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Genetic variation, population structure and mating system in bigleaf maple (acer macrophyllum pursh) Iddrisu, Mohammed Nurudeen 2005

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G E N E T I C V A R I A T I O N , P O P U L A T I O N S T R U C T U R E A N D M A T I N G S Y S T E M IN B I G L E A F M A P L E {ACER MACROPHYLLUM PURSH) by M O H A M M E D N U R U D E E N IDDRISU Ing. For. University of Pinar del R i o , 1993.  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T O F THE REQUIREMENTS FOR THE D E G R E E OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Forestry)  T H E UNIVERSITY O F BRITISH C O L U M B I A  May, 2005  © M o h a m m e d N u r u d e e n Iddrisu, 2 0 0 5  ABSTRACT E c o l o g i c a l characteristics a n d life history traits of long lived w o o d y plants influence their levels of genetic variation. T o e m b a r k upon s o u n d m a n a g e m e n t , utilization a n d c o n s e r v a t i o n of plant s p e c i e s , a thorough understanding of genetic p r o c e s s e s affecting their p e r s i s t e n c e is essential. In this thesis, I studied genetic diversity, population structure, a n d mating s y s t e m a s well a s c o m p a r e d genetic diversity a n d inferred differences in genetic p r o c e s s e s in continuous v e r s u s fragmented populations of bigleaf m a p l e (Acer macrophyllum  Pursh). Bigleaf m a p l e  is o n e of the most abundant hardwood s p e c i e s in the Pacific Northwest a n d its native range e x t e n d s from latitude 3 3 ° N to 5 1 ° N along the Pacific c o a s t of North America. G e n e t i c diversity, estimated using i s o z y m e markers, revealed a m e a n e x p e c t e d heterozygosity (H ) of 0 . 1 5 2 similar to other North A m e r i c a n a n g i o s p e r m E  trees. T h e level of population differentiation w a s moderately low ( F T = 0 . 0 5 4 ) , S  indicating extensive g e n e flow a m o n g populations. E s t i m a t e d outcrossing rates in two populations w e r e high ( 9 5 % ) but significantly less than o n e , with no biparental inbreeding evident. A relatively high level of correlated matings w a s found, consistent with 2 - 5 effective pollen d o n o r s per tree, indicating low adult density a n d limited pollinator d i s p e r s a l . S e e d l i n g a n d adult populations p o s s e s s similar levels of genetic variation regardless of whether populations are fragmented or continuous. H o w e v e r , s e e d l i n g cohorts have higher levels of inbreeding than adult cohorts, on a v e r a g e , in both continuous a n d fragmented populations. A n a l y s i s of spatial genetic structure indicates n o n - r a n d o m distribution of g e n o t y p e s in all three fragmented populations a n d o n e of the three continuous populations. I found a significant positive autocorrelation (p/,= 0 . 2 0 ) a m o n g individuals located up to 1 0 0 m apart in all three  fragmented populations a n d a m o n g individuals located at approximately  100-200  m  apart (p,y = 0 . 1 4 ) in one of three continuous populations. Finally, for quantitative traits, p r o v e n a n c e s a n d families within p r o v e n a n c e s s h o w e d significant genetic variation for height growth a n d bud flush traits, but not for diameter growth. Individual heritabilities for all traits w e r e generally low to moderate (0.15-0.21),  and  F T S  a n d family heritability w a s higher only for bud flush. C o m p a r i s o n of  in this study (mean  Q T= 0.17 S  > mean  F T = 0.09) S  QST  s u g g e s t s the involvement  of selection for different p h e n o t y p e s in different populations of bigleaf m a p l e .  TABLE OF CONTENTS Abstract  ii  Table of Contents  iv  List of Tables  viii  List of Figures  xi  List of A p p e n d i c e s  xii  Acknowledgements  xiii  Dedication  xv  Published papers  xvi  Chapter One G e n e r a l Introduction  1  T h e s i s overview  2  Chapter Two Literature R e v i e w Biology a n d silvics of Acer macrophyllum  4 Pursh  G e n e t i c variation a n d structure in natural populations  4 5  Effects of population size on g e n e t i c variation  6  Effects of population size on mating s y s t e m s  8  Effects of fragmentation on genetic variation in plant populations Effects of fragmentation on spatial genetic structure  10 13  M o l e c u l a r a n d quantitative variation  14  Chapter Three G e n e t i c variation, population structure a n d mating s y s t e m in bigleaf maple [Acer macrophyllum) Introduction  19  Materials a n d methods  20  Isozyme a s s a y  21  Data analysis  22  Results  24  Allele frequency distribution  24  G e n e t i c diversity  24  G e n e t i c structure  25  Mating s y s t e m  26  Discussion  27  G e n e t i c variation  27  Population genetic structure a n d g e n e flow  27  Mating s y s t e m Implications for m a n a g e m e n t a n d conservation  Chapter four  19  ....29 31  Effects of forest fragmentation on genetic variation a n d spatial genetic structure in natural populations of bigleaf m a p l e (Acer macrophyllum)  41  Introduction  41  Materials a n d methods  44  Populations and s a m p l i n g  44  Electrophoresis  45  Data a n a l y s i s  45  G e n e t i c structure  46  Spatial autocorrelation a n a l y s i s  46  Simulations Results  49 50  Allele f r e q u e n c i e s  50  G e n e t i c diversity  50  L e v e l s of inbreeding  51  Bottleneck test  51  G e n e t i c structure  51  Spatial genetic structure  52  Simulations  53  Discussion  54  Effects of fragmentation on genetic variation a n d inbreeding  54  Inbreeding in adults v e r s u s s e e d l i n g s  56  Populations structure  57  Spatial genetic structure  58  C o m p u t e r simulations of fragmentation effects  60  Chapter five G e n e t i c variation a n d population structure in bigleaf m a p l e : a c o m p a r i s o n of a l l o z y m e markers a n d quantitative traits  74  Introduction  74  Materials and m e t h o d s  76  Quantitative traits  76  Data collection..  77  Analysis  77  Isozyme variation  79  Results  80 Quantitative traits  80  M o l e c u l a r genetic variability  81  Discussion  82  Quantitative traits  82  B u d flush  83  G e n e t i c correlations  84  Correlations with climatic v a r i a b l e s  84  FSTVS Q T S  Chapter 6  References  Conclusions  85  95  Major findings  96  Recommendations  98 100  LIST OF TABLES 3.1.  Distribution of allele f r e q u e n c i e s at 10 loci in eight natural mature populations of bigleaf maple (Acer macrophyllum)  3.2.  34  S u m m a r y of genetic diversity within eight mature natural populations of bigleaf maple (Acer macrophyllum)  3.3.  b a s e d o n 10 a l l o z y m e loci  35  Total g e n e diversity (HT), genetic diversity within populations (Hs), e x p e c t e d heterozygosity (H ), 0  alleles per locus (N ), fixation index A  o v e r the total populations (FIT), fixation index within population (F/s), a n d genetic differentiation a m o n g populations (F T) for eight S  mature natural populations of bigleaf maple (Acer  macrophyllum)  at nine polymorphic loci  3.4.  36  E s t i m a t e s of multi-locus outcrossing rates (t ), m  single-locus outcrossing  rates (t ), biparental inbreeding (tm-t ), parental inbreeding coefficients (F) s  s  a n d correlation of paternity a m o n g siblings (r )  37  p  3.5.  C o m p a r i s o n of within-population genetic diversity for Acer  macrophyllum  with a v e r a g e v a l u e s for all plants, w o o d y s p e c i e s , w o o d y a n g i o s p e r m s , a n d for maple s p e c i e s  4.1.  S u m m a r y of population information for adult trees a n d s e e d l i n g s of bigleaf maple Acer macrophyllum  4.2 a.  38  62  A l l e l e f r e q u e n c i e s for nine loci for adults in continuous a n d fragmented populations of Acer macrophyllum  63  4.2 b.  A l l e l e f r e q u e n c i e s for nine loci studied for s e e d l i n g s in continuous a n d fragmented populations of Acer macrophyllum  4.3.  4.4.  64  G e n e t i c diversity estimates for adults a n d s e e d l i n g s in continuous a n d fragmented populations of Acer macrophyllum  65  W i l c o x o n s i g n e d ranked test for recent bottleneck (Cornuet a n d Luikart 1996) in Acer macrophyllum  populations under the Infinite  Alleles Model  66  4.5 a & b. G e n e t i c diversity statistics for the eight polymorphic i s o z y m e loci for (a) continuous populations a n d (b) fragmented populations  4.6.  P a i r w i s e FST between adult fragmented a n d continuous populations of Acer macrophyllum  4.7.  67  68  E x p e c t e d percentage of a l l o z y m e diversity retained o v e r 2 5 0 - y e a r period b a s e d o n computer simulations B O T T L E S I M (Kuo a n d J a n z e n 2003) for adult populations of Acer macrophyllum  in  fragmented a n d continuous forests a s s u m i n g 125-year generation length  5.1.  L o c a t i o n s of bigleaf maple s a m p l e d populations for p r o v e n a n c e trials a n d least s q u a r e m e a n s for growth a n d bud flush traits  5.2.  69  A N O V A results for F approximations for the hypothesis of no family or p r o v e n a n c e effect  88  89  5.3.  C o m p o n e n t s of v a r i a n c e , individual heritabilities (h i), family 2  heritabilities (h f) a n d population differentiation ( Q r ) a m o n g growth 2  S  a n d bud flush traits  5.4.  90  G e n e t i c correlations (above diagonal) a n d family phenotypic correlations (below diagonal) between s e e d l i n g traits for bigleaf maple p r o v e n a n c e s in British C o l u m b i a  5.5.  Correlation coefficients between quantitative traits a n d climatic variables b a s e d on 14 p r o v e n a n c e m e a n s  5.6.  91  G e n e t i c diversity estimates for 14 juvenile populations of Acer macrophyllum  5.7.  91  92  E s t i m a t e s of Wright's F-statistics for eight polymorphic loci in British C o l u m b i a bigleaf maple populations  93  LIST OF FIGURES  2.1.  Native range of Acer macrophyllum  (bigleaf maple)  3.1.  G e o g r a p h i c a l locations of eight Acer macrophyllum  18  mature populations  natural populations  3.2.  4.1.  4.2.  39  U P G M A cluster a n a l y s i s of Nei's genetic d i s t a n c e s b e t w e e n eight mature populations of Acer macrophyllum  40  G e o g r a p h i c a l locations of s a m p l e d bigleaf maple populations  70  Distribution of allele f r e q u e n c i e s for adults (a) a n d s e e d l i n g (b). Filled bars are continuous populations a n d o p e n bars fragmented populations  71  4.3 (a-c). Spatial correlograms of c o a n c e s t r y coefficients (p,y) for continuous populations of Acer macrophyllum.  D a s h e d lines represent upper a n d  lower 9 5 % c o n f i d e n c e limits for p,y under the null hypothesis that g e n o t y p e s are randomly distributed  72  4.3 (d-f). Spatial correlograms of c o a n c e s t r y coefficients (p,y) for f r a g m e n t e d populations of Acer macrophyllum.  D a s h e d lines represent u p p e r a n d  lower 9 5 % c o n f i d e n c e limits for p,y under the null hypothesis that g e n o t y p e s are randomly distributed  5.1.  L o c a t i o n s of s a m p l e d populations of bigleaf m a p l e p r o v e n a n c e trials  73  94  LIST OF APPENDICES  I.  II.  E n z y m e , buffer s y s t e m s a n d recipes for histochemical staining solutions  Allele frequency distribution of ten loci of bigleaf m a p l e p r o v e n a n c e trials  129  130  ACKNOWLEDGEMENTS I would first like to a c k n o w l e d g e with d e e p appreciation the Department of Foreign Affairs a n d International T r a d e for the A w a r d of C a n a d i a n C o m m o n w e a l t h S c h o l a r s h i p through the International C o u n c i l for C a n a d i a n S t u d i e s . F u n d i n g for r e s e a r c h w a s m a d e available initially from the B C Ministry of F o r e s t s , R e s e a r c h B r a n c h through Dr. C h e n g Y i n g a n d c o m p l i m e n t e d by a Natural S c i e n c e s a n d Engineering R e s e a r c h C o u n c i l ( N S E R C ) grant to Dr. Kermit Ritland. T h i s study would not have b e e n c o m p l e t e d without the m u c h n e e d e d fellowship a n d additional funding for r e s e a r c h provided to me by my c o s u p e r v i s o r Dr. S a l l y A i t k e n through the C e n t r e for F o r e s t G e n e C o n s e r v a t i o n via the Forest G e n e t i c s C o u n c i l of B C from the Forest investment A c c o u n t of B C a n d the N S E R C Industry J u n i o r C h a i r in Population G e n e t i c s .  I would like to thank my c o - s u p e r v i s o r s Drs S a l l y A i t k e n a n d Kermit Ritland a n d committee m e m b e r Dr. Jeannette Whitton for their g u i d a n c e , support a n d constructive c o m m e n t s . S p e c i a l thanks go to Dr. S a l l y A i t k e n w h o s p e n t a n extra time o n my draft, challenging me to write c o n c i s e l y a n d e n c o u r a g i n g m e to think critically a n d realistically. M y sincere thanks a l s o g o to Dr. C a r o l Ritland for her initial involvement in my committee, for providing fresh perspective on my r e s e a r c h during the initial s t a g e s , planning my field trips a n d supervising my lab work. T o Dr. C h e n g Y i n g , thanks very m u c h for serving o n my committee, for the g u i d a n c e a n d n u m e r o u s fruitful c o m m e n t s and s u g g e s t i o n s on my r e s e a r c h , for your friendship a n d fatherly advice a n d for providing the quantitative data a n d e n c o u r a g i n g m e to work on bigleaf m a p l e . T o Mr. D o n Pigott for sharing your expertise with m e a n d the great help in collection of s a m p l e s in the field.  I wish to also thank the following: -  My colleagues, Cherdsak Liewlaksaneeyanawin, Charles C h e n , Yanik Berube and H u g h W e l l m a n .  -  Drs. Tongli W a n g , A n d r e a s H a m a n n for their help a n d a d v i c e and P i a S m e t s for being s o o p e n e d to d i s c u s s i n g i s s u e s b e y o n d a c a d e m i c s .  -  M y wife Yanela and children Leandro a n d Neina for their e n o r m o u s sacrifices and support throughout the duration of my program.  -  T o my mother, brothers a n d sister and my e x t e n d e d family m e m b e r s for their e n c o u r a g e m e n t and advice.  DEDICATION  T o the m e m o r y of my late father- M b a Iddi ( G u s h e i - N a a )  "After great pain a formal feeling comes" Emily Dickinson  Chapter One  G E N E R A L INTRODUCTION  F o r e s t s are declining in most regions world-wide, a n d this h a s c a u s e d grave c o n c e r n a m o n g scientists a n d policy m a k e r s throughout our world. M u c h of the world's biodiversity is harbored in forests, particularly tropical forests w h i c h are estimated to contain up to 70 percent of the world's s p e c i e s ( G r o o m 1994). H o w e v e r , over the last two centuries, the exponential growth of h u m a n populations c o u p l e d with growth of cities, industrialization a n d agriculture has led to w i d e s p r e a d destruction a n d degradation of m a n y forested a n d other natural s y s t e m s ( F A O 1997). A p p r o x i m a t e l y half of the world's forest a r e a h a s b e e n c l e a r e d or d e g r a d e d s i n c e the beginning of the H o l o c e n e ( G r o o m b r i d g e a n d J e n k i n s 2002). Currently, about 30 percent of the world's land a r e a is c o v e r e d with forest ( F A O 2001). M a n y of t h e s e forests are partially converted to agricultural or urban u s e , resulting in the loss of s o m e unique characteristics that w e r e previously present. T h e main goal of population genetics is to understand the origin, distribution a n d m a i n t e n a n c e of genetic diversity, which is the raw material for evolutionary c h a n g e (Ledig 1992; Hartl a n d Clark 1997). In small populations in particular, g e n e s undergo genetic drift a n d a s a c o n s e q u e n c e genetic diversity is randomly a n d continuously lost (Vitalis a n d C o u v e t 2001). M o r e o v e r , genetic drift in small populations c a n be more important than selection in determining the fate of n e w alleles (Whitlock 2000). In subdivided populations, the m a i n t e n a n c e of neutral alleles d e p e n d s on the relative strength of local genetic drift a n d the extent of g e n e flow a s a h o m o g e n i z i n g force (Slatkin 1995). C h a n g e s in genetic structure a n d levels of diversity in subdivided or s m a l l populations of forest tree s p e c i e s will d e p e n d on s e v e r a l factors, s u c h a s the magnitude a n d frequency of forest destruction a n d d e g r e e of isolation a m o n g fragmented forests ( B a w a 1994). Forest m a n a g e m e n t practices also affect genetic  diversity ( S a v o l a i n e n a n d Karrkainen 1992; E l - K a s s a b y a n d N a m k o o n g 1994). Harvesting c a n lead to a reduction in stand density, which m a y result in i n c r e a s e d levels of inbreeding a n d a decline in genetic diversity (Murawski a n d Hamrick 1992; Buchert et al. 1997). K n o w l e d g e of mating s y s t e m s , the levels a n d distribution of genetic variation, a n d factors influencing its m a i n t e n a n c e is n e c e s s a r y for effective forest m a n a g e m e n t a n d conservation p r o g r a m s ( E r i k s s o n et a l . 1995). In recent y e a r s , there has b e e n an a c c u m u l a t i o n of data c o n c e r n i n g the patterns of genetic variation in m a n y North A m e r i c a n coniferous tree s p e c i e s . H o w e v e r , relatively little information is available about genetic variation in temperate w o o d y a n g i o s p e r m s . T h i s situation is particularly true for intolerant, early s u c c e s s i o n a l a n d shrubby trees, the majority of which are n o n - c o m m e r c i a l a n d suffer s o m e d e g r e e of habitat fragmentation. T o improve our understanding of the genetic structure of forest trees, it is n e c e s s a r y to b r o a d e n the s c o p e of study to include a n g i o s p e r m s with different mating s y s t e m s , pollination vectors, patterns of d i s p e r s a l , a n d evolutionary histories. Life history a n d e c o l o g i c a l factors that would promote genetic diversity of w o o d y early s u c c e s s i o n a l s p e c i e s , like Acer macrophyllum  (bigleaf maple), are likely to be similar  to those of relatively long-lived s p e c i e s .  Thesis overview In order to e m b a r k on a n y useful conservation program for any s p e c i e s , k n o w l e d g e of how genetic variation is partitioned a m o n g a n d within populations is the first n e c e s s a r y step. T h e m a i n g o a l s of chapter two are: 1) to review the literature on the biology of Acer macrophyllum;  2) to review the b a s i c ideas that  s h a p e our thinking about genetic diversity a n d mating patterns in small a n d subdivided populations; 3) to review the role of evolutionary f o r c e s in explaining genetic differentiation for neutral genetic markers a n d quantitative traits; a n d 4) to a d d r e s s genetic c o n s e q u e n c e s of forest fragmentation a n d spatial genetic structure in natural populations. In chapter three I u s e i s o z y m e markers to  investigate the genetic variation, population structure a n d mating s y s t e m in bigleaf m a p l e . T h i s is a n important b a s i c step to understanding the population g e n e t i c s of this s p e c i e s . I h y p o t h e s i z e d that fragmentation m a y lead to e r o s i o n of genetic variation. In chapter four this is tested by c o m p a r i n g s e e d l i n g a n d mature cohorts in fragmented a n d non-fragmented populations. I also e x a m i n e the extent of spatial structuring within s t a n d s of bigleaf m a p l e trees a n d e x a m i n e how structuring is affected by fragmentation. In chapter five I study the quantitative variation in height, diameter a n d bud flush traits a n d c o m p a r e quantitative genetic differentiation a m o n g populations and genetic differentiation at neutral loci. I then c o n c l u d e by s u m m a r i z i n g the major findings in chapter six, providing specific r e c o m m e n d a t i o n s for m a n a g e m e n t a n d s u g g e s t i n g a r e a s for future r e s e a r c h that will e n h a n c e our k n o w l e d g e for m a n a g e m e n t a n d conservation of bigleaf m a p l e genetic r e s o u r c e s for present a n d future generations.  Chapter two  LITERATURE REVIEW B I O L O G Y A N D SILVICS O F B I G L E A F M A P L E (Acer macrophyllum T h e m a p l e family, A c e r a c e a e , includes two g e n e r a , Dipteronia Dipteronia  Pursh)  a n d Acer.  contains only two s p e c i e s of small trees both native to central C h i n a . Acer  contains about 148 s p e c i e s of small trees a n d s h r u b s that are widely scattered throughout the Northern H e m i s p h e r e but are most abundant in the eastern H i m a l a y a n M o u n t a i n s a n d in central C h i n a ( P e t e r s o n et. al 1999). Thirteen s p e c i e s of m a p l e s are indigenous to the North A m e r i c a , ten of w h i c h are native to C a n a d a (Farrar 1995). T h r e e of C a n a d a ' s ten m a p l e s p e c i e s are native to British C o l u m b i a : Acer macrophyllum  P u r s h (bigleaf maple); Acer glabrum Torr.var. dauglasii  (Douglas or R o c k y Mountain maple); a n d Acer circinatum  (Hook.) Dipple  P u r s h (vine maple).  W h i l e substantial information is available on the silvics, m a n a g e m e n t a n d g e n e t i c s of m a p l e s p e c i e s in eastern North A m e r i c a , the extent to which this information is applicable to bigleaf m a p l e is unknown ( P e t e r s o n et al. 1999). S o m e plant b i o g e o g r a p h e r s initially s u g g e s t e d that b e c a u s e of the isolating effect of P l e i s t o c e n e continental ice s h e e t s on plant distributions (Ritchie 1987), bigleaf m a l e is actually more closely related to s o m e of the A s i a n a n d E u r o p e a n m a p l e s than to the m a p l e s in e a s t e r n North A m e r i c a b a s e d on t a x o n o m i c features (Elias 1980). T h i s c o n c l u s i o n w a s further supported by molecular phylogenetic studies c o n d u c t e d on Acer by A c k e r l e y a n d D o n o g h u e (1998). T h e native range of bigleaf m a p l e e x t e n d s from latitude 33° N to 51° N., mostly within 300 km of the P a c i f i c C o a s t (Fig 1.1) a n d it is the P a c i f i c Northwest's s e c o n d most abundant s p e c i e s of hardwood after red alder ( N i e m i e c et a l . 1995). Bigleaf m a p l e grows over a w i d e range of temperature a n d moisture conditions, from the c o o l , moist maritime climate of C o a s t a l British C o l u m b i a to the w a r m , dry climate of  southern California. It often o c c u r s on c o a r s e gravel soil in mixed s t a n d s with Alnus rubra (red alder), Populus cedar), Pseudotsuga  trichocarpa  menziesii  (black cottonwood), Thuja plicata (western red  (Douglas-fir) a n d Tsuga heterophylla  (western  hemlock) (Farrar 1995). Bigleaf m a p l e is a b l e to produce flowers a n d s e e d s a s early a s 10 y e a r s after germinating from s e e d on o p e n a n d high productivity sites. S e e d crops are p r o d u c e d every year, a n d c a n be prodigious, e s p e c i a l l y in open-grown trees. F l o w e r s of bigleaf m a p l e are relatively s m a l l but insect-pollinated. It is p o l y g a m o u s a n d both staminate a n d perfect flowers are mixed in the s a m e d e n s e cylindrical, r a c e m e s (Minore a n d Z a s a d a 1990). T h e fruit is a double s a m a r a with slightly divergent w i n g s a n d a hairy s e e d c a s e . T h e flowering period is usually from early April to M a y . Fruit ripens by S e p t e m b e r or O c t o b e r a n d s e e d d i s p e r s a l o c c u r s from O c t o b e r through J a n u a r y (Ruth a n d Muerle 1958). S e e d d i s p e r s a l is primarily by wind a n d gravity but d i s p e r s a l by s o m e s m a l l m a m m a l s (mice, w o o d rats, a n d squirrels) a n d birds has b e e n reported ( F o w e l s 1965). S e e d s are not dormant a n d germinate s o o n after d i s p e r s a l . Bigleaf m a p l e is moderately s h a d e tolerant a n d a n excellent s h a d e tree. Its w o o d is known to have g o o d properties for u s e a s furniture but it is neither a s strong nor a s hard a s s u g a r m a p l e ( K e r b e s 1968). T h e w o o d of bigleaf m a p l e is very popular in the piano industry, w h e r e it is the most preferred s p e c i e s for piano f r a m e s . It a l s o h a s s e v e r a l industrial a n d d o m e s t i c u s e s s u c h a s decorative f a c e veneer, container materials, moulding, hardwood flooring, kitchen utensils, pallets, turnery a n d h a r d w o o d plywood, a s well a s for firewood ( K e r b e s 1968; N i e m i e c et a l . 1995).  G E N E T I C VARIATION A N D S T R U C T U R E IN N A T U R A L  POPULATIONS  E c o l o g i c a l characteristics a n d life history of plant s p e c i e s influence levels of genetic variation. Important s p e c i e s characteristics a s s o c i a t e d with levels of variation include t a x o n o m i c status, regional distribution, g e o g r a p h i c range, life form, m o d e of reproduction, s e e d d i s p e r s a l m e c h a n i s m , a n d s u c c e s s i o n a l status (Hamrick a n d G o d t  1989). Plant breeding s y s t e m s are a primary determinant of genetic structure in plant populations, b e c a u s e they alter the probability of random mating a m o n g individuals within a population. O u t c r o s s i n g m e c h a n i s m s reduce the rate of inbreeding and therefore maintain genetic variability within populations ( R i c h a r d s 1986). Selfing tends to d e c r e a s e genetic variation within populations a n d promote genetic differentiation a m o n g populations (Barrett a n d K o h n 1991). Inbreeding c a n o c c u r through either selfing or c o n s a n g u i n e o u s matings (Ritland 1985). M o s t trees are predominantly outcrossing but their reliance on either wind pollination or a wide variety of biotic pollination agents g e n e r a t e s c o n s i d e r a b l e variation in outcrossing rates a n d mating patterns a m o n g individuals (Aizen a n d F e i s i n g e r 1994; Hamrick et al. 1991). G e n e flow o c c u r s through s e e d a n d pollen d i s p e r s a l , a n d d e c r e a s e s the level of genetic differentiation a m o n g populations (Hamrick a n d G o d t 1989). S e e d d i s p e r s a l h a s a greater h o m o g e n i z i n g effect than pollen d i s p e r s a l b e c a u s e s e e d transmits both maternal a n d paternal g e n e s w h e r e a s pollen transmits only paternally inherited g e n e s ( N a s o n a n d Hamrick 1997). Within a given g e o g r a p h i c a l region, s e e m i n g l y large, contiguous populations often consist of s u b p o p u l a t i o n s that are linked along temporal a n d spatial s c a l e s . This network of populations is defined a s meta-population ( H a n s k i 1997). N o n - r a n d o m a s s o c i a t i o n of g e n o t y p e s within meta-populations c r e a t e s further structuring at relatively small s c a l e s ( M u o n a et al. 1991).  Effects of population size on genetic variation S m a l l populations undergo p r o c e s s e s predicted by genetic drift theory a n d population structure m o d e l s (Tempelton et al. 1991). K i m u r a a n d C r o w (1964) d e m o n s t r a t e d that for a diploid population the e x p e c t e d heterozygosity (H ) at e  equilibrium is a direct function of mutation a n d effective population size:  1+  AN  eM  where N  is the effective population s i z e a n d ju is the mutation rate. T h u s the larger  e  the population, the higher the heterozygosity that c a n be maintained, all e l s e being e q u a l . F o r e x a m p l e , Drosophila  populations and m a m m a l s p e c i e s exhibit  heterozygosities of 1 2 % a n d 5-6%, respectively for a l l o z y m e loci with c o r r e s p o n d i n g value of N /u e  of 0.035 a n d 0.015 (Ohta 1992). A m e t a - a n a l y s i s of genetic diversity in  c o m m o n a n d rare plants in the s a m e g e n u s c o n c l u d e s , however, that historically large populations that have recently b e c o m e fragmented m a y still harbor significant genetic diversity despite current small population s i z e . H o w e v e r , in most c a s e s the predicted correlation b e t w e e n genetic diversity a n d population s i z e holds ( G i t z e n d a n n e r a n d Soltis 2000). T h e complexity of random effects of genetic drift on allele f r e q u e n c i e s in finite populations is s u m m a r i z e d by K i m u r a (1955) a n d Wright (1951) in which after one generation of random mating, a population with initial heterozygosity (H ) 0  e x p e c t e d to d e c r e a s e by a proportion of  would be  on a v e r a g e s u c h that in generation t the  e x p e c t e d v a l u e of e x p e c t e d heterozygosity (Hartl a n d Clark 1989) is:  H,  1-  2JV.  H,,  D e c l i n e in population s i z e d u e to deforestation or fragmentation in already s u b d i v i d e d populations further i n c r e a s e s the probability of loss of alleles a n d e n h a n c e s the decline in heterozygosity d u e to genetic drift. H o w e v e r , s u c h effects d o not derive only from direct reduction in effective population s i z e , b e c a u s e the magnitude of genetic drift c a n be predicted a s a s i m p l e function of c e n s u s population s i z e only w h e n the population characteristics meet the a s s u m p t i o n s of the F i s h e r -  Wright drift m o d e l (Caballero 1994). Usually this is not the c a s e (Hartl a n d Clark 1989) a n d N tends to be c o n s i d e r a b l y smaller than the c e n s u s population s i z e N ( F r a n k h a m e  1995). S e v e r a l factors are r e s p o n s i b l e , including u n b a l a n c e d s e x ratio, unequal fecundity a m o n g individuals a n d population size fluctuations ( F a l c o n e r 1989; F u t u y m a 1986; Hartl a n d Clark 1989; Y e h 2000). Aldrich a n d Hamrick (1998) found that reproduction of the tree Symphonia  globulifera  in a 38.5 ha circular plot w a s  d o m i n a t e d by n u m e r o u s small groups of remnant pasture land trees which e x p e r i e n c e d a post-fragmentation i n c r e a s e in fecundity leading to a s e c o n d a r y constriction of the fragmentation bottleneck. Similarly in N e w Z e a l a n d mistletoe {Perexia  tetrapetala)  pollination a n d s e e d set w a s more than four-fold higher in  isolated than continuous forest (Kelly et al. 2000).  Effects of population size on mating systems T h e mating s y s t e m of plant s p e c i e s is a n important biological characteristic b e c a u s e it is a key determinant of genetic variation, genetic structure a n d evolutionary potential of plant populations ( C l e g g 1980; Brown 1990). M o d e s of pollination, population s i z e a n d density, a n d plant a n d floral architecture are all likely to influence mating s y s t e m s ( C l e g g 1980). Plant mating s y s t e m s are characterized by 1) proportion of outcrossing v e r s u s selfing; 2) c o n s a n g u i n e o u s matings, a n d 3) the level of correlated paternity, defined a s the proportion of full-sib pairs a m o n g o u t c r o s s e d maternal p r o g e n i e s (Ritland 1989a). R e d u c t i o n s in population s i z e a n d i n c r e a s e s in the d e g r e e of isolation a n d fragmentation of populations c a n lead to i n c r e a s e s in inbreeding (e.g. Farris a n d Mitton 1984; M u r a w s k i et al. 1994; R a i j m a n n et al. 1994). T h e s e effects may be of particular significance in w o o d y a n g i o s p e r m s b e c a u s e population s i z e s a n d densities c a n be significantly r e d u c e d a s a c o n s e q u e n c e of forest harvesting practices a n d other land u s e practices. In small populations, elevated levels of inbreeding are e x p e c t e d (Barrett a n d K o h n 1991). U n d e r t h e s e conditions, selection c a n purge early-  acting lethal a n d semi-lethal r e c e s s i v e alleles from populations a s they b e c o m e e x p o s e d a s h o m o z y g o t e s ( L a n d e a n d S c h e m s k e 1985; C h a r l e s w o r t h a n d C h a r l e s w o r t h 1987). However, mutations of mild deleterious effects m a y b e c o m e fixed in a p r o c e s s called mutational meltdown (Lynch 1985). T h e level of correlated paternity defines the probability that a s e e d tree d r a w s two m a l e g a m e t e s from the s a m e pollen donor. T h i s c a n be regarded a s the inverse of effective pollination neighbourhood size, a n a l o g o u s to Wright's neighbourhood s i z e , w h e n considering only the d i s p e r s a l v a r i a n c e of m a l e g a m e t e s (Austerlitz a n d S m o u s e 2001). T h e level of correlated paternity, together with setting rate, will determine the d e g r e e of departure from r a n d o m mating and the significance of genetic drift under the isolation-by-distance m o d e l (Ritland 1989a). Correlated mating m a y a l s o influence patterns of selection and competition a m o n g siblings (Karron a n d M a r s h a l l 1990). A m o n g the major factors that m a y e n h a n c e correlated paternity in wind-pollinated plants are pollen limitation (Surles et al. 1990), spatially restricted pollen d i s p e r s a l ( S m o u s e a n d S o r k 2004), a s y n c h r o n o u s floral phenology, u n e q u a l male fecundity ( E r i c k s s o n a n d A d a m s 1989; B u r c z y k a n d Prat 1997), a n d low c o n s p e c i f i c density ( S m o u s e a n d S o r k 2004). R e d u c t i o n s in population s i z e m a y directly affect the mating s y s t e m for three r e a s o n s . First, in small populations the n u m b e r of local compatible m a t e s is r e d u c e d , w h i c h e v e n under random mating will i n c r e a s e the likelihood of correlated mating a n d self-fertilization (Surles et al. 1990). S e c o n d , total pollen availability will d e c r e a s e in s m a l l plant populations, w h i c h m a y result in reduced s e e d set or i n c r e a s e d selfing (Larson a n d Barrett 2000). T h e impact of pollen limitation in wind pollinated s p e c i e s , however, remains unclear (Koenig a n d A s h l e y 2003). Third, a s a c o n s e q u e n c e of the typically leptokurtic s h a p e of the pollen d i s p e r s a l curve in plants (Levin a n d Kerster 1974), pollen pool diversity around individual trees in small populations may be r e d u c e d by the a b s e n c e of a broad spectrum of long-distance pollen d o n o r s ( A d a m s 1992; Ellstrand 1992). E v i d e n c e from experiments on conifer s p e c i e s s u g g e s t s that both the quantity and diversity of available pollen in small s t a n d s may be significantly  lower than in large populations ( S a r v a s 1962; K o s k i 1970, 1973). Little is k n o w n , however, about the p r e c i s e c o n s e q u e n c e s of this potential pollen pool impoverishment for the mating s y s t e m of particular s p e c i e s . M o s t studies dealing with the c o n s e q u e n c e s of s m a l l population s i z e on plant mating s y s t e m s h a v e f o c u s e d on the outcrossing rate a n d reproductive output of insect pollinated s p e c i e s , in w h i c h the interaction between the spatial structure of populations a n d the pollen foraging behaviour of pollinators p o s e s an additional c h a l l e n g e (Levin a n d Kerster 1974; v a n Treuren et al. 1993; H a u s e r a n d L o e s c h c k e 1994; Kennington a n d J a m e s 1997; Routley et a l . 1999). A l t h o u g h no significant effects of small population s i z e w e r e detected in s o m e of t h e s e studies, a g e n e r a l trend towards i n c r e a s e d selfing a n d r e d u c e d s e e d set h a s b e e n o b s e r v e d a s population s i z e s d e c r e a s e .  E F F E C T S O F F R A G M E N T A T I O N O N G E N E T I C VARIATION IN P L A N T POPULATIONS  Habitat d i s t u r b a n c e s c a u s i n g forest fragmentation c a n impact the genetic structure of s p e c i e s . Fragmentation c a n disrupt the pre-existing genetic structure of populations, alter genetic p r o c e s s e s a n d result in a net loss a n d redistribution of genetic diversity. Differentiation a m o n g populations will tend to i n c r e a s e a s o n c e contiguous populations b e c o m e s u b d i v i d e d into s m a l l , isolated fragments (Barrett a n d K o h n 1991). T h e first genetic c o n s e q u e n c e of a reduction in population s i z e is the loss of rare alleles, and over time a concomitant d e c r e a s e in heterozygosity will o c c u r through genetic drift (Barrett a n d K o h n 1991). Additionally, fragmentation c a n impact plant-pollinator a n d plant-seed d i s p e r s e r interactions (Aizen a n d F e i s i n g e r 1994). A shift in l a n d s c a p e pattern is e x p e c t e d to result in altered animal foraging behaviour (Dirzo a n d M i r a n d a 1991). G e n e r a l l y , alterations of pollinator behaviour will tend to limit pollen d i s p e r s a l , i n c r e a s e the level  of inbreeding within populations, reduce the rate of inter-population pollen d i s p e r s a l a n d therefore i n c r e a s e a m o n g - p o p u l a t i o n differentiation ( B a w a 1990). Similarly, a c h a n g e in foraging behaviour of s e e d d i s p e r s e r s could reduce s e e d flow a m o n g existing populations, a n d a l s o d e c r e a s e colonizing e v e n t s , thus reducing the establishment of new populations (Aizen a n d F e i s i n g e r 1994). Recently, two broad a p p r o a c h e s for detecting effects of forest fragmentation on genetic variation have b e e n u s e d : (i) c o m p a r i s o n of fragmented a n d unfragmented (continuous) populations, a n d (ii) a n a l y s i s of relationships between m e a s u r e s of genetic diversity a n d indices of fragmentation (e.g. population s i z e , isolation, or different a g e cohorts). S t u d i e s of t h e s e sorts have p r o d u c e d diverse results. In s e v e r a l c a s e s , important genetic effects h a v e b e e n d e t e c t e d . Fragmentation h a s b e e n a s s o c i a t e d with a decline in allelic richness in a number of c a s e s . F o r e x a m p l e , in 17 fragmented populations of the perennial Swainsona  recta, B u z a et al. (2000) reported  a significant reduction in the p r e s e n c e of rare alleles in small populations. Similar relationships w e r e reported by v a n T r e u r e n et al. (1991) for Salvia pratensis Scabiosa  columbaria  a n d P r o b e r a n d B r o w n (1994) for Eucalyptus  al. (1999) found r e d u c e d allelic diversity in s m a l l populations of leptorrhynchoides,  and  albens. Y o u n g et Rutidosis  a perennial a n d self-incompatible s p e c i e s . Further, they a r g u e d  that the a s s o c i a t i o n of d e c r e a s e d genetic diversity with low s e e d production w a s a c o n s e q u e n c e of parallel reductions in the number of alleles present at loci controlling self-incompatibility (SI). E r o s i o n of allelic r i c h n e s s at SI loci in small populations h a s also b e e n found in the rare l a k e s i d e d a i s y Hymenoxys  acaulis  ( D e M a u r o 1993).  G e n e flow b e t w e e n fragments might restore lost alleles very quickly but only w h e n lost alleles are still present in the post-fragmentation metapopulation a s a w h o l e . For, instance, in a study of Acer macrophyllum  (sugar maple) in C a n a d a , Y o u n g et a l .  (1993) c o m p a r e d genetic variation in eight patchy populations with variation in another eight continuous control populations. T h e y a s s u m e d that genetic variation in the large control populations represented variation in the pre-fragmentation population, a n d that the present patchy populations w e r e derived from o n c e continuous populations. A  c o m p a r i s o n of genetic diversity parameters b e t w e e n fragmented and control populations found genetic diversity (as m e a s u r e d by p e r c e n t a g e polymorphic loci, allelic diversity a n d heterozygosity) w a s not significantly lower in the fragmented populations, nor w a s there any i n c r e a s e in inbreeding. H o w e v e r , the total n u m b e r of alleles w a s six fewer in the fragmented populations, w h i c h w a s attributed to p o s s i b l e founder effects. A s outlined a b o v e , not all m e a s u r e s of genetic diversity are e x p e c t e d to be sensitive to founder effects. H o w e v e r , d e c l i n e s in v a l u e s of e x p e c t e d heterozygosity a n d allelic richness have b e e n reported. F o r e x a m p l e , P r o b e r a n d Brown (1994) d e m o n s t r a t e d that small populations (< 500 reproductive individuals) of  Eucalyptus  albens that w e r e less than 2 5 0 m from a larger population had a higher allelic r i c h n e s s than more isolated small populations. T h e s e results are crucial a s they point out a threshold up to which g e n e flow from a large population c a n maintain genetic diversity, but b e y o n d w h i c h genetic diversity c a n d e c l i n e . Similarly, a significant reduction in genetic diversity a n d i n c r e a s e d genetic differentiation w a s d o c u m e n t e d in fragmented relative to continuous populations of the tropical tree Pithecellobium  elegans  (Hall et  al. 1996). A relatively high correlation between population s i z e a n d genetic diversity w a s a l s o reported by R a i j m a n n et al. (1994) for Gentiana  pneumomanthe.  H o w e v e r in  a n u m b e r of other studies on relatively recently fragmented populations, there w e r e no clear relationships between genetic diversity a n d population s i z e (van T r e u r e n et a l . 1991; F o r e et al. 1992; Y o u n g et al. 1993, 2 0 0 0 ; B u z a et al. 2000). E v i d e n c e for more rapid genetic e r o s i o n in small isolated populations than in less isolated populations w a s reported by D a y a n a n d a n et a l . (1999). T h e y found that genetic d i s t a n c e between adult a n d s e e d l i n g cohorts in fragmented populations of Carapa  guianensis  in C o s t a R i c a w a s greatest in the most isolated population, w h i c h  w a s a l s o the only o n e in w h i c h allelic diversity w a s lower in the adult cohorts. T h e results from t h e s e studies s e e m to s u g g e s t that s p e c i e s with similar life history characteristics s u c h a s those mentioned a b o v e m a y be particularly vulnerable to the  c o n s e q u e n c e s of fragmentation s i n c e they typically exist at low densities a n d are predominantly o u t c r o s s e d (Hamrick a n d G o d t 1989; O ' M a l l e y a n d B a w a 1987). Effects of fragmentation on spatial genetic structure Spatial genetic structure is the n o n - r a n d o m distribution of genetic variation a m o n g sexually reproducing individuals ( M c C a u l e y 1997). T h e spatial distribution of g e n o t y p e s within plant populations is influenced by m a n y e c o l o g i c a l a n d evolutionary p r o c e s s e s s u c h a s limited s e e d a n d pollen d i s p e r s a l (Wright, 1943; S c h o e n a n d Latta, 1989; Bacilieri e t a l . 1994), adult density ( K n o w l e s et al. 1992; Hamrick e t a l . 1993; Hamrick a n d N a s o n , 1996; V e k e m a n s a n d Hardy 2004), colonization a n d disturbance history ( E p p e r s o n a n d C h u n g , 2 0 0 1 ; P a r k e r et a l . 2001), spatial and temporal patterns of seedling establishment (Ellstrand, 1992; S c h n a b e l a n d Hamrick, 1995; P a r k e r e t a l . 2001), differential selection a n d micro-environmental selection (Linhart et a l . 1 9 8 1 ; Slatkin a n d Arter, 1991) a n d forest fragmentation (Doligez a n d J o l y 1997). Of t h e s e factors, probably the most widely studied influence on spatial genetic structure is pattern of g e n e d i s p e r s a l (Hamrick et al. 1993; E n n o s 1994; Hamrick a n d N a s o n 1996). K a l i s z et al. (2001) d e s c r i b e d g e n e r a l s c e n a r i o s of s e e d a n d pollen d i s p e r s a l under which genetic structure could d e v e l o p : (i) If at the s c a l e of investigation, s e e d d i s p e r s a l is localized while pollen d i s p e r s e s long d i s t a n c e s or randomly, spatial clustering of full a n d half-sibs will result in the d e v e l o p m e n t of significant spatial structure in the a b s e n c e of inbreeding (e.g., P e a k a l l a n d Beattie, 1996; K a l i s z et al. 2001). (ii) If pollen d i s p e r s a l is a l s o restricted, this will result in inbreeding thereby reinforcing the buildup of more intense genetic structure (Wright 1943; Barbujani 1987). (iii) In contrast, if s e e d s are widely a n d independently d i s p e r s e d then regardless of whether pollen d i s p e r s e s long or short d i s t a n c e s , s e e d d i s p e r s a l will effectively r a n d o m i z e the spatial distribution of genetic variation within populations (e.g. D e w e y a n d H e y w o o d 1988; Loiselle et al. 1995).  In most studies of spatial genetic structure of tree s p e c i e s with wind d i s p e r s e d s e e d s , whether animal or wind pollinated, m a n y authors h a v e reported either w e a k or no spatial genetic structure (e.g., Acer saccharum et a l . 1993), Quercus 1995); Carapa procera  (Perry a n d K n o w l e s 1991; Y o u n g  spp.(Streiff et a l . 1998), Psychotria  officinalis  (Loiselle et al.  (Doligez a n d J o l y 1996); Pinus s p p (Parker et al. 2 0 0 1 ;  E p p e r s o n et al. 2003); Vitelarria  paradoxa  (Kelly et al. 2004)). Their results e x p l a i n e d  spatial genetic structure by overlapping s e e d s h a d o w s a n d extensive g e n e flow via pollen. W o o d y insect-pollinated s p e c i e s with s e e d s widely d i s p e r s e d by birds a l s o s h o w w e a k genetic structure ( D e w e y a n d H e y w o o d 1988, C h u n g et al. 2000). A lack of spatial structure w a s found for other s p e c i e s by S o k a l a n d O d e n (1978), D o l i g e z a n d Joly (1997), and C h u n g et al. (2000). T h e y attributed their results to extensive g e n e flow, wide s e e d d i s p e r s a l , self incompatibility a n d d i s p e r s a l agents. In c o n c l u s i o n , fragmentation effects on population genetics of forest tree populations are c o m p l e x a n d difficult to predict. Theoretical considerations in particular are p e r h a p s more useful in understanding empirical results rather than predicting t h e m ( Y o u n g et al.1996). H o w e v e r it is worth noting that both theoretical and empirical studies s u g g e s t that fragmentation c a n exert s o m e effects on genetics of fragmented populations.  M O L E C U L A R A N D QUANTITATIVE VARIATION  Forest trees are long-lived, s e s s i l e o r g a n i s m s that are e x p o s e d to large temporal fluctuations in their environmental conditions. C o n s e q u e n t l y , the d e m a n d s p l a c e d on the adaptability of trees are extremely high c o m p a r e d to other o r g a n i s m s . T o fulfill t h e s e d e m a n d s , forest tree s p e c i e s n e e d to maintain large a m o u n t s of genetic variation for the preservation of adaptability a n d survival to s u b s e q u e n t generations (Muller-Starck a n d G r e g o r i u s 1985). O n the other h a n d , the e x i s t e n c e of populations of healthy plants with little or no detectable genetic variation s h o w s that long-term  survival is p o s s i b l e . Without variability, however, s u c h s p e c i e s will be unable to adapt to n e w environmental conditions. Acquisition of sufficient information on the extent a n d pattern of genetic diversity, population differentiation a c r o s s s p e c i e s r a n g e s , a n d the e c o l o g i c a l and genetic relationship a m o n g individuals a n d a m o n g populations, are e s s e n t i a l for establishing guidelines on conservation a n d utilization of genetic r e s o u r c e s ( E r i k s s o n et al. 1995). T h e n e e d to understand genetic structure s t e m s from the n e c e s s i t y to a n s w e r the e s s e n t i a l question of whether o n e population or m a n y different populations will be an effective collection of all the important alleles for a particular s p e c i e s ( B r a d s h a w a n d Stettler 1995). T h e a n s w e r to this question is critical for the efficient m a n a g e m e n t of natural forests or for any effort to restore deforested habitats by reintroduction ( N a m k o o n g 1988). Hereditary b a s i s of differentiation in morphology a n d d e v e l o p m e n t have b e e n d e m o n s t r a t e d in studies of intraspecific variation in quantitative traits beginning over two centuries a g o (see review by Langlet 1971). H o w e v e r , b e c a u s e patterns of g e o g r a p h i c variation within s p e c i e s are influenced by different selective p r e s s u r e s , barriers to g e n e flow a n d genetic drift, the m a i n t e n a n c e of genetic variation in natural populations thus b e c o m e very c o m p l e x (Grant a n d Linhart 1996). T h e importance of genetic drift a n d g e n e flow a s evolutionary f o r c e s in natural populations h a s b e e n thoroughly a d d r e s s e d in studies using m o l e c u l a r markers. H o w e v e r , using s u c h m a r k e r s to determine genetic structure has s e v e r a l limitations in providing information that could be useful to define c o n s e r v a t i o n strategies (Lynch 1996). D e s p i t e t h e s e limitations, m o l e c u l a r markers h a v e b e e n p r o p o s e d a s a n indirect indicator of quantitative genetic variation available for adaptation (Petit et a l . 1998). H o w e v e r W a l d m a n n a n d A n d e r s o n (1998) indicated three major s h o r t c o m i n g s of this a p p r o a c h : (i)  T h e higher mutation rates of quantitative trait c h a r a c t e r s s u g g e s t s that the recovery times after a bottleneck will be shorter for polygenic variation than for single locus p o l y m o r p h i s m .  (ii)  W h e n non-additive v a r i a n c e is high, the e x p e c t e d loss of additive v a r i a n c e c a u s e d by genetic drift follows a different pattern than the reduction in single-locus heterozygosity.  (iii)  T h e effect of small population s i z e on genetic variation is e x p e c t e d to differ for m o n o g e n i c a n d polygenic characters owing to selection having different effects on t h e s e types of characters.  In view of t h e s e differences, planning conservation efforts b a s e d exclusively on marker g e n e loci m a y be m i s l e a d i n g . For this r e a s o n quantitative genetic a n a l y s i s is a n important compliment in studies of s p e c i e s (Lynch 1996; Storfer 1996). C o m p a r i s o n s of d i v e r g e n c e in neutral genetic m a r k e r s (as m e a s u r e d by F T) S  a n d polygenically-controlled quantitative traits (as m e a s u r e d by Q f, S  Wright 1951)  allow for an a s s e s s m e n t of the relative importance of natural selection a n d genetic drift a s a c a u s e of population differentiation in quantitative traits (Spitze 1993; Prout a n d B a r k e r 1993; L o n g a n d S i n g h 1995; P o d o l s k y a n d Hartsford 1995; Bonin et al. 1996; Y a n g et a l . 1996; W a l d m a n n a n d A n d e r s o n 1998; L y n c h et al. 1999; G o n z a l e z M a r t i n e z et al. 2 0 0 2 ; Merila' a n d C r n o k r a k 2 0 0 1 ; M c K a y a n d Latta 2 0 0 2 ; H o w e et al. 2003). Higher d i v e r g e n c e in quantitative traits than in neutral markers ( Q s r > F T) is S  indicative of directional selection favouring different g e n o t y p e s in different populations, w h e r e a s the opposite ( Q s r < F T) s u g g e s t s that the s a m e g e n o t y p e s are favoured in S  different populations, i.e. stabilizing selection. However, if the two m e a s u r e s d o not differ significantly ( Q s r = FST), then patterns of variation for both neutral m a r k e r s a n d quantitative traits are both a s s u m e d to reflect only the actions of genetic drift a n d g e n e flow (Merila a n d C r n o k r a k 2001). C o m p a r a t i v e studies of quantitative trait a n d neutral marker d i v e r g e n c e are relevant from a conservation genetics perspective a s m a n a g e m e n t d e c i s i o n s often rest on population genetic a n a l y s e s c o n d u c t e d with neutral molecular markers (e.g. Moritz 1994; Haig 1998; R e e d a n d F r a n k h a m 2001). F o r instance, operational definitions of evolutionarily significant units ( E S U s ) are b a s e d on d i v e r g e n c e in neutral or nearly neutral markers (Moritz 1994; Moritz et al. 1995). H o w e v e r , quantitative  c h a r a c t e r s are more likely to be related to fitness a n d therefore to population p e r s i s t e n c e (Lynch 1996; Storfer 1996). N e v e r t h e l e s s , the question of whether the levels of variation and the d e g r e e of population differentiation are correlated b e t w e e n neutral genetic markers a n d genetic variation in quantitative traits remains controversial (Pfender et a l . 2 0 0 0 ; Merila a n d C r n o k r a k 2 0 0 1 ; R e e d a n d F r a n k h a m 2 0 0 1 ; Latta a n d M c K a y 2002). In a meta-analysis of 18 studies reporting Q T a n d FST S  v a l u e s for 20 s p e c i e s , Merila a n d C r n o k r a k (2001) did indeed find a positive correlation b e t w e e n the two d i v e r g e n c e indices a c r o s s different studies (see reviews in C r n o k r a k and Merila 2 0 0 2 ; Latta a n d M c K a y 2 0 0 2 ; M c K a y a n d Latta 2 0 0 2 ; H o w e et al. 2003). H o w e v e r , studies c o m p a r i n g the predictive power of neutral markers a s indicators of d i v e r g e n c e in quantitative traits a m o n g populations within s p e c i e s are still lacking (but s e e Steinger et al. 2002), a s are studies examining the sensitivity of Q s r estimates to genotype-environment interactions.  Figure 2 . 1 . Native range of Acer macrophyllum  (Bigleaf maple).  Chapter three  G E N E T I C VARIATION, POPULATION S T R U C T U R E A N D MATING S Y S T E M IN BIGLEAF M A P L E (Acer macrophyllum Pursh)  1  INTRODUCTION S t u d i e s of the genetic variation a n d population structure of w o o d y a n g i o s p e r m s with m o l e c u l a r genetic markers have s h o w n that they p o s s e s s relatively high levels of genetic variation within populations, but little a m o n g population differentiation (Hamrick et al. 1992; L o v e l e s s 1992). G e n e t i c variation e n a b l e s s p e c i e s to survive a n d adapt to changing environments, therefore, information on genetic variation of forest tree s p e c i e s is fundamental to m a n a g e m e n t a n d conservation (Eriksson et a l . 1995). T h e most important determinants of genetic variation are natural selection, mutation, genetic drift, migration a n d the mating s y s t e m (Hartl a n d Clark 1997). H o w e v e r , h u m a n activities s u c h a s deforestation, air pollution and forest fragmentation c a n modify the direction a n d amplitude of t h e s e evolutionary f o r c e s a n d alter genetic variation of natural forest r e s o u r c e s ( L a n d e 1988). M e a s u r e m e n t a n d characterization of this variation, particularly in relation to h u m a n activities, are important first s t e p s towards d e v e l o p i n g strategies to preserve the genetic variation of native forest tree s p e c i e s (Hamrick et al. 1991). Bigleaf m a p l e (Acer macrophyllum  Pursh) o c c u r s along the Pacific c o a s t of  North A m e r i c a , in populations of scattered individuals or a s s m a l l groves, in a s s o c i a t i o n with both conifers a n d b r o a d - l e a v e d trees. Its flowers are p o l y g a m o u s a n d both staminate a n d perfect flowers are mixed in the s a m e d e n s e cylindrical, r a c e m e s (Minore a n d Z a s a d a 1990). Pollination is primarily by insects (Minore a n d Z a s a d a ' A version of this chapter has been pubished. Mohammed N . Iddrisu and Kermit Ritland. Genetic variation, population structure and mating system in bigleaf maple (Acer macrophyllum Pursh). Can. J. Bot. 82: 18171825 (2004).  1990). It is a n early s u c c e s s i o n a l s p e c i e s with consistent s e e d production. T h e s e e d s are double s a m a r a s with slightly divergent w i n g s a n d c a n be d i s s e m i n a t e d by wind for long d i s t a n c e s . Its scattered distribution over the c o a s t a l Pacific Northwest a n d southwestern British C o l u m b i a in rural a r e a s m a k e s it a prime woodlot s p e c i e s (Minore a n d Z a s a d a 1990). In the past, bigleaf m a p l e populations have suffered m u c h habitat disturbance d u e to agricultural practices, a n d the marketing of bigleaf m a p l e w o o d products m a y in the long run lead to an a c c e l e r a t e d loss of its genetic r e s o u r c e s . T h i s could d e c r e a s e the opportunities for the genetic improvement a n d c o n s e r v a t i o n of bigleaf m a p l e . H o w e v e r , there h a s b e e n little study of the genetic structure of bigleaf m a p l e , a n d this is n e e d e d to d e v e l o p a m a n a g e m e n t strategy d e s i g n e d to maintain stable, productive a n d s u s t a i n a b l e forest populations of this species. T h e objectives of this study w e r e to: (1) determine the amount a n d distribution of genetic variation a m o n g bigleaf m a p l e (Acer macrophyllum)  populations, (2)  estimate the mating s y s t e m in two populations from the L o w e r M a i n l a n d of British C o l u m b i a , a n d (3) r e c o m m e n d a strategy for the m a n a g e m e n t and conservation of bigleaf m a p l e genetic r e s o u r c e s . I h y p o t h e s i z e that bigleaf m a p l e is predominantly a n outcrossing s p e c i e s with extensive g e n e flow, a n d therefore, h a s m u c h genetic variation within populations but little population differentiation.  MATERIALS AND METHODS  S e e d s from eight populations representing the range-wide distribution of Acer macrophyllum  w e r e collected. Population locations are given in F i g . 3.1. S e e d s w e r e  collected from 36 adult trees in J e r i c h o a n d 4 0 trees in F r a s e r populations respectively. In e a c h of t h e s e populations, s e e d progenies (progeny arrays) with about 30 s e e d s per mother tree w e r e collected a n d u s e d for estimating outcrossing rates. F o r the rest of the populations s a m p l e d in the southern portion of the s p e c i e s range,  s e e d s w e r e collected from 20 adult trees in e a c h population with the exception of Artie w h e r e only 14 adult trees w e r e s a m p l e d . T h e Artie population w a s at the western e d g e of the s p e c i e s distribution in O r e g o n and no additional trees could be found after travelling 3 km westward towards the coast. Individual s a m p l e d trees w e r e s p a c e d approximately 30 meters apart a s m i n i m u m . A s m u c h a s p o s s i b l e , s e e d s w e r e collected before the first rainfall. If they w e r e d a m p from fog a n d d e w or collected after the first rains, they w e r e dried indoors at room temperature until s e e d s a n d p a p e r s a c k s felt dry, then stored in large plastic g a r b a g e b a g s with holes bored for ventilation at 2-4 °C until germination.  Isozyme assay S e e d s w e r e d e - w i n g e d a n d germinated in Petri d i s h e s on filter p a p e r s m o i s t e n e d with distilled water, then kept in a 4 ° C refrigerator for 5-8 d before s e e d dissection a n d e n z y m e extraction. Individual cotyledons with e m e r g i n g radicles w e r e e x c i s e d a n d p l a c e d in s e p a r a t e wells in microtiter plates for e n z y m e extraction. T h e freshly e x c i s e d material w a s ground in 2-3 drops of extraction buffer: 0.283g g e r m a n i u m dioxide, 25ml_ water, 0.0917g diethyldithiocarbonic acid, 0.1g s o d i u m bisulfate, (0.16M) 2.67ml_ p h o s p h a t e buffer at p H 7, 2 . 6 7 m L D M S O , 1 7 m L 2 phenoxythenol and 0.66ml_ p-mercapthoethanol. T h e extracts w e r e a b s o r b e d onto filter p a p e r w i c k s ( 3 x 1 3 mm), loaded onto 1 2 % starch g e l s . G e l s w e r e c o o l e d overnight to 4 ° C before loading s a m p l e s . S a m p l e w i c k s w e r e r e m o v e d after half a n hour of electrophoresis. T h e voltage w a s then set a n d run from 5 to 7 hours. R e c i p e s for histochemical staining solutions followed Murphy et al. (1996). Buffer s y s t e m s u s e d were: lithium borate p H 8.3, 80 m A ( R i d g e w a y et al. 1970); a n d morpholine citrate p H 8.0, 50 m A (Clayton a n d Tretiak 1972). In all, 33 e n z y m e s y s t e m s w e r e initially s c r e e n e d for p o l y m o r p h i s m a n d 10 putative a l l o z y m e loci for 6 e n z y m e s w e r e resolved clearly a n d consistently, thus s e l e c t e d for a n a l y s i s . T h e s e w e r e glutamic d e h y d r o g e n a s e (GDH;  1 locus), p h o s p h o g l u c o s e i s o m e r a s e (PGI; 2  loci), leucine a m i n o p e t i d a s e (LAP; 2 locus), isocitic d e h y d r o g e n a s e (IDH; 1 locus),  a s p a r a t e a m i n o t r a n s f e r a s e (AAT; 2 loci), a n d 6 - p h o s p h o g l u c o n a t e d e h y d r o g e n a s e (6P G D ; 2 loci). F o r e n z y m e s y s t e m s with multiple loci, the most a n o d a l migrating locus (fastest locus) w a s a s s i g n e d a s 1 a n d other loci w e r e a s s i g n e d increasing n u m b e r s with d e c r e a s i n g migrating d i s t a n c e . A t e a c h locus, the most c o m m o n allele w a s arbitrarily d e s i g n a t e d a s 1 a n d the others 2, a n d s o o n .  Data analysis E s t i m a t e s of the following quantities w e r e obtained: allele f r e q u e n c i e s , m e a n n u m b e r of alleles per locus (A), p e r c e n t a g e polymorphic loci (%P) at 9 9 % criterion, o b s e r v e d heterozygosity (H ) 0  a n d e x p e c t e d heterozygosity (H =1-£pi , 2  E  w h e r e p, the  f r e q u e n c y of the ith allele). T h i s a n a l y s i s w a s performed with B I O S Y S - 2 (William C . B l a c k IV, Department of Microbiology, C o l o r a d o State University), a modified version of the B I O S Y S - 1 program by Swofford a n d S e l a n d e r (1981). Wright's FST (Wright 1965) w a s c o m p u t e d for individual loci of the eight populations. N e i ' s (1978) genetic d i s t a n c e (D) w a s c o m p u t e d b e t w e e n all pairs of populations. A d e n d r o g r a m of genetic relationships a m o n g populations w a s constructed from t h e s e d i s t a n c e s using the unweighted pair group method ( U P G M A , S n e a t h a n d S o k a l 1973), and standard error bars calculated with Ritland's (1989b) genetic d i s t a n c e a n d clustering program ( G D D ) . D e n d r o g a m s plotted using t h e s e p r o c e d u r e s help to v i s u a l i z e the genetic relationship a m o n g populations. In e a c h population, departures of genotypic f r e q u e n c i e s from H a r d y - W e i n b e r g e x p e c t a t i o n s w e r e c h a r a c t e r i z e d by estimating Wright's inbreeding coefficient a s F = 1 -  Hc/H . E  G e n e p o p ( R a y m o n d a n d R o u s s e t 1995) w a s u s e d to estimate P- v a l u e s from e x a c t test of departure from H a r d y - W e i n b e r g equilibrium using the M a r k o v c h a i n method with 1000 iterations ( G u o a n d T h o m p s o n , 1992). Linear regression w a s performed to study the relationship b e t w e e n the e x p e c t e d heterozygosity a n d latitude, heterozygosity a n d elevation, a n d b e t w e e n genetic d i s t a n c e a n d g e o g r a p h i c d i s t a n c e using S - P L U S statistical software ( S - P L U S 6, Insightful, C o r p . 2001). In addition, isolation by d i s t a n c e w a s tested using  R o u s s e t ' s method (1997), w h i c h involves a linear regression of pairwise  FST/(1-FST)  on the natural logarithm of g e o g r a p h i c d i s t a n c e s b e t w e e n populations. S i g n i f i c a n c e w a s tested statistically using the Mantel test (Mantel 1967) in the program IBD version 1.4 ( B o h o n a k 2002). T h i s test a s s e s s e s whether the pairwise g e o g r a p h i c d i s t a n c e matrix correlates with the pairwise genetic d i s t a n c e matrix. T h e d e g r e e of genetic isolation (gene flow) w a s estimated by Nm, the n u m b e r of migrants per generation. Nm w a s estimated by two m e t h o d s , by the relationship between F r  a n d A / a n d by the method of private alleles. F r o m Wright (1951): Nm =  (1-FST)/4F T,  where  S  S  m  FT S  is the proportion of the total genetic diversity a m o n g  populations. I u s e d G e n e p o p ( R a y m o n d a n d R o u s s e t , 1995) to estimate Nm b a s e d on the private alleles (unique alleles found in only o n e population) method d e v e l o p e d by Slatkin (1985), using the f r e q u e n c y a n d distribution of rare alleles a m o n g populations. Mating s y s t e m parameters w e r e estimated using the mixed mating m o d e l of Ritland a n d J a i n (1981), a s implemented in M L T R (Ritland 1990). S i n g l e - l o c u s (f ) s  a n d multi-locus (t ) e s t i m a t e s of population outcrossing rates w e r e estimated for the m  two populations (Jericho a n d Fraser) in w h i c h progeny arrays w e r e s a m p l e d . Maternal g e n o t y p e s w e r e inferred from progeny arrays for t h e s e two populations following B r o w n a n d Allard (1970) and u s e d to estimate genetic diversity p a r a m e t e r s for the northern range of the s p e c i e s distribution. Multilocus outcrossing rates {t ) m  w e r e c o m p a r e d with m e a n single-locus (f ) rates to detect any selfing d u e to biparental s  inbreeding (t -t ). T h e correlation of o u t c r o s s e d paternity (r ) w a s estimated following m  s  p  Ritland's (1989a) sibling pair m o d e l . T h e s e parameters a n d the a v e r a g e s i n g l e - l o c u s inbreeding coefficient of maternal parents (F) w e r e a l s o estimated via the M L T R program (Ritland 1990).  RESULTS  Allele frequency distribution S e e d l i n g g e n o t y p e s w e r e s c o r e d for a total of 10 loci in two e n z y m e s y s t e m s . S o m e loci w e r e apparently m o n o m o r p h i c but w e r e inconsistent in resolution and w e r e e x c l u d e d from the a n a l y s i s (SKD-1,  SKD-2,  MDH-2).  Others w e r e variable but could  not be u s e d for the a n a l y s i s b e c a u s e of overstaining of o n e locus on top of another, e.g. PGM-1,  or b e c a u s e of very faint banding patterns that could not be properly  interpreted, e . g . G-6P-1,  G-6PDH,  ME-1, ACO-1,  ACO-2  and  UGUT-1.  Allele f r e q u e n c i e s for e a c h population are given in T a b l e 3.1. A total of 24 alleles w e r e detected in this study. O n e locus (LAP-2)  w a s m o n o m o r p h i c a c r o s s all  populations s a m p l e d . O f the nine polymorphic loci, 1 locus (AAT-2) w a s polymorphic in only o n e population, 2 loci (6PG-2,  GDH) w e r e polymorphic in two populations, a n d  o n e other l o c u s (LAP-1) w a s polymorphic in six populations. T w o loci (6PG-1, w e r e polymorphic in s e v e n populations a n d the remaining 3 (AAT-1,  PGI-1)  IDH, PGI-2)  were  polymorphic a c r o s s all populations. T w o of the eight populations had a total of 3 private alleles (i.e. alleles found in only o n e population). T h e s e private alleles w e r e unique to the two northern populations (Jericho, AAT-2-2,  AAT-2-3;  F r a s e r , IDH-1-3).  V a r i a b l e loci exhibited just 2 alleles per locus with the exception of J e r i c h o and F r a s e r populations that exhibited 3 alleles at s e v e r a l loci (Table 3.1).  Genetic diversity G e n e t i c variation statistics are s u m m a r i z e d for all populations in T a b l e 3.2. A l l e l e s per locus a v e r a g e d 1.71, a n d ranged from 1.5 (Artie) to 2.2 (Jericho). O n a v e r a g e , 6 1 . 2 % of the loci w e r e polymorphic, ranging from 5 0 % (Artie) to 8 0 % (Jericho). T h e e x p e c t e d heterozygosity within populations ranged from 0.102 (Jericho) to 0.189 (Artie), and a v e r a g e d 0.152 (Table 3.2). A significant negative relationship w a s found b e t w e e n e x p e c t e d heterozygosity a n d latitude (R = 2  0.71; p=<0.05). A n  u n e x p e c t e d relationship w a s a l s o found b e t w e e n e x p e c t e d heterozygosity a n d alleles  per locus (R = 2  loci (R = 2  0.89; p=<0.05), a n d e x p e c t e d heterozygosity a n d percent polymorphic  0.53; p=<0.05).  Genetic structure O b s e r v e d heterozygosities varied from 0.108 to 0.160, with an a v e r a g e of 0.118. T h e m e a n o b s e r v e d heterozygosity w a s 2 2 % lower than the e x p e c t e d heterozygosity (0.152). H a r d y - W e i n b e r g equilibrium w a s rejected for three of the eight populations (P < 0.05), which s h o w e d a deficiency of heterozygotes. O b s e r v e d heterozygosities w e r e found to be slightly lower than the e x p e c t e d v a l u e s within most populations. T h i s heterozygote deficiency is reflected in the m e a n inbreeding coefficient ( F = 0.166). T h e s a m p l e from Siletz had the highest inbreeding coefficient of 0.334, while J e r i c h o had a slight e x c e s s of heterozygosity ( F =-0.050) (Table 3.2). T h e m e a n fixation index ( F  / S ;  suggesting A c e r macrophyllum  Wright 1951) a c r o s s loci a n d populations w a s 0.193, populations have s o m e d e g r e e of inbreeding.  T h e proportion of genetic variation d u e to differences a m o n g populations w a s F T = 0.054 (Table 3.3), indicating that 9 4 . 6 % of the genetic variation resides within S  populations. T h e relatively low v a l u e indicates little population differentiation w h i c h m a y be d u e to high levels of g e n e flow, a s estimated by the indirect method (/V = m  4.39) a n d for the private alleles method (A/ = 4.10). m  N e i ' s genetic d i s t a n c e b e t w e e n populations w a s low a v e r a g i n g 0.011 (SD=0.005) a n d ranging from 0.001 to 0.042. T h e d e n d r o g r a m of genetic relationship a m o n g the eight populations is s h o w n in F i g . 3.2. Significant clusters of populations o c c u r w h e n the length of the s h a d e d portion (thicker bar) is less than half that of (thinner bar); the method for determining the error is b a s e d upon the a m o n g - l o c u s v a r i a n c e of genetic distance b e t w e e n c l a d e s (Ritland 1989b). T h e most genetically distinct population is Artie, which is s e p a r a t e d from the rest of the populations (Fig. 3.2). S e v e r a l statistically significant g r o u p s are evident, but the cluster b e t w e e n Oakville a n d Helmick w a s not statistically significant, a s a large g e o g r a p h i c d i s t a n c e a l s o exists b e t w e e n the 2 populations (228 km). T h e similarity b e t w e e n the J e r i c h o  a n d F r a s e r populations is o b v i o u s in that they both s h a r e the highest v a l u e of allelic diversity, lowest level of heterozygosity a n d with little inbreeding a s c o m p a r e d to the other populations. T h e r e a s o n for their similarity could be that they s h a r e the most c o m m o n recent ancestor. N o significant correlation w a s found between genetic d i s t a n c e s a n d g e o g r a p h i c d i s t a n c e s (Mantel test, r= 0.36, o n e tailed p=0.059).  Mating system E s t i m a t e d multi-locus outcrossing rates for the two populations w e r e high (Table 3.4). S i n g l e - l o c u s outcrossing rates estimate (t ) ranged from 0.939 to 0.942, with a n s  a v e r a g e of 0.94 for the two populations. Multi-locus estimates of outcrossing rates (t ) m  ranged from 0.941 to 0.950 a n d a v e r a g e d 0.945 for the two populations. S i n g l e - l o c u s a n d multi-locus outcrossing estimates for all populations differed significantly from unity ( M ) . Differences in multi-locus a n d s i n g l e - l o c u s {t -t ) c a n indicate biparental m  s  inbreeding, however the difference in this c a s e w a s essentially zero, indicating no biparental inbreeding (Table 3.4). Individual tree outcrossing rate estimates w e r e h e t e r o g e n e o u s in the two populations. Both populations exhibited predominant outcrossing, with a large portion of trees having outcrossing rates e q u a l to or greater than 0.90. Maternal inbreeding coefficients (F) w e r e low a n d did not differ significantly from z e r o in either population. T h e correlation of o u t c r o s s e d paternity r (probability that s i b s s h a r e d the s a m e father) p  for both populations ranged from 0.234 to 0.544 a n d a v e r a g e d 0.389. T h i s v a l u e is high, indicative of few effective pollen d o n o r s (A/ = 1/r = 2.57, s e e Ritland 1989a). ep  p  DISCUSSION Genetic variation C o m p a r e d with the genetic diversity found in other w o o d y a n g i o s p e r m s , Acer macrophyllum  h a s a higher p e r c e n t a g e of polymorphic loci (P= 61.2%) a n d e x p e c t e d  heterozygosity (/-/ =0.152) than a v e r a g e . H o w e v e r , the number of alleles per locus in E  bigleaf m a p l e (A=1.71) is slightly less than that in w o o d y s p e c i e s , on a v e r a g e , a n d slightly higher than the m e a n in all w o o d y a n g i o s p e r m (Table 3.5). Differences in the a m o u n t of genetic variation a m o n g populations, particularly differences in e x p e c t e d hetrozygosity relative to p o l y m o r p h i s m , m a y reflect the action of different genetic p r o c e s s e s . J e r i c h o a n d F r a s e r w e r e the two populations with the lowest levels of heterozygosity, a n d higher p o l y m o r p h i s m , with no e v i d e n c e of deviation from r a n d o m mating. In this c a s e it s e e m s more likely that the low heterozygosity m a y be a reflection of low overall population genetic variation. T h i s is possibly the result of genetic drift or selection in s o m e parts of the northern range of this s p e c i e s . T h e most genetically distinct population is Artie, which is s e p a r a t e d from the rest of the populations (Fig.3.2). T h i s separation in my view is a n o m a l o u s in that there is no clear environmental explanation a s to why it s h o u l d be different. Isozymically, it differs from other populations in having the highest e x p e c t e d heterozygosity (0.189), yet it is the only population w h i c h is invariant at the PGI-1 locus, thereby having the lowest proportion of p o l y m o r p h i s m . In addition, the s a m p l e s i z e of 14 individuals is s m a l l c o m p a r e d to the rest of the populations which could partially be responsible for its genetic distinctiveness.  Population genetic structure and gene flow Differentiation a m o n g populations, a s m e a s u r e d by N e i ' s genetic d i s t a n c e (D), a v e r a g e d 0.011 for the eight populations of bigleaf m a p l e in this study. T h i s v a l u e is similar to that o b s e r v e d in 22 populations of Alnus crispa (D = 0.012, B o u s q u e t et al. 1987) but higher than t h o s e o b s e r v e d for Acer saccharum  (D = 0.003, Perry a n d  K n o w l e s 1989; D = 0.007, Y o u n g et al. 1993). T h e value of genetic distance (0.011) in this study is probably d u e to large g e o g r a p h i c d i s t a n c e s . T h e m e a n g e o g r a p h i c d i s t a n c e between pairs of population w a s 2 5 0 km a n d the largest d i s t a n c e w a s 562 km suggesting a b s e n c e of isolation by d i s t a n c e a c r o s s the s p e c i e s range. M o s t of the sites s a m p l e d w e r e similar in terms of climate a n d e d a p h i c factors. T h e s e factors m a y promote low differentiation a m o n g populations. T h i s result is supported by a positive but non-significant correlation b e t w e e n genetic a n d g e o g r a p h i c d i s t a n c e s . Similar results h a v e b e e n found for Acer saccharum tremuloides  ( Y o u n g et a l . 1993) and  Populus  (Hyun et al. 1987).  T h e level of population differentiation in bigleaf m a p l e , F r = 0.054, is similar to S  that reported for Acer saccharum  (F T= 0.049, Y o u n g et al. 1993; F T = 0.033, Perry S  S  a n d K n o w l e s 1989), Alnus crispa ( F s r = 0.051, B o u s q u e t et al. 1987). T h e s e low v a l u e s reflect extensive g e n e flow via pollen or s e e d , or recent colonization (Huh 1999). S p e c i e s with more pollen or s e e d m o v e m e n t should have less genetic differentiation than s p e c i e s with restricted g e n e flow. In support of t h e s e predictions, F o r e et al. (1992) o b s e r v e d a n a v e r a g e s e e d d i s p e r s a l distance of up to 100 metres for s u g a r m a p l e (Acer saccharum).  C o m m o n life history traits s u c h a s allogamy, wind  d i s p e r s a l of s e e d , high reproductive capacity, longevity a n d s u c c e s s i o n a l behaviour could a c c o u n t for the low differentiation o b s e r v e d . T h e apparent lack of a s s o c i a t i o n b e t w e e n g e o g r a p h i c a n d genetic d i s t a n c e s s o m e w h a t indicated a t e n d e n c y towards isolation by d i s t a n c e but not statistically significant. Similar findings have b e e n found for Alnus rubra (Xie et al. 2002) that h a s similar patchy distribution to Acer macrophyllum  a n d a l s o o c c u p i e s a similar  g e o g r a p h i c range. R e l a t i o n s h i p s between genetic a n d g e o g r a p h i c d i s t a n c e s h a s b e e n o b s e r v e d for s e v e r a l tree s p e c i e s (e.g. Camellia japonica, Tsuga mertensiana,  Ally et a l . 2 0 0 0 ; Pseudotsuga  W e n d e l and P a r k s 1985;  menziesii,  Y e h and O ' M a l l e y 1980),  indicating that for these s p e c i e s isolation by d i s t a n c e m a y be an important factor in population differentiation. However, most studies that have demonstrated s u c h  significant a s s o c i a t i o n s s a m p l e a larger n u m b e r of populations or involve s p e c i e s that cover larger longitudinal or latitudinal distribution than the current study. T h e o b s e r v e d g e o g r a p h i c separation between the two British C o l u m b i a populations (Jericho a n d Fraser) a n d the rest of the populations m a y be of recent origin a s h y p o t h e s i z e d by P i e l o u (1991); it is therefore r e a s o n a b l e to s u g g e s t that strong g e n e flow through pollen a n d s e e d have o v e r c o m e the effects of genetic drift s o that physically s e p a r a t e d small patchy bigleaf m a p l e populations within e a c h g e o g r a p h i c region s h a r e a more or less continuous c o m m o n g e n e pool a s those continuously distributed. F o r instance, Siletz is widely s e p a r a t e d geographically (> 5 0 0 km) from J e r i c h o a n d F r a s e r but is genetically similar.  Mating system High outcrossing rates w e r e found in the two populations s a m p l e d using progeny arrays (Table 3.4). T h e s e outcrossing estimates are similar to other temperate a n g i o s p e r m tree s p e c i e s , e.g. Fagus sylvatica  (mean t = 0.96, R o s s i et al. 1996),  Alnus crispa (t = 0.95, B o u s q u e t et al. 1987), Quercus 2001), a n d Eucalyptus macrophyllum  urophylla  lobata (f = 0.96, S o r k et al.  (t = 0.91, H o u s e a n d Bell 1996). Although Acer  is pollinated by insects, wind pollination c a n not be ruled out. W i n d -  pollination h a s b e e n reported for other North A m e r i c a n Acer s p e c i e s that w e r e originally thought to be only insect pollinated (e.g. Acer grandidentatum, 1982; and Acer saccharum,  B a r k e r et a l .  G a b r i e l a n d Garrett 1984). Interestingly, six of eight  populations of bigleaf m a p l e had a n e x c e s s of h o m o z y g o t e s , a n d the estimated F w a s 0.166. T h e s e results again s u g g e s t that s o m e inbreeding a n d selfing o c c u r s in most populations (Table 3.1). A s s u m i n g inbreeding equilibrium a n d a s s u m i n g all inbreeding is d u e to selfing, this level of inbreeding c a n be explained by a selfing rate of 2F/(1  +  F) = 0.28. S o m e inbreeding in bigleaf m a p l e m a y result from g e i t o n o g a m o u s pollinations by b u m b l e b e e s (Bombus  spp.), through positive assortative mating (i.e., preferential  mating a m o n g similar g e n o t y p e s , Sullivan 1983), or through mating a m o n g relatives.  Acer macrophyllum  h a s perfect a s well a s staminate flowers a n d the pollination  m e c h a n i s m is mainly e n t o m o p h i l o u s (Minore a n d Z a s a d a 1990). T h e m o v e m e n t of pollinators a m o n g adjacent flowers within the crown or b e t w e e n adjacent c r o w n s of related neighbours could c a u s e inbreeding and selfing ( G o n z a l e z - A s t o r g a a n d N u n e z Farfan2001). I found no significant difference between multi-locus (t ) a n d s i n g l e - l o c u s (r ) m  s  e s t i m a t e s of outcrossing rates in either bigleaf m a p l e populations, indicating an a b s e n c e of c o n s a n g u i n e o u s mating. T h i s result is in a g r e e m e n t with the study by S o r k et al. (2001) for Quercus  lobata, w h i c h o c c u r s in o p e n l a n d s c a p e a n d is patchily  distributed, a n d for Stemmadenia  donnell-smithii  by J a m e s et al. (1998). Biparental  inbreeding has b e e n reported for s o m e w o o d y s p e c i e s , including western larch ( E l K a s s a b y a n d J a q u i s h 1998), Fagus sylvatica marginata  ( R o s s i et a l . 1996) and  Eucalyptus  (Millar et al. 2000).  T w o significant results I obtained w e r e that "correlated matings" (the fraction of o u t c r o s s e d sibling pairs that s h a r e the s a m e father) w e r e high, e s p e c i a l l y for the F r a s e r population (r = 0.544, T a b l e 3.5). This correlation is influenced by two factors: p  (i) multiple deposits of pollen from a single m a l e parent, or (ii) repeated mating a m o n g a relatively s m a l l n u m b e r of neighbours nearer to o n e another. A v a l u e near one-half implies that only a few pollen d o n o r s (1-2) must h a v e sired the majority of s e e d s within e a c h tree, while the v a l u e of 0.234 for the J e r i c h o population indicates 4 - 5 effective pollen d o n o r s per tree. T h e lower n u m b e r of effective pollen d o n o r s at F r a s e r could be d u e to features of this habitat, c o m p a r e d to J e r i c h o . F r a s e r is a roadside population which m a y be e x p e c t e d to s h o w r e d u c e d outcrossing rates d u e to d i s t u r b a n c e s . H o w e v e r , the outcrossing rates w e r e similar between the two populations, s u g g e s t i n g that high outcrossing w a s being maintained in the relatively disturbed F r a s e r population d u e to insect or pollen m o v e m e n t along a corridor of trees ( C h a s e et a l . 1996). T h e other significant result w a s that, despite the e v i d e n c e of moderate inbreeding ( F > 0 ) in this s p e c i e s , I found high levels of outcrossing (t = 0.95). /s  A l l o z y m e - b a s e d estimates of outcrossing rates b a s e d upon s e e d progeny could give upwardly b i a s e d estimates of outcrossing if selfing r e d u c e s the germination capacity, or if filled s e e d s are u s e d for estimating outcrossing rates (e.g., the effects of e m b r y o n i c lethals d u e to selfing on s e e d d e v e l o p m e n t and the formation of empty s e e d s are not a c c o u n t e d for; R a j o r a et a l . 2 0 0 0 , 2002). In this study, I u s e d entirely germinated a n d filled s e e d s s i n c e t h e s e yielded interpretable e n z y m e b a n d s . H o w e v e r , this m a y have upwardly b i a s e d estimates of outcrossing. Self-fertilization h a s b e e n found to a d v e r s e l y affect both e m b r y o d e v e l o p m e n t a n d s e e d germination in conifers ( S o r e n s e n 1969). T h i s a l s o holds for s o m e s p e c i e s in the m a p l e family; for e x a m p l e , G a b r i e l (1962) e x a m i n e d the interior of c a r p e l s of s u g a r m a p l e s e e d s {Acer saccharum),  a n d s u g g e s t e d that the low s e e d set after selfing m a y be related primarily  to post-zygotic abortion. In addition, G a b r i e l (1962) noted a reduction in viability of selfed s e e d s c o m p a r e d to o u t c r o s s e d s e e d s , e.g., inbreeding d e p r e s s i o n .  IMPLICATIONS FOR MANAGEMENT AND CONSERVATION  T h e o b s e r v e d genetic differentiation a m o n g bigleaf m a p l e populations probably reflects the c o m b i n e d effects of e c o l o g i c a l , evolutionary a n d b i o g e o g r a p h i c factors s u c h a s pollen a n d s e e d d i s p e r s a l m e c h a n i s m s . C o n s i d e r i n g the relatively large g e o g r a p h i c s c a l e in this study, my results indicate lower than e x p e c t e d levels of differentiation a m o n g populations of bigleaf m a p l e , in light of the fact that most populations in this study h a v e trees patchily distributed, a n d that trees are pollinated by insects. T h i s study a l s o found that bigleaf m a p l e is predominantly outcrossing (t = 0.95) a n d lacks biparental inbreeding. High outcrossing is related to its floral biology a n d characteristics, e.g., protogyny. P e r h a p s the most interesting finding w a s the s m a l l n u m b e r s of effective pollen d o n o r s , w h i c h probably reflects the relatively low density of populations c o u p l e d with limited pollinator m o v e m e n t . Correlated paternity results in an i n c r e a s e d genetic r e l a t e d n e s s of progeny, a n d a d e c r e a s e d genetic  diversity of individual tree p r o g e n i e s , w h i c h m a y limit local adaptive r e s p o n s e s ( J a m e s e t a l . 1998). U n d e r s t a n d i n g genetic diversity a n d population genetic structure is not only crucial in the conservation of s p e c i e s under threat of extinction, but it is a l s o e s s e n t i a l for the m a i n t e n a n c e of healthy populations a n d the breeding of w i d e s p r e a d indigenous tree s p e c i e s (Millar a n d Westfall 1992). T h e genetic variation a n d population genetic structure revealed in this study are instructive for m a k i n g conservation plans a n d d e v e l o p i n g breeding strategies. T h e estimate of  F T= S  0.054 in this study indicated up  to about 9 5 % of the total genetic diversity r e s i d e s within populations. Therefore for s u c h a predominantly outcrossing s p e c i e s (t  m  = 0.95) with insect pollination a n d s e e d  d i s p e r s a l by wind, it m a y be a d v i s a b l e to s a m p l e fewer populations but m o r e individuals per population for breeding p u r p o s e s . M e a s u r e s of genetic diversity b a s e d on n u m b e r of alleles (allelic richness) are important, e s p e c i a l l y in the field of conservation genetics. S i n c e o n e g o a l of a c o n s e r v a t i o n program is to maintain a s m a n y alleles a s p o s s i b l e , c h o i c e s of populations to c o n s e r v e in situ s h o u l d be b a s e d on allelic richness of the population (Marshall a n d B r o w n 1975). In view of this, I s u g g e s t that although a few in situ populations would contain most of the existing genetic variation in the s p e c i e s , it would be e s s e n t i a l to a l s o c o n s i d e r the populations from the northern range; thus, J e r i c h o a n d F r a s e r would be favored. A l t h o u g h inbreeding perse  d o e s not lead to l o s s  of alleles nor alter their f r e q u e n c i e s in a population, it l e a d s to i n c r e a s e d in homozygosity, a n d thus m a y d e c r e a s e the m e a n fitness of the population. T o this e n d , populations displaying extensive inbreeding would not be d e s i r a b l e for future in situ g e n e conservation. T h e lack of correlation between genetic a n d g e o g r a p h i c a l d i s t a n c e s in the s p e c i e s distribution s u g g e s t s that w h e n s a m p l i n g , w e m a y not n e c e s s a r i l y n e e d to s a m p l e sites evenly a c r o s s the s p e c i e s range. A l t h o u g h bigleaf maple is widely distributed without any current threat of extinction, effective in situ conservation a n d r e a s o n a b l e m a n a g e m e n t of its populations in the wild will promote a n d e n h a n c e its  adaptability to changing environments, a n d a l s o sustain its g e n e pool for future genetic improvement.  T a b l e 3.1. Distribution of allele f r e q u e n c i e s at 10 loci in eight natural mature populations of bigleaf m a p l e (Acer  macrophyllum). Populations  Locus  Allele  JERC  FRAS  ARTC  CASC  ELBE  HELM  OAKV  SILE  AAT-1  1  0.942  0.958  0.857  0.900  0.895  0.789  0.833  0.917  2  0.058  0.042  0.143  0.100  0.105  0.211  0.167  0.083  1  0.904  1.000  1.000  1.000  1.000  1.000  1.000  1.000  2  0.077  0.000  0.000  0.000  0.000  0.000  0.000  0.000  3  0.019  0.000  0.000  0.000  0.000  0.000  0.000  0.000  1  0.923  0.854  0.786  0.850  0.850  0.850  0.850  0.850  2  0.077  0.125  0.214  0.150  0.150  0.150  0.150  0.150  3  0.00  0.021  0.000  0.000  0.000  0.000  0.000  0.000  1  0.981  1.000  0.786  0.825  0.825  0.800  0.875  0.850  2  0.019  0.000  0.214  0.175  0.175  0.200  0.125  0.125  1  0.885  0.875  1.000  1.000  1.000  1.000  1.000  1.000  2  0.038  0.063  0.000  0.000  0.000  0.000  0.000  0.000  3  0.077  0.062  0.000  0.000  0.000  0.000  0.000  0.000  1  0.962  0.896  1.000  0.816  0.853  0.789  0.875  0.825  2  0.019  0.042  0.000  0.184  0.147  0.211  0.125  0.175  3  0.019  0.063  0.000  0.000  0.000  0.000  0.000  0.000  1  0.885  0.938  0.714  0.825  0.775  0.875  0.875  0.800  2  0.077  0.021  0.286  0.175  0.225  0.125  0.125  0.200  3  0.038  0.042  0.000  0.000  0.000  0.000  0.000  0.000  1  0.981  0.917  1.000  1.000  1.000  1.000  1.000  1.000  2  0.019  0.083  0.000  0.000  0.000  0.000  0.000  0.000  1  1.000  1.000  0.538  0.825  0.781  0.850  0.850  0.800  2  0.000  0.000  0.462  0.175  0.219  0.150  0.150  0.200  1  1.000  1.000  1.000  1.000  1.000  1.000  1.000  1.000  AAT-2  IDH  6PG-1  6PG-2  PGI-1  PGI-2  GDH  LAP-1  LAP-2  Note: JERC = Jericho; FRAS = Fraser; ARTC = Artie; CASC = Cascadia ; E L B E = Elbe; HELM= Helmick; OAKV = Oakville; SILE = Siletz.  T a b l e 3.2. S u m m a r y of genetic diversity within eight mature natural populations of bigleaf m a p l e (Acer macrophyllum)  POPULATION  N  1. J e r i c h o  40  b a s e d o n 10 a l l o z y m e loci.  A  %P  2.2  80.0  (0.2) 2. F r a s e r  36  2.0  60.0  (0.3) 3. Artie  14  1.5  50.0  (0.2) 4. C a s c a d i a  20  1.6  60.0  (0.2) 5. E l b e  20  1.6  60.0  (0.2) 6. Helmick  20  1.6  60.0  (0.2) 7. Oakville  20  1.6  60.0  (0.2) 8. Siletz  20  1.6  60.0  (0.2)  Mean  23.75  1.71  61.2  Ho 0.108  F  HE  0.102  (0.029)  (0.026)  0.112  0.105  (0.036)  (0.033)  0.160  0.189  (0.060)  (0.066)  0.121  0.164  (0.037)  (0.046)  0.109  0.172  (0.035)  (0.049)  0.118  0.176  (0.038)  (0.049)  0.117  0.148  (0.033)  (0.041)  0.102  0.163  (0.031)  (0.047)  0.118  0.152  -0.050  -0.086  N S  0.105  N S  0  2  2  2  N S  0.285*  0.332*  0.186  N S  E  N S  0.334*  0.166*  Note: N, sample size; A, average number of alleles per locus; %P, percent polymorphic loci; H , observed and heterozygosity ; H , expected heterozygosity; F, inbreeding coefficient; and numbers in parenthesis, standard errors. Exact test of departure from Hardy-Weinberg equilibrium * P < 0.05, not significant after sequential Bonferroni correction (Rice 1989) 0  N y  T a b l e 3.3. Total g e n e diversity e x p e c t e d heterozygosity (H ), 0  populations  (FIT),  (H ),  genetic diversity within populations  T  A  fixation index within population ( F ) , a n d genetic /s  differentiation  S  macrophyllum) at nine polymorphic loci.  Locus  H  AAT-1  0.202  0.202  0.169  2.00  0.116  0.143  0.030  AAT-2  0.024  0.023  0.024  3.00  -0.088  -0.013  0.067  IDH  0.254  0.258  0.222  3.00  0.125  0.134  0.009  6PG-1  0.231  0.226  0.134  2.00  0.396  0.431  0.057  6PG-2  0.059  0.056  0.060  3.00  -0.101  -0.029  0.065  PGI-1  0.219  0.213  0.194  3.00  0.060  0.105  0.048  PGI-2  0.279  0.276  0.180  3.00  0.308  0.335  0.039  GDH  0.025  0.024  0.026  2.00  -0.076  -0.015  0.056  LAP-1  0.277  0.252  0.171  2.00  0.300  0.390  0.128  Mean  0.174  0.170  0.131  2.55  0.193  0.236  0.054  H  s  s  alleles per locus (N ), fixation index over the total  a m o n g populations (F T) for eight mature natural populations of bigleaf m a p l e  T  (H ),  Ho  N  A  Fis  FIT  FST  (Acer  T a b l e 3.4. E s t i m a t e s of multi-locus outcrossing rates (t ), single locus outcrossing m  rates (f ), biparental inbreeding (t -t ), parental inbreeding coefficients (F) a n d s  m  s  correlation of paternity a m o n g siblings (r ). p  Population  t  m  t  t .t  s  m  s  F  r  0.052  0.234 (0.053)  p  0.941  0.939  0.002  (0.057)  (0.008)  (0.052)  (0.010)  Fraser  0.950 (0.067)  0.942 (0.054)  0.008 (0.034)  0.053 (0.009)  0.544 (0.067)  Mean  0.945  0.052  0.389  Jericho  0.940  Note: Standard errors in parentheses.  0.005  T a b l e 3.5. C o m p a r i s o n of within-population genetic diversity for Acer  macrophyllum  with a v e r a g e v a l u e s for all plants, w o o d y s p e c i e s , w o o d y a n g i o s p e r m s , a n d for Acer species. Categories  A  All plant species  1.52  %P  H  E  GST/FST  Reference  34.6  0.113  0.228  Hamrick et al. 1992  Woody species  1.76  49.3  0.148  0.084  Hamrick et al. 1992  Woody angiosperms  1.68  45.1  0.143  0.102  Hamrick et al. 1992  0.012  Fore et al. 1992  0.033  Perry and Knowles 1989  0.049  Young et al. 1993  Acer  saccharum  2.80  87.5  Acer  saccharum  1.95  38.2  Acer  saccharum  2.03  53.7  Acer  platanoides  1.92  53.9  0.128  0.120  Rusanen et al. 2000  Acer  platanoides  2.0  54.5  0.132  0.009  Rusanen et al. 2003  Acer campest re  3.15  100  0.287  -  Acer macrophyllum  1.71  61.2  0.152  0.150 0.110 0.109  Note: S e e tables 3.2 and 3.3 for de finition of variables.  0.054  Bendixen 2001 This study  -130  5 0  -125  1  V  v  M  -120  . v  -115  1  ' ^encho  #  ,  5  0  F r a s e r  -a  48 n  n 48  'Artie if O* 46  y  _<_ |  >;  m  F m PI  •  0  Oakville ° Elbe  5 0 100  j  y  46  n  44  "  |  ( Siletz f O •Heimick I O Cascadia 44  n "130  .125  -120  Figure 3.1. G e o g r a p h i c locations of eight Acer macrophyllum populations.  -115  mature natural  tiMi^MiMmWiM  Hi  ARTIC FRASER JERICHO OAKVILLE HELMICK ELBE SILETZ CASCADIA  Figure 3.2. U P G M A cluster analysis of Nei's genetic distances between eight mature populations of Acer  macrophyllum.  Chapter four  E F F E C T S O F F O R E S T FRAGMENTATION O N G E N E T I C VARIATION AND SPATIAL GENETIC S T R U C T U R E IN N A T U R A L P O P U L A T I O N S OF BIGLEAF M A P L E {Acer macrophyllum Pursh) INTRODUCTION W o r l d w i d e , h u m a n d e v e l o p m e n t is rapidly e n c r o a c h i n g upon a n d subdividing m a n y remaining natural a r e a s . Fragmentation of the l a n d s c a p e p r o d u c e s remnant vegetation p a t c h e s , s u r r o u n d e d by a matrix of different vegetation types or unvegetated land u s e s . Habitat fragmentation, the breaking up of continuous forest into s m a l l e r p a t c h e s , c a n reduce population size a n d i n c r e a s e population isolation ( Y o u n g et a l . 1 9 9 3 ; A n d r e n 1994). It a l s o r e d u c e s the availability of suitable colonization sites for the establishment of n e w populations (Wilcox a n d M u r p h y 1985). S t u d i e s of natural plant populations have s h o w n that population s i z e is a n important factor in determining the amount of genetic variation maintained within sexually mature populations a n d the distribution of this variation a m o n g individuals ( S a m p s o n e t a l . 1988). Habitat fragmentation m a y erode genetic diversity a n d i n c r e a s e population differentiation, affecting population viability in the short or long term ( Y o u n g et a l . 1996). T h e s e effects are d u e mainly to i n c r e a s e d genetic drift a n d inbreeding in habitat fragments with s m a l l c e n s u s s i z e s a n d r e d u c e d g e n e flow b e t w e e n fragments ( Y o u n g et a l . 1996). T h e subdivision c a u s e d by fragmentation will promote local population differentiation if g e n e flow barriers are e s t a b l i s h e d a n d s u b p o p u l a t i o n s diverge d u e to genetic drift ( B a c l e s et a l . 2004). In addition to isolation, the genetic  structure of natural populations prior to fragmentation m a y determine h o w large the impact of fragmentation or habitat loss would be. F o r e x a m p l e , if fragments are large e n o u g h to maintain the genetic structure of the original population, differentiation a m o n g fragments m a y be less m a r k e d . O n the other h a n d , if fragments are small a n d scattered, they will be more likely to contain a b i a s e d s a m p l e of the original genetic variation, a n d differentiation will be further promoted if isolation persists ( N a s o n a n d Hamrick 1997). T h e impact of fragmentation varies a m o n g o r g a n i s m s , d e p e n d i n g on the effects of fragmentation on reproduction, d i s p e r s a l a n d g e n e flow, a n d the original distribution of genetic diversity ( Y o u n g 1996). A n o t h e r potential c o n s e q u e n c e of fragmentation is a c h a n g e in mating s y s t e m . In plant populations in particular, inbreeding c a n i n c r e a s e d u e to either i n c r e a s e d selfpollination or through an i n c r e a s e d probability of mating b e t w e e n individuals sharing recent c o m m o n ancestry (Raijmann et a l . 1994; Y o u n g et a l . 1996). R e s u l t s available from empirical studies of the effects of habitat fragmentation on s e v e r a l a n g i o s p e r m tree s p e c i e s from the tropics have indicated that fragmentation c a n have significant genetic c o n s e q u e n c e s . R e d u c e d population s i z e a n d i n c r e a s e d isolation a s s o c i a t e d with habitat fragmentation m a y c a u s e a reduction in genetic variation, i n c r e a s e d population differentiation b e t w e e n habitat fragments (Wilcove 1987; T e m p l e t o n et a l . 1990; C h a s e et al.1996; White et a l . 1999), a n d i n c r e a s e d inbreeding ( L e e et a l . 2 0 0 0 ; F u c h s et a l . 2003) a s predicted by theory. H o w e v e r , there is a l s o growing empirical e v i d e n c e for e n h a n c e d g e n e flow b e t w e e n isolated trees in forest fragments ( Y o u n g et a l . 1993). It a p p e a r s that the effects of habitat fragmentation o n the genetic behavior of tree s p e c i e s are more varied a n d c o m p l e x than first thought ( Y o u n g 1996; Aldrich a n d Hamrick 1998). Populations of long-lived w o o d y perennials s e e m to be resistant to c h a n g e s in genetic diversity d u e to long generation times, overlapping generations a n d high levels of g e n e flow (White et a l . 1999; M e r w e at a l . 2000). H o w e v e r , genetic l o s s e s are more o b s e r v a b l e in s e e d l i n g cohorts than in adult cohorts b e c a u s e s e e d l i n g s reflect the  genetic effects of r e d u c e d present-day levels of g e n e flow a n d population s i z e (Lee et al. 2000). Bigleaf maple is a c o n s p i c u o u s s p e c i e s in the temperate c o a s t a l rainforests of the Pacific Northwest. It grows in a variety of soils throughout its range a n d it is usually a small to m e d i u m - s i z e d tree. T h e trees are usually scattered or in small g r o v e s in a s s o c i a t i o n with conifers a n d other broad-leaved s p e c i e s . A s this s p e c i e s is c o m m o n l y found in remnant forests surrounded by pasture lands a n d agricultural fields, it is likely that forest fragmentation has divided formerly larger Acer macrophyllum  populations  into s m a l l e r a n d isolated patches in s o m e parts of its range. A c c o r d i n g l y , Acer macrophyllum  populations have e x p e r i e n c e d reductions in effective population size  a n d spatial extent, which m a y have significantly r e d u c e d genetic variation a n d altered population genetic structure. It is therefore h y p o t h e s i z e d that forest patch populations (fragments) of Acer macrophyllum  will h a v e l e s s genetic variation a n d i n c r e a s e d levels  of inbreeding than more continuous populations. In this study, I c o m p a r e d genetic diversity, genetic structure a n d inbreeding level in s e e d l i n g a n d adult cohorts from both fragmented a n d continuous populations. In addition I u s e d c o m p u t e r simulations to forecast the decline in genetic variation due to forest fragmentation. T h e specific questions a d d r e s s e d are: 1. D o e s genetic diversity differ between adult bigleaf m a p l e populations of trees in fragmented a n d continuous forests? 2. Is inbreeding different in s e e d l i n g than that in adult c o h o r t s ? 3.  Is there spatial genetic structure in bigleaf maple p o p u l a t i o n s ?  4. If spatial genetic structure exists, d o e s it differ b e t w e e n continuous a n d fragmented populations? If Acer macrophyllum  genetic variation is being affected by fragmentation, w e  would expect: a) l e s s overall genetic diversity in fragmented forests than in continuous  forests; b) lower inbreeding in continuous than in fragmented l a n d s c a p e s ; c) stronger spatial genetic structure in fragmented than in continuous populations.  MATERIALS AND METHODS Populations and sampling S i x study populations were located on V a n c o u v e r Island (Fig 4.1). T h e s e c o m p r i s e d three populations that o c c u r r e d in a r e a s in which forests have b e e n fragmented over the past 150 y e a r s due to agriculture a n d urban d e v e l o p m e n t , a n d three in relatively continuous forests. T h e intent of the s a m p l e d e s i g n w a s to have a control (continuous populations) against which the genetic effects of fragmentation could be tested. P o p u l a t i o n s s a m p l e d occurred between 20 a n d 150 m in elevation. F o r e a c h population a n d at e a c h s a m p l i n g site, s e v e r a l E a s t - W e s t transects were e s t a b l i s h e d , e a c h approximately 100 m wide. A l o n g t h e s e transects, 50 s e e d l i n g s were collected from the forest floor from e a c h of the six populations, a n d terminal b u d s of lateral s h o o t s were collected from e a c h of 50 adult trees in e a c h population. A s m u c h a s p o s s i b l e , s e e d l i n g a n d mature trees were s a m p l e d at least 30 m apart to avoid s a m p l i n g c l o s e l y related individuals. T h e total a r e a from w h i c h tree s a m p l e s were collected varied widely d u e to the patchy nature a n d variable density of populations, ranging from 79 ha at R o s e w a l l C r e e k to 3 1 2 ha for Nitinat (Table 4.1). After collection, s e e d l i n g a n d bud s a m p l e s were w r a p p e d in a l u m i n u m foil, labeled a n d frozen in liquid nitrogen. U p o n return to University of British C o l u m b i a , s a m p l e s w e r e immediately transferred into a -20°C freezer for the s e e d l i n g s a n d to a 80°C freezer for bud s a m p l e s until electrophoresis.  Electrophoresis Horizontal starch gel electrophoresis w a s u s e d to obtain a l l o z y m e data for s e e d l i n g s a n d adults for the s a m e 10 loci d e s c r i b e d in chapter three, with the exception of L A P - 1 , which w a s m o n o m o r p h i c a c r o s s all adult populations a n d w a s not included in this a n a l y s i s . T h e extraction buffer u s e d to grind e m e r g i n g leaf t i s s u e s from s e e d l i n g s a n d bud t i s s u e s from adult trees w a s the s a m e a s d e s c r i b e d in chapter three.  Data Analysis S t a n d a r d genetic diversity parameters (allele f r e q u e n c i e s , a v e r a g e n u m b e r of alleles per l o c u s (A), o b s e r v e d heterozygosity (Ho), a n d e x p e c t e d heterozygosity (HE) were estimated for both seedling a n d adult cohorts in all populations. T h i s a n a l y s i s w a s performed with B I O S Y S - 2 ( W . C . Black IV, Department of Microbiology, C o l o r a d o State University), a modified version of the B I O S Y S - 1 p r o g r a m , by Swofford a n d S e l a n d e r (1981). Departures in genotype f r e q u e n c i e s from H a r d y - W e i n b e r g expectations w e r e tested using a M a r k o v chain method following the algorithm of G u o a n d T h o m p s o n (1992). E x a c t tests for these departures w e r e c o n d u c t e d at e a c h of the variable loci, a n d linkage disequilibrium w a s tested between pairs of variable loci. T h e inbreeding coefficient F  / s  (Wright 1951) w a s estimated following W e i r a n d C o c k e r h a m  (1984). All calculations a n d tests a b o v e were performed using G e n e p o p (version 3.1 d) ( R a y m o n d a n d R o u s s e t , 1995). I a l s o u s e d B O T T L E N E C K (version 1.2.02) d e s c r i b e d by C o r n u e t a n d Luikart (1996) to test for historical reductions in population s i z e . If a population h a s b e e n through a bottleneck it s h o u l d s h o w a signature of r e d u c e d allelic r i c h n e s s c o m p a r e d to e x p e c t e d heterozygosity, a s rare alleles are lost faster than heterozygosity d e c r e a s e s . After a bottleneck, the e x p e c t e d heterozygosity (HE) c o m p u t e d from allele frequencies for a s a m p l e of g e n e s should be larger than the heterozygosity e x p e c t e d  (H ) eq  b a s e d on the n u m b e r of alleles in the s a m e s a m p l e , a s s u m i n g the population is  at mutation-drift equilibrium (Cornuet and Luikart, 1996). I s o z y m e s are e x p e c t e d to conform to the infinite allele model (IAM), where e a c h new mutation g i v e s rise to a new allele different from all existing o n e s (Kimura a n d C r o w , 1964), thus data were a n a l y z e d under this m o d e l . T o test for a deficiency or e x c e s s in H , the W i l c o x o n E  s i g n e d - r a n k s test w a s u s e d a s it h a s more power than the sign-test a n d c a n be u s e d effectively with fewer loci (Cornuet a n d Luikart, 1996; Piry et a l . 1999).  Genetic structure T h e genetic structure of populations w a s a s s e s s e d a c c o r d i n g to Wright's (1965) F-statistics following W e i r a n d C o c k e r h a m (1984). T h e s e fixation indices w e r e u s e d to m e a s u r e deviations from H a r d y - W e i n b e r g equilibrium attributable to individuals within local populations ( F ) , variation a m o n g populations (F T,) /s  S  a n d variation a m o n g  individuals relative to all populations pooled (FIT). T h e significance of t h e s e parameters w a s tested b a s e d on 1800 permutations of alleles a m o n g individuals within s a m p l e s , g e n o t y p e s a m o n g s a m p l e s , a n d alleles a m o n g s a m p l e s , respectively. M e a n s a n d standard errors were obtained by jackknifing o v e r loci. A boostrap c o n f i d e n c e interval (CI) of 9 5 % w a s c o n s i d e r e d significant w h e n c o n f i d e n c e intervals did not overlap z e r o . T h e s e calculations were m a d e using the program F S T A T (Goudet, 2000).  Spatial autocorrelation analysis of genetic variation Spatial genetic structure within populations w a s a s s e s s e d using C o c k e r h a m ' s (1969) estimates between all possible pairs of individuals at different inter-tree d i s t a n c e s in e a c h population for the adult cohort. T h i s method provides a powerful test of spatial genetic structure (Hardy a n d V e k e m a n s 2002). T h e c o a n c e s t r y coefficient  (p,y) h a s b e e n u s e d in a number of studies recently (e.g., L o i s e l l e et a l . 1 9 9 5 ; P e a k a l l a n d Beattie, 1996; B u r k e et a l . 2 0 0 0 ; K a l i s z et a l . 2 0 0 1 ; P a r k e r et a l . 2 0 0 1 ) . T h e parameter p,j w a s estimated for e a c h distance c l a s s using the software program Spatial Pattern A n a l y s i s of G e n e t i c Diversity ( S P A G e D i ) 1.1 (Hardy a n d V e k e m a n s 2002). T h e software u s e s the estimator d e s c r i b e d by Loiselle et a l . (1995) a s follows:  Pij = M&-P)(Pi-P)  /cp(1-p)  +  2  (8/c + 1 ) °  5  -1  where p, a n d p are the frequencies of h o m o l o g o u s alleles at a l o c u s for individuals / 7  a n d j; p is the m e a n frequency for that allele; a n d k = n(n -1) / 2, the n u m b e r of possible pairs between n individuals located in e a c h d i s t a n c e c l a s s . T h e s e c o n d term in the equation adjusts for bias a s s o c i a t e d with a finite s a m p l e s i z e a n d results in p,y having a n e x p e c t e d value of z e r o for a population in H a r d y - W e i n b e r g equilibrium. T h e results w e r e c o m b i n e d a c r o s s loci to estimate c o a n c e s t r y by weighting the v a l u e s for e a c h locus by its polymorphic index, 2 p,- (1 - pi). F o r a population in H a r d y - W e i n b e r g equilibrium, the c o a n c e s t r y between individuals is a m e a s u r e of the inbreeding coefficient of their hypothetical offspring with e x p e c t e d v a l u e s of 0.25 for pairs of fullsibs, 0.125 for half-sibs, a n d 0.0625 for first c o u s i n s . Individual tree locations w e r e identified by a coordinate grid s y s t e m using a hand-held G l o b a l Positioning S y s t e m instrument ( G P S G a r m i n M o d e l 1 2 X L ) . E a c h tree w a s m a p p e d o n a North-South a n d E a s t - W e s t (x,y respectively) grid using G P S data to construct inter-tree distance matrices for spatial autocorrelation a n a l y s i s . With this procedure, e a c h s c o r e d genotype w a s a s s i g n e d to its c o r r e s p o n d i n g spatial location within e a c h population.  E l e v e n to fourteen d i s t a n c e c l a s s e s w e r e u s e d for the spatial autocorrelation analysis. All populations had the following intervals: 0 - 50 m, 50 - 100 m a n d nine 100 m interval up to 1000 m for all populations. R o s e w a l l h a d two additional distance c l a s s e s (1000 - 1200 m a n d 1200 - 1500 m), a n d M a p l e B a y , Y e l l o w Point, a n d N i t i n a t a n additional four c l a s s e s ; ( 1 0 0 0 - 1200 m, 1 2 0 0 - 1500 m, 1 5 0 0 - 2 0 0 0 m, 2 0 0 0 - 2 5 0 0 m). D i s t a n c e c l a s s e s were c h o s e n s o that e a c h contained at least 30 pairwise c o m p a r i s o n s . T h i s a n a l y s i s tests whether pairs of trees within a specified distance interval exhibit the s a m e alleles more often than e x p e c t e d by c h a n c e under a r a n d o m spatial distribution (Hardy a n d V e k e m a n s 2002). M e a n e s t i m a t e s of c o a n c e s t r y w e r e obtained over all pairs of individuals for the d i s t a n c e c l a s s e s d e s c r i b e d a b o v e . W h e n p,y = 0, there is no genetic correlation b e t w e e n the f r e q u e n c i e s of alleles in individuals at the spatial s c a l e of interest; w h e n p,y > 0, individuals in a given distance c l a s s are more closely related than e x p e c t e d by c h a n c e ; a n d c o n v e r s e l y , w h e n p,y < 0, individuals within a given d i s t a n c e c l a s s are less related than e x p e c t e d by c h a n c e . E s t i m a t e s of c o a n c e s t r y w e r e tested for significance with a randomization procedure that generated populations with a r a n d o m spatial distribution of g e n o t y p e s (i.e. no spatial structure). In e a c h plot, intact multilocus g e n o t y p e s were randomly drawn, with replacement, from the s a m p l e d data a n d a s s i g n e d to points o c c u p i e d by plants; new p,y v a l u e s w e r e then c a l c u l a t e d . T h i s randomization procedure w a s repeated 4 9 9 times for e a c h plot, giving (together with the originally s a m p l e d data) 5 0 0 p,y v a l u e s , from which 9 5 % c o n f i d e n c e intervals were constructed. A p,y estimate falling outside this c o n f i d e n c e limit is c o n s i d e r e d significant. If genetic structure exists, then w e expect a pattern of significant v a l u e s at shorter distance c l a s s e s b e c o m i n g non-significant or negative with i n c r e a s i n g d i s t a n c e . Finally, to test whether the s l o p e (b) of the correlograms obtained for py w a s statistically significant, py estimates w e r e permuted (999 times) with r e s p e c t to the upper b o u n d (m) of e a c h distance c l a s s under the null h y p o t h e s i s b=0.  Simulations I u s e d the simulation program B O T T L E S I M (Kuo a n d J a n z e n  2003)  to estimate  the current levels of genetic variation in fragmented a n d continuous populations, forecast their future genetic diversity levels a n d m a k e r e c o m m e n d a t i o n s with respect to sustainable population s i z e . T h e program allows specification of a n arbitrary population s i z e a n d projects the decline genetic diversity d u e to genetic drift b a s e d on the actual allele f r e q u e n c i e s estimated from the genotypic d a t a input. In order to project the most realistic projections of d e c l i n e in genetic variation for Bigleaf m a p l e , I u s e d the over-lapping generation model of the p r o g r a m . Other parameters during the simulation p r o c e s s were set a s follows: d e g r e e of generation overlap = 1 0 0 (i.e. all individuals start with r a n d o m a g e v a l u e that is within the longevity limit), m o n o e c y with r a n d o m mating a n d selfing reproductive s y s t e m , e x p e c t e d longevity = 1 2 5 y e a r s , a g e of reproductive maturation = 1 0 y e a r s (Minore a n d Z a s a d a 1 9 9 0 ) , n u m b e r of y e a r s simulated = 2 5 0 y e a r s , effective population s i z e s N  E  = 5 0 a n d N E = 1 0 0 respectively for both continuous a n d fragmented populations,  a n d n u m b e r of iterations = 1 0 0 0 . I c o m p a r e d the empirical data to the simulation results in order to determine whether the levels of genetic variation found in fragmented populations will be lower than those in continuous populations. If the fragmented populations s h o w levels of genetic variation that are lower relative to continuous populations, then the empirical data are consistent with the hypothesis that fragmentation affects Acer populations. In contrast, if there is no d e c r e a s e in v a l u e s of A o a n d H  E  macrophyllum respectively  in  fragmented populations relative to continuous populations, then the empirical data are consistent with the hypothesis that fragmentation has not affected populations of A c e r macrophyllum.  RESULTS Allele frequencies Eight of the nine loci w e r e polymorphic in at least o n e of the populations e x a m i n e d for both s e e d l i n g s a n d adults. In all populations s a m p l e d , G D H w a s m o n o m o r p h i c for both adults and s e e d l i n g s . T w o to three alleles w e r e detected for e a c h polymorphic locus, with a total of 18 alleles o b s e r v e d for adults in continuous populations and 20 alleles in fragmented populations (Table 4.2 a). In s e e d l i n g s , a total of 19 alleles w e r e o b s e r v e d in continuous populations and 18 in fragmented populations (Table 4. 2 b). T h e majority of the alleles w e r e c o m m o n a n d distributed widely a c r o s s most populations, but a few rare alleles w e r e private, unique to o n e population, or found only in couple of populations. T h e distribution of allele f r e q u e n c i e s w a s the typical U - s h a p e d , with most alleles either rare or nearing fixation (Fig 4.2a-b). E x a c t test for linkage disequilibrium did not yield a n y significant v a l u e s for s e e d l i n g s or adults in any populations, indicating i n d e p e n d e n c e of loci u s e d in this study.  Genetic diversity In all six populations, both seedling and adult cohorts p o s s e s s e d similar levels of genetic variation regardless of whether populations were fragmented or continuous (Table 4.3). T h e m e a n n u m b e r of alleles per locus for adults a v e r a g e d 1.66 in continuous populations a n d 1.60 in fragmented populations, t h e s e estimates w e r e 1.73 and 1.66, respectively, in s e e d l i n g s . E x p e c t e d heterozygosity w a s slightly higher in adults in fragmented (0.134) than continuous populations (0.120), but slightly lower for s e e d l i n g s (0.130 v e r s u s 0.140). S e e d l i n g s in continuous populations had a slightly higher proportion of polymorphic loci than s e e d l i n g s in fragmented populations or in the adult cohorts (Table 4.3).  Levels of Inbreeding Within all adult populations, genotypic f r e q u e n c i e s s h o w e d significant departures from H a r d y - W e i n b e r g expectations (P<0.05) with a n e x c e s s of h o m o z y g o t e s . In the s e e d l i n g cohort, o n e continuous population (Elk Falls) a n d o n e f r a g m e n t e d population (Maple B a y ) did not deviate significantly from H a r d y - W e i n b e r g expectations. F/s varied c o n s i d e r a b l y a m o n g both loci a n d populations, with overall v a l u e s of 0.20 for adults a n d 0.28 for s e e d l i n g s in continuous populations, a n d 0.25 in adults a n d 0.37 for s e e d l i n g s in fragmented populations, suggesting substantial levels of inbreeding (Table 4.3).  Bottleneck test T h e r e w a s no significant bottleneck signature in a n y of the populations. R e c e n t l y bottlenecked populations should s h o w a m o d e shift of distribution in allele f r e q u e n c i e s s o that alleles in low frequency c l a s s e s (<0.1) b e c o m e l e s s abundant than intermediate a n d high frequency c l a s s e s . T h e bottleneck program did not s h o w a n y significant m o d e shift, thus, all allele frequency distributions w e r e U - s h a p e d (Fig 4 . 2 a b), moreover, the W i l c o x o n test detected more heterozygosity than e x p e c t e d under mutation-drift equilibrium (Table 4.4).  Genetic structure Eight polymorphic loci were consistently s c o r e d , of w h i c h P G I - 2 , L A P - 2 , a n d IDH had high g e n e diversities (HT) in e x c e s s of 2 0 % , s u g g e s t i n g a d e q u a t e variation for a p p r e c i a b l e genetic structure (Table 4.5 a-b). F  / s  a n d F , e s t i m a t e s w e r e positive a n d r  significantly greater than z e r o , suggesting a deficit of h e t e r o z y g o t e s . H o w e v e r , individual loci s h o w e d a great d e a l of variation in their fixation indices. F o r instance,  A A T - 1 , 6 P G - 2 , P G I - 1 , a n d P G I - 2 , s h o w significant e x c e s s of h e t e r o z y g o t e s , while A A T - 2 , IDH, a n d L A P - 2 s h o w a significant deficiency, in a m a n n e r consistent with inbreeding (Table 4 . 5 a-b). T h e r e w a s a low but significant amount of genetic differentiation a m o n g adult populations both in the continuous and the fragmented forests, with a m e a n FST a c r o s s loci of 0.015 ( 9 5 % CI = 0.005 - 0.035) for continuous populations (Table 4.5 a) a n d 0.031 ( 9 5 % CI = 0.010 - 0.056) for fragmented populations (Table 4 . 5 b). Pairwise FST estimates a m o n g populations (Table 4.6) w e r e a l s o low in all c a s e s suggesting extensive g e n e flow between these populations or a recent c o m m o n ancestral population (Table 4.6).  Spatial genetic structure A n a l y s i s of spatial genetic structure revealed significant, positive spatial genetic structure in four of the six populations. All fragmented populations (Fig 4 . 3 d-f) had significant, positive multilocus c o a n c e s t r y (p,y) coefficients at inter-tree d i s t a n c e s of up to 100 m distance for R o s e w a l l a n d M a p l e B a y , a n d up to 2 0 0 m for Y e l l o w Point. F o r instance, in the 5 0 - 1 0 0 m distance c l a s s for these fragmented populations, p,y ranged from 0.13 to 0.30, a v e r a g i n g 0.22. T h i s estimate s u g g e s t s that in fragmented populations of Acer macrophyllum,  trees s a m p l e d up to approximately 100 m apart are  likely to be nearly a s similar a s full-sibs. B e y o n d the 50 - 100 m d i s t a n c e c l a s s , genetic structuring r e m a i n e d significant (a = 0.05) but l e s s p r o n o u n c e d up to approximately 6 0 0 m , then b e c a m e non-significant or negative up to 2 5 0 0 m. Of the continuous populations, only Elk Falls revealed significant spatial genetic structure, with a positive c o a n c e s t r y coefficient (p,y = 0.14) for only the 100 - 2 0 0 m distance c l a s s . T h e overall s l o p e s of the correlograms for all three fragmented populations a n d for Elk Falls w e r e negative a n d significant (P<0.05), indicating spatial genetic structure for these populations.  Simulations E s t i m a t e s for simulations of the o b s e r v e d n u m b e r of alleles (Ao) a n d e x p e c t e d heterozygosity (H ) likely to be retained over a 2 5 0 - y e a r period c o m p a r e d to the E  current levels are s u m m a r i z e d in T a b l e 4 . 7 . T h e o b s e r v e d n u m b e r of alleles decline slightly faster than e x p e c t e d heterozygosity consistent with theoretical predictions (Nei et a l . 1975; C h a k r a b o t y 1980). H o w e v e r the decline is m u c h faster w h e n NE = 50 than when N  E  - 100 (Table 4.7). B a s e d on actual allele f r e q u e n c i e s , over 9 0 % of expected  heterozygosity would be retained for both fragmented a n d continuous populations over two generations (after 2 5 0 years) irrespective of whether NE - 50 or NE =100 (Table 4.7).  DISCUSSION  Effects of fragmentation on genetic variation and inbreeding Habitat fragmentation c a n c a u s e a loss of population genetic variation in two w a y s . First, a transient reduction in population size could result in a substantial loss of alleles (Frankel and S o u l e 1 9 8 1 ; Y o u n g et a l . 1996). T h e extent to which this o c c u r s is d e p e n d e n t on the extent and pattern of forest loss, and its c o i n c i d e n c e with any fines c a l e genetic structure. A n immediate loss of heterozygosity w o u l d , however, only be evident if the population size w a s greatly r e d u c e d . S e c o n d , s u b s e q u e n t to this initial allelic loss, fragmented populations that remain small and isolated for s e v e r a l generations will continue to lose alleles d u e to genetic drift, further reducing levels of genetic variation within the stands (Barrett and K o h n 1 9 9 1 ; Ellstrand a n d E l a m 1993). Heterozygosity is mostly affected by intermediate-frequency alleles (Taggart et al. 1990), w h e r e a s rare alleles are the most likely to be lost in s m a l l or fragmented populations and high frequency alleles are likely to b e c o m e fixed. Nearly all of the differences o b s e r v e d in n u m b e r of alleles in this study w e r e c a u s e d by low frequency alleles (<0.10). ( L A P - 2 , 2 being the o n e exception). T h e r e are three likely explanation for the m a i n t e n a n c e of genetic variation in fragmented populations: (i) T h e r e have been insufficient generations s i n c e fragmentation for detectable loss of diversity through genetic drift and inbreeding or for mutation and genetic drift to generate differences a m o n g populations; (ii) Despite fragmentation, effective population s i z e (A/ ) remains large s o that initial l o s s of e  heterozygosity is very s m a l l , s i n c e the proportionate reduction in e x p e c t e d heterozygosity AH  e  = —!—. E v e n though populations are f r a g m e n t e d , they could still  have h u n d r e d s or e v e n t h o u s a n d s of individuals (depending o n the neighborhood size); (iii) G e n e flow is sufficient a n d there w a s no isolation by d i s t a n c e in fragmented population. I h y p o t h e s i z e d that o n e effect of fragmentation would be d e c r e a s e d heterozygosity a n d p o l y m o r p h i s m in s e e d l i n g s c o m p a r e d to adults in fragmented populations, but did not detect e v i d e n c e d of this. O n the contrary, p o l y m o r p h i s m w a s higher in s e e d l i n g s than adults in both continuous a n d f r a g m e n t e d populations, a n d most alleles found in s e e d l i n g s were c o m m o n a n d w i d e s p r e a d a c r o s s all populations, just a s in adults, s u g g e s t i n g high g e n e flow. G o n z a l e z - A s t o r g a a n d N u n e z - F a r f a n (2001)  found low f r e q u e n c y alleles in s e e d l i n g s of Brongniartia  vazquezii  a  m o n o e c i o u s , animal pollinated shrub in Central M e x i c o , w h i c h w e r e not found in adult populations a n d attributed this to g e n e flow. T h e r e is no reduction of genetic variation in fragmented c o m p a r e d to continuous populations in either adults or s e e d l i n g s , indicating there m a y be substantial g e n e flow a m o n g fragmented populations or that fragmented populations have large effective population s i z e s (Levin a n d Kerster 1974; Y o u n g e t a l . 1996). Similar studies c o n d u c t e d by Y o u n g et al. (1993) o n Acer saccharum  a l s o found  no r e d u c e d genetic variation in fragmented populations c o m p a r e d to continuous populations. C o m b i n e d with higher m e a n levels of p o l y m o r p h i s m in fragmented populations, this indicated i n c r e a s e d g e n e flow m a y be a c o n s e q u e n c e of fragmentation. Similarly, Fore et a l . (1992) c o m p a r e d inter-population genetic d i v e r g e n c e b e t w e e n pre-fragmentation a n d post-fragmentation s e e d l i n g a n d adult cohorts in 1 5 / 4 . saccharum  populations in O h i o , U S A . In their study, genetic  d i v e r g e n c e in post-fragmentation cohorts w a s l e s s than half that of continuous populations, indicating a reduction in genetic differentiation s i n c e fragmentation, a n d suggesting i n c r e a s e d inter-population g e n e flow. It a p p e a r s therefore that maple s p e c i e s m a y be resilient to fragmentation.  T h e results obtained from this study are s o m e w h a t in contrast to similar studies c o n d u c t e d on s o m e a n g i o s p e r m trees s p e c i e s in the tropics that s h o w e d s o m e effects of fragmentation o n the overall genetic structure, (e.g. Swietenia 1999; a n d Spondias  mombin,  humilis; W h i t e et a l .  ( N a s o n a n d Hamrick 1997)).The authors attributed their  results mainly to the d e m o g r a p h i c a n d reproductive characteristics of these s p e c i e s in the tropics. F o r instance, m a n y tropical trees o c c u r at low d e n s i t i e s , are pollinated by a n i m a l s , have high outcrossing rates, a n d have breeding s y s t e m s that involve c o m p l e x m e c h a n i s m s of self-incompatibility ( B a w a 1990; H a m r i c k a n d M u r a w s k i 1990). Inbreeding in adults versus seedlings In this study a high proportion of the a l l o z y m e loci w e r e not in H a r d y - W e i n b e r g equilibrium, with significant inbreeding (F/s) (Table 4.3). Deviations from HardyW e i n b e r g expectations d u e to n o n r a n d o m mating within f r a g m e n t e d populations of either adults or s e e d l i n g s would be e x p e c t e d a c r o s s all loci. H o w e v e r , the high v a l u e s of F/s w e r e not consistent for individual loci or within a particular cohort or population type. F o r instance, adults in all populations had significant inbreeding, but s e e d l i n g s in the Elk Falls a n d M a p l e B a y populations did not differ from H a r d y - W e i n b e r g equilibrium. T w o genetically unlinked loci ( 6 P G - 1 a n d L A P - 2 ) contributed to the overall high estimates of F  / s  in both adults a n d s e e d l i n g s . For, instance if t h e s e two loci are  r e m o v e d from s e e d l i n g s in R o s e w a l l , F/s estimated d r o p s from 0.57 to 0.39, reducing the inbreeding estimate by 3 3 % . T h e r e are two p o s s i b l e explanations for the high F/s o b s e r v e d in this study. First, if population sub-structuring h a s b e e n ignored in s a m p l i n g , the inbreeding coefficient would be o v e r e s t i m a t e d , i.e., the patchy distribution of related individuals m a y generate a W a h l u n d effect (Barbujani 1987). S e c o n d , there m a y be a significant amount of inbreeding occurring in t h e s e populations (see chapter three). Fragmentation, c o u p l e d with l o c a l i z e d pollinator m o v e m e n t a n d s e e d d i s p e r s a l , m a y have resulted in higher correlated paternity or  selfing for t h e s e s a m p l e d populations c o m p a r e d to continuous populations, c a u s i n g a deficiency of heterozygotes. A s s u g g e s t e d by S h e a (1990), inbreeding could a l s o result from differential selection p r e s s u r e s resulting from micro-environmental variations favoring related individuals. W h i l e the e v i d e n c e of inbreeding w a s s h o w n in both continuous a n d fragmented populations for both adults a n d s e e d l i n g s , s e e d l i n g s had higher levels of inbreeding c o m p a r e d to their adult cohorts in four of the six populations (all but Elk Falls a n d M a p l e B a y ) . Positive v a l u e s of F/s at the s e e d l i n g stage m a y be d u e to partial selfing. In Shorea leprosula, dipterocarp with the highest i s o z y m e heterozygosity (H  a n i n s e c t e d pollinated E  = 0.40) e v e r recorded in long  lived plant s p e c i e s (Lee et a l . 2000), a higher inbreeding level found for s e e d l i n g s in natural populations c o m p a r e d to adults w a s attributed to selection against h o m o z y g o t e s between the seedling a n d adults s t a g e s . H o w e v e r in regnans,  Eucalyptus  elevated inbreeding found in natural populations c o m p a r e d to s e e d l i n g s in a  s e e d orchard w a s e x p l a i n e d by spatial genetic structure ( M u o n a et a l . 1990). This a l s o could be the c a s e with A. macrophyllum,  in w h i c h trees in natural populations  s o m e t i m e s exist in c l u m p s . Inbreeding m a y also be d u e to mating b e t w e e n relatives in these clumps.  Population structure M e a n FST estimates indicate w e a k but significant population differentiation in this s p e c i e s , a m o n g both fragmented (FST - 0.031) a n d continuous (FST = 0.015) populations (Table 5.5 a , b). This is consistent with earlier findings of low interpopulation differentiation in a range-wide genetic study of this s p e c i e s (chapter three), a n d s u g g e s t s that s t a n d s s a m p l e d on V a n c o u v e r Island, B C , m a y essentially form single large population with w e a k within-population structure.  Spatial genetic structure T h e d e g r e e of spatial genetic clustering within a population is determined by a variety of genetic a n d d e m o g r a p h i c factors including population s i z e , microenvironmental s e l e c t i o n , s e e d a n d pollen d i s p e r s a l , plant density, temporal variation in population reproductive rates, patterns of competition-induced mortality a n d other s o u r c e s of mortality, spatial s c a l e of g a p formation a n d p o s s i b l y other details of the regeneration p r o c e s s (Frankel et a l . 1995). In particular, the magnitude a n d spatial s c a l e of genetic structure is strongly influenced by s e e d d i s p e r s a l m e c h a n i s m s a n d adult density that characterize individual s p e c i e s (Hamrick et a l . 1 9 9 3 ; Hamrick and N a s o n 1996; D o l i g e z a n d J o l y 1997; K a l i s z e t a l . 2001). Spatial genetic structure in this study revealed apparent differences between continuous a n d fragmented populations. T h e distribution of g e n o t y p e s in all fragmented populations w a s n o n - r a n d o m with significant positive v a l u e s for c o a n c e s t r y (p,y) up to 100 m or 2 0 0 m w h e r e a s in two of the three continuous populations, g e n o t y p e s were distributed randomly (non-significant c o a n c e s t r y estimates) with w e a k or no spatial genetic structure. T h e o b s e r v e d (p,y) v a l u e s for two of the continuous populations are m u c h less than that e x p e c t e d for full or half-sibs at all d i s t a n c e s , s u g g e s t i n g overlapping of s e e d s h a d o w s from maternal parents. T h e strong spatial genetic structure o b s e r v e d in fragmented populations c o u l d be d u e to the high d e g r e e of s e e d production favoring regeneration of s e e d l i n g s in the neighborhood of the mother trees a s s e e d s of bigleaf m a p l e are d i s p e r s e d by wind a n d gravity. In contrast, trees of bigleaf maple that grow in continuous forests d e v e l o p narrow a crown that is supported by a stem free of b r a n c h e s for more than half of its total height due to strong competition for light (Minore a n d Z a s a d a 1990). T h i s habit m a y lead to low s e e d production or a smaller s e e d s h a d o w thereby reducing the n u m b e r of siblings that are likely to d e v e l o p around the n e i g h b o r h o o d of the mother trees (Kelly et a l . 2004). T h e higher s p e c i e s richness (number of s p e c i e s per hectare) of the forest e c o s y s t e m in continuous forests a n d the habitat of d i s p e r s a l a g e n t s m a y  a l s o lead to a reduction in spatial genetic structure in continuous populations. F o r e x a m p l e , s o m e small m a m m a l s s u c h a s mice, w o o d rats, squirrels a n d birds a s reported by F o w e l s (1965), c a n collect fruits from different bigleaf maple trees a n d d i s p e r s e them in the forest. Hamrick et a l . (1993) and Hamrick a n d N a s o n (1996) s u g g e s t e d that plant s p e c i e s with high adult densities have w e a k e r fine-scale genetic structure than s p e c i e s with lower densities. During s a m p l i n g , continuous populations a p p e a r e d to have higher s p e c i e s densities than fragmented populations. T h i s study is consistent with the findings of G a p a r e a n d Aitken (2005) for Picea sitchensis  in w h i c h core  populations with higher densities had no spatial structure, while peripheral populations with lower density h a d strong spatial genetic structure up to 5 0 0 m. In addition, V e k e m a n s a n d Hardy (2004) r e - a n a l y z e d data for six s p e c i e s a n d c o m p a r e d spatial genetic structure for differing population densities with s p e c i e s classified a s low or high density. In e a c h of the six pairwise c o m p a r i s o n s , populations with low densities consistently r e v e a l e d spatial genetic structure. T h e s e findings s u g g e s t that relatively high population density in continuous forests c o m p a r e d to fragmented populations could h a v e a strong influence on spatial genetic structure. T h e lack of spatial genetic structure o b s e r v e d in the two continuous populations (Nitinat a n d Port Alberni) is similar to that o b s e r v e d for a n u m b e r of other tree s p e c i e s (e.g. Pinus contorta ( E p p e r s o n a n d Allard 1989); Picea mariana  ( K n o w l e s 1991);  Pinus banksiana  ( C h u n g et a l . 2000)).  (Xie a n d K n o w l e s 1991); a n d Neolitsea  sericea  In contrast, in s o m e tree s p e c i e s with restricted s e e d d i s p e r s a l , significant spatial genetic structure w a s detected. For, e x a m p l e Quercus Quercus  petraeae  rubra (Sork et a l . 1993); and  (Streiff et a l . 1998) exhibit spatial genetic structure at short spatial  s c a l e s w h i c h w a s attributed to large, gravity d i s p e r s e d s e e d , a s pollen m o v e m e n t by wind is known to be extensive in those s p e c i e s . Strong spatial genetic structure w a s also found in tree s p e c i e s featuring restricted dispersal of both s e e d a n d pollen due to  pollination by s m a l l insects a n d s e e d d i s p e r s a l by gravity, for e x a m p l e , in Eurya emarginata  ( C h u n g et a l . 2000).  T h e high v a l u e s of F/s (Table 4.3) indicate that s o m e populations of bigleaf maple e x p e r i e n c e a p p r e c i a b l e inbreeding. In the four populations w h e r e genetic structure w a s evident, aggregation of g e n o t y p e s most likely resulted from limited s e e d d i s p e r s a l . Similar studies conducted recently by K e v i n et a l . (2004) in two Shorea s p p . attributed spatial genetic structure to limited s e e d d i s p e r s a l .  Computer simulations of fragmentation effects T h e results obtained from the computer simulations s u g g e s t that fragmented populations would maintained well over 9 0 % of their genetic variation o v e r a 2 5 0 - y e a r period e v e n with a n effective population size of N  E  = 5 0 . W h e n the effective  population size is i n c r e a s e d to NE = 100 in the simulation both continuous a n d fragmented populations o n a v e r a g e retained over 9 7 % of e x p e c t e d heterozygosity. T h e s e results s u g g e s t that an effective population s i z e of at least 100 c a n effectively minimize the decline of genetic diversity for bigleaf m a p l e populations. Overall the simulation projections corroborates my earlier findings about m a i n t e n a n c e of genetic variation in fragmented populations a n d might further help explain w h y fragmented populations maintain the s a m e levels of genetic variation a s the continuous populations. It h a s b e e n s u g g e s t e d that fragmentation represents a significant threat to the long-term survival of m a n y plant s p e c i e s (Templeton et a l . 1990; Y o u n g et a l . 1996). In addition, it h a s b e e n argued that erosion in genetic variation is o n e of the important c o n s e q u e n c e s that fragmentation m a y have o n plants s p e c i e s that remain in the smaller p a t c h e s d u e to genetic drift, reduction in g e n e flow, a n d elevated inbreeding (Templeton et a l . 1990; Y o u n g a n d M e r r i a m 1994). In this thesis, using both empirical data a n d simulation projections, I have s h o w n that fragmentation h a s not led to overall reduction in genetic variation nor elevated levels in inbreeding at this time in Acer  macrophyllum  a n d is not likely to in the near future u n l e s s populations are very small  a n d no g e n e flow o c c u r s . Lastly, I a r g u e d that I did not detect a n y significant impacts of fragmentation on the overall genetic variation on bigleaf maple populations. T h i s argument must be taken with caution in view of the fact that with only three fragmented a n d three continuous populations, statistical power w a s weak. In a p o w e r test c o n d u c t e d using the S A S A n a l y s t module ( S A S Inc. 1994) with both a o n e - a n d two- tailed test with a = 0.05, statistical power ranged from 6 % to 1 2 . 5 % . H o w e v e r , no trends w e r e s e e n in overall genetic variation between continuous a n d fragmented populations, s o the lack of significant differences did not s e e m to be a function of the p o w e r of the statistical tests.  T a b l e 4 . 1 . S u m m a r y of population information for adult trees a n d s e e d l i n g s of  Acer  macrophyllum. F o r e s t type  Continuous  Fragmented  Total  Population  S i z e of plot or  N o . of trees  N o . of seedlings  forest a r e a  analysed  analysed  [ha)  Nitinat  312  40  50  Elk Falls  102  50  50  P . Alberni  112  50  50  Maple Bay  170  46  50  Rosewall  79  45  50  Y e l l o w Pt.  278  50  50  281  300  T a b l e 4.2 a . Allele f r e q u e n c i e s for nine loci for adults in c o n t i n u o u s a n d fragmented populations of Acer macrophyllum. Continuous Locus  Alleles  AAT-1  Fragmented  NIT  ELF  PAL  MBY  RWC  YPT  1  0.990  1.000  1.000  0.957  0.978  0.969  2  0.010  0.000  0.000  0.043  0.022  0.031  1  0.857  0.733  0.900  0.910  0.845  0.944  2  0.143  0.244  0.100  0.090  0.131  0.056  4  0.000  0.022  0.000  0.000  0.024  0.000  1  0.900  0.809  0.840  0.716  0.784  0.864  2  0.100  0.191  0.160  0.261  0.216  0.136  4  0.000  0.000  0.000  0.023  0.000  0.000  1  0.944  1.000  0.920  1.000  0.901  1.000  2  0.056  0.000  0.008  0.000  0.099  0.000  1  0.860  0.990  0.920  1.000  0.922  1.000  2  0.140  0.010  0.080  0.000  0.078  0.000  1  1.000  0.970  1.000  1.000  1.000  0.938  2  0.000  0.030  0.000  0.000  0.000  0.063  1  0.850  0.818  0.859  0.744  0.833  0.878  2  0.000  0.000  0.000  0.023  0.000  0.000  3  0.150  0.182  0.141  0.233  0.167  0.122  GDH  1  1.000  1.000  1.000  1.000  1.000  1.000  LAP-2  1  0.949  1.000  0.910  0.837  0.956  0.739  2  0.051  0.000  0.090  0.163  0.044  0.261  AAT-2  IDH  6PG-1  6PG-2  PGM  PGI-2  Note: Populations abbreviations. NIT= Nitinat; ELF = Elk Falls; PAL = Port Alberni; MBY = Maple Bay; RWC = Rosewall Creek; YPT = Yellow Point.  T a b l e 4.2 b. Allele f r e q u e n c i e s for nine loci studied for s e e d l i n g s in continuous and fragmented populations of Acer  macrophyllum.  Continuous  Fragmented  NIT  ELF  PAL  MBY  RWC  YPT  1  0.900  1.000  1.000  0.925  1.000  1.000  2  0.100  0.000  0.000  0.075  0.000  0.000  1  0.906  0.768  0.904  0.780  0.917  0.936  2  0.094  0.232  0.096  0.220  0.083  0.064  1  0.833  0.727  0.800  0.739  0.833  0.811  2  0.167  0.273  0.200  0.261  0.167  0.189  1  0.851  1.000  0.978  1.000  0.851  0.978  2  0.149  0.000  0.022  0.000  0.149  0.022  1  0.956  0.929  0.917  0.952  0.978  0.929  2  0.044  0.071  0.083  0.048  0.022  0.071  1  1.000  0.928  1.000  1.000  1.000  0.901  2  0.000  0.072  0.000  0.000  0.000  0.099  1  0.775  0.761  0.807  0.784  0.788  0.818  2  0.050  0.102  0.023  0.102  0.038  0.023  3  0.175  0.136  0.170  0.114  0.175  0.159  GDH  1  1.000  1.000  1.000  1.000  1.000  1.000  LAP-2  1  0.939  1.000  0.837  1.000  0.959  0.867  2  0.061  0.000  0.153  0.000  0.041  0.133  3  0.000  0.000  0.010  0.000  0.000  0.000  Locus AAT-1  AAT-2  IDH-1  6PG-1  6PG-2  PGI-1  PGI-2  Alleles  T a b l e 4 . 3 . G e n e t i c diversity estimates for adults a n d s e e d l i n g s in continuous a n d fragmented populations of Acer  Populations  N  macrophyllum.  %P  Ho  He  Continuous Nitinat Adults Seedlings  50 47  1.6 1.8  60 70  0.101±0.037 0.091 ±0.042  0.116±0.037 0.142±.0436  0.17* 0.39*  Elk Falls Adults Seedlings  49 47  1.6 1.6  60 50  0.103±0.048 0.124±0.048  0.122±0.053 0.144±0.056  0.25* ,NS 0.100  P. Alberni Adults Seedlings  50 47  1.5 1.9  50 70  0.083±0.030 0.090±0.035  0.113±0.036 0.134±0.043  0.17* 0.36*  1.6 1.8  56.6 63.3  0.095 0.102  0.120 0.140  0.20 0.28  Mean Adults seedlings Fragmented Maple Bay Adults Seedlings  44 47  1.7 1.6  50 50  0.104±0.046 0.109±0.044  0.146±0.055 0.133±0.054  0.34" NS 0.13  Rosewall Adults Seedlings  44 47  1.7 1.8  60 70  0.095±0.036 0.076±0.037  0.130±0.043 0.130±0.042  0.20* 0.57*  Yellow Pt Adults Seedlings  48 47  1.6 1.8  60 70  0.095±0.029 0.074±0.032  0.126±0.042 0.128±0.039  0.22* 0.41*  1.7 1.7  56.6 63.3  0.098 0.086  0.134 0.130  0.25 0^37  Mean Adults seedlings  T a b l e 4.4. W i l c o x o n s i g n e d ranked test for recent bottleneck (Cornuet a n d Luikart 1996) in Acer macrophyllum  populations under the Infinite A l l e l e s M o d e l .  N u m b e r of loci  W i l c o x o n test  with HE e x c e s s Exp  H  E  > Heq  H  < Heq  E  P  Population  P  Nitinat  3.11  0.9609  0.0546  Elk Falls  2.86  0.5781  0.5000  P. Alberni  2.47  0.7187  0.3437  Maple Bay  3.62  0.3711  0.6796  Rosewall Creek  2.79  0.9453  0.0781  Y e l l o w Point  2.99  0.6562  0.4218  Note: Exp = expected number of loci with a heterozygosity excess; H = expected E  Heterozygosity; Heq = heterozygosity expected at mutation drift-equilibrium.  Table 4.5. Genetic diversity statistics for the eight polymorphic isozyme loci for continuous populations (a) and fragmented populations (b). (a)  Hf  H  AAT-1  0.008  0.008  AAT-2  0.279  IDH  Locus  s  F/s  F[  T  FST  -0.002  0.001  0.003  0.272  0.229  0.258  0.038  0.269  0.271  0.626  0. 622  -0.010  6PG-1  0.111  0.108  0. 075  0.098  0.015  6PG-2  0.133  0.130  -0.100  -0.054  0.042  PGI-1  0.020  0.020  -0.020  -0.001  0.019  PGI-2  0.268  0.268  -0.179  -0.186  -0.006  LAP-2  0.215  0.212  0.392  0.402  0.016  Mean  0.170  0.168  0.140  0.149  0.015  0.175  0.171  0.012  (-0.077, 0.480)  (-0.079, 0.486)  SE 95%CI  (-0.005, 0.035)  (b)  Hj  H  AAT-1  0.063  0.063  -0.025  -0.032  -0.007  AAT-2  0.183  0.182  0.517  0.519  0.006  IDH  0.337  0.339  0.595  0. 600  0.010  6PG-1  0.165  0.154  0. 267  0.281  0.045  6PG-2  0.051  0.048  -0.073  0.000  0.068  PGI-1  0.032  0.032  -0.056  -0.002  0.051  PGI-2  0.283  0.283  -0.167  -0.147  0.017  LAP-2  0.270  0.257  0.377  0.421  0.071  Mean  0.174  0.169  0.178  0.204  0.031  0.185  0.179  0.014  Locus  SE 95%CI  s  FJS  (-0.070, 0.507)  FIT  (0.041, 0.518)  FST  (0.010, 0.056)  T a b l e 4.6. P a i r w i s e F T between adult fragmented ( M B Y , R W C Y P T ) a n d S  continuous (NIT, E L F , P A L ) populations of Acer NIT NIT  ELF  PAL  MBY  macrophyllum. RWC YPT  — —  ELF  0.024  PAL  0.009  0.020  MBY  0.031  0.016  0.018  --  RWC  0.004  0.017  0.005  0.013  YPT  0.034  0.029  0.023  0.023  —  — 0.047  Note in parenthesis: Populations abbreviations as in Table 4.2a.  --  T a b l e . 4 . 7 . P e r c e n t a g e of a l l o z y m e diversity retained over 2 5 0 - y e a r period b a s e d on computer simulations B O T T L E S I M (Kuo a n d J a n z e n 2 0 0 3 ) for adult populations of Acer macrophyllum  in fragmented a n d continuous  forests a s s u m i n g 125-year generation length. Populations  N E = 50  A  0  N = 100 E  H  E  AO  H  E  Continuous Nitinat  92.78  94.01  96.15  97.53  Elk Falls  85.88  95.14  88.57  97.23  P. Alberni  92.67  93.71  96.46  98.00  Maple Bay  91.61  95.32  94.95  97.36  Rosewall  91.41  93.77  95.46  97.81  Yellow Pt.  89.60  93.77  96.69  97.08  Fragmented  -130' 52"  -120"  -125"  ®2-"  :  3R7  I' 43'  -130"  -120" km  -125" 0  50  100  Figure 4 . 1 . G e o g r a p h i c locations of bigleaf maple populations s a m p l e d o n V a n c o u v e r Island.  0.45 0.40 c 0.35 O 0.30 '€ 0.25 o Q. 0.20 O 0.15 0.10 0.05 0.00  J l o  o  o  —X  o  o o  cn  p  —k  o  ^  k)  o k>  o  CO  O 4^  o cn  o  o  O  o  co  o  o  o  o  v  k) o  O  cn  o  o  o  05  o  o bo  1  1  1  1  o CD  o CO  o  o  o  o  o  o  o  o  o  o  o  o  6  6  o  o  o  o  o  O  o o o Allele frequency class  o  ro co  CO 4^  4^  cn CD  o CD  o o  09  o o  CD  bo  bo CD  CD —».  o  Figure 4 . 2 . Distribution of allele frequencies for adults (a) a n d s e e d l i n g (b). Filled bars are continuous populations a n d o p e n bars fragmented populations.  w o  c  (0  o  0.30 0.20 0.1 0 0.00 -0.1 0 -0.20  Port A lb e mi  4  5 Ln distance  6 (m)  Figure 4.3 (a-c). Spatial correlograms of coancestry coefficients (p,y) for continuous populations of Acer macrophyllum.  Dashed lines represent upper and lower 95% confidence  limits for pj, under the null hypothesis that genotypes are randomly distributed.  e Maple Bay  Yellow Point  0.30 1 0.20 in w o  0.10 -  (0  0.00  c  o o  -0.10 -0.20 5  6  7  Ln distance (m)  Figure 4.3 (d-f). Spatial correlograms of coancestry coefficients (p/j) for fragmented populations of Acer macrophyllum.  Dashed lines represent upper and lower 95% confidence  limits for p# under the null hypothesis that genotypes are randomly distributed.  Chapter five  GENETIC VARIATION AND POPULATION STRUCTURE IN BIGLEAF MAPLE: A COMPARISON OF ALLOZYME MARKERS AND QUANTITATIVE TRAITS INTRODUCTION  Knowledge of genetic variation and population structure is necessary for understanding and conserving the evolutionary potential of populations (Wright 1951). Patterns of genetic variation can be detected at both among and withinpopulation levels (e.g. Hamrick et al. 1992; Xie and Ying 1996). Levels of genetic variation and degree of genetic control also vary among traits, ages and environments (Mullin et al. 1995; Aitken et al. 1995; W u et al. 1995; Xie and Ying 1996). Langlet (1971) noted that the maintenance of intra-specific variation in natural populations of plants is complex. Patterns of geographic variation result from the joint actions of underlying mechanisms that affect the associations between environmental and genetic heterogeneity, such as different selection pressures, levels of gene flow, and genetic drift (Lindhart and Grant 1996). The use of molecular markers has several limitations in providing information that could be used to define conservation and management strategies (Lynch 1996). This is because the primary aim of conservation genetics is to quantify and maintain the evolutionary potential of a species. For this reason, studies should include an assessment of genetic variation for traits affecting fitness, many of which are polygenic (Petit et al. 2000). Most molecular genetic markers are considered selectively neutral, while the pattern of quantitative trait variation is likely to be driven by environmental factors resulting in different selection pressures in different locations (Petit et al. 2000; Lynch et al. 1999). A  comparison of molecular and quantitative measures of genetic variation allows insights into the different modes of evolution in sub-divided populations. Studies of patterns of genetic differentiation of quantitative traits are not uncommon in forest trees. However, only a fraction of these allow for the estimation of Qsr, the parameter estimating the portion of total quantitative genetic variation due to among-population differences (Spitze 1993). It is expensive to conduct sufficiently large common garden experiments with trees to include both populations and families within populations to obtain among and within-population genetic diversity estimates. A s a result, the availability of such estimates in the literature is biased towards small, short-lived organisms (Ritland 2000). Studies that have reported joint estimates of quantitative (Qsr) and molecular (Fsr) estimates of genetic variation among populations in plants include, menziesii (Rehfeldt 1978; Campbell 1986), Populus  Pseudotsuga  balsamifera  (Riemennschneider et al. 1992), Picea glauca (Li et al. 1992; Jaramillo-Correa et al. 2001), Daphnia obtusa (Spitze 1993; Lynch et al.1999), Clarkia  dudleyana  (Podolsky and Holtsford 1995), Pinus contorta ssp. latifolia (Yang et al. 1996), Populus tremuloides  (Thomas et al. 1997), Quercus petraeae (Kremer et al. 1997),  Cerastium arvense (Quiroga et al. 2002), and Pinus pinaster (Gonzalez-Martinez et al. 2002). Merila and Crnokrak (2002), in their meta-analysis comparing such studies of genetic differentiation at marker loci and quantitative traits, found that the degree of genetic differentiation coding quantitative traits (Q r) typically S  exceeds that of presumably neutral genetic markers (F r)- These results have S  been attributed to the role of differential natural selection among populations in determining the population genetic structure of quantitative traits. In forests of the Pacific Northwest of North America, bigleaf maple {Acer macrophyllum)  is an important component of biodiversity, and a species of growing  economic importance. However, breeding programs have not yet been initiated. C o m m o n garden experiments including provenance trials are being conducted to screen genetic variation in natural populations and to allow selection of the best available genotypes for reforestation or for breeding (Wright 1976). In addition,  provenance research also aims to define the genetic and environmental components of phenotypic variation between trees from different geographic regions (Morgenstern 1997). In this chapter, I use a provenance/progeny common garden experiment to estimate quantitative genetic parameters, and compare genetic differentiation among populations at allozyme loci with quantitative variation.  MATERIALS AND METHODS  In 1995 and 1996, seeds from 14 populations were collected from across the portion of the species range of distribution in British Columbia by the Ministry of Forests (Table 5.1 and Figure 5.1). Nine out of the 14 populations were located on Vancouver Island. Populations selected ranged from 48°22' to 50°21' N latitude, 121°23' to 126°35' W longitude, and 14 to 600 m elevation (Table 5.1).  QUANTITATIVE TRAITS  Seeds from 148 open-pollinated families from 14 provenances in total were sown in 614 Styroblocks® in mixture of peat and vermiculite in early December 1995, and maintained at 5.2°C minimum and 10°C maximum temperature in a greenhouse. Regular misting four times per day continued throughout the germination period. Germination started in mid-January, 1996. Germination of all provenances was nearly complete by the end of February. Seedlings were fertilized in February and March and moved to a cooler greenhouse for acclimatization on March 18. Overall, only about 45.3% of the seedlings germinated. Germination rates ranged from 8-86% among population and 0-100% among families. However there was no clear geographic pattern in germination rate.  The common garden test was planted from April 29 to May 1, 1997 at Surrey, British Columbia. The experiment was laid out in a split-plot design in four randomized blocks with provenances as main plots, and five-tree family rows as subplots. A total of 2925 seedlings were planted. Maintenance of the experiment to ensure high survival and good seedling growth included weeding, watering and fencing against deer.  Data collection Height growth was measured at the end of the second year in the field (1998), third (1999) and fourth year (2000). Diameter was measured for all provenances at the end of the growing season in year three (1999). Phenological data (bud flush) was monitored and recorded two to three times a week from March 2002 to mid-May 2002. Julian bud flush data was defined by when the first unfolded leaf was observed. With the exception of bud flush, data measurements of height and diameter were made available to me from the B C Ministry of Forests.  Analysis Analysis of variance (ANOVA) was conducted using P R O C G L M ( S A S Institute Inc. 1990) for height, diameter and bud flush traits using the following general linear models; Y |= n+ ijk  Bi + Pj + PBj, + F ( P ) , + F ( P ) B k(l  k(ij)  +8IP)  Where: Y = measurement of seedling / from family k in provenance j in block / u, = overall mean Bj = effect of block i Pj = effect of provenance j PBjj = interaction effect of block with provenance F(P) (j) = effect of family within provenance k  F(P)B jj) = interaction effect of block with family within provenance k(  e = experimental error  (1)  All effects in the model were assumed to be random. Variance components for all traits were estimated using the P R O C V A R C O M P ( M E T H O D = R E M L ) procedure ( S A S Institute Inc. 1990). In addition, the G L M procedure type III sums of squares ( S A S , 1990) was used to estimate the proper F-test for family and provenance effects with the null hypotheses (Ho): No family or provenance effects. I used provenance-by-block interaction (PB) as the error term to test provenance effect and family within provenance-by-block interaction (F(P)B) as the error term to test for family effect. The amount of genetic variation in growth traits and bud flush was quantified by estimating the family variances and testing their significance (P < 0.05). Individual tree and family heritabilities were estimated as follows:  Individual heritability:  h = 3o  Family heritability:  h f = O F ( P / 0" F<P) + O B F<P) lb + a ln  2  2  2 F ( P )  2  / o  2 F ( P )  +o  2  2  (P) + O E  (2)  2  B X F  (3)  2  2  E  X  All variables are defined above with the exception of b and n which are number of blocks and number of trees in plot respectively. For estimating individual heritability, the additive genetic variance was estimated as three times the family variance (instead of four times the family variance) as suitable for half-sib progenies. It is assumed that Acer macrophyllum  open-pollinated progenies are  more closely related than half-sibs in view of the high inbreeding in this species and the relatively low number of effective pollen parents (Iddrisu and Ritland 2004). The standard errors of heritability estimates were calculated following Dickerson (1969). To assess the associations among traits for both growth and bud phenology (bud flush), genetic correlations (r ) between pairs of traits were calculated g  following Falconer (1989) as follows: r = Cov g  F(x  ,  y)  / (o  2 F x  o  2 F y  )  where Cov (x,y) is the family covariance between traits x and y, and o F  (4)  1 / 2  2 F x  and o  are their corresponding family variances. C o v , y ) was calculated using the F(x  following relationship:  2 F y  Cov  , = (a x y) - a 2  F(x  y)  F (  2 F x  +  - a  2 F y  ) /2  (5)  Phenotypic correlations for each pair of traits, as well as correlations between traits and geographic and climatic variables were estimated as Pearson's product moment correlations using the P R O C C O R R procedure ( S A S Institute Inc. 1990). Climatic data were obtained using a method developed by Hamann and W a n g (2005).  Wright's (1951) F-statistics provide a useful measure of the level of population genetic structure at neutral marker loci by quantifying the proportion of total allelic variation found within versus among populations. Similarly, population differentiation for quantitative traits can be estimated using QST (Spitze 1993) which is analogous to the F T estimate for marker loci. It is estimated a s : S  QST=  o  2  /(2a Gw a GB) 2  G B  +  (6)  2  where O G B is the among population component of variance and o 2  2 G  w  the within  population component of variance. The neutral expectation for Q S T is equivalent to F s r f o r selectively neutral genetic markers (Lande 1992).  ISOZYME VARIATION  Vegetative buds were collected in February 2001 from two of the four blocks (1460 trees) from all 14 populations in the common garden experiment. Buds were stored at -80°C until analyzed by isozyme electrophoresis. The trees sampled from each population for isozyme analysis were the s a m e as those used for quantitative genetic analysis. Electrophoresis buffer systems and loci a s s a y e d are those described in chapter three.  Genetic data analyses were performed using B I O S Y S - 2 , a modified version of the BIOSYS-1 program (Swofford and Selander 1981). The following parameters were estimated: allele frequencies, mean number of alleles per locus (A), percent of loci that were polymorphic (%P) (with the most common allele having a frequency of 9 9 % or less), observed heterozygosity (Ho) and expected heterozygosity (H = 1- Ip, , where p, is the frequency of the ith allele). To 2  E  investigate the extent of population structuring and differentiation, Fsr (Wright 1965) was estimated for individual loci across the 14 populations.  RESULTS  Quantitative traits Provenances differed significantly in growth traits (p<0.001). All growth traits had similar patterns of variation at all ages. Provenance means for height, diameter and bud flush are presented in Table 5.1. The highest growth rates were observed in trees from Hope, Squamish, Port Alberni and Qualicum. The difference between the most productive provenance (Hope) and the least productive (Woss) in terms of height was about 36%.  The first bud flush was recorded on Julian day 105 day. All buds in the trial completely flushed by the 1 2 9 day. In general, flushing was variable among trees th  within provenances. W o s s , Sayward and Owl, the three northernmost provenances (Table 5.1) flushed first and Metchosin, the southernmost provenance, flushed last, this latitudinal trend was weak.  For height growth and bud flush, block, population and family within population effects were all highly significant (p<0.001) (Table 5.2). The family variance for height increase slightly from 1% of the total variance for height-2 to 2.3% for height-4 (Table 5.3).  Estimates of individual and family heritability for height were relatively low, ranging from 0.15 to 0.18. Family heritability ranged from 0.37 for height-2 to 0.40 for height-4 (Table 5.3). Timing of bud flush had the highest heritability estimate both for individuals (0.21) and families (0.91) (Table 5.3). Estimates of QST values varied from 0.12 for bud flush to 0.26 for height-2 and averaged 0.17. For height, QST values seemed to decrease with age (Table 5.3).  There were strong genetic correlations among heights at all ages (Table 5.4). Diameter was also strongly correlated with height growth. Timing of bud flush was weakly and negatively correlated with all growth traits. Phenotypic correlations were strongly correlated among growth traits at all ages and significant at P = 0.01 (Table 5.4). Height growth in all years was correlated with degree days above 5°C and bud flush was mainly correlated with continentality (Table 5.5).  Molecular genetic variability Eight of the 10 loci analysed were polymorphic in at least one population. In all populations, G D H and LAP-1 were monomorphic. The percent of loci that were polymorphic (%P) varied among populations between 3 0 % and 6 0 % , averaging 43.5%. The mean number of alleles per locus (A) ranged from 1.3 to 1.5, averaging 1.37 (Table 5.6). The expected heterozygosity within populations ranged from 0.071 to 0.134 and averaged 0.127 across the 14 populations studied. The proportion of inter-population genetic differentiation among populations ( F T ) indicated that the vast majority of total variation resided within populations, S  with approximately 9% of the total variation occurring among populations (Table 5.7). Locus specific estimates ranged from 0.0653 for 6 P G D - 1 to 0.2243 for L A P - 2 .  DISCUSSION QUANTITATIVE TRAITS  The provenances of Acer macrophyllum  sampled did not exhibit high  germination rate under nursery conditions, with an overall mean of 4 5 . 3 % and a range from 0% to 86% among provenances. The low germination rate, assuming seeds were healthy and well handled, may suggest that growth conditions in the nursery were not optimal and under such conditions seedlings may not fully express genetic variation at an early age (Bongarten and Hanover 1985). In addition, seedling growth can suffer following transplanting from the nursery, which could also impact expression of genetic differences in early stages (Namkoong and Conkle 1976; Camussi et al. 1995). Provenances like Hope and Chilliwack that showed higher germination rates in the nursery (greenhouse) also showed higher height and diameter growth (Table 5.1). Genetic variation in growth traits for bigleaf maple seedlings both among and within provenances was detected at an early age. Both provenance and family variance components were significant for bud flush and height at all ages but not for diameter. This pattern is similar to that reported for young lodgepole pine (Wu et al. 1995). Although provenance, block and family within population effects were significant for height, the largest variance component was block by family within provenance interaction (Table 5.3). The narrow-sense heritabilties for individual (h ) and family (h ) were 2  2  f  moderate and remained stable with age for height (Table 5.3). The individual heritability estimate for height growth in bigleaf maple in this study is on the low side compared with those reported for forest trees by Cornelius (1994). Franklin (1979) found diminishing heritability estimates for height growth with age, as competition increased with canopy closure. Other studies, however, have reported different age trends of individual heritability for height growth. For instance, Cotterill and Dean (1988) observed an increase in individual heritability for radiata pine (Pinus radiate) following thinning, followed by a decrease. On the contrary, Xie  and Ying (1996) reported a decrease followed by an increase after thinning a lodgepole pine (Pinus contorta) early selection test. It is therefore difficult to find a consistent pattern for heritabilities for growth traits with age or silvicultural treatment. Genetic parameters for quantitative traits need to be interpreted with caution, as they are applicable only to the defined base population, reference unit of selection and specific environments where studies are performed (Zobel 1984).  Bud flush Several studies of bud flush phenology have reported that it is under moderate to strong genetic control (reviewed in Howe et al. 2003). For example, Howe et al. (2000) and Bradshaw and Stettler (1995) reported that heritability for bud flush was moderate for F hybrid poplar. Other studies indicate that bud flush 2  is under strong genetic control in Douglas-fir (Aitken and A d a m s 1997), in Populus trichocarpa  (Thomas et al. 1997) as well as in other angiosperm and coniferous  tree species (Bongarten and Hanover 1985; Chuine et al. 2000) than in bigleaf maple. In this study, bud burst phenology for bigleaf maple varied significantly among families with moderate estimates for heritability. Notwithstanding, geographically based patterns of genetic variation have been observed for bud flush (e.g., Howe et al. 2000). For some species, trees from northern locations and high elevations will tend to flush earlier than those from southern locations, especially in common garden tests, because they have been exposed to shorter frost free seasons in their native environment, leading to selection of genotypes that have either a lower chilling requirements to break bud dormancy, or a lower heat sum or threshold temperature to initiate growth therefore begin growing earlier in a common garden than populations from milder climates in the spring (Farmer 1993). For example, northern provenances (Owl, Sayward and W o s s ) flushed slightly earlier than the southern provenances on average. If chilling requirements are met, bud flush is mainly in response to heat accumulation in the spring (Lavender 1981). In this study, it is presumed that, chilling requirements were met  and thus bud flush timing differences among families may reflect different heat sums or threshold temperature required for bud flush (Li and A d a m s 1993). This result corroborates the findings of Perry and W u (1960) from another maple {Acer rubrum), in which buds from northern provenances flushed earlier than southern provenances or at the same time, depending on the temperature. The test site (Surrey, B C ) experienced mild winter temperatures, and according to Hunter and Lechowitz (1992), under such natural conditions, the lack of chilling temperature will be less important than the lack of forcing temperature as an agent to speed up bud flush.  Genetic correlations There were strong genetic correlations observed among growth traits (Table 5.4) . These high genetic and phenotypic correlations could be due to either pleiotropy or maternal effects (contribution of the maternal parent to the offspring phenotype via some mechanism other than the transmission of genes, e.g. seed size). The presence of maternal effects can bias estimates of seedling genetic variance, heritability and genetic correlations, especially for height (Lambeth 1980). Hence, it would be useful to study growth patterns of bigleaf maple seedlings over more growing seasons, to investigate the extent of maternal effects and age trend in genetic control of growth traits (Lambeth 1980). High age-to-age genetic correlations between growth traits detected in this study suggest that selection for fast growing trees can be done at the early ages. However the interval between ages two, three and four is too short a time to realize significant changes in family ranks with tree age for the tested families. Therefore, caution should be taken when interpreting such genetic correlations, since they might be lower over long intervals (Rweyengeza et al. 2003).  Correlations with climatic variables Correlations between geographic and climatic variable were moderate (Table 5.5) which may reflect the capacity of bigleaf maple to adapt to varying  environmental conditions (Jaramillo-Correa et al. 2001). Height growth was significantly correlated with mean annual temperature, and degree days above 5°C (DD5) and bud flush correlated with temperature differential (TD). Since differentiation in quantitative traits (Qsf- see below) is observed for these traits, according to Jaramillo-Correa et al. (2001) these quantitative traits may be under differential selection in response to regional differences in climatic factors. For instance, the mean annual temperature (MAT) and degree days above 5°C (DD5) for Owl average 4.79°C and 891 respectively whiles that in Hope was 10.41°C and 2022. A s one would expect, trees from the milder climate (Hope) exhibited higher growth rates than Owl (Table 5.1).  F  S T  vs  QST  Over the past few years, joint estimates of differentiation for quantitative traits and for molecular marker loci have shown two main patterns. S o m e species, such as Daphnia obtusa (Lynch et al. 1999) and Arabidopsis  thaliana (Kuittinen et al.  1997), have a quantitative population structure essentially identical to that for molecular markers suggesting genetic structure for both quantitative and genetic markers is determined by drift and gene flow, whereas other plant species such as Quercus petraeae (Kremer et al. 1997) or Clarkia dudleyana  (Podolsky and  Holtsford 1995) have highly divergent populations to quantitative traits. The latter pattern is found in coniferous species. Y a n g et al. (1996) found differences between allozyme  (F T= S  0.019) and quantitative genetic differentiation for specific  gravity (Qsr = 0.133), stem diameter (Qsr = 0.166), stem height (Qsr = 0.195) and branch length (Qsr= 0.161) in Pinus contorta.  In this study, estimates of Qsr for  five quantitative traits varied from (Qsr = 0.12) in bud flush, diameter, and height-4 to (Qsr = 0.26) for height-2 (Table 5.3). By comparing estimates of differentiation from quantitative traits (QST) and isozymes (Fsr) we can examine whether evolutionary processes involved in quantitative and isozyme variation in Acer macrophyllum  are similar or not. In  meta-analyses of published results that compared population structure in markers  with that of quantitative traits, Mckay and Latta (2002) and Merila and Crnorkak (2001) found that mean QST is typically larger than but poorly correlated with mean F  across 29 species of plants, vertebrates and invertebrates. Spitze (1993)  S T  suggested three possible outcomes from the comparison of F T and Q f. 1) If QST S  S  > F T, the implication is that natural selection rather than genetic drift alone must S  have been involved in shaping or favouring different phenotypes in different populations; 2) if FST = QST, then genetic drift alone could be responsible in the population divergence and this could be evident in smaller populations; and 3) if QST < F T, then it is most likely that natural selection is convergent in that the same S  phenotypes are favoured in different populations. Comparison of average estimates of Q  s r  and F T in this study (QST = 0.17 > F T = 0.09) according to S  S  Merila and Crnokrak (2001) provides evidence of involvement of differential selection in shaping phenotypic variation in different populations. Growth traits such as height have been reported to be under differential selection in Pinus contorta (Yang et al. 1999) and Picea glauca (Jaramillo-Correa et al. 2001) because individual trees must grow rapidly to escape suppression from competition from neighbouring trees yet have a sufficiently conservative growth pattern to avoid frost injury, the risk of which varies locally. Acer macrophyllum  is  an early successional species, relatively shade tolerant and growing across a wide range of sites and climatic conditions. Differential adaptation to regional and local patterns of precipitation, temperature and other climatic variables s e e m s to be the explanation for the divergence in these traits. Notwithstanding, it is worth noting that, geographic and environmental scale of sampling will affect the magnitude of QST-  S o m e studies using isozyme markers have reported greater differences between QST and F s r t h a n the current study (e.g., Prout and Barker 1993; Spitze 1993; Long and Singh 1995; Y a n g et al. 1996; and Waldmann and Anderson 1998). For several other tree species, Q S T values are relatively low for timing of bud flush but high for growth cessation or timing of bud set (Howe et al. 2003).  Merila and Crnorkak (2001) and Latta and Mckay (2002) have reviewed the basic assumption underlying comparative studies of population genetic structure which included assumption of neutrality of allozymes in these comparative studies. It is worth noting that, in some instances genetic variances within populations have been overestimated because of non-genetic (maternal) effects which could lead to a downward bias of Q r (Waldmann and Anderson 1998). One way to resolve this, S  as proposed by Merila and Crnorkak (2001), would be to compare the consistency of QST estimates and direct measures of selection in different populations for different traits.  Table 5.1. Bigleaf maple populations sampled for provenance trials, and least square means for growth and bud flush traits (with standard errors in parenthesis). Population  LAT  Metchosin  48.36  123.55  40  135.5 (0.04)  184.9 (0.05)  231.4 (0.09)  32.5 (0.01)  122.5 (0.05)  Maple Bay  48.83  123.63  14  132.2 (0.03)  179.5 (0.04)  240.6 (0.06  30.7 (0.01)  120.8 (0.120)  Chilliwack  49.15  122.00  142  135.9 (0.04)  181.6 (0.04)  236.6 (0.05)  28.6 (0.01)  121.7 (0.10)  P.AIberni  49.26  124.85  15  139.8 (0.03)  192.2 (0.05)  263.2 (0.05)  34.2 (0.01)  121.9 (0.07)  Qualicum  49.33  124.36  80  138.2 (0.03)  194.5 (0.04)  243.9 (0.05)  32.7 (0.01)  120.2 (0.06)  Hope  49.36  123.38  90  160.2 (0.03)  216.8 (0.04)  247.5 (0.04)  33.5 (0.01)  120.6 (0.20)  Courtenay  49.66  125.03  70  126.8 (0.03)  174.9 (0.05)  225.8 (0.05)  30.1 (0.01)  120.9 (0.04)  Gold River  49.75  124.73  200  112.2 (0.03)  162.1 (0.05)  201.0 (0.05)  31.4 (0.01)  121.5 (0.8)  Squamish  49.78  123.13  50  143.4 (0.03)  193.2 (0.06)  246.3 (0.05)  32.3 (0.01)  120.7 (0.05)  Lang Bay  49.78  124.36  25  136.3 (0.03)  186.6 (0.04)  236.7 (0.05)  30.2 (0.01)  120.3 (0.07)  Cowichan  49.81  124.21  200  116.0 (0.03)  166.8 (0.05)  233.8 (0.04)  31.9 (0.01)  121.0 (0.20)  Woss  50.21  126.58  160  95.8 (0.03)  147.6 (0.05)  235.5 (0.06)  25.1 (0.01)  120.2 (0.03)  Sayward  50.31  125.93  50  131.5 (0.04)  180.0 (0.04)  237.9 (0.06)  33.2 (0.01)  119.6 (0.05)  Owl  50.35  124.73  118.9 (0.04)  171.8 (0.04  213.0 (0.05)  28.6 (0.01)  118.5 (0.05)  LONG  ELEV  600  HT-2  HT-3  HT-4  DIA  BF  Note: LAT = latitude (°N), L O N G = longitude f W ) , ELEV = elevation (m), HT-2 = second year height (cm), HT-3 = third year height (cm), HT-4 = forth year height (cm), DIA = third year diameter (cm), B F = bud flush (Julian days).  Table 5.2. A N O V A results for F approximations for the hypothesis of no family or provenance effect.  Trait  Source  DF  SS  MS  HT-2  B P BxP F(P) BxF(P)  3 13 39 134 386  58.24 62.78 53.11 87.42 160.12  19.41 4.83 1.36 0.65 0.41  154.85 38.52 10.86 5.2 3.31  0.0001 0.0011 0.0001 0.0004 0.0001  HT-3  B P BxP F(P) BxF(P)  3 13 39 134 386  112.85 71.96 90.60 159.79 291.72  37.62 5.54 2.32 1.19 0.76  210.32 30.95 12.99 6.67 4.23  0.0001 0.0183 0.0001 0.0004 0.0001  HT-4  B P BxP F(P) BxF(P)  3 13 39 134 383  250.22 77.85 120.45 169.69 466.50  83.41 5.99 3.09 1.27 1.22  283.01 20.32 10.48 4.30 4.13  0.0001 0.0455 0.0001 0.0383 0.0001  DIA  B P BxP F(P) BxF(P)  3 13 39 134 386  2.30 1.59 2.69 4.36 7.04  0.77 0.12 0.07 0.03 0.02  126.01 20.05 11.33 5.35 3.00  0.0001 0.0841 0.0001 0.0001 0.0001  BF  B P BxP F(P) BxF(P)  3 13 39 134 386  822.71 12625.82 15395.30 17591.27 46934.67  274.24 971.22 394.75 131.28 121.59  7.61 26.96 10.96 3.64 3.38  0.0001 0.0150 0.0001 0.0285 0.0001  F  Pr> F  Note: DF = degree of freedom, S S = sum of squares, MS = mean sum of squares, F = F-value approximation, Pr > F= probability of greater F-values occurring by chance.  Table 5.3. Components of variance, individual heritabilities (h j), family heritabilities (h ) and 2  2  f  population differentiation (QST) among growth and bud flush traits.  Trait  B  P  HT-2 HT-3 HT-4 DIA BF  0.028 0.054 0.093 0.001 0.032  0.022 0.021 0.019 0.004 0.086  +  +:  BxP  F(P)  BxF(P)  E  h  0.019 0.031 0.036 0.001 0.058  0.010 0.019 0.023 0.005 0.110  0.075 0.144 0.165 0.004 0.210  0.127 0.184 0.190 0.001 0.780  0.15(0.06) 0.17(0.02) 0.18(0.05)  h  2  -  0.29 (0.01)  Not significant, all other variables are significant for all effects at the P<0.001.  2 f  0.37 (0.04) 0.38 (0.01) 0.40 (0.01)  -  0.91 (0.02)  QST  0.26 0.16 0.12 0.12 0.12  Table 5.4. Genetic correlations (above diagonal) and family phenotypic correlations (below diagonal) between seedling traits for bigleaf maple provenances in British Columbia.  Trait  HT-2  HT-2 HT-3 HT-4 DIA BF  0.95 0.89 0.69 -0.32  HT-3  HT-4  0.99  0.94 0.97  0.93 0.70 -0.41  0.64 -0.39  DIA  BF  0.78 0.79 0.75  -0.19 -0.22 -0.15 -0.11  -0.20  Table 5.5. Correlation coefficients between quantitative traits and climatic variables based on 14 provenance means. HT-2  LAT ELEV MAT TD MAP AHM DD5 + +  + +  -0.47 -0.44 0.58* -0.24 -0.15 0.23 0.62*  HT-3  -0.42 -0.38 0.51 -0.25 -0.15 0.21 0.54*  HT-4  DIA  -0.22 -0.46 0.32 -0.10 0.20 -0.10 0.33  -0.35 -0.42 0.45 -0.27 -0.23 0.28 0.42  BF  0.45 -0.05 -0.15 0.55* 0.25 -0.36 -0.13  Note: * Significant at P<0.05 after sequential Bonferroni adjustment (Rice 1989). ^Abbreviations as in table 5.1. MAT= mean annual temperature, TD = temperature differential, MAP = mean annual precipitation, AHM = annual heat: moisture index, DD5 = degree days above 5°C.  Table 5.6. Genetic diversity estimates for 14 juvenile populations of Acer Population  A  macrophyllum.  %P  Ho  H  E  Courtenay Hope Sayward Squamish Cowichan Metchosin Maple Mt Owl Woss Pt. Alberni Qualicum Lang B a y Chilliwack Gold River  1.43 1.43 1.31 1.37 1.37 1.37 1.43 1.37 1.50 1.31 1.37 1.31 1.31 1.37  50 50 40 40 50 50 60 40 50 30 40 30 30 50  0.103 0.101 0.090 0.090 0.090 0.080 0.110 0.103 0.096 0.112 0.092 0.079 0.109 0.121  0.126 0.134 0.127 0.128 0.119 0.133 0.111 0.123 0.138 0.127 0.119 0.119 0.148 0.137  Mean  1.37  43.5  0.098  0.127  Table 5.7. Estimates of Wright's F-statistics for eight polymorphic loci in British Columbia bigleaf maple populations. Locus  AAT-1  F  F  IS  0.721  IT  FST  0.747  0.091  AAT-2  0.050  0.124  0.106  IDH  0.103  0.101  0.106  6PG-1  0.314  0.370  0.085  6PG-2  0.314  0.391  0.126  PGI-1  0.110  0.203  0.108  PGI-2  -0.123  0.114  0.146  LAP-2  0.291  0.322  0.068  Mean  0.222  0.301  0.090  -1 20"  -125'  -130' 52'  52'  Owl  50"  50'  1-  Gol  Ccu PJO.ua "  .  Hope  43'  -130'-  -120'  -125' 0  50 100  Figure 5.1. Locations of sampled populations of bigleaf maple provenance trials.  Chapter Six  CONCLUSIONS  In British C o l u m b i a there is a trend towards greater understanding and utilization of a n g i o s p e r m trees, b e c a u s e of their important contributions to the diversity a n d sustainability of British C o l u m b i a ' s forest e c o s y s t e m s a s well a s the value of their w o o d . R e s p o n s i b l e m a n a g e m e n t and utilization of this hardwood resource could provide e m p l o y m e n t opportunities in forestry a n d v a l u e - a d d e d sectors. In addition, these trees are a desirable e c o s y s t e m c o m p o n e n t , adding to the structural a n d s p e c i e s diversity of British C o l u m b i a ' s forests. Forest fragmentation is a growing problem b e c a u s e of h u m a n population growth a n d land u s e conversion of forests. Therefore, what w e e n c o u n t e r today in s o m e a r e a s are small patches of original habitat for s p e c i e s restoration a n d conservation of genetic diversity. In this s c e n a r i o , a thorough understanding of genetic p r o c e s s e s affecting g e n e s , individuals and populations, a n d thus affecting the persistence of this s p e c i e s in modified l a n d s c a p e s , is e s s e n t i a l for designing s o u n d conservation practices. M y study h a s contributed towards our understanding of s o m e important c o m p o n e n t s of genetics of bigleaf m a p l e . B y d o c u m e n t i n g genetic variation and population structure, both at the quantitative and molecular levels, investigating the mating s y s t e m , a n d c o m p a r i n g genetic diversity a n d genetic p r o c e s s e s in continuous v e r s u s fragmented populations of bigleaf m a p l e , I have provided information n e e d e d to m a n a g e a n d c o n s e r v e this species.  Major findings In chapter three I s h o w e d that natural populations of bigleaf m a p l e harbour moderate levels of genetic variation. H o w e v e r , at the northern range of the s p e c i e s distribution (Jericho and F r a s e r populations), p o l y m o r p h i s m w a s high yet e x p e c t e d hetrozygosities were low c o m p a r e d to more southern populations. In addition there w a s no e v i d e n c e of deviation from r a n d o m mating in northern populations, in contrast to populations from the southern portion of the range, which had substantial inbreeding in three out of the six populations.  Inbreeding  in bigleaf maple m a y result from g e i t o n o g a m o u s pollinations by b u m b l e b e e s (Bombus  spp), from assortative mating, or from mating a m o n g relatives. In  addition, a s pollination is mainly by insects, the m o v e m e n t of pollinators a m o n g adjacent flowers within a crown or between adjacent c r o w n s of related neighbours would a l s o c a u s e inbreeding or selfing. T h e low heterozygosity, however, m a y reflect overall low genetic variation at the s p e c i e s northern range d u e to genetic drift or founder effects during postglacial recolonization. A c r o s s the s a m p l e d range, populations are only w e a k l y differentiated, s u g g e s t i n g extensive g e n e flow or recent d i v e r g e n c e from a c o m m o n ancestral population. O n e alternate hypothesis to explain the low differentiation a m o n g populations m a y be the e c o l o g i c a l similarity between most of the sites s a m p l e d . T h i s is supported by the non-significant correlation between g e o g r a p h i c a n d genetic distances. A n a n a l y s i s of mating s y s t e m found that bigleaf maple populations are predominantly outcrossing with no e v i d e n c e of biparental inbreeding. T h i s result w a s s o m e w h a t surprising s i n c e most populations have significant levels of inbreeding ( F s > 0 ) . H o w e v e r , this estimate of o u t c r o s s i n g rates m a y be b i a s e d t  upwards in view of the fact I u s e d entirely germinated or filled s e e d s w h i c h probably did not a c c o u n t for embryonic lethals d u e to selfing. A n o t h e r interesting finding is the e v i d e n c e of few pollen donors per s e e d parent, yet the m a i n t e n a n c e of high outcrossing rates.  G e n e t i c study of the effects of fragmentation o n plant s p e c i e s s o far have s h o w n that forest fragmentation c a n significantly affect population genetic p r o c e s s e s . R e s u l t s from my study s u g g e s t that both s e e d l i n g a n d adult cohorts in six populations p o s s e s s similar levels of genetic variation r e g a r d l e s s of whether population habitats were classified a s fragmented or continuous. T h i s finding s u g g e s t s extensive g e n e flow a m o n g populations of bigleaf m a p l e . Furthermore, the m a i n t e n a n c e of genetic variation in fragmented populations could be attributed to the fact that there have not b e e n a sufficient number of generations s i n c e fragmentation to generate a detectable loss of diversity d u e to genetic drift a n d inbreeding. T h e most important finding, however, is the e v i d e n c e of spatial structuring of g e n o t y p e s within all three fragmented populations a s well a s one continuous population. I attribute the clumping of genetically similar individuals mainly to limited s e e d d i s p e r s a l resulting in individuals in clusters being more related than e x p e c t e d by c h a n c e . In this study, spatial genetic structure a p p e a r e d to be affected by s p e c i e s density. Coincidentally, the two populations that did not s h o w spatial genetic structure s e e m to have higher population density than the four populations that s h o w e d spatial genetic structure. G e n e t i c variation in s e e d l i n g growth a n d bud p h e n o l o g y w a s a l s o detected both a m o n g populations, a n d a m o n g families within populations. T h e substantial within-population variation o b s e r v e d in this study, c o u p l e d with the moderate heritabilities a n d moderate genetic correlations a m o n g growth traits a n d bud flush, s u g g e s t a n opportunity for genetic improvement and early selection for these traits. In addition, the significant correlation between quantitative traits a n d climatic variables in this study s e e m s to s u g g e s t that bigleaf maple h a s a d a p t e d to varying environmental conditions, with natural selection favouring different p h e n o t y p e s in different environments.  Recommendations  T h e r e is no doubt that m a n a g e m e n t of fragmented populations of plant s p e c i e s has b e c o m e a n important element of biological c o n s e r v a t i o n , a n d this i s s u e will continue to grow. M a n y of the plant s p e c i e s that are currently r e c o g n i s e d a s threatened are restricted to small habitat fragments a n d in situ conservation of large contiguous populations within a relatively pristine environment is no longer feasible. But the g o o d n e w s with bigleaf maple is that historic levels of genetic variation have thus far persisted a n d it d o e s not a p p e a r that the s p e c i e s is in n e e d of immediate conservation attention. Having s a i d this, integrating our results with other findings, o n e i s s u e that r e m a i n s contentious is whether genetic p r o c e s s e s , primarily genetic erosion a n d inbreeding, actually play a significant role in reducing the viability of small fragmented populations c o m p a r e d to the risk of habitat l o s s a n d a s s o c i a t e d d e m o g r a p h i c factors. Future studies that e x a m i n e the effects of fragmentation o n plant populations should s e e k a s t a n d a r d i z e d a p p r o a c h to e x a m i n e habitat subdivision effects. B a s e d on my findings of how fragmentation affects genetic variation a n d spatial genetic structure, mating s y s t e m , a n d genetic variation at both the quantitative a n d molecular levels, the following guidelines are r e c o m m e n d e d :  1. W h e r e e c o n o m i c a l l y feasible, c o m p a r e original un-fragmented (continuous) populations a n d fragmented populations, a s fragmentation is a population level p r o c e s s a n d not a n individual b a s e d o n e . If original habitats d o not exist, a n a l y z e a n u m b e r of sites or fragments from s m a l l e s t a n d more isolated to largest l e s s isolated. Ideally, o n e should s a m p l e a sufficient n u m b e r of sites to have the statistical power to separate out the effect of s i z e a n d isolation.  2.  R e s e a r c h e r s should f o c u s on critical a s p e c t s of the biology of the plants to be studied a s they c a n provide e v i d e n c e of how surviving individuals are  p a s s i n g o n their g e n e s to the next generation or the potential for s e e d and pollen d i s p e r s a l in a m o n g fragments.  3.  G e n e t i c diversity m e a s u r e s should be carefully s t a n d a r d i z e d b e c a u s e these m e a s u r e s are very sensitive to s a m p l e s i z e s . F r o m a n analytical standpoint, n e w tools are n e e d e d to detect c h a n g e s e s p e c i a l l y in mating patterns a n d d i s p e r s a l rates of plant populations in h u m a n d o m i n a t e d l a n d s c a p e s that would otherwise g o undetected. Hypervariable c o d o m i n a n t markers s u c h a s microsatellites are highly r e c o m m e n d e d to s h e d more light on the total genetic structure a n d variability of bigleaf m a p l e .  4. 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G . , a n d B r o w n , A . H . D . 1999. Paternal bottlenecks in fragmented populations of the e n d a n g e r e d g r a s s l a n d daisy Rutidosis leptorryhynchoides. G e n e t i c a l R e s e a r c h , 7 3 : 111-117.  Y o u n g , A . G . , a n d B o y l e , T . J . 2 0 0 0 . Forest fragmentation. In: F o r e s t C o n s e r v a t i o n G e n e t i c s : Principles and Practice. Y o u n g , A . , B o s h i e r . D. and B o y l e , T. (eds). C S I R O , Australia, pp. 123-134.  Z a s a d a , J . C . , T a p p e i n e r II, J . C . , and M a x , T . A . 1990. Viability of bigleaf maple s e e d s after storage. W e s t e r n J o u r n a l of A p p l i e d Forestry, 5: 5 2 - 5 5 . Z o b e l , B., a n d Talbert, J 1984. A p p l i e d Forest tree Improvement. J o h n W i l e y and S o n s . N e w York. 5 0 5 pp.  Appendix I Enzyme, buffer systems and recipes for histochemical staining solutions.  Enzyme  Aspirate Aminotransferase (AAT)  6Phosphogluonate dehydrogenase (6-PGD)  Isocitric dehydrogenase (IDH)  Phosphoglucose isomerase (PGI)  #of loci  Gel buffer  2  Sodium Borate (Ridgeway)  2  Sodium Borate (Ridgeway)  1  Sodium Borate (Ridgeway)  Morpholine 2  Stain components 50 ml 0.2 M Tris-HCL pH 8.0 1 mg Pyridoxal 5-phosphate 200 mg L-Aspartic acid 100 mg Ketoglutaric acid 200 mg Fast Blue B B salt 50 ml 0.2 M Tris-HCI pH 8.0 10 mg Phosphogluconic acid 1 ml N A D P 1 ml MTT 1 ml P M S 50 ml 0.2 M Tris-HCI pH 8.0 100 ml DL-lsocitric acid 1 ml N A D P 1 ml MTT 1 ml P M S 50 ml 0.2 M Tris-HCI pH 8.0 25 mg Fructose-6-phosphate 1 ml N A D P 1 ml MTT 1 ml P M S 1 ml MgCI 2  Leucine Aminopeptidase  2  Morpholine  1  Morpholine  (LAP) Glutamete Dehydrogenase (GDH)  50 ml Aminopeptidase buffer pH 6.0 0.4% L-Leucine 30 mg B-naphtylamide 20 mg Black K salt 50 ml 0.1 M Tris-HCI pH 8.0 400 mg Glutamic acid 3 ml N A D P 3 ml MTT 3 ml P M S  Appendix  II.  Allele f r e q u e n c y distribution of ten loci of bigleaf m a p l e p r o v e n a n c e trials.  Locus  POP1  AAT-1 (N)  POP2  POP3  POP4  POP5  POP6  POP7  1 2  89 0.719 0.281  63 0.849 0.151  48 0.882 0.118  79 0.715 0.285  85 0.235 0.765  82 0.634 0.366  67 0.743 0.257  1 2  88 1.000 0.000  62 1.000 0.000  47 1.000 0.000  80 1.000 0.000  85 1.000 0.000  86 1.000 0.000  61 1.000 0.000  1 2  96 0.708 0.292  72 0.861 0.139  51 0.686 0.314  70 0.700 0.300  79 0.184 0.816  84 0.673 0.327  64 0.706 0.294  1 2  90 0.822 0.178  73 0.747 0.253  46 0.685 0.315  78 0.763 0.237  88 0.42 0.58  80 0.587 0.412  59 0.795 0.205  1 2  92 0.891 0.109  70 0.786 0.214  45 1.000 0.000  78 0.904 0.096  91 1.000 0.000  87 1.000 0.000  60 1.000 0.000  1 2  88 1.000 0.000  72 1.000 0.000  50 1.000 0.000  80 1.000 0.000  91 0.835 0.165  88 0.966 0.034  60 0.975 0.025  1 2  96 1.000 0.000  72 1.000 0.000  51 1.000 0.000  80 1.000 0.000  91 1.000 0.000  89 1.000 0.000  61 0.878 0.122  1 2  82 1.000 1.000  73 1.000 1.000  52 1.000 0.000  80 1.000 0.000  92 1.000 0.000  89 1.000 0.000  63 1.000 1.000  1 2  87 1.000 1.000  72 1.000 1.000  50 1.000 0.000  79 1.000 0.000  89 1.000 0.000  80 1.000 0.000  63 1.000 1.000  1 2  84 0.917 0.083  71 0.782 0.218  47 0.543 0.457  80 0.781 0.219  89 0.966 0.034  84 0.821 0.179  66 1.000 0.000  AAT-2 (N)  IDH (N)  6PG-1 (N)  6PG-2 (N)  PGM (N)  PGI-2 (N)  GDH (N)  LAP-1 (N)  LAP2 (N)  A p p e n d i x II (con't).  Locus  POP8  AAT-1 (N)  POP9  POP10  POP11  POP12  POP13  POP14  1 2  65 0.777 0.223  102 0.647 0.353  61 0.746 0.254  79 0.734 0.266  65 0.615 0.385  47 0.649 0.351  53 0.557 0.443  1 2  70 1.000 0.000  101 1.000 0.000  61 1.000 0.000  80 0.975 0.025  65 1.000 0.000  50 1.000 0.000  53 0.745 0.255  1 2  70 0.779 0.221  102 0.642 0.353  54 0.611 0.389  74 0.649 0.351  62 0.589 0.411  45 0.678 0.322  50 0.340 0.660  1 2  69 0.812 0.188  104 0.702 0.298  59 0.737 0.263  78 0.840 0.160  65 0.808 0.192  51 0.608 0.392  45 0.733 0.267  1 2  73 0.863 0.137  106 0.915 0.085  59 0.822 0.178  80 0.850 0.150  64 0.844 0.156  50 0.610 0.390  44 1.000 0.000  1 2  72 1.000 0.000  107 0.930 0.070  60 1.000 0.000  80 1.000 0.000  60 1.000 0.000  50 1.000 0.000  48 1.000 0.000  1 2  72 0.847 0.153  106 0.882 0.118  60 1.000 0.000  80 1.000 0.000  60 1.000 0.000  50 1.000 0.000  46 1.000 0.000  1 2  72 1.000 0.000  106 1.000 0.000  60 1.000 0.000  80 1.000 0.000  62 1.000 0.000  50 1.000 0.000  49 1.000 1.000  1 2  71 1.000 0.000  101 1.000 0.000  50 1.000 0.000  79 1.000 0.000  62 1.000 0.000  50 1.000 0.000  45 1.000 1.000  1 2  72 1.000 0.000  104 1.000 0.000  61 1.000 0.000  78 1.000 0.000  61 1.000 0.000  49 1.000 0.000  44 1.000 0.000  AAT-2 (N)  IDH (N)  6PG-1 (N)  6PG-2 (N)  PGI-1 (N)  PGI-2 (N)  GDH (N)  LAP-1 (N)  LAP2 (N)  

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