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Distyly, pollen flow and seed set in Menyanthes trifoliata (Menyanthaceae) Christy, Nancy Lynne 1987

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D1STYLY, P O L L E N  FLOW  AND  SEED  SET  IN MENYANTHES  TR1FOLIATA  (MENYANTHACEAE) by NANCY LYNNE  CHRISTY  B . S c , The University of British Columbia, A THESIS SUBMITTED  IN P A R T I A L  1983  FULFILMENT  T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF MASTER OF SCIENCE  in  THE F A C U L T Y OF G R A D U A T E  STUDIES  Department of Botany  We accept this thesis as conforming to the required standard  THE UNIVERSITY  OF BRITISH  March  COLUMBIA  1987  ® Nancy Lynne Christy,  1987  OF  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that, copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department of Botany The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V 6 T 1W5 Date: March 1987  ABSTRACT The  influence of variation  examined  in style  in six populations  length on pollen flow  of Menyanthes  trifoliata  and seed  set was  in southwestern  British  Columbia to evaluate Ganders' hypothesis that morphological distyly increases the fecundity of a diallelic self-incompatible plant. In five populations, Menyanthes distylous  and  morphological  self-incompatible. The  sixth  population  consisted  was  of pins and  homostyles (thrums with unusually long styles). In each population  morph frequency, pollen frequency, the composition  of stigmatic pollen loads and  seed  set were estimated. Results from the six populations demonstrate that the  size  and composition  flowering  of stigmatic pollen loads fluctuates erratically  season.  Pins  and  thrums  experienced  during the  disassortative  pollination,  assortative pollination and random pollination at different times during the season. Homostyles were always assortatively pollinated. Among populations there was a high correlation between morph frequency of  stigmatic  morphological  pollen  loads. However, in anisoplethic populations  Comparison of the composition thrums  revealed  that  flowers of Menyanthes and  the reciprocal  of stigmatic pollen loads of homostyles with those  the separation  of stigmas  and anthers  seed  in distylous  reduces the number of incompatible pollen grains received, and anthers  appears to increase the  pollen grains received. Seed set in the six populations of  was always below the potential maximum. Among  was a high correlation between the number of compatible and  Menyanthes  pollen received by the opposite floral form.  placement of stigmas  number of compatible Menyanthes  of  distyly seems to compensate for the rarity of one floral form, by  increasing the proportion of compatible  of  (pollen frequency) and the composition  populations there  pollen grains received  set. In at least one population, pollen availability was a major factor  ii  limiting  seed  Menyanthes  as  set, but well.  other  Thrums  factors  are probably  set significantly  more  influencing seeds  seed  set in  per capsule  than  homostyles. In populations of Menyanthes,  the reciprocal placement of stigmas and  anthers  to homostylous  in distylous  flowers  (compared  flowers) increases the  amount of compatible pollen deposited on stigmas, and this increase is associated with greater fecundity.  iii  TABLE OF CONTENTS ABSTRACT  ii  LIST OF TABLES  v  LIST OF FIGURES  vi  ACKNOWLEDGEMENT  viii  I. INTRODUCTION  1  II. FLORAL MORPHOLOGY ! A. INTRODUCTION B. MATERIALS AND METHODS C. RESULTS D. DISCUSSION III.  6 6 10 13 22  EXPERIMENTAL POLLINATIONS A. INTRODUCTION B. MATERIALS AND METHODS C. RESULTS D. DISCUSSION  28 28 31 33 37  IV. POLLEN FLOW A. INTRODUCTION B. MATERIALS AND METHODS C. RESULTS D. DISCUSSION  42 42 45 53 76  V. SEED A. B. C. D.  SET INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION  ;  82 82 84 87 101  VI. CONCLUSIONS  107  BIBLIOGRAPHY  114  iv  L I S T  O F  T A B L E S  1. Floral characters of pins, thrums and homostyles of Menyanthes 2. Variance components of floral characters of Menyanthes  Seed  set  following  self,  geitonogamous,  intramorph  pollinations of pins and thrums of Menyanthes  15  pin and thrum 20 and  intermorph  trifoliata  34  5. Representation of floral morphs in six populations of Menyanthes of  pin, thrum  pollen  trifoliata.  6.  Frequency  7.  Percentage of pin and thrum (or homostyle) pollen participating compatible pollinations in six populations of Menyanthes trifoliata  populations of Menyanthes  and homostyle  ... 14  trifoliata  3. Number and percentage of pollen grains removed from flowers of Menyanthes trifoliata by pollinators 4.  trifoliata.  produced  in six  trifoliata  56  in  8. Percentage of pollen on stigmas of pin, thrum and homostyle flowers of Menyanthes trifoliata from six populations 9.  ... 54  Composition of stigmatic pollen loads of pin, thrum flowers of Menyanthes trifoliata from six populations  57 58  and homostyle 63  10. Expected and observed pollen frequencies on stigmas of pin and thrum flowers of Menyanthes trifoliata from population B L 2  71  11. Expected and observed pollen frequencies on stigmas of pin and thrum flowers of Menyanthes trifoliata from population B L 3  72  12. Expected and observed pollen frequencies on stigmas of pin and thrum flowers of Menyanthes trifoliata from population P C I  73  13. 14.  Expected and observed pollen frequencies on stigmas of pin and homostyle flowers of Menyanthes trifoliata from population S L 1  74  Seed set and expected seed set in pins, thrums Menyanthes trifoliata from six populations  89  and homostyles of  15. Seed set, percentage fruit set and percentage seed set in pins, and homostyles of Menyanthes trifoliata from nine populations  v  thrums 99  LIST OF 1. Typical habitat of Menyanthes  FIGURES  trifoliata  7  2. Emergence of an inflorescence of Menyanthes 3. Distribution map of Menyanthes  trifoliata  trifoliata.  9  in southwestern British Columbia. .. 12  4. Longitudinal sections of pin and thrum flowers of Menyanthes  trifoliata  17  5. Longitudinal sections of pin, thrum and homostyle flowers of Menyanthes  trifoliata  18  6. Pin and thrum plants of Menyanthes  trifoliata  7.  on  growing in a growth chamber.  '. 32 The  effects  Menyanthes  8.  of  pollination  intensity  seed set  in  pins  and  thrums  trifoliata  Populations of Menyanthes Vancouver, B.C  trifoliata  at  Beaver Lake  in  Stanley  Park, 47  9. Populations of Menyanthes trifoliata located in the Pinecrest area, 0.9 km north of Daisy Lake canal on B.C. Hwy 99 10. 11.  of 36  Populations of Menyanthes Provincial Park  trifoliata  Populations of Menyanthes Pinecrest area  trifoliata  at  Stump  Lake  in  Alice  48  Lake 49  located  in  small  ponds  in  the 50  12.  The percentage of compatible pollen deposited on stigmas of pins, thrums and homostyles in six populations of Menyanthes trifoliata plotted against the percentage of compatible pollen produced in each population 60  13.  The number of pollen grains deposited on stigmas of pin flowers of Menyanthes trifoliata in population B L 2  and thrum  The number of pollen grains deposited on stigmas of pin flowers of Menyanthes trifoliata in population B L 3  and thrum  The number of pollen grains deposited on stigmas of pin flowers of Menyanthes trifoliata in population B L 4  and thrum  The number of pollen grains deposited on stigmas of pin flowers of Menyanthes trifoliata in population P C I  and thrum  The number of pollen grains deposited on stigmas of pin flowers of Menyanthes trifoliata in population PC2  and thrum  14. 15. 16.  17.  vi  65 66 67  68  69  18. The number of pollen grains deposited on stigmas of pin and homostyle flowers of Menyanthes trifoliata in population S L l 19.  20. 21.  22.  Seedlings of Menyanthes the Pinecrest area  The  number  The  number  The  number  Menyanthes  25.  The  number  Menyanthes  26.  The  number  Menyanthes  27.  in the mud in ponds in 77 Meadow  in Cypress 86  The number of seeds set per capsule in pins, thrums and homostyles of Menyanthes trifoliata plotted against the number of compatible pollen grains deposited on stigmas  Menyanthes  24.  germinating  Populations of Menyanthes trifoliata at Strachan Provincial Park in West Vancouver, B.C  Menyanthes  23.  trifoliata  The number Menyanthes  of  seeds  trifoliata  of of of of of  pins  and  thrums  set  per  set  per  set  per  set  per  92  capsule  in  pins  and  thrums  of 93  capsule  in  pins  and  thrums  of 94  capsule  in  pins  and  thrums  of 95  capsule  set per capsule  in population S L l  vii  88  of  in  pins  and  thrums  of  in population P C 2  seeds  trifoliata  in  in population P C I  seeds  trifoliata  capsule  in population B L 4  seeds  trifoliata  per  in population B L 3  seeds  trifoliata  set  in population B L 2  seeds  trifoliata  70  96  in  pins  and homostyles  of 97  ACKNOWLEDGEMENT  I  thank  my  major  professor,  Dr. Fred  Ganders  for inspiration, advice and  financial support. Special thanks are due to Ms. Sue Dreier, Dr. Helen Kennedy and  Dr. Jack Maze, and to my committee members Dr. Gary Bradfield and Dr.  Wilf Schofield. I am  also grateful to my  endless patience and love.  viii  husband  John  and my  family for  To J.A.C. who  rescued me  from countless muddy abysses.  ix  I. INTRODUCTION  As reviewed by Ganders (1979), distyly is a relatively rare plant breeding system in which a single locus, diallelic self-incompatibility linked  with  certain  consisting of two and anthers flowers other  floral  dimorphisms.  type  has flowers  pollen  from  in  in  populations  of  plants  with  two  floral  forms.  stamens and is called the  short  One type  has  "pin" form. The  styles and long stamens and is called the  Pollen from pin anthers is compatible with only thrum stigmas, thrum  dimorphisms  stigmatic  results  are reciprocally positioned in the  form.  differences  This  morphologically distinguishable mating groups in which stigmas  with long styles and short  "thrum" while  floral  system is genetically  anthers  often  pollen  is  compatible  associated  size  and  with  the  morphology,  papillae. Those distylous  species  with  in  only  pin  style/stamen  pollen  production  which  pin  visibly distinguishable provide a unique opportunity  for  stigmas.  Other  dimorphism and  length  and thrum  pollen  are of are  the study of pollen flow  in self-incompatible plants.  Distyly 1979,  Neisess  named first  Clusius to  would  in  studies the  system  only  occur was  in  25  of  from  families  first  reported  of Primula  (vanDijk  relationship  using Primula,  result  (legitimate  to  1984), but  establish  incompatibility pollinations  is known  distylous  between plants.  in  of  flowering  1583  by  a Flemish  1943). Darwin the  floral  Through  a  plants  (1862),  dimorphisms series  of  he was able to show that fertilization pollinations  between  flowers  of  the  (Ganders botanist was  the  and  the  experimental and seed set opposite  form  pollinations). Darwin (1877) suggested that the reciprocal placement of  1  INTRODUCTION / 2 stigmas and anthers in the two floral forms increased the amount of compatible pollen deposited  on stigmas  by  pollinators  since the  portion  of the  pollinator's  body where pollen from one form was deposited would correspond to the position of  the  stigma  disassortative that the  of  the  opposite  floral  pollination  between  pins  adaptive  form. and  significance of distyly  This  thrums.  was to  would  result  Darwin  reduce  in  (1877)  pollen  phenotypic hypothesized  wastage  leaving  metabolites for other purposes.  More most  of  1970a, and  recent  the  pollen  1970b,  Schou  studies of pollen flow produced in  1971,  1981;  1976,  Rama  distylous  1979,  Swamy  in  distylous  plant  populations  1980a; Ganders and  Bir  plants  have shown  is  wasted  1974; Weller  Bahadur  1984;  that  (Ornduff  1980; Philipp  Nicholls  1985).  An  average of 1% of the pollen produced in populations was deposited on conspecific stigmas in these studies. Contrary that  distyly  may  not  be an  to Darwin's hypothesis, these studies indicate  effective  mechanism for  Examination of the composition of stigmatic  reducing  pollen  pollen loads of distylous plants has  shown that most of the pollen that was deposited on stigmas was (Levin  1968;  Ornduff  1975,  1978;  Ganders  1979;  Oleson  1979;  incompatible Lewis  Schou 1983), but this is not surprising given the type of incompatibility present.  Most  self-incompatible  plants  wastage.  have  a  multi-allelic  1982; system  incompatiblity  mechanism, with several mating groups in each population. But with the diallelic self-incompatibility mating  groups.  morphs  in  system found With  vegetative  pins and thrums  complete reproduction  in  distylous  plants,  self-incompatibility or  survivorship,  populations and the  no  have only  differences  equilibrium  two  between  frequency  of  is 1:1 and is reached in one generation (Ganders 1979). With  INTRODUCTION / 3 a 1:1 ratio, pollen from one-half of the population is incompatible with any given stigma (Ganders 1979).  In assessing the adaptive significance of disytly, Ganders (1974) suggested that  more  emphasis should  be placed on  the  pollen  received by  the  female,  rather than the pollen wasted by the male. Since the chances of the stigma of a diallelic self-incompatible plant receiving incompatible mechanism that would increase the number  pollen are so great,  any  of compatible pollen grains received  would increase the fecundit3' of the plant. Because of the reciprocal placement of stigmas and anthers in flowers of distylous plants, it is possible that they would receive a greater proportion of compatible pollen than a diallelic self-incompatible plant  lacking the floral dimorphism. Ganders (1974)  significance  of  distyty  is  to  increase  fecundity  suggested that the adaptive  by  increasing the  number  of  compatible pollinations received.  Ganders' hypothesis assumes that seed set in distylous plants  is  limited  by a lack of compatible pollinations. As reviewed by Shore and Barrett  (1984),  many factors have been shown to influence seed set in self-incompatible plants. These  include:  spatial  pattern,  environmental resource  the and  quantity number  conditions  availability  for  and of  during fruit  influence of these factors  efficiency mating  of  groups  pollinators present  pollination, fertilization and seed maturation;  on seed set in distylous  present; in  the  the  density,  population;  and embryo  development;  and pollen availability. plants  the  is not known.  The Since  stigmas of distylous plants often receive a small fraction of the pollen produced in the population, and because their chances of receiving incompatible pollen are  INTRODUCTION / 4 so great,  it  is plausible that a lack of compatible pollinations may  limit  their  seed set.  The purpose of the present study was to determine the effect of variation in style length on pollen flow in the distylous perennial Menyanthes trifoliata L., and  to  investigate  the  relationship  grains received and the number conjunction  with  pollen  flow  between  the  number  of  compatible  pollen  of seeds set. Seed set data were collected in data,  in  order  to  determine  whether  pollen  availability was a major factor influencing seed set in Menyanthes. The influence of variation in morph frequency on pollen flow in populations of Menyanthes was also examined.  In  most populations  positioned unusual  in  the  two  population  an opportunity the  floral  of  homostyles (thrums  of Menyanthes, stigmas forms  (pins  and  Menyanthes consisting  and anthers  thrums). of  pins  The and  are reciprocally  occurrence  of  an  morphological  long  with styles the same length as their own stamens) provided  to test Darwin's (1877) theory that morphological distyly increases  proportion  of  compatible  pollen  received  by  the  stigmas  of  diallelic  self-incompatible plants. By comparing the stigmatic pollen loads of homostyles to those  of  thrums  from  other  reciprocal position of stigmas compatible  pollen  correspond  to  .the  received. number  populations, and anthers If,  as of  it  was  possible to  had any effect  Ganders  compatible  test  on the  (1974)  assumes,  pollen  grains  whether  the  proportion  seed  received,  set  of  does  and  if  morphological distyly does increase the proportion of compatible pollen received by stigmas of diallelic self-incompatible plants, then this should result in higher seed  INTRODUCTION / 5 set in thrums than in self-incompatible homostyles.  II. FLORAL MORPHOLOGY  A. INTRODUCTION  Menyanthes circumpolar  trifoliata  distribution  is  a  between  distylous,  40 ° N  aquatic  and the  perennial  Arctic  herb,  with  circle, and north of  Arctic circle into Greenland, Norway, Alaska and Siberia (Hewett  1964). It  a the  grows  at elevations ranging from sea level to 3200 meters and inhabits bogs, marshes, lakes and rivers (Figure 1) (Hewett  1964). Water levels in its habitat can range  from a few centimeters (often drying out by the end of summer) to depths of 2 meters or more.  Menyanthes has submerged horizontal stems with adventitious roots  arising  at the nodes. The leaves are emergent, with sheathing petioles and ternate, obovate back  leaflets  each  4-12  winter  cm long  and  (Hitchcock  growth  resumes  and Cronquist each  spring  1973). The leaves die  from  axillary  buds.  inflorescence is a terminal, leafless raceme with up to 40 flowers (Hewett It  is  winter  formed  at  (Hewett  the  end of  each growing  season  and  15-30 mm  in diameter  corolla is white  with pinkish tips and covered with white  surface  limb.  corolla sagittate  the  lobes.  remains  The  1964).  dormant  over  1964). The flowers have a fused calyx with five ovate lobes and  a sympetalous, campanulate corolla  of  with  The  There  are  five  filaments  are  white  epipetalous and  and contain bright orange-yellow  the  (Fernald  hairs along the  stamens, anthers  pollen. The pistil  alternate are  inner  with  purplish  the  brown,  is composed of  or three carpels and has a unilocular ovary with parietal placentation.  6  1950). The  two  FLORAL MORPHOLOGY / 7 FIGURE 1. Typical habitat of Menyanthes trifoliata. A small pond located nearby Stump Lake in Alice Lake Provincial Park, southwestern British Columbia.  FLORAL MORPHOLOGY In  populations  Columbia,  growth  of  of  Menyanthes trifoliata  leaves and  the  found  in  inflorescence begins  southwestern in  / 8  British  February  at  low  elevations, and in June at high elevations. Initially, one or two leaves elongate, become emergent and expand, followed by the elongation and emergence of  the  inflorescence (Figure 2). Flowering sometimes begins as early as April, but most often occurs during the month of May (July at higher elevations).  The  fruit  is  a  round  capsule approximately  1 cm  in  persistent style and dehisces along one or two sutures (Hewett be  dispersed by  capsule dehiscence or  withers, falls in the water this  study  may  and rots. In  Red-wing Blackbirds were  diameter,  has a  1964). Seeds may  be released as the  infructescence  one population (Beaver Lake) used for  observed pecking at the  fruits  and eating  the seeds within.  Menyanthes is a monotypic genus in the Menyanthaceae. Distyly occurs in three  other  1979). Ornduff Villarsia,  Little  genera in work  (1966,  this  has  1982,  family:  been  1986)  Fauria,  done  on  and Barrett  Nymphoides, and  the  distylous  (1980)  have  Villarsia  members  of  (Ganders  this  family;  studied Nymphoides and  and Ganders (1979) has presented some data on Fauria crista-galli and  Menyanthes trifoliata. Distyly in Menyanthes was first studied by Darwin The  (1877).  population described by Darwin was one that consisted of an equal number  of pins and thrums. Although some variation in floral characters was found, the flowers species.  of Menyanthes exhibited the Pin  pistils  were  1.5  times  same dimorphisms found in other longer  than  thrum  pistils,  distylous  while  stamens were 2 times longer than pin stamens. Pin stigmas were generally  thrum  FLORAL MORPHOLOGY / 9 FIGURE  2. Emergence of an inflorescence of Menyanthes  trifoliata.  F L O R A L M O R P H O L O G Y / 10 larger  than  thrum  stigmas, and thrum  anthers  were  larger  than  pin  anthers.  Although exact sizes for the pollen grains were not given, the diameter of thrum pollen was found to average 1.2 times larger than pin pollen.  Distyly reported  in  Menyanthes was  next  discussed  by  Warming  (1886)  who  that both distylous and homostylous forms occurred in Greenland. This  report has since been misquoted, first by Knuth in 1899 and then by Baker in 1959; both stated that Menyanthes was distylous throughout Greenland  where  it  was exclusive^ homostylous.  Knuth  the world, except in  (1899)  examined some  distylous flowers of Menyanthes, and noted that the stigmatic papillae were longer in pins, and that thrum  pollen was larger  than  pin pollen. Pollen grain  sizes  were given as 26x50 um for pin, and 63x120 Mm for thrum. Since the turn of the century, the onlj' mention of distyly pins  of  Menyanthes produced  (Ganders  1979).  dimorphisms distyly  in  Other  1.34x  than  these  in Menyanthes has been a report  more few  reports  in Menyanthes trifoliata, there this  genus.  between  pins  and  breeding  system  This  thrums  of  chapter in  floral  pollen  anther  concerning  than  did  some of  thrums  the  floral  has been no comprehensive study  presents characters  Menyanthes trifoliata.  per  that  data  that  associated  Lnterpopulational  quantify with  the  variation  of  differences distylous in  these  characters was also examined.  B. M A T E R I A L S  AND  METHODS  Populations of Menyanthes trifoliata were  studied for  floral  Beaver Lake in Stanley Park, Vancouver, British Columbia, in  morphology  in  Stump Lake in  FLORAL MORPHOLOGY / Alice  Lake  Provincial  Park,  13  km  north  of  Squamish, on  British  11  Columbia  Highway 99, and in ponds at Pinecrest, 0.9 km north of Daisy Lake Canal, on British Columbia Highway 99 (Figure 3).  In  each  morphological  population  25-30  measurements.  A  flowers  total  of  of  each  11  form  different  were  floral  collected  for  characters  were  measured (Table 1). Petal length, stamen length and style length were measured from the base of the ovary to the tip of the structure. removal  and  acetolyzed (Erdtman  pollen  and  diameter  then  were  suspended in  estimated a  1:1  from  mixture  Pollen production, pollen  pollen of  samples  lactophenol  that  and  were  glycerin  1960). Collections were made from each population and samples of pin  and thrum pollen were treated undehisced  anthers  separately. Pollen production was measured from  collected just  before  flower  buds  opened. Approximately  40  flower buds of each form were collected and their anthers pooled. The number of pollen  grains  present  was  counted  using  were counted for each collection (Lloyd pollen  was  removed . from  the  anthers  a  haemocytometer,  and  20  replicates  1965). In order to determine how of  Menyanthes flowers  by  much  pollinators,  shrivelled anthers were collected from approximately 40 flowers of each form just before flowers wilted. The number of pollen grains remaining in the anthers was counted using a haemocytometer  (20 replicates) and this number subtracted from  corresponding pollen production numbers  (Ornduff  1980a, 1980b). Pollen diameter  was determined using collections of ten anthers from open flowers of each form. The longest diameter of 300 pollen grains of each form, in each population, was measured  using  a  Vickers  M-22  microscope  with  an  image  splitting  module  (Ganders 1976). Ovule number was counted from several hundred ovaries of each  FLORAL MORPHOLOGY  /  12  F I G U R E 3. Distribution of Menyanthes trifoliata in southwestern British Columbia. Pie diagrams represent estimates of the frequency of floral morphs in each population. Each circle represents a separate population found at each locality. In some cases, populations were located in the same lake (localities: 6, 8, and 9), and in others populations were found in separate lakes or ponds (localities: 2 and 5). The frequency of each floral morph is represented as a portion of the pie diagram. The frequency of pins in each population is represented in white, the frequency of thrums in black, and the frequency of homostyles in stripes. Morph frequencies were estimated from nearest-neighbour counts in some populations (2, 5, 7, 9 and 10), or from counting a representative sample of plants in the population (3, 6 and 8). No estimate of morph frequencies were available for populations 1 or 4.  1 Whistler S w a m p 2  Pinecrest PC1 2  3  3 Stanley  Lake  4 Roadside ponds 11.2 km N of B r o h m Lake 5 S t u m p Lake SL1  2  6 B r o w n i n g Lake  Q • 7 Strachan Meadow, Cypress Mtn. #  SM1  8 Hollyburn  Mtn.  • o •  •  ' o Blue Lost L. F i r s t Gentian L. L. 9 Beaver O  P  0  thrums  ^  homostyles  i n  s  Lake  0 9 • BL2  3  4  10 L a d n e r M a r s h  FLORAL MORPHOLOGY  / 13  form from each population. The average number of flowers per inflorescence was counted from approximately  Data were analyzed  200 inflorescences of each form from each population.  using the non-parametric Median  test to determine  whether significant differences existed between forms in the different characters measured. Tests of significance with ANOVA did  not conform  to the underlying  could not be used because the data  assumptions  of equality of variance and  normality. However, a nested ANOVA was used to partition variation observed in floral characters into three sources: differences between forms, differences between populations  and differences between individuals  (the residual  factor) (Sokal and  Rohlf 1981).  C. RESULTS  Results from measurements of floral characters showed that in two out of three  populations  Menyanthes trifoliata  sampled were  (Beaver  Lake  distylous (Table  and  Pinecrest)  1), but that  the  many  flowers  of  of the floral  characters displayed a considerable amount of variation at the populational and individual level (Table 2). The third population (Stump Lake), consisted of pins and (morphological) long homostyles.  Up for  to 85% of the variation in style and stamen lengths was accounted  by differences between pins and thrums  (Table  2). At Beaver  Lake and  Pinecrest, anthers and stigmas were reciprocal!}' positioned in the two floral  TABLE  1. F l o r a l  characters  Lake,  Pinecrest  and Stump  of p i n s , Lake.  Character Petal  F1ora1  length  (mtn) Stamen  length  (mm) Style  length  (mm) Difference  thrums  Values a r e  and homostyles of the mean and  morph  Menyanthes trifoliata  standard d e v i a t i o n  Beaver  Lake  from  three p o p u l a t i o n s :  ( i n parentheses). P i necies t  Sample Stump  Lake  pin  16. 1 ( 1.02)  16.2  (1.21)  12.5  (0.92)  thrum  14.1  15.8  (0.83)  12.8  (0.85)*  pin  9.7 (0.56)  9.1  thrum  13.2  13.9  (1.13)  (1.12)  pin  15.4  thrum  8.5  ( 1.09)  (0.81)  13.2 8.2  (0.87) (0.64) (1.21)  7.4  (0.76)  11.7  (0.56)*  11.5  ( 1 . 18)  (0.53)  9.9  (0.64)'  (0.72)  between  stamen and s t y l e  pin  5.7  (0.78)  4.1  (0.77)  4.0  l e n g t h (mm)  thrum  4.7 (0.81)  5.8  (0.58)  1 . 8 (0.61)•  Anther  pin  2.0  thrum  2.1  length  (mm) Pollen per  production  f1ower  (0.22)  2.5  (0.14)  2 . 1 (0.09)  3.0  (0.25)  2.5  (0.11)*  pin  17,408  (6,309)  15,539  (1,838)  17,783  thrum  10.617  (3.268)  18.095  (1,753)  9,990  (6,604) (1.754)*  Po11 en d i ameter  pin  4 1.3 (2.90)  4 1.6 (2.80)  4 1.7  (2.30)  (pm)  thrum  47.8  50. 1 (3. 10)  47 . 4  (2.90)*  Ovary  length  (mm)  (3.17)  pin  3.4 (0.44')  2.5  (0.41)  2.4  (0.36)  thrum  2.9  3.0  (0.21)  2.9  (0.22)*  (0.39)  Ovu1e number  pin  37.4  (13.38)  30. 5 (6.81)  17.2  (5.93)  per  thrum  30.2  ( 12.88 )  29.3  20. 1  (5.43)*  Number o f  pin  2.3  (0.47)  2.6  (0.49)  2.7  s t i gma  thrum  2.0  (0.19)  2.3  (0.45)  2.0*  f1ower  1obes  (8.52 )  (0.48)  Number of f l o w e r s  pin  20.6  (3.62)  15.1  < 2.87)  28. 5  (3.46)  per i n f l o r e s c e n c e  thrum  21.3  (4.56)  15.2  (3.09)  27 . 2  (3.94)*  * homostyle  Beaver  s i z e s are given  TABLE  2. V a r i a n c e  variation  which  components  of  is attributable  floral to  Stamen Style Anther  in  interpopulational  Population  Character  Petal  characters  length length  Pollen  production  Pollen  diameter  variation.  Values  are  the percentage of  Intermorph v a r i a t i o n and  Morph  Individual  10  39  14  76  10  85  12  60  22  18  36  53  1 1  58  40  Ovary  length  19  27  54  Ovule  number  38  4  58  25  58  Number Stigma  observed  variation.  Individual  51  length length  Menyanthes trifoliata.  r o  of lobes  3 o  to Number per  of flowers inflorescence  62  36  X  o tr O O  1  F L O R A L M O R P H O L O G Y / 16 forms  (Figure 4). The difference in length between stamens and styles averaged  4.9 mm On  (s.d. = 1.13)  average, the  in pin flowers, and 5.2 mm  stigmas  of  one floral  form  (s.d. = 0.88)  were  at  in thrum  the  flowers.  same level  as  the  middle of the anther sacs of the opposite floral form (Table 1).  At  Stump Lake the  pin flowers  difference  averaged 4.0 mm  in length  between stamens and styles  (s.d. = 0.72), similar  to that found  in pin  in  flowers  from the other  populations. Morphologically, the rest of the flowers from Stump  Lake  appeared  to  were  the  same  length  (s.d. = 0.61)  longer  stigmas  the  at  be long as  than  than  same level  same length pin  the  as pin  stamens,  style  pin  The  stamens  styles,  but  own  styles  (Figure  as the  middle  of  their  average of 2.5 mm higher the  homostyles.  of  only  an  5).  their  the  homostyle  average  This  own  anther  length,  not  stamen  styles were length,  was  1.8  placed  mm  homostyle  sacs,  than pin stamens. Because homostyle  styles, and homostyle  of  flowers  and  an  stamens were  considerably thought  to  longer be  the  variable factor. The homostyles had longer styles than normal thrums.  At smaller  Beaver  in  significantly diameter  Lake  diameter  than  (p< 0.001)  of pin  and  Pinecrest,  thrum  smaller  pollen grains  in  pin  pollen  pollen, while diameter  averaged  at  than  41.5  um  was  significantly  Stump Lake, homostyle  pin  (p< 0.001) pollen was  pollen. The  (s.d = 2.69), while  the  longest longest  diameter of thrum pollen averaged 48.9  um (s.d. = 3.27). In  each population  size  overlapped  (Table  ranges  for  the  two  pollen  types  populations, average pollen diameters were similar  slightly except for  1).  the  Between  thrum pollen from  Pinecrest, which was on average 2.3 um larger in diameter than thrum pollen  FLORAL MORPHOLOGY  / 17  FIGURE 4. Longitudinal sections of pin and thrum flowers of Menyanthes trifoliata from Pinecrest in southwestern British Columbia showing reciprocal placement of stigmas and anthers in the two floral forms.  FIGURE  5.  Longitudinal  sections  of p i n , homostyle  and thrum  flowers  of  Menyanthes  trifoliata.  F L O R A L M O R P H O L O G Y / 19 from Beaver Lake (Table 1). Pollen from homostyles at Stump Lake was similar in  size  (47.4  um,  s.d. = 2.90)  to  thrum  pollen  from  Beaver Lake  um,  (47.8  s.d. = 3.17).  A total of 53% of the variation in pollen production was accounted for by differences variation  between  morphs,  but  i n pollen production  there  was  also  between populations  a  considerable  (36%)  (Table  amount  of  2). Pin flowers  produced more pollen than thrum flowers at Beaver Lake and homostyle flowers at Stump Lake, but at Pinecrest, thrum flowers produced more pollen than  pin  flowers (Table 1). At Pinecrest, thrum flowers had 1.65 times more pollen grains removed by  pollinators  than  did pin flowers. A t  Beaver Lake, pin flowers  had  1.67 times more of their pollen removed than did thrum flowers (Table 3).  Within  each  population,  thrum  anthers  (and  homostyle  anthers)  were  significantly longer than pin anthers (Stump Lake and Pinecrest p<0.001, Beaver Lake p<0.05),  but the variation among populations (60%) was greater than  the  variation between morphs (22%) (Table 2). Pin and thrum anthers from Pinecrest were  the  (s.d. = 0.14)  largest in  of  the  three  pins, and 3.0 mm  populations. (s.d. = 0.25)  Anther  length  i n thrums.  averaged  Anthers  2.5  mm  from Beaver  Lake were the smallest where anther length in pins was 2.0 mm, and averaged 2.1 mm (s.d. = 0.22) in thrums.  Petal length also varied more among populations than among individuals or between morphs  (Table 2). Petals from  Pinecrest were the longest, while  those  from Stump Lake were the shortest. Beaver Lake was the only population where  TABLE 3. Number and percentage of p o l l e n g r a i n s removed from p i n and thrum flowers of Menyanthes trifoliata by pollinators. Values are the mean and standard d e v i a t i o n ( i n parentheses), and represent the amount of p o l l e n removed from one flower ( f i v e anthers per f l o w e r ) .  Average Population  Floral Morph  Beaver  pin  41  thrum  GO  pin  23  10.14 1  thrum  18  16,732  Lake  Pinecrest  number  of p o l l e n grains removed from flower  13,396  (9,257)  8,037 (3,022)  Average percentage of p o l l e n grains removed from flower  64.7 ( 18.47) 70.8  (8.15)  (3,300)  62.8  (14.00)  (1,091)  90.4  (3.88)  o so > O so X  O  o o  o to  F L O R A L M O R P H O L O G Y / 21 there  was  thrums; length  a  pin  petals  difference  petal length  was  averaged  significant  only  14.1  16.2 mm  from  averaged mm  (p< 0.001)  in  16.1  (s.d. = 1.02), whereas thrum  mm  (s.d. = 1.13).  At  Lake  averaged  12.5  size  Pinecrest,  (s.d. = 1.21), and in thrums  Stump  petal  petal  15.8 mm  mm  in  between  pins  length  and petal  in  pins  long (s.d. = 0.83). Pin  length  (s.d. = 0.92),  while  homostyle petal length averaged 12.8 mm (s.d = 0.85).  The remaining three floral characters, ovary length, ovule number and the number Ovary  of  stigma  length  homostyles  lobes were  varied  within  most  significantly  populations,  variable between  but  this  at  the individual  pins  and  variation  level (Table  thrums  was  not  and  pins  consistent  2). and  among  populations (Table 1). Homostyle ovaries were larger than pin ovaries in flowers from  Stump  Lake  (pin:  2.4  mm,  s.d. = 0.36;  homostyle:  2.9  mm,  s.d. = 0.22;  p<0.001). At Pinecrest, thrum ovaries were significantly larger than pin ovaries (pin: 2.5 mm, s.d. = 0.41; thrum:  3.0 mm, s.d. = 0.21; p<0.001), but the reverse  was true at Beaver Lake (pin: 3.4 mm, s.d. = 0.44; thrum: p<0.001). Ovule number was significantly  2.9 mm, s.d. = 0.39;  different between pins and homostyles  at Stump Lake (p<0.001), and pins and thrums at Beaver Lake (p<0.001), but there was no significant difference in ovule number between forms at Pinecrest. In  those populations where there was a significant difference  ovule  number,  this  difference  Lake,  ovary  length  Stump averaged Beaver 37.4  17.2 Lake,  (s.d. = 5.93) ovary  (s.d. = 3 3.38)  length  for  pins,  corresponded was  for  greater  pins,  and  was greater and  to  30.2  differences  in  between forms  in  ovary  At  for  homostyles  and  20.1  (s.d. = 5.43)  for  for  pins, and ovule  (s.d. = 12.88)  for  length.  ovule  number  homostyles. number  thrums.  There  At  averaged was  no  F L O R A L M O R P H O L O G Y / 22 significant difference between pins and thrums  or between populations in stigma  lobe number.  While  the  number of flowers per  inflorescence did not  vary  significantly  between morphs, a total of 62% of the variation in the number of flowers per inflorescence Plants  at  number  was  accounted  Stump  for  Lake had  averaged 28.5  by  the  differences  most  flowers  (s.d. = 3.46), and for  between per  populations  (Table  2).  pins  this  inflorescence, for  homostyles 27.2  (s.d. = 3.94). Plants  from Pinecrest had the fewest, for pins this number averaged 15.1 (s.d. = 2.87), and for thrums  15.2 (s.d. = 3.09). In general, it appeared that inflorescences from  Pinecrest had large flowers, few in number, while those from Stump Lake had smaller but more numerous flowers.  D. DISCUSSION  Distyly 1979).  is  Besides  known the  to  occur in  reciprocal  24  placement  distylous plants have several other  found  in  distylous  of  of  anthers  flowering and  plants  stigmas,  (Ganders  flowers  of  morphological characters in common. Flowers  of Menyanthes trifoliata share some, but commonly  families  not  all of these characteristics. As is  plants, corollas of Menyanthes are  salverform and sympetalous, with a short floral tube  small,  regular,  (Ganders 1979). In  some  distylous species, thrums have a larger corolla than pins (Ganders 1979), but in Menyanthes, petal length varied more between populations than between forms. the  one  population  (Beaver  Lake)  where  there  between the petal length of pins and thrums,  was  a  significant  In  difference  the pins had longer petals. The  F L O R A L M O R P H O L O G Y / 23 present study confirms Darwin's (1877) report that in Menyanthes thrum anthers are  larger  several  than  other  (Ornduff  pin  anthers.  distylous  1966,  species  1982; Ganders  Larger  thrum  including 1979).  anther  other  size  members  However,  in  has been reported of  the  in  Menyanthaceae  Menyanthes interpopulational  variation in anther size is greater than intermorph variation.  The pollen size dimorphism in Menyanthes was consistent with that found in other distylous species; pin pollen was smaller in diameter than thrum pollen. The  pollen  diameters  for  pins  and  thrums  of  Menyanthes determined  in  the  present study (pin = 41.5 urn, s.d. = 2.69; thrum = 48.9 um, s.d. = 3.27) were similar to  those  s.d. = 0.57).  given In  by  Nilsson  (1973)  (pin = 33.5  agreement with Darwin's  jum, s.d. = 0.54;  (1877)  findings,  thrum = 40.4 Mm,  results  of the  present  study showed that thrum pollen was an average of 1.2 times larger in diameter than pin pollen. This is in contrast to the much larger sizes reported by Knuth (1899)  and later quoted by Hewett (1964)  (pin = 26x50 um,  thrum = 63xl20 Mm).  According to Knuth's measurements, thrum pollen was 2.4 times larger than pin pollen.  Reasons  for  the  differences  between  Knuth's  (1899)  measurements  of  pollen sizes and those found in the present study are not known.  In Menyanthes, the number of pollen grains produced by pins and thrums varied  considerably  among  thrum pollen production  populations.  The  at Beaver Lake was  ratio  of  pin  pollen  production  1.64 while at Pinecrest the  to  ratio  was 0.86. A t Stump Lake, the ratio of pin pollen production to homostyle pollen production was 1.78. In most distylous species studied, pins produced more pollen than thrums,  and this  difference  is correlated with a difference  in pollen size  F L O R A L M O R P H O L O G Y / 24 (Ganders  1979). Linum perenne (Nicholls  1986)  and  two  species  (Ganders  1979) are the only known distylous taxa besides Menyanthes in which  higher thrum pollen production has been reported. Because of the  of  Amsinckia  interpopulational  variation in pollen production in Menyanthes it is difficult to correlate differences in pollen production between pins and thrums  with any developmental features.  In Menyanthes, variation in pollen production does not correspond well to variation in anther size or pollen size.  In  those distylous taxa in which pollen removal by pollinators has been  measured, a greater number of pollen grains were removed from thrum anthers than  pins  anthers  (Ornduff  1980a,  1980b).  Ornduff  suggested  because thrum anthers were more accessible to pollinators Menyanthes, the varied  number  that  this  than pin anthers.  of pollen grains removed from pin and thrum  considerably between  populations.  At  was  Pinecrest, thrum  flowers  In  anthers  had  1.65  times more pollen grains removed than did pin flowers, but at Beaver Lake, pin flowers had 1.67 times more of their pollen removed than did thrum flowers. So that in Menyanthes, anther the  number  of  pollen  removal  Beaver  Lake  pollen did  and  accessibility may  grains  removed  by  not have been a factor pollinators.  correspond to intermorph Pinecrest, the  morph  variation  which  Intermorph in  pollen  produced more  influencing  variation  in  production.  At  pollen  also  had  more pollen removed by pollinators.  While dimorphisms characters  flowers  of  characteristic variation  Menyanthes of  between  the  trifoliata  distylous  populations  exhibited  many  of  the  breeding system, in ' some of was  greater  than  variation  floral these  between  FLORAL MORPHOLOGY / 25 morphs.  By  far  the most  significant  example  of interpopulational variation in  floral morphology in Menyanthes is the variation in style length found in flowers from Stump Lake. At Beaver Lake and Pinecrest, there is a strong reciprocal relationship between style length and stamen length in pins and thrums. In these two  populations, stigmas of one morph are at the same level as the middle of  the anther sacs of the opposite morph. While pins from Stump Lake exhibit a similar degree of separation of their stigmas and anthers as is found in flowers from other populations, the rest of the flowers from Stump Lake appear to be long homostyles. Their stamens are the same length as the pin styles, but the homostyle styles are longer than those expected  in the thrum form. Homostyle  stigmas are at the same level as their own anther sacs.  Interpopulational  variation in style  length (and  been found in thrums from three species of Villarsia,  stamen  length) has  another distylous genus in  the Menyanthaceae (Ornduff 1986). Three populations of V. parnassiifolia, V.  capitata  populations  and  two of  pins appeared  V. lasiosperma were normal. In six  also  studied,  and  in each  two of  of these  of these populations, stamens of the  other floral form (referred to as "thrums" by Ornduff) were shorter than the pin styles and at the same level as  or longer than pin stamens. Style length of  these thrums varied amoung populations from above the distal tip of their own stamens, to below the proximal tip. In four populations (three of V. parnassiifolia and  one of V. capitata), the thrums appeared  to be short homostyles. Only in  one population (V. lasiosperma), was there a reciprocal relationship between style and  stamen length in the two floral forms. Ornduff (1986) concluded that in the  populations of Villarsia  that he studied, thrum flowers  were highly variable in  FLORAL MORPHOLOGY / 26 both  style and stamen length. He suggested  homostylous  were  merely  a result  that those flowers that  of morphological  appeared  variation, but offered no  evidence to support this.  As reviewed by Ganders  (1979),  most homostyles  in distylous species are  a result of a crossover in the supergene controlling the compatibility reaction and the stamen-style length polymorphism. Such a crossover results in flowers with the pistil  of one form  genetic homostyles  and the stamens of the opposite form,  are self-compatible. Self-incompatible homostyles  so that true are very rare,  but two examples have been reported. In both cases, the homostylous appearance has  been  attributed  to morphological  variation,  either  elongated  pin stamens  {Primula farinosa) or elongated thrum styles (Mitchella repens).  In  order  to determine  whether  a  homostyle  is a  result  of genetic  recombination or morphological variation, one must self-pollinate the homostyle to test for self-compatibility. Unfortunately this has not been done for Menyanthes homostyles seem  from  to have  Stump Lake. However as in Villarsia, a  great  capacity  for morphological  flowers of Menyanthes  variation.  In  subsequent  chapters, evidence is presented to support the view that the homostyles at Stump Lake are not true genetic homostyles, but are a result of morphological variation, that is, they are thrums with elongated styles.  The discovery of a homostylous form of Menyanthes at Stump Lake is the first  report  homostyles  of homostyly  in this  species since Warming's  in Greenland. Whether the homostyles  (1886) report of  found by Warming were true  FLORAL MORPHOLOGY / 27 genetic homost^des, or simply a result of morphological variation  is not known.  However the morphological variation in style length in flowers from Stump Lake is a significant discovery, because it provides an (1877) theory  that morphological  distyly  opportunity to test Darwin's  increases the proportion of compatible  pollen received by a diallelic self-incompatible plant. If one assumes that the only difference between the Stump Lake homostyles and thrums from other populations is the length of their styles, any difference in the proportion of compatible pollen received by style length.  thrums and  homostyles must be a result of the variation  in their  III. E X P E R I M E N T A L POLLINATIONS  A. INTRODUCTION  This pollination chamber.  chapter  describes  programs The  first  using was  the  results  Menyanthes  to  determine  from  two  trifoliata whether  types  plants a  of  grown  experimental in  a  self-incompatibility  growth  system  is  associated with morphological distyly in Menyanthes trifoliata. The second was to quantify the relationship between the number of compatible, pollen grains deposited on a stigma (pollination intensity) and the number of seeds set.  Distylous  plants  typically  have  a  relatively  inefficient  self-incompatibility  mechanism controlled by two alleles at one locus, which is genetically linked with the loci controlling the floral pofymorphisms (Ganders 1979). The strength of the incompatibilty  reaction  self-compatible  taxa  pyramidata;  and  been reported  all  varies  are  in  known:  different  distylous  species,  and  even  some  some species of Oxalis and Hedyotis, Melochia  species of Amsinckia.  More  recently,  self-compatibility  has  in distylous Salvia brandegei (Neisess 1984) and Cryptantha flava  (Casper 1985).  Data on self-incompatibility  systems are available for only two of the four  distylous genera in the Menyanthaceae: Nymphoides (Ornduff and  Villarsia  Nymphoides  (Ornduff  (Ornduff indica  1966)  and  1982).  (Ornduff  Villarsia  Controlled 1966;  crossing programs  Barrett  capitata (Ornduff  28  1980),  1982)  1966; Barrett have  Nymphoides  have  the  shown  1980) that  humboldtiana  same diallelic  E X P E R I M E N T A L P O L L I N A T I O N S / 29 self-incompatibility the  system found in other  self-incompatibility  substantial  system  seed set (Ornduff  morphological  characteristics  self-incompatibility  distylous plants. In Nymphoides peltata  was weak,  and illegitimate  1966). To date, of  distyly  in  crosses  no one has tested  Menyanthes are  resulted whether  associated  system. The only evidence of self-incompatibility  in the  with  a  in Menyanthes  comes from Darwin's (1877) report of a population of Menyanthes which consisted entirely  of thrums. In this  contain  any viable  that  seed.  population, the capsules expanded, but they  Darwin  (1877)  Menyanthes was self-incompatible.  suggested that In  the  present  this  did not  was an indication  study  a  experimental  pollination program was designed to test for the presence of a self-incompatibility system in Menyanthes trifoliata.  In another  order  to  further  our understanding  aspect of the pollination/fertilization  relationship between pollination  intensity  of breeding  systems  process warrants  in  plants,  investigation. The  (the number of compatible pollen grains  deposited on a stigma) and seed set is an aspect of the reproductive biology of plants that has been virtually to quantify  this relationship  1978;  Bertin  1978; Snow  Shore  and Barrett  ignored. Only recently have attempts  been made  (Akamine and Girolami 1959; Silander and Primack 1982; McDade  1984; Guth  and Weller  1983; Schemske and Fenster 1983; 1986). These studies indicate  that  there may not always be a 1:1 relationship between the number of compatible pollen grains received and the number  of seeds set, and this  relationship may  vary among species.  For the best results, the relationship between pollination intensity and seed  E X P E R I M E N T A L P O L L I N A T I O N S / 30 set  should  be  determined  under  field  conditions.  Ideally,  seed set  should  be  measured in the same flowers in which the number of compatible pollen grains received has also been determined. McDade (1983) used a hand lens to count the number  of  pollen  grains  deposited  on  stigmas  of  day  non-heterostylous species Tricanthera gigantea then later set,  old  flowers  of  the  measured fruit and seed  but only pollen from stigmas with fewer than eight pollen grains could be  reliably counted. This method is not very practical in distylous plants. In  order  to distinguish between the compatible and incompatible pollen grains deposited on a stigma, the stigma must be removed and its pollen counted under a compound microscope. But once the stigma and its pollen have been removed, so are the chances of effecting fertilization pollinate  flowers  determine  with  seed set.  a  and seed set. A n alternative  known  This  number  method  was  of  compatible  used by  Snow  method is to hand  pollen  grains  (1982)  on  and  field  then grown  Passiflora uitifolia, but she was unable to determine the exact number of pollen grains  applied to  stigmas, so results  were  only  approximate.  This  method  is  performed more easily under the controlled conditions of a pollinator free growth chamber Barrett  or  greenhouse,  and  this  was  the  procedure  followed  by  Shore  and  (1984), Guth and Weller (1986) and in the present study. Results from  these experimental pollinations provide an estimate of how many compatible pollen grains  must  be  deposited  on  a  stigma  in  order  to  fertilize  each  ovule  in  Menyanthes trifoliata. These results serve as a basis for an investigation into the relationship  between  the  number  of  compatible  pollen  grains  received and  number of seeds set in natural populations, of Menyanthes . (Chapter 5).  the  E X P E R I M E N T A L P O L L I N A T I O N S / 31 B. MATERIALS AND  Pin  METHODS  and thrum  of Menyanthes  plants  trifoliata  transplanted  from  Beaver  Lake, Lost Lake and Pinecrest (Figure 3), were potted and placed in trays full of water  in pollinator-free  growth  chambers (Figure  6). Four different  types  of  experimental pollinations were performed. A total of 276 flowers were pollinated; 118 flowers were  were  pollinated  (geitonogamous  selfed with pollen from with  pollen  pollination).  from  other  Fifty-four  their  own anthers, while  flowers  intra-morph  on  the  same  cross  71  inflorescence  pollinations  (between  different plants of the same form) and 33 inter-morph cross pollinations flowers of different  forms)  were made. Nine pin plants  and four  flowers  (between  thrum  plants  were recipients of all four crossing regimes.  Pollen dehisced  was  anthers  transferred from  the  by  pollen  removing, donor  and  with  jeweller's  rubbing  the  forceps,  anther  freshly  across  the  recipient stigma until all visible traces of the pollen had been transferred. Fruits were allowed to mature, but harvested before dehiscence in order to count how many seeds had been set.  Seventy-eight number the  experimental  pollinations  were  made  in  which  the  of compatible pollen grains was determined before transferring  recipient  stigma. First,  a random numbers table was  used to  exact  them  to  select some  number of compatible pollen grains between 1 and 99. Then, a dehisced anther from ' a donor plant was removed with jeweller's forceps and brushed against a glass slide to deposit its pollen on the slide. Under a dissecting microscope, a  EXPERIMENTAL POLLINATIONS / 32 FIGURE 6. Pin and thrum plants of Menyanthes trifoliata growing in a growth chamber.  EXPERIMENTAL POLLINATIONS / 33 needle was used to count out the required number of pollen grains and then to transfer them  to the stigma of the recipient plant (Shore and Barrett  1984).  Fruits were allowed to mature and seed set was determined. A total of 29 pin flowers (on 12 separate plants), and 35 thrum flowers (on 11 separate plants) were used as pollen recipients.  C. RESULTS  A  considerable amount of variation was noted in the fertility of individual  plants of Menyanthes Some plants failed applied  to their  used  for both  of the experimental pollination programs.  to set seed regardless of the amount of compatible pollen  stigmas,  and these  plants  were  excluded  from  the results.  Reasons for this variation in fertility are not known.  The  controlled  that Menyanthes  crossing program  trifoliata  provided the first experimental evidence  possesses the same type of self-incompatibility system as  other distylous plants. While self, geitonogamous and intra-morph crosses resulted in less than 2% seed set and averaged and  thrums  alwa3 s r  resulted  0.75% (s.d. = 0.78), crosses between pins  in substantial  seed  production averaging 43.7%  (s.d. = 12.9) (Table 4).  Observations of seed set in natural rnonomorphic  populations of Menyanthes  support these results. Two populations which consisted only of thrums located at Strachan Meadow and at Pinecrest, and one composed entirely of morphological homostyles located at Stump Lake (Figure 3), all had seed set lower than 4%  TABLE 4. Seed s e t f o l l o w i n g s e l f , ge1tonogamous, intramorph and Intermorph p o l l i n a t i o n of p i n s and thrums of Menyanthes trifoliata. P l a n t s were grown from c u t t i n g s c o l l e c t e d from Beaver Lake, P i n e c r e s t and Lost Lake ( F i g u r e 3 shows locations). All pollinations were performed in a growth chamber. Values a r e the mean and standard d e v i a t i o n ( i n parentheses ) .  Average Number Type  of  of  p o l 1 i n a t ion  flowers  pol11nated  number of seeds set p e r pol1inatIon  Average percentage seed set per pol11nat1on  Intrap 1 ant pin  se 1 f  thrum pin  self geitonogamous  thrum  geitonogamous  83  0.30  (0.94)  1.30 (4.36)  35  0.03  (0.16)  0.09  (0.53)  40  0.47  (1.50)  2.00  (6.16)  41  0.09  (0.53)  0.36  (1.99)  Interplant  X  W S> H  Intramorph  H >  pin x p i n thrum x  thrum  45  0. 16 (0.56) 0  9  0.73  (2.60) 0  >  Intermorph pin x  *d o r  thrum  23  13.80  thrum x p i n  10  8.00  (8.95) (6.21)  52.80 (29.47)  O w  34.50 (28.50) CO  E X P E R I M E N T A L P O L L I N A T I O N S / 35 (Chapter 5, Table 15). A t Strachan Meadows in 1983, the capsules expanded to at least one half the size of mature fruits, but contained only unfertilized ovules. In the same population in 1985, the capsules did not expand to such an extent and were  heavily  infested  with  aphids. Average .seed set for  1985 was  0.01  seeds/capsule (s.d. = 0.20).  Results from pollination intensity experiments show that in Menyanthes, an increase  in  the  number  of  compatible  pollen  grains  received  by  a  stigma  generally resulted in an increase in the number of seeds set, but there was a considerable amount of variation in seed set at each level of pollination (Figure  7).  The  number  of  seeds set  was  always  less than  the  intensity  number  of  compatible pollen grains received.  In nine flowers (on separate plants) very high levels of compatible pollen resulted in very fertilized after  low seed set. For example, in one flower only one ovule was  98 compatible pollen grains were deposited on its  stigma. These  flowers were not excluded from the dataset because other flowers on the same plants  had  infertile,  much  higher  seed  fertility,  indicating  that  the  plants  were  not  but were capable of seed production. Inclusion of these flowers in  the  data set doubled the calculated average number of pollen grains required to set each  seed.  (including  The the  average  nine  number  flowers:  of  n = 78)  pollen was  grains 13.5  required  (s.d. = 19.82),  to  set  each seed  compared  to  an  average of 7.7 pollen grains per seed set (s.d = 7.45) when the results from those nine flowers were excluded (n = 69). The median number of pollen grains per seed set gives a more accurate indication of the relationship between pollination  FIGURE 7. The e f f e c t s of p o l l i n a t i o n I n t e n s i t y on seed set in pins and thrums of Menyanthes trifoliata. Individual pin f l o w e r s are represented as c l o s e d c i r c l e s , thrum flowers as open c i r c l e s . Average number of ovules per ovary i s 25.3 (s.d.=7.55). Broken l i n e represents the least squares r e g r e s s i o n of seed set on p o l l i n a t i o n i n t e n s i t y for a l l datapoints. C o r r e l a t i o n : r=0.37, p<0.01. S o l i d l i n e represents the l e a s t squares regression excluding nine d a t a p o i n t s r e p r e s e n t 11ng f l o w e r s with extremely low seed f e r t i l i t y (see text f o r e x p l a n a t i o n ) . C o r r e l a t i o n : r=0.57. p<0.01.  50 6 0 70 8 0 Number of Compatible Pollen G r a i n s Deposited on S t i g m a s  9 0 100  >  o z  CO  E X P E R I M E N T A L P O L L I N A T I O N S / 37 intensity  and  seed  set  in  Menyanthes than  the  mean  number.  The  median  number of compatible pollen grains required to fertilize each ovule was 6.0 with the nine low seed set flowers included in the dataset, and 5.0 when they  were  excluded. The small difference between the two medians indicates that the results from the  nine  flowers  with  very  low  seed set were  whole data set. Results from pins and thrums  not  characteristic  were similar; for  of  the  pins 6.0 was  the median number (n = 37) of compatible pollen grains required to set each seed, while for thrums the median was 5.3 (n = 41). The number of compatible pollen grains applied to stigmas ranged from 1-99, but the lowest number of compatible pollen grains that resulted in seed set was three.  Two relationship resultant  linear between  regressions the  number  were of  performed compatible  in pollen  seed set. Regression lines from the two  order  to  evaluate  grains  received and  the the  analyses are shown in Figure  7: the broken line represents the regression of seed set on pollination  intensity  for all data points (n = 73, r = 0.37, p<0.01), while the solid line represents the regression of all data points except the nine flowers that had very low seed set (n = 69,  r = 0.57,  p<0.01).  Results from  these  analyses show  that  there  is  a  significant correlation between pollination intensity and seed set. The correlation is not especially high, which underscores the great variation in the results.  D.  DISCUSSION  As in other distylous members of the Menyanthaceae, morphological distyly is  associated  with  a  self-incompatibility  system  in  Menyanthes trifoliata.  In  E X P E R I M E N T A L P O L L I N A T I O N S / 38 Menyanthes, seed set levels for similar  intra-morph  and inter-morph  to those found for Nymphoides humboldtiana (Ornduff  indica (Barrett using  self,  1980) and Villarsia capitata (Ornduff  own-form  thrums.  In  pollen  resulted  inter-morph  in  less  pollinations  than  pins  crosses were  1966), Nymphiodes  1982). In Menyanthes, crosses  2% seed set  tended  to  in  set  both  more  pins seed  and (13.8  seeds/capsule, s.d. = 8.95) than thrums (8.0 seeds/capsule, s.d. = 6.21).  In thrums  most  distylous  following  Primula  spp.  legitimate  (Dowrick  ulmifolia (Barrett  species studied,  seed set  hand-pollinations  1956),  was  (Barrett  Eichhornia  similar  1980).  crassipes  1978), and Nymphoides indica (Barrett  In  for a  (Barrett  pins  few  and  species,  1977),  Turnera  1980), seed set following  hand-pollination was lower in pins. Dowrick (1956) attributed lower pin seed set in Primula  obconica to the smaller area of conducting tissue available for pollen  tube growth.  contrast to this, reports of lower seed set in thrums  following  hand-pollinations  in  Villarsia  capitata (Ornduff  1982) and in Menyanthes trifoliata in the present study indicate  that  the  In  area  of  Nymphoides  stylar  indica  conducting  (Reddy  tissue  and  is  Bahadur  probably  1976),  not  a  major  factor  influencing seed set in these species.  In pollination  Menyanthes both  pin  was  the  less  than  and  thrum  potential  pollinations, only eight resulted in greater  seed  maximum.  set  following  Out  of  33  inter-morph inter-morph  than 80% seed set. This may be a  result of resource availability, if the plants grown in growth chambers were not as  healthy  Variation  as among  those  in  natural  individuals  in  populations seed  and/or  more pollen  ovules  may  fertility  have  may  also  aborted. be  a  E X P E R I M E N T A L P O L L I N A T I O N S / 39 contributing  factor.  Variation  ulmifolia (Shore and Barrett seed regardless of  how  in  fertility  was  found  in  hand-pollinated  Turnera  1984). As in Menyanthes, some plants failed to set  much  compatible  pollen  was  applied to  their  stigmas.  Barrett (1980) was able to demonstrate significant difference among individuals of Nymphoides indica in average seed fertility, but no differences in pollen fertility were apparent. More data are necessary before a similar analysis of variation in seed and pollen fertility can be performed in Menyanthes.  The observation of expanded capsules in the monomorphic thrum population at Strachan Meadow is very puzzling. Darwin in  a monomorphic  thrum population  located at  (1877) Kew  made similar observations Gardens. Darwin  reported  that fruit set (i.e., expanded capsules) occurred without the production of viable seed. In  most natural populations of Menyanthes that I observed, fruit set does  not occur without seed set, however parthenocarpy does occur in another distylous member of the  Menyanthaceae, Fauria  crista-galli (Griffiths  and Ganders 1983).  Why  it would occur only in some populations of Menyanthes and not in others,  and  vary  among  years  is  not  understood.  In  Menyanthes, caution  should  exercised when fruit set is used as an indication of successful fertilization;  be fruit  set should be measured in conjunction with seed set.  Results  from the  pollination  show that there was not a  intensity  experiments in Menyanthes trifoliata  1:1 relationship between the  number  of compatible  pollinations received and seed set. The number of seeds set was always lower than the number of compatible pollen grains received, and the median number of compatible pollen grains required to fertilize each ovule was 6.0. Similar results  E X P E R I M E N T A L P O L L I N A T I O N S / 40 were  found  for  distylous  Turnera  ulmifolia  ulmifolia, there was a considerable amount set at each level of pollination the  number  of compatible  pollinations  set was always below the compatible  pollen  intensity,  grains  and  of variation but  Barrett  1984).  each  T.  in general, seed set increased as  maximum, and it  fertilize  In  in the number of seeds  increased (Shore and Barrett  potential to  (Shore  ovule  1984).  Seed  took an average of  (Shore  and  Barrett  2-7  1984).  Similarily, in Tricanthera gigantea, at least eight pollen grains were necessary for fruit and seed set (McDade 1983).  Other  studies  have  (Akamine  and  Girolami  Passiflora  uitifolia (Snow  shown  of  pollen  pollination intensity.  for  several  species  1959), Oenothera fruticosa (Silander  number  grains  of compatible  effecting  fertilization  1978),  and Weller  1986)},  pollinations decreased  {Passiflora edulis  and Primack  1982), and Oxalis magnifica (Guth  seed set increased as the efficiency  that  increased, but at  higher  In Oenothera fruiticosa there was close to a 1:1  the  levels  of  relationship  of compatible pollen grains to seed set when the number of pollen grains applied to  stigmas  (Silander  was and  below  one  Primack  third  1978).  magnifica when pollination  the A  number  similar  required  for  relationship  was  was  Girolami  required  (1959)  to  set each seed (Guth  reported  a  similar  edulis; at low pollination intensities pollen  grains  were  required  (1776 pollen grains/stigma)  found  seed set in  Oxalis  levels were below 35 pollen grains per stigma  and Weller 1986). A t higher levels of pollination intensity, grain  maximum  for  decrease  and in  Weller pollen  more than one pollen 1986).  efficiency  (189 pollen grains/stigma)  each fertilized  ovule, but  (Guth  Akamine  and  for Passiflora  an average of 2.2 at  higher  intensities  an average of 7.3 pollen grains were required.  With  E X P E R I M E N T A L P O L L I N A T I O N S / 41 an  average of  350 ovules per  flower,  maximum  even at the highest pollination intensity  seed set was never achieved  levels (Akamine and Girolami 1959). In  Passiflora vitifolia an average of 1.6 pollen grains were required to set each seed when the number of compatible pollen grains per stigma was less than 100, but more pollen was required at higher pollination intensity levels (Snow 1982).  Because  so  make  few  studies  of  pollination  any  generalizations  difficult  of  number  of compatible pollen grains  intensity  concerning  the  have  been  done,  relationship  it  between  a stigma receives and the number  is the  of seed  set. Results from the few published studies and from Menyanthes show that the number of compatible pollen grains required for each fertilized ovule varies among species  and  between pollen  is  frequently  compatible grains  intensity.  This  pollen  effecting is not  not  a  grains  1:1  In  some  and seed set is not  fertilization particularily  ratio.  decreases  at  species the linear, the  higher  levels  surprising, because competition  relationship efficiencj'  of  of  pollination  among pollen  grains might be expected at higher pollination intensities. More work needs to be done  to  provide  a better  intensity and seed set.  understanding  of  the  relationship  between  pollination  IV. P O L L E N  FLOW  A. INTRODUCTION  In  1877 Darwin  compatible  pollen  hypothesized that distyly  deposited  on  stigmas  anthers and stigmas in the two the  pollinator's  body  where  should increase the amount  because of  floral  placement  of  forms. He suggested that the portion  of  pollen from  one floral  the  reciprocal  of  morph  was deposited  would  come in contact with the stigma of the opposite floral morph. This would ensure that pins were pollinated with thrum pollen and that thrums were pollinated with pin pollen (disassortative  pollination).  Few studies of pollen flow in distylous species have been able to this  hypothesis. Studies have shown that stigmas of distylous plants  support  have  high  levels of incompatible pollen deposited on them, and that pollen flow between pins and thrums  is asymmetric  pin  receive more  stigmas  (Ganders pollen  stigmas (Ganders 1979; Ornduff 1983;  Schou  1983; Rama  receive  more  incompatible  1979;  Ornduff  1980a,  (pin  and  general, it  thrum  pollen  has been found combined)  than  that thrum  1980a, 1980b; Weller 1980; Lewis 1982; Casper  Swamy pollen  1979). In  and  than  1980b; Weller  Bir  Bahadur  compatible  1984),  pollen  1980; Lewis  that  (Ganders  1982;  Schou  pin  stigmas  1979; Oleson 1983; Nicholls  1985), and that thrum stigmas receive more compatible than incompatible pollen (Ganders 1979; Ornduff  However,  1980a, 1980b; Lewis 1982; Schou 1983).  examination  of  total  stigmatic  42  pollen  loads  is  not  the  most  P O L L E N F L O W / 43 effective method of testing whether flowers are disassortatively pollinated (Ganders 1974). This is because the intraflower  selfing component of stigmatic pollen loads  can mask any disassortative pollen flow by creating an excess of own-form pollen on the stigma. Distyly does not affect the amount of intraflower  selfing a flower  undergoes,  but through  the  own-form  pollen  opposite-form  disassortative  derived  pollen  from  received.  pollination  other With  should  flowers,  and  reduce  increase  100% efficiency  and  the  amount  of  amount  of  intraflower  selfing  excluded, all the pollen on stigmas should be of the opposite form. With random pollination  (expected if distyly  has no effect  on pollen flow),  the composition of  stigmatic pollen loads would be the same as the frequency of pollen produced in the  population.  The intraflower  component  of a  stigmatic  pollen  load  can be  eliminated through emasculation. By comparing observed pollen loads from stigmas of emasculated flowers with expected pollen loads based on random pollination, it is  possible  Ganders  to  determine  (1974)  demonstrate  is  whether  one of  significant  the  levels  flowers few  are being  researchers  of disassortative  disassortatively  who  pollination  used in  this  pollinated. method  a distylous  to  plant  {Jepsonia heterandra).  While pollination  in  Ganders' a  (1974)  distylous  study  species,  documented it  did not  the occurrence of disassortative test  whether  the  disassortative  pollination was a result of the stamen/style length dimorphism. However, Ganders (1979) was able to demonstrate that in a population of Lithospermum californicum small differences in the degree of separation between the stigmas and anthers of pin  flowers  influenced  the  proportion  of  compatible  pollen  they  received. Pin  flowers with a larger spatial separation between stigmas and anthers received a  P O L L E N F L O W / 44 higher percentage of compatible pollen than the other pin flowers.  Ideally,  the  relationship  between  morphological  distyly  and  disassortative  pollination could be tested by comparing the composition of stigmatic pollen loads of distylous flowers, with the composition of pollen loads from otherwise identical diallelic  self-incompatible  Darwin  hypothesized (1877),  compatible  pollen  flowers  lacking  the  stamen/style  morphological distyly  received by  a diallelic  dimorphism.  does increase the  self-incompatible  plant,  If,  amount  as of  then stigmas of  distylous. flowers would be expected to receive a greater proportion of compatible pollen  than  the  stigmas  of  the  homomorphic  The r- occurrence  flowers.  unusual population of Menyanthes trifoliata consisting of pins and homostyles provided an opportunity length  of their  from  other  to test  styles, these homostyles of  Menyanthes.  pin/homostyle  population  to  population, it  was possible to investigate  pollen  By  flow  in  morphologically  comparing a  pollen  similarily  the effect  an  (morphological)  Darwin's hypothesis. Except for  are identical  populations  of  to  thrums  flow  structured  the  in  the  pin/thrum  of morphological distyly  on  the composition of stigmatic pollen loads.  Most populations  studies in  which  of  pollen  the  flow  two  in  floral  distylous  morphs  species  occur  in  have a  1:1  been ratio,  based on and  are  randomly distributed in the population. The previously described asymmetric pollen flow patterns  are typical of such populations. Because of the  clonal nature  of  Menyanthes, there is a considerable amount of variation in floral morph frequency among  populations.  Menyanthes  were  Pollen compared  flow in  patterns order  to  from  six  investigate  different the  populations  influence  of  of  morph  POLLEN FLOW / 45 frequency  on the composition  of stigmatic  populations studied, the morph frequency  pollen loads. In four  of the  six  deviated significantly from the expected  1:1 ratio.  In previous  studies of pollen flow  in distylous species, populations were  sampled on one date during the flowering season. This approach assumes that there  is no seasonal  seasonal  changes  variation  in pollen flow. Only  in pollen flow  one study  has measured  in a distylous species. In Primula  vulgaris,  stigmatic pollen loads were collected on two dates, one at the beginning of the flowering season, the other at the end (Ornduff 1979). Stigmas collected at the end of the season had twice as many pollen grains deposited on them as those collected  at the beginning  of the season. The relative proportions of pin  and  thrum pollen deposited on pin stigmas was the same at the beginning and end of the flowering season, but on thrum stigmas there was a threefold increase in the  proportion of pin pollen received between  results indicate that in populations of Primula of stigmatic pollen loads are not constant variation Menyanthes  in the size  and/or  composition  the two collection dates. These vulgaris,  over  the size and composition  the flowering season. Seasonal  of the stigmatic  was examined in six populations of Menyanthes  pollen  loads of  by collecting stigmas  at regular intervals during the flowering season.  B. MATERIALS AND  METHODS  In the spring and summer  of 1985, samples of stigmatic pollen loads  were collected from flowers of Menyanthes  trifoliata  at 2-3 day intervals during  P O L L E N F L O W / 46 the  flowering  within the  season. Stigmas were collected from six study  three  populations described in  Chapter 2. Three  areas, all located areas were chosen  from Beaver Lake and are referred to here as B L 2 , B L 3 and B L 4 (Figure 8). A t Pinecrest, two study sites were chosen and are designated as P C I and PC2 (Figure 9). One study SLl  (Figure  area was chosen at Stump Lake and is referred to as  10). Study areas B L 2 , B L 3 , B L 4 , S L l and P C I all represented a  subset of a larger population. Study area PC2 included all the inflorescences in a single  population  (Figure  11).  (Extensive  clonal growth  in  Menyanthes made  it  impossible to determine the number of genets in each population, so the number of inflorescences was counted instead.)  At  each  determined  and  study  site,  deviations  the  from  frequency the  of  the  expected isoplethic  two (1:1)  floral  morphs  morph  ratio  was were  analyzed using the G-statistic (Sokal and Rohlf 1981). A nearest neighbour count was performed and a 2x2 contingency table used to determine whether the floral  morphs  monomorphic proportion  were randomly clumps  (Pielou  distributed  among one another  or  two  segregated into  1961). Pollen frequencies were calculated from  of pin and thrum (or  homostyle)  the  pollen produced in each population,  adjusted for the frequency of pin and thrum (or homostyle) inflorscences growing in each study area (Ganders 1979).  During each visit to each study area, 40 stigmas per floral morph were collected from intact flowers using jeweller's forceps. Stigmas were collected from flowers  that  had  been  exposed  to  pollinators  for  2-3  days.  Stigmas  Menyanthes trifoliata flowers usually remain receptive for 3-4 days. The anthers  of  POLLEN FLOW / 47 FIGURE 8. Population of Menyanthes trifoliata at Beaver Lake in Stanley Park, Vancouver, B.C. Stippled area indicates extent of population. Shaded areas indicate study areas BL2, BL3 and BL4 where pollen flow and seed set data were collected.  POLLEN FLOW / 48 FIGURE 9. Populations of Menyanthes trifoliata located in the Pinecrest area, 0.9 km north of Daisy Lake canal on B.C. Hwy. 99. Stippled areas indicate extent of populations. Shaded areas indicate study areas PCI, PC2 and PC3.  POLLEN FLOW / 49 FIGURE 10. Population of Menyanthes trifoliata at Stump Lake in Alice Lake Provincial Park in southwestern B.C. Stippled area indicates extent of population. Shaded areas indicate study areas S L l and SL2. Study area SL2 was a small pond separated from Stump Lake by forest, and includes all plants in that population.  FIGURE 11. P o p u l a t i o n s of ponds were separated from where Menyanthes occurred. the p l a n t s growing i n one  /Menyanthes trifoliata l o c a t e d in small ponds i n the P i n e c r e s t area. In t h i s l o c a t i o n small each other by p o r t i o n s of a Sphagnum bog. S t i p p l e d area i n d i c a t e s the area i n each pond Shaded areas i n d i c a t e study areas PC1 and PC2. Study area PC 1 i n c l u d e a l a r g e p o r t i o n of pond. Study area PC2 included a l l the p l a n t s growing i n another pond.  O  P O L L E N F L O W / 51 are wilted  by  the  third  day  of  anthesis, and by  the  fourth  day  the  corolla  begins to wilt. Those flowers with wilted anthers but fresh corollas were chosen as flowers that had been exposed to  pollinators  for  three  days, ensuring that  stigmas were collected from flowers at the same stage of maturity. collected from flowers of plants selected by chance in the  Stigmas were  study area. Pin and  thrum (or homostyle) stigmas were collected separately and pooled for each floral morph.  Pollen  samples  from  stigmas  were  obtained  by  collections of stigmas and suspending the pollen in a 1:1 and glycerin (Erdtman  acetolyzing mixture  replicates of each sample were counted (Lloyd  pollen  classes  slightly,  the  point  of of  pooled  of lactophenol  1960). Pollen counts were made using a haemocytometer  and twenty size  the  the  two  minimum  floral  morphs  for  overlap between  each  the  1965). Because the population  two  size  overlapped  classes  of each  population was chosen as a cutoff point. For pollen from Beaver Lake the cutoff point was 43.0 jum, for Stump Lake 44.0 fim,  and for Pinecrest 45.0 um.  In  order to determine the number of pin and thrum (or homostyle) pollen grains in each sample of pooled stigmas, an image splitting microscope was set at the cutoff point. pollen grains, producing either  module  on a Vickers M22  The module sheared the image of the  double images  that  overlapped when  the  pollen  grain was larger than the cutoff point, or two discrete images when the pollen grain  was  smaller  than  the  cutoff  point.  The  number  of  larger  and smaller  pollen grains was determined for each sample and these numbers were used to estimate the number of pin and thrum (or homostyle) pollen grains present using the following formulae (Ganders 1976):  POLLEN FLOW / 52  T  = S - a (L +  S)  b - a and P =  L +  S - T  T=  number of thrum (or homostyle) pollen grains  P=  number of pin pollen grains  L=  number of pollen grains larger than the cutoff point  S=  number of pollen grains smaller than the cutoff point  a=  frequency of pin pollen grains smaller than the cutoff point in the measured pin pollen size distribution  b=  frequency of thrum  (or homostyle) pollen grains larger than the  cutoff point in the measured thrum pollen size distribution  Stigmas were also collected from emasculated flowers. Emasculations were performed  by  removing,  with jeweller's  forceps, all the  anthers  from  newly  opened flowers (Ganders 1974). Only those flowers with undehisced anthers were used. Flowers for emasculation were chosen by  chance, and  after emasculation  were tagged with the date, and their stigmas collected three days later. Stigmas were  processed  as  described  above, and  the  number  of pin and  thrum (or  homostyle) pollen grains determined using the same formulae.  Stigmatic counted on stigmas  pollen  loads from  both  intact  and  emasculated  a per flower basis. Pollen loads of pin and  were  treated  separately  for each  study  thrum  area. Because  flowers were (or homostyle) of the  slight  overlap in pollen sizes between floral morphs, estimates of the number of pin  P O L L E N F L O W / 53 and thrum (or homostyle) pollen grains deposited on stigmas may not be exact, but should provide a general indication of the proportion of the two pollen types present. Means were calculated for combined. Unless  otherwise  flowering  not  season,  to  individual  specified, results  collection dates and for refer  to  averages over  all dates the  whole  averages of individual collection dates. Mean stigmatic  pollen loads were compared to pollen frequencies for each study area in order to determine how the proportion of pin and thrum (or homostyle) pollen on stigmas compared to the proportion of pin and thrum (or homostyle) pollen produced in the  study  morph  area.  Pollen frequencies  frequencies, because they  were  used  for  this  represented a more  comparison  instead  accurate estimate  much pin and thrum (or homostyle) pollen was available for pollination  of  of how  in each  population.  C. RESULTS  The total number of inflorescences within each study area ranged from 86 (PC2) to 433 (BL3) (Table 5). Based on the results of tests using the G-statistic, only B L 3 and P C I had the expected isoplethic (1:1) ratio of floral morphs that is usually found in distylous plant areas  were  BL4,  to  anisoplethic 91:9%  populations  with morph  pins:thrums  at  ratios PC2.  (Table 5). The other  ranging Nearest  from  four  study  7:93% pins:thrums  neighbour  counts  and  at 2x2  contingency tables showed that B L 2 and B L 4 were the only two study sites in which four  pins  and thrums  were randomly  sites, B L 3 , S L l , P C I  clumps.  and  distributed  P C 2 , plants  (Table  were  5). In  grouped  into  the  remaining  monomorphic  TABLE 5. R e p r e s e n t a t i o n of f l o r a l morphs In s i x p o p u l a t i o n s of Menyanthes trifoliata in southwestern B r i t i s h Columb1 a . G v a l u e s i n d i c a t e d e v i a t i o n s from the expected 1:1 morph frequency. X' values i n d i c a t e d e v i a t i o n s from the random d i s t r i b u t i o n of f l o r a l morphs in the p o p u l a t i o n (based on nearest expected neighbour c o u n t s ) .  Inflorescences  in  population  F1ora1 Population  morph  Number  Total  BL2  p1n thrum  205 71  276  74 . 3 25.7  67 . 89  BL3  pin  218  433  50.0  thrum  215  BL4  pin thrum  14 197  211  PC 1  pin  100 89  189  thrum PC2  pin thrum  78 8  86  90. 7 9.3  SL 1  pin homostyle  212  289  73.4  77  Percentage  G  (df=1)  P  X  1  (df=3)  P  <0.001  O. 32  NS  0.03  NS  133.27  <0.001  6.6 93.4  189.49  <0.001  2 . 25  NS  52.9  0.53  NS  24.88  <0.001  63 . 74  <0.001  45 .06  <0.001  65.58  <0.001  30.91  <0.001  50.0  47 . 1  26.6  ,o  tw 1  O 3  P O L L E N F L O W / 55 Because the number of pollen grains produced per flower floral  morphs, the  frequencj'  of  pin  and thrum  (or  pin  varied between  and homostyle)  pollen  produced in each population was not exactly the same as the frequency of pin and  thrum  (or  pin  and  homostyle)  inflorescences  in  each  population.  Pollen  frequencies ranged from 8:92% pimthrum pollen (BL4) to 90:10% pimthrum pollen (PC2)  (Table 6). A t  pollen  than  thrum  sites B L 2 , B L 3 and PC2 there was significantly pollen  produced  in  the  frequency of pin pollen was significantly  population.  greater  Similarily,  at  more  pin  SLl  the  than that of homostyle pollen.  Site B L 4 was the only study area in which the frequency of thrum pollen was significantly  greater  than  the  frequency  of pin  pollen. A t  P C I , there  was no  significant difference in pin and thrum pollen frequencies.  Approximately  1% of the pollen produced in each population was deposited  on conspecific stigmas (Table  7). The proportions  pollen deposited on stigmas of flowers studied were very  of compatible and incompatible  of Menyanthes from the  similar to pollen frequencies in the population  six  populations  (Table 8).  At  B L 2 , B L 3 , B L 4 and P C 2 where there was a significant difference between floral morphs  in  the  percentage  of  compatible  pollen  received,  corresponded to differences in pollen frequencies in the population BL2,  BL3  (p< 0.001)  and  P C 2 , the  frequency  of  pin  pollen  was  this  difference  (Table 8).  significantly  than thrum pollen, and thrum stigmas received a significantly  (BL2: p<0.01,  B L 3 p<0.01,  At  greater higher  PC2 p<0.05) percentage of compatible pollen than  pins (Table 8). Similarly, at B L 4 the frequency of thrum pollen in the population was  significantly  greater  (p< 0.001),  and  pins  received  a  significantly  higher  (p<0.01) percentage of compatible pollen than thrums. A t P C I , the frequency of  TABLE 6. Frequency of p i n , thrum and homostyle p o l l e n produced In s i x p o p u l a t i o n s of Menyanthes trifoliata. Pollen frequencies were c a l c u l a t e d from the p r o d u c t i o n of p i n and thrum (or p i n and homostyle) p o l l e n a d j u s t e d f o r the frequency of the two f l o r a l morphs i n the p o p u l a t i o n . G values I n d i c a t e d e v i a t i o n s from a 1:1 p o l l e n frequency In the p o p u l a t i o n . P o l l e n p r o d u c t i o n : N=20 flowers per f l o r a l morph i n each p o p u l a t i o n .  Popu1 at ion  Floral morph  Pol 1 en product 1 on per flower  BL2  pin  28.282  thrum  14,980  p1n  16,266 (3452) 8,898 (2451)  BL3  thrum  Percentage of pol1 en produced in anthers  65 . 4 (676)  34 .6 66 . 7 33.. 3  Morph frequency in popu1 at 1 on  Pol 1 en frequency in popu1 at 1 on  G (df=1)  P  52.37  <0 .001  0..667 0.. 333  1 1 .38  <0. .001  42 .67  <0. ,001  0 .743 0 . 257  0 .845  0.. 500 0..500  0.. 155  pin  11, 1 10  53 .4  9,693 (2168)  46 .6  0..066 0,,934  0..075  thrum PC 1  pin thrum  14,732 (1688) 17,149 (885)  46. 2 53 8  0. 529 0..471  0..491 0.. 509  0.38  PC2  pin  17,153  46.. 2 53 .8  0. 893 0,. 107  <0. 001  19,986  0..907 0. 093  70.57  thrum pin homosty1e  17,783 9,990 (1754)  64 ..0 36 ..0  0..734 0. 266  0. 831 0.. 169  46.62  <0 .001  BL4  SL1  0,.925 NS  o  3  TABLE 7. Percentage of p i n and thrum p o p u l a t i o n s of Menyanthes trifoliata.  (or homostyle) p o l l e n  participating  Percentage p1n pol1 en part 1 d p a t ing in compatible pol1 inat ions  Populat ion  In  compatible p o l l i n a t i o n s  1n s i x  Percentage thrum po11 en participating In compatible p o l 1 i n a t ions  BL2  0.46  0. 75  BL3  0.97  1.12  BL4  0. 70  3.22  PC 1  0.58  0.40  PC2  0.41  0. 30  SL 1  0. 1 1  0. 19*  0.50  0. 99  X for a l l study areas comb 1ned * percentage pollen  Tl O  tr  1  homostyle  O  3  TABLE 8. Percentage of p o l l e n d e p o s i t e d on stigmas of p i n , thrum and homostyle f l o w e r s of Menyanthes trifoliata from six p o p u l a t i o n s . Values a r e the mean and standard d e v i a t i o n ( i n p a r e n t h e s e s ) . Means are c a l c u l a t e d from samples collected at r e g u l a r i n t e r v a l s d u r i n g the f l o w e r i n g season, N r e p r e s e n t s the t o t a l number of stigmas collected during the whole f l o w e r i n g season.  N  Percentage of p i n pollen  Percentage of thrum pollen  Population  Stigma  Pollen frequency  BL2  pi n thrum  0 .845 0. . 155  190 175  73 .0 (9 .90) 61 . 4 (14 .38)  27 . 0 (9.90) 38. 6 ( 14.38)  BL3  p1n  0 .677 0 . 333  354  thrum  333  69 .6 (19 . 18) 57 .5 (20 .40)  30. 4 (19.18) 42 . 5 (20.40)  BL4  p1n thrum  0. 075 0. 925  151 320  32 .7 ( 19..85) 31 . 1 ( 14.. 15)  67. 3 (19.85) 68 . 9 ( 14.15)  PC 1  p1n thrum  0. 491  202  0. 509  205  69 .9 ( 14..08) 48 . 4 ( 12.51)  30. 1 (14.08) 51 . 6 (12.51)  PC2  p1n thrum  0. 893 O. 107  163 43  81 2 ( 13.43) 69 2 (31 .60) .  18. 4 ( 13.43) 30. 8 (31.60)  SL 1  pin  0. 831  245  85 . 3 (14. 98)  14.7 '*  (14.98)  homosty1e  0. 169  160  24 . 0 (30. 30)  76.0*  (30.30)  Tl  * homostyle p o l l e n  o  f f w z  Tl  f O  3  oo  P O L L E N F L O W / 59 pin and thrum pollen in the population was equal, and there was no significant difference between floral morphs in the percentage of compatible pollen received.  There percentage  was  of  a  very  compatible  high  pollen  correlation deposited  (r = 0.93,  on  p<0.001)  stigmas  and  the  between  the  percentage  of  compatible pollen produced in each population. A s the frequency of thrum pollen produced  increased among  populations,  so did  deposited on pin stigmas in those populations  the  percentage  (Figure  of  thrum  pollen  12). The same was true  for pin pollen on thrum stigmas. However, this relationship was not proportional. In the five populations where one form of pollen was more abundant than other, stigmas of the rarer floral compatible  pollen  than  expected  produced in the population) more abundant floral  morph received a slightly (based on the  (Table 8). In  percentage  the  lower percentage of of  compatible  pollen  the same populations, stigmas of the  morph received a slightly  higher  percentage of compatible  pollen than expected.  As was found in other populations of Menyanthes, at S L 1 the percentage of  compatible  similar  to the  S L 1 were  the  was markedly (Figure much  pollen  deposited  on  pin  stigmas  (14.7%,  very  Homostyles  from  the  only  the composition of stigmatic  in  which  (0.169).  was  percentage produced in flowers  population  s.d= 14.98)  pollen loads  different from the frequency of pollen produced in the  population  12, Table 8). The frequency of pin pollen produced at SL1 (0.831) was greater  stigmas  should  than be  that  of  homostyle  expected to  have  pollen received  (0.169); an  on  excess  this of  basis  homostjde  compatible  However, homostyle stigmas received an average of only 24.0% (s.d. =  pollen.  30.30)  POLLEN FLOW / 60 FIGURE 12. The percentage of compatible pollen deposited on stigmas of pins, thrums and homostyles in six populations of Menyanthes trifoliata plotted against the percentage of compatible pollen produced in each population. Each point represents one floral morph from each of the six populations: BL2, BL3, BL4, PCI, PC2 and S L l . Closed circles represent pins, open circles represent thrums and the triangle represents homostyles. The line is the least squares regression for pins and thrums (homostyle was excluded). Correlation: r = 0.93, p<0.0001.  -j——i  1  1  1  1  1 r  20 40 60 80 % Compatible Pollen Produced in P o p u l a t i o n  P O L L E N F L O W / 61 compatible pollen.  Study area PC2 consisted of a pin/thrum population structured similarly to the pin/homostyle population at S L l . A t P C 2 , the frequency of pin pollen (0.893) was  much  greater  than  the  frequency  of  thrum  pollen  (0.107),  and  the  distribution of floral morphs in the population was non-random. The proportion of compatible  pollen  corresponded  well  deposited with  on  the  pin  stigmas  frequency  of  at  PC2  compatible  (18.4%,  pollen  in  s.d= 13.43)  the  population  (0.107), and was very similar to that found for pin stigmas at S L l . Similarly, the proportion of compatible pollen deposited on thrum stigmas from PC2 (69.2%, s.d = 31.60) corresponded well with the frequency of compatible pollen produced in the population (0.893), and was much greater than that received by pin stigmas. However, there was a remarkable difference between the proportion of compatible pollen  received by  thrums  from  PC2 and  homostyles  from  S L l ; the  thrums  received 45.2 percentage points more compatible pollen than the homosytles.  Although  there  was  a  very  high  correlation  between  the  percentage  of  compatible pollen deposited on stigmas of Menyanthes and pollen frequency in the population,  the  correlation between  the  numbers  of compatible  and  incompatible  pollen grains received and pollen frequency was not as high (r = 0.69, p<0.05). In  all  study  areas  except  PCI,  the  frequency  of  one  pollen  form  was  significantly greater than the frequency of the other (Table 6), yet sites B L 3 and BL4  were  between BL3,  the  floral  where  only  study  morphs  in  pin  pollen  areas in the  which  number  frequency  of  was  there  was  compatible higher  a  significant  difference  pollen grains received. A t  (0.677),  thrums  received  a  P O L L E N F L O W / 62 significantly  higher  (p<0.05)  number of compatible pollen grains than pins and  pins received a significantly higher (p<0.05) number of incompatible pollen grains than thrums (Table 9). A t B L 4 , where thrum pollen was more abundant (0.925), pins received a significantly  higher  (p<0.05) number of compatible pollen grains  than thrums, but there was no significant difference between floral morphs in the number of incompatible pollen grains received (Table 9). A t P C I , where pin and thrum pollen frequencies were equal, there  was no significant difference between  floral morphs in the number of compatible or incompatible pollen grains received. In the remaining three study areas (BL2, P C 2 , S L l ) , pin pollen was much more abundant  than  the  other  pollen  form,  yet  there  was  no  significant  difference  between floral morphs (within each population) in the number of compatible pollen grains received. In addition, at PC2 and S L l there was no significant difference between  floral  However,  morphs  at  BL2  in  pins  the  number  received  a  of  incompatible  significantly  pollen  higher  grains  (p<0.05)  received.  number  of  incompatible pollen grains than thrums. A t B L 2 , the lack of correlation between the  number  of  compatible  pollen  grains  received  and  pollen  frequency  was  probably a result of differences in the size of pollen loads between the two  floral  morphs. At B L 2 , the frequency of pin pollen in the population was 0.845, and thrum (69.7%)  stigmas  received  a  significantly  percentage  of  compatible  pollen  than pins (27.0%). But, pin pollen loads were almost twice the size of  thrum pollen loads, consequently the received  higher  by  the  two  received at least four  floral times  morphs  actual number was  similar.  of compatible pollen grains  Even  though  thrums  as much compatible pollen as pins, this  at PC2 difference  was not statistically significant, perhaps as a result of small sample sizes.  TABLE 9. Composition of s t i g m a t i c p o l l e n loads of p i n , thrum and homostyle flowers of Menyanthes trifoliata from s i x populations. Values are the mean and standard d e v i a t i o n (1n p a r e n t h e s e s ) . Means a r e c a l c u l a t e d from samples c o l l e c t e d at regular Intervals during the f l o w e r i n g season. N represents the t o t a l number of stigmas c o l l e c t e d d u r i n g the whole f l o w e r i n g season.  Number of thrum p o l l e n grains  (143.25) (85.96)  113.1 (76.51) 86.7 (67.50)  Population  Stigma  BL2  pin thrum  190 175  290.5 130.2  BL3  p1n  354  257.8  (130.77)  thrum  333  172.6  (89.70)  pin thrum  151 320  152.5 77.7  (99.05) (53.94)  thrum  202 205  70.5  PC2  p1n thrum  163 43  118.9 168.1  SL 1  pi n  245  homostyle  160  BL4  PC 1  pin  N  Number of pin pollen grains  136.3  (108.93)  Total number pollen  of grains  403.6 216.9  192.63) 126.56)  99.9 (57.54)  357.7  128.81 )  129.7 (95.83)  302.3  1 16.74)  313.5 154.6  466.O 232.3  410.34)  (325.83) (75.53)  119.85)  59.6  (54.25)  195.9  159.75)  75.3  (45.28)  145.8  1 14.03)  (101.10) (174.52)  38.9 (59.98) 74.8 (23.46)  157.8 242.9  154.03) 197.90)  204.9  (46.56)  39.9*  244.9  57.65)  104.3  (68.17)  329.5* (228.23)  433.8  192.20)  (70.03)  (44.40)  homostyle p o l l e n  O  3 OS CO  P O L L E N F L O W / 64 Comparison of the actual number of compatible pollen grains deposited on stigmas of PC2 thrums an  average of  1.6  and SL1 homostyles revealed that the thrums  times  more  compatible  pollen  grains  than  the  received  homostyles.  However, the number of incompatible pollen grains deposited on stigmas of S L 1 homostyles was  4.4  (Pins from the two  times  greater  than  the  number  received by  P C 2 thrums.  populations received a nearly identical number of compatible  pollen grains (PC2: 38.9, s.d. = 59.98; S L 1 : 39.9, s.d. = 44.40).)  Patterns  of pollen deposition on stigmas over the  flowering  season were  very erratic (Figures 13-18). There was a great deal of variation in the size and composition of stigmatic pollen loads during the flowering season, and there were few patterns the only  two  in common among the six study areas. Sites P C I and P C 2 were populationsn that showed similar pollen deposition patterns  in the  two floral morphs during the flowering season (Figures 16 and 17). For pins and thrums  from  number  of pollen grains  end  the  of  BL3, BL4, PCI  season.  and  P C 2 , and  deposited on stigmas  However,  for  pins  and  homostyles seemed to  thrums  from  SL1 the  total  decrease towards  from  BL2  total  the  stigmatic  pollen loads increased in size towards the end of the flowering season. For pins at S L 1 , the total number of pollen grains received was similar at the beginning and at the end of the season.  Results  were  highly  variable  from  the  four  stud}'  areas  in  which  emasculations were performed. There was considerable variation in the composition of  stigmatic  pollen loads among the  different floral  morphs  on different- dates,  and in different populations (Tables 10-13). A t B L 2 , the composition of stigmatic  POLLEN FLOW / 65 FIGURE 13. The number of pollen grains deposited on stigmas of pin and thrum flowers of Menyanthes trifoliata in population BL2 during the flowering season of 1985. For each collection date, the number of pin and thrum pollen grains deposited on stigmas was determined from pooled samples of approximately 40 stigmas per floral morph. Closed circles and solid lines represent the number of pin pollen grains deposited on stigmas. Open circles and broken lines represent the number of thrum pollen grains deposited on stigmas.  Pi n S t i g m a s 600500 to 4 0 0 GM  <  300 -  LO  200-  z O Q LLI  1-  100 0 -  i  5/16  U) o  i  19  o_  1  1  22 2 4 DATE  r  28  IU  a  z < rr O z  Thrum Stigmas 600 500 -  LLI  _l _l  o  Q_  LL  o cr  400300 200 -  LU  m ZD Z  1000 -  i  5/16  19  1  1  r  22 2 4 2 8 DATE  POLLEN FLOW / 66 FIGURE 14. The number of pollen grains deposited on stigmas of pin and thrum flowers of Menyanthes trifoliata in population BL3 during the flowering season of 1985. For each collection date, the number of pin and thrum pollen grains deposited on stigmas was determined from pooled samples of approximately 40 stigmas per floral morph. Closed circles and solid lines represent the number of pin pollen grains deposited on stigmas. Open circles and broken lines represent the number of thrum pollen grains deposited on stigmas.  Pin Stigmas  if)  <  o «to z O Q LU  f-  Ln O  5/19  T  1  1  22  24  28  CL LU Q  CO Z  r  i  1  1  r  6/1 4 DATE  6  12  15  18  1  1  1  Thrum Stigmas  < rr CD  z  LU  O o_ LL  O  ce  LU CO  ZD 1  5/19  1  22  1  24  1  28  1  r—  6/1 4 DATE  T  1  8  12  15 18  POLLEN FLOW / 67 FIGURE 15. The number of pollen grains deposited on stigmas of pin and thrum flowers of Menyanthes trifoliata in population BL4 during the flowering season of 1985. For each collection date, the number of pin and thrum pollen grains deposited on stigmas was determined from pooled samples of approximately 40 stigmas per floral morph. Closed circles and solid lines represent the number of pin pollen grains deposited on stigmas. Open circles and broken lines represent the number of thrum pollen grains deposited on stigmas.  Pin Stigmas  T  5/22  r  24 2 8  <  cr CD  6/1 4 8 DATE  -i  12  1  1  r  15 18 21  Thrum Stigmas  LU  O 400 CL LL  O  300 H  cr 2 0 0 -| LU  | z  100o  1  5/22  1  1  1  1  2 4 2 8 6/1 4 8 DATE  1  1  r  12 15 18  21  POLLEN FLOW / 68 FIGURE 16. The number of pollen grains deposited on stigmas of pin and thrum flowers of Menyanthes trifoliata in population PCI during the flowering season of 1985. For each collection date, the number of pin and thrum pollen grains deposited on stigmas was determined from pooled samples of approximately 40 stigmas per floral morph. Closed circles and solid lines represent the number of pin pollen grains deposited on stigmas. Open circles and broken lines represent the number of thrum pollen grains deposited on stigmas.  Pin S t i g m a s 500  -I  2  4 0 0 -|  O  300H  CO  200-  Z  o  100 -  LU  O  o  h00  O  6/3  Q_ LU Q 00  <  or  Thrum Stigmas  O LU _l _l  O CL LL  o cc LU CO  ZD Z  6/3  8  T  1  r  11 15 18 DATE  21  POLLEN FLOW / 69 FIGURE 17. The number of pollen grains deposited on stigmas of pin and thrum flowers of Menyanthes trifoliata in population PC2 during the flowering season of 1985. For each collection date, the number of pin and thrum pollen grains deposited on stigmas was determined from pooled samples of approximately 40 stigmas per floral morph. Closed circles and solid lines represent the number of pin pollen grains deposited on stigmas. Open circles and broken lines represent the number of thrum pollen grains deposited on stigmas.  Pin Stigmas to  <  o 10  z O Q LU  h-  00  O  6 / 3  8  11  1 5  18  D A T E  CL LU Q CO  z < cr O  Thrum Stigmas  6 / 3  8  11 D A T E  21  POLLEN FLOW / 70 FIGURE 18. The number of pollen grains deposited on stigmas of pin and homostyle flowers of Menyanthes trifoliata in population S L l during the flowering season of 1985. For each collection date, the number of pin and homostyle pollen grains deposited on stigmas was determined from pooled samples of approximately 40 stigmas per floral morph. Closed circles and solid lines represent the number of pin pollen grains deposited on stigmas. Open circles and broken lines represent the number of homostyle pollen grains deposited on stigmas.  ^  < O  Pin Stigmas  I 400-  5/18  21 2 4 2 6 31 DATE  TABLE 10. Expected and observed p o l l e n f r e q u e n c i e s on stigmas of p i n and thrum flowers of Menyanthes trifoliata population BL2. N=10 stigmas per f l o r a l morph f o r each c o l l e c t i o n d a t e . Expected values a r e based on random pollination and a r e c a l c u l a t e d from the frequency of p o l l e n produced i n the p o p u l a t i o n . X values i n d i c a t e the deviation of observed p o l l e n g r a i n d e p o s i t i o n from random p o l l i n a t i o n . P o l l i n a t i o n regime: R= random p o l l i n a t i o n , significant assortatlve pollination. J  Pol 1 en type Floral morph  Date  P1n  5/16  thrum  Pin  Thrum  Obs . Exp.  146 .4  20.0 25.9  1 .57  140.6  5/19  Obs . Exp.  164 .0 161 .7  27 .9 29.7  0.14  5/ 16  Obs . Exp.  93 . 2 253 .4  206 . 7  653 . 2  46.5  Obs . Exp.  143.5 151 .O  35 . 2 27.7  5/19  X  !  (df=1)  2.4  Pollination regime  TABLE 11. Expected and observed p o l l e n f r e q u e n c i e s on stigmas of p i n and thrum flowers of Menyanthes trifoliata from p o p u l a t i o n BL3. N=10 stigmas per f l o r a l morph f o r each c o l l e c t i o n date. Expected values a r e based on random pollination and a r e c a l c u l a t e d from the frequency of p o l l e n produced i n the p o p u l a t i o n . X' values i n d i c a t e the deviation of observed p o l l e n g r a i n d e p o s i t i o n from random p o l l i n a t i o n . P o l l i n a t i o n regime: R= random p o l l i n a t i o n , D= significant d i s a s s o r t a t i v e p o l l i n a t i o n , A= s i g n i f i c a n t a s s o r t a t l v e p o l l i n a t i o n . C a l c u l a t i o n s of percentage efficiency of d i s s a s o r t a t i v e p o l l i n a t i o n explained in text.  Pol 1 en type Pol 1(nation  F1ora1 morph  Date  Pin  5/16  5/19  Thrum  X'  (df=1)  P1n  Thrum  Obs . Exp.  202 . 2 157.3  32.6 77 .5  38.9  Obs . Exp.  357 .6  12.7  146 . 3  248. 1  122 . 1  5/22  Obs . Exp.  37 . 7 34.8  14.2 17.1  0.98  5/24  Obs .  40.7  94 .9  84. 1  Exp.  90.9  44.7  Obs . Exp.  179. 1 202.6  123 . 3 99.8  8.2  Obs . Exp.  366.7  46.5 136.4  88.5  276.8  5/22  Obs. Exp.  58.8 53.6  21.1 26.4  1 .5  5/24  Obs .  6.9  18.0  12.5  Exp.  16.7  8.2  5/16  5/19  regime  Percentage e f f i c i e n c y of disassortative p o l 1 i n a t ion  55 . 2  65.9  O f f  w f O  3 to  TABLE 12. Expected and observed p o l l e n f r e q u e n c i e s on stigmas of p i n and thrum flowers of Menyanthes trifoilata from population PC 1 . N=10 stigmas per f l o r a l morph f o r each c o l l e c t i o n d a t e . Expected values a r e based on random pollination and a r e c a l c u l a t e d from the frequency of p o l l e n produced i n the p o p u l a t i o n . X' values I n d i c a t e the deviation of observed p o l l e n g r a i n d e p o s i t i o n from random p o l l i n a t i o n . P o l l i n a t i o n regime: R= random p o l l i n a t i o n , D= significant d i s a s s o r t a t 1ve p o l l i n a t i o n , A= s i g n i f i c a n t a s s o r t a t l v e p o l l i n a t i o n . C a l c u l a t i o n s of percentage efficiency of d i s s a s o r t a t i v e p o l l i n a t i o n explained in text.  Pol 1 en type Pol 1inat1on  F1 o r a l morph  Date  Pin  6/8  6/11  Thrum  X  1  (df=1)  regime  Percentage e f f i c i e n c y of di s a s s o r t a t 1 v e pol11 n a t i o n  P in  Thrum  Obs . Exp.  353.9 315.0  287 . 7 326 .6  9.4  Obs . Exp.  437 . 7  332.3 391 .9  18 . 5  378 . 1  6/15  Obs . Exp.  118.3 124.8  135.9 129.4  0.65  6/18  Obs.  10.2  18.7  19.4  121.4  182.7  Exp.  149.3  154.8  Obs . Exp.  255.6 212.8  177 .0 220.6  16 .9  Obs .  173 .8  127.9  8.8  16.7  Exp.  148 . 1  153.6  6/15  Obs. Exp.  189.8 150.0  1 15.7 155 .6  20.8  25.6  6/18  Obs.  56.7  15. 1  42.8  Exp.  39.3  23.3 40. 7  6/8  6/11  TJ O f f  z Tl f O 3 CO  TABLE 13. Expected and observed p o l l e n frequencies on stigmas of p i n and homostyle flowers of Menyanthes trifoliata from p o p u l a t i o n SL1. N=10 stigmas per f l o r a l morph f o r each c o l l e c t i o n d a t e . Expected values a r e based on random pollination and a r e c a l c u a l t e d from the frequency of p o l l e n produced i n the p o p u l a t i o n . X' values i n d i c a t e the deviation of observed p o l l e n g r a i n d e p o s i t i o n from random p o l l i n a t i o n . P o l l i n a t i o n regime: R= random p o l l i n a t i o n , D= significant d i s a s s o r t a t i v e p o l l i n a t i o n , A= s i g n i f i c a n t a s s o r t a t i v e p o l l i n a t i o n . C a l c u l a t i o n s of percentage efficiency of d i s s a s o r t a t 1ve p o l l i n a t i o n e x p l a i n e d in text.  Pol 1 en type Floral morph  Date  Pin  5/18  Homostyle  Pin  Homosty1e  Obs.  2.0  1.0  Exp.  2.5  2.5  5/21  Obs. Exp.  52.4 52.2  5/24  Obs. Exp.  Obs. Exp.  49.9 85.3  52.8 17.4  5/18  (df=1 )  Pol 1inat ion regime  0.35  R  10.5 10.G  0.002  R  40.G  30.0  33.0  58.7  11.9  8G.7  0  Percentage e f f i c i e n c y of disassortative p o l 1 i n a t ion  30.8  A HJ  O 5/21  5/24  Obs.  9.1  24.8  Exp.  28.2  5.7  Obs.  161.8  Exp.  200.3  79.2 40.7  76.9  A  tT  1  2  43.8  A  f  O 3  4^  P O L L E N F L O W / 75 loads  of  pins  was  not  significantly  different  than  that  pollination (Table 10). The same was true for thrums on the other  date they  Pin  from  stigmas  significant  BL3  amount  disassortatively  received a significant  of  were  randomly  assortative  pollinated on the  pollination  fourth  (Table  random  at B L 2 on one date, but  amount  pollinated  expected from  of assortative  on  on  one  two  date,  pollination. received  dates,  11). Thrums  and  a  were  stigmas from  BL3  received a significant amount of assortative pollination on three dates, and were randomly random thrum dates  pollinated on the fourth. and  P C I , pin stigmas experienced assortative,  disassortaive pollinations  stigmas  received a significant  sampled. A t  pollinated,  At  whereas  S L 1 , pin  on  amount  stigmas  homostyles  different  (Table  12).  However,  of disassortative pollination on  were  received  dates  either  a  randomly  significant  or  all  disassortatively  amount  of  assortative  pollination on all dates measured (Table 13).  The formula promoting  E  =  disassortative  (o - r)/(d pollination  - r) in  measures the  emasculated  efficiency  flowers,  of distyly  where  E  is  in the  disassortative component of the pollen load, o is the observed frequency of one of the pollen forms, r is the expected frequency of that pollen form with random pollination,  and  disassortative pollination) the  four  d  is  pollination  to  the  expected  frequency  of  (Ganders 1974). Values of E  that  pollen  range  from  100% (complete disassortative pollination),  populations  of Menyanthes in  which  significant  form 0%  with  (random  and were calculated for levels of disassortative  pollination were detected (BL2, B L 3 , P C I and S L l ) (Tables 10-13). There was a considerable experienced  amount on  of  different  variation dates  in  the  throughout  degree the  of  flowering  disassortative season.  pollination  Values ranged  P O L L E N F L O W / 76 from  16.7% to  (s.d = 20.48) for  65.9%, and thrums  averaged  34.9%  (s.d= 18.59)  for  pins  and 34.1%  on the occasions when the flowers were disassortatively  pollinated (homostyles were not disassortatively pollinated).  D.  DISCUSSION  None  of  the  population structure distributed  six  populations  of  Menyanthes studied  of a distylous plant: a 1:1 ratio of  among one  another.  Price  Pontederia cordata, a combination  and  Barrett  floral  (1984)  of clonal reproduction  had  typical  morphs randomly  have  and  the  found  pollinator  that  in  behavior  resulted in a 75% chance that plants growing next to each other would be of the  same form.  Menyanthes, the  While  little  clonal nature  is  known  of the  of  pollinator  activity  species is a major  in  factor  populations influencing  of the  population structure. Hewett (1964) concluded that vegetative propagation was the most  important  method  of  reproduction  in  Menyanthes, and  he  stated  that  seedlings had not been reported from nature. I did find seedlings of Menyanthes germinating in the mud in the ponds at Pinecrest (Figure  19), but it appeared  that vegetative propagation was a much more common form of reproduction.  The low percentage of pollen (<1%)  participating in legitimate  pollinations  in populations of Menyanthes is similar to that found in other distylous species (Onrduff  1970a, 1970b, 1971, 1976, 1979, 1980a; Ganders 1974; Weller 1980;  Philipp and Schou 1981; Rama Swamy and Bir Bahadur 1984; Nicholls 1985), and seems to be a common feature  of distylous breeding systems. Since more  than 60% of the pollen produced in populations of Menyanthes was removed from  P O L L E N F L O W / 77 F I G U R E 19. Seedlings of Menyanthes trifoliata germinating in the mud near ponds in the Pinecrest area of southwestern British Columbia.  P O L L E N F L O W / 78 anthers by pollinators (Table 3), the low percentage of Menyanthes pollen reaching conspecific stigmas was not the result of a lack of pollinator activity.  The bulk  of the pollen produced in populations of Menyanthes must have been lost through vagaries of the pollination process.  In  flowers  of  Menyanthes trifoliata,  a  considerable amount  of  variation  existed among populations, between floral morphs and among individual collection dates  in  patterns  the  size  and composition of  fluctuated  erratically  stigmatic  during  the  pollen  flowering  loads. Pollen deposition  season,  and  were  very  different among the six populations studied. Within each study area, flowers from both  floral  morphs  experienced  either  random,  assortative  or  disassortative  pollination depending on the date the samples were collected. The magnitude of disassorative pollination varied from 16.7% to 65.9%. Thrums from P C I were the only flowers that received a significant amount of disassortative pollination on all dates  measured, while  homostyles  from  SL1 received a  significant  amount  of  assortative pollinatation on all dates measured.  Glover  and Barrett  (1986)  found similar intermorph  variation in the composition of stigmatic tristylous  Pontederia cordata. The  pollinated in  all  four  and  interpopulational  pollen loads of emasculated flowers of  long-styled  floral  populations examined, but  morph  the  type  was  disassortatively  of pollination  regime  experienced by the mid-styled and short-styled morph varied among populations. The mid-styled morph experienced both disassortative and random pollination and the  short-styled  disassortative  and  morph  experienced  assortative  all  pollination.  three  pollination  Among  those  regimes: flowers  random,  that  were  P O L L E N F L O W / 79 disassortatively pollinated, the degree of disassortative pollination ranged 7.1% to 39.3%.  Individual been  found  Linum  in  variation  in the composition of stigmatic  populations  of  distylous  pollen loads has also  Hedyotis caerulea (Ornduff  tenuifolium (Nicholls 1985). Ornduff (1980b) found that in H.  1980b)  and  caerulea up  to 21% of the stigmas held no pollen at all. Results from these studies of pollen flow in various heterostylous species (including Menyanthes trifoliata), indicate that pollen flow is not as uniform  among individuals or during the flowering  season  as was once assumed. The size and composition of stigmatic pollen loads appears to  be  highly  variable  among  individuals  of  each floral  morph,  between  floral  morphs, among different days during the flowering season and among populations. These results suggest that erratic pollen deposition may be. a common feature of pollen flow in distylous plant populations.  Despite Menyanthes populations  the  apparent  trifoliata, of  some  erratic  nature  general  Menyanthes studied,  of  trends  there  was  pollen  were a  flow  evident.  high  in  populations  Among  correlation  of  the  six  between  the  frequency of pollen produced in the population and the composition of stigmatic pollen loads. Richards and Ibrahim the proportion  (1978)  found a similar  relationship  of pin and thrum pollen deposited on stigmas and the frequency  of floral morphs in populations of Primula veris. However, Ornduff 1980b,  between  (1976, 1978,  1986) was unable to find a similar relationship in the various distylous  species he studied.  P O L L E N F L O W / 80 In populations of Menyanthes, the high correlation between pollen frequency in the population and the composition of stigmatic pollen loads indicates that in general,  pollen  pollination.  flow  was  close  to  what  However,  this  relationship  would  was  be  not  expected  proportional.  with In  random  anisoplethic  populations of Menyanthes, stigmas of the more abundant floral morph received a slightly  higher  percentage of compatible pollen than  expected based on random  pollination. Whereas, stigmas of the rarer floral morph received a slightly percentage  of  compatible  pollen  than  expected.  This  is  significant  lower  because  it  suggests that morphological distyly is successful in compensating for the rarity of one floral form by increasing the proportion of compatible pollen received by the opposite floral form. Distyly seems to buffer  the effects of drastic differences in  morph or pollen frequency in the population.  Comparison of stigmatic pollen loads of the homostyles from S L l to pollen loads of thrums compatible percentage  from P C 2 revealed a remarkable difference in the proportion of  pollen  received.  points  more  Thrums  compatible  from  PC2  pollen than  received  the  approximately  homostyles  from  45  S L 2 . But,  when the actual number of compatible pollen grains received by the thrums and homostyles  was  approximately major  more  1.6  difference  was in  the  than  thrums.  compared, the times in  number four  more  stigmatic  difference compatible  pollen  of incompatible  times  the  was  number  not  pollen  as  great.  grains  than  the  thrums  loads between  Thrums  received  homostyles.  The  and homostyles  pollen grains received. Homostyles received of  incompatible  pollen  grains  received by  POLLEN FLOW / 81 Except for the difference in style length, the thrums and homostyles were morphologically identical. They both grew in similarly structured populations. The composition of pin stigmatic pollen loads was indicating  that  pollen  flow, patterns  were  Therefore, differences in the composition thrums and homostyles  very similar in both populations similar  in  the  two  of stigmatic pollen loads between the  can be attributed to the difference in style length found  in the two floral morphs. Because stigmas and anthers in homostyle close to each  populations.  flowers are  other, they received a much higher number of own-form pollen  grains than did thrums. In addition, the reciprocal stamen/style dimorphism in the pin/thrum population resulted in thrum stigmas receiving a higher number of compatible pollen grains than homostyle anthers  in  pin  and  thrum  stigmas. The in Menyanthes  flowers  incompatible pollen grains received, and  separation of stigmas reduces  the reciprocal placement  the  number  of stigmas  and of and  anthers does appear to increase the number of compatible pollen grains received. These  results  provide  quantitative  hypothesis that morphological distyly  evidence  in  support  of  Darwin's  (1877)  increases the amount of compatible pollen  deposited on stigmas of diallelic self-incompatible plants.  V. SEED  SET  A. INTRODUCTION  With  the  diallelic self-incompatibility  mechanism found in distylous  plants,  the chances of a stigma receiving incompatible pollen are great. If one assumes that seed set is limited by a lack of compatible pollinations, then any mechanism that would increase the amount of compatible pollen received should increase the fecundity  of the  plant.  placement of stigmas  Darwin  (1877)  and anthers,  suggested that because of the  flowers  of distylous  plants  reciprocal  should receive a  greater amount of compatible pollen than flowers of a diallelic plant lacking the floral dimorphism.  Ganders (1974) hypothesized that morphological distyly increase number  in  fecundity  of compatible  of  a  diallelic  self-incompatible  might result in an  plant  by  increasing  the  pollen grains received. A n unusual population of Mitchella  repens consisting of pins, thrums and self-incompatible homostyles allowed Ganders (1975a)  to  homostyle  test flowers  this  hypothesis.  were  identical  to  Except  for  the  thrum  the  length  flowers.  any difference in seed set that existed between thrums attributed compatible significantly  to  the  pollen higher  influence grains  of  the  stamen/style  received. Ganders'  seed set  than  the  of  homostyles  the  and homostyles could be  show and  styles,  Ganders suggested that  dimorphism  results  their  on  that he  the the  number  of  thrums  had  suggested that  this  indicated that in diallelic self-incompatible plants, "distyly may be associated with a greater reproductive fitness than homostyly".  82  SEED SET / 83 Although  Ganders  (1975a) was  flowers were more fecund than  able to demonstrate  that  the distylous  the homostylous flowers, he lacked the pollen  flow data to confirm that differences in seed set between the two floral morphs resulted  from  differences in the number  of compatible  pollen  grains received.  Many factors have been shown to influence seed set in self-incompatible plants: the quantity and efficiency of pollinators, the density, spatial pattern and number of mating  groups  fertilization  and  in the population, environmental conditions during pollination, embryo  development, resource  availability  for fruit  and seed  maturation, and pollen availability (Shore and Barrett 1984). Without pollen flow data, it is impossible to determine  whether the observed difference in seed set  between pins and homostyles of Mitchella repens resulted from pollen limitation or from some other factor.  I investigated  the relationship between  the number  of compatible pollen  grains received and seed set in six populations of Menyanthes trifoliata in order to determine whether pollen availability was a major factor influencing seed set. For each floral morph . in  each  study  area, the number  of compatible pollen  grains received (Chapter 4) was compared to the number of seeds set.  Results  from pollination intensity experiments (Chapter 3) showed that an average of six compatible Menyanthes. determine  pollen  grains  are required  Using this number  to fertilize  each  ovule  as a basis for comparison,  in flowers of  it was possible to  whether observed seed set in Menyanthes corresponded to the number  of compatible pollen grains received. In one population, an attempt was made to increase seed set by increasing the number of compatible pollen grains deposited on stigmas. Flowers were hand-pollinated with large amounts of compatible pollen,  S E E D S E T / 84 and then the resultant seed set was compared to seed set in naturally flowers  pollinated  in the same population.  In  addition,  seed  set  of  homostyles  in  a  pin/homostyle  population  of  Menyanthes was compared to seed set of thrums in a pin/thrum population. Like the  homostyles  homostylous  in  flowers  the  population  of  incompatibility  homostyles  styles. In  Menyanthes are behavior.  Menyanthes thrums  Mitchella  repens (Ganders  of Menyanthes are morphologically  except for the length of their homostyles  of  received  (Chapter  1.6  4), and I  of  times attribute  identical to thrum  addition, evidence (below)  self-incompatible  Examination  1975a),  and  stigmatic  more this  equivalent pollen  compatible difference  to  the  to  suggests, distyly  is  associated with  a greater  thrums  revealed  grains  in that  than  the  presence of  the  stamen/style dimorphism in flowers from the pin/thrum population. If, (1974)  flowers  indicates that  loads  pollen  the  reproductive  as Ganders fitness  than  homostjdy, then the higher number of compatible pollen grains observed on thrum stigmas should be associated with higher seed set.  B. MATERIALS AND  I which  METHODS  collected seed set data in the  pollen  flow  data were  collected  same six populations  of Menyanthes in  (BL2, B L 3 , B L 4 , P C I , PC2 and S L l ) .  Seed set data were collected from flowers tagged on the same days that stigmas were  collected for  pollen  flow  analysis; this  gave an estimate  of seed set for  flowers pollinated during the same period as those from which stigmas had been collected. Ten to twenty flowers per floral morph, at the same stage of anthesis  S E E D S E T / 85 as those from  which stigmas were collected, were tagged with the date. Some  flowers were selected from the same inflorescence from which stigmas had been collected, and others were on separate inflorescences. Inflorescences with tagged flowers  were  flagged with  fluorescent  surveyors' tape  for  fruits of the tagged flowers were allowed to mature for  easy relocation.  The  4-5 weeks and collected  prior to dehiscence. Percentage seed set was determined by counting the number of seeds and the number of aborted ovules in each fruit. Approximately 60 fruits per  floral  morph  were  collected for  each population  during  the  course of  the  flowering season.  Bulk  samples of  Approximately PCI,  were  collected  from  addition, bulk  populations: one from  the  fruit  samples were  populations.  SMI  collected from  three  Stump Lake area (SL2), one from  Pinecrest area (PC3) and one at Strachan Meadow (SMI) Populations  nine  300 fruits per floral morph were collected from B L 2 , B L 3 , B L 4 ,  P C 2 and S L 1 . In  monomorphic  undehisced fruits  and PC3 were both monomorphic for  (Figures 9,  thrums,  while  the  10, 20). SL2 was  monomorphic for homostyles. Up to 3255 fruits were collected from each of the three monomorphic populations.  In  an attempt to increase seed set, large  amounts  of compatible  pollen  were applied by hand to receptive stigmas of flowers growing in the SL1 study area. Recently dehisced anthers from  flowers of the opposite form  were  rubbed  on stigmas until all pollen had been deposited (Weller 1980). A total of 14 pin flowers and 14 homostyle flowers were hand-pollinated. These flowers were tagged with the date, their fruits collected 4-5 weeks later and percentage seed set was  SEED SET / 86 FIGURE 20. Populations of Menyanthes trifoliata at Strachan Meadow in Cypress Provincial Park in West Vancouver, B.C. Populations were found in a series of small ponds scattered throughout the meadow and along the edges of Yew Lake. Stippled area indicates the location and extent of populations. Shaded areas indicate study area SMI where seed set data were collected.  STRACHAN MEADOW  LEGEND H  M. t r i f o l i a t a  |  Study area  A  N - S M1  50m  S E E D S E T / 87 determined.  A l l calculations of seed set and percentage seed set were determined on a per fruit basis. Averages of seed set and percentage seed set were calculated for pins and thrums specific  (or  collections  homostyles) in each population, and wherever  dates  during  the  season.  Unless otherwise  possible for  specified, results  refer to averages for the whole flowering season rather than individual collection dates.  C.  RESULTS  In  the  correlation grains  six  populations  Menyanthes studied,  (r = 0.88, p<0.001) between the  deposited on stigmas  number  of  of  seeds set  per  of  average number  pins, thrums  capsule  there  (Figure  was  a  very  of compatible pollen  and homostyles- and the 21).  On  high  average,  pins  average  from  BL4  received the highest number of compatible pollen grains of any floral morph from the six populations studied and had the highest seed set (Table PCI  and PC2 received the lowest number  14). Pins from  of compatible pollen grains and had  the lowest seed set.  At and  B L 2 , B L 3 , and P C I  thrums  difference  in  existed  the  number  between  of  pins  no significant seeds and  set.  difference Similarly,  homostyles  in  the  appeared between pins at  SL1  no  significant  number  of  seeds set.  However, at B L 4 and PC2 the average number of seeds set differed  significantly  between floral morphs. A t B L 4 , pin seed set was significantly higher than thrum  SEED SET / 88 FIGURE 21. The number of seeds set per capsule in pins, thrums and homostyles of Menyanthes trifoliata plotted against the number of compatible pollen grains deposited on stigmas. Each point represents one floral morph from each of the six populations studied: BL2, BL3, BL4, PCl, PC2 and SL1. Closed circles represent pins, open circles represent thrums and the triangle represents homostyles. The line is the least squares regression of seed set on the number of compatible pollen grains received. Correlation: r = 0.88, p<0.001.  Number of Compatible Pollen G r a i n s Deposited on Stigmas  TABLE 14. Seed s e t and percentage seed set in p i n s , thrums and homostyles of Menyanthes trifoliata from s i x populations. Values are the mean and standard d e v i a t i o n ( i n p a r e n t h e s e s ) . Means a r e c a l c u l a t e d from samples collected at regular i n t e r v a l s during the f l o w e r i n g season. r values represent the c o r r e l a t i o n between the number of compatible p o l l e n g r a i n s d e p o s i t e d on stigmas and the number of seeds s e t f o r i n d i v i d u a l c o l l e c t i o n dates d u r i n g the flowering season (see F i g u r e s 22-27 f o r d a t a ) .  Populat ion  Floral morph  Number of compat i b l e pol1 en g r a i n s per stigma  Seed  set  Percentage  per  capsule  seed  (5.04)  set  pin thrum  113.1 130.2  30 35  7.0 8. 3  (4.43)  33.5 34 . 2  (30.15) (26.31 )  -0.97 -0.55  pin  99.9  71  6.4  (3.97)  25.2  (27.84)  - 0 . 34  thrum  172.6  72  10.3  (7.32)  30. 6  (27.32)  0. 48  p1n  313.5 77.7  31 108  (4.07)  thrum  9.5  (4.79)  71.1 48.8  (24.05) (38.86)  -0.09 0.45  pin  59 .6  74  3O  (2.13)  -0.67  70.5  106  8.0  (5. 19)  10. 1 28 . 1  (20.58)  thrum PC2  pin thrum  38.9 168 . 1  56 25  3.9 16.0  (2.76) (7.07)  7.1 45.7  (18.88) (29.86)  -0.77 -0.60  SL1  pin homosty1e  39.9  86  5.8  (2.12)  34. 1  (38.23)  0.87  104 . 3  61  5. 1  (2.79)  31 .8  (31.23)  -0.87  BL2  BL3  BL4  PC 1  18.8  (32.09)  0. 22  CO  H w o w H  oo CO  SEED SET / 90 seed set (p< 0.001) and this difference corresponded (p<0.05) between  floral  morphs  in the number  to a significant difference  of compatible  pollen grains  received (Table 14). At PC2, thrum seed set was significantly higher (p<0.05) and thrums received at least four times more compatible pollen grains than pins. (The difference in the number  of compatible pollen grains received by the two  floral morphs was not significant, probably as a result of small sample sizes.) At BL2,  PCI  and S L l where there was  no significant  difference between floral  morphs in seed set, neither was the number of compatible pollen grains received by the two floral morphs significantly different. At BL3, seed set was similar for the two floral morphs even though compatible  pollen  population  to that  than  pins.  thnums recieved significantly more (p<0.05)  Comparison  in the similarly  of seed  structured  set in the  pin/thrum  pin/homostyle  population at PC2,  revealed that thrums were more fecund than homostyles (Table 14). Thrum seed set  at PC2  averaged  16.0 seeds per capsule (s.d. = 7.07), and was significantly  greater (p<0.05) than seed set in the homostyles from S L l which averaged 5.1 seeds per capsule (s.d. = 2.79).  In  general, patterns of seasonal  Menyanthes  seed  set in the six populations of  examined were strikingly erratic. Within each  study area, seed set  fluctuated considerably between collection dates. However, among populations and floral  morphs  there appeared  to be a general trend towards lower  towards  the end  of the flowering season.  seasonal  averages  of seed  set and  Although  the number  seed set  the correlation between  of compatible  pollen grains  received was very high, within each population, there was little or no correlation between the number  of seeds set and the number  of compatible pollen grains  S E E D S E T / 91 received on specific dates during the flowering season (Table 14, Figures 22-27). The average number of compatible pollen grains received on each collection date was compared to seed set averages calculated (1) with tagged fruits that failed to set seed (heavy solid line), and (2) without failed fruits (lighter solid line), but neither corresponded very well to the seasonal pollen deposition pattern. The basic difference that appeared between the two of  magnitude.  significant  Pins  from  positive  SLl  correlation  were  sets of seed set calculations was one  the  (r = 0.87,  only  plants  p<0.05)  in  which  between  there  seasonal  was  seed  a set  patterns and the deposition of compatible pollen (Table 14, Figure 27).  Calculation of seasonal averages of percentage seed set for  pins,  thrums  and homostyles of Menyanthes revealed that in the six populations examined, seed set was always below the potential the  highest  percentage considerably  percentage  seed set lower  for  seed all  set  other  maximum averaging plants  in  (Table  14). Pins  71.1% the  averaging 29.9% (s.d. = 12.58). A t  six  from B L 4 had  (s.d. = 24.05). populations  BL4, PCI  However,  studied  was  and P C 2 , there  was a significant difference between pins and thrums in the percentage of seeds set. A t B L 4 , percentage seed set was significantly higher (p<0.01) in pins than in  thrums.  thrums  At  PCI  and P C 2 , percentage  (PCI: p<0.001,  P C 2 : p<0.001). A t  seed set was  significantly  higher  in  B L 2 , B L 3 and S L l there was no  significant difference between floral morphs in percentage seed set.  Results from pollination intensity  experiments (Chapter  3) showed that in  flowers of Menyanthes an average of six compatible pollen grains are required to fertilize each ovule. Comparison of the average number of seeds set on specific  SEED SET / 92 FIGURE 22. The number of seeds set per capsule in pins and thrums of Menyanthes trifoliata in population BL2 during the flowering season of 1985. Solid lines represent average seed set per capsule calculated (1) with fruits that failed to set seed (heavy solid line) and (2) without failed fruits (lighter solid line). N= 10-20 capsules per floral morph for each collection date. Broken line represents average number of compatible pollen grains deposited on stigmas collected on the same dates that flowers were tagged for seed set data. N = 40 stigmas per floral morph for each collection date.  m  E  Pin F l o w e r s  -400  r  tn  i  i_  5/16 19 22 24 2 8 DATE  c 0  1  c o o_  Thrum F l o w e r s  T  1  1  1  r  5/16 19 22 2 4 2 8 DATE  SEED SET / 93 FIGURE 23. The number of seeds set per capsule in pins and thrums of Menyanthes trifoliata in population BL3 during the flowering season of 1985. Solid lines represent average seed set per capsule calculated (1) with fruits that failed to set seed (heavy solid line) and (2) without failed fruits (lighter solid line). N= 10-20 capsules per floral morph for each collection date. Broken line represents average number of compatible pollen grains deposited on stigmas collected on the same dates that flowers were tagged for seed set data. N = 40 stigmas per floral morph for each collection date.  Pin Flowers  fx  E  O)  tn  O  o. a 100 D  in c a> i_ O c  H  0  T  5/1618  1  1  1  1  2 2 2 4 2 8 6/1 4 DATE  1  8  r~ - l —  12  15  -f18  o o_ 600  -  Thrum F l o w e r s  5/16 19  15 18  0  SEED SET / 94 FIGURE 24. The number of seeds set per capsule in pins and thrums of Menyanthes trifoliata in population BL4 during the flowering season of 1985. Solid lines represent average seed set per capsule calculated (1) with fruits that failed to set seed (heavy solid line) and (2) without failed fruits (lighter solid line). N= 10-20 capsules per floral morph for each collection date. Broken line represents average number of compatible pollen grains deposited on stigmas collected on the same dates that flowers were tagged for seed set data. N = 40 stigmas per floral morph for each collection date.  g  300  I"  200  £  100  co  o c O  1 1 1—I r 5/22 24 28 6/1 4 DATE  T — i — r — i — i — i — i — i — r 5/22 24 28 6/1 4 8 11 15 18 DATE  SEED SET / 95 FIGURE 25. The number of seeds set per capsule in pins and thrums of in population PCI during the flowering season of 1985. Solid lines represent average seed set per capsule calculated (1) with fruits that failed to set seed (heavy solid line) and (2) without failed fruits (lighter solid line). N= 10-20 capsules per floral morph for each collection date. Broken line represents average number of compatible pollen grains deposited on stigmas collected on the same dates that flowers were tagged for seed set data. N = 40 stigmas per floral morph for each collection date.  Menyanthes trifoliata  i O c  DATE  o CL  Thrum Flowers  6/3 8  11 15 18 21 DATE  SEED SET / 96 FIGURE 26. The number of seeds set per capsule in pins and thrums of Menyanthes trifoliata in population PC2 during the flowering season of 1985. Solid lines represent average seed set per capsule calculated (1) with fruits that failed to set seed (heavy solid line) and (2) without failed fruits (lighter solid line). N= 10-20 capsules per floral morph for each collection date. Broken line represents average number of compatible pollen grains deposited on stigmas collected on the same dates that flowers were tagged for seed set data. N = 40 stigmas per floral morph for each collection date.  u, E  6/3 8  11 15 18 21 DATE  ro  i_ c O o  Q_  mpati i  Thrum F l o w e r s Cn R  - 20  400o u 300o E  z  200100/•N  u -  / / /  J  /  CL  - 15-g  X  V  ' 1  ->  - 10 n _ T3  1  \ , \ ' \ ;  - 5 v. c 0 R  1  1  1  6 / 3 8 11 DATE  SEED SET / 97 FIGURE 27. The number of seeds set per capsule in pins and homostyles of Menyanthes trifoliata in population S L l during the flowering season of 1985. Solid lines represent average seed set per capsule calculated (1) with fruits that failed to set seed (heavy solid line) and (2) without failed fruits (lighter solid line). N= 10-20 capsules per floral morph for each collection date. Broken line represents average number of compatible pollen grains deposited on stigmas collected on the same dates that flowers were tagged for seed set data. N = 40 stigmas per floral morph for each collection date.  Pin F l o w e r s CO  I/)  c  flj 1_  5/18 21 24 26 DATE  O c  o D.  Homostyle Flowers  XJ  - i — i — i  i  i  5/18 21 24 26 31 DATE  S E E D SET / 98 dates during the grains  flowering  received  revealed  season with the average number of compatible pollen that  on  some  collection  dates,  some  floral  morphs  received fewer than six compatible pollen grains per seed set. This indicates that seed  set  in  Menyanthes may  during part of the flowering  be limited  by  a  lack  of  compatible  pollinations  season. Pins from S L l were the only plants that  received fewer than six compatible pollen grains per seed set during most of the flowering  season.  At were Pin  S L l , pin  and homostyle  hand-pollinated seed  (s.d. = 4.47)  set  in  11.7  compatible  hand-pollinated  compared  naturally-pollinated averaged  with  to  an  flowers. seeds  seed set was much higher pollen flowers  average  For per  of  than  in  averaged 5.8  capsule  (s.d. = 3.26),  flowers  naturally-pollinated 14.3  seeds per  homost3'les, seed set  in  in  seeds  capsule  per  flowers. capsule  (s.d. = 2.12)  hand-pollinated  whereas  that  seed  in  flowers set  in  naturally-pollinated flowers averaged 5.1 seeds per capsule (s.d. = 2.79). Percentage seed set in pins was 47.5 percentage points higher with supplemental pollination averaging 81.6% (s.d. = 21.55) compared to an average of 34.1% (s.d. = 38.23) naturally-pollinated  flowers.  percentage points higher  For  homostyles  percentage  in hand-pollinated flowers  seed  set  was  in  37.8  averaging 69.6% (s.d. = 14.19)  compared to an average 31.8% (s.d. = 31.23) in naturally-pollinated flowers.  Percentage fruit set w as calculated from bulk samples of undehisced fruits. r  At  B L 2 , E L 3 , B L 4 and S L l percentage fruit  morphs  in  each population  (Table  set was similar  15). However, at  PCI  for  both  floral  and P C 2 percentage  fruit set was 2-3 times greater in thrums than in pins. Percentage fruit set was  TABLE 15. Seed s e t . percentage f r u i t set and percentage seed set in p i n s , thrums and homostyles of Menyanthes trifol iat a from nine p o p u l a t i o n s in southwestern B r i t i s h Columbia. Values are the mean and standard deviation ( i n p a r e n t h e s e s ) . Means are c a l c u l a t e d from bulk samples of undehlsced f r u i t s .  Floral Population  morph  Percentage N  fruit  set  Seed set per c a p s u l e  Percentage seed  set  D i morph i c 36.4  (29.51)  39.7  (30.33)  6.1 (10.60) 8.2 ( 1 1 .28)  16.9 27.2  (27.71) (31.00)  63.6  11.8  (12.27)  45.1  (41.11)  78.5  12.6  (9.78)  51.7  (37.02)  29. 1 64 . 1  3.4 9.6  (7.29) (10.78)  11.3 32.3  (23.59) (33.64)  1.7  (5.54)  5.6 (17.13)  8.8  (10.79)  25.7  (29.78)  (6.71) (5.61 )  25.5 18.4  (36.41) (27.67)  pin  248  83.4  9.6  thrum  258  78 . 3  10.5  BL3  pin thrum  306 241  40. 2 58.5  BL4  p1n thrum  173 332  PC 1  pin thrum  726 943  PC2  pin  709  17.9  thrum  120  55.8  pin homostyle  436 472  46.8 38.6  4.4 3.7  BL2  SL1  (8.06) (8.78)  Monomorph i c  CO  W  PC3  thrum  1236  18 .O  0.79  (1.90)  SM 1  thrum  3225  0. 1  0.01  (0.43)  SL2  homosty1e  232  2.0  0.05  (0.20)  3.9  (8.80)  w o CO  w 0.2(1.90)  CO  co  S E E D S E T / 100 highest at B L 2 and lowest in the monomorphic population S M I .  Within each population, seed set averages calculated from bulk samples of undehisced fruits (Table 15) were similar to seasonal seed set averages in tagged flowers for  (Table  14). Sample sizes of bulk seed set were much larger than those  seasonal seed set averages, and revealed some different  number  of seeds set by  the two  floral  morphs  relationships in  in each population. With  the bulk  seed set averages, thrum seed set was significantly greater than pin seed set at BL3  (p<0.05) and P C I (p<0.001). On the other hand, there was no significant  difference in seed set between pins and thrums populations  from B L 4 . For the other  (BL2, PC2 and S L l ) , the relationships between the  three  average number  of seeds set by the two floral morphs were the same as those found in seasonal seed set averages.  Percentage seed set calculated from bulk samples of undehisced fruits was also  similar  to  seasonal  percentage  seed  set  values  and  averaged  27.9%  (s.d. = 13.77) (Table 15). At B L 3 , P C I and P C 2 , percentage seed set (calculated from  bulk  p<0.01,  samples)  PCI:  was  p<0.001,  significantly  higher  P C 2 : p<0.001).  in  At  thrums  SLl  than  percentage  in  pins  seed  (BL3:  set  was  significantly higher (p<0.05) in pins than in homostyles. A t B L 2 and B L 4 , there was no significant difference in percentage seed set between floral morphs.  Bulk  samples  of  undehisced  monomorphic populations: P C 3 , S M I seed  set in  the  monomorphic  fruits  were  also  collected  from  three  and S L 2 . Average seed set and percentage  populations  was  much  lower  than  that  for  the  S E E D S E T / 101 same floral  morphs growing in dimorphic  populations (Table  15). A t P C 3 , S M I  and S L 2 , seed set averaged less than one seed per capsule, and percentage seed set was less than 4%.  D. DISCUSSION  There are conflicting reports in the literature concerning pollen limited seed set  in  distjdous  Lithospermum  plants.  In  some  caroliniense (Weller  1980),  Palicourea fendleri (Sobrevila et al Linum  tenuifolium (Nicholls  species {Pulmonaria Cratoxylum  obscura (Ornduff formosum  (Lewis  1983), Hedyotis caerulea (Ornduff  1979), 1982),  1980b) and  1986)}, seed set is limited by a lack of compatible  pollinations. But, in other species seed set appears to be limited by factors other than  pollen  grandiflora  (Schou  availability. (Ornduff  1983)},  In  populations  1976),  seed set  Cryptantha  was far  of  flava  below  three  distylous  (Casper  the  potential  1983)  species {Amsinckia and  Primula elatior  maximum.  However,  the  average number of compatible pollen grains deposited on stigmas of these species was higher than the number of ovules per ovary. Casper (1983) suggested that in Cryptantha flava, embryo abortion was responsible for the observed low seed set.  In  the  six  dimorphic  always below the potential (calculated from bulk Among  populations,  populations  of  Menyanthes studied,  was  maximum. The percentage of seeds set per capsule  samples of undehisced fruit) there  seed set  was  a  very  high  averaged 27.9% (s.d. = 13.77).  correlation  between  the  average  number of seeds set by each floral morph during the whole flowering season and  SEED SET the average number of compatible within most populations deposition  on  specific  deposition and  dates  during  no the  stigmas. However,  correlation between seed set and pollen flowering  season. Because  both pollen  seed set fluctuated so erratically during the season, the average  number of compatible was  there was  pollen grains deposited on  / 102  pollen grains received per seed set (on each collection date)  highly variable. Within each population, seed set appeared to be limited by  pollen availability on some dates but not on others. But  the lack of correlation  between  patterns  daily  seed  set  patterns  and  pollen deposition  makes  these  results difficult to evaluate.  Pins from  SLl  were the only plants in which there was  positive correlation between the daily seed set pattern and deposition pattern. The seed  set in pins  measured. This  from  average number of compatible SLl  was  less  than  six on  suggests that seed set in these  and  a significant  the daily pollen  pollen grains received per four  plants was  out  of five dates  limited by pollen  availability. Results from hand-pollinations of pins and homostyles at S L l support this conclusion. Seed set in pin flowers from S L l that were hand-pollinated with large amounts of compatible  pollen was  47.5  percentage points higher than seed  set in naturally-pollinated flowers. Similarly, seed set in homostyle flowers from SLl  that were hand-pollinated was  37.8  percentage points higher than seed set  in flowers that were naturally-pollinated on the same days. Yet, comparison of daily seed set and  pollen deposition patterns in homostyles revealed no apparent  pollen limitation.  In  populations of Hedyotis caerulea (Ornduff 1980b) and  Linum tenuifolium  SEED SET (Nicholls 19S6), there was  a considerable amount of variation in the number of  pollen grains deposited on individual stigmas. In Hedyotis caerulea, up  compatible to 21%  of the stigmas  species, seed  set was  held no limited  pollen at all (Ornduff  by  flowers, but in others there was ovules. Although examined, the suggests  / 103  pollen  erratic  loads  a  lack of compatible  of daily  stigmas  pollen  that in Menyanthes there ma}'  number of compatible  pollinations  two  in some  ample pollen available for the fertilization of  of individual  nature  1980b). In these  be  flow a  of Menyanthes were  not  patterns during the  season  great deal of variation  in the  pollen grains deposited on individual stigmas. Even though  seed set data were collected from flowers pollinated on the same days as flowers collected for pollen flow data, the composition of their stigmatic pollen loads have been  very  received very few  different. If seed compatible  set data  were collected  pollen grains, and  from  may  flowers that  pollen flow data were collected  from flowers that received an ample number of compatible  pollen grains, pollen  flow data would not correlate very well with seed set data. It is possible that in populations of Menyanthes, the lack of correlation between seed set and  the  number of compatible pollen grains received. on individual collection dates is partly a  result  of variation  in the  composition  of individual  stigmatic pollen loads.  Results from hand-pollinations at S L l indicate that pollen availability may had  have  a greater influence on seed set in homostyles than daily patterns of pollen  deposition and seed set suggest.  Even  though  seed  set  in  hand-pollinated  flowers  of Menyanthes  was  substantially higher than seed set in naturally pollinated flowers, maximum seed set  was  never  obtained  in  supplementally  pollinated  flowers.  The  highest  S E E D S E T / 104 percentage seed set that was obtained with hand pollination was 95%. However, percentage  seed set in  and in homostyle  hand-pollinated  flowers  averaged  pin  flowers  averaged 81.6% (s.d. = 21.55)  69.6% (s.d. = 14.19).  Supplemental  pollination  has been found to increase seed set in two other distylous species, Lithospermum caroliniense (Weller  1980)  and  Palicourea fendleri  (Sobrevila  Lithospermum caroliniense, seed set was increased by supplemental  pollination,  but,  et  al.  1983).  19 percentage points  as in Menyanthes, maximum  In with  seed set was  never  obtained. Because many factors have been shown to influence seed set in plants, it  is  probably  Menyanthes compatible  unrealistic  there  is  pollen  to  a  grains  think  strong  that  only  correlation  received  and  one  factor  between  average  limits  the  average  seed set,  and  population a lack of compatible pollen seems to be a major set.  However,  it  seems evident  that  factors  other  than  seed  in  at  factor  pollen  set.  In  number  of  least  one  limiting seed  availability  are  influencing seed set in Menyanthes trifoliata.  Comparison populations  of  True  genetic  (Ganders  have  stamens  morph.  Long  homostyles are  set  1979). of one  homostyles  have pin  morphologically  homostyles, their  in  monomorphic  homostyles  morphological  supergene the  seed  Menyanthes  self-incompatible homostyles.  of  suggests  homostyles,  homostyles  result  have  stamens equivalent  morph, thrum  to  long  a  are  and the  pistils.  and  self-compatible in  self-compatible  and  pin  of  the  are  genetic distyly  because  they  the  opposite  floral  pistils,  whereas  short  Stump  Lake  Homostyles If  (SLl)  homostyles  crossover  pistils  homostyles.  dimorphic  these  than  from  stamens  and thrum  that  rather  Genetic homostyles floral  (SL2)  they  stigmas would have the same compatiblity  from were  recombinant  relationships as pin  S E E D S E T / 105 stigmas.  Pin pollen  would  be incompatible  on their  stigmas.  Only  pollen  thrum flowers or from their own anthers would be compatible on their and  self-pollination  homostyle  stigmas  would to  result  their  in  own  substantial  anthers  seed  would  set.  almost  from  stigmas,  The  proximity  guarantee  that  of  some  self-pollination would occur.  Examination extremely  of  stigmatic  pollen  high numbers of own-form  loads  of  homostyles  from  SLl  revealed  pollen grains. Presumably, homostyles  from  S L 2 , a population consisting entirely of homostyles, would receive similar numbers of own-form pollen grains. Yet, seed set in SL2 averaged 0.05 seeds per capsule (s.d. = 0.43).  At  S L l , where homostyles were growing with pins, homostyle seed  set averaged 5.1 seeds per capsule (s.d. = 2.70). These results indicate that Stump Lake  homostyles  pollen)  for  are  seed set  hand-pollination  self-incompatible to  with  occur.  large  and  Higher  amounts  require  seed of  set  pin  intermorph in  SLl  pollen  pollen  flow  homostyles  supports  this  (pin  following conclusion.  Homostyle stigmas from Stump Lake have the same compatiblity  relationships as  thrum  appear  flowers.  self-incompatible.  They  are  compatible  These  observations,  with  combined  pin with  pollen  and  the  observed  to  morphological  variation in Menyanthes flowers, support the view that the homostyles from are  not  true  genetic  homostyles,  but  self-incompatible  be  morphological  SLl  homostyles  that are equivalent to thrums with unusually long styles.  Comparison of seed set in the pin/homostyle population at S L l to that in the  similarly  structured  pin/thrum  population  at  PC2 shows  more fecund than homostyles. Seed set in PC2 thrums  that thrums  were  averaged 16.0 seeds per  S E E D S E T / 106 capsule  (s.d. = 7.07),  compared  to  an  average  of  5.1  seeds  set  per  (s.d. = 2.79) in S L l homostyles. These results agree with those found for  capsule thrums  and homostyles of Mitchella repens (Ganders 1975a). In Menyanthes, there was a high correlation between the average number of compatible pollen grains deposited on stigmas and average seed set. Average seed set in homostyles was higher in flowers that were hand pollinated with large amounts of compatible pollen than in naturally  pollinated flowers. This indicates that seed set in homostyle flowers  was limited to some degree by a lack of compatible pollinations. The higher seed set in PC2 thrums corresponded to the receipt of a higher number of compatible pollen  grains;  homostyles.  PC2  thrums  These results  received  support  1.6  times  Gander's (1979)  more  compatible  hypothesis that  pollen  than  morphological  distyly is associated with greater fecundity, and demonstrate that higher seed set in  thrums  compared to  homostyles resulted from thrums  compatible pollen grains than homostyles.  having received more  VI. CONCLUSIONS  This study was  designed  to investigate  the distylous breeding system of  Menyanthes trifoliata in order to determine (1) the effect  of variation in morph  frequency on pollen flow of a distylous plant, (2) whether morphological distyly increases  the  amount of compatible  self-incompatible  plant,  and  if so  pollen deposited  (3)  whether  on stigmas  an increase  of a diallelic  in the  amount of  compatible pollen received is associated with an increase in fecundity.  In  two out of three populations  examined (Beaver  Lake and Pinecrest),  flowers of Menyanthes trifoliata exhibited the typical floral dimorphisms associated with  a  distylous  breeding  system.  Stigmas  and  anthers  were  reciprocally  positioned in the two floral forms, and thrum pollen was significantly larger than pin  pollen.  dimorphic than  Other  in  some  floral  characters  distylous  such  species,  as  displayed  anther more  length  and  petal  length,  interpopulational variation  intermorph variation in flowers of Menyanthes. A series  of experimental  pollinations revealed that in Menyanthes, morphological distyly is associated with a strong self-incompatibility system,  and that an average of six compatible pollen  grains are required to fertilize each ovule.  The and  long  third population of Menyanthes studied (Stump Lake) consisted of pins homostyles.  Although  these  homostyles  were  not  tested  for  self-compatibility  through self-pollination, other evidence suggests that they were  self-incompatible  morphological  homostyles  rather  than  self-compatible  genetic  homostyles. If the homostyles at Stump Lake resulted from a genetic crossover  107  C O N C L U S I O N S / 108 in  the  distyly  supergene,  their  stigmas  would  have  the  same  compatibility  relationships as pin stigmas, and would be compatible with pollen from their own anthers  (Ganders  1979).  Examination  of  homostyle  stigmatic  pollen  Menyanthes revealed that they receive a high number of own-form probably then  the  as a result high  of  number  self-pollination. of  own-form  If  the  pollen  homostyles  grains  were  of  homostyles  averaged  0.05  seeds  self-compatible,  deposited on stigmas  per  capsule  in  pollen grains,  result in substantial seed set. Nevertheless, seed set in a population entirely  loads  should  consisiting  (s.d. = 0.20).  In  comparison, homostyle seed set in the pin/homostyle population averaged 5.1 seeds per capsule (s.d. = 2.79); this indicates that intermorph seed set. Increased homostyle  pollen flow is required for  seed set following hand-pollination  with pin  pollen  (11.7 seeds per capsule, s.d. = 3.26) supports the view that the homostyle stigmas have the  same compatibility  self-incompatible  relationships  as thrum  stigmas, and are  homostyles  equivalent  to  morphological  thrums  with  therefore unusually  long styles.  An  average  of  1%  of  the  pollen  produced  in  populations  of  Menyanthes trifoliata is deposited on conspecific stigmas.  Comparison of seasonal  pollen  of  flow  patterns  in  the  six  dimorphic  populations  Menyanthes studied,  revealed that in general, pollen flow is very erratic. Within each population, the size and composition of stigmatic flowering  season.  Pins  and  pollen loads fluctuated  thrums  experienced  considerabty during  random,  disassortative  the and  assortative pollination at different times during the flowering season. The level of disassortative assortatively  pollination pollinated  varied on  all  from  16.7% -  dates  measured,  65.9%.  Homostyle  probably  as  a  stigmas result  of  were the  C O N C L U S I O N S / 109 proximity of their stigmas to their own anthers.  Within each population, floral morph frequency (or more accurately, pollen morph  frequency)  had a very  strong influence  on the  composition of  stigmatic  pollen loads averaged for the whole season. The percentage of compatible pollen deposited  on  homostyles)  stigmas  was  of  highly  both  floral  correlated  morphs  (r = 0.93,  in  each  p< 0.001)  population  with  compatible pollen produced in the population. However this proportional. slightly  Within  more  produced  in  each population,  the  more  abundant  the  (excluding  percentage  of  relationship was not floral  morph  received  compatible pollen than  expected based on the freqeunc3' of pollen  the  is  population.  This  significant  because  it  suggests  that  morphological distyly compensates for the rarity of one floral morph bj' increasing the  proportion  of compatible  pollen received by  the  opposite floral  morph.  The  actual number of compatible pollen grains deposited on stigmas did not correlate as  strongly  probably  with  resulted  pollen in  frequency  part  from  in  the  population  differences  between  (r=0.69,  floral  p<0.05).  morphs  in  the  This total  number of pollen grains deposited on stigmas.  Comparison of stigmatic pollen loads from the pin/homostyle population to those of thrums thrums  received  from a similarly a  much  structured  higher  percentage  pin/thrum population revealed that of  compatible  pollen  than  the  homostyles. Thrums received an average of 69.2% (s.d. = 31.60) compatible pollen, whereas homostyles received an average of 24.0% (s.d. = 30.30) compatible pollen. The  difference  between  homostyles  and  thrums  in  the  actual  number  of  compatible pollen grains deposited on stigmas was not as great. Thrums received  CONCLUSIONS / 110 an average of 1.6 times more compatible number of incompatible times  greater than  pollen than homostyles. However, the  pollen grains deposited on homostyle stigmas  the number  received by thrums. Except  was  4.4  for differences in  style length, homostyle flowers were morphologically identical to thrum flowers. Both  grew  in populations  differences observed pollen  with  a  similar  morph  frequencies. Therefore, the  between thrums and homostyles in composition  loads can be attributed  to the presence  of the reciprocal  of stigmatic stamen/style  dimorphism in the pin/thrum population.  In populations of diallelic self-incompatible plants where there are only two mating groups, the chances of a stigma Darwin (1877) hypothesized and  anthers  that because of the reciprocal placement of stigmas of compatible  pollen  the amount of compatible  pollen  received by a homomorphic diallelic self-incompatible plant. Results from my  study  deposited  of  on stigmas  pollen  support distyly  in flowers  receiving incompatible pollen are great.  flow  of this  of distylous  would  plants, the amount  be greater than  in Menyanthes trifoliata provide hypothesis.  In some  increases the amount  some  populations  of compatible  quantitative evidence in  of Menyanthes,  pollen  deposited  on  morphological stigmas, and  reduces the amount of incompatible pollen received.  Among the six dimorphic very  high  compatible set. seed  correlation  (r = 0.88,  populations of Menyanthes studied, there was a p< 0.001) between  pollen grains deposited on stigmas  the  average  number  of  and the average number of seeds  However, within each population there was little or no correlation between set and the number of compatible  grains received on individual collection  C O N C L U S I O N S / 111 dates during the  flowering  season. Pins from S L l were the only plants in which  there was a significant positive correlation between the two (r = 0.83, p<0.05).  In  the  six  dimorphic  populations  of  Menyanthes studied,  seed set  was  always below the potential maximum. A n average of 27.9% (s.d. = 13.77) of the ovules in  each ovary  were  fertilized.  Comparison of the  number  of  compatible  pollen grains received to the number of seeds set revealed that on some dates during  the  flowering  season,  fewer  than  six  compatible  pollen  grains  were  received per seed set. This indicates that, in populations of Menyanthes, seed set may  be  limited  by  pollen  availability  during  part  of  the  flowering  season.  However the lack of correlation between seed set and the number of compatible pollen  grains  received on  individual  dates  during  the  flowering  season  makes  evaluation of these results difficult.  At S L l , pins and homostyles were hand-pollinated with large amounts of compatible pollen on the same dates in which seed set data were collected from naturally-pollinated flowers. Seed set in hand-pollinated flowers was an average of 42.7  percentage  points  higher  than  seed  set  in  naturally-pollinated  flowers  indicating that seed set was limited by pollen availability in this population. For SLl  pins these results  are not  surprising because comparison of their seasonal  seed set and pollen deposition patterns revealed that they received fewer than six compatible pollen grains per seed set during most of the  flowering  season. For  homostyles a similar comparison revealed that homostyle seed set was not limited by pollen availability. Results from hand-pollinations show that pollen may  have  had  a  greater  influence  on  seed  set  in  homostyles  availability than  the  C O N C L U S I O N S / 112 comparison suggested between  of  seasonal pollen  that  in  seed  individual variation  populations  set  and  collection in  the  the  dates  of  during of  and  seed  set  patterns  Menyanthes trifoliata,  number  composition  sampling techniques may even with  flow  the  of  compatible  flowering  individual  have contributed  supplemental pollination,  pollen  season  stigmatic to  this  the  is  pollen  indicate. lack  of  I  have  correlation  grains  received  on  partly  a  of  result  loads. In  addition,  lack of correlation. However,  seed set in pins and homostyles from S L l  never reached its potential maximum, and this suggests that in addition to pollen availabilty, other factors influenced seed set in populations of Menyanthes.  Comparison of homostyle seed set in the pin/homostyle population to thrum seed  set  in  the  pin/thrum  population  showed  that  the  higher  number  of  compatible pollen grains, deposited on thrum stigmas was associated with higher fecundity.  Thrums  set  an  homostyles. Ganders (1974) fecundity  of  a  diallelic  average  of  10.9  more  seeds  per  capsule  hypothesized that morphological distyly  self-incompaitble  plant  by  increasing  compatible pollen grains deposited on stigmas. Results from my  the  increases the number  of  stud}' of pollen  flow and seed set in populations of Menyanthes provide quantitative support of this hypothesis. In  than  evidence in  populations of Menyanthes trifoliata, the reciprocal  placement of stigmas and anthers  in distylous flowers increases the amount  of  compatible pollen deposited on stigmas, and this increase in compatible pollen is associated with higher seed set.  This investigation has provided considerable quantitative  information on the  influence of morphological distyly and variation in morph frequency on pollen flow  CONCLUSIONS / 113 and  seed  patterns The  set. In addition, this study have been examined  is the first in which  at regular  intervals during  methods used in this study have revealed  in populations of distylous plants. In future placed  on  the investigation  of this  deposition  the flowering season.  the erratic nature of pollen flow studies, more emphasis should be  phenomenon  understanding of the distylous mating system.  pollen  in order  to improve our  BIBLIOGRAPHY Akamine, E . K . , and G. Girolami.  1959. Pollination  and fruit set in the yellow  passion fruit. Tech. Bull. Hawaii Exp. Stn. No. 39. Baker, H . G . 1959. The contribution  of autecological and genecological studies to  our knowledge of the past migrations of plants. Amer. Nat. 93: 255-272. Barrett,  S.C.H.  1977. 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