . • , 55 • • • 27 • • • 29 - v i Table XVI Table XVII Table XVIII Table XIX Table XX Table XXI Table XXII Table XXIII Table XXIV Table XXV Mean flower sizes and sample sizes of the populations graphed i n Fig. 17 Mean flower sizes and sample sizes of the populations graphed i n Fig. 18 Mean flower sizes and sample sizes of the populations graphed in F i g . 19 Mean flower sizes and sample sizes of the populations graphed i n Fig. 20 Mean flower sizes and sample sizes of the populations graphed in Fig . 2 1 Mean flower sizes and sample sizes of the populations graphed i n Fig. 22 Mean flower sizes and sample sizes of the populations graphed i n Fig . 23 Mean flower sizes and sample sizes of the populations graphed in Fig. 24 Data sheet for population _________ Summary of the variation among the populations studied . . . . • ..• - v i j -L i s t o f F i g u r e s and I l l u s t r a t i o n s Page F i g u r e 1 Map o f the s t u d y a r e a showing t h e l o c a l -i t i e s from which seeds were c o l l e c t e d • • • 6 F i g u r e 2 C o l l i n s i a s growing i n 5 - i n c h p o t s • • • • • 8 F i g u r e 3 C o l l i n s i a s growing c l o s e l y spaced i n s h a l l o w f l a t s • • • • • • • • • • • • • • • 8 F i g u r e k P l a n t s w i t h and "without p u r p l e antho-c y a n i n s p o t s on the upper e p i d e r m i s o f the l e a v e s • • • • • • • • • • • • • • • • 11 F i g u r e 5 P l a n t showing the f a i n t - s p o t t i n g c h a r a c t e r • • • • • • • • • • • • • • • • • 12 F i g u r e 6 P l a n t showing the h e a v y - s p o t t i n g c h a r a c t e r • • • • • • • • • • • • • • • • • Ik F i g u r e 7 C a r l o s I s l a n d p l a n t s showing the s i l v e r y - l e a f c h a r a c t e r • • • • • • • • • • 25 F i g u r e 8 C a r l o s I s l a n d p l a n t s showing normal green l e a v e s • • • • • • • • • • • • • • • 25 F i g u r e 9 S i l v e r y , green and i n t e r m e d i a t e ' p l a n t s • 31 F i g u r e 10 A magenta f l o w e r from the E l k F a l l s p o p u l a t i o n • • • • • • • • • • • • • • • • 35 F i g u r e 11 An a l b i n o f l o w e r from the Mt» Douglas P a r k p o p u l a t i o n • • • • • • • • • • • • • • 35 F i g u r e 12 A p o p u l a t i o n w i t h a n t h o c y a n i n pigments i n the l e a v e s • • • • • • • • • • • • • • • 37 F i g u r e 13 The Mt, Douglas P a r k p o p u l a t i o n w i t h y e l l o w l e a v e s • • • • • • • • • • • • • • • 37 F i g u r e Ik The F 2 g e n e r a t i o n i n the c r o s s between white and magenta-flowered p l a n t s , showing s e g r e g a t i o n f o r the t h r e e f l o w e r c o l o u r s • k? F i g u r e 15 The v a r i a t i o n i n f l o w e r s i z e between the f i v e p o p u l a t i o n s used i n s t u d y . From l e f t t o r i g h t : E l k F a l l s , J a c k P o i n t , Nanoose H i l l , B o t a n i e V a l l e y , Lindeman Lake • • • • 3k F i g u r e 16 A C o l l j n s i a f l o w e r showing the a n g l e a t which measurements were taken • • • • • 56 - v i i i -Page Figure 1? Graph comparing flower sizes of the five populations i n the f i r s t (parental) generation • • • • • • • • • • • • • • • • • 58 Figure 18 Graph comparing flower sizes of the five populations i n the second (S ,) generation • 60 Figure 19 Graph comparing flower sizes of the five populations i n the third (S^) generation • • 62 Figure 20 Graph comparing the flower sizes of Lindeman Lake (#6) and Jack Point (#11) populations with a possible hybrid between them • • • • • • • • • • • • • • • • 65 Figure 21 Graph comparing the flower sizes of the hybrids between populations from Botanie Valley (#22) and Nanoose H i l l (#9) with their parental populations • • • • • • • • • 67 Figure 22 Graph comparing the flower sizes of the hybrids between populations from Botanie Valley (#22) and Jack Point (#11) with their parental populations • • • • • • • • • 69 Figure 23 Graph comparing the flower sizes of hybrids between populations from Lindeman Lake (#22) and Elk F a l l s (#17) with their parental populations • • • • • • • • • • • • 71 Figure 24 Graph comparing the flower sizes of hybrids between populations from Mt. Douglas Park (#2) and Elk F a l l s (#17) with their parental populations • • • • • • • • • • • • 74 Figure 25 A pollen mother c e l l from the Nanoose H i l l population showing a chromosome number of 2n s 14 II 80 Figure 26 A pollen mother c e l l from the Carlos Island population showing a chromosome number of 2n = 14 II 80 Figure 27 The leaf characters of the Botanie Valley population • • • • • • • • • • • • • 87 Figure 28 The leaf characters of the Jack Point population • • • • • • • • • • • • • • • • • 87 Acknowledgement I would like to express my gratitude to my director, Dr. F. R. Ganders and to my committee, Dr. C. J . Marchant and Dr. A, J . F. G r i f f i t h s for their willing assistance, discussion and criticism during the research and their help i n preparing the manuscript; to Dr. K. I. Beamish, Dr. C. 0. Person and Dr, B. A. Bohm for advice and assistance with various parts of this thesis; to Mr. Ken Carey and Dr. W. B. Schofield for collecting Collinsia seeds; and to the Department of Botany for the use of equipment and f a c i l i t i e s . I would also l i k e to acknowledge receipt of two National Research Council Scholarships (1975/76 and 1976/77) and thank Mrs. Lorraine Wiebe for typing the manuscript. Finally, I would li k e to thank my husband, Eric, for a l l his encouragement and i n f i n i t e patience. C h a p t e r 1 I n t r o d u c t i o n T a x o n o m y T h e b l u e - f l o w e r e d C o l l i n s i a s i n s o u t h w e s t e r n B r i t i s h C o l u m b i a a n d n o r t h w e s t e r n W a s h i n g t o n h a v e g e n e r a l l y b e e n d i v i d e d i n t o t w o s p e c i e s . T h e l a r g e r - f l o w e r e d p l a n t s a r e u s u a l l y c o n s i d e r e d C, g r a n d -i f l o r a D o u g l , e x L i n d l , a n d t h e s m a l l e r - f l o w e r e d p l a n t s a r e p l a c e d i n C , p a r v i f l o r a D o u g l , e x L i n d l , ( A b r a m s , 1951; H i t c h c o c k e t a l , , 1959; T a y l o r a n d M a c B r y d e , 1977)* C , g r a n d i f l o r a w a s o r i g i n a l l y d e s c r i b e d b y L i n d l e y ( 1 8 2 7 ) f r o m g a r d e n s p e c i m e n s g r o w n f r o m s e e d s c o l l e c t e d b y D a v i d D o u g l a s i n t h e v i c i n i t y o f t h e C o l u m b i a R i v e r , C ^ p a r v i -f l o r a w a s d e s c r i b e d i n t h e same y e a r , a g a i n b y L i n d l e y , f r o m g a r d e n s p e c i m e n s g r o w n f r o m s e e d s c o l l e c t e d b y D o u g l a s a t " t h e d r y b a n k s o f t h e C o l u m b i a R i v e r , a t t h e d i s t a n c e o f a n h u n d r e d m i l e s a n d m o r e f r o m t h e o c e a n " , N e w s o m (1929), i n h e r m o n o g r a p h o f t h e g e n u s m a i n t a i n e d t h e t w o s p e c i e s a n d f u r t h e r r e c o g n i z e d t w o v a r i e t i e s o f C_, g r a n d i f l o r a ; C , g r a n d i f l o r a v a r , t y p i c a a n d C± g r a n d i f l o r a v a r , p u s i l l a G r a y , v a r i e t y p u s i l l a h a v i n g s m a l l e r f l o w e r s t h a n t y p i c a . V a r i e t y p u s i l l a h a s a l s o b e e n g i v e n s u b s p e c i f i c r a n k ( P i p e r , 1906), P e c k (1961) f o l l o w e d H o w e l l (1903) a n d r e c o g n i z e d pusilla a t t h e s p e c i f i c l e v e l : £_t Pusilla ( G r a y ) H o w e l l , H i t c h c o c k e t a l . (1959) a n d P e n n e l l ( i n A b r a m s , 1 9 5 D c o n s i d e r e d pusilla t o b e a s y n o n y m o f C , p a r v i f l o r a « a l t h o u g h P e n n e l l r e c o g n i z e d t w o v a r i e t i e s o f p a r v i f l o r a . A l l o f t h e r e c e n t f l o r a s o f t h e w e s t c o a s t o f N o r t h A m e r i c a h a v e r e c o g n i z e d t w o s p e c i e s i n t h i s g r o u p , a l t h o u g h t h e y d o n o t a l w a y s a g r e e o n how t o s e p a r a t e t h e s e e n t i t i e s . There has been l i t t l e uniformity among authors over where to draw the line between Cj, grandiflora and C, parviflora, This con-fusion i s reinforced by the condition in nature. One finds a con-tinuous series of intergrading populations from the smallest flowered parviflora to the largest flowered grand!flora. This fact was noted by Newsom (1929) but she maintained the two species on the basis that "the, two extremes dif f e r greatly' 1. However, after hybridization and morphological studies, I have come to the conclusion that this C o l l i n s i a group containing grandiflora. parviflora and pusi l l a i s only one highly variable species. The name C, parvlflora has priority over the name £• grandiflora. Therefore I w i l l use the name C, parviflora to refer to the whole group unless otherwise specified. Cytogenetics Garber and his students have published a considerable amount of work on the genus C o l l i n s i a , Most of the work i s cytogenetic, some' i s morphological and some i s chemical. However C± parviflora« which he divides into parvlflora and grand!flora, has not been examined except for basic chromosome counts. He reported a chromosome number of n=7 for both species (Garber, 1956, 1958b), However, Taylor and Mulligan (1968) reported that £• parviflora i n the Queen Charlotte Islands had a chromosome number of n=14. The chromosome counts obtained from the populations i n this study agree with Taylor and Mulligan rather than Garber, Unfortunately, Garber does not give the l o c a l i t i e s of his samples nor does he cite voucher specimens. - 3 -Genetics Newsom (1929) divided the genus i n t o two groups based on the length of the p e d i c e l s . One group of species has s e s s i l e flowers, congested i n t o whorls, the other group has pediceled flowers that are s o l i t a r y or i n whorls, Garber (1958a) divided the species i n t o two groups according to whether they exhibited high or low chiasmata frequencies, and these groups correlated well with the*pediceled flower group and the s e s s i l e flower group r e s p e c t i v e l y . He also d i s -tinguished between " v a r i a b l e " and "uniform" populations (Garber, 1974), Although he does not define p r e c i s e l y what he means by these terms i n h i s discussion of sessile-flowered, he states that "unpublished data i n d i c a t e that these (sessile-flowered) species share a large number of phenotypes, each c o n t r o l l e d by a s i n g l e gene d i f f e r e n c e , not yet observed,,, i n the species with d i s t i n c t l y pediceled flowers" (Garber, 1958b). On the basis of the above three c h a r a c t e r i s t i c s , Garber places C. g r a n d i f l o r a and C_» p a r v i f l o r a i n t o h i s Group I I which contains plants with pediceled flowers, uniform populations and high chias-mata frequency. The c o l l i n s i a s i n southwestern B r i t i s h Columbia and northwestern Washington are d e f i n i t e l y pediceled. There are no data on the chiasmata frequency. However, the populations are not very uniform. Many populations show considerable v a r i a t i o n i n l e a f s i z e , shape, pubescence and colouring, flower s i z e and colouring, e t c . No doubt many of these characters are g e n e t i c a l l y c o n t r o l l e d . I invest i g a t e d a number of these polymorphisms to determine the genetic mechanisms c o n t r o l l i n g them. - k -One common polymorphism i s the presence or absence of dark purple spots on the upper surface of the leaves. This character was found to actually include two separate characters. A gene system producing large, heavy spots sometimes covering most of the leaf was found to be a simple dominant. The other character appeared as a few tiny dots on one or a few leaves of the plant. Its genetic mechanism was not positively determined but the results of a study by Gr i f f i t h s et a l . (1977) suggests that a single incompletely penetrant gene i s involved. Another leaf polymorphism i s the presence or absence of a silvery sheen on the early leaves. This was also shown to be controlled by a single gene with a dominant a l l e l e for the silvery sheen. The inheritance of flower colour i s more complex. Three different flower colours were found i n the parviflora populations studied. These were magenta and white i n addition to the normal blue. The results were.not conclusive but suggested the following genetic interpretation. One gene (A) controlled the production of the magenta pigment from the colourless precursor. Another gene (B) controlled the production of the blue pigment from the magenta pigment. A homo-zygous recessive (aa) at the f i r s t step resulted in a white-flowered plant and a homozygous recessive (bb) at the second step resulted i n a magenta-flowered plant. This relatively simple Mendelian system waB, however, complicated by selection against the recessive a l l e l e s in the gamete or zygote which distorted the expected 3*1 ratios. The genetic control of flower size i s of special interest because Cj, parviflora and C. grandiflora are separated mainly on the - 5 -basis of that character. As expected f o r a continuously varying character such as flower s i z e the inheritance mechanism i s probably polygenic. However i t was not possible to determine exactly how many genes are involved. Materials and Methods C. p a r v i f l o r a i s a good organism f o r genetic studies since i t grows well under a r t i f i c i a l conditions and, being an annual, has a r e l a t i v e l y short l i f e c y c l e . The populations of plants used i n t h i s study were grown from seed c o l l e c t e d from various places i n southwestern B r i t i s h Columbia and northwestern Washington. Figure 1 i s a map of the area showing the l o c a l i t i e s from which seeds were c o l l e c t e d . For convenience, the populations used i n the cross i n g experiments were assigned numbers. These population numbers are l i s t e d i n Table I . Seeds were c o l l e c t e d from mature brown capsules and stored i n paper packets u n t i l needed. Seeds were sown between pieces of moist f i l t e r paper i n p e t r i plates and placed i n a r e f r i g e r a t o r at 5 ° - 9°C u n t i l the seeds germinated and the r a d i c l e s were about 1-2 cm long. This u s u a l l y took about two to three weeks. They were then planted i n f l a t s or pots of s o i l . The parental and F^ generations were grown i n 5 inch pots ( F i g . 2) and were large vigourous plants, but since the number of i n d i v i d u a l s i n the F 2 generation was usu a l l y much l a r g e r , i t was planted c l o s e l y spaced i n shallow f l a t s ( F i g . 3 )« As a r e s u l t , the plants were often smaller and l e s s vigourous but t h i s did not a f f e c t the study because s i z e and vigour were not measured and the - 6 -Table I Population numbers of the Collinsia populations used in the crossing experiments Population number #2 #6 #9 #11 #17 #22 #25 Source of seeds (Locality) Mt« Douglas Park, Vancouver Island Lindeman Lake, B. C, Nanoose H i l l , Vancouver Island Jack Point, Vancouver Island Elk F a l l s , Vancouver Island Botanie Valley, B, C. Carlos Island, B. C. - 8 -F i g . 3 C o l l i n s i a s growing c l o s e l y spaced i n shallow f l a t s - 9 -characters studied were expressed i n the small plants as well as the large ones. Three to four weeks were required to grow seedlings i n s o i l to the rosette stage where leaf polymorphisms could be evaluated and another four to five weeks before most of the plants started to flower. A l l plants were grown in growth chambers under a cool, long day regime of 16 hours light at 20°C and 8 hours dark at 10°C. This regime proved best for good vegetative growth and flowering and the low temperature ensured maximum expression of the spotting character. Controlled cross-pollination i s quite d i f f i c u l t because the flowers are small and w i l l self-pollinate readily. They have to be emasculated in the bud stage before the pollen i s mature and before the anthers can dehisce either naturally or accidentally with handling. The buds are only about 2-3 mm long, and must be handled under the dissecting microscope i n order to ensure that the anthers are com-pletely removed and the style and stigma are not damaged. An additional complication i s that occasionally one of the anthers w i l l dehisce i n the closed bud stage and scatter pollen over the stigma. However, i n a successfully emasculated bud, the stigma w i l l reach maturity i n 3 to 5 days and can then be cross-pollinated. This was accomplished by removing mature anthers from a flower of the male parent plant and brushing them over the receptive surface of the stigma. - 10 -Chapter 2 Leaf Spot Polymorphism Introduction One of the most obvious polymorphisms found within many of the populations of Collinsia parviflora on Vancouver Island i s the pres-ence or absence of purple anthocyanin spots on the upper epidermis of the leaves (Fig. 4). These spots vary in size, number and shape both among and within the individual plants and are usually found only on the earlier leaves of the plant and sometimes on the cotyledons. The anthocyanin spots usually fade and disappear as the plant gets older or as the temperature ris e s . For example, wild collected spotted plants transferred to a heated greenhouse lost their spots within three days. Gorsic (1957) reported a similar polymorphism in his genetic studies of (Lj, heterophylla Buist. He called i t dark-dotted (Ld) and described i t as a transient cotyledon and leaf character i n which the "upper surfaces of cotyledons and leaves show maroon spots of various numbers and sizes". He found this character to be inherited as a simple dominant. Gr i f f i t h s et a l . , 1 9 7 7 » in working with populations of £. grand-i f l o r a Lindl. in southwestern Br i t i s h Columbia and northwestern Washington discovered two genetically distinct spotting systems, a faint spotting system (F) and a heavy spotting system (H). There i s considerable variation within each system and the ranges of the degree of spotting within the two classes probably overlap. However, plants with the faint degree of spotting usually have only a few of their leaves bearing a small number of tiny dots, about 0 . 5 mm in diameter (Fig. 5 ) . The precise genetic determination of the faint spots was not conclusively established but the results suggested that a single - 11 -F i g , k Plants with and without purple anthocyanin spots on the upper epidermis of the leaves - 12 Fig, 5 Plant showing the faint-spotting character - 1 3 -incompletely penetrant gene i s involved. Plants with a heavy degree of spotting u s u a l l y have blotches of various shapes and s i z e s on a l l or most of the leaves and on the cotyledons ( F i g , 6), The amount of blotching can vary considerably from a large blotch covering almost the e n t i r e l e a f to a few small spots near the base. The genetic determination of heavy spotting i s d i s t i n c t from that of f a i n t spotting and maintains i t s inheritance pattern even when the f a i n t - s p o t t i n g gene i s present. The spotting system investigated i n t h i s study was the heavy spotting one. Crosses were set up to determine the exact inheritance pattern f o r t h i s p a r t i c u l a r polymorphism. Materials and Methods In order to study the genetics of the spotting system, an exper-imental population containing both spotted and unspotted plants was used. Reciprocal crosses were set up between spotted and unspotted plants from a population of plants (population #11) grown from seeds c o l l e c t e d from Jack Point ( F i g , 1 ) , Spotted and unspotted plants were also allowed to s e l f . In a d d i t i o n , r e c i p r o c a l crosses were made between the spotted plants of population #11 and plants from another population (population #9), derived from seeds c o l l e c t e d from Nanoose H i l l ( F i g , 1 ) , that contained only unspotted plants. These unspotted plants were a l s o allowed to s e l f . In order to get s u f f i c i e n t progeny from the crosses, more than one plant was involved i n each cross. Therefore, both homozygous and heterozygous parents could have been involved i n any p a r t i c u l a r cross or s e l f . F i g , 6 Plant showing the heavy-spotting character - 15 Seeds from the c r o s s - p o l l i n a t i o n s were c o l l e c t e d , germinated and grown to the rosette stage when they were c l a s s i f i e d and tagged fo r the presence or absence of spotting i n the ea r l y leaves. The tagging at t h i s stage i s very important since the spotting fades as the plant gets older. The plants were then l e f t to s e l f n a t u r a l l y and the seed from each of these plants was c o l l e c t e d and grown i n d i v i d u a l l y to be scored. Results The r e s u l t s of the a r t i f i c i a l crosses i . e , the F^ plants and the r e s u l t s of the s e l f - p o l l i n a t i o n s , i , e , the S^ plants are summarized i n Table I I , These r e s u l t s i n d i c a t e that the absence of spots i s a true-breeding character i n populations from both Nanoose H i l l and Jack Point, The spotted plants on the other hand, produced both spotted and unspotted progeny when s e l f e d , i n d i c a t i n g that a si n g l e gene system may be involved and that at l e a s t some of the plants involved were heterozygous f o r spotting and that the spotted c h a r a c t e r i s t i c i s dominant over the unspotted one. The i n d i c a t i o n that the spotted character i s dominant i s r e i n -forced by the r e c i p r o c a l crosses. When the spotted plants are used e i t h e r as male or as female parents and crossed with unspotted plants the r e s u l t i n g progeny are mostly spotted. A l l of the spotted F-^  progeny would be expected to be heterozygous, containing spotted a l l e l e s from t h e i r spotted parents and unspotted a l l e l e s from t h e i r unspotted parents. S.. and F, progeny of the crosses involving leaf spots Table II Pollination regime e P r°Seay ° Spotted Unspotted Population #11; spotted selfed 18 5 Population #11; unspotted selfed 0 22 Population #9; unspotted selfed 0 13 #11 spotted (9) x #11 unspotted (cf) 26 15 #11 unspotted (?) x #11 spotted (o") 22 7 #11 spotted (9) x #9 unspotted (cf) 13 11 #9 unspotted (?) x #11 spotted (cr) 15 19 - 17 -Seeds were c o l l e c t e d from each of the and S^ plants that reached maturity (some were k i l l e d by aphids and mildew) and each family was grown and scored i n d i v i d u a l l y to determine the genetic make-up of the parent and to determine the r a t i o of spotted plants to unspotted plants i n the progeny of the heterozygotes. Most of f a m i l i e s grown from spotted F^ and S^ plants segregated i n t o spotted and unspotted plants i n approximate y.l r a t i o s confirming that they were heterozygotes and that a s i n g l e gene with a dominant a l l e l e f o r the presence of heavy spotting and a recessive a l l e l e f o r the absence of heavy spotting controls t h i s p a r t i c u l a r polymorphism. The a c t u a l r a t i o s are summarized i n Table I I I . Most of.the heterozygotes seem to f i t the 3:1 r a t i o w ell and confirm that heavy spotting i s i n h e r i t e d as a simple dominant. The F 2 progeny r e s u l t i n g from the #11 spotted (9) x #9 unspotted (cf) cross, however, shows a s l i g h t l y s i g n i f i c a n t deviation from the expected r a t i o due to a greater number of unspotted progeny than expected. This i s t y p i c a l of the behaviour of the f a i n t - s p o t t i n g a l l e l e ( G r i f f i t h s et a l . , 1977) and can probably be a t t r i b u t e d to one or more fai n t - s p o t t e d plants having been mistaken f o r a heavy-spotted p l a n t . Table IV summarizes the F 2 and progeny of a l l of the un-spotted plants r e s u l t i n g from both cross and s e l f - f e r t i l i z a t i o n . I f unspotted plants are r e a l l y homozygous recessives a l l of the progeny of unspotted plants would be expected to be unspotted. This i s not e n t i r e l y the case. Although the vast majority of the progeny are unspotted, there are a few anomalies. However, since these anomalies are few and do not seem to f i t any p a r t i c u l a r r a t i o , i t i s Table III S 2 and F 2 progeny of heterozygous spotted S 1 and F 1 plants Origin of spotted and F^ plants # Of S 2 & F 2 families Number of S 2 & F 2 individuals Spotted Unspotted X 2 (1 d.f.) Population #11; spotted self #11 spotted (?) x #11 unspotted (d1) 10 #11 unspotted (?) x #11 spotted (c?) 18 #11 spotted (?) x #9 unspotted (c?) 12 #9 unspotted (9) x #11 spotted (<?) 12 261 619 466 250 584 100 184 132 111 21? 1 . 4 0 ; P= 0 . 5 - 0 . 1 deviation not significant 1 . 8 7 ; P= 0 . 5 - 0 . 1 deviation not significant 2 . 7 3 ; P= 0 . 1 - 0 . 0 5 deviation not significant 6 . 3 6 ; P= 0 . 0 2 5 - 0 . 0 1 deviation significant 1.875 P= 0 . 5 - 0 . 1 deviation not significant Table IV S 2 and F 2 progeny of unspotted and F^ plants Origin of unspotted S^ and F^ plants # of S 2 & F 2 families # of S 2 & F 2 Individuals Spotted Unspotted Population #11; spotted selfed Population #11; unspotted selfed Population #9; unspotted selfed #11 spotted (9) x #11 unspotted (<?) #11 unspotted (9) x #11 spotted (c?) #11 spotted (9) x #9 unspotted (c?) #9 unspotted (?) x #11 spotted Q?) 3 3 10 13 7 10 7 5 o o 3 40 1 2 312 265 404 1,087 698 685 417 20 -p r o b a b l e t h a t t h e y a r e n o t a r e s u l t o f t h e h e a v y - s p o t t i n g g e n e t i c s y s t e m . Some, f o r e x a m p l e , were p r o b a b l y c o n t a m i n a n t s r e s u l t i n g when t h e s e e d s were p l a n t e d . The f a m i l i e s were sown s i d e by s i d e i n p l a s t i c " f l a t s a n d a s e e d f r o m a s p o t t e d f a m i l y c o u l d q u i t e e a s i l y have f l o a t e d o v e r t h e b a r r i e r t o an u n s p o t t e d f a m i l y when t h e y were b e i n g w a t e r e d . T h i s i s p r o b a b l y t h e c a s e i n t h e c r o s s #11 s p o t t e d x #9 u n s p o t t e d (&) where o u t o f 10 f a m i l i e s , 9 showed o n l y u n s p o t t e d p l a n t s a n d 1 f a m i l y h a d a s i n g l e s p o t t e d p l a n t g r o w i n g c l o s e t o t h e b a r r i e r between t h e u n s p o t t e d f a m i l y and a s p o t t e d o n e . The same argument c a n e x p l a i n most o f t h e o t h e r a n o m a l i e s . H o w e v e r , one F 2 f a m i l y grown from an F ^ p l a n t f rom t h e c r o s s , #11 u n s p o t t e d ( § ) x #11 s p o t t e d fcT) h a d 37 s p o t t e d and 94 u n s p o t t e d F 2 p l a n t s . T h i s i s t o o l a r g e a number o f s p o t t e d p l a n t s t o be e x -p l a i n e d s i m p l y a s c o n t a m i n a t i o n o f a s t r i c t l y u n s p o t t e d f a m i l y . B u t t h i s i s a l s o t o o s m a l l a number o f s p o t t e d p l a n t s t o be e x p l a i n e d as n o r m a l 3*1 s e g r e g a t i o n o f a h e t e r o z y g o t e s p o t t e d p l a n t . The p a r e n t p l a n t o f t h i s f a m i l y was o r i g i n a l l y l a b e l l e d ' s p o t t e d * b u t because o f t h e h i g h p e r c e n t a g e o f u n s p o t t e d p l a n t s i t was p r o b a b l y n o t s p o t t e d , a t l e a s t n o t h e a v i l y - s p o t t e d . I t seems more r e a s o n a b l e t o c o n s i d e r t h i s a f a i n t - s p o t t e d p l a n t . I n g e n e r a l , o b v i o u s l y f a i n t - s p o t t e d p l a n t s were s c o r e d as ' u n s p o t t e d ' b u t t h e amount o f s p o t t i n g i n t h i s f a m i l y f a l l s i n t o t h e r a n g e o f o v e r l a p between t h e h e a v y and f a i n t -s p o t t i n g c h a r a c t e r s and so was m i s i n t e r p r e t e d . I f t h e above e x p l a n a t i o n s f o r t h e a n o m a l i e s a r e a c c e p t e d , u n -s p o t t e d p l a n t s c a n be c o n s i d e r e d t o be homozygous f o r t h e r e c e s s i v e c h a r a c t e r , a b s e n c e o f h e a v y s p o t s . 21 -Some of the families scored also showed a l l spotted and no unspotted progeny indicating that the parent plants were homozygous for the dominant character, heavy-spotting. The results are summarized i n Table V. According to these results, six of the S^ plants resulting from the selfing of spotted plants from population #11 (Jack Point) were homozygotes. This was not unexpected. But, a l l of the plants resulting from the #11 spotted ($) x #11 unspotted (cf) cross should have been heterozygotes. This apparent anomaly can be explained by noting that the seed parents i n the controlled cross were spotted plants. An imperfect emasculation can result i n a s e l f - f e r t i l i z a t i o n producing a homozygous spotted plant. Since emasculation of Collinsia  parviflora buds i s so d i f f i c u l t , these accidental selfs are not unusual. The 6 unspotted plants found i n these 2 families are probably the result of seeds floating i n from the adjacent unspotted families. Discussion It.can be concluded from the above results that the heavy-spotting polymorphism i n £. parviflora i s controlled by a single Hendelian gene with a dominant a l l e l e for the presence of heavy spotting and a recessive a l l e l e for the absence of heavy spotting. Another.allele, that for faint-spotting, may be involved at this locus ( G r i f f i t h s et al.) but the actual inheritance pattern of this particular character remains i n doubt and requires further research. Table V S 2 and F 2 progeny of homozygous spotted and plants Origin of spotted and F^ plants # of S 2 & F 2 families # of S 2 and F 2 individuals Spotted Unspotted Population.#11; spotted selfed 491 0 #11 spotted (9) x #11 unspotted (cf) 2 93 6 - 23 -I t i s i n t e r e s t i n g to note that the heavy-spotting gene (H) i n C. p a r v i f l o r a shows the same type of inheritance pattern as the dark-dotted gene (Ld) that Gorsic (1957) described i n heterophylla. This s i m i l a r i t y i n inheritance pattern as well as the s i m i l a r i t y i n hi s d e s c r i p t i o n of the dark-dotted character makes i t appealing to conjecture that the genes involved i n both species may be homologous. However, there i s no c l e a r evidence that t h i s i s the case. Now that the genetic determination of the heavy-spotting character is.understood, i t can be used to determine outcrossing rates using progeny t e s t s within populations of C_» p a r v i f l o r a . G r i f f i t h s et a l . (1977)» i n surveying populations of C o l l i n s i a i n southwestern B r i t i s h Columbia and northwestern Washington found the spotting character to be quite common i n the area between Crofton and L i t t l e River on Vancouver Island and i n those populations i n which i t does not occur n a t u r a l l y i t could e a s i l y be introduced since d i f f e r e n t populations of p a r v i f l o r a are able to interbreed (see Chapter 7)* Spotted plants can be distinguished i n the f i e l d with r e l a t i v e ease, e s p e c i a l l y the strongly heavy-spotted ones, as long as they are scored and marked when i n the rosette stage. The f a i n t - s p o t t i n g phenotype can be a complication when the degree of spotting f a l l s i n t o the range of overlap between the two types of sp o t t i n g . But since e r r o r s i n scoring could occur i n e i t h e r d i r e c t i o n , a large enough sample could eliminate any major deviations. Therefore, the heavy-spotting gene could become a use f u l t o o l i n determining breeding systems and outcrossing rates within populations of C o l l i n s i a p a r v i f l o r a . / - 24 -C h a p t e r 3 S i l v e r y L e a f Polymorphism I n t r o d u c t i o n A n other i n t e r e s t i n g l e a f and c o t y l e d o n polymorphism was found i n a p o p u l a t i o n o f CL* p a r v i f l o r a grown from seeds c o l l e c t e d on C a r l o s I s l a n d , B. C. ( F i g , 1), I n t h i s p o p u l a t i o n some o f the p l a n t s had a w r i n k l e d upper s u r f a c e and s i l v e r y sheen on t h e i r e a r l i e r l e a v e s and sometimes on t h e i r c o t y l e d o n s ( F i g . 7). Other p l a n t s had normal n o n - w r i n k l e d , green l e a v e s and c o t y l e d o n s ( F i g . 8). I n c r o s s - s e c t i o n t h e s e green l e a v e s l o o k e d normal w i t h a t y p i c a l , c l o s e l y - p a c k e d p a l i s a d e c e l l l a y e r . The s i l v e r y l e a v e s , however, had a v e r y l o o s e l y packed p a l i s a d e l a y e r , f u l l o f a i r p o c k e t s . When a drop o f water was added t o a f r e s h l y c u t s i l v e r y l e a f c r o s s - s e c t i o n , the e p i d e r m a l c e l l l a y e r would f l o a t away from the r e s t o f the l e a f s e c t i o n as the p a l i s a d e l a y e r broke up. L i k e the s p o t t e d - l e a f c h a r a c t e r , the s i l v e r y - l e a f c h a r a c t e r i s t r a n s i e n t and g e n e r a l l y seems t o fade o r be masked by a n t h o c y a n i n pigment as the p l a n t a ges. U n l i k e the s p o t t e d - l e a f c h a r a c t e r , how-ev e r , the s i l v e r y - l e a f c h a r a c t e r i s v e r y d i f f i c u l t t o d i s t i n g u i s h i n n a t u r e , p o s s i b l y due t o masking by the a n t h o c y a n i n pigments i n the l e a v e s . M a t e r i a l s and Methods The procedure used t o determine the i n h e r i t a n c e p a t t e r n f o r the s i l v e r y - l e a f polymorphism was v e r y s i m i l a r t o t h a t used f o r the s p o t t e d - l e a f polymorphism. S i l v e r y and green p l a n t s were chosen from p o p u l a t i o n #25 which was grown from seeds c o l l e c t e d on C a r l o s Fig, 8 Carlos Island plants showing normal green leaves - 26 -Island and reciprocal crosses were set up between them. Silvery plants from population #25 were also reciprocally crossed with plants from population #11 (Jack Point), which contained no silvery plants (Fig. l ) . A l l of the parent plants were also allowed to s e l f . Controlled crossing using the Carlos Island plants as seed parents was especially d i f f i c u l t . Not only were the buds small and d i f f i c u l t to,work with, the pedicels were so short that the flowers were borne very close to the stem and almost hidden among the leaves. This made them extremely d i f f i c u l t to manipulate while they were being emasculated. Therefore, more than one plant was used i n each cross and self and both homozygotes and heterozygotes may have been involved. The F^ and S^ plants were grown to the rosette stage, scored and tagged before the silvery character faded. Seed from each of these plants was then grown and scored individually. Two other plants brought from Carlos Island were crossed i n an attempt to study the inheritance of flower colour. The results were inconclusive as far as flower colour was concerned but the F^ and offspring included silvery and green-leafed plants and so were used for the assessment of the silvery-leaf character. Results The two plants from Carlos Island used in flower colour crosses showed good silvery-leaf results as mentioned above. The results of the cross-fertilizations and s e l f - f e r t i l i z a t i o n s of the two Carlos Island plants are summarized i n Table VI. Table VI S, and F, progeny of the crosses involving the silvery-leaf character Pollination regime Plant A selfed Plant B selfed Plant A (?) x Plant B (cT) Plant B (9) x Plant A (cf) Population #25; silvery-leafed, selfed Population #25; non-silvery, selfed #25 silvery (9) x #25 non-silvery (cT) #25 non-silvery (?) x #25 silvery (cf) #25 silvery (?) x #11 non-silvery (d1) #11 non-silvery ($) x #25 silvery (cf) # of individuals Silvery Green 22 0 0 34 7 0 12 0 15 0 0 14 5 0 0 16 5 0 15 7 28 These data i n d i c a t e that a s i n g l e gene system i s probably i n -volved and that plant A was a homozygous s i l v e r y - l e a f e d plant and that plant B was a homozygous green-leafed p l a n t . The r e c i p r o c a l crosses i n d i c a t e that the s i l v e r y - l e a f e d character i s dominant over the n o n - s i l v e r y - l e a f e d character. Table VI a l s o summarizes F^ and S^ progeny from population #25 (Carlos Island) and population #11 (Jack Point, n o n - s i l v e r y ) . The s i l v e r y - l e a f e d plants and the non-silvery-leafed plants allowed to s e l f - f e r t i l i z e y i e l d e d a l l s i l v e r y and a l l green progeny r e s p e c t i v e l y , i n d i c a t i n g they are homozygotes. These data agree with the r e s u l t s discussed above. However, the r e s u l t s of the cross-f e r t i l i z a t i o n s are not as expected. The cross, #25 non-silvery (9) x #25 s i l v e r y (c?) y i e l d e d only non-silvery-leafed o f f s p r i n g instead of the heterozygous s i l v e r y - l e a f e d o f f s p r i n g that were expected. These no n - s i l v e r y - l e a f e d plants are probably the r e s u l t s of a c c i d e n t a l s e l f s . Since emasculating and c r o s s - p o l l i n a t i n g the flowers of population #25 i s so d i f f i c u l t , a c c i d e n t a l s e l f i n g can probably occur quite e a s i l y . I f t h i s i s the case, the other crosses i n v o l v i n g seed parents from population #25 are also suspect and the s i l v e r y progeny from these crosses may also be the r e s u l t of accidental s e l f i n g . These suspicions were confirmed i n the next generation when a l l of the F., progeny r e s u l t i n g from crosses where a s i l v e r y -l e a f e d plant was used as the o r i g i n a l seed parents were s i l v e r y . This i n d i c a t e s that a l l the s i l v e r y F^ progeny were the r e s u l t of ac c i d e n t a l s e l f i n g . The F^, and S^ progeny are summarized i n Table V II, Table VII S P and Fp progeny of the crosses involving the silvery-leaf character Origin of and F.^  plants Population #25; silvery, selfed Population #25; non-silvery, selfed #25 silvery (9) x #25 non-silvery (c?) #25 non-silvery (9) x #25 silvery (d1) #25 silvery (9) x #11 non-silvery (c?) of S 2 & F 2 # of Sg. & F 2 individuals families Silvery Green 12 1 ,203 0 Ik 0 1 ,156 5 765 0 8 0 630 if 220 0 - 30 -The one cross that appears to have been successful i s #11 non-silvery (9) x #25 silvery (cf). The flowers from population #11 (Jack Point) can be emasculated and cross-fertilized quite successfully as shown in the spotted-leaf experiment. The 7 green-leafed progeny i n the F^ generation (Table VI) may be the result of a cross with a heterozygous silvery plant but since a l l of the s e l f -f e r t i l i z a t i o n s (accidental or otherwise) seemed to indicate only homozygotes, i t i s more probable that these progeny resulted from the accidental selfing of one or more flowers of the seed plants. However, the 15 silvery offspring of this cross can only be heterozygotes and the result of successful crossing since population #11 contains no silvery-leafed genes. These silvery offspring were i n i t i a l l y scored as 'intermediate 1 since they were not as clearly si l v e r as the plants resulting from the s e l f - f e r t i l i z a t i o n s (Fig. 9 ) . This made i t attractive to postulate that heterozygotes could be distinguished from homozygotes by the degree to which the silvery character i s expressed. However, when the F 2 progeny of these •intermediate' F^ plants were grown, the silvery plants did not segregate into the two categories, silvery and intermediate as expected, but stayed mostly i n the intermediate category with almost no plants showing the clearly silvery leaf of the population #25 plants. This would indicate that the 'intermediate' expression i s due to the modification of the silvery gene by the population #11 genotype rather than to the effects of heterozygosity. The F 2 progeny of the #11 green (9) x #25 silvery (cf) cross are summarized i n Table VIII. - 31 -F i g , 9 S i l v e r y , g r e e n and • i n t e r m e d i a t e 1 p l a n t s Table VIII Fp progeny of the #11 green (?) x #25 silvery (cf-) cross Phenotype of F, # of F . families Non-silvery 7 h 932 Silvery (intermediate) 13 1,026 347 0.05$ P= 0 . 9 - 0 . 5 deviation not significant # of F 2 individuals Y 2 . - x Silvery Green A v x a , I , ; 33 -The non-silvery plants bred true as expected. The If silvery plants were probably contaminants from neighbouring silvery families. The silvery plants segregating into an approximately 3*1 ratio indicates that the silvery-leafed character i s a simple dominant. Discussion The silvery-leaf polymorphism found on Carlos Island i s con-tr o l l e d by a single gene with a dominant a l l e l e for presence of the silvery-leafed character and a recessive a l l e l e for the absence of i t . The dominant a l l e l e i s very strongly expressed and very obvious in plants grown under a r t i f i c i a l conditions. Unfortunately however, the presence of the silvery-leafed character i s d i f f i c u l t to identify i n nature. So far, this silvery-leaf polymorphism has not been found i n any populations other than on Carlos Island, Controlled crossing with plants grown from seed from Jack Point indicate that other genotypes can modify the expression of this gene. - 34 -Chapter k Flower Colour Mutants Introduction The flowers of both the large and small-flowered £_ parviflora are blue with varying amounts of magenta and white i n the upper l i p . The varying intensity of pigment from plant to plant and i n different parts of the flower produces different shades of colour ranging from light blue to dark purple. In his original description, Lindley (1827) describes Collinsia grandiflora as "one of the most beautiful hardy annuals with which we are acquainted, covering the ground with a carpet, as i t were, of blue, purple and white, during the months of June and July". A l l of the populations studied had normal blue flowers, however, two populations from Vancouver Island also contained plants with atypical flower colour. Some of the plants from Elk F a l l s , a population of large-flowered Cj, parviflora, had pink or magenta flowers (Fig. 10) and some of the plants from Mt. Douglas Park, a population of small-flowered £. parviflora, had flowers that were completely white (Fig. 11). These two unusual flower colour mutants had not been reported in the area of this study but St. John (1956) reports these colours to be present i n two small-flowered ("parviflora") populations i n southeastern Washington and formally recognizes them as formae. Forma alba English i s described as having white corollas and forma rosea Warren i s described as having rose-mallow corollas. Harborne (1967) states that "a general ... characteristic of white flower mutants of coloured forms i s their unthriftiness as plants". This was clearly true of the white flowered C o l l i n s i a . - 35 -F i g . 11 An albino flower from the Mt Douglas Park population - 36 -The white-flowered population, although i t survived well under ideal culture conditions was more drastically affected by aphid attack, fungus infection and drying out than the normal blue-flowered populations* It i s interesting to note that aphids damaged the white flowers quite badly, often hampering successful c r o s s - f e r t i l -izations but did not seem to attack those flowers containing antho-cyanins, indicating perhaps that one of the functions of anthocyanin pigments i n parviflora i s protection from insect pests or that the character i s linked with another chemical character. It was observed that plants with blue or magenta flowers often had purple pigment on the underside of leaves. This pigment also tinged the upper surfaces of leaves in a l l except the white-flowered plants as the plants aged (Fig, 12), Pigment production could be considerably speeded up by increasing the light intensity or by an aphid attack. Greater leaf pigment production under higher light intensities i s probably related to the fact that light has been shown to be important for i n i t i a t i n g or stimulating flavonoid synthesis (Harborne, 1967), Injury can also stimulate anthocyanin synthesis or even i n i t i a t e pigment formation in tissues that usually do not contain any antho-cyanin colour (Bopp, 1959), This was the case when aphids infested a population. Then purple pigment was produced in the leaves even i n the seedling stage. Plants with white flowers did not produce any anthocyanin and when the plants aged or were infected by aphids or fungus the leaves turned yellow (Fig, 13), - 57 -F i g , 13 The Mt, Douglas Park population with yellow leaves — 38 — Through crossing experiments, I attempted to determine the inheritance of these flower colour mutants i n £. parviflora. Materials and Methods Population #2 was used as the source of white-flowered plants. It was grown from seed collected from white-flowered plants at Mt. Douglas Park (Fig. 1). Population #17 grown from seed collected i n Elk F a l l s (Fig. l ) was the source of both blue and magenta-flowered plants. In addition two more populations of blue-flowered plants were used; population #9 (Nanoose H i l l ) and population #11 (Jack Point). Some plants from each population were allowed to self for two generations and others were used i n the crosses.. The following crosses were set up between blue and white-flowered plants: #9 blue (9) x #2 white (cf) #2 white (9) x #9 blue (cf) #11 blue (?) x #2 white (cf) #2 white (9) x #11 blue (cf) The crosses between blue and magenta-flowered plants were: #17 blue (9) x #17 magenta (cf) #17 magenta (9) x #17 blue (cf) 39 -And the crosses between white and magenta-flowered plants were: #2 white (?) x #17 magenta (cf) #17 magenta (?) x #2 white (c?) In a d d i t i o n , two sets of crosses from the flower s i z e study showed flower colour segregations i n the F 2 generation and so were used to supplement the data from the above-mentioned crosses* In the crosses #17 (?) x #22 (cf); #22 (9) x #17 (cf), both blue and magenta-flowered plants from population #17 were used so that i n the F 2 generations some of the f a m i l i e s showed a segregation f o r flower colour* The other set of supplementary data came from the cross #22 (9) x #9 (c?)* This was also a cross used i n the flower s i z e study* Both populations were blue-flowered* Some of the F 2 f a m i l i e s from t h i s cross showed segregation f o r blue and white flower colour* This was completely unexpected since no other cross or s e l f - f e r t i l -i z a t i o n of e i t h e r of these two populations produced any white-flow-ered plants* One possible explanation f o r t h i s segregation i s that there was a mutation i n one of the parent plants which was passed on through t h i s p a r t i c u l a r c ross. Another i s that the white flower gene i s present i n one of the populations i n such low frequencies that i t i s r a r e l y i n i t s homozygous condition and therefore r a r e l y detected. I f t h i s were the case, one of the o r i g i n a l parents of the cross would have been heterozygous f o r the white flower gene* Whatever the reason, the white flower gene was present i n seven of the F 2 f a m i l i e s and so could be used i n determining i t s inheritance pattern* ko -The cross-fertilization methods, growing conditions and scoring methods were basically the same as for the studies discussed in previous chapters. In most crosses more than one plant was used as pollen parents and seed parents. However, i n crosses involving the Elk Fall s population, only one pollen parent and one seed parent were used i n each case, since the Elk Fall s flowers are very large and easy to manipulate* Seeds from the crosses and selfs were collected, grown, scored for flower colour and tagged* Seed from each F^ and S^ plant was then collected and grown separately* The F 2 and S 2 families were scored separately to determine the F^ and S^ genotypes and eliminate any contaminants and then the frequencies were totalled to get a more accurate ratio* The F 2 generation of the blue and white coloured crosses was attacked by aphids and most of the plants were k i l l e d i n the seedling stage* However, a tentative count was made without flowers, based on the observation that white-flowered plants turn yellowish and blue-flowered plants turn purplish when damaged by aphids* The plants that remained green were not included i n the count* Results The results from the plants that were allowed to se l f are summarized i n Table IX* The plants from a l l the populations appear to breed true for flower colour* This large amount of homozygosity i s expected in populations #9 and #11 since the magenta and white flower colour mutants are probably not present. The high frequency of homozygosity Table IX S 1 and S 2 progeny of the populations used in the crosses involving flower colour Population (allowed to self) Flower colour of parent plants # and flower colour of individuals # and flower colour of S 2 individuals #2 Mt. Douglas Park #9 Nanoose H i l l #11 Jack Point #17 Elk Fal l s #17 Elk Fal l s White Blue Blue Magenta Blue 20 plants - a l l white 13 plants - a l l blue ifl7 plants - a l l white i+Ok plants - a l l blue 19 plants - a l l blue 1,169 plants - a l l blue 3 plants - a l l magenta (S^ died before seed set) 30 plants - a l l blue 56 plants - a l l blue - 2f2 -i n population #1? i s not s u r p r i s i n g e i t h e r since the o r i g i n a l sample was very small, only four or f i v e p l a n t s . And the white-flowered plants from population #2 appear to be homozygous r e c e s s i v e . The fact that a l l of the plants l e f t to s e l f were homozygous in d i c a t e s that a l l or most of the plants used i n the crosses were probably also homozygous. Crossing data supports t h i s i n a l l of the crosses except #22 blue (9) x #9 blue (cf) which was discussed above. The o f f s p r i n g of the cross between blue and white-flowered plants were a l l blue except f o r 3 white-flowered plants which r e s u l t e d from the cross #2 white (9) x #11 blue (cf) and these were probably the product of an a c c i d e n t a l s e l f of the #2 white seed parent rather than a cross with a heterozygous population #11 p l a n t . In the F 2 gener-a t i o n there was segregation f o r both blue and white-flowered p l a n t s . The r e s u l t s of these crosses are summarized i n Table X. The t o t a l F 2 score f o r a l l of these crosses i s 92 blue : 32 white, an almost perfect 3:1 r a t i o . This simple Mendelian r a t i o makes i t a t t r a c t i v e to postulate a s i n g l e gene but the data were obtained from a t e n t a t i v e count of dying seedlings and hence the experiment should be repeated to substantiate t h i s r a t i o . The other cross r e s u l t i n g i n the segregation of blueand white-flowered plants was #22 blue (9) x #9 blue (cf). In t h i s cross, seven F 2 f a m i l i e s showed segregation i n t o blue and white-flowered progeny but they d i d not conform to the expected 3 blue : 1 white r a t i o . The t o t a l score f o r these f a m i l i e s i s 403 blue : 41 white (Table X I ) . Although these data do not f i t a common Mendelian r a t i o they are more r e l i a b l e than the 3:1 r a t i o obtained from the dying Table X and F 2 progeny resulting from crosses between blue-flowered and white-flowered plants Cross # of F, individuals # of F~ individuals Blue White Blue White #9 blue (9) x #2 white (cf) 9 0 15 k #2 white (?) x #9 blue (c?) 7 0 9 2 #11 blue (?) x #2 white (c?) 31 0 36 10 #2 white (9) x #11 blue (<*) 11 3* 32 _l£ Total 58 3 92 32 •accidental selfs Table XI F 2 progeny of the #22 blue (9) x #9 blue (cr) that showed segregation for flower colour Colour of the F, plants # of individuals i n each family which produced each family Blue White 1 . Blue 89 5 2 . Blue 32 9 3 . Blue 59 6 4 . Blue 61 8 5 . Blue 70 5 6 . Blue 38 2 1 2 . Blue 6 Total 403 41 - 45 -seedling count discussed above* I t i s reasonable to assume that t h i s seedling count underestimated the number of blue-flowered progeny since only the unhealthy seedlings whose leaves had turned colour were scored* And since white-flowered plants are l e s s hardy and more severely a f f e c t e d by aphids, most or a l l of the healthy and s t i l l green seedlings omitted from the a n a l y s i s were probably blue-flowered plants* Had they been scored, the f i n a l r a t i o would probably have shown an unexpectedly high proportion of the dominant blue-flowered F 2 o f f s p r i n g . In the crosses between blue and magenta-flowered plants the F^ plants were a l l blue-flowered, i n d i c a t i n g that blue flowers are dominant over magenta flowers as w e l l as white. In the F 2 generation the f a m i l i e s showed segregation f o r blue and magenta flowers. How-ever, the r a t i o was again an anomalous one, 50 blue : 6 magenta. These r e s u l t s are summarized i n Table X I I . Thus the magenta flower character, l i k e the white flower character, i s transmitted at a frequency that i s much lower than expected f o r simple s i n g l e gene in h e r i t a n c e . The cross between magenta and white-flowered plants produced a l l blue-flowered F.^  progeny except f o r one white contaminant proving that white and magenta flower colour are not a l l e l i c . And the F 2 generation showed segregation f o r blue, magenta and white flowers ( F i g . 1 4 ) . The r e s u l t s are summarized i n Table X I I I . Table XII F ^ and F^ progeny of the crosses between blue-flowered plants and magenta-flowered plants Pollination regime # and colour of F -L individuals # of F 2 individuals (by individual family) Blue Magenta #17 blue (9) x #17 magenta (cf) 11 blue 1 5 1 0 0 0 #17 magenta (9) x #17 blue (cf) #22 blue (9) x #17 magenta (<f) #17 magenta (?) x #22 blue (cf) 3 blue 1 blue 2 blue 12 6 20 3 1 Total 17 blue 50 - 47 -F i g , 14 The F 2 generation i n the cross between white and magenta-flowered plants, showing segregation for the three flower colours Table XIII F^ and F 2 progeny of the crosses between white-flowered and magenta-flowered plants # and flower colour of # of F 2 individuals Pollination regime t h e p i n ^ y i , ^ ^ B l u e Magenta White #17 magenta (?) x #2 white (cf») 10 blue 18 #2 white (?) x #17 magenta (c?) 65 blue 148 16 12 (1 white)* Total 75 blue 166 18 13 (1 white)* •accidental self - 49 -Discussion These data suggest that at least two l o c i are involved, with one gene (A) regulating the production of magenta pigment from a colourless precursor and a second gene (B) regulating the production of a blue pigment from the magenta one* i . e . colourless precursor ^ e n e A—> magenta g e n e B — ^ blue If this i s the case, the white-flowered plant has the genotype aaBB and the magenta-flowered plant has the genotype AAbb* The hybrid i s AaBb and blue* And the blue-flowered plants used i n the crosses were a l l AABB* However, using the above hypothesis, the F 2 ratio for the crosses between blue-flowered and white-flowered plants would be expected to be 3 blue : 1 white. But the actual ratio was 403 blue : 41 white. The crosses between blue and magenta would also be expected to yield an F-> ratio of 3:1 instead of the unusual 50 blue : 6 magenta ratio that was actually produced* This very low frequency of the recessive a l l e l e also occurred in the crosses between white and magenta-flowered plants. The expected ratio would be 9 blue : 3 magenta : 4 white. The actual ratio was 166 blue : 18 magenta : 13 white. One of the simplest ways to explain these data i s to postulate that only two genes are actually involved but that there i s a selective pressure against the recessive allele s i n the formation of gametes or against the homozygous recessive condition i n the zygote or young seedling. - 5© -This is not the only possible explanation for this unusual ratio. It may be a much more complex system involving more than two loci and various gene interactions. But a system of three loci can-not explain a l l of the information and there are not sufficient data to postulate more genes. Therefore at this point the simplest hypothesis is the one discussed above. However, i t must be further tested to determine whether or not selection against the recessive alleles is actually taking place. If the hypothesis proved false, more complex genetic determinants would have to be investigated. Although the inheritance patterns for the flower colour poly-morphisms have not been conclusively determined, this study has provided a feasible working hypothesis as a basis for initiation of further studies into the problem. - 51 -Chapter 5 Inheritance of Flower Size Introduction Flower size i s the main character used to separate £. parviflora and C. grandiflora i n most floras and in Newsom's (1929) monograph of the genus. The original descriptions (Lindley, 182?) do not give any actual measurements but later authors divide the two species into two very precise and measurable size classes. However, they do not always agree on the range i n flower size of each species, the degree of overlap between the two or the point at which the two species are separated. Table XIV summarizes how various authors have treated this character. The dividing line between grandiflora and parviflora has been drawn seemingly a r b i t r a r i l y at different flower sizes by different authors, Abrams (1951) includes i n C_, parviflora those plants with corollas less than 10 mm long whereas Davis (1952) states that C. parviflora has corollas less than 6 mm long and £• grandi-flora has corollas between 6,5 and 9 nim long. Only Peck (1961) and St. John (1956) have formally recognized any region of overlap between the two species i n their keys and descriptions. However, Newsom (1929) does state in her text that "i n studying these two closely a l l i e d species, one finds a continuous series of intergradation from the largest-flowered grandiflora to the smallest-flowered parviflora". Other authors, notably Gilkey and Dennis (1967), make the two size classes appear to be quite disti n c t . This i s not the case i n nature. In fact there seems to be a more or less continuous range i n flower size among the populations Table XIV Author Peck (1961) Davis (1952) St. John (1956) Henry (1915) Anderson (1961) Munz & Keck (1959) Lindley (1827) Newsom (1929) Hitchcock et a l . (1959) Piper (1906) Piper & Beattie (1915) Gilkey & Dennis (1967) Abrams (195D Corolla length classes assigned to C. parviflora and C. grandiflora by various authors C. grandiflora C. parviflora „ . » Total corolla length (mm) Region of flora e 1 2 3 k 5 6 7 8 9 10.11 12 13 14 15 16 17 18 19 1 1 i 1 Oregon I—. 1 Idaho h •• ' 1 S.E. Washington 1 1 Southern B.C. +• 1 ' ' Alaska *" ^ California "H 1 ' Orig. descript. no size estimate Monograph \ T ' ' Pacific N.W. I- 1 ' f Washington ' ^ ' ' 1 1 I 1 Pacific N.W. I - i Pacific States J 1 1 - 53 -observed in this study. And many of them occupy the regions of division chosen by various authors and so are d i f f i c u l t to assign confidently to one species or another. Because of the importance placed on flower size as a key character and because of the differing opinions among various authors using this character, i t was of special interest to examine variation in flower size both among and within populations i n this study and to determine the genetic mechanism controlling i t . Materials and Methods Flower size varies considerably among populations of C. parvi-f l o r a i n southwestern B r i t i s h Columbia and northwestern Washington. For example, flowers from the Lindeman Lake population are usually 4-5 mm whereas flowers from Elk F a l l s are about 10-1? mm. However, the flower size within any particular population i s usually relatively uniform and populations grown from seeds collected i n different areas can often be visually distinguished merely on the basis of flower size. For this particular study five populations were chosen on the basis of flower size, ranging from the population with the largest observed flowers to the population with the smallest observed flowers and with each population v i s i b l y different from the others i n the size of i t s flowers (Fig. 15). Three of these populations f e l l within the C. grandiflora range according to Hitchcock and Cronquist (1973) and two f e l l within the C. parviflora range. See Table XV. The flowers of the Lindeman Lake population were very small and d i f f i c u l t to work with. Emasculation of the tiny buds was not - 54 -Fig. 15 The variation in flower size between the five populations used in study. From l e f t to right: Elk F a l l s , Jack Point, Nanoose H i l l , Botanie Valley, Lindeman Lake Table XV Populations of C. parviflora used i n the study of flower size variation Total corolla length Locality Population number Species according to Hitchcock & Cronquist (1973) 10 8 7 6 k 17 mm 10 mm 8 mm 7 mm 5 mm Elk F a l l s Jack Point Nanoose H i l l Botanie Valley Lindeman Lake 17 11 9 22 6 C« grandiflora C, grandiflora  C. grandiflora C. parviflora C, parviflora - 56 -possible and so they were only used as a pollen parent. Plants from population #6 were crossed with plants of each of the other populations. The Botanie Valley population had slightly larger flowers than those from Lindeman Lake and so was less d i f f i c u l t to work with. Reciprocal crosses wereset up between plants from this population and plants from each of the "(_. grandiflora" populations. Some plants from each population were also allowed to s e l f . Seeds from the cross-fertilizations and s e l f - f e r t i l i z a t i o n s were grown to pro-duce the F-^  and S^ generation which was i n turn allowed to self to produce the F 2 and S 2 generation. A sample of flowers was measured for each of the .original parental populations as well as each of the F l ' S l ' F2 a j x^ i S2 Populations except for the S^ generation of popul-ation #11 (Jack Point). To minimize the effect of differing flower angles the measure-ment taken was from the t i p of the keel to the top of the saccate or gibbous swelling at the base of the corolla tube (See Fig. 16). Fig . 16 A Collinsia flower showing the angle at which measure-ments were taken The measurements were taken to the nearest 0.5 mm. These were later rounded off to the nearest mm in order to simplify the graphing. Samples of three flowers per plant were taken (whenever possible) from up to twenty plants in each parental, S^._ and F^ population. In 57 -the and generation three flowers (whenever possible) per plant were taken from up to ten plants per family and up to nine families from any particular self or cross. These samples were analyzed and compared by totaling the number of flowers i n each size class (to the nearest mm) in the sample and converting them to percentages. The percentages were then graphed. In addition to each graph, a table was set up indicating the mean flower size and the sample size of each population involved. One of the crosses set up for flower colour (population #2 x population #17) also showed interesting results as far as flower size was concerned. Population #2 (Mt, Douglas Park) had "parviflora" flowers and population #17 (Elk Falls) had very large "grandiflora" flowers. The hybrids were obviously intermediate. Samples were measured, analyzed and compared in the same way as the crosses dis-cussed above. But because this was not originally designed as a cross to determine the inheritance of flower size there i s no sample for the original parental population from Mt, Douglas Park, Results The graphs for flower size of the five populations showed a certain amount of overlap between the populations and some shifting of the histograms from one generation to the next but the peak of each histogram was clearly separate from a l l the others (Figs, 17, 18, 19). The mean flower sizes were also different for each of the populations (Tables XVI, XVII, XVIII), The shifting of the histograms Figur< 17 Graph c omp arinj j ric wer sizes < r the f i v e populations in 1 the i i "irst (parenta] /) a •eneration • o 8 u It £_ / o •3 / c / \ / / \ #22 J i re 1 \ 4-= f < » +• «w 40 1 M #13 » 2 4 J . 8 flowe. 11II L c t ii :e • ( 1( rar ) 12 14 1 1 6 Table XVI Mean flower sizes and sample sizes of the populations graphed i n Fig, 1? Population x flower size ( # Q f p l a ^ f e g Z * f f l o w e r s ) Lindeman Lake 4»0 mm 5 15 Botanie Valley 4*5 mm 9 2? Nanoose H i l l 6,0 mm 5 15 Jack Point 7,0 min 6 18 Elk Falls 10,5 mm 18 53 Table XVII Mean flower sizes and sample sizes of the populations graphed i n Fi g . 18 _ , . . - . Sample size Population x flower size ( # Q f p l a n t s ) ( # o f f l o w e r e ) Lindeman Lake 3 . 0 mm 15 45 Botanie Valley 4»0 mm 9 26 Nanoose H i l l 5*0 mm 11 33 Elk Falls 13 .0 mm 17 40 Figure 1 9 Graph comparing flower s i z e s of the f i v e populations i n the flower s i z e (mm) Table XVIII Mean flower sizes and sample sizes of the populations graphed in Fi g . 19 - „n . Sample size Population x flower size ( # Q f p l a n t e ) ( # o f flowers) Lindeman Lake 2.5 mm 50 121 Botanie Valley if.5 mm 72 188 Nanoose H i l l 6.0 mm 72 186 Jack Point 7.5 mm 40 102 Elk F a l l s 11.5 mm 60 164 from one generation to the next may simply be due to differences i n the age of the plants when the flowers were measured. I t was ob-served, p a r t i c u l a r l y i n population #17, that the plants produced smaller flowers as they aged. These s h i f t s could also be due to environmental f a c t o r s . However, since the plants were a l l grown under uniform growth chamber conditions, the environmental e f f e c t s would be minimal. The crosses i n v o l v i n g the Lindeman Lake population (#6) as a polle n parent were not very s u c c e s s f u l . Very few seeds were set and most of those seemed to be contaminants since the supposed 'hybrids' resembled t h e i r seed parents. This was l a r g e l y a t e c h n i c a l problem since the flowers from t h i s population were very small and had very l i t t l e p o l l e n . Most of the poll e n was u s u a l l y l o s t i n the attempt to t r a n s f e r i t onto another stigma. However, at l e a s t one or two pro-geny appear to have been genuine hybrids because i n the F^ generation one family from the cross #11 (9) x #6 (6*) had flowers of an i n t e r -mediate s i z e (See F i g , 20) and one family ( c o n s i s t i n g of a s i n g l e plant) from the cross #9 (9) x #6 ( c f ) , had flowers i n the s i z e range of population #6, i . e . 2,5 - 3-5 mm. The crosses using the Botanie V a l l e y population (#22) for the " p a r v i f l o r a " parent were more su c c e s s f u l . Hybrids were r e a d i l y formed and the F.^  and F 2 hybrid peaks were intermediate to the parental peaks i n most cases ( F i g s , 21, 22, 23), The mean flower s i z e s of the hybrids were also intermediate between the parents (Tables XX, XXI, XXII), And the F g generation showed a greater v a r i a t i o n i n flower s i z e than the F n generation, suggesting segreg-Figure > 5rai )h L C 0 m rini tt t lower sizei 3 < >f Lindeman ike ( #6 ) > ack P nt L] »opul ions ( pa ie h£ i J 01 • I at : )( ) with a ] i •Vi c m L -r> 0 0 n (1) 1 \ ft hi ill o o w i cr V / nv 1 i i j 0 0 5 1 c 2 _ .• ••' 4 •\ / 6 % i 'lower siz 3 { 10 [mn L) 12 14 16 Table XIX Mean flower sizes and sample sizes of the populations graphed in Fig, 20 Population x flower size Sample size (# of plants) (# of flowers) Lindeman Lake parental if,0 mm 15 Jack Point parental 7,0 mm 18 Possible F 2 hybrid 6,0 mm 10 21 Figure 21 100 Graph comparing the flower s i z e s of the hybrids between populations from Botanie V a l l e y (#22) and Nanoose H i l l (#9) with t h e i r parental populations flower s i z e (mm) Table XX Mean flower sizes and sample sizes of the populations graphed i n Fig. 21 Population x flower size Sample size (# of plants) (# of flowers) Botanie Valley parental F x hybrid F 2 hybrid Nanoose H i l l parental if*5 mm 4»5 mm 5.5 mm 6.0 mm 9 18 160 5 27 53 380 15 100 80 CO i -o 60 H (0 +> O +» © k0 20 Figure 22 Graph comparing the flower sizes of the hybrids between populations from Botanie Valley (#22) and Jack Point (#11) with their parent populations #22 parental 8 I ON VC 1 .0 ] ll > Ik im) -... Table XXI Mean flower sizes and sample sizes of the populations graphed i n Fig, 22 Population x flower size Sample size (# of plants) (# of flowei Botanie Valley parental F x hybrid F 2 hybrid Jack Point parental 4,5 mm 5,5 mm 6,5 mm 7,0 mm 9 18 120 6 27 49 290 18 Figure 23 Graph comparing the flower s i z e s of hybrids between populations from flower s i z e (mm) ^ T J T T J T T l T t l I M I I I I I H - H - l 1 1 1 1 1 1 1 M 1 I I I I I I M H H + H ^ B Table XXII Mean flower sizes and sample sizes of the populations graphed i n Fi g . 23 Population Botanie Valley parental F1 hybrid F 2 hybrid Elk Fal l s parental - „ . Sample size x flower size ( # Q f p l a n t s ) ( # o f f i 0 W e r s ) 4»5 mm 9 27 8,5 mm 11 33 7.5 mm 107 187 10.5 mm 18 53 - 73 -ation for the genes determining flower size (Figs, 21, 22, 23), This would be expected i f flower size is polygenically controlled. The cross between population #17 and population #22 produced similar results (Fig, 24; Table XXIII), These results indicate that the character of flower size i s controlled by polygenic or quantitative inheritance though i t is not clear how many genes are actually involved. Discussion The graphs (Figs, 17, 18, 19) confirmed previous observations that flower size varies between populations and yet is relatively uniform within any particular population. The amount of overlap between the different populations, especially between "grandiflora" and "parviflora" populations weakens the case for using flower size as a key character especially considering that this was a relatively small sample of populations chosen particularly on the basis of their clearly different flower size distributions, A more extensive col-lection of populations would undoubtedly blur the taxonomic dis-tinctions even more. In addition, the "grandiflora" and "parviflora" populations can interbreed quite readily. This clearly indicates that they are not good biological species in the sense of Mayr (1957) and that there is only one biological species, £, parviflora.involved. However, i f the morphological characters of £, grandiflora and C, parviflora are sufficiently distinct and different, they could be separated at the level of taxonomic species as distinguished from biological species F i g n r s of 9 ] K >J «• Graph comparing the flowei 1 s i z e ids 3 1 between 1 copulation B fron fbr l n .00 Mt • Dougla (#2 ) sine 1 El k F a witl J l l o FX/; r ] jarem; popuiax-xons 80 r r-t /ft s r-l «H > n 1 H / \ l OS +> • O *» <* o 40 20 c > 1 _ «L 6 / / _ / / / / I 1 / / i \ l c \ F 3 •wer 2 \ \ s i z F l \ \ y hybrit \ \ T VT \ \ H N "••-...\ i t [mm) 1 V-#17 ] 12 parental 14 Lc i Table XXIII Mean flower s i z e s and sample s i z e s of the populations graphed i n F i g . 24 Population x flower size Mt. Douglas Park S1 F^ hybrid F 2 hybrid Elk F a l l s parental 4.5 mm 9.0 mm 7«5 mm 10.5 mm Sample s i z e (# of plants) (# of flowers) 18 20 154 18 47 70 388 53 - 76 -by Cain (1954) and Grant (1963» 1971)• T h i s cannot be done with the character of flower size which shows a continuous variation, however other morphological characters w i l l be examined i n Chapter 7 i n order to determine whether the recognition of two or more taxonomic specieB can be j u s t i f i e d . - 77 -Chapter 6 Chromosome Numbers Introduction Garber and his students have done extensive work on the chromo-somes of Collinsia and their studies indicate that with one exception, the species a l l have seven bivalents at metaphase I (Garber, 1956, 1958a, 1958b; Ahloowalia and Garber, 1961; Garber and Dhillon, 1962, Hayhome and Garber, 1968; Garber, 1974)• The one exceptional species i s C, torreyi with a count of 21 bivalents, making i t a hexaploid. It was at f i r s t considered the " f i r s t polyploid to be encountered in the genus" (Garber, 1958b) but later was said to have been "erroneously identified as a member of Collinsia (since) no tetraploid species has been found i n the genus" (Garber, 1974)• This casts some doubt on the identity of the plants for which Garber has reported chromosome numbers, especially since there are no voucher specimens* However, Taylor and Mulligan (1968) i n their Flora of the Queen Charlotte Islands, give the chromosome number of £, parviflora as n=14, making i t a tetraploid, Garber's chromosome counts for C, parviflora (1956) and C^ grand-i f l o r a (1958b) are both n=7 but the parviflora count came from only two pollen mother c e l l s and the grandiflora count appears to have come from only one plant, Taylor and Mulligan took their count from a population on Haida Pt,, Graham Island but Garber made no mention as to the source of his populations. Because of these conflicting reports I examined six populations with different flower sizes to determine their chromosome number. The objectives were to determine i f there were differences i n number in different populations, i f both tetraploids and diploids existed i n - 78 -t h i s a r e a o f s t u d y , and whether s m a l l and l a r g e - f l o w e r e d p o p u l a t i o n s d i f f e r e d i n chromosome n u m b e r . M a t e r i a l s and Methods P l a n t s were grown from s e e d c o l l e c t e d a t t h e f o l l o w i n g l o c a l i t i e s (See F i g . 1): it P o p u l a t i o n #6 - L i n d e m a n L a k e P o p u l a t i o n #22 - B o t a n i e V a l l e y P o p u l a t i o n #9 - Nanoose H i l l P o p u l a t i o n #25 - C a r l o s I s l a n d P o p u l a t i o n #11 - J a c k P o i n t P o p u l a t i o n #17 - E l k F a l l s Buds were f i x e d i n a 6:3?2 s o l u t i o n o f e t h a n o l , c h l o r o f o r m and p r o p i o n i c a c i d and t h e n s t a i n e d i n a l c o h o l i c h y d r o c h l o r i c a c i d - c a r m i n e (Snow, 1963). T h e s e buds were t h e n d i s s e c t e d , t h e a n t h e r s removed a n d s q u a s h e d . S i n c e t h e a n t h e r s mature a t d i f f e r e n t t i m e s and a l l o f t h e c e l l s i n a s i n g l e a n t h e r do n o t d i v i d e s y n c h r o n o u s l y , a bud w i t h t e t r a d s o f t e n a l s o c o n t a i n e d a few p o l l e n mother c e l l s a t l a t e p r o -phase o r metaphase I . The chromosomes t e n d e d t o be q u i t e s t i c k y and were d i f f i c u l t t o c o u n t b u t a t l e a s t one c e l l w i t h d i s t i n g u i s h a b l e chromosomes was f o u n d and p h o t o g r a p h e d f o r e a c h p o p u l a t i o n . V o u c h e r s a r e d e p o s i t e d a t t h e 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 . R e s u l t s The f o l l o w i n g c o u n t s were o b t a i n e d from t h e p o p u l a t i o n s s t u d i e d : P o p u l a t i o n #6 - L i n d e m a n L a k e 2n = 14 I I P o p u l a t i o n #22 - B o t a n i e V a l l e y 2n = 14 I I - 79 -Population #9 - Nanoose H i l l 2n = 14 II (See Fig. 25) Population #25 - Carlos Island 2n = 14 II (See Fig . 26) Population #11 - Jack Point 2n = 14 II Population #17 - Elk Fa l l s 2n = 14 II Discussion A l l of the populations surveyed for chromosome number were tetraploids with a count of 2n = 14 I I . This agrees with Taylor and Mulligan's (1968) count for C_. parviflora from the Graham Island population and suggests that C. parviflora i s tetraploid i n Britis h Columbia. None of the populations studied had a chromosome number of n=7 as reported by Garber (1956, 1958b). It i s possible that C^ parvi-flora consists of both diploid and tetraploid populations and Garber obtained his plants from diploid populations. However, i t i s also possible that C^ parviflora i s a s t r i c t l y tetraploid species and the material from which Garber obtained his counts was misidentified as parviflora and £. grandiflora. It i s unfortunate that he does not state the source of his seeds. Both large-flowered and small-flowered populations had a chromo-some number of 2n = 14 I I , supporting the inclusion of both large and small-flowered plants in the same species. Since large and small-flowered plants formed f e r t i l e hybrids, this result was not unexpected. - 8 0 Fig. 25 A pollen mother c e l l from the Nanoose H i l l population showing a chromosome number of 2n = 14 II (mag, x 1,650) Fig, 26 A pollen mother c e l l from the Carlos Island population showing a chromosome number of 2n = 14 II (mag. x 1,900) - 81 Chapter 7 Morphology and Taxonomy Introduction I t was established i n Chapter 5 that £ . g r a n d i f l o r a and C. p a r v i f l o r a are i n t e r f e r t i l e and therefore not separate b i o l o g i c a l species i n the sense of Mayr (1957) and Grant (1963)» However, t h i s i s not s u f f i c i e n t reason to ignore the nomenclatural d i s t i n c t i o n between the two. I t may be u s e f u l i n p r a c t i c a l taxonomic work to consider them to be two taxonomic species (Grant, 1971) which express morphological d i f f e r e n c e s rather than c r o s s a b i l i t y or i n t e r f e r t i l i t y r e l a t i o n s h i p s . The following morphological study was conducted i n an attempt to determine whether the " g r a n d i f l o r a " and " p a r v i f l o r a " groups are s u f f i c i e n t l y d i f f e r e n t and d i s t i n c t to be considered separate taxonomic species. M a t e r i a l s and Methods Sixteen populations were grown from seed c o l l e c t e d from the following l o c a l i t i e s (See F i g . l ) : Vancouver Island. B. C. Thetis Lake - population I - population I I M i l l H i l l - population I - population I I Mt. Douglas Park - population I - population I I Nanoose H i l l - population IV - population I I I - 82 -Nile Creek L i t t l e River Crofton - population I - population II Rathtrevor Park Sechelt Peninsula. B. C. Lund Irvine's North Washington. U.S.A. Anacortes In addition five of the populations used i n the genetic studies were used in the Sg generation: #6 Lindeman Lake, B. C. Vancouver Island, B. C. #2 Mt. Douglas Park - (population III) #9 Nanoose H i l l - (population I) #11 Jack Point #17 Elk F a l l s A l l of the above populations were planted i n shallow f l a t s i n growth chambers and allowed to reach maturity, at which time veget-ative and f l o r a l characters were measured. The vegetative and f l o r a l characteristics of £• grandiflora and C. parviflora. especially those that reportedly differed between the two groups, were summarized on a data sheet (Table XXIV). Each 83 -Table XXIV Data Sheet for Population Stem Leaves erect, ascending, other branched, unbranched glandular, puberulent, glabrate, other t a l l oblong, ovate, spatulate, other obtuse, acute, other serrulate, entire, entire-revolute, other glabrate, puberulent, glandular, other sessile, sub-sessile, petiolate upper whorls becoming linear smaller bracteolate i n inflorescence i purple underside ___________ long; wide Flowers Pedicels Calyx flowers i n whorls rarely solitary, commonly solitary below, never solitary puberulent, glandular, glabrate, other times as short as flowers long glabrate, puberulent, glandular, other membranous below ________ scabrous-margined __________ times length of corolla long lobes: subulate, linear-lanceolate, other acuminate, other wide - 84 -Corolla strongly declined, declined, almost erect forms 0 angle with pedieel long upper lips colour _ _ _ _ _ _ long sinus deep lobes: obovate, spatulate, retuse, dilate, crisp-crenulate, recurved, erect, other wide lower l i p : colour wide corolla tube: colour long saccate, gibbous, other sparsely bearded, glabrous, other Comments: - 85 -population was then surveyed for the presence or absence of each possible character. Corolla size was measured as the total length of the corolla rather than the measurement used in Chapter 5 in order to make the measurements comparable to those of other workers. The data were then summarized in Table XXV to include a l l those characters that showed variation among the different popul-ations. In this form the data could be examined to determine any patterns or obvious differences which could be used to divide the populations into two or more groups. Results a) Stem In the populations studied, the stem height ranged from 2.4 cm (Rathtrevor) to 25 cm (Nanoose H i l l I). Newsom (1929) describes C. parviflora stems as ascending or erect as compared to £j, grandi-flora stems which are almost always erect. However, the populations in this study cannot be separated on this basis since a l l plants were erect. They were also a l l puberulent. The only stem character that varied was branching. Most populations showed branching but the absence of branching could not be correlated with any other character. b) Leaves The greatest amount of variation within and between populations was found in the leaves. Leaves varied in size, shape, leaf margin, pubescence, pigmentation and length of petioles (See Figs. 27, 28). However, this variation was often as great or greater within a - 8 6 -Table XXV Summary of the v a r i a t i o n among the populations studied . . • • A. «_ __> A* • t. I_l i_l 1—4 • I. -^t m ft* «•** «-t* Ua PT* FT I—«• ct <+ H- P* P 4 H* • • • H CD CD P H ft cr H O O O _ H- H- ffl O O O W CD OJ 5 r r < | 0 & t J c t H O O I » r t H c H J B F- o a . | 3 * H H i , M » o * + ' c o cu o o Hrjfcj O <+_ ct- d- O H P O O P CD *t> a i H D o o » i » O r k i i i B M - O CD CD H' P P ct- 4 CD CD <D H CO H- <t M ffl W CD „ - , „ , „ „ . . . 0) P M e H H H B H ' J I < H H H H H H H « < + 4 M • «) rTd) H < S » !B p O M CD M M CO 01 CO M 4 H 4 H • ct- <D M M M p* V) M M M £ 0 P H* £ If) W IS H H H P R-CD + + + + + + + + + + + + + + + + + + + + + + + + 4- + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + •*• + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + ++ ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + • + + + + + + + + + • + + + + + + + + + + + + + + + + + + + + + + ++ +• + + ++ + + + + + + • + + + '++ + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + * + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + •»•+ + + + + + + + + + +•+ + + + + + + + + + + + + + + + + + + + + + + + + stem branched leaves oblong leaves ovate leaves acute leaves obtuse leaves crenate leaves lobed leaves s e r r u l a t e leaves toothed leaves undulate leaves puberulent leaves glabrate leaves sub-sessile purple underside flowers s o l i t a r y below pedicels 1-2 x flower length pedicels 1/2-1 x flower length calyx glabrate calyx puberulent <- 1/2 c o r o l l a length > 1/2 c o r o l l a length c o r o l l a strongly declined ( c o r o l l a declined ( 45°) c o r o l l a almost erect tube saccate tube gibbous c o r o l l a limb ^. tube c o r o l l a limb ^ tube c o r o l l a k mm c o r o l l a 5 mm c o r o l l a 6 mm c o r o l l a 7 mm c o r o l l a 8 mm c o r o l l a 9 mm c o r o l l a 10 mm c o r o l l a 7" 10 mm - 15 mm 90°) - 87 -Fig, 27 The leaf characters of the Botanie Valley population Fig. 28 The leaf characters of the Jack Point population - 88 particular population than "between populations and i t i s not possible to use leaf characters as a means for separating the populations into different taxa. c) Flowers The number of flowers per whorl has been used as a diagnostic character separating the two species. Newsom (1929) describes C. parviflora as having "2-5 flowers i n a whorl above, usually solitary below". By contrast, she describes C. grandiflora as having "flowers 3-7 at a node, rarely solitary". As can be seen in Table XXV, a l l populations except Nanoose H i l l IV had flowers "usually solitary below". As for the upper whorls, a l l populations had flowers in 2's and only three populations had whorls with more than 5 flowers. These were Mt. Douglas Park III, Nanoose H i l l I and Elk Fal l s and include both "parviflora" and "grandiflora"-size flowers. d) Pedicels A l l of the populations had puberulent pedicels. Hitchcock et a l . (1959) describe Cj_ grandiflora flowers as being shorter pediceled. According to Table XXV, this does not appear to be the case. e) Calyx The presence or absence of puberulence on the calyx varies between and within the populations and does not seem to be a diagnostic character. But the length of the calyx compared to that of the corolla was used as a diagnostic character i n Newsom1s (1929) monograph. She states that i n C_» parviflora the calyx i s "from half - 89 -as long to almost equal the corolla length" and i n C_» grandiflora the calyx i s "1/3 - 1/2 the corolla length". In the populations studied, only two (Irvine's North, Elk Falls) had calyces which were less than 1/2 the corolla length. These are both very large-flowered populations but other large-flowered populations (e.g. Jack Point) did not show this characteristic. f) Corollas In addition to corolla size C_, parviflora i s said to diff e r from C. grandiflora i n the "possession of an almost erect and gibbous corolla tube i n contrast to a declined and saccate tube" (Newsom, 1929). The populations were studied for the angle of the corolla and whether the corolla was saccate or gibbous. Most of the populations examined had a corolla tube declined at about a 45° angle. Six pop-ulations had almost erect corollas but since these were found in both larger flowered- and small flowered-populations, there i s no very good correlation between small flowers and erect corollas. Three populations contained plants with corollas that were strongly declined (about 90°)• Two of these were the very large-flowered populations from Irvine's North and Elk F a l l s . The other was a smaller-flowered population from M i l l H i l l I I . There i s also no clear pattern i n the distribution of saccate and gibbous tubes. Many populations had both types of corolla tube present, usually with their smaller flowers being gibbous and their larger ones saccate. Again, the gibbous corolla tube does not correlate with almost erect flowers. There does however seem to be a general trend toward gibbous corolla tubes i n the smaller-flowered populations and saccate corolla tubes i n the larger-flowered populations. Another corolla character used by some authors to distinguish the two species i s the relative size of the corolla limb versus the corolla tube (Piper, 1906; Henry, 1915; Peck, 1961), In C_, parviflora the corolla tube i s said to be longer than the limb and i n grandi  flora the limb i s not longer than the tube. In the populations studied, a l l were of the "parviflora" type i n that the tube was longer than the limb, g) Flower size There was a continuous range of flower sizes from 4 mm to 15 mm without any clear break. I f the populations were to be divided into two or more groups on the basis of flower size there would be con-siderable overlap between the two classes no matter where the line was drawn. Discussion Although there i s considerable variation i n vegetative charact-e r i s t i c s among and within the populations studied, there were no correlations among the characters that would support dividing the group into two species. The f l o r a l characteristics are taxonomically more useful. There i s a general trend towards the association of saccate corolla tubes with larger flowers and gibbous corolla tubes with smaller flowers. - 91 -There i s also a trend towards an association of declined (45°) to almost erect corolla tubes with smaller flowers and declined (45°) to strongly declined (90°) corolla tubes with larger flowers. How-ever, neither of these trends can be used to make a clear-cut division into two species. The calyx character i s more clear cut. In most populations, the calyx i s greater than 1/2 the corolla length. In two of the largest-flowered populations, however, the calyx length i s less than 1/2 of the corolla length. Using this character, i t i s possible to separate these two populations from the rest. These two populations also both have strongly declined corollas and saccate corolla tubes but these three characters are not entirely independent of one another. If calyces stay the same size but corollas are longer, the calyx w i l l automatically be less than 1/2 the corolla length. And since the difference between saccate and gibbous i s a matter of degree, i f the corolla as a whole i s smaller, i t might be expected to have smaller gibbosity. The angle of the corolla w i l l also strongly influence the degree of gibbosity. These characters are probably a l l different expressions of just one difference ~ smaller versus larger corollas. Therefore, the separation of these two populations from the rest i s not sufficient to ju s t i f y putting them into a separate taxonomic species. Therefore I propose to place a l l the entities now considered to be C_ grandiflora Dougl, ex L i n d l . and C&. parviflora Doug Vex Lind l . into a single species; _C_. parviflora Dougl. ex L i n d l . since this name has published p r i o r i t y . In addition, I propose to erect two sub-92 -species, C, parviflora ssp, parviflora and C, parviflora ssp, grandiflora (Dougl,.ex Lindl,) Krause to express the morphological differences discussed above. Although these two subspecies are somewhat arbitrary, they do recognize the two extremes i n flower size that were formerly given species status. And they maintain the terms "parviflora" and "grandiflora" to distinguish these extremes. - 93 C o l l i n s i a parviflora Dougl, ex Lindl, Bot, Reg, 13 : p, 1082 1827, ssp, parviflora Anthirrhinum tenellum Pursh, F l , Am, Sept. 421, 1814, Linaria tenella F, G, Dietr, V o l l s t , Lexik, Gaertn, Nachtr, 4:408, 1818, Col l i n s i a tenella Piper. Contr, U.S. Nat. Herb, 11:496, 1906, Not C, tenella Benth. 1846. C. minima Nutt. Journ. Acad. Phila. 7:47, 1834. C. parviflora var. minima M. E. Jones, Contr. West. Bot. 12:69. 1908. C. grandiflora var. pus i l l a Gray, Syn. F l . 21:256. 1878. C. p u s i l l a Howell F l . N.W. Am. 506. 1901. C. grandiflora ssp. pus i l l a Piper, Contr. U.S. Nat. Herb. 11:496. 1906. C. breviflora Suksd. W. Amer. S c i . 12:54. 1901. C. multiflora Howell, F l . N.W. Amer. 506. 1901. C. d i e h l i i M. E. Jones, Contrib. West. Bot. 12:68. 1908. C. parviflora var. d i e h l i i Pennell i n Abrams, 111. F l . Pac. St. 3:778. 1951. C. parviflora forma alba English i n St. John, F l . S.E. Wash. 370. 1956. C. parviflora forma rosea Warren, Proc. B i o l . Soc. Wash. 41:197. 1928. Stems ascending or erect, branched or unbranched, glabrate to puberulent, 2.4-40 cm t a l l ; leaves various, serrulate to entire, ovate to lance-linear, obtuse to acute, labrate to puberulent, petiolate to occasionally sub-sessile, often purplish below, 0.6-5 cm long, 0.3-1.9 cm wide, becoming smaller and bractiform i n inflorescence; flowers i n whorls, 2-7 at a node, often solitary below; pedicels puberulent to glandular-pubescent; calyx membranous below, puberulent to glabrate, scabrous margined, from 1/2 as long to almost equal the corolla length; calyx lobes subulate to linear-lanceolate, acuminate, ca, 1 mm wide; corolla usually declined (ca 45°) to almost erect, 4-10 mm i n length, various shades of blue to purple, sometimes with white or whitish upper l i p , rarely a l l white or magenta; f i l a -ments stout, glabrous; stigma 2-lobed; capsule 3-5 mm long, slightly exceeded by calyx tips; seeds, usually 4-6, round-oblong, thick, smooth, reddish-brown or brown. Colli n s i a parviflora Dougl. ex Li n d l . ssp. grandiflora (Dougl. ex Lindl.) Krause. stat. n. £ i grandiflora Dougl. ex L i n d l . Bot. Reg. 13:pi. 1107. 1827. C. grandiflora var. nana Gray, Proc. Am. Acad. 8:394. 1872. ! - 9 4 -Similar to parviflora ssp, parviflora; corollas 9-17 mm long; strongly declined (ca 90°); saccate corolla tube; calyx less than 1/2 the corolla length. By placing the entire group into one species with two subspecies, both biological relationships and morphological differences are ex-pressed. The single species expresses the i n t e r f e r t i l i t y as well as the continuous nature of the variation within the group. But the recognition of two subspecies, though somewhat a r t i f i c i a l , recognizes the fact that i n spite of the seemingly continuous variation, the two extremes are very different. - 95 -Bibliography Abrams, L. 1951. Illustrated Flora of the Pacific States. Vol. I I I . Stanford University Press, Stanford, California. Ahloowalia, B.S. and Garber, E.D. 1961. The genus Collinsia XIII. Cytogenetic studies of interspecific hybrids involving species with pediceled flowers. Botanical Gazette 122: 228-232. Anderson, J.P. 1961. Flora of Alaska and adjacent parts of Canada. Iowa State University Press, Amer. Iowa. Bopp, M. 1959* Uber die bildung von anthocyan und leucoanthocyan an wundrandern. Zeitschrift fur Botanik 197-215* Cain, A.J. 1954* Animal Species and their Evolution. Hutchinson and Co. Ltd., London. Davis, Ray J . 1952. Flora of Idaho. Wm. C. Brown Co. Dubuque, Iowa. Garber, E.D. 1956. The genus Collinsia I. Chromosome number and chiasma frequency of species in the two sections. Botanical Gazette 118: 71-73» Garber, E.D. 1958a. The genus Collinsia I I I . The significance of chiasmata frequencies as a cytotaxonomic tool. Madrono 1_±: 172-176. . 1958b. The genus Coll i n s i a VII. Additional chromo-some numbers and chiasmata frequencies. Botanical Gazette 120: 55-56. . 1974. C o l l i n s i a . 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