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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 i n populations of C o l l i n s i a p a r v i f l o r a Dougl.ex L i n d l . (Scrophulariaceae)  by  Sc.  Gerda Rosa Krause (Hon.), U n i v e r s i t y of B r i t i s h Columbia, 1975  A thesis submitted i n p a r t i a l f u l f i l l m e n t of the requirements for the degree of Master of Science  in The Faculty of Graduate Studies Department of Botany  We accept t h i s thesis as conforming to the required standard  The U n i v e r s i t y of B r i t i s h Columbia Gerda Rosa Krause, 1978  In  presenting  an  advanced degree  the I  Library  further  for  shall  agree  scholarly  by  his  of  this  written  thesis  in  at  University  the  make  that  thesis  partial  freely  permission  for  It  is  financial  for  gain  of of  British  of  of  Columbia,  British  Columbia  for  extensive by  the  understood  permission.  University  fulfilment  available  p u r p o s e s may be g r a n t e d  2075 Wesbrook P l a c e Vancouver, Canada V 6 T 1W5  Date  it  representatives.  Department The  this  shall  requirements  reference copying  Head o f  that  not  the  of  I  agree  and  be a l l o w e d  or  that  study.  this  thesis  my D e p a r t m e n t  copying  for  or  publication  without  my  - i i-  Abstract The C o l l i n s i a populations i n southwestern B r i t i s h Columbia and northwestern  Washington show considerable v a r i a t i o n i n l e a f and  flower characters both within and between populations.  One  of the  purposes of t h i s study was to determine the genetic mechanisms cont r o l l i n g some of these characters. Two  l e a f polymorphisms were studied.  absence of purple anthocyanin  They were the presence or  spots on the surface of the leaves and  the presence or absence of a s i l v e r y sheen, also on the surface of the leaves. two a l l e l e s .  Each was shown to be c o n t r o l l e d by a single gene with The spotted l e a f character was dominant over the un-  spotted l e a f character and the s i l v e r y sheen was dominant over the normal green l e a f character. Flower size was also studied and shown to be controlled by polygenic inheritance. Two mutant flower colours, white and magenta, are found i n t h i s region of study, i n 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 c o n t r o l l i n g the pro-  duction of the magenta pigment from the colourless precursor and  one  c o n t r o l l i n g the production of the blue pigment from the magenta one. Another purpose of t h i s work wa6 number of the C o l l i n s i a s i n t h i s area.  to determine the chromosome Both d i p l o i d (n=7)  t e t r a p l o i d (n=l*f) counts have been reported. studied and a l l were found to be  tetraploid.  and  Six populations were  - iii -  F i n a l l y , the c y t o l o g i c a l and genetic data were used i n conjunction with morphological data to revise the taxonomy of the C o l l i n s i a s i n t h i s region of study.  Most authors divide them i n t o  two species, £ . g r a n d i f l o r a Dougl, ex L i n d l , and ex L i n d l ,  p a r v i f l o r a Dougl,  However t h i s study indicates that they are only one  highly variable species, C_, parvi f l o r a .  - iv Table  of  Contents Page  Abstract Table of Contents L i s t of Tables L i s t of F i g u r e s and I l l u s t r a t i o n s Acknowledgement Chapter a) b) c) d)  1  Chapter a) b) c) d)  2  Chapter a) b) c) d)  3  Chapter a) fc) c) d)  k  Chapter a) b) c) d)  5  i i iv v vii ix  Introduction Taxonomy Cytogenetics Genetics M a t e r i a l s and Methods  1 2 3 5  L e a f Spot Polymorphism Introduction M a t e r i a l s and Methods Results Discussion  10 13 15 21  S i l v e r y L e a f Polymorphism Introduction M a t e r i a l s and Methods Results Discussion Flower Colour Mutants Introduction M a t e r i a l and Methods Results Discussion Inheritance of Flower Introduction M a t e r i a l s and Methods Results Discussion  2 2  4 2If 6 33  3k 38 ifO k9 Size 51 53 57 73  Chapter 6 Chromosome C o u n t s a) Introduction fc) M a t e r i a l s a n d M e t h o d s c) Results d) Discussion  77 78 78 79  Chapter a) b) c) d)  81 81 85 90  7  Taxonomy Introduction M a t e r i a l s and Methods Results Discussion  Bibliography Vita  - v List  of Tables Page  Table I  Table I I  P o p u l a t i o n numbers o f the C o l l i n s i a p o p u l a t i o n s used i n the c r o s s i n g experiments • • • • • • • • • • • • • • • • Si and F^ progeny o f the c r o s s e s i n v o l v i n g the s p o t t e d l e a f c h a r a c t e r  • • •  7  16  S 2 and Fp progeny o f h e t e r o z y g o u s s p o t t e d S^ and F^ p l a n t s • • • • • • • • •  18  Sg and Fg progeny o f u n s p o t t e d S^ and Fx p l a n t s • • • • • • •  19  Sg and Fg progeny o f homozygous s p o t t e d S, and F, p l a n t s • • • • • • • • • • • • •  22  S^ and F]_ progeny o f the c r o s s e s i n v o l v i n g the s i l v e r y - l e a f c h a r a c t e r  • • •  27  Table VII  S and F progeny o f the c r o s s e s i n v o l v i n g the s i l v e r y - l e a f c h a r a c t e r  • • •  29  Table V I I I  F progeny o f the #11 green (9) x #25 s i l v e r y (d ) c r o s s • • • • • • •  Table I I I T a b l e IV Table V T a b l e VI  2  2  2  1  T a b l e IX  Table X  T a b l e XI  Table XII  Table XIII T a b l e XIV  T a b l e XV  S^ and So progeny o f the p o p u l a t i o n s used i n t h e c r o s s e s i n v o l v i n g f l o w e r colour  41  F^ and Fp progeny r e s u l t i n g from c r o s s e s between b l u e - f l o w e r e d and w h i t e - f l o w e r e d plants • • • • • • • • • • • • • • • • • •  43  F progeny o f the #22 (?) x #9 b l u e (<3) c r o s s t h a t showed s e g r e g a t i o n f o r f l o w e r colour • • • • • • • • • • • • • • • • • •  kk  F^ and F progeny o f the c r o s s e s between b l u e - f l o w e r e d p l a n t s and magenta-flowered plants • • • • • « • • • • • • • • • • • •  46  F i and F progeny o f the c r o s s e s between w h i t e - f l o w e r e d magenta-flowered p l a n t s • •  48  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  P o p u l a t i o n s o f C. b a r v i f l o r a used i n the study o f f l o w e r s i z e v a r i a t i o n . . • ,  55  2  2  2  - vi  Table XVI  Mean flower s i z e s and sample s i z e s of the populations graphed i n F i g . 17  Table XVII  Mean flower s i z e s and sample s i z e s of the populations graphed i n F i g . 18  Table XVIII  Mean flower s i z e s and sample s i z e s of the populations graphed i n F i g . 19  Table XIX  Mean flower s i z e s and sample s i z e s of the populations graphed i n F i g . 2 0  Table XX  Mean flower s i z e s and sample s i z e s of the populations graphed i n F i g . 2 1  Table XXI  Mean flower s i z e s and sample s i z e s of the populations graphed i n F i g . 2 2  Table XXII  Mean flower s i z e s and sample s i z e s of the populations graphed i n F i g . 2 3  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 . 2 4  Table XXIV  Data sheet f o r population  Table XXV  Summary of the v a r i a t i o n among the populations studied . . . . • ..•  _________  - vij  List  of Figures  -  and  Illustrations Page  Figure  1  Map o f t h e s t u d y a r e a s h o w i n g t h e l o c a l i t i e s f r o m w h i c h s e e d s were c o l l e c t e d • • •  6 8  Figure  2  Collinsias  Figure  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 • • • • • • • • • • • • • • •  8  P l a n t s w i t h a n d "without p u r p l e a n t h o c y a n i n s p o t s on t h e u p p e r e p i d e r m i s o f the leaves • • • • • • • • • • • • • • • •  11  P l a n t showing the f a i n t - s p o t t i n g character • • • • • • • • • • • • • • • • •  12  Figure  Figure  k  5  growing i n 5-inch  pots  • • • • •  Figure  6  P l a n t showing the h e a v y - s p o t t i n g character • • • • • • • • • • • • • • • • •  Ik  Figure  7  C a r l o s I s l a n d p l a n t s showing the silvery-leaf character • • • • • • • • • •  25  Figure  8  Carlos Island plants green leaves  9  Silvery,  Figure  10  Figure  11  A magenta f l o w e r from population • • • • • An a l b i n o f l o w e r f r o m Park population • • •  Figure  Figure  Figure  Figure  12  13  Ik  15  16  normal  • • • • • • • • • • • • • • •  Figure  Figure  showing  g r e e n and  intermediate' the • • the • •  plants  25 •  Elk Falls • • • • • • • • • Mt» Douglas • • • • • • • • •  31  35 35  A population with anthocyanin pigments i n the leaves • • • • • • • • • • • • • • •  37  The Mt, Douglas P a r k p o p u l a t i o n w i t h yellow leaves • • • • • • • • • • • • • • •  37  The F g e n e r a t i o n i n t h e c r o s s between white and magenta-flowered p l a n t s , showing segregation f o r the three flower colours •  k?  The v a r i a t i o n i n f l o w e r s i z e between t h e f i v e populations used i n study. From l e f t to 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  2  A C o l l j n s i a flower showing the angle a t w h i c h measurements were t a k e n • • • • •  56  - viii Page Figure 1?  Figure 18 Figure 19 Figure 20  Figure 21  Figure 22  Figure 23  Figure 24  Graph comparing flower sizes of the f i v e populations i n the f i r s t (parental) generation • • • • • • • • • • • • • • • • • Graph comparing flower sizes of the f i v e populations i n the second ( S , ) generation  •  60  Graph comparing flower sizes of the f i v e populations i n the t h i r d (S^) generation • •  62  Graph comparing the flower s i z e s of Lindeman Lake (#6) and Jack Point (#11) populations with a possible hybrid between them • • • • • • • • • • • • • • • •  65  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 • • • • • • • • •  67  Graph comparing the flower s i z e s of the hybrids between populations from Botanie V a l l e y (#22) and Jack Point (#11) with t h e i r parental populations • • • • • • • • •  69  Graph comparing the flower sizes of hybrids between populations from Lindeman Lake (#22) and E l k F a l l s (#17) with t h e i r parental populations • • • • • • • • • • • •  71  Graph comparing the flower s i z e s of hybrids between populations from Mt. Douglas Park (#2) and E l k F l l s (#17) with t h e i r parental populations • • • • • • • • • • • •  74  A pollen mother c e l l from the Nanoose H i l l population showing a chromosome number of 2n s 14 I I  80  A pollen mother c e l l from the Carlos Island population showing a chromosome number of 2n = 14 II  80  The l e a f characters of the Botanie V a l l e y population • • • • • • • • • • • • •  87  The l e a f characters of the Jack Point population • • • • • • • • • • • • • • • • •  87  a  Figure 25  Figure 2 6  Figure 27 Figure 28  58  Acknowledgement  I would l i k e to express my gratitude to my d i r e c t o r , 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 t h e i r w i l l i n g assistance, discussion and c r i t i c i s m during the research and t h e i r help i n preparing the manuscript; to Dr. K. I . Beamish, Dr. C. 0. Person and Dr, B. A. Bohm f o r advice and assistance with various parts of t h i s t h e s i s ; to Mr. Ken Carey and Dr. W. B. S c h o f i e l d f o r c o l l e c t i n g C o l l i n s i a 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 f o r typing the manuscript.  Finally,  I would l i k e to thank my husband, E r i c , for a l l h i s encouragement and i n f i n i t e patience.  Chapter  Introduction  1  Taxonomy  The and  blue-flowered  northwestern  species. iflora C,  The  Taylor  Lindley  by  David  of  the  more  (1827)  grown  the  Newsom species C,  also  followed  £_t (in  and  from  (1929), i n further  been  recent  two  species separate  at  year,  collected the  was  into  plants  by  et  seeds  by L i n d l e y ,  of  an  at  "the  grand-  placed a l , ,  originally  from  Douglas  two C,  are  in  1959;  described  collected  Columbia River,  again  distance  Columbia  considered  Hitchcock  grown  the  her  monograph two  a n d C±  smaller  of  C^  parvi-  from  garden  dry  hundred miles  195D  floras  Howell,  rank  recognized of  this  these  the  banks and  pusilla  to  be  entities.  varieties  coast  although  of  maintained  var,  pusilla  1906), at  the  Peck  do n o t  (1961)  parviflora«  C,  parviflora.  always  level:  Pennell  N o r t h A m e r i c a have  they  pusilla  specific  synonym o f  of  two  Gray,  Variety  (1959) a n d a  the  C_, g r a n d i f l o r a ;  typica.  pusilla a l .  two  of  (Piper,  et  west  group,  than  Hitchcock  considered  genus  grandiflora  flowers  subspecific  the  varieties  (1903) a n d r e c o g n i z e d  (Gray)  in  grandiflora  of  divided  usually  1951;  specimens  same  typica  given  Pennell  the  C,  recognized  var,  Howell  Abrams,  the  been  smaller-flowered  vicinity  seeds  p u s i l l a having  Pusilla  although  to  i n  are  (Abrams,  garden  the  generally  British  ocean",  grandiflora  variety has  in  Columbia River,  from  the  1977)*  from  described  specimens  and  southwestern  plants  D o u g l , ex L i n d l ,  Douglas  was  have  larger-flowered  and MacBryde,  by  flora  Washington  D o u g l , ex L i n d l ,  parviflora  Collinsias in  A l l  of  recognized  agree  on  how  There has been l i t t l e uniformity among authors over where to draw the l i n e between Cj, g r a n d i f l o r a and C, p a r v i f l o r a , fusion i s r e i n f o r c e d by the condition i n nature.  This con-  One finds a con-  tinuous s e r i e s of intergrading populations from the smallest p a r v i f l o r a to the largest flowered grand!flora. by Newsom (1929) but she maintained  flowered  This fact was noted  the two species on the basis that  "the, two extremes d i f f e r greatly' . However, a f t e r h y b r i d i z a t i o n and 1  morphological  studies, I have come to the conclusion that t h i s  C o l l i n s i a group containing g r a n d i f l o r a . p a r v i f l o r a and p u s i l l a i s only one highly v a r i a b l e species.  The name C, p a r v l f l o r a has p r i o r i t y  over the name £• g r a n d i f l o r a . Therefore I w i l l use the name C, p a r v i f l o r a to r e f e r to the whole group unless otherwise  specified.  Cytogenetics Garber and h i s students have published a considerable amount of work on the genus C o l l i n s i a , morphological  Most of the work i s cytogenetic, some' i s  and some i s chemical.  However C± parviflora« which he  divides i n t o p a r v l f l o r a 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 £• p a r v i f l o r a i n the Queen Charlotte Islands had a chromosome number of n=14.  The chromosome counts obtained from the  populations i n t h i s 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 h i s  samples nor does he c i t e voucher specimens.  -  3  -  Genetics Newsom (1929) d i v i d e d the genus i n t o two l e n g t h o f the p e d i c e l s . congested  i n t o whorls,  One  group o f s p e c i e s has  sessile  flowers,  Garber (1958a) d i v i d e d the s p e c i e s i n t o  groups a c c o r d i n g t o whether they e x h i b i t e d h i g h or low  f r e q u e n c i e s , and  these groups c o r r e l a t e d w e l l w i t h  chiasmata  the*pediceled  f l o w e r group and the s e s s i l e f l o w e r group r e s p e c t i v e l y . He t i n g u i s h e d between " v a r i a b l e " and " u n i f o r m " Although  the  the o t h e r group has p e d i c e l e d f l o w e r s t h a t  a r e s o l i t a r y or i n whorls, two  groups based on  populations  also dis-  (Garber,  he does not d e f i n e p r e c i s e l y what he means by these terms i n  h i s d i s c u s s i o n o f s e s s i l e - f l o w e r e d , he s t a t e s t h a t " u n p u b l i s h e d i n d i c a t e t h a t these  observed,,,  On  data  ( s e s s i l e - f l o w e r e d ) s p e c i e s share a l a r g e number o f  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  (Garber,  1974),  yet  i n the s p e c i e s w i t h d i s t i n c t l y p e d i c e l e d f l o w e r s "  1958b). the b a s i s o f the above t h r e e c h a r a c t e r i s t i c s , Garber p l a c e s  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 c o n t a i n s p l a n t s with p e d i c e l e d flowers, uniform mata f r e q u e n c y . northwestern  The  c o l l i n s i a s i n southwestern B r i t i s h Columbia  Washington a r e d e f i n i t e l y p e d i c e l e d .  on the chiasmata f r e q u e n c y . uniform.  p o p u l a t i o n s and h i g h c h i a s -  There a r e no  However, the p o p u l a t i o n s a r e not  Many p o p u l a t i o n s show c o n s i d e r a b l e v a r i a t i o n i n l e a f  shape, pubescence and  c o l o u r i n g , f l o w e r s i z e and  data  very size,  colouring, etc.  doubt many of these c h a r a c t e r s are g e n e t i c a l l y c o n t r o l l e d .  and  No  I  i n v e s t i g a t e d a number o f these polymorphisms t o determine the g e n e t i c mechanisms c o n t r o l l i n g them.  -  One  k  -  common polymorphism i s the presence or absence of dark  purple spots on the upper surface of the leaves. was  This character  found to a c t u a l l y include two separate characters.  A gene system  producing large, heavy spots sometimes covering most of the l e a f 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.  I t s genetic mechanism  was not p o s i t i v e l y determined but the r e s u l t s of a study by G r i f f i t h s et a l . ( 1 9 7 7 ) suggests that a single incompletely penetrant  gene i s  involved. Another l e a f polymorphism i s the presence or absence of a s i l v e r y sheen on the e a r l y leaves.  This was a l s o shown to be c o n t r o l l e d by a  single gene with a dominant a l l e l e f o r the s i l v e r y sheen. The inheritance of flower colour i s more complex. flower colours were found i n the  Three d i f f e r e n t  p a r v i f l o r a populations studied.  These were magenta and white i n addition to the normal blue.  The  r e s u l t s were.not conclusive but suggested the following genetic interpretation.  One gene (A) c o n t r o l l e d the production of the magenta  pigment from the c o l o u r l e s s precursor.  Another gene (B) c o n t r o l l e d  the production of the blue pigment from the magenta pigment.  A homo-  zygous recessive (aa) a t the f i r s t step r e s u l t e d i n a white-flowered plant and a homozygous recessive (bb) a t the second step resulted i n a magenta-flowered plant. waB,  This r e l a t i v e l y simple Mendelian system  however, complicated by s e l e c t i o n against the recessive a l l e l e s  i n the gamete or zygote which d i s t o r t e d the expected 3*1 r a t i o s . The genetic c o n t r o l of flower s i z e i s of s p e c i a l i n t e r e s t because Cj, p a r v i f l o r a and C. g r a n d i f l o r a are separated mainly on the  -  basis of that character.  5 -  As expected f o r a c o n t i n u o u s l y  varying  c h a r a c t e r such as f l o w e r s i z e the i n h e r i t a n c e mechanism i s p r o b a b l y polygenic.  However i t was n o t p o s s i b l e t o determine e x a c t l y how many  genes a r e i n v o l v e d .  M a t e r i a l s and Methods C. p a r v i f l o r a i s a good organism f o r g e n e t i c s t u d i e s s i n c e i t grows w e l l under a r t i f i c i a l  c o n d i t i o n s and, b e i n g 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  o f p l a n t s used i n t h i s study were grown from seed  c o l l e c t e d from v a r i o u s p l a c e s i n southwestern B r i t i s h Columbia and n o r t h w e s t e r n Washington.  F i g u r e 1 i s a map o f the a r e a 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 .  F o r c o n v e n i e n c e , the  p o p u l a t i o n s used i n the c r o s s i n g experiments were a s s i g n e d  numbers.  These p o p u l a t i o n numbers a r e l i s t e d i n Table I . Seeds were c o l l e c t e d from mature brown c a p s u l e s and s t o r e d i n paper p a c k e t s u n t i l needed. filter  Seeds were sown between p i e c e s o f m o i s t  paper i n p e t r i p l a t e s and p l a c e d i n a r e f r i g e r a t o r a t 5 ° - 9°C  u n t i l t h e seeds germinated and the r a d i c l e s were about 1-2 cm l o n g . T h i s u s u a l l y took about two t o t h r e e weeks.  They were then p l a n t e d  in  f l a t s or pots o f s o i l .  The p a r e n t a l and F^ g e n e r a t i o n s  in  5 i n c h p o t s ( F i g . 2 ) and were l a r g e v i g o u r o u s  the number o f i n d i v i d u a l s i n the F it the  2  generation  was p l a n t e d c l o s e l y spaced i n s h a l l o w  flats  were grown  p l a n t s , but s i n c e  was u s u a l l y much l a r g e r , (Fig. 3)«  p l a n t s were o f t e n s m a l l e r and l e s s v i g o u r o u s  As a r e s u l t ,  but t h i s d i d n o t  a f f e c t t h e study because s i z e and v i g o u r were n o t measured and the  - 6 -  Table I Population numbers of the C o l l i n s i a  populations  used i n the crossing experiments  Population  number  Source of seeds (Locality)  #2  Mt« Douglas Park, Vancouver Island  #6  Lindeman Lake, B. C,  #9  Nanoose H i l l , Vancouver Island  #11  Jack Point, Vancouver Island  #17  Elk F a l l s , Vancouver Island  #22  Botanie V a l l e y , B, C.  #25  Carlos Island, B. C.  - 8 -  Fig. 3  C o l l i n s i a s growing c l o s e l y i n shallow f l a t s  spaced  - 9 -  characters studied were expressed large ones.  i n the small plants as well as the  Three to four weeks were required to grow seedlings i n  s o i l to the rosette stage where l e a f polymorphisms could be  evaluated  and another four to f i v e weeks before most of the plants started to flower. A l l plants were grown i n growth chambers under a c o o l , long day regime of 16 hours l i g h t 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 c r o s s - p o l l i n a t i o n i s quite d i f f i c u l t because the flowers are small and w i l l s e l f - p o l l i n a t e r e a d i l y .  They have to be  emasculated i n the bud stage before the pollen i s mature and  before  the anthers can dehisce e i t h e r n a t u r a l l y or a c c i d e n t a l l y with handling. The buds are only about 2-3 mm  long, and must be handled under the  d i s s e c t i n g microscope i n order to ensure that the anthers are comp l e t e l y removed and the s t y l e and stigma are not damaged.  An  a d d i t i o n a l complication i s that occasionally one of the anthers dehisce i n the closed bud stage and scatter p o l l e n over the  will  stigma.  However, i n a s u c c e s s f u l l y emasculated bud, the stigma w i l l reach maturity i n 3 to 5 days and can then be c r o s s - p o l l i n a t e d . 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.  -  Chapter 2  10  -  Leaf Spot Polymorphism  Introduction One  of the most obvious polymorphisms found within many of the  populations of C o l l i n s i a p a r v i f l o r a on Vancouver Island i s the presence or absence of purple anthocyanin the leaves ( F i g . 4 ) .  spots on the upper epidermis of  These spots vary i n s i z e , number and shape both  among and within the i n d i v i d u a l plants and are usually found only on the e a r l i e r leaves of the plant and sometimes on the cotyledons. anthocyanin  The  spots usually fade and disappear as the plant gets older  or as the temperature r i s e s .  For example, wild c o l l e c t e d spotted  plants transferred to a heated greenhouse l o s t t h e i r spots within three days. Gorsic ( 1 9 5 7 ) reported a s i m i l a r polymorphism i n h i s genetic studies of (Lj, heterophylla B u i s t .  He c a l l e d i t dark-dotted  (Ld) and  described i t as a transient cotyledon and l e a f character i n which the "upper surfaces of cotyledons and leaves show maroon spots of various numbers and s i z e s " .  He found t h i s character to be i n h e r i t e d as a  simple dominant. G r i f f i t h s et a l . , 1 9 7 7 » i n working with populations of £. grandi f l o r a L i n d l . i n southwestern B r i t i s h Columbia and  northwestern  Washington discovered two g e n e t i c a l l y d i s t i n c t spotting systems, a f a i n t spotting system (F) and a heavy spotting system (H).  There i s  considerable v a r i a t i o n within each system and the ranges of the degree of spotting within the two classes probably overlap.  However, plants  with the f a i n t degree of spotting u s u a l l y have only a few of t h e i r leaves bearing a small number of t i n y dots, about 0 . 5 mm (Fig. 5 ) .  i n diameter  The precise genetic determination of the f a i n t spots  not conclusively established but the r e s u l t s suggested  was  that a single  - 11 -  Fig, k  P l a n t s w i t h and without anthocyanin  purple  s p o t s on the upper  e p i d e r m i s o f the  leaves  -  Fig, 5  12  Plant showing the character  faint-spotting  -  incompletely  penetrant  13  -  gene i s i n v o l v e d .  P l a n t s with a heavy degree  o f s p o t t i n g u s u a l l y have b l o t c h e s o f v a r i o u s shapes and or most of the l e a v e s and  on the c o t y l e d o n s  b l o t c h i n g can v a r y c o n s i d e r a b l y the e n t i r e l e a f t o a few determination  ( F i g , 6),  s i z e s on a l l The  amount o f  from a l a r g e b l o t c h c o v e r i n g  s m a l l s p o t s near the base.  o f heavy s p o t t i n g i s d i s t i n c t  The  almost  genetic  from t h a t of f a i n t  spotting  and m a i n t a i n s i t s i n h e r i t a n c e p a t t e r n even when the f a i n t - s p o t t i n g gene i s The  present. s p o t t i n g system i n v e s t i g a t e d i n t h i s study was  s p o t t i n g one.  the heavy  C r o s s e s were s e t up t o determine the exact  pattern for t h i s p a r t i c u l a r  inheritance  polymorphism.  M a t e r i a l s and Methods In o r d e r t o study the g e n e t i c s of the s p o t t i n g system, an imental used.  population  c o n t a i n i n g both s p o t t e d and u n s p o t t e d p l a n t s  R e c i p r o c a l c r o s s e s were s e t up between s p o t t e d and  p l a n t s from a p o p u l a t i o n  of p l a n t s ( p o p u l a t i o n #11) (Fig, 1),  c o l l e c t e d from J a c k P o i n t were a l s o a l l o w e d  to s e l f .  Spotted  Hill  ( p o p u l a t i o n #9),  ( F i g , 1),  to  unspotted  grown from seeds  unspotted  plants  and  p l a n t s from another  d e r i v e d from seeds c o l l e c t e d from Nanoose  that contained  p l a n t s were a l s o a l l o w e d  was  In a d d i t i o n , r e c i p r o c a l c r o s s e s were made  between the s p o t t e d p l a n t s of p o p u l a t i o n #11 population  and  exper-  only unspotted p l a n t s .  These u n s p o t t e d  self.  I n o r d e r t o get s u f f i c i e n t progeny from the c r o s s e s , more than one  p l a n t was  i n v o l v e d i n each c r o s s .  and heterozygous p a r e n t s c r o s s or  self.  Therefore,  both homozygous  c o u l d have been i n v o l v e d i n any  particular  Fig, 6  P l a n t showing the character  heavy-spotting  - 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 t o the r o s e t t e stage when t h e y were c l a s s i f i e d and tagged for  t h e presence o r absence  of s p o t t i n g i n the e a r l y l e a v e s .  The  t a g g i n g a t t h i s s t a g e i s v e r y important s i n c e the s p o t t i n g fades a s the p l a n t g e t s o l d e r .  The p l a n t s were then l e f t  to s e l f  naturally  and the seed from each o f these p l a n t s was c o l l e c t e d and grown i n d i v i d u a l l y t o be s c o r e d .  Results The r e s u l t s o f the a r t i f i c i a l  c r o s s e s i . e , t h e F ^ p l a n t s and  the r e s u l t s o f t h e s e l f - p o l l i n a t i o n s , i , e , the S^ p l a n t s a r e summarized i n T a b l e I I , These r e s u l t s i n d i c a t e t h a t the absence  o f spots i s a t r u e -  b r e e d i n g c h a r a c t e r i n p o p u l a t i o n s from both Nanoose H i l l Point,  and J a c k  The s p o t t e d p l a n t s on t h e o t h e r hand, produced both s p o t t e d  and u n s p o t t e d progeny when s e l f e d , i n d i c a t i n g t h a t a s i n g l e gene system may be i n v o l v e d and t h a t a t l e a s t some o f the p l a n t s i n v o l v e d were h e t e r o z y g o u s  f o r s p o t t i n g and t h a t the s p o t t e d c h a r a c t e r i s t i c  i s dominant over t h e u n s p o t t e d one. The i n d i c a t i o n t h a t t h e s p o t t e d c h a r a c t e r i s dominant i s r e i n f o r c e d by t h e r e c i p r o c a l c r o s s e s .  When t h e s p o t t e d p l a n t s a r e used  e i t h e r as male o r a s female p a r e n t s and c r o s s e d w i t h u n s p o t t e d p l a n t s the r e s u l t i n g progeny a r e m o s t l y s p o t t e d .  A l l o f the s p o t t e d F-^  progeny would be expected t o be h e t e r o z y g o u s , c o n t a i n i n g s p o t t e d a l l e l e s from t h e i r s p o t t e d p a r e n t s and u n s p o t t e d a l l e l e s from unspotted parents.  their  Table I I S.. and F, progeny of the crosses involving l e a f spots  P o l l i n a t i o n regime °  Pr e  Spotted  Population #11; spotted selfed  °Seay Unspotted  18  5  Population #11; unspotted selfed  0  22  Population #9; unspotted selfed  0  13  (cf)  26  15  (?) x #11 spotted (o")  22  7  (cf)  13  11  (?) x #11 spotted (cr)  15  19  #11 spotted (9) x #11 unspotted #11 unspotted  #11 spotted (9) x #9 unspotted #9 unspotted  -  17  -  Seeds were c o l l e c t e d from each o f the  and S^ p l a n t s t h a t  reached m a t u r i t y (some were k i l l e d by a p h i d s and mildew) and f a m i l y was  grown and s c o r e d i n d i v i d u a l l y t o determine  make-up o f the p a r e n t and t o determine to u n s p o t t e d p l a n t s i n the progeny  each  the g e n e t i c  the r a t i o o f s p o t t e d p l a n t s  o f the h e t e r o z y g o t e s .  Most o f f a m i l i e s grown from s p o t t e d F^ and S^ p l a n t s s e g r e g a t e d i n t o s p o t t e d and u n s p o t t e d p l a n t s i n approximate  y.l r a t i o s c o n f i r m i n g  t h a t they were h e t e r o z y g o t e s and t h a t a s i n g l e gene w i t h a dominant allele  f o r the presence o f heavy s p o t t i n g and a r e c e s s i v e a l l e l e f o r  the absence  o f heavy s p o t t i n g c o n t r o l s 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 a r e summarized i n T a b l e I I I . Most o f . t h e h e t e r o z y g o t e s seem t o f i t the 3:1 r a t i o w e l l  and  c o n f i r m t h a t heavy s p o t t i n g i s i n h e r i t e d as a simple dominant. F  2  progeny  The  r e s u l t i n g from the #11 s p o t t e d ( 9 ) x #9 u n s p o t t e d (cf)  c r o s s , however, shows a s l i g h t l y s i g n i f i c a n t d e v i a t i o n from the expected r a t i o due t o a g r e a t e r number o f u n s p o t t e d progeny expected. allele  T h i s i s t y p i c a l o f the b e h a v i o u r o f the  faint-spotting  ( G r i f f i t h s e t a l . , 1 9 7 7 ) and can p r o b a b l y be a t t r i b u t e d t o  one o r more f a i n t - s p o t t e d p l a n t s h a v i n g been mistaken spotted  than  f o r a heavy-  plant.  T a b l e IV summarizes the F  2  and  progeny  s p o t t e d p l a n t s r e s u l t i n g from both c r o s s and  o f a l l o f the  un-  self-fertilization.  I f u n s p o t t e d p l a n t s a r e r e a l l y homozygous r e c e s s i v e s a l l o f the progeny  o f u n s p o t t e d p l a n t s would be expected t o be u n s p o t t e d .  i s not e n t i r e l y the c a s e . are  A l t h o u g h the v a s t m a j o r i t y o f the  u n s p o t t e d , t h e r e a r e a few a n o m a l i e s .  This  progeny  However, s i n c e these  anomalies are few and do not seem t o f i t any p a r t i c u l a r r a t i o , i t i s  Table I I I S  2  and F  2  progeny of heterozygous spotted S  # Of S  O r i g i n of spotted and F^ plants  2  & F  families  Population #11; spotted s e l f  2  1  Number of S  and F  1  plants  & F  2  individuals  2  Spotted  Unspotted  X  261  100  1.40; P=0.5-0.1 deviation not significant  2  (1 d.f.)  #11 spotted (?) x #11 unspotted (d )  10  619  184  1.87; P=0.5-0.1 deviation not significant  #11 unspotted  18  466  132  2.73; P=0.1-0.05 deviation not significant  #11 spotted (?) x #9 unspotted (c?)  12  250  111  6.36; P=0.025-0.01 deviation significant  #9 unspotted  12  584  21?  1.875 P = 0 . 5 - 0 . 1 deviation not significant  1  (?) x #11 spotted (c?)  (9) x #11 spotted (<?)  Table IV S  2  and F  progeny of unspotted  2  # of S  Origin of unspotted S^ and F^ plants  and F^ plants  2  & F  families  2  # of S  2  & F  2  Individuals  Spotted  Unspotted  Population #11;  spotted selfed  3  5  312  Population #11;  unspotted  3  o  265  10  o  404  (<?)  13  3  1,087  spotted (c?)  7  40  698  spotted (9) x #9 unspotted (c?)  10  1  685  Q?)  7  2  417  Population #9; unspotted  selfed selfed  #11 spotted (9) x #11 unspotted #11 unspotted #11  #9 unspotted  (9) x #11  (?) x #11 spotted  20 -  probable  that  they  system.  Some,  when t h e  s e e d s were  plastic"flats have  floated  for  over  plants  between  argument  can  2  one F  unspotted plants.  (§)  This  plained  s i m p l y as  this  also  is  n o r m a l 3*1 plant the at  out  of  too  x #11  least a  not  family  falls into  If spotted  fcT)  scored  h a d 37  of unspotted plants  as  spotted  but  the  plants  character,  c a n be c o n s i d e r e d t o  absence  of heavy  spots.  the  unspotted to  be  amount  The  anomalies  But  but  to  as  parent  because  of  spotted, consider  faint-spotted  of spotting i n  the  ex-  family.  plant.  obviously  misinterpreted. for  cross,  was p r o b a b l y n o t  c h a r a c t e r s a n d s o was explanations  the  be e x p l a i n e d  'spotted*  o v e r l a p between  above  to  seems more r e a s o n a b l e  general,  'unspotted' range  it  a n d 94  unspotted  of  the  the  In  to  T h e same  plants  plants  originally labelled  plant.  unspotted  from the  spotted spotted  spotted  of a heterozygote  It  spotted  growing close one.  were  anomalies.  of a s t r i c t l y  s m a l l a number o f  heavily-spotted.  were  other  c r o s s #11  spotted  in  easily  9 showed o n l y  plant  a number o f  contamination  faint-spotted  spotting  large  f a m i l y was  plants  families,  spotted  the  by s i d e  f a m i l y when t h e y  case i n the  f a m i l y and a  spotted  too  segregation  this  of  resulting  family could quite  an u n s p o t t e d  o f 10  genetic  sown s i d e  f a m i l y grown from an F ^ p l a n t  2  high percentage  this  to  unspotted  is  heavy-spotting  probably contaminants  p r o b a b l y the  where  the  the  T h e f a m i l i e s were  barrier is  of  from a s p o t t e d  e x p l a i n most  However,  F  seed  the  (&)  were  and 1 f a m i l y had a s i n g l e  barrier  #11  a result  planted.  This  unspotted  not  example,  and a  being watered. x #9  are  heavy and  are  be h o m o z y g o u s f o r  accepted, the  this  faint-  un-  recessive  21 Some of the f a m i l i e s scored also showed a l l spotted and  no  unspotted progeny i n d i c a t i n g that the parent plants were homozygous for the dominant character, heavy-spotting. summarized i n Table  The r e s u l t s are  V.  According to these r e s u l t s , s i x of the S^ plants r e s u l t i n g from the s e l f i n g of spotted plants from population #11 homozygotes.  This was not unexpected.  r e s u l t i n g from the #11  spotted ($) x #11  have been heterozygotes.  (Jack Point) were  But, a l l of the plants unspotted  (cf) cross should  This apparent anomaly can be explained by  noting that the seed parents i n the c o n t r o l l e d cross were spotted plants.  An imperfect emasculation  can r e s u l t 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 C o l l i n s i a  p a r v i f l o r a buds i s so d i f f i c u l t , these a c c i d e n t a l s e l f s are not unusual.  The 6 unspotted  plants found i n these 2 f a m i l i e s are  probably the r e s u l t of seeds f l o a t i n g i n from the adjacent  unspotted  families.  Discussion It.can be concluded  from the above r e s u l t s that the heavy-  spotting polymorphism i n £ . p a r v i f l o r a i s c o n t r o l l e d by a s i n g l e 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 f o r the absence of heavy spotting. Another.allele, that for f a i n t - s p o t t i n g , may  be involved at t h i s  locus ( G r i f f i t h s et a l . ) but the a c t u a l inheritance pattern of t h i s p a r t i c u l a r character remains i n doubt and requires further research.  Table V S  2  and F  2  progeny of homozygous spotted  O r i g i n of spotted and F^ plants  # of S  spotted (9) x #11  unspotted (cf)  & F  families  Population.#11; spotted selfed  #11  2  2  2  and  plants  # of S  2  and F  2  individuals  Spotted  Unspotted  491  0  93  6  - 23 -  I t i s i n t e r e s t i n g t o note t h a t the h e a v y - s p o t t i n g  gene (H) i n  C. p a r v i f l o r a shows the same type o f i n h e r i t a n c e p a t t e r n as t h e darkd o t t e d gene (Ld) t h a t G o r s i c  (1957) d e s c r i b e d i n  heterophylla.  T h i s s i m i l a r i t y i n i n h e r i t a n c e p a t t e r n as w e l l a s t h e s i m i l a r i t y i n his  d e s c r i p t i o n o f the d a r k - d o t t e d  c h a r a c t e r makes i t a p p e a l i n g t o  c o n j e c t u r e t h a t t h e genes i n v o l v e d i n both s p e c i e s may be homologous. However, t h e r e i s no c l e a r evidence  that t h i s i s the case.  Now t h a t t h e g e n e t i c d e t e r m i n a t i o n character is.understood,  o f the heavy-spotting  i t can be used t o determine o u t c r o s s i n g  r a t e s u s i n g progeny t e s t s w i t h i n p o p u l a t i o n s  o f C_» p a r v i f l o r a .  G r i f f i t h s e t a l . (1977)» i n s u r v e y i n g p o p u l a t i o n s  of C o l l i n s i a i n  southwestern B r i t i s h Columbia and n o r t h w e s t e r n Washington found t h e s p o t t i n g c h a r a c t e r t o be q u i t e common i n the a r e a between C r o f t o n and L i t t l e R i v e r on Vancouver I s l a n d and i n those  populations i n  which i t does n o t occur n a t u r a l l y i t c o u l d e a s i l y be i n t r o d u c e d since d i f f e r e n t populations (see Chapter 7)*  Spotted  of  p a r v i f l o r a are able t o interbreed  p l a n t s can be d i s t i n g u i s h e d i n t h e f i e l d  w i t h r e l a t i v e ease, e s p e c i a l l y the s t r o n g l y h e a v y - s p o t t e d  ones, as  l o n g a s they a r e s c o r e d and marked when i n t h e r o s e t t e s t a g e .  The  f a i n t - s p o t t i n g phenotype can be a c o m p l i c a t i o n when the degree o f s p o t t i n g f a l l s i n t o t h e range o f o v e r l a p between the two types o f spotting.  But s i n c e e r r o r s i n s c o r i n g c o u l d o c c u r i n e i t h e r d i r e c t i o n ,  a l a r g e enough sample c o u l d e l i m i n a t e any major d e v i a t i o n s . the h e a v y - s p o t t i n g breeding  Therefore,  gene c o u l d become a u s e f u l t o o l i n d e t e r m i n i n g  systems and o u t c r o s s i n g r a t e s w i t h i n p o p u l a t i o n s o f  Collinsia parviflora. /  -  Chapter 3  24  -  S i l v e r y Leaf  Polymorphism  Introduction  Another i n t e r e s t i n g l e a f and cotyledon in  a population  Island,  B . C. ( F i g , 1),  a wrinkled and  o f CL* p a r v i f l o r a grown  upper  In this  surface  sometimes on t h e i r  cotyledons  non-wrinkled, green leaves these  green leaves  palisade  cell  looked  layer.  packed p a l i s a d e  and cotyledons  full  palisade  transient  ever,  away f r o m  the spotted-leaf and g e n e r a l l y  as the plant  had normal  In cross-section  closely-packed  however, had a v e r y  of a i r pockets.  loosely  When a d r o p o f w a t e r was  l e a f cross-section, the epidermal  cell  the r e s t o f the l e a f section as the  possibly  character,  seems t o f a d e  ages.  the s i l v e r y - l e a f  nature,  ( F i g . 8).  leaves  l a y e r broke up.  Like  pigment  float  leaves,  earlier  Other p l a n t s  normal with a t y p i c a l ,  added t o a f r e s h l y c u t s i l v e r y l a y e r would  some o f t h e p l a n t s h a d  s h e e n on t h e i r  ( F i g . 7).  The s i l v e r y  layer,  f r o m s e e d s c o l l e c t e d on C a r l o s  population  and s i l v e r y  p o l y m o r p h i s m was f o u n d  Unlike  character  the s i l v e r y - l e a f  character i s  o r be m a s k e d b y a n t h o c y a n i n  the spotted-leaf  i s very  difficult  character,  how-  to distinguish i n  due t o m a s k i n g b y t h e a n t h o c y a n i n p i g m e n t s i n t h e  leaves.  Materials  The  and Methods  procedure used  t o determine the i n h e r i t a n c e  silvery-leaf  p o l y m o r p h i s m was v e r y  spotted-leaf  polymorphism.  from p o p u l a t i o n  #25  s i m i l a r to that  used  S i l v e r y and green p l a n t s  w h i c h was grown f r o m  pattern  for the  f o r the  were  chosen  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 r e c i p r o c a l crosses were set up between them. plants from population #25 were also r e c i p r o c a l l y crossed  Silvery with  plants from population #11 (Jack P o i n t ) , which contained no s i l v e r y plants ( F i g . 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 e s p e c i a l l y 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 s e l f 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 s i l v e r y character faded.  Seed from each of these  plants was then grown and scored i n d i v i d u a l l y . Two other plants brought from Carlos Island were crossed i n an attempt to study the inheritance of flower colour.  The r e s u l t s  were inconclusive as f a r as flower colour was concerned but the F^ and  o f f s p r i n g included s i l v e r y and green-leafed plants and so were  used f o r the assessment of the s i l v e r y - l e a f character.  Results The two plants from Carlos Island used i n flower colour crosses showed good s i l v e r y - l e a f r e s u l t s as mentioned above.  The r e s u l t s of  the c r o s s - f e r t i l i z a t i o n s 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 V I .  Table VI S, and F, progeny of the crosses involving the s i l v e r y - l e a f  character  # of i n d i v i d u a l s Silvery Green  P o l l i n a t i o n regime  Plant A s e l f e d  22  0  Plant B s e l f e d  0  34  Plant A (?) x Plant B (cT)  7  0  12  0  15  0  Population #25; non-silvery, s e l f e d  0  14  #25 s i l v e r y (9)  x #25 non-silvery (cT)  5  0  #25 non-silvery (?) x #25 s i l v e r y (cf)  0  16  #25 s i l v e r y (?) x #11 non-silvery (d )  5  0  #11 non-silvery ($) x #25 s i l v e r y (cf)  15  7  Plant B (9)  x Plant A (cf)  Population #25; s i l v e r y - l e a f e d , s e l f e d  1  28  These d a t a i n d i c a t e t h a t a s i n g l e gene system i s p r o b a b l y i n volved  and t h a t p l a n t A was a homozygous s i l v e r y - l e a f e d p l a n t and  t h a t p l a n t B was a homozygous g r e e n - l e a f e d  plant.  The r e c i p r o c a l  c r o s s e s i n d i c a t e t h a t the s i l v e r y - l e a f e d c h a r a c t e r the n o n - s i l v e r y - l e a f e d  i s dominant over  character. #25  T a b l e VI a l s o summarizes F^ and S^ progeny from p o p u l a t i o n ( C a r l o s I s l a n d ) and p o p u l a t i o n The  #11 (Jack P o i n t , n o n - s i l v e r y ) .  s i l v e r y - l e a f e d p l a n t s and t h e n o n - s i l v e r y - l e a f e d  allowed t o 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 t h e y a r e homozygotes. the r e s u l t s d i s c u s s e d  above.  These d a t a agree w i t h  However, the r e s u l t s o f the c r o s s -  f e r t i l i z a t i o n s a r e n o t as e x p e c t e d . #25 s i l v e r y  plants  The c r o s s , #25 n o n - s i l v e r y (9)  x  (c?) y i e l d e d o n l y n o n - s i l v e r y - l e a f e d o f f s p r i n g i n s t e a d o f  the h e t e r o z y g o u s s i l v e r y - l e a f e d o f f s p r i n g t h a t were e x p e c t e d .  These  n o n - s i l v e r y - l e a f e d p l a n t s are probably the r e s u l t s o f a c c i d e n t a l selfs.  Since  e m a s c u l a t i n g and c r o s s - p o l l i n a t i n g the f l o w e r s o f  #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 p r o b a b l y  population  occur q u i t e e a s i l y .  I f t h i s i s t h e c a s e , the o t h e r  seed p a r e n t s from p o p u l a t i o n  #25 a r e a l s o suspect  crosses  involving  and the s i l v e r y  progeny from t h e s e c r o s s e s may a l s o be the r e s u l t o f a c c i d e n t a l selfing.  These s u s p i c i o n s were c o n f i r m e d i n t h e next  generation  when a l l o f the F., progeny r e s u l t i n g from c r o s s e s where a s i l v e r y l e a f e d p l a n t was used as the o r i g i n a l seed p a r e n t s were s i l v e r y . T h i s i n d i c a t e s t h a t a l l t h e s i l v e r y F^ progeny were the r e s u l t o f accidental selfing. Table V I I ,  The F^, and S^ progeny a r e summarized i n  Table VII S  P  and Fp progeny of the crosses involving the s i l v e r y - l e a f character  O r i g i n of  of S & F families  and F.^ plants  2  2  # of Sg. & F Silvery  2  individuals Green  Population #25; s i l v e r y , s e l f e d  12  1,203  0  Population #25; non-silvery, s e l f e d  Ik  0  1,156  #25 s i l v e r y (9) x #25 non-silvery (c?)  5  765  0  #25 non-silvery (9) x #25 s i l v e r y (d )  8  0  630  #25 s i l v e r y (9) x #11 non-silvery (c?)  if  220  0  1  - 30 -  The one cross that appears to have been successful i s #11 non-silvery (9)  x #25 s i l v e r y (cf).  The flowers from population  #11 (Jack Point) can be emasculated and c r o s s - f e r t i l i z e d quite s u c c e s s f u l l y as shown i n the spotted-leaf experiment. leafed progeny i n the F^ generation (Table VI) may  The 7 green-  be the r e s u l t of  a cross with a heterozygous s i l v e r y 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 i n d i c a t e only homozygotes, i t i s more probable that these progeny r e s u l t e d from the a c c i d e n t a l s e l f i n g of one or more flowers of the seed p l a n t s . However, the 15 s i l v e r y o f f s p r i n g of t h i s cross can only be heterozygotes  and the r e s u l t of successful crossing since population  #11 contains no s i l v e r y - l e a f e d genes. i n i t i a l l y scored as 'intermediate  1  These s i l v e r y o f f s p r i n g were  since they were not as c l e a r l y  s i l v e r as the plants r e s u l t i n g from the s e l f - f e r t i l i z a t i o n s ( F i g . 9 ) . This made i t a t t r a c t i v e to postulate that heterozygotes  could be  distinguished from homozygotes by the degree to which the s i l v e r y character i s expressed.  However, when the F  2  progeny of these  •intermediate' F^ plants were grown, the s i l v e r y plants d i d not segregate i n t o the two categories, s i l v e r y and intermediate  as  expected, but stayed mostly i n the intermediate category with almost no plants showing the c l e a r l y s i l v e r y l e a f of the population plants.  #25  This would i n d i c a t e that the 'intermediate' expression i s  due to the modification of the s i l v e r y gene by the population #11 genotype rather than to the e f f e c t s of heterozygosity. The F  2  progeny of the #11 green (9)  are summarized i n Table V I I I .  x #25 s i l v e r y (cf) cross  - 31 -  Fig, 9  S i l v e r y , g r e e n and plants  •intermediate  1  Table VIII Fp progeny of the #11 green (?) x #25 s i l v e r y (cf-) cross  Phenotype of F,  Non-silvery  Silvery  (intermediate)  # of F . families  # of F i n d i v i d u a l s Silvery Green  Y  2  7  h  932  13  1,026  347  A  2  . - x v x  a  ,  I  ,  ;  0.05$ P = 0 . 9 - 0 . 5 deviation not significant  33 -  The non-silvery plants bred true as expected. plants were probably contaminants  The If s i l v e r y  from neighbouring s i l v e r y  The s i l v e r y plants segregating i n t o an approximately 3*1 i n d i c a t e s that the s i l v e r y - l e a f e d  families.  ratio  character i s a simple dominant.  Discussion The s i l v e r y - l e a f polymorphism found on Carlos Island i s cont r o l l e d by a single gene with a dominant a l l e l e f o r presence of the s i l v e r y - l e a f e d character and a recessive a l l e l e f o r the absence of it.  The dominant a l l e l e i s very strongly expressed and very obvious  i n plants grown under a r t i f i c i a l conditions. Unfortunately however, the presence of the s i l v e r y - l e a f e d character i s d i f f i c u l t to i d e n t i f y i n nature. So f a r , t h i s s i l v e r y - l e a f polymorphism has not been found i n any populations other than on Carlos I s l a n d ,  Controlled crossing  with plants grown from seed from Jack Point i n d i c a t e that other genotypes can modify the expression of t h i s gene.  - 34 -  Chapter k  Flower Colour Mutants  Introduction The flowers of both the large and small-flowered £_ p a r v i f l o r a are blue with varying amounts of magenta and white i n the upper l i p . The varying i n t e n s i t y of pigment from plant to plant and i n d i f f e r e n t parts of the flower produces d i f f e r e n t shades of colour ranging from l i g h t blue to dark purple.  In h i s o r i g i n a l d e s c r i p t i o n , Lindley  (1827) describes C o l l i n s i a g r a n d i f l o r a as "one  of the most b e a u t i f u l  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 J u l y " . A l l of the populations studied had normal blue flowers, however, two populations from Vancouver Island also contained plants with a t y p i c a l flower colour.  Some of the plants from Elk F a l l s , a  population of large-flowered Cj, p a r v i f l o r a , had pink or magenta flowers ( F i g . 10) and some of the plants from Mt. Douglas Park, a population of small-flowered £. p a r v i f l o r a , had flowers that were completely white ( F i g . 11). These two unusual flower colour mutants had not been reported i n the area of t h i s study but S t . 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 c o r o l l a s and forma rosea Warren i s described as having rose-mallow c o r o l l a s . Harborne (1967) states that "a general ... c h a r a c t e r i s t i c of white flower mutants of coloured forms i s t h e i r u n t h r i f t i n e s s as plants".  This was c l e a r l y true of the white flowered C o l l i n s i a .  - 35 -  F i g . 11  An a l b i n o f l o w e r from the Mt Douglas Park p o p u l a t i o n  - 36 -  The white-flowered  population, although i t survived well under i d e a l  culture conditions was more d r a s t i c a l l y affected by aphid attack, fungus i n f e c t i o n and drying out than the normal populations*  blue-flowered  I t i s i n t e r e s t i n g to note that aphids damaged the  white flowers quite badly, often hampering successful c r o s s - f e r t i l i z a t i o n s but d i d not seem to attack those flowers containing anthocyanins, i n d i c a t i n g perhaps that one of the functions of anthocyanin pigments i n  p a r v i f l o r a i s protection from insect pests or that the  character i s l i n k e d with another chemical  character.  I t 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 i n a l l except the white-flowered plants as the plants aged ( F i g , 12),  Pigment production could be  considerably speeded up by increasing the l i g h t i n t e n s i t y or by an aphid attack. Greater l e a f pigment production under higher l i g h t i n t e n s i t i e s i s probably r e l a t e d to the fact that l i g h t has been shown to be important f o r 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 i n tissues that usually do not contain any anthocyanin colour (Bopp, 1959), a population.  This was the case when aphids i n f e s t e d  Then purple pigment was produced i n the leaves even  i n the seedling stage. Plants with white flowers d i d not produce any anthocyanin and when the plants aged or were i n f e c t e d by aphids or fungus the leaves turned yellow ( F i g , 13),  - 57 -  F i g , 13  The Mt, Douglas P a r k p o p u l a t i o n with yellow  leaves  — 38 —  Through crossing experiments,  I attempted to determine the  inheritance of these flower colour mutants i n £ . p a r v i f l o r a .  Materials and Methods Population #2 was used as the source of white-flowered p l a n t s . It was grown from seed c o l l e c t e d from white-flowered plants at Mt. Douglas Park ( F i g . 1 ) . Population #17 grown from seed c o l l e c t e d i n Elk F a l l s ( F i g . l ) was the source of both blue and magenta-flowered plants.  In a d d i t i o n 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 s e l f f o r  two generations and others were used i n the crosses.. The following crosses were set up between blue and whiteflowered plants: (9)  x  #2 white (cf)  (9)  x  #9 blue (cf)  #11 blue (?)  x  #2 white (cf)  (9)  x  #11 blue (cf)  #9 blue #2 white  #2 white  The crosses between blue and magenta-flowered plants were: #17 blue (9)  x  #17 magenta (9)  #17 magenta (cf) x #17 blue (cf)  39 -  And  the c r o s s e s between white and magenta-flowered p l a n t s were: #2 white ( ? )  x  #17 magenta (cf)  #17 magenta ( ? ) x  #2 white (c?)  I n a d d i t i o n , two s e t s o f c r o s s e s from the f l o w e r s i z e showed f l o w e r c o l o u r s e g r e g a t i o n s i n the F  2  study  g e n e r a t i o n and so were  used t o supplement the d a t a from t h e above-mentioned c r o s s e s * the c r o s s e s #17 ( ? ) x #22  (cf); #22  In  (9) x #17 (cf), both b l u e and  magenta-flowered p l a n t s from p o p u l a t i o n #17 were used so t h a t i n the F  2  g e n e r a t i o n s some o f t h e f a m i l i e s showed a s e g r e g a t i o n f o r  flower colour* The #22  o t h e r s e t o f supplementary d a t a came from the c r o s s  (9) x #9  study*  (c?)*  T h i s was a l s o a c r o s s used i n t h e f l o w e r s i z e  Both p o p u l a t i o n s were b l u e - f l o w e r e d *  Some o f t h e F  2  families  from t h i s c r o s s showed s e g r e g a t i o n f o r blue and white f l o w e r c o l o u r * T h i s was c o m p l e t e l y unexpected s i n c e no o t h e r c r o s s o r s e l f - f e r t i l i z a t i o n o f e i t h e r o f these two p o p u l a t i o n s produced any w h i t e - f l o w ered p l a n t s *  One p o s s i b l e e x p l a n a t i o n f o r t h i s s e g r e g a t i o n i s t h a t  t h e r e was a mutation through  i n one o f the parent p l a n t s which was passed on  this particular cross.  Another i s t h a t the white  flower  gene i s p r e s e n t i n one o f the p o p u l a t i o n s i n such low f r e q u e n c i e s t h a t i t i s r a r e l y i n i t s homozygous c o n d i t i o n and t h e r e f o r e r a r e l y detected.  I f t h i s were t h e c a s e , one o f the o r i g i n a l p a r e n t s o f  the c r o s s would have been heterozygous  f o r t h e white f l o w e r gene*  Whatever the r e a s o n , the white f l o w e r gene was p r e s e n t i n seven o f the F  2  f a m i l i e s and so c o u l d be used i n d e t e r m i n i n g i t s i n h e r i t a n c e  pattern*  ko -  The c r o s s - f e r t i l i z a t i o n methods, growing conditions and scoring methods were b a s i c a l l y the same as f o r the studies discussed i n previous chapters.  In most crosses more than one plant was used  as p o l l e n parents and seed parents.  However, i n crosses i n v o l v i n g  the E l k F a l l s population, only one p o l l e n parent and one seed parent were used i n each case, since the E l k F a l l s flowers are very large and easy to manipulate*  Seeds from the crosses and s e l f s were  c o l l e c t e d , grown, scored f o r flower colour and tagged*  Seed from  each F^ and S^ plant was then c o l l e c t e d and grown separately* F  2  and S  2  The  f a m i l i e s were scored separately to determine the F^ and S^  genotypes and eliminate any contaminants and then the frequencies were t o t a l l e d to get a more accurate r a t i o * 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 p u r p l i s h when damaged by aphids*  The plants  that remained green were not included i n the count*  Results The r e s u l t s from the plants that were allowed to s e l f are summarized i n Table IX* The plants from a l l the populations appear to breed true f o r flower colour*  This large amount of homozygosity i s expected i n  populations # 9 and # 1 1 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  i n the crosses involving flower colour  # and flower colour of S i n d i v i d u a l s  Population (allowed to s e l f )  Flower colour of parent plants  # and flower colour of individuals  #2 Mt. Douglas Park  White  20 plants - a l l white  ifl7 plants - a l l white  #9  Nanoose H i l l  Blue  13 plants - a l l blue  i+Ok plants - a l l blue  #11  Jack Point  Blue  19  #17  Elk F a l l s  Magenta  #17  Elk F a l l s  Blue  plants - a l l blue  3 plants - a l l magenta 30 plants - a l l blue  2  1,169  plants - a l l blue  (S^ died before seed set) 56  plants - a l l blue  -  i n p o p u l a t i o n #1? was  2f2  -  i s not s u r p r i s i n g e i t h e r s i n c e the o r i g i n a l sample  v e r y s m a l l , o n l y f o u r or f i v e p l a n t s .  p l a n t s from p o p u l a t i o n #2  And  the  white-flowered  appear t o be homozygous r e c e s s i v e .  The  f a c t t h a t a l l o f the p l a n t s l e f t t o s e l f were homozygous i n d i c a t e s t h a t a l l o r most o f the p l a n t s used i n the c r o s s e s were p r o b a b l y homozygous. #22  C r o s s i n g d a t a s u p p o r t s t h i s i n a l l o f the c r o s s e s except  blue (9) x #9 The  b l u e (cf) which was  d i s c u s s e d above.  o f f s p r i n g o f the c r o s s between b l u e and  p l a n t s were a l l b l u e except from the c r o s s #2  white  (9) x #11  a c r o s s w i t h a heterozygous a t i o n t h e r e was  white-flowered  f o r 3 w h i t e - f l o w e r e d p l a n t s which r e s u l t e d b l u e (cf) and these were p r o b a b l y  p r o d u c t o f an a c c i d e n t a l s e l f o f the #2  white  p o p u l a t i o n #11  total F  the  seed parent r a t h e r than  plant.  I n the F  gener-  2  s e g r e g a t i o n f o r both b l u e and w h i t e - f l o w e r e d p l a n t s .  The r e s u l t s o f these c r o s s e s are summarized i n T a b l e The  also  2  X.  s c o r e f o r a l l o f these c r o s s e s i s 92 b l u e :  white, an almost p e r f e c t 3:1  ratio.  T h i s simple M e n d e l i a n  32  ratio  makes i t a t t r a c t i v e t o p o s t u l a t e a s i n g l e gene but the d a t a were o b t a i n e d from a t e n t a t i v e count o f d y i n g s e e d l i n g s and hence the experiment The  s h o u l d be r e p e a t e d t o s u b s t a n t i a t e t h i s  o t h e r c r o s s r e s u l t i n g i n the s e g r e g a t i o n o f blueand  f l o w e r e d p l a n t s was seven F  2  ratio.  #22  b l u e (9) x #9  b l u e (cf).  white-  In t h i s c r o s s ,  f a m i l i e s showed s e g r e g a t i o n i n t o b l u e and  white-flowered  progeny but they d i d not conform t o the expected 3 b l u e : 1  white  ratio.  white  The  (Table X I ) .  t o t a l s c o r e f o r these f a m i l i e s i s 403 Although  blue : 41  these d a t a do not f i t a common Mendelian  they are more r e l i a b l e than the 3:1  ratio  r a t i o o b t a i n e d from the d y i n g  Table X and F  2  progeny r e s u l t i n g from crosses between  blue-flowered and white-flowered plants  Cross  # of F, i n d i v i d u a l s Blue White  # of F~ i n d i v i d u a l s Blue White  x #2 white (cf)  9  0  15  k  #2 white (?) x #9 blue (c?)  7  0  9  2  31  0  36  10  11  3*  32  _l£  58  3  92  32  #9 blue (9)  #11  blue (?) x #2 white (c?)  #2 white (9)  x #11  Total •accidental  selfs  blue (<*)  Table XI F  2  progeny of the #22 blue (9) x #9 blue (cr) that showed segregation f o r flower colour  Colour of the F, plants which produced each family  # of i n d i v i d u a l s i n 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  12.  Blue Total  6 403  41  - 45 s e e d l i n g count  d i s c u s s e d above*  s e e d l i n g count u n d e r e s t i m a t e d s i n c e only the unhealthy were s c o r e d *  I t i s reasonable  t o assume t h a t t h i s  the number o f b l u e - f l o w e r e d progeny  s e e d l i n g s whose l e a v e s had t u r n e d c o l o u r  And s i n c e w h i t e - f l o w e r e d  p l a n t s a r e l e s s hardy and more  s e v e r e l y a f f e c t e d by a p h i d s , most o r a l l o f t h e h e a l t h y and green s e e d l i n g s o m i t t e d from the a n a l y s i s were p r o b a b l y plants*  2  blue-flowered  Had they been s c o r e d , t h e f i n a l r a t i o would p r o b a b l y have  shown an u n e x p e c t e d l y F  still  h i g h p r o p o r t i o n o f the dominant b l u e - f l o w e r e d  offspring. I n t h e c r o s s e s between b l u e and magenta-flowered p l a n t s t h e F^  p l a n t s were a l l b l u e - f l o w e r e d , i n d i c a t i n g t h a t b l u e f l o w e r s a r e dominant over magenta f l o w e r s as w e l l as w h i t e .  I n the F  2  generation  the f a m i l i e s showed s e g r e g a t i o n f o r b l u e and magenta f l o w e r s .  How-  e v e r , t h e r a t i o was a g a i n an anomalous one, 50 b l u e : 6 magenta. These r e s u l t s a r e summarized i n T a b l e X I I . Thus t h e magenta f l o w e r c h a r a c t e r , l i k e t h e white f l o w e r c h a r a c t e r , i s t r a n s m i t t e d a t a frequency  t h a t i s much lower than expected  f o r simple s i n g l e gene  inheritance. The  c r o s s between magenta and w h i t e - f l o w e r e d  a l l b l u e - f l o w e r e d F.^ progeny except  p l a n t s produced  f o r one white contaminant p r o v i n g  t h a t white and magenta f l o w e r c o l o u r a r e n o t a l l e l i c .  And the F  g e n e r a t i o n showed s e g r e g a t i o n f o r b l u e , magenta and white (Fig.  1 4 ) . The r e s u l t s a r e summarized i n T a b l e X I I I .  2  flowers  Table XII F ^ and F^ progeny of the crosses between blue-flowered plants and magenta-flowered plants  P o l l i n a t i o n regime  #17 blue (9)  x #17 magenta (cf)  # and colour of F-L i n d i v i d u a l s  11 blue  # of F i n d i v i d u a l s (by i n d i v i d u a l family) Blue Magenta 2  1 5 1  x #17 blue (cf)  3 blue  x #17 magenta (<f)  1 blue  12  #17 magenta (?) x #22 blue (cf)  2 blue  6 20  17 blue  50  #17 magenta (9) #22 blue (9)  Total  0 0 0  3 1  - 47 -  F i g , 14  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 flower c o l o u r s  Table XIII F^ and F  2  progeny of the crosses between  white-flowered and magenta-flowered plants  P o l l i n a t i o n regime  # and flower colour of i n ^ y i , ^ ^ t  h  e  p  # of F i n d i v i d u a l s Magenta White 2  B  l  u  e  #17 magenta (?) x #2 white (cf»)  10 blue  18  #2 white (?) x #17 magenta (c?)  65 blue  148  16  12  75 blue  166  18  13  Total  (1 white)*  (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* colourless precursor  ^  e n e  A  —>  i.e. magenta  g  e  n  e  B  —^  blue  I f t h i s i s the case, the white-flowered plant has the genotype aaBB and the magenta-flowered plant has the genotype AAbb* hybrid i s AaBb and blue*  The  And the blue-flowered plants used i n the  crosses were a l l AABB* However, using the above hypothesis, the F  2  r a t i o for the crosses  between blue-flowered and white-flowered plants would be expected to be 3 blue : 1 white.  But the actual r a t i o was 403 blue : 41  white.  The crosses between blue and magenta would also be expected to y i e l d an F-> r a t i o of 3:1  instead of the unusual 50 blue : 6 magenta r a t i o  that was a c t u a l l y produced*  This very low frequency of the recessive  a l l e l e a l s o occurred i n the crosses between white and magentaflowered p l a n t s . 4 white. One  The expected r a t i o would be 9 blue : 3 magenta :  The a c t u a l r a t i o was 166 blue : 18 magenta : 13 white. of the simplest ways to explain these data i s to postulate  that only two genes are a c t u a l l y involved but that there i s a s e l e c t i v e pressure against the recessive a l l e l e s i n the formation of gametes or against the homozygous recessive condition i n the zygote or young seedling.  - 5© -  This i s not the only possible explanation for this unusual ratio.  It may be a much more complex system involving more than two  l o c i and various gene interactions.  But a system of three l o c i 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 i s the one discussed above. However, i t must be further tested to determine whether or not selection against the recessive alleles i s 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 polymorphisms have not been conclusively determined, this study has provided a feasible working hypothesis as a basis for i n i t i a t i o n of further studies into the problem.  - 51 Chapter 5  Inheritance of Flower Size  Introduction Flower size i s the main character used to separate £ . p a r v i f l o r a and C. g r a n d i f l o r a i n most f l o r a s and i n Newsom's (1929) monograph of the genus.  The o r i g i n a l descriptions (Lindley, 182?) do not  give any a c t u a l measurements but l a t e r authors divide the two species i n t o two very precise and measurable size c l a s s e s . 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 t h i s character. The d i v i d i n g l i n e between  g r a n d i f l o r a and  p a r v i f l o r a has  been drawn seemingly a r b i t r a r i l y at d i f f e r e n t flower s i z e s by d i f f e r e n t authors,  Abrams (1951) includes i n C_, p a r v i f l o r a those  plants with c o r o l l a s l e s s than 10 mm long whereas Davis (1952) states that C. p a r v i f l o r a has c o r o l l a s l e s s than 6 mm long and £• grandif l o r a has c o r o l l a s between 6,5 and 9 nim long. Only Peck (1961) and S t . John (1956) have formally recognized any region of overlap between the two species i n t h e i r keys and descriptions.  However, Newsom (1929) does state i n her text that  " i n studying these two c l o s e l y a l l i e d species, one finds a continuous s e r i e s of intergradation from the largest-flowered g r a n d i f l o r a to the smallest-flowered p a r v i f l o r a " .  Other authors, notably Gilkey  and Dennis (1967), make the two size classes appear to be quite distinct. This i s not the case i n nature. more or l e s s continuous  In fact there seems to be a  range i n flower size among the populations  Table XIV Corolla length classes assigned to C. p a r v i f l o r a and C. grandiflora by various authors C. g r a n d i f l o r a C. p a r v i f l o r a Author  „ . » Region of f l o r a  T o t a l c o r o l l a length e  1  2  k  3  5  6  7  9 10.11 12 13 14 15 16 17 18 19  1 I—. 1  Peck (1961)  Oregon  Davis (1952)  Idaho  h  S t . John (1956)  S.E. Washington  1  Henry (1915)  Southern B.C.  +•  1  Anderson (1961)  Alaska  *"  ^  Munz & Keck (1959)  California  Lindley (1827)  Orig. descript.  Newsom (1929)  Monograph  \  Hitchcock et a l . (1959)  P a c i f i c N.W.  I-  Piper (1906) Piper & Beattie (1915) Gilkey & Dennis (1967)  Washington  Abrams (195D  8  1  i  1  •• '  1  1  '  "H  ' '  1  no size estimate T  '  '  1 '  '  ^  f  ' 1  P a c i f i c N.W. P a c i f i c States  (mm)  '  I  1  I - i J  1  1 1  - 53 observed i n t h i s study.  And many of them occupy the regions of  d i v i s i o n 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 s i z e as a key character and because of the d i f f e r i n g opinions among various authors using t h i s character, i t was of s p e c i a l i n t e r e s t to examine v a r i a t i o n i n flower size both among and within populations i n t h i s study and to determine the genetic mechanism c o n t r o l l i n g i t .  Materials and Methods Flower size varies considerably among populations of C. p a r v i 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 E l k F a l l s are about 10-1?  mm.  However,  the flower size within any p a r t i c u l a r population i s usually r e l a t i v e l y uniform and populations grown from seeds c o l l e c t e d i n d i f f e r e n t areas can often be v i s u a l l y distinguished merely on the basis of flower size.  For t h i s p a r t i c u l a r study f i v e populations were chosen on the  basis of flower s i z e , 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 d i f f e r e n t from the others i n the s i z e of i t s flowers ( F i g . 15).  Three of these populations f e l l within the  C. g r a n d i f l o r a range according to Hitchcock and Cronquist (1973) and two f e l l within the C. p a r v i f l o r a 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 t i n y buds was  not  - 54 -  F i g . 15  The v a r i a t i o n i n flower size between the f i v e populations used i n study. right:  From l e f t to  E l k F a l l s , Jack Point,  Nanoose H i l l , Botanie Lindeman Lake  Valley,  Table XV Populations of C. p a r v i f l o r a used i n the study of flower size v a r i a t i o n  Total c o r o l l a length  Locality  Population number  Species according to Hitchcock & Cronquist (1973)  10  17 mm  Elk F a l l s  17  C«  8  10 mm  Jack Point  11  C, g r a n d i f l o r a  7  8 mm  Nanoose H i l l  9  C. g r a n d i f l o r a  6  7 mm  Botanie  k  5 mm  Lindeman Lake  Valley  grandiflora  22  C. p a r v i f l o r a  6  C, p a r v i f l o r a  - 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 V a l l e y population had s l i g h t l y l a r g e r flowers than those from Lindeman Lake and so was l e s s d i f f i c u l t to work with. Reciprocal crosses wereset up between plants from t h i s population and plants from each of the "(_. g r a n d i f l o r a " populations. plants from each population were a l s o allowed to s e l f .  Some  Seeds from  the c r o s s - f e r t i l i z a t i o n s and s e l f - f e r t i l i z a t i o n s were grown to produce the F-^ and S^ generation which was i n turn allowed to s e l f 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'  F  2  ation #11  ajx  ^ 2 i S  Populations except for the S^ generation of popul-  (Jack P o i n t ) .  To minimize the e f f e c t of d i f f e r i n g flower angles the measurement taken was  from the t i p of the keel to the top of the saccate or  gibbous swelling at the base of the c o r o l l a tube (See F i g . 16).  F i g . 16  A C o l l i n s i a flower showing the angle at which measurements were taken  The measurements were taken to the nearest 0.5 rounded o f f to the nearest mm  mm.  These were l a t e r  i n order to s i m p l i f y the  graphing.  Samples of three flowers per plant were taken (whenever possible) from up to twenty plants i n 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 p a r t i c u l a r s e l f or cross. These samples were analyzed and compared by t o t a l i n g the number of flowers i n each size c l a s s (to the nearest mm) converting them to percentages.  The percentages  i n the sample and were then graphed.  In addition to each graph, a table was set up i n d i c a t i n g the mean flower s i z e and the sample s i z e of each population involved. One  of the crosses set up for flower colour (population #2 x  population #17) size was  a l s o showed i n t e r e s t i n g r e s u l t s as far as flower  concerned.  Population #2 (Mt, Douglas Park) had  flowers and population #17 flowers.  "parviflora"  (Elk F a l l s ) had very large " g r a n d i f l o r a "  The hybrids were obviously intermediate.  Samples were  measured, analyzed and compared i n the same way as the crosses d i s cussed above.  But because t h i s was not o r i g i n a l l y designed as a cross  to determine the inheritance of flower size there i s no sample for the o r i g i n a l parental population from Mt, Douglas Park,  Results The graphs f o r flower size of the f i v e populations showed a c e r t a i n amount of overlap between the populations and some s h i f t i n g of the histograms from one generation to the next but the peak of each histogram was c l e a r l y separate from a l l the others ( F i g s , 17, 18, 19). The mean flower s i z e s were also d i f f e r e n t for each of the populations (Tables XVI, XVII, XVIII),  The s h i f t i n g of the histograms  Graph c omp arinj  jric wer  sizes  <  Figur< 17 r the f i v e populations in 1thei i " i r s t (parenta] /)  a•eneration  •  o  8u It  /  £_  o  /  c•3  /  \ \ \  1 re  4-=  #22  / / J  i <  f  +• «w  » 1  40  M  #13  2  1()  8  4  flowe.c tii :e J.  11II L  •  »  12  14  16  ( rar  1  Table XVI Mean flower sizes and sample s i z e s of the populations graphed i n F i g , 1?  Population  x flower size  (  #  Q  f  p  l  a  ^ f  e  g  Z  *  f  f  l  o  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  18  53  Elk F a l l s  10,5 mm  w  e  r  s  )  Table XVII Mean flower sizes and sample s i z e s of the populations graphed i n F i g . 18  _ , . . Population  . x flower size  Lindeman Lake  3 . 0 mm  15  45  Botanie Valley  4»0 mm  9  26  Nanoose H i l l  5*0 mm  11  33  1 3 . 0 mm  17  40  Elk F a l l s  Sample s i z e ( #  Q  f  p  l  a  n  t  s  )  ( #  o  f  f l o w e r e  )  Figure 1 9 Graph comparing f l o w e r s i z e s o f the f i v e p o p u l a t i o n s i n the  flower s i z e  (mm)  Table XVIII Mean flower sizes and sample s i z e s of the populations graphed i n F i g . 19  Population  - „ . x flower size n  ( #  Q  f  p  l  a  n  t  e  Sample s i z e ( flowers)  )  #  o  f  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 the age  g e n e r a t i o n t o the next may  s i m p l y be due  o f the p l a n t s when the f l o w e r s  s e r v e d , p a r t i c u l a r l y i n p o p u l a t i o n #17, s m a l l e r f l o w e r s as they aged. environmental  factors.  were  to d i f f e r e n c e s i n  measured.  I t was  t h a t the p l a n t s produced  These s h i f t s c o u l d a l s o be due  The  to  However, s i n c e the p l a n t s were a l l grown  under u n i f o r m growth chamber c o n d i t i o n s , the environmental would be  ob-  effects  minimal. c r o s s e s i n v o l v i n g the Lindeman Lake p o p u l a t i o n (#6)  p o l l e n parent were not v e r y s u c c e s s f u l . V e r y  as a  few seeds were s e t and  most o f those seemed t o be contaminants s i n c e the supposed ' h y b r i d s ' resembled t h e i r seed p a r e n t s .  T h i s was  l a r g e l y a t e c h n i c a l problem  s i n c e the f l o w e r s from t h i s p o p u l a t i o n were v e r y s m a l l and had little  pollen.  Most of the p o l l e n was  t r a n s f e r i t onto another  stigma.  u s u a l l y l o s t i n the attempt t o  However, a t l e a s t one  or two  geny appear to have been genuine h y b r i d s because i n the F^ one  f a m i l y from the c r o s s #11  mediate s i z e  (See F i g , 20)  p l a n t ) from the c r o s s #9 o f p o p u l a t i o n #6, The  (9) x #6  and one  (9) x #6  i . e . 2,5  - 3-5  very  (6*) had  pro-  generation  f l o w e r s o f an  inter-  f a m i l y ( c o n s i s t i n g of a s i n g l e ( c f ) , had  f l o w e r s i n the s i z e range  mm.  c r o s s e s u s i n g the B o t a n i e V a l l e y p o p u l a t i o n (#22)  f o r the  " p a r v i f l o r a " p a r e n t were more s u c c e s s f u l . H y b r i d s were r e a d i l y formed and the F.^ and F  2  h y b r i d peaks were i n t e r m e d i a t e t o the  p a r e n t a l peaks i n most c a s e s ( F i g s , 21,  22,  23),  The mean f l o w e r  s i z e s o f the h y b r i d s were a l s o i n t e r m e d i a t e between the ( T a b l e s XX,  XXI, X X I I ) ,  And  the F  g e n e r a t i o n showed a g r e a t e r  g  v a r i a t i o n i n f l o w e r s i z e than the F  parents  n  generation, suggesting  segreg-  Figure (5rai)hL C 0m pa rini  :  >  ttie tlower s i z e i 3 <>f Lindeman h£ike (#6 ) iL  with a ]  )()  i J ack P 01 nt  >  L]• I»opulat ions  •Vi c m  -  r>  00  n (1) ft  o  1\ o  hiill  w  i  / c  crV  nv i  i  1  j  5  1  0  /  20  •\  % ••'  _ .•  4  10  6 i'lower  s i z 3 {[mnL)  12  14  16  Table XIX Mean flower sizes and sample s i z e s of the populations graphed i n F i g , 20  Sample size (# of plants) (# of flowers)  Population  x flower size  Lindeman Lake parental  if,0 mm  15  Jack Point parental  7,0 mm  18  Possible F  6,0 mm  2  hybrid  10  21  Figure  21  Graph comparing the f l o w e r s i z e s o f t h e h y b r i d s between p o p u l a t i o n s  100  B o t a n i e V a l l e y (#22)  and Nanoose H i l l  (#9)  flower s i z e  from  with t h e i r p a r e n t a l populations  (mm)  Table XX Mean flower sizes and sample s i z e s of the populations graphed i n F i g . 21  Population  x flower size  Sample s i z e (# of plants) (# of flowers)  Botanie V a l l e y parental  if*5 mm  9  27  F  x  hybrid  4»5 mm  18  53  F  hybrid  5.5 mm  160  380  2  6.0 mm  5  15  Nanoose H i l l parental  Figure 22 Graph comparing the flower s i z e s of the hybrids between populations from 100  Botanie Valley (#22)  and Jack Point (#11)  with t h e i r parent  populations  80  CO  i o  60 I  H(0  ON VC  +> O +» ©  1  k0  #22 parental  20  8  .0  ] ll>  Ik  im)  -  ...  Table XXI Mean flower sizes and sample s i z e s of the populations graphed i n F i g , 22  Population  x flower size  Sample size (# of plants) (# of flowei  Botanie V a l l e y parental  4,5 mm  9  27  F  x  hybrid  5,5 mm  18  49  F  2  hybrid  6,5 mm  120  290  7,0 mm  6  18  Jack Point parental  Figure Graph comparing the f l o w e r s i z e s  23  o f h y b r i d s between p o p u l a t i o n s  flower s i z e  (mm)  ^TJTTJTTlTtl IMIIIII H - H - l 1 1 1 1 1 1 1 M 1 I I I I I I M H H + H ^ B  from  Table XXII Mean flower sizes and sample s i z e s of the populations graphed i n F i g . 23  Population  - „ . x flower size  ( #  Q  f  p  l  a  n  t  s  Sample size i ers)  )  ( #  o  f  f  0 W  Botanie V a l l e y parental  4»5 mm  9  27  F  hybrid  8,5 mm  11  33  hybrid  7.5 mm  107  187  10.5 mm  18  53  1  F  2  E l k F a l l s parental  - 73 -  ation for the genes determining flower size (Figs, 21, 22, 23), This would be expected i f flower size i s 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 i s 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 i s 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 distinctions even more. In addition, the "grandiflora" and "parviflora" populations can interbreed quite readily.  This clearly indicates that they are not  good biological species i n the sense of Mayr (1957) and that there i s 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 9 >J«• Graph comparing the n.00  s i z e s o f ]K fbr ids 3 1between 1c o p u l a t i o n B fron l  1  (#2 ) sine1 E l k F a l l o  Mt • Dougla  J  flowei  FX/;  witl  r ]jarem; popuiax-xons  80  r  r-t /ft  s  r-l «H  H OS  >  1  n  l  / \  +> O  *» <* o  •  i  40  F  / /  /  i  \  F  2  /  \  /  \  T VT \  /  c>  6  1  H \3 1 l c •wer s i z  _  «L  _  \ y  I  /  #17 ]p a r e n t a l  \ \ \  /  20  hybrit 1  l  \  N "••-...\  it [mm)  V-  12  14  Lc  Table XXIII Mean f l o w e r s i z e s and sample  sizes  o f the p o p u l a t i o n s graphed i n F i g . 24  x flower  Population  Mt. Douglas Park  S  1  size  Sample s i z e (# o f p l a n t s ) (# o f f l o w e r s )  4.5  mm  18  47  F^  hybrid  9.0  mm  20  70  F  hybrid  7«5 mm  154  388  18  53  2  Elk F a l l s parental  10.5  mm  - 76 -  by Cain (1954) and Grant (1963» 1971)•  T  h i s cannot be done with the  character of flower s i z e which shows a continuous v a r i a t i o n , 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 h i s students have done extensive work on the chromosomes of C o l l i n s i a and t h e i r 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; Hayhome and Garber, 1968; Garber, 1974)•  Garber and D h i l l o n ,  1962,  The one exceptional species  i s C, t o r r e y i with a count of 21 bivalents, making i t a hexaploid. I t was at f i r s t considered the " f i r s t polyploid to be encountered i n the genus" (Garber, 1958b) but l a t e r was said to have been "erroneously i d e n t i f i e d as a member of C o l l i n s i a (since) no t e t r a p l o i d species has been found i n the genus" (Garber, 1974)• the  This casts some doubt on  i d e n t i t y of the plants for which Garber has reported chromosome  numbers, e s p e c i a l l y since there are no voucher specimens*  However,  Taylor and Mulligan (1968) i n t h e i r F l o r a of the Queen Charlotte Islands, give the chromosome number of £, p a r v i f l o r a as n=14,  making  i t a tetraploid, Garber's chromosome counts for C, p a r v i f l o r a (1956) and C^ grandi f l o r a (1958b) are both n=7 but the p a r v i f l o r a 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 t h e i r count from  a population on Haida Pt,, Graham Island but Garber made no mention as to the source of h i s populations. Because of these c o n f l i c t i n g reports I examined s i x populations with d i f f e r e n t flower sizes to determine t h e i r chromosome number. The objectives were to determine i f there were differences i n number i n d i f f e r e n t populations, i f both t e t r a p l o i d s and d i p l o i d s existed i n  - 78 -  this  area  differed  of  study,  and whether  i n chromosome  s m a l l and l a r g e - f l o w e r e d  populations  number.  M a t e r i a l s and Methods  Plants localities  w e r e grown f r o m s e e d (See  Fig.  collected  at  the  following  1):  it P o p u l a t i o n #6  -  P o p u l a t i o n #22  -  P o p u l a t i o n #9  cells  -  Jack  P o p u l a t i o n #17  -  Elk  phase  the  anther  also  were d i f f i c u l t t o  are  Island  Point F lls a  s o l u t i o n of  ethanol,  in alcoholic  a n t h e r s mature  was  do n o t  contained  o r metaphase I .  chromosomes  stained  a  count  a  at  different  few p o l l e n m o t h e r  at  least  tended  one c e l l  found and photographed  deposited at  the  for  the U n i v e r s i t y of B r i t i s h  to  times  cells  each  and a l l  of  the  a bud w i t h at  be q u i t e  with  acid-carmine  a n t h e r s removed and  divide synchronously,  T h e chromosomes but  c h l o r o f o r m and  hydrochloric  T h e s e b u d s were t h e n d i s s e c t e d ,  Since  often  6:3?2  V lley Hill  P o p u l a t i o n #11  i n a single  tetrads  Nanoose Carlos  a c i d and then  squashed.  -  1963).  (Snow,  Botanie  P o p u l a t i o n #25  B u d s were f i x e d i n a propionic  Lindeman Lake  late  pro-  sticky  and  distinguishable  population.  Vouchers  Columbia.  Results The  f o l l o w i n g counts  were o b t a i n e d  P o p u l a t i o n #6 2n = 14 I I P o p u l a t i o n #22 2n = 14 I I  from the  Lindeman Lake  -  Botanie  Valley  populations  studied:  -  79 -  Population #9 - Nanoose H i l l 2n = 14 I I (See F i g . 25) Population #25 - Carlos Island 2n = 14 I I (See F i g . 26) Population #11 - Jack Point 2n = 14 I I Population #17 - E l k F a l l s 2n = 14 I I  Discussion A l l o f the populations surveyed f o r chromosome number were t e t r a p l o i d s with a count of 2n = 14 I I .  This agrees with Taylor  and Mulligan's (1968) count for C_. p a r v i f l o r a from the Graham Island population and suggests that C. p a r v i f l o r a i s t e t r a p l o i d i n B r i t i s h Columbia. None of the populations studied had a chromosome number of n=7 as reported by Garber ( 1 9 5 6 , 1958b).  I t i s possible that C^ p a r v i -  f l o r a c o n s i s t s of both d i p l o i d and t e t r a p l o i d populations and Garber obtained h i s plants from d i p l o i d populations.  However, i t i s also  possible that C^ p a r v i f l o r a i s a s t r i c t l y t e t r a p l o i d species and the material from which Garber obtained h i s counts was m i s i d e n t i f i e d as p a r v i f l o r a and £ . g r a n d i f l o r a .  I t i s unfortunate that he does  not state the source of h i s seeds. Both large-flowered and small-flowered populations had a chromosome number of 2n = 14 I I , supporting the i n c l u s i o n of both large and small-flowered plants i n the same species.  Since large and small-  flowered plants formed f e r t i l e hybrids, t h i s r e s u l t was not unexpected.  -  Fig.  25  80  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 I I (mag. x 1,900)  - 81 Chapter 7  Morphology and Taxonomy  Introduction It  was  e s t a b l i s h e d i n Chapter 5 t h a t £ . g r a n d i f l o r a and  C. p a r v i f l o r a a r e i n t e r f e r t i l e and  t h e r e f o r e not s e p a r a t e  s p e c i e s i n the sense o f Mayr (1957) and Grant (1963)»  biological  However, t h i s  i s not s u f f i c i e n t reason  t o i g n o r e the n o m e n c l a t u r a l  between the two.  be u s e f u l i n p r a c t i c a l taxonomic work t o  I t may  c o n s i d e r them t o be two morphological relationships.  taxonomic s p e c i e s (Grant, 1971)  d i f f e r e n c e s r a t h e r than c r o s s a b i l i t y o r The  distinction  following morphological  study was  which  express  interfertility conducted i n an  attempt t o 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 a r e 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 t o be c o n s i d e r e d  separate  taxonomic s p e c i e s .  M a t e r i a l s and Methods S i x t e e n p o p u l a t i o n s were grown from seed c o l l e c t e d from following l o c a l i t i e s  (See F i g . l ) :  Vancouver I s l a n d . B.  C.  T h e t i s Lake - p o p u l a t i o n I - population II M i l l H i l l - population I - population I I Mt.  Douglas P a r k - p o p u l a t i o n I - population I I  Nanoose H i l l - p o p u l a t i o n IV - population I I I  the  - 82 -  N i l e Creek L i t t l e River Crofton - population I - population I I Rathtrevor Park Sechelt Peninsula. B. C. Lund Irvine's North Washington. U.S.A. Anacortes In addition f i v e of the populations used i n the genetic studies were used i n 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 vegeta t i v e and f l o r a l characters were measured. The vegetative and f l o r a l c h a r a c t e r i s t i c s of £• g r a n d i f l o r a and C. p a r v i f l o r a . e s p e c i a l l y those that reportedly d i f f e r e d between the two groups, were summarized on a data sheet (Table XXIV).  Each  83  -  Table XXIV Data Sheet f o r Population  Stem  erect, ascending, other branched, unbranched glandular, puberulent, glabrate, other tall  Leaves  oblong, ovate, spatulate, other obtuse, acute, other serrulate, e n t i r e , entire-revolute, other glabrate, puberulent, glandular, other s e s s i l e , sub-sessile, petiolate upper whorls becoming l i n e a r smaller bracteolate i n inflorescence purple underside  i  ___________  long;  wide  flowers i n whorls  Flowers  r a r e l y s o l i t a r y , commonly s o l i t a r y below, never s o l i t a r y Pedicels  puberulent, glandular, glabrate, other times as short as flowers long  Calyx  glabrate, puberulent, glandular, other membranous below scabrous-margined  ________ __________  times length of c o r o l l a long lobes:  subulate, l i n e a r - l a n c e o l a t e , other acuminate, other wide  - 84 -  Corolla  strongly declined, declined, almost erect forms  0  angle with pedieel  long upper l i p s colour  ______ long  sinus lobes:  deep obovate, spatulate, retuse, d i l a t e , crisp-crenulate, recurved, erect, other wide  lower l i p : colour wide c o r o l l a 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 i n Chapter 5 i n order to make the measurements comparable to those of other workers. The data were then summarized i n Table XXV to include a l l those characters that showed variation among the different populations.  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 2 5 cm (Nanoose H i l l I ) . Newsom (1929) describes C. parviflora stems as ascending or erect as compared to £j, grandiflora 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.  varied was branching.  The only stem character that  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 i n the leaves.  Leaves varied i n 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  -86T a b l e XXV Summary o f t h e v a r i a t i o n among t h e p o p u l a t i o n s s t u d i e d  . . • • A. «_ __> A* • t. I_l i_l 1 — 4 • I. ct^-t<+ m «•** «-t* Ua PT* FT — I «• H-ft* P* P H*  rr<|0&tJctHOOI»rtHcHJB Fo 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 i i i B M - O CD CD H' P P ct4 CD CD <D k  H  0)  CO H-  <t  P M «<+  M  ffl  W CD  4  •  • • H CD CD P H f t cr H O O O _ H- H- ffl O O O W CD OJ 5 £ 0 P H* £ If) W IS H H H P „ - , „ , „ „ . . .  e HHHBH'JI < H H H H H H H 4 M • «) rTd) H < S » !B p M CD M M CO 01 CO M H 4 H • <D M M M V) M M M  O  4 ct-  p*  + 4- +  + + ++  + + + + ++  + ++ ++  + +  RCD  + + +  + + ++ + ++  ++  + + + + + +  ++  +  + +  + + +  +++  +  + + •*• + + + + + + + + + + + + + + + + + + +  + + ++ + + + ++ + + + + + + + ++ +  +  +  +  +  + + + + +  + + +  +  + + ++ + + +++ +  +  +  +  +  +  +  + ++ + + ++ + ++  ++  +  +  + ++ + + + + + + + + + ++ ++ + ++ + + + + + + + + + ++ + ++ + + + + •++ + + +  + + + +  + ++ ++++  +  +  +  ++++  + + + + ++++ +  + +  +• + + + +• + + ++ + + ++++ • +  + ++ + + ' + + + + + + + + + + + ++ + + + + + + + + +  +  +  + +++++++++ +++++++ + +* + + ++ + + + + + + + ++ + + ++ + + +  +  + + + +  + +  + + + + + + + + + + + + + ++ + + + + + + + + + + + +  + ++ + + + ++  + + + + + + + + + + + •»•+ +  + + + + + + + + +•+ + + + + + + + + + +++ + ++ ++ + + + + ++  +  stem branched leaves leaves leaves leaves leaves leaves leaves leaves leaves leaves leaves leaves purple  oblong ovate acute obtuse crenate lobed serrulate toothed undulate puberulent glabrate sub-sessile underside  f l o w e r s s o l i t a r y below p e d i c e l s 1-2 x f l o w e r l e n g t h p e d i c e l s 1/2-1 x flower length calyx glabrate c a l y x puberulent <- 1/2  corolla  length  corolla strongly declined ( o re oc ll li an e d le( n g t h 45°) c>o r o1/2 lla cd c o r o l l a almost e r e c t tube s a c c a t e tube gibbous c o r o l l a l i m b ^. tube c o r o l l a l i m b ^ tube corolla corolla corolla corolla corolla corolla corolla corolla  k mm 5 mm 6 mm 7 mm 8 mm 9 mm 10 mm 7" 10 mm - 15  mm  90°)  -  F i g , 27  87  -  The l e a f characters of the  Botanie  V a l l e y population  F i g . 28  The l e a f characters of the Jack Point population  - 88  p a r t i c u l a r population than "between populations and i t i s not possible to use l e a f characters as a means for separating the populations into d i f f e r e n t taxa. c)  Flowers The number of flowers per whorl has been used as a diagnostic  character separating the two species.  Newsom (1929) describes  C. p a r v i f l o r a as having "2-5 flowers i n a whorl above, u s u a l l y s o l i t a r y below".  By contrast, she describes C. g r a n d i f l o r a as having  "flowers 3-7 at a node, r a r e l y s o l i t a r y " .  As can be seen i n Table XXV,  a l l populations except Nanoose H i l l IV had flowers "usually s o l i t a r y below".  As for the upper whorls, a l l populations had flowers i n 2's  and only three populations had whorls with more than 5 flowers. These were Mt. Douglas Park I I I , Nanoose H i l l I and Elk F a l l s and include both " p a r v i f l o r a " and " g r a n d i f l o r a " - s i z e flowers. d)  Pedicels A l l of the populations had puberulent p e d i c e l s .  Hitchcock  et a l . (1959) describe Cj_ g r a n d i f l o r a flowers as being shorter pediceled.  According to Table XXV, t h i s does not appear to be the  case. e)  Calyx The presence or absence of puberulence on the calyx v a r i e s  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 c o r o l l a was used as a diagnostic character i n Newsom s (1929) 1  monograph.  She states that i n C_» p a r v i f l o r a the calyx i s "from h a l f  - 89 -  as long to almost equal the c o r o l l a length" and i n C_» g r a n d i f l o r a the calyx i s "1/3 - 1/2 the c o r o l l a length".  In the populations studied,  only two (Irvine's North, E l k F a l l s ) had calyces which were l e s s than 1/2 the c o r o l l a length.  These are both very large-flowered populations  but other large-flowered populations (e.g. Jack Point) d i d not show this characteristic. f)  Corollas In addition to c o r o l l a s i z e C_, p a r v i f l o r a i s said to d i f f e r from  C. g r a n d i f l o r a i n the "possession of an almost erect and gibbous c o r o l l a tube i n contrast to a declined and saccate tube" (Newsom,  1929). The populations were studied for the angle of the c o r o l l a and whether the c o r o l l a was saccate or gibbous.  Most of the populations  examined had a c o r o l l a tube declined at about a 45° angle.  S i x pop-  u l a t i o n s had almost erect c o r o l l a s but since these were found i n both larger flowered- and small flowered-populations, there i s no very good c o r r e l a t i o n between small flowers and erect c o r o l l a s . Three populations contained plants with c o r o l l a s that were strongly declined (about 90°)•  Two of these were the very large-flowered  populations from Irvine's North and E l k 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 d i s t r i b u t i o n o f saccate and gibbous tubes.  Many populations had both types of c o r o l l a tube  present, u s u a l l y with t h e i r smaller flowers being gibbous and t h e i r larger ones saccate.  Again, the gibbous c o r o l l a tube does not  correlate with almost erect flowers.  There does however seem to be a general trend toward gibbous c o r o l l a tubes i n the smaller-flowered populations and saccate c o r o l l a tubes i n the larger-flowered populations. Another c o r o l l a character used by some authors to d i s t i n g u i s h the two species i s the r e l a t i v e size of the c o r o l l a limb versus the c o r o l l a tube (Piper, 1906;  Henry, 1915;  Peck, 1961),  In C_, p a r v i f l o r a  the c o r o l l a tube i s said to be longer than the limb and i n f l o r a the limb i s not longer than the tube.  grandi  In the populations  studied, a l l were of the " p a r v i f l o r a " type i n that the tube was longer than the limb, g)  Flower size There was a continuous range of flower s i z e s from 4 mm  without any c l e a r break.  to 15  mm  I f the populations were to be divided i n t o  two or more groups on the basis of flower size there would be considerable overlap between the two classes no matter where the l i n e was drawn.  Discussion Although there i s considerable v a r i a t i o n i n vegetative characte r i s t i c s among and within the populations studied, there were no c o r r e l a t i o n s among the characters that would support d i v i d i n g the group i n t o two species. The  f l o r a l c h a r a c t e r i s t i c s are taxonomically more u s e f u l . There  i s a general trend towards the association of saccate c o r o l l a tubes with l a r g e r flowers and gibbous c o r o l l a tubes with smaller flowers.  - 91 -  There i s also a trend towards an association of declined (45°)  to  almost erect c o r o l l a tubes with smaller flowers and declined  (45°)  to strongly declined (90°)  c o r o l l a tubes with l a r g e r flowers.  How-  ever, neither of these trends can be used to make a clear-cut d i v i s i o n i n t o two species. The calyx character i s more c l e a r cut. the calyx i s greater than 1/2  In most populations,  the c o r o l l a length.  In two of the  largest-flowered populations, however, the calyx length i s l e s s than 1/2  of the c o r o l l a length.  Using t h i s character, i t i s possible to  separate these two populations from the r e s t .  These two  populations  a l s o both have strongly declined c o r o l l a s and saccate c o r o l l a tubes but these three characters are not e n t i r e l y independent of one another.  I f calyces stay the same s i z e but c o r o l l a s are longer, the  calyx w i l l automatically be l e s s than 1/2  the c o r o l l a length.  And  since the difference between saccate and gibbous i s a matter of degree, i f the c o r o l l a as a whole i s smaller, i t might be expected to have smaller g i b b o s i t y .  The angle of the c o r o l l a w i l l also  strongly influence the degree of gibbosity.  These characters are  probably a l l d i f f e r e n t expressions of just one difference ~ versus l a r g e r c o r o l l a s .  Therefore, the separation of these  smaller two  populations from the rest i s not s u f f i c i e n t to j u s t i f y putting them i n t o a separate taxonomic species. Therefore I propose to place a l l the e n t i t i e s now  considered to  be C_ g r a n d i f l o r a Dougl, ex L i n d l . and C&. p a r v i f l o r a Doug V e x  Lindl.  i n t o a s i n g l e species; _C_. p a r v i f l o r a Dougl. ex L i n d l . since t h i s name has published p r i o r i t y .  In addition, I propose to erect two  sub-  92 -  species,  C, p a r v i f l o r a ssp, p a r v i f l o r a and C, p a r v i f l o r a ssp,  g r a n d i f l o r a (Dougl,.ex L i n d l , ) Krause to express the morphological differences discussed above. Although these two subspecies are somewhat a r b i t r a r y , they do recognize the two extremes i n flower s i z e that were formerly given species status.  And they maintain the terms " p a r v i f l o r a " and  " g r a n d i f l o r a " to d i s t i n g u i s h these extremes.  - 93  C o l l i n s i a p a r v i f l o r a Dougl, ex L i n d l , Bot, Reg, 13 : p, 1082 1827, ssp, p a r v i f l o r a Anthirrhinum tenellum Pursh, F l , Am, Sept. 4 2 1 , 1814, L i n a r i a t e n e l l a F, G, D i e t r , V o l l s t , Lexik, Gaertn, Nachtr, 4:408, 1818, C o l l i n s i a t e n e l l a Piper. Contr, U.S. Nat. Herb, 11:496, 1906, Not C, t e n e l l a Benth. 1846. C. minima Nutt. Journ. Acad. P h i l a . 7:47, 1834. C. p a r v i f l o r a var. minima M. E. Jones, Contr. West. Bot. 12:69. 1908. C. g r a n d i f l o r a var. p u s 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. g r a n d i f l o r a ssp. p u s i l l a Piper, Contr. U.S. Nat. Herb. 11:496. 1906. C. b r e v i f l o r a Suksd. W. Amer. S c i . 12:54. 1901. C. m u l t i f l o r a 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. p a r v i f l o r a var. d i e h l i i Pennell i n Abrams, 111. F l . Pac. S t . 3:778. 1951. C. p a r v i f l o r a forma alba English i n S t . John, F l . S.E. Wash. 370. 1956. C. p a r v i f l o r a 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 e n t i r e , ovate to l a n c e - l i n e a r , obtuse to acute, labrate to puberulent, p e t i o l a t e to occasionally sub-sessile, often purplish below, 0.65 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 s o l i t a r y below; pedicels puberulent to glandular-pubescent; calyx membranous below, puberulent to glabrate, scabrous margined, from 1/2 as long to almost equal the c o r o l l a length; calyx lobes subulate to l i n e a r - l a n c e o l a t e , acuminate, c a , 1 mm wide; c o r o l l a 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 , r a r e l y a l l white or magenta; f i l a ments stout, glabrous; stigma 2-lobed; capsule 3-5 mm long, s l i g h t l y exceeded by calyx t i p s ; seeds, usually 4-6, round-oblong, thick, smooth, reddish-brown or brown.  C o l l i n s i a p a r v i f l o r a Dougl. ex L i n d l . ssp. g r a n d i f l o r a (Dougl. ex L i n d l . ) Krause. s t a t . n. £ i g r a n d i f l o r a Dougl. ex L i n d l . Bot. Reg. 1 3 : p i . 1107. 1827. C. g r a n d i f l o r a var. nana Gray, Proc. Am. Acad. 8:394. 1872. !  -94  -  S i m i l a r to p a r v i f l o r a ssp, p a r v i f l o r a ; c o r o l l a s 9-17 mm long; strongly declined (ca 90°); saccate c o r o l l a tube; calyx l e s s than 1/2 the c o r o l l a length.  By placing the entire group i n t o one species with two subspecies, both b i o l o g i c a l r e l a t i o n s h i p s and morphological differences are expressed.  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 v a r i a t i o n 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 v a r i a t i o n , the two extremes are very d i f f e r e n t .  - 95 Bibliography  Abrams, L.  1951. I l l u s t r a t e d F l o r a of the P a c i f i c States. V o l . I I I . Stanford U n i v e r s i t y Press, Stanford, California.  Ahloowalia, B.S. and Garber, E.D. 1961. The genus C o l l i n s i a X I I I . Cytogenetic studies of i n t e r s p e c i f i c hybrids i n v o l v i n g species with pediceled flowers. Botanical Gazette 122: 228-232. Anderson, J.P. 1961. F l o r a of Alaska and adjacent parts of Canada. Iowa State U n i v e r s i t y Press, Amer. Iowa. Bopp, M.  1959* Uber die bildung von anthocyan und an wundrandern. Z e i t s c h r i f t fur Botanik  Cain, A.J.  1954* Animal Species and t h e i r E v o l u t i o n . Hutchinson and Co. L t d . , London.  Davis, Ray J . 1952. F l o r a of Idaho. Dubuque, Iowa.  Wm.  leucoanthocyan 197-215*  C. Brown Co.  Garber, E.D. 1956. The genus C o l l i n s i a I . Chromosome number and chiasma frequency of species i n the two sections. Botanical Gazette 118: 71-73» Garber, E.D. 1958a. The genus C o l l i n s i a I I I . The s i g n i f i c a n c e of chiasmata frequencies as a cytotaxonomic t o o l . Madrono 1_±: 172-176. . 1958b. The genus C o l l i n s i a VII. A d d i t i o n a l chromosome numbers and chiasmata frequencies. Botanical Gazette 120: 55-56. . 1974. C o l l i n s i a . In: Handbook of Genetics. R.C. King (ed) V o l . 2: 333-361. Plenum Press, New Garber, E.D. and D h i l l o n , T.S. 1962. The genus C o l l i n s i a Cytogenetic studies of i n t e r s p e c i f i c hybrids. Botanical Gazette 12_>: 291-298.  York.  XX.  Gilkey, Helen M. and Dennis, La Rea J . 1967. Handbook of northwestern plants. Oregon State U n i v e r s i t y Bookstores, Inc., C o r v a l l i s , Oregon. Gorsic, J . 1957* The genus C o l l i n s i a V. Genetic studies i n £ 4 heterophylla. Botanical Gazette 118(3): 208-223. Grant, V.  1963. The o r i g i n of adaptations. Press, New York.  Columbia U n i v e r s i t y  . 1971. Plant s p e c i a t i o n . Columbia U n i v e r s i t y Press, New York.  - 96 -  G r i f f i t h s , A.J.F., Krause, G. and Ganders, F.R. 1977. A l e a f spot polymorphism i n C o l l i n s i a grandiflora (Scrophulariaceae). Canadian Jour* of Botany _5_5: 654-661. Harborne, J.B. 1967. Comparative biochemistry of the flavenoids. Academic Press, London. Hayhome, Barbara A. and Garber, E.D. 1968. The genus C o l l i n s i a XXIX. P r e f e r e n t i a l p a i r i n g i n d i p l o i d , t r i p l o i d and t e t r a p l o i d i n t e r s p e c i f i c hybrids i n v o l v i n g stricta x Cj, concolor and r e l a t e d species. Cytologia 33(2): 246-255. Henry, J.K. 1915* F l o r a of southern B r i t i s h Columbia and Vancouver I s l a n d . W.J. Gage & Co. L t d . , Toronto. Hitchcock, C L . and Cronquist, A. 1973. F l o r a of the P a c i f i c Northwest. U n i v e r s i t y of Washington-Press, S e a t t l e . Hitchcock, C.L., Cronquist, A., Ownbey, M. and Thompson, J.W. 1959. Vascular plants of the P a c i f i c Northwest. Part 4. Ericaceae to Campanulaceae. U n i v e r s i t y of Washington Press, S e a t t l e . Howell, T.  1903. Oregon.  F l o r a of northwestern America.  Lindley, J . 1827. C o l l i n s i a p a r v i f l o r a . 13_: p i . 1082. • 1827. C o l l i n s i a g r a n d i f l o r a . 13_: p l . 1107. Mayr, E.  Portland,  Botanical Register Botanical r e g i s t e r  1957. Species concepts and d e f i n i t i o n s . In: The species problem, E. Mayr (ed). American Assoc. Adv. Science, Washington.  Munz, P h i l i p A. and Keck, D.D. 1959. A California flora. Univ. of C a l i f o r n i a Press, Berkeley. , Newsom, V.M. 1929. A r e v i s i o n of the genus C o l l i n s i a . Botanical Gazette 87.: 260-301. Peck, M.E.  1961. A manual of the higher plants of Oregon. Binfords and Mort, Portland, Oregon.  Piper, Charles V. 1906. Contributions from the United States National Herbarium. (Vol. X I ) . F l o r a of the State of Washington. Washington Government P r i n t i n g O f f i c e .  - 97 -  Piper, Charles V, and B e a t t i e , R.K. 191% F l o r a of the northwest coast. The New Era P r i n t i n g Co., Lancaster, Pa. Snow, Richard. 1963* A l c o h o l i c hydrochloric acid-carmine as a s t a i n for chromosomes i n squash preparations. Stain Technology 38(1): 9-13« S t . John, Harold. 1956. F l o r a of southwestern Washington and of adjacent Idaho. State College of Washington Press, Pullman, Washington. Taylor, R.L. and MacBryde, B. 1977* Vascular plants of B r i t i s h Columbia. A d e s c r i p t i v e resource inventory. Technical B u l l e t i n No. U Botanical Garden. U n i v e r s i t y of B r i t i s h Columbia. U.B.C. Press. Vancouver, B.C. m  Taylor, R.L. and Mulligan, G.A. 1968. F l o r a of the Queen Charlotte Islands. Part 2. Research Branch, Canada Dept. of,Agriculture Monograph No. k»  

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