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Morphological variation in a biotically patchy environment : evidence from a pasture population of Trifolium… Evans, Richard C. 1986

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MORPHOLOGICAL BIOTICALLY  VARIATION IN A  PATCHY  ENVIRONMENT:  E V I D E N C E F R O M A P A S T U R E P O P U L A T I O N O F TRIFOLIUM  REPENS  by R I C H A R D C. E V A N S A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY  OF GRADUATE  STUDEES  Botany Department  We accept this thesis as conforming to the required standard  THE UNIVERSITY  O F BRITISH  COLUMBIA  October, 1986 © Richard C. Evans, 1986  L.  In p r e s e n t i n g  this thesis  r e q u i r e m e n t s f o r an of B r i t i s h it  Library  shall  for reference  and  study.  I  for extensive copying of  for  h i s or  her  copying or  f i n a n c i a l gain  be  g r a n t e d by  s h a l l not  be  _ Botany of  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada  V6T 1Y3  15  nr.tnhPr,  1 Qfifi  of  Columbia  make  further this  thesis  head o f  this  my  It is thesis  a l l o w e d w i t h o u t my  permission.  Department  the  representatives. publication  the  University  the  f o r s c h o l a r l y p u r p o s e s may understood that  the  I agree that  agree t h a t permission d e p a r t m e n t o r by  f u l f i l m e n t of  advanced degree a t  Columbia,  freely available  in partial  written  ABSTRACT The  relationship  heterogeneity  between  was studied  morphological  variability  in a pasture  population  and  biotic  of Trifolium  environmental repens. It had  been argued that the unexpectedly high levels of variation in T. repens could be maintained by diversifying selection. The mosaic of neighbours (perennial grasses) with which T. repens coexists constitutes a prominent element of biotic patchiness that  may  lead  to  sorting  T.  among  repens  genotypes  on  the  basis  of  neighbour-specific compatibilities.  A  variation  study  was conducted  on a set of 400 individuals  of T. repens  collected on a neighbour-specific basis from a 43 year old pasture and grown for one  season  under  common  garden  conditions.  A  significant  proportion  of the  variation in a set of twelve morphological characters was accounted for by the neighbour with which the individual of T. repens had been growing in the field. The actual amount of variation accounted for, however, was low (6-19%). It was concluded that although diversifying selection could be operating in the pasture, it is not of primary importance in the maintenance of variation in this population.  A  repeat  study  was carried out after  common garden. None of the earlier were  retained.  I  concluded  that  the plants had spent two years among-neighbour  the original  differences carried over from the pasture.  ii  differences  results  reflected  in the  in morphology developmental  TABLE OF CONTENTS Abstract  ii  List of Tables  iv  Acknowledgements  v  Preface  vi  I. Overview  1  A. Variation in Trifolium repens  B. C. D. E. F.  Genotype dynamics and variation in clonal populations Environmental heterogeneity and diversifying selection Environmental heterogeneity in pastures The clonal growth habit and environmental heterogeneity Diversifying selection in the pasture  II. Common garden study, 1982 A. Materials and Methods 1. The Pasture 2. Collection and Propagation of Material 3. Assessment of variation B. Analyses and results C. Discussion III. Common garden study, 1984 A. Carry-over effects and the common garden method B. Materials and methods C. Analyses and results D. Discussion IV. Diversifying selection and Trifolium repens: a reconsideration References  1  2 6 8 12 15 17 17 17 20 22 25 37 47 47 51 53 60 66 72  iii  List of Tables Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table  I. Composition of the "Highland Forage M i x " used in seeding a pasture in 1977 18 II. List of species present in the study site, a 42 year old pasture in the lower Fraser Valley in British Columbia 19 H I . List of characters measured in the 1982 common garden study of variation in a pasture population of Trifolium repens 23 IV. Stratification of the sample population of Trifolium repens by original neighbours 25 V . Tests of homogeneity of variances for twelve morphological characters of Trifolium repens. Raw and log transformed data from the 1982 common garden study. ... 27 V I . Summary of analyses of variance for 12 morphological characters from four neighbour-specific groups of Trifolium repens. Data from the 1982 common garden study 29 VII. Variance components for four morphological characters from a pasture population of Trifolium repens. Data from the 1982 common garden study 31 VIII. Correlations among 12 morphological characters of Trifolium repens. Data from the 1982 common garden study 32 I X . Principal components analysis of 12 morphological characters of Trifolium repens. Data from the 1982 common garden study 35 X . Summary of analysis of variance of principal component scores for four neighbour-specific groups of Trifolium repens. Data from 1982 common garden study 35 X I . List of characters measured in the 1984 common garden study of variation in a pasture population of Trifolium repens 52 X I I . Summary of analyses of variance for 12 morphological characters from four neighbour-specific groups of Trifolium repens. Data from the 1984 common garden study 54 XIII. Variance components for four morphological characters from a pasture population of Trifolium repens. Data from the 1984 common garden study 55 X V . Principal components analysis of 12 morphological characters of Trifolium repens. Data from the 1984 common garden study 58 X V I . Summary of analysis of variance of principal component scores for four neighbour-specific groups of Trifolium repens. Data from 1984 common garden study 59  iv  ACKNOWLEDGEMENTS I  would  like  to  extend  my  sincerest  thanks  to  my  supervisor,  Dr.  Roy  Turkington for his guidance and enthusiasm. I  am  also grateful to my commitee  members; Dr. Jack  Maze  (scepticism)  Dr. Judy Meyers (optimism) for playing the antipodes of ecology, and Dr. Ganders  for  his  part  in  the  final  acts  and  for  his  tolerance  of last  and Fred  minute  impositions. I  wish  notably  to  acknowledge  Lonnie  the  Aarssen,  counsel  Roberta  and encouragement Parish,  Loyal  of my  Mehrhoff,  Rob  fellow  students,  Scagel,  John  Spence, and Gary Armstrong. The financial support provided by Dr. Turkington and by the Botany  Department  was greatly appreciated. I also wish to thank  Mr.  Bill  Chard and Ms. M a r y  Chard for the access to  their pastures. Finally,  I am especially  grateful to my  and perseverance.  v  wife, Janet,  for  her continued  support  PREFACE An  important area of emphasis  Turesson  1922)  populations Walters  (see  has  reviews  at  the  level  populations (Harper stemmed  of  study  of  genetic  Heslop-Harrison  the  Langlet  in  (since  species  1971;  and  Briggs  &  attention has been drawn to the relationships  between  individuals  level  desire  speciation). The synthetic  variation  1964;  and  patterns  of  1967, 1982; Dirzo & Sarukhan  from  phenomena may  the  by  1984). Recently,  events  has  been  in plant ecology over the last 60 years  to  understand  variation  at  the  1984). Much of this interest  evolutionary  patterns  theory of evolution holds that large scale  be explained  by  reference  to processes  (especially evolutionary  affecting individuals  small populations. This postulated connection between population-level events species-level patterns has never been clearly  of  established, and is currently  in and  under  debate.  There are, however, clear connections between individual-level events and patterns of local 1976,  (micro-)evolution  1984;  proposed  to  migration) variation be  more  species.  Hamrick account  can  also  (Jain  &  Bradshaw  1982; Turkington for  among-species  be  applied  to  within populations. Features convincingly Population-level  tied  to  &  1966; Bradshaw Aarssen  patterns  the  (natural  organization  of populations  individual-level  demography  and  1984).  and  Processes  dynamics  and communities  thus  Antonovics, originally  selection, genetic  processes  genetics  1972;  of  drift, genetic  can  often  than  can  features  of  retain  their  interest  for  ecologists despite the lack of a definite linkage with species-level evolution.  Much  of  the  interest  in  genetic  variation  vi  in  populations  has  centered  on the  amount of variation and its maintenance (Lewontin 1974a; Brown 1979; Hamrick, Linhart & Mitton 1979; Ennos 1983). Spieth (1979), for example has referred to the explanation of the high levels of variation within populations as "the central problem in population genetics". A potential contributing factor i n the maintenance of  variation  involves  environmental  (Hedrick, Ginevan & Ewing attention because  heterogeneity  1976; Ennos  it is clearly  and diversifying  selection  1983). Diversifying selection has drawn  involved in the fine-scale differentiation  of plant  populations distributed across sharp environmental transitions such as from heavy metal polluted soil near mines to non-polluted pastures (Jain & Bradshaw 1966; Antonovics, Bradshaw & Turner  1971). Even finer scale differentiation  associated  with mosaic environments has also been demonstrated (Snaydon & Davies 1972, 1976, that  1982; Turkington & Harper environmental  heterogeneity  1979c). These  and diversifying  results  support the suggestion  selection  might  be commonly  involved with the maintenance of variation in plant populations.  Diversifying  selection  has been  specifically  invoked  as  a  possible  general  explanation for the maintenance of variation in populations of Trifolium repens in permanent because  pastures  the high  (Burdon levels  1980a).  of variation  These  populations  recorded  in them  have  been  appear  of interest  to contradict  expectations for variation in primarily clonal populations.  The work to be reported here investigates the possibility that variation for a set of morphological characters in a pasture population of T. repens is maintained by diversifying selection associated with a mosaic of patches of grass. In the first part  of the thesis,  the arguments  leading to the prediction of maintenance of  vii  variation  by  diversifying  selection in  T.  repens are reviewed. The  results  of a  common garden study of the relationship between morphological variation and the distribution  of  the  grasses  is  then  reported.  The  second  part  of  the  thesis  investigates the possibility that the patterns of variation detected in the common garden represented developmental effects carried over from the field rather  than  genetically based variation. Finally, some of the original arguments on diversifying selection in T. repens populations are reconsidered.  viii  I. OVERVIEW  A.  VARIATION  IN TRIFOLIUM REPENS.  Trifolium repens L., white clover, has been of widespread interest to population biologists (Turkington & Aarssen 1984; Turkington 1985). This common component of pastures and lawns has been extensively importance  as a forage species (Lowe  studied because of its economic  1970; Wilson 1978; Sackville-Hamilton  1980). Many of these studies have considered within-population variation, and have consistently shown that pasture populations of white clover contain genetic or quantitative variation for a wide array of characters (Burdon 1983; Turkington & Burdon 1983). Characters studied have included several polymorphic traits such as leaf marks (Brewbaker Harper  1976), cyanogenic  1955; Carnahan et al. 1955; Corkill 1971; Cahn & glycosides (Corkill 1952; Jones 1966; Angseesing &  Angseesing 1973), incompatibility alleles (Atwood 1942), and isozymes (Gliddon & Trathan 1985). Quantitative variation has beeen shown for physiological characters including disease resistance (Burdon 1980a,b), susceptibility to Rhizobium infection (Mytton 1975), and response to nutrients (Snaydon 1962a; Snaydon & Bradshaw 1962b, 1969). Common garden studies have shown variation in morphology for numerous characters, and in phenology and reproduction (Burdon 1980a; Aarssen &  Turkington  1985c).  performance-related (Burdon  1985c)  variation  has  also  been  shown for  characters such as growth rate, rate of stolon  and Harper  Turkington  Quantitative  1980),  flower  and response  to  production growth  (Burdon  with  1980a;  different  Aarssen &  pasture  (Turkington & Harper 1979c; Turkington 1979; Gliddon & Trathan 1985).  1  extension  grasses  Overview / 2 The demonstration that a particular plant population is highly variable should not be  surprising.  Reviews  shown that plants broad  groups  Hamrick,  of reported  variation  have  as a group contain as much or more variation than  other  of organisms  Linhart  &  such  Mitton  levels  of genetic  as vertebrates  1979; Hamrick  highly  variable  widespread Its  populations.  geographical  mode  Trifolium  distribution  of reproduction  or invertebrates  1982).  comparisons it might be predicted that Trifolium  From  (Brown 1979;  similar  coarse-level  repens, in particular, would have  repens is  (Burdon  (allozyme)  a  long-lived  1983; Turkington  is outcrossing enforced  perennial  with  & Burdon  by self-incompatibility  a  1983). (Atwood  1942). These characters have been consistently associated with the most variable of plant species (Hamrick, Linhart & Mitton 1979; Loveless & Hamrick  Variation predict  T.  in that  variability.  a  repens has drawn clonal  Therefore,  perennial  attention  living  in  because,  pastures  theoretically, would  have  1984).  we might low  the observed high levels of variation in these  genetic  populations  require either a special explanation or modifications to the prediction.  B. GENOTYPE  DYNAMICS  One of the characteristic dominant cloning.  plants  have  Vegetative  AND VARIATION features  of pasture  a well developed  reproduction  among  IN CLONAL communities  capacity pasture  tillering as in many tufted grasses (e.g., Lolium and  stolon  or rhizome  repens, Ranunculus of these  plants  formation  POPULATIONS. is that most of the  for vegetative plants  takes  reproduction or  two basic  forms,  perenne and Dactylis glomerata)  as in creeping  herbs  and grasses  (e.g.,  T.  repens, and Poa compressa). Studies of the population dynamics  have  shown  that  cloning  represents  the predominant  mode of  Overview / 3 reproduction. Seedling establishment is rare in the closed pasture except on disturbances  (Harberd  vegetation  1961a; Turkington et al. 1979; Parish, in  Turkington 1985). Generally, the establishment of clonally produced copies (ramets) of existing genetic individuals (genets) vastly exceeds the establishment of new sexually produced genets. These observations lead to a common assumption that the input of new genets has a minor effect on the population biology of clonal perennials such as T. repens in established pastures.  Contingent on the validity of this assumption, some predictions about genotype dynamics can be made. In recently established pastures, clones will be small and numerous and clonal diversity will be high. Maintenance of this diversity, as the pasture ages, will be a function of the persistence of members of the original cohort.  Although each genet may, in theory, have an indefinite lifespan, in  reality, its persistence will depend on the continual establishment of new ramets as existing ramets senesce. Ramets of pasture perennials typically have short lifespans,  <  1 year  in T. repens (Chapman  1983;  Thorhallsdottir  1983;  Turkington 1983a) and 1.2-2.1 years in Ranunculus repens (Sarukhan & Harper 1973). Because of this rapid turnover of ramets, the sizes (numbers of ramets) of clones will be subject to rapid fluctuations. Any condition which depresses ramet production within a clone can cause an immediate decline in the size of a clone. Continued low ramet production will eventually lead to death of the clone.  In an older pasture, populations of ramets may be regulated at constant densities (Harper  1977).  This  has been  noted  Ranunculus repens (Sarukhan & Harper  for the clonal 1973)  grassland  perennials  and Prunella vulgaris (Schmid &  Overview / 4 Harper  1985).  expansion  If  in size  the entire of some  ramet  clones  population  must  then  is  under  density  be accompanied  regulation,  by declines in  others. A n y differences among genets in competitive ability, as demonstrated for T. repens by Aarssen (1983), may find expression as differences in relative sizes of clones. Eventually, a few vigorous clonal families might steadily expand at the expense of less competitive lines and come to dominate the population. A s less competitive clones decline in size, some will certainly be lost from the population. Such clonal dominance has been predicted on theoretical grounds (Williams and  might  be particularly  likely  in pasture  vegetation  which  1975),  is expected to  commonly experience density limitation (Donald 1963; Snaydon 1978).  Even  without  competition  from  the random  simply  a certain base failure  rate  of genets  of extinction  to replace  might  themselves.  simulation based on demographic data for the clonal Ranunculus  be expected A  computer  repens predicted  an exponential decline in the number of original genets in the population in the absence  of competition  through  stochastic  seedling  recruitment,  (Soane  or directional the  &  Watkinson processes,  genotypic  1979). if they  diversity  of  Whether  genets  are not replaced  the  population  will  are lost through become  progressively depleted, eventually consisting of a few very old, very large clones (Harberd 1961a, 1962, 1963).  Numerous  studies on population dynamics of pasture perennials have documented  a steady, often exponential decline in numbers of genets within a cohort (Langer, Ryle & Jewiss  1964; Antonovics  1972; Kays  & Harper  1974). Langer, Ryle &  Jewiss (1964) observed ramet and genet dynamics in swards of Phleum pratense  Overview / 5 and Festuca pratensis for three years  after establishment  and noted that  "..the  swards, which initially  contained many plants with few tillers, were changed to  swards  with many  have  of few plants extremely  including  sand  low genotypic dune  tillers".  diversity.  populations  In addition,  A series  of T.  some  of studies  repens, (Harberd  clonal  populations  on clonal  1961a,  species,  1962,  1963)  demonstrated population dominance by a small number of widely distributed clones of  great  age. Similar (Oinonen  Pteridium  low diversity  clonal  1967) and Spartina  populations  (Silander  have  1979).  been  reported in  Comparisons  among  populations from pastures of different ages suggest that genotype depletion is a continuing  process.  McNeilly  and Roose  (1984)  found  36-43  different  isozyme  genotypes of Lolium perenne per 0.25 m in three ten year old pastures but only 2  5 genotypes per 0.25 m in a 40 year old pasture. Clearly, clonal populations in J  some  circumstances  do exhibit  genotypic  depletion.  Such  depletion  should be  reflected in overall levels of genetically determined variability, including the types of characters which have been studied for T. repens.  The observation of high levels of variation in white clover populations, however, has  been interpreted  (Harper  1978; Burdon  as contradictory  to the expectation  of genotypic  depletion  1980a; Biilow-Olsen, Sackville-Hamilton & Hutchings  1984).  Studies on clonal diversity in British pastures have found no evidence that clonal depletion  is a dominant  polymorphism, distribution frequency  found  of these clones.  trend. Cahn  at least morphs  3-4 morphs  showed  In the same  and Harper  mark  per 100 sq cm. The frequency  no evidence  pasture,  (1976), using the leaf  Gliddon  of dominance by a few high and Trathan  (1985)  identified  48-50 isozyme phenotypes per square metre. Burdon (1980a) found that each of  Overview / 6 50  ramets  collected  from  the same  pasture  were  phenotypically  distinct and  concluded that each represented a different genet.  Aarssen four  and Turkington  pastures  variation  were  declined  across  in British again  (1985c) Columbia  high,  morphological  variation  in a series of  (aged 4, 23, 42, and 67 years). Levels of  although  the age series  declines in variances  studied  variances  for both  for many  of their  characters  T. repens and Lolium perenne. These  were accompanied, however, by parallel declines in means.  Coefficients  of variation  interpreted,  then,  did not follow  as reflecting  shifts  the pattern.  in mean  values  These rather  results than  can be  declines in  actual levels of genotypic diversity.  In some pastures at least, the expectation of low variability has not been met. This  has prompted  observed  in these  the question; what highly  clonal  maintains  populations?  the high  levels  One popular  of variation  suggestion  involves  environmental heterogeneity and natural selection. It was proposed for T. repens by Turkington and Harper to explain the generally  (1979c) and Burdon (1980a), and by Hamrick high  levels  of variation  in plant  populations,  (1982) and  by  Snaydon (1985) for variation in pasture species.  C.  ENVIRONMENTAL  HETEROGENEITY  AND  DIVERSIFYING  SELECTION. The term natural selection is derived from the observation that in a population some  individuals  than  others.  survive  Differential  while  others do not, or some leave more  survivorship  and/or  reproduction  (fitness)  descendants may be  Overview / 7 randomly  distributed  attributes  of  among individuals  individuals.  or may  Natural  be  nonrandom  selection  refers  with  to  respect  the  to  events  (organism-environment interactions) which cause nonrandom patterns in fitness. As a  consequence  variants  or  of  natural  types  selection,  (nucleotide  relative  sequences,  frequencies  proteins,  of  genetically  character  states,  based  etc.)  may  change, i.e., evolution by natural selection may occur.  One theoretically environments  prominent  (Levins  source of natural  1968; Hedrick,  Ginevan  selection &  Ewing  lies  in the  structure  1976). Theoretically  of an  environment is termed homogeneous when it presents a single "selective regime" with  respect  to  attributes  of  the  individuals  living  in  it.  That  is,  the  most  successful individuals in a homogeneous environment will be of a single type. If the attributes involved have a genetic basis, the relative frequency of this type will increase. Population-wide  variation for the attributes  will decline along with  the frequency of alternative types.  Alternatively, "microhabitats" are  different  a  heterogeneous  each presenting enough  successful across  no single  environment  is  composed  a distinct  selective  type  have  will  the  of  a  regime. If the flexibility  to  of  microhabitats the  most  the entire range. In this case, a set of different types  may  persist, each representing the most successful type in one of the Population-wide  variety  be  microhabitats.  variation will be maintained at a level reflecting the number of  types and the differences among them. The pattern of natural selection associated with heterogeneous environments is termed diversifying (or disruptive) in contrast with stabilizing or directional selection in homogeneous environments.  Overview / 8 The  prediction  of  depletion  of  variation  in  clonal  populations  matches  the  expectation for variation in general in a homogeneous environment. If the pasture environment is homogeneous  (or irrelevant)  to clovers, the most successful  lines  would be of a single type or a random sample. Either of these cases leads to the  expected  loss  of  homogeneous,  elements  these  soils  include  Harper  clones. of  Although  fine-scale  (Snaydon  pastures  patchiness  1985),  are  topography,  may easy  and  appear to  superficially  identify.  vegetation  Some  of  (Turkington  &  1979a). If patchiness constitutes environmental heterogeneity, depletion of  variation may be counteracted by diversifying selection. Burdon (1980a) specifically proposed that diversifying selection associated with vegetation patchiness maintains the high levels of variation in T. repens populations.  D. ENVIRONMENTAL The basic  existence to  selection.  of  any A  HETEROGENEITY  environmental  argument  study  that  IN  heterogeneity variation  of diversifying  selection  is  PASTURES. or  differentiated  being begins  maintained with  the  microhabitats by  is  diversifying  identification  of  a  pattern of patchiness in the environment followed by the demonstration that the patchiness represents an actual heterogeneity. This requires that performances  of  individuals in the field show environment-specific responses.  There is no doubt that the pasture  environment contains elements  of diversity.  These may be easily recognizable, such as the patterns of distribution of different species, or variations in topography. Other, more subtle elements may be found in the movements of grazing animals, soil types and availabilities of nutrients, or distributions of microorganisms. Because any of these elements might  reasonably  Overview / 9 be expected potentially  to have  define  an impact  on the lives  and deaths  a set of microhabitats. Variation  of plants,  each can  in the soil environment, for  example, has been shown to represent environmental heterogeneity for T. repens (Snaydon 1962 a,b; Snaydon & Bradshaw 1962a).  Although  abiotic  patchiness  exists  in  pastures  (e.g.,  soils),  the  diversifying  selection arguments, as applied to T. repens have been based primarily on biotic heterogeneity. This has been identified specifically as "the identity, age, and size of neighbours  (associated plants) and the degree to which they compete for the  same resources" (Burdon 1980a). The most prominent of these neighbours are the perennial grasses  T. repens dominate  which, along with  pasture communities in  Great Britain and North America. The local composition of a sward changes from dominance by each grass alone through various mixtures of two or more. This mosaic of variation presents an easily recognisable pattern of habitat variation.  The  dense  and continuous  nature  of pasture  vegetation ensures  that T. repens  plants must exist in close association with the grasses. We might then predict strong selection on T. repens individuals (persistence,  growth,  and reproduction  grasses. In addition, the lives by  different  neighbours,  for characters  in association  and deaths of plants  (Donald  1963; Harper  influencing compatibilities  with  neighbours)  are influenced  1977).  with the  differentially  The neighbour  mosaic  therefore represents a potential source for diversifying selection on T. repens for characters  influencing compatibilities  with  specific  species  explain the maintenance of variation in Trifolium the  anthropocentric  "easily  recognised  pattern"  of grasses. Its use to  repens, however, requires translates  to  an  that actual  Overview / 10 heterogeneity in the environments of clovers.  The pasture grasses differ in a number of characters which could influence the compatibilities  of  T.  repens  with  them.  These  include  characters  such  as  morphology, resource use, and seasonality in growth patterns. In one pasture in British  Columbia,  for example,  the grasses  include  three  tufted,  patch-forming  species {Lolium perenne L., Holcus lanatus L., and Dactylis glomerata L.) and one stoloniferous, spreading species  (Poa compressa L.). Among the tufted species D.  glomerata tends to form small dense clumps whereas L. perenne and H.  lanatus  patches tend to be larger but less exclusive. In addition, the grass patches may differ in a variety of ways which might have indirect effects on clovers. These include attractiveness  and palatability  to grazers, and differences  in habitat for  small grazing mammals and invertebrates.  Numerous  studies  pasture grasses Harper  have  considered  aspects  of the responses  (review in Chestnutt & Lowe  1977) grew  ramets  of  Trifolium  densities of L. perenne and Agrostis  combination  with  also  a difference  in the magnitude of the effect depending on which  was  used.  strongly  density  that  of neighbours,  branching  in  T.  there was neighbour  repens was  influenced by the physical structure of grass patches. Stolon branching  and adventitious (H.  showed  varying  While dry weight, flowering, and  all declined  (1985)  increasing  clover to  (1960, cited in  stolon production  Solangaarachchi  with  1970). Clatworthy  repens in  capillaris.  of white  lanatus  rooting occurred at a lower rate in densely  and A.  capillaris)  relative  to more  loosely  structured  structured  patches  patches  (L.  perenne and Cynosurus cristatus). This was attributed both to physical impedence  Overview / 11 and to changes in light quantity and quality.  Turkington (1983 a,b) studied the demography of leaves and flowers of T. repens grown in plots of several different grass species. In these experiments structures  of the leaf and flower  dry weight leaf  production all responded  and flower  (Turkington One  populations,  flux  rates  were  strongly less  the age  stolon extension rates, and total to the identity  influenced.  In  of the neighbours;  a  related  experiment  1983c), dry weight allocations to various plant parts were measured.  of the test individuals  environment.  The other  showed little  showed  a  response to changes  differential  response  in  in its neighbour its  allocation  to  inflorescences, stolons, and in its total dry weight production.  Differential responses of clover to grass neighbours have also been demonstrated in  the  field.  transplanted different ramets  Turkington ramets  concurrent  al.  (1979)  of T. repens into  grasses. They among  et  and Turkington patches  of pasture  found significant differences  the four  neighbour  greenhouse experiment  environments.  and Harper dominated  (1979c) by four  in dry weight production of Comparable  (Turkington & Harper  results  from  a  1979c) strengthened the  conclusion that the response was primarily a neighbour effect rather than a soil effect.  In  many  responsive and Harper  cases,  therefore,  to changes  and for  some  in its neighbours.  (1979c) in particular  support  response  measures,  The in situ transplants the interpretation  T.  repens is  of Turkington  that the mosaic of  grass neighbours constitutes a heterogeneous environment for clovers as well as  Overview / 12 for  ecologists.  populations Harper  Several  are variable  (1979c),  habitats responses  of these for these  in addition  for clovers,  studies  also  to the grasses.  have  "neighbour  to demonstrating  found  also  shown  relationships". that  pasture  T. repens individuals  that  The results  of Turkington  (1983c)  T. repens  that  Turkington and grasses differed also  differ as in their  demonstrated  differences among individuals in responses to neighbours.  The  identification  of  a  likely  environmental  heterogeneity  plus  related  environment-specific responses suggests that the potential for diversifying selection does exist. Therefore, some associated potential for maintenance of variation might also be expected.  E.  THE  CLONAL  GROWTH  HABIT  AND  ENVIRONMENTAL  HETEROGENEITY Additional support for the diversifying  selection argument has been drawn from  consideration of the growth habit of T. repens. The modular construction, which is typical of plants,  finds  T. repens. The clover units  (rooted  existence  nodes  its extreme expression in stoloniferous herbs such as  plant  is composed of a branched  or ramets).  and, in fact,  Each  will typically  is  potentially  become physically  sequence capable  of functional  of independent  independent through the  deaths of adjacent ramets or the breakage of stolon connections. While the entire T. repens clone can persist indefinitely, the ramet is transitory, persisting usually for less than one year (mean lifespan about six months, Thorhallsdottir 1983).  The  size  (number  of  ramets)  of  a  clone  will  respond  rapidly  to any  Overview / 13 environmental condition which influences the lives and deaths of ramets. In an environment  which is generally  unfavourable,  or in which new ramets  are not  consistently produced, a clone may go quickly to extinction. As noted earlier, this contributes to the expectation of genotype depletion within a population of clones in a  homogeneous  clovers,  however,  environment. the  clonal  In  an  habit  environment  may  enhance  which  the  is  heterogeneous  potential  for  for  diversifying  selection.  Through Clones  the can  growth migrate  produced more than  of  stolons,  rapidly;  a  T.  repens individuals  single  plant  started  movement,  separated.  different  During  from  spread a  2  horizontally.  cm  stolon  tip  100 new stolon branches for a total of over 20 metres of  stolon after five months (Evans unpublished). As for  can  its  parts  of the  lifetime,  a consequence of this capacity  same clone will commonly  an  individual  genet  may  become  widely  encounter  (and  reencounter) the entire range of available habitats in a pasture.  As  an  ramets  expanding may  encounters by may  clone  respond differently  a favourable  branching. respond  encounters  second  apex  by  dying,  or  branching or rooting (Harvey Newton  1986).  For  which  by  example,  spatially  in different  microhabitat  A  a  may  heterogeneous  microhabitats.  stolon apex  its  which  respond by producing new ramets or  encounters  continuing  A  environment,  to  a  grow  less  favourable  through  the  microhabitat area  without  1979, cited in Newton 1986; Solangaarachchi 1985; clover  stolons  which  encounter  tightly  clumped  patches of Dactylis glomerata commonly grow through the patch without rooting.  Overview / 14 As  a  result  (patches),  of  differential  the distribution  production  of a clone  of  ramets  may come  suitable patches. In addition, if the clover  in  different  to reflect  microhabitats  the distribution  of  population is composed of genotypes  which differ in their habitat suitabilities, the distributions of different clones may diverge. In a heterogeneous environment then, clovers may be sorted by habitat suitabilities both within and among clones. This process has been referred to in a  anthropocentric  written  "the  sense  growth  as "tracking". Harper form  of  clover  allows  (1977, p766), for example, has individual  genets  to  migrate  continuously through the sward, tracking local favourable areas". Salzman  (1985)  has demonstrated tracking of soil types by clones of Ambrosia psilostachya.  Plants  grown in a gradient of saline to nonsaline soil showed differential rhizome growth into nonsaline soil.  The result of this environmental sorting (or tracking) would amount, of course, to diversifying which  selection and could clearly  are likely  to persist  act to increase the number of genotypes  in a pasture.  As discussed  earlier,  the neighbour  mosaic potentially provides the habitat heterogeneity within which such a pattern of responses might be expected. As noted by Burdon (1980a) "...a genet may spread until it encounters a set of biotic factors to which it is less well adapted ... A t this point the original successful genet is supplanted by other genets which are better adapted to the different set of conditions. It is through this interaction of biotic forces at  the  local  environment  genotypes is maintained."  level  that  the  high  diversity  of  clover  Overview / 15 F. DIVERSIFYING  SELECTION  IN THE  PASTURE.  If such a process is operating at any level other than ecological speculation there should  be evidence  of it! Environmental  sorting  of clones  on the basis  of  neighbour relationships and ramet responses should be reflected in the distribution of  clones.  That  is,  there  should  be a  relationship  between  the  neighbour  compatibilities of T. repens individuals in the field and their physical neighbours. Evidence  for such a relationship  using reciprocal  transplants.  They  was found by Turkington and Harper collected  T. repens growing in close  (1979c) physical  association with each of four pasture grasses. Ramets of these individuals grown  in combination  significantly  with  all four  of the grasses.  The test  ramets  were  produced  more dry weight when grown in combination with the same grass  with which they had originally been associated in the field. These results  were  obtained  flats.  both  from  transplants  into  the pasture  and into  greenhouse  Further evidence of such fine-scale biotic differentiation was obtained by Aarssen and Turkington  (1985b). They  repeated  the experiment  T. repens collected  with  from patches of a single grass species, Lolium perenne. The results were similar, clovers  performed  individual  with  best  which  when they  grown  in combination  had originally  with  been associated  the same  L.perenne  in the field. Similar  results, also using L.perenne and T. repens were obtained by Gliddon and Trathan (1985) working in a pasture in N . Wales.  These results suggest that clover genotypes are sorted in the field on the basis of  their  compatibilities  with  their  neighbours.  That  is,  neighbour-specific  diversifying selection does appear to be occurring in these pastures. The results also support the suggestion that such diversifying selection could be counteracting  Overview / 16 the genotypic depletion expected for a clonal population. Turkington and  Harper  (1979c) concluded "The  simplest  interpretation  of  the  observations  is  that  the  genetic  diversity of white clover in this old pasture is maintained in part by diversifying selection from the variety of neighbouring grass species".  None of these variation for and  by  studies have, however,  morphological characters  Aarssen  and  Turkington  specifically  addressed  such as were  (1985b).  the maintenance  studied by  Neighbour-specific  Burdon  of  (1980a)  differentiation  was  demonstrated for only a single character, dry weight production. The work to be reported  here  neighbour-specific  will  extend  patterns  morphological characters.  these in  the  studies  by  distribution  looking of  for  evidence  variation  for  of a  similar set  of  II. C O M M O N G A R D E N S T U D Y , 1982  A . MATERIALS  AND METHODS  1. The P a s t u r e The material used in this study was collected from a pasture on the farm of Bill and Mary  Chard, 25704 Fraser Highway, Aldergrove, British Columbia (SW  1/4 Sec. 25, Twp. 10). The farm is located in the Fraser Valley in the Coastal Douglas F i r biogeoclimatic zone (Krajina 1965). This farm has been the site of a number  of  other  studies  including  Aarssen  (1983),  Aarssen  and  Turkington  (1985a,b,c), Parish (in Turkington 1985).  The  farm  century.  has been managed  The pasture  is grazed  for dairy  production  intermittently  from  since  the beginning of the  spring or midsummer  late fall by a herd of 20 to 30 cows. The cows are rarely  present  December and April, because of unfavorable weather conditions. Typically,  until  between grazing  is also delayed until after a hay crop is harvested in midsummer. The pasture receives no fertilizers other than animal excretions, occasionally supplemented with barnyard manure. A conversion to beef cattle in 1983 was not accompanied by any alteration in management.  The pasture was first cleared about 1900 and was last ploughed and seeded in 1939. The seed mixture used was composed of 5-10% Trifolium repens, 15-20% Dactylis glomerata, and 70-80% Buckerfield's "Highland" forage mix. Although the exact composition of the Highland forage mix used in 1939 is unavailable, it has  17  Common garden study, 1982 / 18 not  changed  much  over  the  years  (Richardson  Seed  Company,  personal  communication) and that used in a recently established pasture (1977) is given in Table I.  Table I. Composition of the "Highland Forage M i x " used in seeding a pasture in 1977.  The  pasture  herbaceous Lolium these  Dactylis glomerata  45%  Trifolium  20%  Lolium perenne  15%  Lolium multiflorum  10%  Phleum pratense  5%  Trifolium  repens  2%  Ladino clover (T. repens)  3%  community  species  (Table  is presently  composed  of  15  grasses  and 17  II). The most common of these are Trifolium  other repens,  perenne, Holcus lanatus, Dactylis glomerata, and Poa compressa. Together, five  Percentage  species  constitute  cover by species  Parish, unpublished).  approximately  75% of the total  vegetation  cover.  varies both seasonally and annually (Aarssen 1983;  Common garden study, 1982 / Table II. List of species present in the study site, a 42 year old pasture in lower Fraser Valley in British Columbia.  GRASSES  NON-GRASSES  Agropyron repens (L.) Beauv.  Achillea  Agrostis alba L.  Carex spp.  Alopecuris geniculatus L.  Cerastium vulgatum L.  Alopecuris pratensis L.  Cirsium  Anthoxanthum  Hypochoeris radicata  odoratum L.  millefolium  L.  arvense (L.)Scop. L.  Dactylis glomerata L.  Juncus spp.  Festuca pratensis Huds.  Medicago lupulina  Festuca rubra L.  Plantago lanceolata L.  Glyceria declinata Breb.  Plantago major L.  Holcus lanatus L.  Ranunculus  Lolium  multiflorum  Lam.  L.  acris L.  Rumex acetosella L.  Lolium perenne L.  Rumex crispus L.  Phleum pratensc L.  Rumex obtusifolius L.  Poa  compressa L.  Stellaria  Poa  trivialis  Taraxacum officinale Weber  L.  Data of R. Parish (personal communication)  media (L.)Vill.  Trifolium  pratense L.  Trifolium  repens L.  Common garden study, 1982 / 20 A l l four of the common grasses are perennial. Vegetative replication in three (L. perenne, H.  lanatus,  and D. glomerata) is by tillering,  compressa) by stolons.  A l l exhibit  varying  degrees  and in the other  of patchiness,  (P.  up to nearly  100% local cover. Patches dominated by L. perenne or H. lanatus may exceed one  square  metre,  while  D. glomerata and P.  compressa patches  are usually  smaller. D. glomerata patches are the smallest, usually less than .25 m very  dense  clumps.  The sizes  and locations  of patches  2  do not appear  but are to be  stable either annually or seasonally. Percentage cover within patches also appears to vary seasonally.  2. Collection and Propagation of Material Material patches  for the study  of each of the grasses  compressa) were cover  was collected on M a y 17 and 18, 1982. One hundred  identified  visually.  of one of the four  consistently  met for L.  (L. perenne, H. lanatus,  grasses  perenne,  Ideally, over H.  a patch an area  lanatus,  D. glomerata, and P.  consisted  of at least 75%  of .5 m . This  and P.  2  goal was  compressa patches;  D.  glomerata patches tended to be smaller in area but had very high percent cover of D. glomerata. One ramet of Trifolium  repens was collected from each of the  400 patches. This gave a replication rate of one hundred genets per neighbour. Replication  was by patches  rather  than  by repeated  collections  per patch, in  order to minimize the problem of genet duplication (Harberd 1961b). Each ramet consisted of a 4 cm section of stolon apex, including the apical bud and the first node with its leaves. Any roots remaining on the node were removed.  Only stolons which were actually rooted in the patch were used, and preference  Common garden study, 1982 / 21 was given to stolons with several rooted nodes and/or branches within the patch. However, Where  of D. glomerata.  few rooted clover stolons were found within patches  none were available, a stolon growing through the patch and rooted on  both sides was chosen.  The collection of 400 ramets was propagated in a common garden at the Plant Sciences Field Station on the University of British Columbia campus. Each ramet was  planted  planting,  separately  ramets  production. neighbour  were  The pots of  the  in  a  six  treated  with  Rootone  arranged  in  groups  potted  ramets  were  clovers.  The  inch  pot with  "field  rooting  station  hormone  Before  to stimulate  corresponding were  soil".  allowed  to  the a  root  original  six  week  establishment period. In July, 1982 a fresh cutting was taken from each pot and replanted in a second pot in the same manner as before. The second set of pots was given one fertilization with 1 teaspoon per pot of N P K 20-20-20. During the establishment period, 26 out of the original  400 ramets  had died. These  were  replaced with cuttings from a stock of extra ramets which had been collected at the same time as the originals and maintained in the same manner.  After a further ten weeks, the second generation of ramets was harvested (Sept, 1982). The entire plants were removed from the pots and soil washed from the roots. (approx.  Because  of  the  four  weeks),  length  of  the plants  time  were  required  stored  in  for a  taking  cold-room  measurememts while  awaiting  processing. Toward the end of the processing period, it was noticed that many of the  plants  had continued to grow,  slowly,  while  in the cold-room. It was not  possible to estimate the amount of growth, but there was no reason to believe  Common garden study, 1982 / 22 that it was nonrandomly distributed among the four groups of clovers. Therefore, this  growth  contributor  was considered to among-groups  to  be  part  variation.  of  Each  experimental  plant  error  was assessed  and not  for a set of  characters reflecting mostly sizes and numbers of parts. The character based on the characters  used in the variation studies of Burdon  a  set was  (1980a), and  Aarssen and Turkington (1985c) (Table III).  3. Assessment of variation  a. Numbers of Parts Each further rising  plant  was separated  subdivided directly  from  into  root  by its primary the main  and shoot  stolons.  taproot.  A  material.  These count  were  Shoot  identified  was made  material was as  branches  of the number of  primary stolons (PSTOL#) and of the total number of stolons (TSTOL#) including secondary  and tertiary  branches  along with  the primaries.  The count  did not  include dormant or unelongated buds (less than 10mm). The number of internodes on the longest primary stolon was also counted (INODE#).  b. Sizes of Parts The length of the longest primary  stolon (PSTOL.L)  and the longest  secondary  stolon derived from it (SSTOL.L) were measured. Three internodes were measured on the longest stolon (INODE.L). The choice of internodes excluded the actively elongating stolon tip and unelongated internodes  at the stolon base. Five  leaves  were selected from each plant. These were taken from the third to fifth nodes below, the apex and from as many different  stolons as possible. The length of  Common garden study, 1982 /  Table III. List of characters measured in the 1982 common garden study of variation in a pasture population of Trifolium repens.  UNITS OF MEASUREMENT  CHARACTER Root dry weight  RTWT  0.01 gm  a,b  Shoot dry weight  SHTWT  0.01 gm  a,b  Total dry weight  TWT  0.01 gm  a,b  Primary stolon number  PSTOL#  Total stolon number  TSTOL#  Internode number  INODE#  Length of primary stolon  PSTOL.L  5 mm  a,b  Length of secondary stolon  SSTOL.L  5 mm  Internode length  INODE.L  0.5 mm  a,b  Petiole length  PET.L  1 mm  a,b  Leaflet width  LF.W  0.5 mm  a.b  Leaflet length  LF.L  0.5 mm  Leaf marks: white chevrons  LFMK.W  red flecks  LFMK.R  a = character used in the variation study of Aarssen and Turkington(1985a). b = character used in the variation study of Burdon (1980a).  Common garden study, 1982 / 24 each petiole (PET.L) from the top of the stipule attachment to the base of the leaflets, and the length and width of each terminal leaflet (LF.L,  L F . W ) were  measured. The leaves were collected and pressed just prior to harvesting of the plants. three  Measurements leaf  characters  were  made  on the pressed  were  not affected  leaves.  by the growth  For this that  reason, the  occurred  during  storage in the cold-room.  c. Weights Root and shoot material was dried separately for at least three days at 100 C. Root and shoot dry weights (RTWT, SHTWT)  were measured directly  and total  dry weights (TWT) derived from them.  d. Leaf marks Each plant was scored for two types of leaf markings, red flecks (LFMK.R) and white they  chevrons  (LFMK.W).  are genetically  These  independent  were  treated  as separate  (Carnahan et al.  characters  1955; Corkill  because  1971). The red  flecks were recorded as present or absent, and the white chevrons classified into types et al.  which  were  known to be genetically  1955). Because  classification  the expression  of phenotypes  distinct  of leaf mark  was done independently  (Brewbaker genotypes by three  1955; Carnahan is often  unclear,  observers  on two  occasions. Plants for which a consensus could not be reached were classified as "indistinct".  Common garden study, 1982 / 25  B. ANALYSES AND RESULTS Statistical Data  analyses  Analysis  of the data were  System  (MIDAS)  performed  (Fox  and  using the Michigan  Guire  1976)  on  the  Interactive  Ahmdal  5840  computer at the University of British Columbia Computing Center.  The data for each character were stratified according to the original neighbour of each clover. The data set thus had four strata corresponding to the four common grasses;  Lolium  (TDAC),  and Poa  perenne  (TLOL),  Holcus  compressa (TPOA).  lanatus,  Each  stratum  (THOL), had  Dactylis  about  100  glomerata T. repens  individuals (Table IV).  Table IV.  Stratification of the sample population of Trifolium neighbours.  repens by  ORIGINAL NEIGHBOUR  STRATUM  Lolium perenne  TLOL  96  Holcus lanatus  THOL  100  Dactylis glomerata  TDAC  93  Poa compressa  TPOA  95  original  N U M B E R OF INDIVIDUALS  The within group (stratum) variances for all characters were highly heterogeneous (Table  V). The  data were  transformed  to base  ten logarithms  (LOG 10)  (Sokol  Common garden study, 1982 / 26 and  Rohlf  1981). Heterogeneity  was  reduced  in  most  cases  and  homoscedasity  was obtained for five characters (Table V). Analyses of variance (ANOVA) carried  out  on  either  the  raw  or  transformed  data  as  appropriate  were  (by  the  carried  out  (original neighbour)  was  homogeneity of variances criterion).  The  test  for  the  presence  using analyses  of variance  treated  as  random  random  effects  groups  when  present certain  a  situation that  their  causes involved  neighbour-specific  (ANOVA).  effect  model is the  of  (Model  used for of  The II  stratification  ANOVA),(Sokal  the analysis  differences  quantitative,  expression  differentiation  under  and  Rohlf  of variation  within  and  are  unclear.  among  group  highly  plastic  common  garden  means  characters. conditions  indicator of their expression in the field (Briggs and Walters and  Rutledge  1986). For  this  was  reason, comparisons  of means  1981).  It  was  The  among The  was a  not  reliable  1984; Mitchell-Olds would  have  been  inappropriate and possibly misleading. The use of a fixed effects model (Model I) including comparisons among means would be appropriate for the analysis of data from reciprocal transplants in the actual field environment.  Any neighbour-specific differentiation in these characters should be reflected in the data as phenotype-environment correlation. Such a correlation would be indicated by  the  presence  of a significant  added variance  component in the A N O V A  (a  significant portion of the total variance lying in the among-groups component). In a  Model  I  ANOVA  added  variance  components  are  detected  by  testing  the  among-group mean squares against the error (within-groups) mean squares. In ten out of the twelve characters a significant added variance component was  detected  Common garden study, 1982 / 27  Table V. Tests of homogeneity of variances for twelve morphological characters of Trifolium repens. Raw and log transformed data from the 1982 common garden study.  TRANSFORMED D A T A (LOG 10) F  CHARACTER  RAW DATA F  RTWT  16.36 **  11.14 **  SHTWT  11.02 **  5.56 **  TWT  10.26 **  5.65 **  PSTOL#  6.95 **  3.87 **  TSTOL#  4.95 **  7.01 **  INODE#  3.31 *  5.68 **  PSTOL.L  4.69 **  4.00 **  SSTOL.L  4.02 **  2.38 N S  INODE.L  4.56 **  2.36  NS  PET.L  7.97 **  1.64  NS  LF.W  5.84 **  3.68 *  LF.L  7.18 **  3.78 *  Columns give F-statistic and significances for test of homogeneity of variances of four neighbour-specific groups. ** = p<0.01 * = p<0.05 N S = no significant heterogeneity among variances  Common garden study, 1982 / 28  (Table  VI).  This  means  that  for  ten  characters  the  population  showed  neighbour-specific differentiation. For two characters, INODE# and P S T O L . L , there were no added variance components, hence no evidence of differentiation.  The  proportion  components (Table  of  the  total  variances  represented  by  the  added  (percent variation among-groups) were calculated for these  VI).  For  the  ten  characters  that  showed  differentiation,  variance characters  the  percent  variation among-groups ranged from 2.02% (SSTOL#) to 20.19% (PET.L) with a mean  of  population  7.93%. These which  is  amounts  reflect the  "explained" by  amount  the type  of variation  of grass  in  the clover  the  was  clover growing  with and are measures of the strength of the biotic differentiation.  The  data  individual  for  four  (PET.L,  of  the  LF.W,  characters  LF.L  included  several  measurements  (five leaves per individual)  for  and I N O D E . L  each (three  per individual)). In these cases, a within-individual mean square can be calculated and  used  to  estimate  (within-neighbours) model  ANOVA  estimated  from  variance. using  the  them  using  a  variance  Mean  squares  ANOVAR the  component were  program  method  of  representing  obtained and  Falconer  from  variance (1981).  among-genets an  heirarchical  components  were  Significant  added  variance components (among-genets) were detected for all four characters. The % variance among-genets ranged from 45.1 for L F . L  to 73.5 for I N O D E . L  VII). The % variances among-neighbours were also estimated from the  (Table  ANOVAR  Common garden study, 1982 / 29 Table V I . Summary of analyses of variance for 12 morphological characters from four neighbour-specific groups of Trifolium repens. Data from the 1982 common garden study.  n  MS  MS A  S E  SIG  J  % VAR(A)  A  RTWT  96.0  0.559  0.096  0.0048  **  4.80  SHTWT  96.5  0.686  0.117  0.0059  **  4.81  TWT  95.8  0.645  0.101  0.0057  **  5.33  PSTOL#  96.5  0.245  0.036  0.0021  **  5.73  TSTOL#  96.8  1610  442  12.06  *  2.65  INODE#  96.8  14.8  9.07  0.0596  NS  0.65  PSTOL.L  96.8  0.042  0.026  0.0001  NS  0.66  SSTOL.L  94.3  0.119  0.041  0.0008  *  2.02  INODE.L  96.8  0.078  0.023  0.0006  *  2.42  PET.L  98.0  0.461  0.018  0.0045  **  20.19  LF.W  89.8  0.062  0.005  0.0006  **  11.75  LF.L  89.8  0.145  0.006  0.0016  **  19.61  n = mean # of individuals/group MS  A  = among-groups mean square  MS^ = error mean square S  1  A  = added variance component = ( M S  % VAR(A)  A  - MS  = % variance among-groups = S  2  A  E  )/n  /( S  2  A  + MS  ) E  Sig = Level of significance from F-test for presence of added variance components ** = p<0.01 * = p<0.05 N S = not significant  Common garden study, 1982 / 30 results and were comparable with those that had been obtained from the M I D A S program  (Table  VI).  Differences  between  the  two  can  be  attributed  to  the  subsetting of cases necessary to obtain a balanced data set for A N O V A R and to the reduction of the repeated measures to means for the M I D A S analysis.  Some of the characters retained significant amounts of heterogeneity of variances even  after  transformation  (Table  V).  This  situation  formally  violates  the  assumptions of analysis of variance, although the effects often do not alter the results obtained (Sokal and Rohlf 1981). To check the A N O V A characters,  they  were  reanalyzed  using  the  results for these  Kruskal-Wallis  test  which  is  independent of means and variances (Sokal and Rohlf 1981). The results of the Kruskal-Wallis  test  exactly  paralleled  case in which the A N O V A  the  ANOVA  results  (Table  VI). In every  detected a significant added variance component, the  Kruskal-Wallis test indicated a significant difference among groups.  Because the leaf mark  characters  were categorical rather  than continuous,  they  were analyzed using the nonparametric Kruskal-Wallis test. This method analyzes ranked  observations,  testing  for  significant  rankings. Neither of the characters, L F M K . W  differences  among-groups  nor L F M K . R ,  in  the  showed any evidence  of neighbour-specific differentiation.  Most  of  the  characters  in  the  study  involved  sizes  and  numbers  of  parts.  Because it might be reasonably expected that larger plants would also have more stolons, larger leaves, etc, it is likely that the characters vary in parallel. If this were the case, the analyses of these characters  would not be independent  and  Common garden study, 1982 / 31 Table VII. Variance components for four morphological characters from a pasture population of Trifolium repens. Data from the 1982 common garden study.  Neighbour  Genet  Error  PET.L  17.0 (**)  50.9 (**)  32.1  LF.W  8.5 (**)  56.6 (**)  27.5  LF.L  14.1 (**)  45.1 (**)  27.5  2.7 (**)  73.5 (**)  23.8  INODE.L  Entries give % variation accounted for by each source of variation and a test for the presence of an added variance component. ** = p < . 0 1 Sources of variation: Neighbour = among four neighbour-specific groups Genet = among-Trifolium repens individuals (within groups) Error = among-measurements (within-individuals)  the use of a series of univariate A N O V A s might not be appropriate. Conclusions based on such an analysis would certainly be weaker than if based on analysis of independent characters.  The  relationships  calculating analysis  among  correlation  show  clearly  the  coefficients that  morphological (Table  the characters  VIII).  characters  were  The results  of the  are not independent.  positive, and all except three are significant (p<0.01).  investigated  by  correlation  A l l values are  Common garden study, 1982 / 32  Table VIII. Correlations among 12 morphological characters of Trifolium repens. Data from the 1982 common garden study.  RTWT SHTWT TWT PSTOL# TSTOL# INODE# PSTOL.L SSTOL.L INODE.L PET.L LF.W LF.L  INODE.L PET.L LF.W LF.L  1.000 0.832 0.919 0.610 0.742 0.269 0.432 0.545 0.390 0.523 0.356 0.367  1.000 0.983 0.560 0.801 0.306 0.599 0.720 0.583 0.683 0.422 0.448  RT WT  SHT WT  1.000 0.583 0.428 0.481  1.000 0.568 0.581  1.000 0.850  1.000  PET.L  LF.W  LF.L  INODE L  1.000 0.598 0.813 0.306 0.567 0.690 0.514 0.625 0.417 0.439 TWT  1.000 0.662  0.068 0.167 0.273 0.227 0.249 0.162 0.200  1.000 0.282 0.380 0.558 0.319 0.400 0.273 0.307  1.000 0.515 0.384 0.192  0.109 0.067 0.082  PSTOL  TSTOL  INODE  #  #  #  1.000 0.825 0.814 0.532 0.412 0.461  1.000 0.710 0.529 0.395 0.440  PSTOL L  SSTOL L  Table entries are product moment correlation coefficients. Characters with coefficients greater than 0.139 are significantly correlated (p<.01) Coefficients indicating no significant correlation are printed in dark type, n  = 373  Common garden study, 1982 / 33 This high level of intercorrelation suggests that pattern in the data set might be best represented by a single character, probably reflecting plant sizes. Analysis of additional characters  correlated  with  the first would be redundant,  interpretable information. For example, P S T O L . L r=0.825  suggesting that these variables  and T W T because  are also highly  it  is  are highly  intercorrelated  completely  determined  and S S T O L . L  RTWT  are correlated at  redundant. RTWT,  (r>0.80). T W T  by  adding little  and  is obviously  SHTWT.  SHTWT, redundant  Analysis  of  highly correlated characters such as these is only a little more informative  ten than  an analysis of the same character ten times.  A method of dealing with a set of highly correlated characters is to reduce them to  a  smaller  number  principal components components,  are  of  uncorrected  analysis  analogous  variables  using  (PCA). The new, composite to  coordinate  axes  in  a  multivariate  method,  variables, or  principal  multidimensional  space.  The  principal components are derived from the correlation structure among all of the original  characters.  Each  component describes  an independent  trend of  variation  across the entire data set.  The location of an individual in multivariate space is defined by its position on the  principal  component  axes.  Position  on  each  axis  is  determined  by  a  combination of its scores for all of the original characters. Each character score is  weighted  by  the  amount  that  the  character  contributes  to  the  trend  of  variation being described by that axis. The patterns of variation associated with the  various  character  PCA  axes  weightings,  the  can  sometimes  amount  be  of variation  interpreted  by  explained  by  inspection the  axis,  of  the  and  the  Common garden study, 1982 / 34 original correlation matrix.  The P C A  axes represent a set of independent, composite characters. They can,  therefore, be analyzed individually in the same manner as were the original ten characters but without the ambiguities that were introduced by the high levels of intercorrelation.  A principal components analysis was performed on the data (Table IX). The first component (PCA1) represents the most prominent trend in the data set (54% of the total variation). This axis was influenced in the same direction by all of the original variables. strong multivariate  This  is typical for  association  an axis  among the  describing variation  variables  supports  the  in size. pattern  The noted  from the correlation analysis; these plants are primarily distinguished by size, all of the measured variables  increasing or decreasing together. Plant  weights  and  stolon numbers contribute most strongly to the pattern.  An A N O V A original  was performed on P C A l . As was the case for the majority of the  characters,  a significant  added variance  component  was  detected  (Table  X). The interpretation based on the earlier A N O V A s is thus supported. When all twelve characters are reduced to one multivariate character representing the most prominent trend in the data, the neighbour-specific differentiation is retained. The percent  variance  among-groups  for  PCAl  was  9.73%.  This  result  is  also  comparable to those obtained from the original characters.  The  second  component  (PCA2)  represents  the  next  most  prominent  trend  in  Common garden study, 1982 / 35  Table IX. Principal components analysis of 12 morphological characters of Trifolium repens. Data from the 1982 common garden study.  AXIS PCAl  PCA2  PCA3  PCA4  PCA5  % VARIATION CUMULATIVE  54.36 54.36  14.32 • 68.68  10.97 79.65  6.18 85.83  4.32 90.15  RTWT SHTWT TWT PSTOL# TSTOL# INODE# PSTOL.L SSTOL.L INODE.L PET.L LF.W LF.L  0.322 0.362 0.363 0.215 0.300 0.150 0.303 0.325 0.281 0.288 0.234 0.248  -0.287 -0.178 -0.220 -0.426 -0.361 -0.053 0.244 0.095 0.292 0.225 0.404 0.397  0.106 0.022 0.050 0.253 0.063 -0.598 -0.403 -0.277 -0.188 0.168 0.373 0.344  -0.068 0.041 0.007 0.079 -0.093 -0.668 0.121 0.190 0.479 0.156 -0.363 -0.310  -0.232 -0.224 -0.235 0.627 0.082 0.030 0.161 0.064 0.276 -0.524 0.123 0.208  Table entries are coefficients for each character for the first five principal components (axes). % V A R I A T I O N is the amount of variation in the multivariate data set which is explained by each axis.  T A B L E X . Summary of analysis of variance of principal component scores for  Common garden study, 1982 / 36 four neighbour-specific groups of Trifolium repens. Data from 1982 common garden study.  AXIS  MS  MS  S  SIG  2  %VAR(A)  A  n  PCA 1  66.16  6.03  0.650  9.73  PCA 2  5.67  1.69  0.043  2.49  PCA 3  13.02  1.22  0.128  9.46  PCA 4  0.04  0.75  PCA 5  2.13  0.50  NS 0.018  3.36  = mean # of individuals/group  MS MS S  2  A  A £  = among-groups mean square = error mean square  = added variance component = ( M S  % VAR(A)  A  - M S „ )/n  = % variance among-groups = S  E  2  A  /( S  2  A  0.00  + MS  ) E  Sig = Level of significance from F-test for presence of added variance components ** = p<0.01 * = p<0.05 N S = not significant  Common garden study, 1982 / 37 variation  (14.3% of the total  associated  positively  with  variance,  stolon  for a total of 68.7%). This  lengths  and leaf  sizes  axis was  and negatively  with  weights and stolon numbers. This suggests that when the influence of plant size is removed, there is a negative relationship between stolon numbers and stolon lengths. Such a pattern is reminiscent of the guerrilla-phalanx dichotomy in stolon structure  described  long,  Lovett-Doust  (1981)  Ranunculus  for  (1985) for T. repens. Some clovers  Solangaarachchi few,  by  unbranched  stolons  while  others  repens  (guerrillas)  (phalanx)  and by  tend to have a  have  more  numerous,  shorter, more highly branched stolons with shorter internodes. A N O V A detected a significant variation  accounted for 11.0%, 6.2%, and 4.3% of the variance, respectively,  for a five  of  relationships added  scores  (Table  component explaining 2.5% of the components  total  axis  added variance  fifth  axis  in  (p<.01)  on P C A 2  90.2% (Table  among  variance  X ) . The third,  IX). There  the characters  components  were  fourth,  were  and these present  and  no obvious three  on P C A 3  axes.  patterns  in the  Significant  (p<.01)  and P C A 5  with  percent  variation-among groups of 9.5% (PCA3) and 3.4% (PCA5) (Table X). A N O V A on PCA4  detected  no added  variance  component.  Again,  the multivariate  analysis  generally  (for four out of five components) supports the interpretations from the  analyses  of  the  original  characters,  that  this  population  is  differentiated  morphologically on a neighbour-specific basis.  C.  DISCUSSION  The  results  Trifolium out  of . this  study  repens population  of twelve  characters,  show  that  is distributed a significant  some  morphological  variation  on a neighbour-specific (p<.05)  added variance  in  this  basis. For ten component was  Common garden study, 1982 / 38 detected when variation was partitioned  according to the species  of grass  with  which the Trifolium repens individuals had been growing in the field (Table VI). When  the twelve  characters  were  reduced  to principle  components  significant  neighbour-specific variation was also detected on two out of the first three axes (Table IX). The results of Turkington and Harper Turkington clovers  (1985c)  relating variation in performance  to neighbour  characters.  This  (1979c) and of Aarssen and  relationships  pattern  are paralleled  is consistent  with  (dry weight  by variation  the expectations  production) of  in morphological  of Burdon  (1980a)  that morphological variation was being maintained by neighbour-specific diversifying selection. Aarssen  These  also  and Turkington  populations were  results  confirm  (1985c)  the  that  there  of T. repens. For the four  taken,  there  was a  highly  findings  of Burdon  (1980a)  is a lot of variation  characters  on which  significant  (p<.01)  and of  in pasture  repeated  measures  variance  component  representing differences among individuals.  Although  variation  influenced  by clover-grass  interactions,  characters  in determining  neighbour  possible,  using  involvement  in  this  this  type  of a character  set of  of  morphological evidence  characters  appears  for any direct  compatibilities  nonmanipulative  role  is circumstantial. study,  to  to be of these  It is not  distinguish  direct  from passive correlation with the actual determining  characters. For example, a highly significant (p<.01) proportion of the variation in petiole length (PET.L) was accounted for by the identity of neighbours. This is not  sufficient,  neighbour persistence  however,  compatibilities. of clover  to establish  petiole  length  as a major  determinant of  Although petiole length has been related to differential  types  in swards  of different  heights  and under  different  Common garden study, 1982 / 39 grazing intensities  (Rhodes  &  Harris  1979; Davies  1973),  such  an  explanation  does not seem likely in this pasture. There are no consistent differences the  grass  patches  in  either  height  or  grazing  intensity  (Parish,  among personal  communication). Given the data from this study it is as likely that petiole length is merely correlated with neighbour compatibility  as that it is a determinant of  it. Furthermore, because I have no data on the expressions of these characters in  the  field,  I  have  no  grounds  for  placing  ecological  interpretations  on  the  common garden measurements. Evidence addressing both of these criticisms could be gained from reciprocal transplant experiments using clovers with a range character  combinations.  Because  much  of  the  causal  basis  of  of  neighbour  relationships is likely to be physiological (Turkington 1985), demonstrating a direct role for morphological characters would be difficult.  Whether  these  relationships, That  characters  there  is, there  are  directly  is, nevertheless,  is more  a  or  passively  involved  neighbour-specific  variation in this  with  component  population, for  neighbour  to variation.  these characters,  than  would be expected in a population coexisting with a monoculture of one of the four grasses. Variation is probably maintained at a higher level in this biotically heterogeneous  pasture  than  it  would  be  in  a  similar  but  more  homogeneous  pasture.  The demonstration that a neighbour-specific component to variation exists answers only part of the question of maintenance how  much  of  the  population-wide  of variation. We also want to know  variation  is  acounted  for  by  the  neighbour-specific pattern. The relative importance of neighbours as a diversifying  Common garden study, 1982 / 40 influence  should  classification  be reflected  into  neighbour  in the  amount of variation  groups  (%  variation,  accounted for  Table  VI).  These  by  the  amounts  indicate that neighbour-specific diversifying selection is not playing a major role in the maintenance of morphological variation in this population. Only for the three leaf characters,  petiole  length  (PET.L),  leaflet  width  (LF.W), and leaflet  length  (LF.L) was more than 6% of the total variation accounted for. The multivariate analysis reflects the same pattern with less than 10% of the variation accounted for by A N O V A of the first principle component scores (Table X).  This means that although neighbour-specific an effect the effect is not strong. Over remains  unexplained.  There  is  only  diversifying selection may be having  90% of the variation in the data set  6-7% more  morphological  variation  in  this  population than would be expected for a population in a pasture composed of a single grass species. This can be compared with the 45-70% variance  explained  by  variation  differences  among-individuals  within the patches  than there  (within-patches). is  There  is  much  among them. Thus, this  more  population would be  highly variable even if there were no differences among the patches.  This  conclusion  components  must  from  be qualified, however, because common  garden  data  the  may  estimation be  of  variance  complicated  by  genotype-environment interactions (Mitchell-Olds & Rutledge 1986). The variance in field-collected  data  environmental  component  patches.  The  point  for  of  these reflecting the  quantitative  characters  the  effects  of  common  garden  method  would  growing is  in to  include different minimize  an grass this  environmental component of variation and so isolate the genetic component which  Common garden study, 1982 / 41 represents the among-neighbours  differentiation. The procedure assumes,  however,  that different environments will have the same effect on each group of clovers. If the differences among groups are not constant among environments (if there is genotype-environment interaction), differentiation will not be reliably estimated from measurements  taken in any common environment  (Lewontin  1974b; Mitchell-Olds  & Rutledge 1986). Even if the common environment is comparable to one of the field environments, the method is restricted to one of "local" analysis  (Lewontin  1974b).  In contrast to the common garden design, reciprocal transplant experiments make no  assumptions  about  genotype-environment  interactions  and  also  surmount  the  local analysis problem if transplants are carried out across a representative range of  environments.  method  for  For  these  investigating  reasons,  variation  reciprocal  when  transplants  are  genotype-environment  the  preferred  interactions  are  expected.  In the case of T. repens, the neighbour relationships detected by Turkington and Harper  (1979c)  clearly  rule  assumption  originally  designed  experiment. Transplants were set out in May,  on  genotype-environment  a reciprocal transplant experiment would have allowed for stronger was  focussed  no  relationships,  study  study  of  Because  This  present  the  interactions.  conclusions.  the  out  around  the  a  same  reciprocal  neighbour  transplant  1983 but initial mortality was so  high (>80% after two weeks) that the experiment was abandoned.  It is not possible to determine what effect interactions between genotypes and the  Common garden study, 1982 / 42 common garden might have had on the estimation of variance components in this data  set.  It  does  neighbour-specific environment  not  seem  likely,  differentiation  interaction.  On  was  the  however,  that  the  entirely  a  product  hand,  any  portion  other  highly  of  genotype-common  of the  among-groups  variance  which does not represent additive genetic variance further  already  small  amount  of  variation  explained  by  significant  reduces  neighbour-specific  the  diversifying  selection.  Numerous cases of morphological differences across environmental boundaries have been recorded in herbaceous plant populations (e.g., Watson Silander  &  Antonovics  1979;  Scheiner  &  Snaydon & Davies 1976, 1982; Seliskar cases  transplants  sub-populations.  have  By  provided  implication,  Goodnight  1985; Silander  evidence  diversifying  of  1984;  Antlfinger  1974; 1981;  1985). In many of these  genetic  selection  1969; Linhart  differentiation  could often  be  among  involved  in  the maintenance of morphological variation. It is also noteworthy that in several of these  studies, no evidence  Goodnight  1984; Antlfinger  for  genetic  1981; Seliskar  differentiation  was  found  (Scheiner  &  1985). The magnitude of the role of  diversifying selection in maintenance of variation has seldom been investigated. Of the  studies  which  showed  morphological  estimate  of the amount of variance  (Silander  &  reciprocally 21  Antonovics  associated  Silander  only  one  with environmental  1985). Ramets  included  an  heterogeneity  of Spariina patens were  transplanted into adjacent salt marsh, dune, and swale habitats. For  morphological  differentiation with  1979;  differentiation,  characters,  among  habitats  the was  18.5% among genets, and  median 4.9%  %  (range  59.8% within  variation from  accounted  0-34.1%).  This  habitats. These results  for  by  compared are  quite  Common garden study, 1982 / 43 comparable  to  the  present  results  for  Trifolium  repens.  Again,  although  diversifying selection appears to be commonly operating in plant populations, the extent of its role  in maintenance  of variation  is not clear,  and may not be  large.  The  low proportion of variation  preclude  the possibility  associated  with  other  associated with the neighbour mosaic does not  that additional variation could be explained by selection heterogeneities.  One other  element  of patchiness  in the  pasture environment which could constitute a heterogeneity is soil type. Numerous examples  of local  scale  differentiation  of plant  populations  heterogeneity have been recorded (Jain & Bradshaw &  Turner  an  1971; Snaydon & Davies  agent of diversifying  associated  1966; Antonovics,  with  soil  Bradshaw  1976, 1982). The role of soil patchiness as  selection on T. repens has been reported by Snaydon  (1962, 1971).  The ecological role of soil patchiness in pastures was played down by Turkington and  Harper  variability  (1979a), by Burdon  for soil  distributions.  factors  The reciprocal  (1980a),  and Aarssen  and few correlations transplant  between  experiments  (1983) soil  who found low type  of Turkington  and species and Harper  (1979c), however, do show evidence that both vegetation and soils are associated with  differentiation  in T. ripens.  Trifolium  repens individuals  transplanted  into  plots from which the grasses had been removed showed site-specific differentiation paralleling  the neighbour-specific  differentiation  of individuals  transplanted  into  undisturbed vegetation. Because the neighbour-specific differentiation remained when soil heterogeneity was removed in a glasshouse experiment, it is not likely  that  Common garden study, 1982 / 44 the effect was primarily soil based, as suggested by Snaydon (1985).  Additional  elements  heterogeneities  (e.g.,  of  patchiness  vegetation  could  density,  be  proposed  topography,  as  environmental  pathogens,  invertebrate  grazers, and soil microorganisms). A s with soils some of these could potentially be associated with diversifying selection on T. repens populations. Considering the low  proportion  of variation  accounted  for by  such  a  prominant  element of  patchiness as the neighbour mosaic, however, it seems probable that these other factors would play a similarly minor role in maintenance of variation.  It  appears  complete  unlikely  that  explanation  appropriate  maintenance  based  to reconsider  on  in T. repens will  of variation  diversifying  the potential  role  selection.  played  Therefore,  find  it  a  seems of new  by the introduction  genetic variation into the population. A s has been discussed, the expectation of genotype depletion in clonal populations is tied to the assumption that the rate of  replenishment  through  seedling  establishment  is  observations on pasture perennials have consistently seedling  (genet)  proposed  role  establishment  for diversifying  is  rare  selection  relative  negligible.  Studies  and  supported or confirmed that  to  ramet  was to retard  establishment. The  the rate  of  genotype  depletion to a level that would be offset by the occasional successful seedling. The crucial question, however, of genet input relative to genet extinction has not been  commonly  quantifying  examined,  genet extinction  presumably  due to the difficulty  in a stoloniferous  species.  of observing or  The modelling  study by  Soane and Watkinson (1979) did address this question using demographic data for pasture  populations  of Ranunculus  repens, a  clonal  perennial  with  a  similar  Common garden study, 1982 / 45 growth  form  expected  to T.  decline  repens. Although  in numbers  their  of original  computer  genets,  simulation  it also  confirmed the  demonstrated  that  a  recruitment rate of only 3% of the existing numbers of genotypes was sufficient to  offset  the estimated  simulation  rate  did not provide  of genet  loss  any evidence  and so maintain  for selective  diversity.  maintenance  of  Their clonal  diversity. They concluded that "even a very low rate of genetic recruitment through seedling establishment is sufficient to maintain a large diversity of clonal families within small areas. Consequently, it may not always be necessary to search for disruptive forces to explain diversity within the field." Soane  and Watkinson's  replacement  plays  (1979)  a negligible  study role  suggests  that  the assumption  in the maintenance  that  of diversity  genet  in clonal  pasture perennials might be unrealistic. A recent study of seedling establishment on disturbances, conducted on the same pasture that  recruitment  of T.  repens may also  generally recognized (Parish, in Turkington successful  T. repens seedlings  were  as the present study,  be more 1985). After  frequent  than  indicated has been  two years in which no  detected, in 1985 seedlings  appeared  at a  average of 20 per disturbance. After three months, five T. repens seedlings per m  2  (averaged across the entire pasture) were surviving, representing a potentially  massive genotypic input into the population. If this is not a unique event, such episodic recruitment could easily account for the genotypic diversity in T. repens populations, again without resorting to complex, selective explanations. The present results are consistent with this interpretation. Much more of the variation in this population is explained by the establishment of morphologically different individuals than by any subsequent sorting of them through neighbour relationships.  Common garden study, 1982 / 46 In addition to seedling recruitment, another potential source of genetic variation in clonal populations is somatic mutation. Although the process is largely unexplored, clonal herbs  may  be particularly  likely  to accumulate  genetic diversity  through  somatic mutations. A non-lethal mutation which occurs in any active meristem of a clone may be perpetuated through all of the daughter ramets produced from it. A  clonal line may, over a period of years, evolve into a mosaic of genotypes.  Such  within-individual  Slobodchikoff  1981)  variation  and  in  has  been  Hamamelis  documented  virginiana  in  (Gill  Populus  &  (Whitham  Halverson  resistance to insect herbivory. Within clone variation in rate of tiller by  Lolium  Hayward  perenne has &  Slobodchikoff  Thomas (1981)  been demonstrated 1965;  and  Shimamoto  Antolin  and  using &  selection  Hayward  Strobeck  experiments 1975).  (1985)  1984)  for  production (Breese,  Whitham  suggest  &  that  and the  accumulation of somatic mutations may represent an important source of genetic variation within clonal populations.  III. COMMON G A R D E N STUDY, 1984  A. CARRY-OVER  EFFECTS  AND THE COMMON  GARDEN  METHOD  The common garden technique has been a fundamental tool of genecology since its inception (Heslop-Harrison 1964; Langlet 1971). Its importance is reflected in a recent quote by Bradshaw (1984) who stated that "The crucial step forward was taken when investigators such as Kerner (1891) and especially Turesson (1922) appreciated the value of the common garden technique to remove direct effects of differing environments and reveal underlying genetically determined variation, an approach so simple that it seems extraordinary it had not been used by Darwin". However,  simplicity does not necessarily  equate with reliability. Genecological  studies which depend, as the present one did, on the common garden method can be criticized on the grounds that the patterns of variation detected do not have a clear genetic basis. At best, variation can be resolved into "genetic" and environmental components but without more formal genetic techniques even this cannot be taken with confidence. Heritability studies using seed progeny or transplants into a variety of environments can often support stronger conclusions.  One of the largest difficulties with the common garden method is the uncertainty over the amount of variation that is carried over from the field environment to the  garden.  The expression  of many  morphological  characters  is strongly  influenced by the environment in which the plant lives. Genetic interpretation of differences  among plants  impossible.  The basic  taken  directly  from their  original environments is  assumption of the common garden method is that a  common environment will influence all of the plants in the same manner. The 47  Common garden study, 1984 / 48 environmental  or  developmental  component' of variation  will then be minimized.  Remaining differences among the plants will reflect only genetic differences. The effect of genotype-environment interactions on this assumption of no environmental variation was validity  of  discussed earlier.  this  assumption.  The  When  problem of carry-over transplants  are  is  collected  also for  tied to the  a  study,  the  resource levels and physiological state of the material will reflect to some degree the conditions influences  under which the plant was growing. If such a preexisting state  the  plants'  performance  in  the  garden,  transplants  from  different  environments may express morphological differences which have no genetic basis. Interpretation  of such  patterns  will  be  confounded  by  variation  "carried  over"  from the field. Even when seeds are used in place of transplants the problem of carry-over may remain. Because the size and condition of a seed can influence its performance, the environment under which it was produced may  precondition  it and confound a variation study. Such "maternal effects" have been documented (Schaal 1984).  The usual method of addressing carry-over effects in transplanted material is to give  the  plants  an establishment  period during which  their  ostensibly become adjusted to the garden environment and  physiological  field-derived  are used up. This approach is particularly well suited to vegetatively species like Trifolium an entire plant. A  states  resources replicating  repens because a transplanted stolon tip can be grown into subsequent cutting taken from this plant will be formed of  tissue grown entirely under the garden conditions. The "second generation" plant grown from this cutting is expected to have lost most of the field effects carried over by its parent. This cloning process can be repeated for as many generations  Common garden study, 1984 / 49 as is thought to be necessary. Obviously, the length of the conditioning period or the  number  of cloning  investigators  have  generations  used a variety  required  will  vary  among species.  of conditioning treatments,  usually  Different supporting  their choice by rationalization rather than with data. In fact, carry-over  effects  and conditioning treatments have not been widely investigated (but see Warwick & Briggs  1978; Akeroyd & Briggs 1983; Mackenzie 1985) and are often ignored  in common garden studies.  The patterns of variation described in Part I were those of a set of clones in their  second  clones  generation  had undergone  measurements  were  in the common a  made,  ten  week  garden.  The original  conditioning  the second  generation  period. clones  (first  By  the  had spent  generation) time  the  an entire  season in the common garden. This treatment was comparable to those used on T. repens in the earlier relationship  study  of  competition Turkington  studies and  of Aarssen  Harper  (1983),  (1979c),  the neighbour  and  the  edaphic  differentiation studies of Snaydon (1962b) and Snaydon & Bradshaw (1962a,b).  Despite  the consensus of previous investigators, some doubt remained about the  adequacy  of the conditioning  morphological  change  treatment.  (plasticity)  of T. repens to undergo  The capacity  in response  to environmental  change  is  well  known. Brougham et al. (1978) noted that such changes could be persistent and suggested caution in the interpretation of experimental results when T. rvpens is grown  in  component  different of  environmental  environments.  variation variation,  detected the  Obviously, in  small  the  if  the  1982  portion  of  among-neighbour  study  included  variation  groups persistent  attributed  to  Common garden study, 1984 / 50 neighbour-specific diversifying selection would have been an overestimate.  The  second part of this  research  was  undertaken  to determine  if the  original  pattern would be retained under a longer conditioning period. Specifically, if the striking neighbour-specific it should not conditioning incomplete case,  6how  period  of  a  differences  much decay over a longer period. Alternatively, if the original was  inadequate,  the  differentiation  loss of developmental responses  additional  expression  differentiation did, in fact, represent genetic  time  under  new  set  of  common  might  simply  carried over from the  garden  developmental  conditions  responses,  morphological convergence rather than differentiation.  would this  time  reflect  field. allow  In  the this  further  reflected  as  Common garden study, 1984 / 51 B.  MATERIALS  On July  AND  METHODS  28, 1984 a fresh set of stolon tip cuttings was taken from the  376  surviving clones and replanted in the same manner as described previously.  By  this time, the clones had been in the common garden for 27 months (from May, 1982) and had undergone two more generations of repropagation for a total of four generations since the original collection. This  set of ramets was given the  same period of growth (ten weeks) as the 1982 set. The plants were harvested in the fall of  1984  and a set of characters, similar, but not identical to the  original set measured (Table XI). Because of the problem of the harvested plants continuing to grow while stored in the cold room, an extended harvest was used. Plants  were  harvested  at a rate that allowed immediate  processing in the lab  without the need for cold storage. The harvest was begun on Oct 1, 1984 and completed on Nov 10, 1984.  Several changes were made in the character set mostly to remove ambiguities in the interpretation of characters. The original character  "primary  stolon number"  (PSTOL#) was eliminated because of difficulty in identification of primary stolons. In many cases, a prominent stolon having secondary and tertiary branches had developed from the base of a primary stolon rather than directly from the main taproot. was  To  above  define ground  this  as  rather  a secondary  stolon simply  than  seemed  below  because its branch  arbitrary.  The  point  alternative  of  including such branches as primaries was not feasible because of the impossibility of defining a consistent criterion for recognizing the point at which a secondary branch becomes a primary. For these reasons, only the total number of stolons (TSTOL#)  was  recorded. The  character  "internode  number"  (INODE#)  was  also  Common garden study, 1984 / 52 Table XI. List of characters measured in the 1984 common garden study of variation in a pasture population of Trifolium repens.  UNITS OF MEASUREMENT  CHARACTER Root dry weight  RTWT  0.01 gm  Shoot dry weight  SHTWT  0.01 gm  Total dry weight  TWT  0.01 gm  Total stolon number  TSTOL#  Length of primary stolon  PSTOL.L  5 mm  Total stolon length  TSTOL.L  5 mm  Internode length  INODE.L  0.5 mm  Petiole length  PET.L  1 mm  Leaflet width  LF.W  0.5 mm  Leaflet length  LF.L  0.5 mm  eliminated because of the difficulty of counting the first few internodes which are commonly  unelongated.  unelongated structure  internodes,  of the  Because the  of  character  stolon. "Internode  the  highly  seemed  length"  to  variable have  (INODE.L)  little was  number  of  relationship  these to  the  expanded to include  three internodes on each of three stolons for a total of nine measurements plant. One additional measurement, total stolon length (TSTOL.L) was recorded.  per  Common garden study, 1984 / 53 C. ANALYSES  Analyses the  AND  RESULTS  of variance were performed on the data in the same manner  1982 data  (Table  XII). Because  none  of the variables  showed  as for  significant  levels of heterogeneity among variances, no transformations were used.  The analyses provided no evidence of differentiation for any of the characters. In no  case  proportion  was  a  significant  of  the  variance  added  variance  explained  was  component for  detected.  PSTOL.L  (0.17%).  The  highest  The  highly  significant differentiation detected in 1982 had completely disappeared.  Among-genet characters  (within-neighbour)  variance components were calculated for the four  with repeated measurements  (PET.L,  L F . W , L F . L , and INODE.L.  In  contrast to the among-neighbour variance components, the among-genet components remained large (40%-70%) and highly significant (Table XIII). Because the number of measurements  of I N O D E . L  had been increased  to three  on each  of three  stolons per individual an among-stolon (within-genet) variance component could also be calculated. This component was also highly the variance  in INODE.L.  Thus,  significant and explained 30% of  for this character,  there  was more  difference  among stolons than there had ever been among neighbours for any character.  The correlation structure was similar to that of the 1982 data set (Table XIV). A l l correlations were positive and all except L F . W and L F . L with TSTOL# were significant  (p<0.01).  Correlations  among  the  seven  characters  which  were  measured in both years tended to decline from 1982 to 1984. Correlations among the weights, TWT, RTWT, and S H T W T remained high, however.  Common garden study, 1984 / 54 Table XII. Summary of analyses of variance for 12 morphological characters from four neighbour-specific groups of Trifolium repens. Data from the 1984 common garden study.  n  MS  MS  S A  SIG  %VARC  2  A  E  RTWT  92.3  0.265  0.460  -  NS  0.00  SHTWT  92.5  1.11  1.37  -  NS  0.00  TWT  92.3  2.13  3.23  -  NS  0.00  TSTOL#  92.3  50.8  80.4  -  NS  0.00  TSTOL.L  90.5  455000  474000  -  NS  0.00  PSTOL.L  92.3  6140  5300  9.09  NS  0.17  INODE.L  89.0  26.2  31.9  NS  0.00  PET.L  91.8  96.4  97.1  -  NS  0.00  LF.L  91.3  1.61  2.04  -  NS  0.00  LF.W  91.3  1.08  2.89  _ -  NS  0.00  n = mean # of individuals/group MS  = among-groups mean square A  MS = error mean square S = added variance component = ( M S - MS )/n A A E %VAR(A) = % variance among-groups = S /( S + MS ) A A E Sig = Level of significance from F-test for presence of added variance components. ** = p<0.01 * = p<0.05 N S = not significant :  £  y  2  2  Common garden study, 1984 / 55  Table XIII. Variance components for four morphological characters from a pasture population of Trifolium repens. Data from the 1984 common garden study.  NEIGHBOUR  GENET  ERROR  PET.L  0.0 (NS)  40.3 (**)  59.7  LF.W  0.0 (NS)  70.7 (**)  29.3  LF.L  1.3 (NS)  70.6 (**)  28.1  INODE.L  0.0 (NS)  49.7 (**)  20.1  STOLON  30.2 (**)  Entries give % variation accounted for by each source of variation and a test for the presence of an added variance component. ** = p < . 0 1 Sources of variation: Neighbour = among four neighbour-specific groups Genet = among-Trifolium repens individuals (within-groups) Error = among-measurements (within-individuals)(within-stolons for INODE.L) Stolon = among stolons (within-individuals) (for I N O D E . L only)  Common garden study, 1984 / 56  Table X I V . Correlations among 12 morphological characters of Trifolium repens. Data from the 1984 common garden study.  RTWT  1.000  SHTWT  0.867  1.000  TWT  0.945  0.982  1.000  TSTOL#  0.723  0.760  0.771  1.000  TSTOL.L  0.733  0.880  0.853  0.836  1.000  PSTOL.L  0.354  0.561  0.501  0.235  0.596  1.000  INODE.L  0.278  0.438  0.392  0.205  0.488  0.811  1.000  PET.L  0.326  0.500  0.451  0.235  0.380  0.477  0.518  1.000  LF.L  0.187  0.219  0.214  0.104  0.142  0.148  0.216  0.316  1.000  LF.W  0.211  0.223  0.226  0.136  0.170  0.184  0.235  0.302  0.837  RTWT  SHT  TWT  TSTOL TSTOL PSTOL INODE PET.L  WT  #  L  L  LF.L  L  Table entries are product moment correlation coefficients. Characters with coefficients greater than 0.139 are significantly correlated (p<.01) Coefficients indicating no significant correlation are printed in dark type, n  = 342  Common garden study, 1984 / 57 Because  the  variables  remained  highly  intercorrelated,  a  principal  components  analysis was performed (Table X V ) . As for the 1982 data, the first axis ( P C A l , 53.4% of the total variation) was most strongly influenced by plant weights and stolon numbers. A N O V A  on P C A l  produced the same result as the A N O V A s on  the original variables, no added variance components, only 0.69% of the variance accounted for, and no evidence of differentiation (Table XVI).  The  second  influenced by  and the  third  axes  relatively  (18.3% low  and  correlations  13.7%  of  between  the LF.L  total  variation)  and L F . W  other characters. The stolon structure pattern noted on P C A 2  were  and  the  in 1982 appeared  again but was less distinct. The fourth and fifth axes accounted for 5.8% and 3.7% of the total variation respectively  for a  five  axis total of 94.9%. As  in  1982, there were no obvious patterns on the last two axes. A N O V A s on PCA2-5 again produced no evidence of differentiation (Table XVI).  Common garden study, 1984 / 58  Table X V . Principal components analysis of 12 morphological characters of Trifolium repens. Data from the 1984 common garden study.  AXIS PCA1  PCA2  PCA3  PCA4  PCA5  % VARIATION CUMULATIVE  53.42 53.42  18.30 71.72  13.72 85.44  5.84 91.28  3.75 94.93  RTWT SHTWT TWT TSTOL# TSTOL.L PSTOL.L INODE.L PET.L LF.L LF.W  0.366 0.412 0.408 0.331 0.392 0.291 0.262 0.255 0.150 0.159  0.192 0.135 0.161 0.273 0.172 -0.144 -0.238 -0.262 -0.583 -0.574  0.228 0.062 0.126 0.262 -0.009 -0.522 -0.534 -0.236 0.356 0.347  -0.059 -0.084 -0.077 0.097 0.208 0.303 0.245 -0.861 0.090 0.171  -0.514 -0.168 -0.304 0.638 0.357 -0.170 0.087 0.207 -0.002 0.027  Table entries are coefficients for each character for the first five principal components (axes). % V A R I A T I O N is the amount of variation in the multivariate data set which is explained by each axis.  Common garden study, 1984 / 59  Table X V I . Summary of analysis of variance of principal component scores for four neighbour-specific groups of Trifolium repens. Data from 1984 common garden study.  AXIS  MS  MS  S  SIG  2  % VAR(A)  A  NS  0.69  1.844  NS  0.00  0.460  1.380  NS  0.00  PCA 4  1.480  0.577  NS  1.80  PCA 5  0.353  0.365  NS  0.00  PCA 1  8.487  5.315  PCA 2  0.285  PCA 3  0.037  0.011  n = mean # of individuals/group MS MS S  2  A  A E  = among-groups mean square = error mean square  = added variance component = ( M S  - MS  A  %VAR(A) = % variance among-groups = S  2  A  E  /( S  2  A  )/n + MS^ ) E  Sig = Level of significance from F-test for presence of added variance components. ** = p<0.01 * = p<0.05 N S = not significant  Common garden study, 1984 / 60  D.  DISCUSSION  The extreme plasticity of Trifolium repens is well known (Broughham et al. 1978; Hill  1977) In fact, Bradshaw (1965) referred to the petiole of T. repens as one  of the most  plastic  plant  showed the strongest  organs  known. It is noteworthy  differentiation  that  of any of the measured  petiole  length  characters  in the  first part of this study. It should not be surprising that T. repens would respond plastically  to changes  surprising  that  were  retained  however, there  some  in its neighbour of the plastic  for a season  was that  had been  environment.  effects  produced  in the common  the carry-over  effects  6-7% differentiation  garden.  would have  among  It should  also  in different What  not be  environments  was unexpected,  been so extreme.  neighbour-groups  after  Where  one season,  after two years none remained. The clear conclusion is that there is no evidence at  all  in  this  differentiation  data  detected  set  for  morphological  in the first  part  differentiation.  of the study  simply  The apparent reflected the  carry-over of environment-specific developmental adjustments (phenotypic plasticity). Over the 30 months of the study, the plants converged morphologically as they readjusted developmentally to the common garden environment. Presumably, if the plants were transplanted back to the field, the neighbour-specific pattern would be regenerated.  The morphological convergence among neighbour-groups did not extend to variation among individuals. The among-genets component of variance did not change much between years.  Coefficients  of variation  were also similar  in both years.  Thus,  the convergence represented shifts in mean values for the neighbour-groups rather  Common garden study, 1984 / 61 than an overall decline in variation.  A  number  of  studies  on differentiation  have  also  demonstrated  morphological  convergence among plants transplanted to a common environment. Watson (1969) collected Potentilla Molinia  meadow.  immediately  after  erecta from two adjacent habitats, an Agrostis pasture and a Lengths  of  stems  and  basal  internodes  collection  and again  after  two years  were  measured  in a common  garden.  Populations from the two habitats differed in the initial measurements by =70% for  both  Despite  characters. the  Two years  convergence,  the  later,  the differences  populations  had declined  remained  to =30%.  significantly  (p<.01)  differentiated, supporting the conclusion that they were genetically different.  Warwick  and Briggs (1979) collected Achillea  lanceolata, P. major, and Prunella different  lawn  immediately  and grassland after  among-habitat  collection  types  millefolium,  Bellis perennis, Plantago  vulgaris from 40 populations representing four  habitat showed  types.  Leaf  significant  and among-populations  sizes  and widths,  (p<.05)  (within-habitat  measured  differentiation types)  for  both  all  five  species. After one year in. a common garden, only one of the species, P. major, retained the among-habitat differentiation. A l l retained significant  among-individual  and  among-population  among-population  variation.  After  a  second  year,  the  differences in P. lanceolata had also disappeared.  Akeroyd and Briggs (1983) collected Rumex crispus over two years from 23 sites representing characters  five at  different  the time  habitat of  types.  collection  Measurements  showed  significant  of seven  morphological  differentiation  (p<.05)  Common garden study, 1984 / 62 among-habitat types)  for  plants  collected  types  all  for  four  characters  seven. Measurements in  the  first  year  after  and one  showed  among-population year  in  convergence  a  (within-habitat  common  garden  among-populations  on but  divergence among-habitat types. For the second year's collection, the results were opposite, convergence both among-habitat types and among-populations. The results of Akeroyd and Briggs (1983) show that the pattern of phenotypic response to a common  garden  may  be  unpredictable.  Both  divergence  and  convergence  were  noted as well as different responses among years.  Seliskar  (1985) reciprocally  transects  between  transplanted ramets of five salt marsh species along  mudflat  transplanted vertically  and  upland  habitats.  After  one  year,  the  ramets  along transects had converged morphologically with  ramets  transplanted between comparable points on different transects.  In these studies, convergence has been noted over periods varying from 6 months to 2 years. In the present study it occurred before 30 months. The conclusion is that  if  measurements  on  common  garden  material  are  made  within  the  first  season after collection, carry-over effects may not have had time to dissipate and differentiation may be overestimated.  The time scale for morphological convergence noted in the above studies fits well with  the  concept  of  plasticity  as  reversible,  developmental  responses  to  environmental changes occurring within the lifetime of the individual, or in the case  of  (Bradshaw  clonal  herbs  such  as  T.  repens, within  the  lifetime  of  the  ramet  1965). This time scale is linked to the largely intuitive treatment of  Common garden study, 1984 / 63 carry-over effects in genecological work on perennial herbs. After to  a  common  environment,  plants  are generally  given  transplantation  "conditioning"  periods  roughly corresponding to the lifetime of a ramet. The conditioning period is often measured as numbers of "tissue turnovers" or "cloning generations". It is tacitly assumed that any persistent effects of the field environment will be lost with the death of the original material and that development of second generation ramets will be entirely  under the influence of the common environment.  second  generation  doubt,  experimenters  additional Silander  time  ramets  thus  often  choose  for conditioning  studies  which  have  not reflect  carry-over  effects.  to err on the conservative  (e.g.,  (1979), 2 years for Spartina  18 months for Carex aquatilis; The  should  Snaydon  2 years  side  When in and add  for T. repens;  patens; Shaver, Chapin & Billings (1979),  Turkington  documented  (1971),  Phenotypes of  (1983a,b), 17 months for T. repens).  convergence  suggest  that  such  extended  periods are usually  sufficient to minimize carry-over. Pilot studies on carry-over  are  leaving  rare,  however,  removed  any  effects  of  most  investigators  preconditioning  by  to "assume the different  that field  this  treatment  environments"  (Shaver, Chapin & Billings 1979).  One experimental study of carry-over effects was conducted by Mackenzie (1985). Ramets  cloned  laboratory  from  Ranunculus  environments,  grown  repens for three  genets months  common environment for six months. Highly  were  transplanted  into  three  transplanted  to a  (p<.01) differences  were  and then  significant  found wtf/ttn-genets for ramets subjected to the different treatments. These results demonstrate  that  plastic  effects  that  develop  longer to disappear (at least six months).  quickly  (three  months)  may take  Common garden study, 1984 / 64 In the case of T. repens, the short lifespan of the ramet makes it convenient to incorporate Thus,  several  a number  (and  other  generations of studies  perennial  conditioning  3 months,  Trathan  (1985),  turnover  of differentiation  herbs)  have  but incorporating  (1962b),  of tissue  several  Turkington  5 months,  been  tissue  to experimental  and variation  conducted  using  turnovers.  and Harper  Aarssen  prior  (1979c),  and Turkington  in Trifolium repens  only  brief  These 3  work.  periods of  include  months,  (1985c),  Snaydon  Gliddon and  4 months, and  Aarssen and Turkington (1985b), no conditioning. Burdon (1980a) did not report any conditioning period. Similarly brief periods were used for Bellis perennis and Prunella  vulgaris  among-habitat  by Schmid  (1985), (7 weeks). Lovett-Doust  (1981c) found  that  increased in the common garden for R. repens and  differentiation  thus used no conditioning period. These studies have not generally reported the actual number of tissue turnovers used but generally refer to periods of cloning or of repeated division, implying that several tissue generations had taken place. The  present  beyond more  results  two cloning effectively  generations. reflected should  show  in T. repens can last  that  carry-over  effects  generations.  Therefore,  an extended  minimize  carry-over  than  an  period  extended  of time  number  of  Some of the results based on short conditioning periods  carry-over be  noted  effects that  and thus while  all  may have of  the  overestimated  studies  cited  well should  cloning  may have  differentiation. It above  used  brief  pre-experimental conditioning periods, the two which demonstrated neighbour-specific differentiation Turkington conditions studies,  in Trifolium  1985b)  and Harper  both allowed extended periods  (one year) then,  repens (Turkington  before  may have  taking  been  less  Aarssen and  of growth under  measurements. influenced  1979c;  The results  by carry-over  experimental  of these two  than  the others  Common garden study, 1984 / 65 mentioned. Therefore, their conclusions  are not necessarily  brought into  question  by the present results. It should also be noted that these two studies looked at performances of T.  repens (dry weight production) in the presence of the actual  neighbours (i.e., neighbour-compatibilities were measured directly). Because there is no evidence linking the morphological characters studied here to performances, the lack of neighbour-specific morphological differentiation does not necessarily imply a lack of differentiation for neighbour compatibilities.  The implication of my results for genecological work is that short term studies with  perennial  herbs  may  not  give  reliable  evidence  on  differentiation.  This  applies both to common garden and reciprocal transplant studies. The control of carry-over effects will require more objective treatment than intuitively numbers  of tissue  turnovers.  The  process of plastic  adjustment to  generated  experimental  environments can be followed by periodic recording of characters beginning at the time of collection. Final measurements convergence is complete.  should be delayed until  after  the  initial  IV. DIVERSIFYING SELECTION AND  TRIFOLIUM  REPENS:  A  RECONSIDERATION These results have confirmed that there is considerable morphological variation in T.  repens populations  which  is  stable  under  extended  periods  in a  common  garden, and which may be genetically based. They offer no support however, for the hypothesis that genetically based morphological variation in this population is being  maintained  by  neighbour-specific  diversifying  selection.  There  was no  evidence for spatial correlation between the patterns of distribution of the grasses and the distribution of persistent variation in the morphology of clovers. Although the data from the first season in the common garden showed a neighbour-specific pattern, little of the population-wide later  proved  to be ephemeral.  variation was accounted for, and even this  While  T.  repens individuals  are  influenced by  changes in their neighbour environment, the effects of neighbours may not extend to  ramet  transitory  dynamics. adjustments  establishment  Responses, in  and survival  at the morphological  physiology of ramets.  level,  and development, Thus,  there  does  not  apparently changes  not appear  reflect in the  to be a  strong linkage beween neighbour relationships and population-wide genetic patterns in morphology.  In light of the importance  often attributed to environmental heterogeneity  diversifying  Hamrick  influence  (e.g.,  1980; Hedrick,  Ginevan  &  as a  Ewing 1976;  Ennos 1983), it seems appropriate to consider why such a prominent element of patchiness  as  the  neighbour  mosaic  did  not  turn  out  to  constitute  an  environmental heterogeneity. Why is morphological variation in this population not strongly shaped by neighbour-specific diversifying selection? The relationship among  66  Diversifying selection and Trifolium. repens: a reconsideration / 67 environmental  heterogeneity,  has been analysed  theoretically  Dickinson & Antonovics in  a heterogeneous  individuals  environment  spend  (Levins  selection,  entire  lives  of variation  Ginevan & Ewing 1976;  suggested that diversifying  will be most effective  among microhabitats  their  and maintenance  1969; Hedrick,  1973). Models have  when selective potentials and  diversifying  in maintaining  selection variation  (patches) are high and consistent,  in a single  patch  (i.e.,  have  coarse  grained environments).  Bradshaw (1972) has argued that plants are commonly subject to strong selective potentials. This is, in part, a consequence of their immobility and a reflection of the fact that most plants do have coarse-grained environments. Certainly, plant borne  populations out by  differentiated Bradshaw selection  will  be subject  the large  number  in concordance  1966; Snaydon  with &  may be cumulative,  to diversifying of cases  selection.  in  which  an environmental  Davies especially  plant  expectation populations  heterogeneity  1976, 1982). where  This  is are  (e.g., Jain  For perennials,  the conditions  many  effects  & of  of a patch are  permanent or repeated annually.  The expectation of among-neighbour selection in T. repens was supported by the results of competition experiments. However, the evidence on the effectiveness of such selection is equivocal. Plot and greenhouse studies have commonly, but not invariably  shown significant neighbour-specific differences in performance  measures  such as dry weight or stolon production. Turkington (1983c), for example, found that one of two T. repens individuals responded differently to different neighbours, but  the other  did not. Solangaarachchi  (1985)  found  that  performances  of T.  Diversifying selection and Trifolium repens differed when  among  measurements  neighbours were  on  when  dry  stolon  weights  repens: a reconsideration / 68  lengths  of  were  stolons  or  measured shoots.  but  In  not  addition,  rankings of neighbours on the basis of their effects on the performances of T. repens  have  sometimes  Solangaarachchi grown  in  (1985)  been  inconsistent  found  experimental  plots  between  significantly of Lolium  greater  and stolon  perenne or  plots of Holcus lanatus or Agrvstis capillaris.  even  within  lengths  Cynosurus  studies.  T. repens  in  cristatus than  in  The transplants of Turkington et al.  (1979) into natural plots of the same four grasses produced a similar ranking. The concurrent transplants of Turkington and Harper (1979c) into patches of the same grasses in the same field, however, produced a reversal in the rankings of L. perenne and A.  capillaris.  Consideration of the patch structure of the pasture generates further doubt as to the likelihood of its being a source of effective diversifying selection. Any  single  survey of this pasture is likely to reveal distinct patches of some or all of the four grasses. Such was the case in May, 1982 when the material for this study was  collected.  located. these  One  Repeated patches  composition  surveys  are  of  hundred  not  patches  patches  across  stable can  of each  seasons  (Parish, vary  or  of the years,  personal  widely  four  grasses  however,  the  year  easily  demonstrate  communication).  through  were  as  The a  size result  that and of  differences in the active growing times of the grasses. Additionally, the neighbour mosaic varies across years. A survey in May, collection  date),  failed  to  identify  a  single  1983, (one year after the original  patch  dominated  by  Poa  compressa  (personal observation). Similarly, in 1984 Holcus lanatus was nearly absent from the  pasture  (Parish,  personal  communication).  Such  wide  variation  in  species  Diversifying selection and Trifolium repens: a reconsideration / 69 composition is not unusual for permanent pastures (Thorhallsdottir 1985).  As  a  relationships  consequence,  any selective  potential  associated  may fluctuate even within the lifetimes  1983; Snaydon with  neighbour  of clover ramets.  Snaydon  (1985) has argued that this temporal variation should enhance the effectiveness of diversifying selection in maintaining variation in pasture populations. Theoretical models,  however,  selection  will  suggest  be diluted  the opposite, by such  that  the effectiveness  inconsistencies  in strength  of  diversifying  and direction of  selection (Ennos 1983).  The  second  selection  condition  requires  for  that  effective  maintenance  the environments  of  of  variation  individuals  by  diversifying  be coarse-grained.  A  consequence of the modular construction of T. repens and its stoloniferous habit is that the environmental grain of a genet can be different than that for a ramet. Because a genet is composed of a number of stolons which may commonly grow across patch boundaries, the environment of a genet will usually be fine grained. In fact, a long lived T. repens genet probably encounters much of the range of microhabitats in a pasture. Thus, the persistence of genets may not depend on conditions within any single patch. In contrast, a ramet does spend its entire life on a single spot. If conditions at that spot do not change during that period of time,  the ramet  will  replication  will  possibility  of strong  have  be dependent  a  coarse-grained on conditions  patch-specific  selection  environment. at  that  among  Its persistence and  spot.  ramets  Thus that  there could  is  a  lead to  sorting of genets among patches and a reduced rate of genet depletion. It is not clear,  however, just  Trifolium  how coarse-grained  repens ramets  do not normally  the environments become  of ramets  physically  really are.  independent  of the  Diversifying selection and Trifolium parent  ramet  until after  they  have become  repens: a reconsideration / 70  well established  (Harvey  1970). It  has been demonstrated in various clonal plants that daughter ramets can draw physiological support from their parents 1983; Mackenzie single  patch,  (Lovett-Doust  1981a; Hartnett & Bazzaz  1985). Instead of being dependent on the conditions  the performances  of T. repens genets  may actually  within a  reflect the  integration of the conditions across several patches. Thus ramets may not have the  coarse-grained  environment  that  would  be compatible  with  a  diversifying  selection scenario. The evidence for clonal integration in T. repens, however, is not  conclusive.  Harvey  (1970)  showed  that  photosynthates  moved  only  in the  direction of stolon apices. This suggests that integration would occur only within stolon branches. Ramets contrast,  on different  Solangaarachchi  formation)  in one part  (1985)  stolons  found  would not support each other. In  that  of an interconnected  manipulations  (prevention  clone infuenced  growth  of root  and stolon  branching in other parts of the clone.  Most of the experiments whch have supported the expectation of neighbour-specific selection among ramets have been based on the survival and growth of excised ramets.  If, as seems  ramets  is  narrower  performances  as  than  of excised  These experiments mosaic  likely,  the "regeneration  that  ramets  of ramets  niche"  with  may not equate  (Grubb  intact with  stolon those  1977) of excised connections, the of intact  ramets.  may, then, have overestimated the strength of the neighbour  an agent  of diversifying  selection.  One relevant  experiment was  performed by Turkington (1983a) who planted ramets of T. repens at the centers of hexagons  of patches  of different  neighbours  including  four  pasture  grasses.  Growth of the T. repens ramets into the patches was monitored. Final percent  Diversifying selection and Trifolium repens: a reconsideration / 71 cover of T. repens did not vary significantly among the four grass patches. For initial invasion, at least, the differences among grass patches may be irrelevent to established T. repens plants.  Because the effectiveness of among-neighbour selection on T. repens ramets may be diluted by instability in patch structure and by among-patch integration, the neighbour mosaic may not really have represented a likely source of diversifying selection  for T.  repens populations.  Bazzaz  and Sultan  (in press)  have  also  proposed that clonal integration and temporal environmental instability will buffer clonal genets  against  selection from their  biotic environment.  They  also  suggest  that it may be unrealistic to expect extensive selective elimination of genets from (1983) noted that T. repens ramets  clonal populations. Thorhallsdottir  commonly  become established on small gaps within patches. She suggested that these gaps were more important than the identity of the patch for the establishment of T. repens ramets (and therefore for the persistence of genets). These reinforce variation  the earlier in  unnecessary.  pasture  conclusion  that  populations  of  a selective T.  explanation  repens may be  considerations  for maintenance of both  unlikely  and  REFERENCES  Aarssen, L. W, (1983). Interactions and coexistance of species in pasture community evolution. Ph.D. Thesis, University of British Columbia. Aarssen, L. W. & Turkington, R. 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