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LIBRARY
Michigan State
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This is to certify that the

dissertation entitled

Importance of Hybridization and Ecological Differentiation
in the success of Lithrum Salicaria in North America.

presented by
Jaimie Melissa Houghton—Thompson

has been accepted towards fulfillment
of the requirements for

Doctoral degree in Genetics

Major professor

Date December Q, 2991

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IMPORTANCE OF HYBRIDIZATION AND ECOLOGICAL DIFFERENTIATION IN
THE SUCCESS OF LY THRUli/I SALICARIA IN NORTH AMERICA

By

Jaimie Melissa Houghton-Thompson

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Program in Genetics

2001

ABSTRACT

IMPORTANCE OF HYBRIDIZATION AND ECOLOGICAL DIFFERENTIATION IN
THE SUCCESS OF LY THRUA/I SALICARIA IN NORTH AMERICA

By

Jaimie Melissa Houghton-Thompson

Lythrum salicaria is much more invasive in North America than its native Europe
(Batra et al.1986). One possibility for how it became more invasive is that it hybridized
with a close relative in North America, such as L. alatum, and gained genes that made it
better adapted to its new environment. This hypothesis was first tested by searching for
morphological evidence of hybridization. By examining a variety of purple loosestrife
populations across the northeastern United States, several traits were found that are not
present in European populations of purple loosestrife but are found in North American
winged loosestrife. These unique morphs found in North American purple loosestrife
suggest hybridization between the two species. In support of this, we identified
intermediate sized L. salicaria where the two species grow sympatrically and could have
hybridized.

Amplified fragment length polymorphism (AFLP) markers were then used to
further analyze the relationship between L. salicaria and L. alatum, and search for
evidence of hybridization. Twenty-seven North American and eleven European
populations of L. salicaria and L. alatum were screened with 5 primer pairs, and then
eight Michigan populations (selected for allopatry or sympatry between the two species)
were screened with an additional 18 primer pairs. When the resulting patterns of

molecular diversity were examined, North American L. salicaria clustered more closely

to European L. salicaria than to North American L. alatum, and no evidence of inter-
species hybridization was found. However, a considerable amount of differentiation was
observed among the North American L. salicaria. This differentiation may be the real

reason why L. salicaria became so successfiil in North America.

I dedicate this work to my parents, William and Jean Houghton, who have always
believed I could do anything I set my mind to, and my fiance, Matthew Thompson, who
was always there to listen when I thought I couldn't go on.

iv

ACKNOWLEDGEMENTS

First, I would like to thank my advisor, Dr. Jim Hancock. Without his tremendous
knowledge, support, belief in my ability, and more than occasional push in the right

direction, this never would have been accomplished.

I would also like to thank the members of my committee: Dr. Harold Prince, Dr. Barbara
Sears, Dr. James Smith, and Dr. Patrick Venta, who have always been willing to guide

my work and help me find direction.

None of this would ever been accomplished without the help, support, and humor of the
members of the Hancock lab, past and present, including Pete Callow, Chad Osborn,
Sedat Serce (including his enormous knowledge of statistics and computer formattingl),

Cholani Webadde, Rebekah F leis, Chris Owens, Joan Stroher, and Chrislyn Drake.

Fellow students in the Genetics Program, especially Sheldon Leung and Ribka Bedilu,

have also made this journey a more interesting, and sometimes even productive one.

Finally, I would like to thank my closest friends, Sarah Byers and Megan Nunez-Lane,
and my family, including my parents, William and Jean Houghton, my brothers Cory and
Patrick Houghton, members of my extended family including June, Buddy, Brian and
Ryan Horr, Willard Blaisdell, and Arlene Tebbetts, and my fiance, Matthew Thompson.

Without all of your love and support, this would not have been nearly as fun!

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... viii

LIST OF FIGURES ............................................................................................................. x
CHAPTER 1

THE IMPLICATIONS OF POLYPLOIDY 1N LY T HR UM SALICARIA ............................ 1

Introduction .............................................................................................................. 1

Lythrum in North America ....................................................................................... 3

Type of polyploidy in Lythrum ................................................................................ 5

Role of hybridization and polyploidy in Lythrum .................................................. 1 1
CHAPTER 2

MORPHOLOGICAL DIVERSITY IN NORTH AMERICAN LY THR W ..................... 14

Introduction ............................................................................................................ 14

Materials and Methods ........................................................................................... 16

Population surveys ..................................................................................... 16

Common garden experiment ...................................................................... 21

Results .................................................................................................................... 22

Discussion .............................................................................................................. 37
CHAPTER 3

MOLECULAR DIVERSITY IN EUROPEAN AND NORTH AMERICAN

LY T HRW ................................................................................................ 41

Introduction ............................................................................................................ 41

The utility of AF LP analysis .................................................................................. 42

Materials and Methods ........................................................................................... 44

Plant collection ........................................................................................... 44

DNA extraction .......................................................................................... 45

DNA preparation for AF LP analysis ......................................................... 46

AFLP reactions .......................................................................................... 47

Polyacrylamide gel electrophoresis ........................................................... 48

Silver staining technique ............................................................................ 48

Exposure of film ........................................................................................ 49

Scoring the gel ........................................................................................... 49

Statistical Analyses .................................................................................... 49

Results .................................................................................................................... 50

Study with all populations ......................................................................... 50

Further study of Michigan populations ...................................................... 54

Discussion .............................................................................................................. 59

APPENDIX ........................................................................................................................ 63

LITERATURE CITED ...................................................................................................... 88

vii

LIST OF TABLES

Table 1: Taxonomic characteristics that separate North American winged loosestrife
(Lythrum alatum) and Eurasian purple loosestrife (Lythrum salicaria) (Graham, 1975)..15

Table 2: Location and species found at each study site ................................................... 18
Table 3: Loosestrife population size and type of environment at each study site ........... 19

Table 4: Prevalence of L. alatum diagnostic traits in populations of L. salicaria and L.
alatum surveyed in North America. For qualitative traits, the percentage of L. salicaria
carrying L. alatum traits is reported. For quantitative traits, the mean and range of each
trait are denoted .................................................................................................................. 23

Table 5: ANOVA of plant height in 14 populations of Lythrum alatum and 16
populations of L. salicaria in North America .................................................................... 24

Table 6: ANOVA of leaf length, width, and length/width ratio in 9 populations of
Lythrum alatum and 12 populations of L. salicaria in North America ............................. 24

Table 7: ANOVA of seedlings produced per flower in four populations of Lythrum
salicaria in Michigan. Two of the populations were sympatric to L. alatum, two were
allopatric. Number of seedlings was determined for 10 flowers each of 10 clones from
each population .................................................................................................................. 33

Table 8: Height and leaf characteristics of four populations of Lythrum salicaria grown
from seed in a common greenhouse at Michigan State University, East Lansing, MI. Two
of the populations were sympatric with L. alatum [Harsen's Island (HIS) and Sheep Farm
B (SFB)], and two were allopatric [Lake Lansing (LLA) and Quanicassee B (QWB)].
Values are given for natural populations and seedlings grown in a greenhouse. Mean
values were compared using SAS (Cary, NC). Bold values are significant at P<0.05 ..... 33

Table 9: Summary of AFLP variation in 15 populations of Lythrum salicaria and 12
populations of L. alatum in North America, 11 European populations of L. salicaria, and
6 cultivars. Included are number of fragments for each primer, with numbers unique to
each species and number shared between species ............................................................. 51

Table 10: Summary of AFLP variation in four populations of Lythrum salicaria and four
populations of L. alatum in Michigan. Included are number of fragments for each primer,
with numbers unique to each species and number shared between species ...................... 55

Table 11: AFLP data for global experiment. Msel primer for all combinations is CAG,

EcoRl primer is indicated. Molecular weight of fragment is indicated after primer name.
Presence of fragment is indicated with 1, absence with 0. North American purple

viii

loosestrife is bold, European purple loosestrife is italicized, and cultivars are
underlined ............................................................................................ 63

Table 12: AFLP data for extended Michigan experiment. Population is indicated, and
primer pair is indicated in the following manner: the first two letters of the primer
indication is the last two letters of the Msel primer, the last two letters of the primer
indication is the last two letters of the EcoRI primer (the first letter of all Msel primers is
C, the first letter of all EcoRl primers is 0). For example, ACAG is M-CAC/E-AAG...79

ix

LIST OF FIGURES

Figure l: Crossing evidence for type of tetraploidy of purple loosestrife. A is the result
of crosses if purple loosestrife is an allotetraploid, B is the case when it is an
autotetraploid. Experimental findings of full fertility of hybrids indicates that purple
loosestrife rs an autopolyploid ........................................................................................... .9

Figure 2: Inheritance patterns in purple x winged loosestrife hybrids where there is little
or extensive chromosomal divergence. With little chromosomal divergence, the hybrid
exhibits tetrasomic inheritance and backcrosses easily to purple loosestrife. With
extensive divergence, meiotic irregularities develop and backcrossing is difficult ........... 10

Figure 3: Locations of Lythrum salicaria, L. alatum, and mixed populations surveyed in
this study. L. salicaria is denoted with a diamond, L. alatum with a circle, and mixed
sites with a triangle. States are indicated by abbreviation .............................................. 20

Figure 4: Distribution in height of Lythrum salicaria and L. alatum from 30 North
American populations. L. salicaria is denoted in black, L. alatum in white ................... 25

Figure 5: Mean heights of Lythrum salicaria and L. alatum populations in
Massachusetts, Michigan, Ohio, and Wisconsin. Populations from the same state are
placed next to each other. L. salicaria is denoted in black, L. alatum in white, and states
are denoted along the x-axis. Striped bars indicated sympatric populations of L. salicaria
and L. alatum ..................................................................................................................... 26

Figure 6: Distribution of leaf length of Lythrum salicaria and L. alatum from 30 North
American populations surveyed. L. salicaria is denoted in black, L. alatum in white ..... 27

Figure 7: Mean leaf length of Lythrum salicaria and L. alatum in different populations
in Michigan and Wisconsin. Populations from the same state are placed next to each
other. L. salicaria is denoted in black, L. alatum in white, and states are denoted along
the x-axis. Striped bars indicate sympatric populations of L. salicaria and L. alatum ..... 28

Figure 8: Distribution of leaf ratios (length/width) of Lythrum salicaria and L. alatum

from 21 North American populations surveyed. L. salicaria is denoted in black, L.
alatum in white .................................................................................................................. 29

Figure 9: Mean leaf ratios (length/width) of Lythrum salicaria and L. alatum in 12
Michigan and 9 Wisconsin populations. Populations from the same state are placed next
to each other. L. salicaria is denoted in black, L. alatum in white, and states are denoted

along the x-axis. Striped bars indicate sympatric populations of L. salicaria and L.
alatum ................................................................................................................................ 30

Figure 10: Distribution in height of all allopatric Lythrum salicaria and L. alatum
populations (bottom panels) compared to four sympatric populations of L. salicaria
(Harsen's Island, Sheep Farm B, Ottawa National Wildlife Refuge, and Kildeer) skewed
towards L. alatum morphology .......................................................................................... 34

Figure 11: Distribution in leaf length in all allopatric L. salicaria and L. alatum (top and
bottom times) compared to two sympatric populations of L. salicaria (Harsen's Island,
Sheep Farm B) skewed towards L. alatum morphology ................................................... 35

Figure 12: Distribution in leaf length/width ratios in all allopatric Lythrum salicaria and
L. alatum (top and bottom frames) compared to leaf length/width ratios in two sympatric
populations of L. salicaria (Harsen's Island and Sheep Farm B) skewed towards L.
alatum morphology ............................................................................................................ 36

Figure 13: Principle component analysis of L. salicaria and L. alatum populations. All
variable morphological traits were included in this analysis (leaf placement, length,
width, length/width ratio, plant height, and flower number per leaf axil). Region of
origin, species, and sympatry vs. allopatry are denoted. Two populations which appear
intermediate are labeled by name ...................................................................................... 37

Figure 14: Principle Component Analysis for all populations included in study. North
American L. salicaria is indicated by filled circles, European L. salicaria by filled
triangles, cultivars of L. salicaria by open triangles, and L. alatum by open circles.
Percentage that each principle component contributed to overall variation is indicated in
parentheses on the appropriate axis ................................................................................... 52

Figure 15: Cluster analysis of L. salicaria and L. alatum, including all populations
studied in analysis. The top cluster includes all L. salicaria, and the lower cluster
includes all L. alatum. The top cluster can be firrther broken into North American L.
salicaria, cultivars, and European L. salicaria. Abbreviation for state of origin is listed
before each North American population name... . .. .......................................................... 53

Figure 16: Principle component analysis of the 8 Michigan populations more
extensively studied. L. salicaria populations are on the lefi, L. alatum populations on the
right. Percentage that each principle component contributed to overall variation is
indicated in parentheses on the appropriate axis ................................................................ 56

Figure 17: Cluster analysis of the eight Michigan populations of L. salicaria and L.
alatum more intensively studied. The top cluster includes all L. salicaria populations, the
lower cluster includes all L. alatum populations ............................................................... 57

Figure 18: An example of a polyacrylamide gel run in the Michigan experiment. Purple
loosestrife is on the lefi, winged loosestrife is on the right. Some markers are present in
purple loosestrife and not winged loosestrife, and vice versa, and some markers are
present in both species ............................................................................ 58

CHAPTER 1

THE MPLICATIONS OF POLYPLOIDY 1N L YTHRUM SALICARIA

Introduction

It has been suggested that some exotic species are preadapted to become invasive
if they are released from natural predators in their new environment (Blossey et al.,
1992). As a result, the control of invasives has moved more and more into the arena of
biological control, operating under the assumption that if a critical natural control agent
can be found, the fecundity of the invasive species, and their ability to invade, will be
severely hampered if not eliminated. Recent studies, however, ask if invasives are truly
‘born’ and become invasive in a new environment simply because of the lack of natural
enemies, or if they are ‘made’ and their invasive ability evolves after colonization
(Ellster and Schierenbeck, 2000).

There is support for both of these hypotheses. Some invasive species have been
controlled by the introduction of a biological control agent (Dodd, 1959; Huffaker and
Kennett, 1959), suggesting that the release from biotic pressure was the main factor
allowing invasion to occur. However, only a small fraction of species, when taken out of
their natural environment and introduced to a new ecosystem, do develop invasiveness,
and even for species that do become invasive, there is often a considerable lag time
between their first introduction and their becoming invasive (Ewel et al., 1999). If
release from biotic pressure was the only prerequisite to invasiveness, the spread of the

species should occur quickly after the release from predators. Ellstrand and Schierenbeck

(2000) have proposed that the reason for this delay may often be that interspecific
hybridization with native species must occur, allowing invasiveness to evolve after the
introduction of new genes. Abbott (1992) observed that interspecific hybridization
involving a non-native and a native or non-native species has led to the development of
numerous new sexually reproducing species. The models of Ellstrand and Schierenbeck
extend this idea to include previously-isolated, allopatric populations of sexually
reproducing species as well.

However, species can also evolve invasiveness without an influx of genes via
hybridization. Considerable ecological differentiation has been observed in many native
species, and it is certainly conceivable that such differentiation could occur in an
introduced species if its founders carried sufficient genetic variability. Polyploidy may
be an excellent way for this variability to be stored. Several aspects of polyploidy
contribute to their success as a whole, including their polyphyletic origin, which
incorporates high levels of genetic diversity into the species from recurrent formation
from multiple parent populations (Soltis and Soltis, 2000). As a result, polyploids have
higher levels of heterozygosity than their diploid progenitors and less inbreeding
depression. The genetic variability found in polyploids can be further assorted through
genomic rearrangement, and in the case of autopolyploids, tetrasomic inheritance.

An underlying theme in our laboratory is the investigation of how polyploidy,
especially autopolyploidy, plays a role in a species’ survival and evolutionary history. In
my project, that question was addressed by testing for interactions between an introduced,
invasive autopolyploid, Lythrum salicaria (purple loosestrife) and a closely related native

diploid species to explore if introgression has occurred, which could explain the

increased invasiveness of the introduced autopolyploid. The alternative hypothesis was
that genetic variability already present in L. salicaria reassorted and allowed previously
unrevealed characteristics to be expressed, expanding its ability to adapt and invade. L.
salicaria (purple loosestrife) is a highly invasive, exotic, autotetraploid plant that has
been the focus of recent attention because of its ability to become the dominant species in

wetlands, replacing many native species (Cutright, 1986; Batra et al., 1986).

Lyth_r_ym in North America

Purple loosestrife is a tall, perennial, autotetraploid plant found in wetland
habitats. It is native to Eurasia, but was introduced into North America in the early
1800’s (Pursh, 1814) both accidentally through ship ballast and purposely through seed
sales (Mack, 1991). It is considered invasive and a noxious weed in several states due to
its ability to eliminate pre-existing native species in wetlands (Cutright, 1986). Over the
last century, its range has spread westward from New England and it has now
successfully established itself as far west as the Pacific Northwest (Stuckey, 1980).
Although it is aggressive in North America, it is not in its native Eurasia (Batra et al.,
1986)

Purple loosestrife was initially introduced to North America in the early 1800’s,
specifically in New England, but it was not recognized as invasive until the 1930’s, when
it began to form monospecific stands in the floodplain pastures of the St. Lawrence River
in Quebec (Louis-Marie, 1944). Since that time, it has followed a distinct pattern of

invasion across the US. Typically, it remains unobtrusive for a long period (at least 20

years) followed by a brief period (of less than 3 years) in which it becomes dominant in
many parts of that region (Stuckey, 1980). One possibility why purple loosestrife is much
more dominant in North America than Eurasia may be that favorable genes have
introgressed from a close relative native in North America. If this is the case, the most
likely candidate is Lythrum alatum (winged loosestrife), a widespread diploid species that
is closely related to purple loosestrife, not found in Eurasia, and has habitat overlap at its
outer edges with purple loosestrife, but is typically found in drier areas. There are 11
known species of Lythrum native to North America, but L. alatum is the most
widespread. Winged loosestrife is a shorter, less showy species than purple loosestrife
(Blackwell, 1970), and grows in wet meadows as a sub-dominant (Cody, 1978).

There is evidence that the genomes of winged and purple loosestrife are
compatible, as some cultivars of purple loosestrife were generated by breeders by
crossing the two species (Anderson and Ascher, 1993). Most of these cultivars are self-
sterile, but recent work has shown that these cultivars are fiilly fertile when crossed with
the wild species of purple loosestrife (Lindgren and Clay, 1993; Anderson and Ascher,
1993; Ottenbreit and Staniforth, 1994). Purple loosestrife is self incompatible and
heterostylous (Darwin, 1877) and as a result the cultivars are self-sterile.

While no direct evidence of hybridization in the wild has been documented
between winged and purple loosestrife, bees and butterflies have been observed to move
between these species when they grow sympatrically (Levin, 1970), and hybridization
could occur between the two species via unreduced gametes (Anderson and Ascher,
1993). Morphological characters thought to be unique to winged loosestrife have also

been observed in purple loosestrife populations (Anderson and Ascher, 1994) and the

hybrids produced by breeders have been shown to be highly fertile tetraploids, which can

readily backcross to the tetraploid purple loosestrife.

Type of polyploidy in Lythrum

There are two major types of polyploids: autopolyploids and allopolyploids.
Autopolyploidy arises when only one type of chromosome set is doubled, while
allopolyploidy occurs when two nonhomologous genomes are united. Most polyploids
are thought to originate through unreduced gametes (Harlan and deWet, 1975). Until
recently (Soltis and Soltis, 1993, 1995), autopolyploidy was considered to be much rarer
in natural populations than allopolyploidy. The traditional view of autopolyploidy was
that it is generally maladaptive: that there would be multivalent pairing of chromosomes
and this would lead to reduced pollen and seed fertility because of unbalanced
chromosome segregation at meiosis (Stebbins, 1950, 1971). Successful establishment of
an autopolyploid was therefore seen as unlikely, particularly since their adaptations were
predicted to be very similar to their parent species (Levin, 1983; Soltis and Rieseberg,
1986). These assumptions have now been tested in a wide range of polyploid species
using molecular markers, and segregation ratios have indicated the reverse —
autopolyploids are often highly fertile. In many autopolyploids, multiple homologues
actually pair in random assortments of bivalents rather than in multivalents, and as a
result pairing relationships are normal (Soltis and Rieseberg, 1986; Krebs and Hancock,

1989; Samuel et al. 1990; On et al., 1998). Autopolyploidy is much more common than

previously thought because many autopolyploids were misidentified as allopolyploids,
based on bivalent pairing, but this bivalent pairing is random with tetrasomic inheritance.

Molecular studies of autopolyploid species have revealed three important
characters that may confer an evolutionary advantage over their diploid progenitors:
enzyme multiplicity, increased heterozygosity, and increased allelic diversity (Soltis and
Soltis, 1993). This increased biochemical diversity may confer a fitness advantage to
polyploids (Levin, 1983), especially autotetraploids because of the tetrasomic inheritance
they exhibit. Fertility is high in many autopolyploid species because the multiple
homologues pair in random assortments of bivalents rather than in multivalents (Qu et
al., 1998). A number of studies comparing genetic variation (as a function of
polymorphic loci and mean observed heterozygosity) have shown autotetraploids to be
more genetically variable than related diploid species (Shore, 1991; Soltis and Soltis,
1989; Lumaet, 1986).

Autopolyploidy could contribute greatly to the success of an introduced
autotetraploid species if its diploid progenitor (or close relative) is located in the area of
introduction. An alien autotetraploid species can cross with a closely related diploid
species with little chromosomal divergence if the diploid produces unreduced gametes,
since the unification of the reduced gamete from the tetraploid with the unreduced one
from the diploid would result in tetraploid F1 progeny with normal tetrasomic pairing
association, as shown in Figure 18. Through backcrossing, advantageous genes from the
diploid genome could become incorporated into the tetraploid genome. Over time, this
could allow genes to be injected into the tetraploid genome that allow the alien species to

further adapt to the new environment.

Alternatively, autopolyploidy in L. salicaria may have allowed unique adaptions
to arise via reassortment once exposed to a new environment. The increased level of
heterozygosity generally observed in polyploids may have given some traits the plasticity
needed to adapt to a new habitat. Heterozygosity is fixed in allopolyploids, due to the
fact that the two ancestral genomes do not pair with each other in the polyploid. In
autopolyploidy, with pairing freely allowed among all chromosome sets, reassortment
into new ecotypes suited for a wider range of habitats is possible. Genetic variability
already present in L. salicaria may have reassorted without hybridization into new
morphologies and adaptive types. Several species of Lythrum in Europe are diploid, and
some carry similar traits to L. alatum (Tutin et al., 1968). Some of these species could be
the natural progenitors of L. salicaria, although this possibility has not been tested. If
these species are the original progenitors to the tetraploid L. salicaria, seemingly new
traits in L. salicaria could actually have lain hidden in the ancient polyploid genome and
reassorted when L. salicaria encountered new habitats in North America. The presence
of new habitats in North America may have presented the opportunity for these genes to
reassort and become phenotypically expressed.

Researchers have referred to L. salicaria as an allotetraploid because it is thought
to be an interspecific hybrid (Strefeler et al. 1996), but at the chromosome level, it
probably acts as an autopolyploid. This is supported by the discovery that the inheritance
patterns of style length are tetrasomic (Fisher, 1949). Breeding evidence that purple and
winged loosestrife can intercross, and the fact that these cultivar hybrids can backcross to
purple loosestrife (Ottenbreit and Staniforth, 1994), are also compelling evidence that

purple loosestrife is an autopolyploid. If, chromosomally, it acted as an allopolyploid,

the two sets of chromosomes in the purple loosestrife gametes would be incompatible and
would cause significant fertility reductions in an interspecific cross (Figure 2). The most
conclusive evidence that the hybrid acts as an autotetraploid comes from crossing studies
where tetraploid purple loosestrife was hybridized with diploid winged loosestrife, and
the resulting tetraploid F1 hybrids produced via unreduced gametes (in the diploid) were
completely fertile (Ottenbreit and Staniforth, 1994). A known purple and winged
loosestrife hybrid, the cultivar Morden Gleam, was crossed with purple loosestrife and no
significant reduction in the fertility of progeny was found when compared to crosses
among purple loosestrife. Therefore, several lines of evidence support the hypothesis that
L. salicaria is an autopolyploid.

The F1 hybrid between purple and winged loosestrife must be an autopolyploid
rather than an allopolyploid, as the unification of a purple loosestrife gamete with an
unreduced winged loosestrife gamete in the case of allopolyploidy would result in an
unfertile hybrid that had two chromosome sets from the winged gamete that could freely
pair, but two sets of chromosomes from the purple gamete that could not pair with each
other. Depending on chromosomal divergence between winged and purple loosestrife,
either one or none of the purple loosestrife chromosomes could pair with the winged
chromosomes (Figure 1). This would result in reduced fertility of the hybrid due to
meiotic irregularities, leading to the production of numerous partially aneuploid and/or
triploid gametes with poor viability, and backcross hybrids would likely have low vigor
and meiotic irregularities. The only way for a hybrid between purple and winged
loosestrife to be fiilly fertile when backcrossed to a purple loosestrife plant is if all four

chromosomes of each set in the hybrid can interchangeably pair with each other at

meiosis (Figure 2). This would result in tetrasomic inheritance (four alleles at a locus)
(Fisher, 1944, 1949). In the case of an autopolyploid, there would be little chromosomal
divergence between winged and purple loosestrife, and the chromosomes could pair in a
balanced fashion at meiosis resulting in high fecundity. If there is as little chromosomal
divergence between the two species, as illustrated by this example, it is not hard to

imagine a pathway for the movement of information fiom one genome to the other.

 

A: If purple loosestrife is an allotetraploid:

Purple loosestrife gamete x unreduced winged loosestrife gamete = Hybrid

In hybrids: L3 can pair with L3, but L1 and L2 cannot pair with each other.
L1 or L2 may be able to pair with L3, depending on how divergent the
chromosomes are.

Some hybrids would have meiotic irregularities (partially aneuploid and/or
triplo id gametes).

B: If purple loosestrife is an autotetraploid:

69

Purple loosestrife gamete xunreduced winged loosestrife gamete = Hybrid

In hybrids: L3 can pair with L3, and L1 can pair with L]. Forms balanced
L1 L3 gametes. L1 may be able to pair with L3, depending on how diverged
the chromosomes are. If there is little divergence, gametes can backcross

to purple loosestrife (L1 L1) gametes to form L1 L1 L1 L3 hybrids.

 

 

 

Figure 1: Crossing evidence for type of tetraploidy of purple loosestrife. A is
the result of crosses if purple loosestrife is an allotetraploid, B is the case when it is an
autotetraploid. Experimental findings of full fertility of hybrids indicates that purple
loosestrife is an autopolyploid.

 

Extensive Chromosomal Divergence Little Chromosomal Divergence

6)

Purple gamete Winged Garnete Purple Garnete Winged Garnete
(unreduced) (unreduced)
Resultant Hybrid Resultant Hybrid
Gametes formed Gametes formed

Backcross to Purple Loosestrife (L1 L1) lBackcross to Purple Loosestrife (L1 L1)

L1 L1 .

 

Some of the Gametes Formed Gametes Formed
(Reduced fertility due to pairing (Full fertility)
irregularities)

 

 

 

Figure 2: Inheritance patterns in purple x winged loosestrife hybrids where there is little
or extensive chromosomal divergence. With little chromosomal divergence, the hybrid
exhibits tetrasomic inheritance and backcrosses easily to purple loosestrife. With
extensive divergence, meiotic irregularities develop and backcrossing is difficult.

10

Role of hybridization ananolyploidy in Lythrum

In recent years, the role of interspecific hybridization in plant evolution has
received renewed attention. The importance of interspecific hybridization was long
suspected utilizing morphological markers (Anderson, 1949; Riley, 193 8; Heiser, 1947
and 1949), and with the advent of molecular markers, considerable direct evidence has
accumulated that genes have been exchanged between closely related species of the same
ploidy (Arnold et al., 1990, 1992 and 1993; Rieseberg and Warner, 1987; Rieseberg et
al., 1988; Rieseberg and Ellstrand, 1993). In some cases, substantial genomic
reorganizations have occurred after hybridization resulting in speciation (Arnold, 1993;
Rieseberg et al., 1993; Rieseberg, 1995).

Recently, a number of plant families were identified as “hotspots” for
hybridization (Ellstrand et al. , 1996). Although Lythraceae, the family that includes
Lythrum, was not included in this discussion, its closest relative, Onagraceae, was, and
this family was identified as one of the families with an unusually high level of natural
hybridization. Lythrum also fits all three criteria that were identified to increase the
likelihood of hybridization: perennial habit, outcrossing breeding system, and
reproductive modes that can stabilize hybridity (e. g. clonal reproduction).

Our original hypothesis was that there was hybridization occurring between
winged and purple loosestrife as it moved across North America, via unreduced gametes,
that allowed genes from the winged loosestrife genome to be incorporated into purple
loosestrife populations. These incorporated genes then allowed purple loosestrife to more

rapidly adapt to North American habitats. We based this hypothesis on previous

11

morphological (Anderson and Ascher, 1994) and isozyme (Strefeler eta1., 1996a, 1996b)
studies conducted in Minnesota that indicated that there had been movement of traits
fiom winged loosestrife into purple loosestrife populations. The alternative hypothesis
was that the appearance of new traits and adaptation to new habitats of purple loosestrife
in North America was due to genetic reassortment in the polyploid, unveiling previously
untapped invasive potential. My project addressed these hypotheses in three ways. First,
by characterizing both purple loosestrife and winged loosestrife on both a regional level
and species level, using genetic [amplified fragment length polymorphism (AFLP)] and
morphological markers, and searching for evidence of either movement of genetic
material from winged to purple loosestrife or extensive differentiation without
hybridization. Second, by observing putative hybrid populations in a controlled
environment and determining if the morphological patterns observed in the field have a
genetic basis. Third, by obtaining plant material from European populations and
discovering whether North American populations of purple loosestrife have diverged
from European ones.

This project allowed us to study whether autopolyploidy and interspecific
hybridization can play a critical evolutionary role in a species’ adaptation to a new
environment. To date, ecotypic variation in an autopolyploid species that arose through
sexual interaction with a diploid carrying the same genome has not been documented, and
levels of genetic differentiation in autopolyploids have only rarely been described, with
or without interspecific hybridization. Showing that polyploidy and natural hybridization

can be a dynamic process in evolutionary change, especially as a mechanism for

12

increasing invasiveness, would further our understanding of how plants evolve invasive
properties such as weediness, and can help in developing strategies to control them.
Understanding the mechanism of invasion of a polyploid alien species can also be
critical to determining methods of biological control, particularly when natural
hybridization could lead to the transfer of resistance. Many studies have been performed
to determine methods for biological control of purple loosestrife, specifically looking at
herbivores that feed on purple loosestrife in its native Eurasia (Blossey, 1995). Based on
host specificity, three herbivore species have been chosen: Galerucella calmariensis (L.)
and G. pusiIIa (Dull), leaf feeding beetles, and Hylobius transversovittatus Goeze, a root
feeding weevil (Kok et al. 1992a, b). In no-choice tests, the leaf feeding beetles were
found to oviposition as well as feed on winged loosestrife, although in choice tests,
purple loosestrife was preferred (Kok et al., 1992). If hybridization has taken place
between purple and winged loosestrife, that could have an significant effect on the
susceptibility of both purple and winged loosestrife to Eurasian predators. Two effects
are possible: 1) the hybrid could be less susceptible to Eurasian predators than purple
loosestrife is in Eurasia, or 2) the hybrid could provide an evolutionary “bridge” for the
Eurasian predators to adapt to feeding on winged loosestrife. Documentation of
hybridization occurring in the wild between these species will allow environmental
managers to make more informed decisions about biological control agents. If purple
loosestrife in North America becomes more resistant to Eurasian predators than the
Eurasian purple loosestrife, much time and money could be wasted breeding and
releasing insects that will have little or no effect. Additionally, if the Eurasian predators

are more effective predators of F1 hybrids, our native species may be put at risk.

13

CHAPTER 2

MORPHOLOGICAL DIVERSITY IN NORTH AMERICAN LY 1' HR UM

Introduction

Lythrum salicaria, purple loosestrife, was introduced to North America from
Eurasia in the early 1800’s and now ranges from New England to the Pacific Northwest.
Its closest relative is L. alatum, more commonly known as winged loosestrife, a shorter,
less showy diploid species that is native to North America and is not present in Eurasia
(Blackwell, 1970). Winged loosestrife grows in wet meadows as a sub-dominant (Cody,
1978), and its preferred habitat overlaps extensively with purple loosestrife.

Purple and winged loosestrife can intercross, and they probably share a common
genome. Purple loosestrife has been identified as an autotetraploid by previous
researchers (Fisher, 1949) by determining that inheritance patterns of style length were
tetrasomic. Purple and winged loosestrife have been successfully hybridized by breeders,
and these cultivar hybrids can be successfully backcrossed to purple loosestrife
(Ottenbreit and Staniforth, 1994). Winged loosestrife is a diploid species, therefore the
most likely way for these two species to intercross is through an unreduced gamete in
winged loosestrife fertilizing a natural diploid gamete in purple loosestrife. The resultant
hybrid would be tetraploid, and could backcross to purple loosestrife. Unreduced
gametes have been shown to be important in the evolution of numerous autopolyploid
species including blueberry (Qu et al., 1998), potato (Lam, 1974) and alfalfa (V eronesi et

aL,1986)

l4

There are several taxonomic characters that are found in winged loosestrife that
are not present in European populations of purple loosestrife (Graham, 1975; Table 1).
All of these morphological characters have been anecdotally observed in purple
loosestrife populations in Minnesota (Anderson and Aster, 1994), but similar searches
have not been made across the entire range of purple loosestrife. The research described
herein sought to determine whether winged loosestrife traits occur elsewhere in purple
loosestrife populations across the northeastern United States, following its original
invasion pattern. We found that winged loosestrife traits do appear in purple loosestrife
all across its North American range, and that some purple loosestrife populations which
are sympatric with winged loosestrife have heights and leaf lengths intermediate to most
other winged and purple populations. These observations are consistent with the
possibility that purple and winged loosestrife have hybridized in North America, although
the possibility can not be excluded that purple loosestrife has evolved traits similar to

those found in winged loosestrife without hybridization.

Table 1: Taxonomic characteristics that separate North American winged loosestrife
(Lythrum alatum) and Eurasian purple loosestrife (Lythrum salicaria) (Graham, 1975).

 

 

Lythrum alatum Lythrum salicaria
1 to 2 flowers per leaf axil 4 or more flowers per leaf axil
leaves alternately placed leaves placed opposite or whorled
glabrous, oblong calyx Pubescent calyx
distylous Tristylous
dwarf, less than 3 feet tall 4 feet tall or more

Oblong-Ovate to Linear-Lanceolate Leaves Lanceolate leaves

 

15

Materials and Methods

Population surveys

Lythrum salicaria and L. alatum populations were surveyed afier flowering in the
months of July and August. Michigan populations were surveyed in August, 1997, Ohio
populations in July, 1998, and Massachusetts and Wisconsin populations in July, 1999.
Distribution of collection sites by map location are outlined in Figure 3, and details for
each population (region, species, name, abbreviation) are found in Table 2. Population _
size estimates and short descriptions of the habitats of each population are listed in Table
3. The main difference in population habitats was water depth. Populations ranged from
permanent flooding at depths greater than 30 cm, to no history of flooding whatsoever,
with all available water determined by rainfall.

L. salicaria clones were surveyed in a transect across the width of each
population, at a distance of ten meters from each other, to minimize the possibility of
repeat sampling of the same clone. In general, individual clones were distinct, forming
colonies of 20 - 25 individual shoots. An attempt was made to sample at least 50 clones
from each population, although some populations were too limited to collect this many
samples without duplication. L. alatum clones were also surveyed 10 meters apart in the
larger populations, but in the smaller populations, plants were examined as close as 1 m,
as L. alatum plants do not exhibit the same degree of vegetative underground growth as
L. salicaria, so distinguishing between different plants was much easier. In the large

populations, 50 clones were surveyed at regular intervals, but in the small populations, all

16

distinct clones were surveyed. Total numbers of clones surveyed in both L. salicaria and
L. alatum populations are listed in Table 3.

A total of six taxonomic traits (Table 1) were surveyed in each clone. Flower
number was counted in each leaf axil along a random shoot in each clone and the average
number of flowers per leaf axil was recorded. Placement of leaves along the stem was
also noted as either alternate, opposite, or whorled. The calyx was rated as either
glabrous or pubescent, by examining all calyxes along another random shoot in each
clone. Style length was recorded on 10 randomly selected flowers on each clone to
determine if there was deviation from distyly in Lythrum alatum or tristyly in Lythrum
salicaria. The height of the tallest shoot of each clone was also measured. Leaf length
and width of two to three randomly selected leaves on each plant was measured for use in

determining leaf shape, via ratios (length / width).

17

Table 2: Location and species found at each study site.

 

 

State Species Site Abbr. Latitude Longitude

Michigan Lsalicaria Lake Lansing LLA 42:44:59 N 084:24:02 W
Michigan L.salicaria Crow Island St. Game Area CIA 43:28:11 N 083:54: 14 W
Michigan L.salicaria Shiawassee R. St. Game Area SRA 43:23: 13 N 083:57:58 W
Michigan L.salicaria Quanicassee Wildlife Area B QWB 43:35:00 N 083:40:51 W
Michigan Lsalicaria Quanicassee Wildlife Area A QWA 43:35:00 N 083:40:51 W
Michigan Lsalicaria Sheep Farm B SFB 43:39:13 N 083:27z58 W
Michigan Lsaltbaria Harsen's Island HIS 42:35:22 N 082:35:]9 W
Michigan L.alatum Sheep Farm A SFA 43:39:13 N 083:27z58 W
Michigan L. alatum Algonac State Park ASP 42:37:06 N 082:31:52 W
Michigan L. alatum Wildfowl Bay WFB 43:53:00 N 083:22:00 W
Michigan Lalatum Rose Island Railroad RIR 43:46:58 N 083:25253 W
Michigan L. alatum Harsen's Island I-IIW 42:35:22 N 082:35: 19 W
Wisconsin Lsalicaria Wee Know School WKS 43:06:18 N 0882031 W
Wisconsin L.salicaria Bark River BR] 43:04:50 N 088:15:40 W
Wisconsin Lsalicaria Duck Creek DCR 44:33:43 N 088:04:09 W
Wisconsin Lsalicaria Senior Citizen Center SCC 42:54:38 N 087:5]:38 W
Wisconsin Lsalicaria Tichigan Lake TLP 42:49:44 N 088:11:51 W
Wisconsin Lalatum Janesville JAN 42:40:58 N 089:0]:07 W
Wisconsin Lalatum Herbarium Preserve HPR 43:04:53 N 088:54z42 W
Wisconsin Lalatum Tichigan Lake TLW 42:49:44 N 088:11:51 W
Wisconsin L. alatum Nature Conservancy NCO 42:30:44 N 087:48:33 W
Ohio L.salicaria Ottawa Natl Wildlife Refuge ONP 41:36:56 N 083: 12:58 W
Ohio Lsalicaria Kildeer KIL 41:02:39 N 083:39:00 W
Ohio Lalatran Kitty Todd A KTA 41:34:46 N 083:37:02 W
Ohio Lalatum Kitty Todd B KTB 41:34:46 N 083:37:02 W
Ohio Lalatwn Kildeer KIW 41 :02:39 N 083:39:00 W
Ohio Lalatum Ottawa Natl Wildlife Refuge ONW 41:36:56 N 083:12:58 W
Massachusetts L.salicaria Field Farm FFA 42:42:43 N 073:12: 15 W
Massachusetts Lsalicaria West Pittsfield WPI 42:25:51 N 073:18:37 W
Massachusetts L. alatum Sheflield SHE 42:06:37 N 073:21z20 W

 

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20

Common garden experiment

Seed was collected from 8 Michigan populations ( 4 L. salicaria and 4 L. alatum)
in late September of 2000. Four purple loosestrife and four winged loosestrife
populations were selected (HIS, SFB, LLA, QWB, HIW, SFA, ASP, and WFB). These
populations were chosen to represent two allopatric and two sympatric populations of
each species. The sympatric populations of purple loosestrife chosen (HIS and SFB)
were previously determined to have intermediate heights and leaf lengths in the field
studies. A randomly selected shoot from each of 20 random clones was collected along a
transect across each population. Ten flowers were chosen fiom each shoot, and their
seed were spread onto moistened soil in a covered tray and allowed to germinate under
artificial light. After germination, seedlings were transplanted to 10 cm. pots and grown
in a single greenhouse at Michigan State University, East Lansing MI, under 12 hour day
lengths supplemented with artificial light. They were each fertilized with 2 tablespoons
Osmocote (The Scotts Company, Marysville, OH) after transplantation and watered
daily. Plants were arranged on three benches in a completely randomized block design
(bench being the blocking factor), and allowed to grow until flowering (approximately 8
weeks). At flowering, the same traits were measured that varied in the field (height,
flower number, style length, and leaf size). Height was recorded for the tallest shoot in
each plant. Number of flowers per leaf axil were counted for 10 - 20 axils in each plant
and the most common number was recorded. Styly was measured on a random shoot and
recorded as long, mid, or short. Leaf size was measured on a randomly selected fully

expanded leaf of each plant.

21

Univariate statistics, Analysis of Variance (AN OVA), and Principle Component
Analysis on all data were performed using the SAS statistical software package (Cary,

NC).

Results

All the L. alatum clones had the taxonomic traits considered diagnostic for L.
alatum. Among the L. salicaria clones, the calyx and stny traits were always true for L.
salicaria; however, the height of L. salicaria clones varied widely on a population level,
from x=74.8 to 173.6 cm. The purple loosestrife populations HIS (x=107.5 cm), ONP
(109.5 cm), KIL (74.8 cm) and SF B (96.7 cm) had mean heights significantly closer to
typical L. alatum (x= 50.4 - 68.1 cm) than L. salicaria (Table 4, Figure 5). All four of
these purple loosestrife populations were also sympatric to winged loosestrife. Similarly,
leaf length in the L. salicaria populations HIS (x= 26.4 mm) and SFB (41.4 cm) were
also much closer to the typical mean leaf length of L. alatum (x= 8.0 - 22.4 mm) than L.
salicaria (x= 26.4 - 78.9 mm) (Figures 6 and 7). Length/width ratios were significantly
smaller in L. alatum (x= 3.33 - 3.93) than L. salicaria (x= 3.26 - 5.63), but there were no
significant differences among the individual species populations (Figures 8 and 9). A
varying percentage of clones in all L. salicaria populations exhibited the traits thought to
be diagnostic for L. alatum, alternate leaf placement along the stem and fewer than four
flowers per leaf axil (Table 4). ANOVAS for height, leaf length, leaf width, and leaf

length/width ratio are in Tables 5 and 7.

22

 

 

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Table 5: AN OVA of plant height in 14 populations of Lythrum alatum and 16

populations of L. salicaria in North America.

 

 

Source DF SS MS F P Var. Comp1 % Var.2

Replication 50 91129.365 1822.587 4.91 <.0001 81.08681 2
Species 1 1009008099 1009008099 2718.42 <.0001 3069.6 78
Pop(Species) 28 346023 .656 123 57.988 33 .29 <.0001 547.67895 14
Error 915 339624.253 371.174 218.3909 6
Total 994 2344528.422 3916.7567 100

 

1Variance in each component
2 % of variance in each component

Table 6: AN OVA of leaf length, width, and length/width ratio in 9 populations of
Lythrum alatum and 12 populations of L. salicaria in North America.

 

 

 

 

 

 

 

 

Length:

Source DF SS MS F P Var. Comp] % Var.2
Replication 49 7509 153 1.04 0.4104 -2.33 0.0
Species 1 185761 187561 1267.62 <0.001 978.02 77.0
Pop(Species) 19 83526 4396 29.71 <0.001 161.63 12.7
Error 571 84487 147 132.69 10.4
Total 640 453791 1270.01 100.0
Width:

Source DF SS MS F P Var. Comp] % Var.2
Replication 49 275 5.6 0.89 0.6794 0.002 0.0
Species 1 5831 5831 927.58 <0.001 30.36 72.7
Pop(Species) 19 2920 153 24.45 <0.001 6.49 15.5
Error 571 3589 6.2 4.91 11.8
Total 640 15529 41.762 100.0
Ratio:

Source DF SS MS F P Var. Comp1 %Var.2
Replication 49 66 1 .34 1 .01 0.4638 -0.01 0.0
Species 1 182 181 136.16 <0.001 0.9 37.3
Pop(Species) 19 127 6.7 5.01 <0.001 0.12 5.0
Error 571 762 1.34 1.4 58.1
Total 640 121 1 2.41 100.0

 

:Variance in each component
% of variance in each component

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Each of the ‘intermediate’ populations of purple loosestrife was compared to
allopatric purple and winged loosestrife individually for height (Figure 10), leaf length
(Figure 11), and leaf length/width ratio (Figure 12). Principle component analysis was
then used to look at all traits simultaneously, and the Harsen's Island (HIS) and Sheep
Farm B (SFB) populations of L. salicaria appeared to be intermediate between all the
other L. salicaria and L. alatum populations (Figure 13).

There were significant differences (P< 0.05) in the rate of germination of seeds
from the population LLA, which was significantly higher than the other purple loosestrife
populations collected from seed. Unfortunately, none of the L. alatum populations
produced seed that germinated at all. This may be due to a dormancy requirement for L.
alatum that was not met. However, germination of HIS was still lower than the other
purple loosestrife populations collected, as in the field, with some families having
germination rates that averaged less than 1 seedling per flower, but this trend was non-
significant when compared to the other populations. ANOVA of germination is in Table
7.

HIS and SFB had significantly shorter heights and smaller leaf values than the
other two purple loosestrife populations in the greenhouse (Table 8). Although absolute
values of traits differed significantly between naturally occurring and greenhouse grown
plants from the same populations, the overall trend of relationships between populations
for each trait was preserved. However, few L. salicaria plants carried the other two L.
alatum traits found in the field. All four purple loosestrife populations exhibited

overwhelming numbers of plants (>95%) with opposite leaf placement vs. either alternate

31

 

(the diagnostic for winged loosestrife) or whorled, and four or more flowers per leaf axil

compared to fewer than two flowers (the diagnostic for winged loosestrife).

Table 7: AN OVA of seedlings produced per flower in four populations
of Lythrum salicaria in Michigan. Two of the populations were
sympatric to L. alatum, two were allopatn'c. Number of seedlings was
determined for 10 flowers each of 10 clones from each population.

 

 

Source DF SS MS F P
Replication 9 5016 557 1.05 0.4285
Population 3 46308 15436 29.08 <0.001
Error 27 14332 530

Total 39 65657

 

32

Table 8: Height and leaf characteristics of four populations of Lythrum salicaria grown
fiom seed in a common greenhouse at Michigan State University, East Lansing, MI. Two
of the populations were sympatric with L. alatum [Harsen's Island (HIS) and Sheep Farm
B (SFB)], and two were allopatric [Lake Lansing (LLA) and Quanicassee B (QWB)].
Values are given for natural populations and seedlings grown in a greenhouse. Mean
values were compared using SAS (Cary, NC). Bold values are significant at P=0.05.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Natural populations: Greenhouse populations:
Height:

Mean LLA QWB SFB Mean LLA QWB SFB

107.5 HIS <.0001 0.0118 0.151 67.9 HIS <.0001 <.0001 0.508

173.6 LLA <.0001 <.0001 87.7 LLA <.0001 <.0001

126.3 QWB <.0001 77.3 QWB <.0001

96.7 SFB 69.2 SFB

Leaf Length:
Mean LLA QWB SFB Mean LLA QWB SFB
26.4 HIS <.0001 <.0001 0.0028 69.5 HIS 0.0005 0.0064 0.0185

48 LLA 0.0003 0.0547 76.2 LLA 0.3043 <.0001

62.1 QWB <.0001 74.6 QWB <.0001
41.4 SFB 65 SFB

Leaf Width:

Mean LLA QWB SFB Mean LLA QWB SFB
8.2 H18 0.4939 <.0001 0.3485 21.1 HIS 0.0929 0.0022 0.2828
8.8 LLA <.0001 0.7029 19.9 LLA <.0001 0.4563
11.7 QWB <.0001 23.4 QWB <.0001

9 SFB 20.4 SFB
Leaf Length/Width Ratio:

Mean LLA QWB SFB Mean LLA QWB SFB
3.3 HIS <.0001 <.0001 0.0006 3.4 HIS <.0001 0.7588 0.9574
5.5 LLA 0.9139 0.0079 4.1 LLA <.0001 <.0001
5.4 QWB 0.0236 3.3 QWB 0.7596
4.7 SFB 3 .4 SFB

 

 

33

 

 

 

 

 

 

 

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Figure 11: Distribution in leaf length in all allopatric L. salicaria and L. alatum (top and
bottom frames) compared to two sympatric populations of L. salicaria (Harsen's Island,
Sheep Farm B) skewed towards L. alatum morphology.

35

 

Allopatric L. salicaria

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 12: Distribution in leaf length/width ratios in all allopatric Lythrum salicaria and
L. alatum (top and bottom fi'ames) compared to leaf length/width ratios in two sympmc
populations of L. salicaria (Harsen‘s Island and Sheep Farm B) skewed towards L.
alatum morphology.

36

 

 

 

 

3
o Allopatric L salicaria
I Sympatfic L salicaria
2 _ o Allopatn'c L alatum
c1 Sympatn'c L alatum
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Figure 13: Principle component analysis of L. salicaria and L. alatum populations. All
variable morphological traits were included in this analysis (leaf placement, length,
width, length/width ratio, plant height, and flower number per leaf axil). Region of
origin, species, and sympatry vs. allopatry are denoted. Two populations which appear
intermediate are labeled by name.

Discussion

Most of the North American L. salicaria carried at least a few L. alatum traits that
have not been described in Eurasian L. salicaria. This suggests that the two species may

have hybridized in North America, supporting the morphological evidence in Minnesota

37

described by Anderson and Ascher (1994), but on a much larger geographic scale. None
of the L. alatum populations carried any of the L. salicaria traits, which is not surprising,
since the theoretical hybrid would be tetraploid and only capable of backcrossing to
purple loosestrife populations, unless triploids exist (which is rare in plant species).
Screening 20 random winged loosestrife plants, unreduced gametes were produced at a
rate of 0.35% (70 unreduced gametes in 20,000 pollen grains screened, data not shown).
Because the L. alatum traits appear across the entire range of L. salicaria, such a
hybridization would have had to occur several times throughout the history of the two
species, or the hybridization occurred early in the establishment of purple loosestrife and
spread across North America.

The possibility of multiple hybridizations is supported by the two L. salicaria
populations (HIS and SFB) which were intermediate in height and leaf ratio between the
typical L. salicaria and L. alatum populations in both the native field and common
greenhouse. This intermediacy may be evidence that they are hybrid swarms, suggesting
that interspecific hybridization is ongoing, although it remains possible that purple
loosestrife is evolving a more xeric ecotype favoring winged loosestrife habitats.
Interestingly, most of the purple loosestrife plants grown in the common greenhouse had
the leaf placement and flower numbers typical of purple loosestrife, even though plants of
both typical purple and typical winged loosestrife traits were collected in the field for use
in this study. These traits must have a strong environmental component.

Purple loosestrife was not considered invasive until well after its establishment in
North America, specifically when it began to form monospecific stands in the floodplain

pastures of the St. Lawrence River in Quebec in the 1930’s (Louis-Marie, 1944). The

38

 

subsequent invasions of purple loosestrife have followed a distinct pattern, a few plants
appear in a region, the plant stays a very minor member of the community for 20 - 40
years, then in a very short interval (approximately 5 years) becomes the dominant
member of the wetland community (Stuckey, 1980). Ellstrand and Schierenbeck (2000)
have suggested that some exotic invasive plants only become invasive afier hybridization
with a native species, and one of the examples they site is L. salicaria, using isozyme
evidence provided by Strefeler et al. (1996). This isozyme evidence was not conclusive,
which is why I chose to do further, more in depth studies. My morphological data lends
further support to the hybridization hypothesis. The delays in invasion may occur
because purple loosestrife must gain and reassort genetic material from winged
loosestrife to help it adapt to new North American habitats.

Not knowing the genetic basis for any of the morphological traits studied, it
remains possible that invasiveness in purple loosestrife arose through genetic divergence
without interspecific hybridization, or new mutations have arisen in purple loosestrife
genetic material after its arrival in North America. It is also possible that an ecotype is
evolving in purple loosestrife that is better adapted to the drier habitats of winged
loosestrife through reassortment of its own genetic variability. In the next chapter, we
will use AFLP markers to show that, indeed, ecological differentiation without
interspecific hybridization is the most likely basis of L. salicaria's ecological success in
North America.

Knowing whether hybridization or ecological differentiation has played the
primary role in purple loosestrife’s success in North America could be critical to

determining methods of control. Much interest has been devoted to methods for

39

biological control of purple loosestrife, specifically searching for herbivores that feed on
purple loosestrife in its native Eurasia (Blossey, 1995). Based on host specificity, three
herbivore species have been chosen: Galerucella calmariensis (L) and G. pusilla
(Duff), leaf feeding beetles, and Hylobius transversovittatus Goeze, a root feeding
weevil (Kok et al. 1992a, b). In no-choice tests, the leaf feeding beetles were found to
oviposition as well as feed on winged loosestrife, although in choice tests, purple
loosestrife was preferred (Kok et al., 1992).

If hybridization has taken place between purple and winged loosestrife, it could
have altered the susceptibility of purple loosestrife to Eurasian predators. Two effects are
possible. First, the hybrid could be less susceptible to Eurasian predators than purple
loosestrife is in Eurasia and pass that resistance on to purple loosestrife populations.
Alternatively, the hybrid could provide an evolutionary “bridge” for the Eurasian
predators to adapt to feeding on winged loosestrife. Documentation of hybridization
occurring in the wild between these species will allow environmental managers to make
more informed decisions about biological control agents. If purple loosestrife in North
America becomes more resistant to Eurasian predators than the Eurasian purple
loosestrife, much time and money could be wasted breeding and releasing insects that
will have little or no effect. Additionally, if the Eurasian predators are more effective

predators of F1 hybrids, our native species may be put at risk.

40

CHAPTER 3
MOLECULAR DIVERSITY IN EUROPEAN AND NORTH AMERICAN

LYTHRUM

Introduction

Lythrum salicaria became an invasive, noxious weed after arriving in North
America, but it is not considered invasive in its native environment (Batra et 01.1986).
One possibility for how it became invasive is that it hybridized with a close relative in
North America, such as L. alatum, and gained genes that made it better adapted to this
new environment. In Chapter 2, I tested this hypothesis by searching for morphological
evidence of hybridization. By examining a variety of purple loosestrife populations
across the northeastern United States, I found several traits that are not present in
European populations of purple loosestrife but are found in North American winged
loosestrife. These unique morphs found in North American purple loosestrife suggest
hybridization between the two species (Chapter 2; Anderson and Ascher, 1994; Strefeler
et al., 1996). In support of this, I identified intermediate sized L. salicaria where the two
species grow sympatrically and could have hybridized (Chapter 2).

However, it is also possible that the genetic variability already present in L.
salicaria has reassorted without hybridization into new morphologies and adaptive types.
This might have been enhanced by its polyploid nature. There are several aspects of
polyploids that can contribute to their success. They can hybridize with their diploid

progenitors via unreduced gametes, or form recurrently fiom multiple parent populations.

41

Their polyphyletic origin also can lead to the incorporation of high levels of genetic
diversity (Soltis and Soltis, 2000). As a result, polyploids have higher levels of
heterozygosity than their diploid progenitors and less inbreeding depression. The high
level of genetic variability found in polyploids can be fithher assorted through genomic
rearrangement, and in the case of autopolyploids, tetrasomic inheritance. All of these
factors are quite applicable to L. salicaria and may have contributed its success as it
encountered new habitats across North America.

We used amplified fragment length polymorphism (AFLP) markers to fiirther
analyze the relationship between L. salicaria and L. alatum, and search for evidence of
hybridization. We screened all of our North American and European populations of L.
salicaria and L. alatum with 5 primer pairs, and then evaluated eight Michigan
populations (selected for allopatry or sympatry between the two species) with an
additional 18 primer pairs, to determine the genetic relationship between L. salicaria and
L. alatum in North America, and North American L. salicaria to European L. salicaria.
We found that there is no molecular evidence of hybridization in North America between
the two species. However, we did find that North American L. salicaria has
differentiated from European L. salicaria, and this differentiation may be the real reason

that L. salicaria has been so successful in North America.

The utility of AFLP analysis

Molecular markers are useful in determining genetic evolution and changes within
an organism. Morphological traits are controlled by a wide range of gene numbers and

frequently have large environmental components, making them often misleading in

42

 

evolutionary studies. By using molecular markers, the changes across an entire genome
can be studied with no extra weight assigned to any single trait or change (Tohme et al.,

1996)

Amplified fiagment length polymorphism (AFLP) analysis, as developed by Vos
et al. (1995), has fast become a favored method of DNA marker analysis. This technique
combines the reliability of RFLP markers with the time efficiency of PCR—based markers
and yields at least 10 times more genetic loci per primer pair than RFLP or RAPD
analysis in many crop species (Tohme et al., 1996). The resulting DNA fingerprint
yields a large number of genetic markers, and the multiplex ratio (the number of
information points analyzed per experiment) is higher than for other commonly used
markers (RFLPs, RAPDs, or SSRs) (Powell et al., 1996). AFLPs have been used in
many species to study evolution and genetic relationships, with some examples including
soybean (Maughan et al., 1996), lettuce (Hill et al., 1996), tea (Paul et al., 1997), tef (Bai

et al., 1999), olive (Angiolillo etal., 1999), and cassava (Roa et al., 1997).

AFLP analysis has been used to determine both genetic similarity and average
amount of polymorphism in a number of different species groups. Comparing difl‘erent
cultivated and natural olive species, Angiollio et a1. (1999) found the average percentage
of polymorphism to be between 51% for one primer combination and 83% for another.
Vroh et al. (1999) compared upland cotton to the wild species and found that genetic
similarity between the two groups ranged from 29.5% to 43.2%. Within closely related
tef accessions, however, Bai et al. (1999) detected a very low level of polymorphism
(18%), but still detected differentiation within these closely related individuals. One

common denominator for AFLP analysis is its ability to discern relationships in even the

43

closest of related individuals; this made it ideal for searching for hybridization between L.
salicaria and L. alatum, because the closeness of the relationship between the two species

was not previously known, and could be quite high.

Materials and Methods

Plant collection

Plant tissue was collected from each clone of L. salicaria and L. alatum surveyed
in the previous morphological studies. Approximately 10 young green leaves were
collected from every clone in early to late June before full growth and flowering
occurred. The leaves were placed in a ziploc bag with enough silica gel to completely
cover the leaf tissue. Bags were then labeled as to population and individual, sealed, and
stored at room temperature for several months until the DNA was extracted. Number of
individuals collected from each population corresponds with numbers surveyed in the
previous morphological study, outlined in Table 4.

European samples of L. salicaria were obtained from Dr. Bernd Blossey at
Cornell University. Seed had been collected from 11 European populations and grown in
a common garden at Cornell University. These populations originated from Germany,
England, Ireland, Austria, and Finland. Four to six leaves were collected from five
clones in each European population, covered with silica gel, and shipped to Michigan
State University.

Six cultivars of L. salicaria were purchased based on availability. Three clones

each of Robert, Roseum Superbaurn, and Purple Spires were purchased from Wrenwood

44

 

of Berkeley Springs (Berkeley Springs, WV). Three clones each of Happy, Morden's
Gleam, and Morden's Pink were purchased from Bluestone Perennials (Madison, OH).

New leaves were collected from each plant the morning that DNA was to be extracted.

DNA extraction

DNA was extracted following the protocol of Doyle and Doyle (1990) with
modification. Three to four leaves from each clone (approximately 1 g) were placed in a
sterile mortar, liquid nitrogen was added, and the leaves were then ground to a fine
powder. CTAB buffer [2% w/v hexadecyltrimethylammonium bromide (CTAB), 1.4 M
NaCl, 0.2% v/v 2-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl (pH 8.0)] was
added 800 uL at a time twice for a total of 1600 uL, and the material was ground into a
slurry. The resulting homogenate was poured into two 1.6 mL tubes and gently mixed by
inversion. The tubes were placed in a water bath at 60°C for 30 — 60 minutes, with
periodic mixing by inversion. The tubes were then removed fi'om the water bath and
allowed to return to room temperature. Equal volumes of Chloroform: Isoamyl alcohol
(24: 1) were added to each tube (approximately 800 uL), and the tubes were inverted to
gently mix them. The tubes were centrifuged at 14,000 x g for 5 minutes, and then the
top aqueous layer was transferred to a new tube. Equal volume (approximately 800 uL)
of ethanol acetate (4 % 3 M NaAc and 96 % EtOH) was added to each tube, gently
mixed, then the DNA was allowed to precipitate for 10 to 30 minutes at room
temperature. The tubes were centrifirged at 14,000 x g for 5 minutes, and the
supernatant was gently poured or pipetted off, saving the pellet. The pellet was washed

in 70% EtOH, and then spun for 5 minutes at 14,000 x g. The tube was then left open

45

 

overnight in a sterile laminar flow hood, to allow the pellet to dry thoroughly, before

being resuspended in 50 uL TE.

DNA preparation for AFLP analysis

Gibco BRL-Life Technology (Rockville, MD) reagents were used for all
digestions, ligations, and other AFLP preparations and experiments. First, 200 ng of
DNA were digested in 5 uL of 5X Restriction digest buffer, 2 uL of EcoRl/Mse I
restriction enzyme solution, and an appropriate amount of autoclaved ddI-120 to bring the
final volume to 20 uL per reaction. This solution was held at 37°C for 2 hours, then the
enzymes were deactivated at 70°C for 15 minutes. Next, ligations were performed by
adding 19.2 uL of the Adaptor/Ligation solution (containing Gibco patented extensions to
ligate to the sticky ends of our digested DNA) and 0.8 uL of T4 DNA ligase directly to
each digestion. This was incubated at 20°C for 3 hours. Afier ligations, 10 uL of each
solution was pipetted into a new tube and diluted 1:10 with 90 uL of 0.1 M Tris/EDTA
(TE). The remaining ligation solution was stored at —20°C. The diluted ligation solution
was then used for the pre-amplification reaction.

For each preamplification reaction, 20.0 uL of Pre Amp Primer Mix 1, 2.5 uL 10X
PCR Buffer, 1.0 uL 50 mM MgC12, 0.5 uL Taq DNA polymerase, and 0.5 uL of the
diluted template DNA from the ligation reaction were mixed together in a tube, and then
held in a Perkin Elmer 9600 PCR machine for 20 cycles of the following program: 94°C
for 30 seconds, 56°C for 1 minute, and 72°C for 1 minute. The program finished with
72°C for 10 minutes and then a 4°C soak. 5 uL of the preamplified reaction was then run

on a 2% Tris, Boric Acid, and EDTA (TBE) gel, and photographed to score for relative

46

 

DNA concentration. The brightness of the DNA smear was used to determine the final
dilution of the sample. Bright, clearly visualized pre-amplified reactions were diluted
1:20; while faint, identifiable but not clearly visualized pre-amplified reactions were
diluted 1:10, and absent smears were lefi undiluted. These diluted samples were then

stored at —20°C pending AFLP amplification reactions.

AFLP reactions

Initial screens included 10 individuals from each of the North American purple
and winged populations, as well as 5 individuals fiom each European population and 3
samples of each cultivar. The 320 samples were divided into four sets of 80, with two
positive controls (Arabidopsis and Tomato DNA) and two negative controls (ddH20)
included. Five primer pairs were selected to use in this study, which are listed in Table 9.
Each reaction consisted of 0.5 uL EcoRl primer, 4.5 uL Msel primer and dNTP mixture,
2.5 uL 10X PCR buffer, 0.8 uL 50 mM MgC12, 0.5 uL Taq polymerase, 13.7 uL ddH20,
and 2.5 uL diluted template DNA for a total reaction volume of 25 uL. Reactions were
performed in a Perkin Elmer 9600 PCR thermocycler set with the following parameters:
35 cycles of 94°C for 30 seconds, 56°C for 30 seconds, 72°C for 1 minute, then 72°C for
10 minutes and a 4°C soak. The reaction solutions were then stored at —20°C until being
run on a gel.

Eight populations of Lythrum in Michigan were selected for fithher analysis: 1)
four populations of L. salicaria, two sympatric with L. alatum and two allopatric, and 2)
four populations of L. alatum, two sympatric with L. salicaria and two allopatric. The

sympatric populations of L. salicaria were chosen because they were shorter and had

47

smaller leaves than the other L. salicaria populations, even in the greenhouse. Five
samples fiom each of the species populations were analyzed with 18 primer pairs (Table

10) following the above protocol.

Polyacrylamide gel electrophoresis

A 5% acrylamide gel (12% acrylamidezbis, 1X TBE, 40X Urea w/v) was poured
into a BioRad Sequi-Gen GT Sequencing Cell (Hercules, CA) and polymerized by
adding 700 uL 10X ammonium persulfate and 200 uL TEMED per 150 mL acrylamide
solution. Formamide buffer containing xylenol and bromphenol indicators were added to
the DNA reaction solutions, and the samples were denatured by heating to 96°C for 5
minutes, then stored on ice to preserve denaturization while loading into the gel. The gel
was loaded through a 96 well 0.4 mm comb, with Gibco 10 bp and 25 bp ladders on both
ends. The gel was run at 85 W with a variable voltage (1500 — 1700 average) for
approximately 3 hours (when the xylenol color marker was approximately 10 cm from
the bottom of the gel). One glass plate was then removed from the gel, and the gel was

fixed, stained and developed on the other glass plate.

Silver staining technique

Fix/Stop (10% acetic acid), staining (1% silver nitrate and 0.6% forrnaldhyde),
and developing (0.03g/mL sodium carbonate, 0.6% fonnaldhyde, and 0.002 mg/ml
sodium thiosulfate) solutions were prepared from the protocol in the Promega kit
(Madison, WI). The gel was placed in the fix/stop solution for 20 minutes (until the dye

band disappeared), and washed three times in ddH20 for 2 minutes each. The gel was

48

 

then shaken in the staining solution for 1 hour, washed in ddH20 for 10 seconds, and
placed in ice cold developing solution for 5 minutes, until bands appeared and were
clearly identifiable, but background staining was still low. The reaction was then stopped
by the addition of the fix/ stop solution, the gel was shaken for 5 minutes, and rinsed in

ddH20 for 5 minutes. The gel was air dried overnight.

Exposure of APC Film

The gel was placed face up on a light box. Under red light, the film was aligned
on the gel. The white light box was then turned on for 50 — 180 seconds, depending the
darkness of the gel bands and background staining. The film was developed using the

Kodak X-omat processor (Rochester, NY).

Scoring the gel

The gel was placed on a white light box and bands were scored for presence or
absence in each lane. Results were compiled for every band that was clearly scorable,
based on intensity of the band and smearing of the gel samples. This process was

repeated for every primer combination run in both experiments.

Statistical Analyses

SAS (Cary, NC) and NTSYSpc (Setauket, NY) were used for all statistical
analyses, including Cluster Analysis and Principle Component Analysis. The first three
principle components were then represented graphically (Figures 14 and 16). Cluster

analysis was performed with UPGMA similarity coefficients.

49

mm
Study with all populations

Five primer pairs were used to study 10 individuals fi'om each population, as well
as five individuals from each European population and three samples from each cultivar.
Sixty-four total fi'agments were observed, with 17 of those unique to L. salicaria and 12
unique to L. alatum. Of the remaining 35 fi'agments, 23 were monomorphic in all
species, and 12 were polymorphic. However, no fragment appeared in both North
American L. salicaria and L. alatum that was not also present in some proportion of the
European L. salicaria (Table 9).

Three distinct groups were observed in the principle component analysis (Figure
14): L. salicaria from North America, L. salicaria from Europe, and L. alatum. The
cultivars formed a distinct subgroup within the European L. salicaria. This result was
borne out by cluster analysis (Figure 15), where two major clusters were evident, one of
L. salicaria and one of L. alatum. The native North American L. salicaria cluster
monophyletically, forming a distinct cluster separate from the European L. salicaria and
the cultivars of L. salicaria, suggesting that the evolutionary relationships between the
North American L. salicaria are more recent than to the European L. salicaria. For the
most part, individuals from the same North American populations clustered together.
There was a clinal trend within the North American L. salicaria, with many of the
populations from the same region clustering together; however, this relationship was

violated with the Ohio and Massachusetts populations.

50

 

 

Table 9: Summary of AFLP variation in 15 populations of Lythrum salicaria and 12
populations of L. alatum in North America, 11 European populations of L. salicaria, and
6 cultivars. Included are number of fragments for each primer, with numbers unique to
each species and number shared between species.

 

# Frag. Unique to

 

Primer Comb. L. salicaria L. alatum # Frag. in both Total # Frag.
M-CAG/E-ACT 0 l 6 7
M-CAG/E-AGG 5 14 21
M-CAG/E-AAG 2 3 6 11
M-CAG/E-ACG 5 1 6 12
M-CAG/E-ACC 5 5 3 13
Total 17 12 35 64

 

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53

Further stuajr of Michigan populations

Eighteen additional primer pairs were selected to study five individuals from each
of eight Michigan populations. Using the additional eighteen primer pairs, 216 total
fragments were observed, with 76 of those unique to L. salicaria and 86 unique to L.
alatum. Of the remaining 54 fragments, all were monomorphic in both species (Table
10).

Both principle component analysis and cluster analysis split the populations into
two separate groups based solely on species (Figures 16 and 17). The sympatric
populations of L. salicaria that appeared morphologically intermediate to L. alatum did
not appear intermediate using the molecular markers. They clustered within L. salicaria,
separate from L. alatum. Individuals from the same population always clustered together.

An example of a gel run in this experiment is in Figure 18.

54

Table 10: Summary of AFLP variation in four populations of Lythrum salicaria and four
populations of L. alatum in Michigan. Included are number of fragments for each primer,
with numbers unique to each species and number shared between species.

 

# Frag. Unique to:
Primer Combination L. salicaria L. alatum # Frag. In both Total # Frag.

 

M—CAC/E-AAG 2 1 2 5
M-CAC/E-AAC l 4 2 7
M-CAA/E-AAG 3 2 4 9
M-CAA/E-AAC l l 0 2
M—CAC/E-AGG 2 3 1 6
M-CAT/E-AAG 4 1 3 8
M-CTC/E-ACT 3 4 8 l S
M-CTC/E-ACC 3 6 2 1 l
M-CTC/E-AGC 9 1 0 l 20
M-CTG/E-ACC 9 1 0 3 22
M-CTG/E-AGC 3 12 5 20
M-CTT/E-AGC 2 1 l 6 1 6 52
M-CTA/E-AGG l 0 0 1
M-CTA/E-ACT l 1 0 2
M-CAT/E-AAC 6 l 1 8
M-CAT/E-AGG 1 2 2 5
M-CTA/E-AAG 5 6 2 13
M-CTA/E-AAC 1 6 2 9
M-CAG/E-ACT l 2 4 7
M-CAG/E-AGG 5 3 1 3 2 1
M-CAG/E-AAG 4 4 3 1 1
M—CAG/E-ACG 6 1 5 1 2
M-CAG/E-ACC 5 5 3 l 3
Total 97 101 83 28 1

 

55

 

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57

 

 

 

Figure 18: An example of a polyacrylamide gel run in the Michigan experiment. Purple
loosestrife is on the left, winged loosestrife is on the right. Some markers are present in
purple loosestrife and not winged loosestrife, and vice versa, and some markers are
present in both species.

58

Discussion

Unlike the morphological results, the molecular data provide no evidence of
hybridization between the two Lythrum species. The North American purple loosestrife
have clearly diverged from the native species in Europe, but there is no evidence that a
hybridization between purple and winged loosestrife in North America contributed to that
differentiation. When patterns of molecular diversity were examined, North American
purple loosestrife formed its own distinct cluster within the larger purple loosestrife
cluster, with the European and cultivar samples clustering outside the North American
purple loosestrife cluster.

Not only was there difference in European and North American purple loosestrife,
but there was also an east-west cline observed within the North American purple
loosestrife. Samples from the same population, by and large, grouped together, and
populations from the same region also grouped into the same subgroups within the
clusters with few exceptions. This suggests that over the course of invading North
America, purple loosestrife has gone through considerable evolutionary change, and have
significantly diverged from the European populations. It is not known if this is solely the
result of selection in unique environments or partially due to founder effects of a
restricted sample.

Character state analysis of all molecular markers revealed one marker from the
global experiment that could be consistent with hybridization, for it was present in some
of the North American purple loosestrife, and some, but not all, of the winged loosestrife,

and none of the European purple loosestrife (AGG325c). However, even that pattern is

59

suspect when determining hybridization, for it is present in Massachusetts purple
loosestrife, but not in Massachusetts winged loosestrife. Two markers are present in all
winged loosestrife and a few North American purple loosestrife (AGG300, AAG475),
but are also present in some of the European purple loosestrife, aruging against a hybrid
origin of these markers in purple loosestrife, and nine markers are present in all winged
loosestrife but none of the purple loosestrife, which argues against a hybridization event.

All the commercially available cultivars were also examined. Included in this
group were Morden's Gleam and Morden's Pink, which are claimed to be first or second
generation hybrids of L. salicaria x L. alatum. In both the principle component analysis
and cluster analysis, all of the cultivars, including the two putative hybrids, clustered
more closely to European purple loosestrife than North American purple loosestrife or
winged loosestrife. This suggests that these cultivars have not extensively hybridized
with North American purple loosestrife, and the integrity of the cultivars in nursery stock
remains. It also suggests that the reported hybrid ancestry of the Morden cultivars is
unlikely. Visually, we could see no morphological differences between the Morden
cultivars and wild purple loosestrife populations.

In an often cited paper, Strefeler (1996) examined isozyme diversity in 11
Minnesota populations of purple loosestrife, one New Jersey purple loosestrife
population, and three Minnesota winged loosestrife populations, and concluded that
winged and purple loosestrife could have hybridized in North America. These
conclusions were based on the fact that L. salicaria and L. alatum shared many
allozymes; however, no species specific winged loosestrife allozymes were found to have

moved into purple loosestrife individuals, and their cluster analysis clearly distinguished

60

 

the L. salicaria from L alatum individuals. It is our contention that their evidence does not
support the conclusion that the two species have hybridized.

Strefler et al. (1996) suggested that firrther analysis was needed with a larger
number of winged loosestrife populations to "adequately define the role of L. alatum in
the evolution of weedy purple loosestrife". Our molecular analysis did just this, as it
surveyed 13 populations of purple loosestrife and 12 populations of winged loosestrife,
spread over four states, as well aseleven populations of European purple loosestrife and
six purple loosestrife cultivars. The first molecular experiment included 64 total bands,
51 of which were polymorphic, and 14 of which were specific to winged loosestrife. The
second experiment, with a smaller number of populations (four of each species) included
an additional 216 bands, 162 of which were polymorphic, and 86 which were specific to
winged loosestrife. Within this broad, comprehensive survey, there was no evidence of
hybridization between these two species. So, if there has been no hybridization
between purple and winged loosestrife, why does purple loosestrife in North America
exhibit traits not previously seen in Europe? Some of the traits previously observed in
the purple loosestrife in Minnesota (Anderson and Aster, 1994) specifically glabrous vs.
pubescent calyx and distyly vs. tristyly, were not observed in our survey. Also, within
the common greenhouse experiment, some of the variable traits (leaf placement and
flowers per leaf axil) almost completely disappeared, which indicates that there must be
an environmental component to these traits. There were ‘interrnediate’ populations of
purple loosestrife for height, flower and leaf traits in our screening across the
northeastern geographic range on the morphological level, but these populations did not

show intermediacy in their molecular markers. It may be that purple loosestrife is

61

evolving a shorter genotype that is adapted to the more xeric habitats favored by winged
loosestrife in North America.

Polyploidy in L. salicaria may have played a role in its ability to adapt to new
habitats in North America. The increased level of heterozygosity generally observed in
polyploids may have preadapted L. salicaria with sufi'rcient plasticity to fill many
habitats. This may explain why purple loosestrife is found in a much wider range of
habitats, specifically water availability, in North America as compared to its native
Eurasia. Being autopolyploid, it also has the potential to carry high levels of genomic
diversity, which could be reassorted through tetrasomic inheritance. Several species of
Lythrum in Europe are diploid and some exhibit similar traits to L. alatum. Some of these
species could be the natural progenitors of L. salicaria. For example, L. borystem'cum is
a dwarf species with either alternate or opposite leaf placement, and L. portula’s flowers
are placed solitarily in leaf axils (Tutin et al., 1968). If some of these species are the
original progenitors to the tetraploid L. salicaria, these seemingly new traits could
actually be hidden as recessives in the ancient polyploid genome and reassorted when L.
salicaria encountered new habitats in North America.

In conclusion, while purple loosestrife did not appear to hybridize with winged
loosestrife in North America, it does seem that there has been significant evolution within
purple loosestrife since its original arrival in North America. This evolution has allowed
purple loosestrife in North America to invade new habitats previously inaccessible to the

species, and may very well have contributed to the overall success as an invader of alien

habitats.

62

APPENDIX

AFLP data for global experiment. Msel primer for all combinations is CAG,

EcoRl primer is indicated. Molecular weight of fragment is indicated afier primer name.

Table 11

Presence of fragment is indicated with l, absence with 0. North American purple

loosestrife is bold, European purple loosestrife is italicized, and cultivars are underlined.

AAG650
AAG700
AAGI30
AAGI40
AAG150
AGGlooS
AGGI95
AGG220
AGGZSO
AGG270
AGGZooO
AGGZ9O
AGG300
AGG325
AGG325
AGG325
AGG350
AGG400
AGG400
AGG450
AGG450
AGG480
AGG480
AGG480
AGG480
AGGSOO
ACT190
ACT240
ACT270
ACT350
ACT375
ACT450
ACTSOO

 

000

C
b
a

d
C
b
a
d
C
b
a

000000000000000000000000000000000000000
000000000000000000000000000000000000000
lllllllllllllllllllll111111111000000000
000000000000000000000000000000111111111
lllllllllllllllllllll11111111111111.1111
lllllllllllllllllllll111111111111111111
l111111lllllllllllllllllllll111111111111
lllllllllllllllllllllll1.111111111111111
lllllllllllllllllllll11111111111111.1111
111111111111II.1.1111111111111111...111111111
00000000000000000000000000000000000000
1111111111110111111010100011111111.1111
00000000000000000000000000000000000000
00000000000000000000000000000000000000

1111111111llllllllllllllllllllllll1111

l

1111111111111111111111111111111111111]
00000000000000000000000000000000000000
1111111111111111111111111111111111.1111

llllllllllllllllllllllllllllllllllllll

1110110010

llllllllllllllllllll11111111111111.1111

llllllllllllllllllllllllllllllllllllll

l

111111111111111111111111111111111111.1111
l1111111111111111111111.111111111111111].
111111lll111ll111llllllllllllllllllllll
l1111111111111lllllllllllllllllllll1111
000000000000000000000000000000111111111
l11111111111111111111111111111111111111
lllllllllllllllllllll11111111111111.1111
111111111111111llllllllllllllllllllllll1|.
l11111111111111111111111111111111111111
O00000000000000000000000000000000000000
1111111lllllllllllllllllllll11111111111

000000000000000000000000000000000000000

 

 

63

Table 11 continued

AAG650
AAG700
AAGI3O
AAGl40
AAGISO
AGGlooS
AGG195
AGGZZO
AGGZSO
AGG270
AGGZooO
AGGZ9O
AGG300
AGG325
AGG325
AGG325
AGG350
AGG400
AGG400
AGG450
AGG450
AGG480
AGG480
AGG480
AGG480
AGGSOO

 

000

abc

abcdabab

0000000000000000000000000000000111111111111111
0000000000000000000000000000000111111111111111
0000000000000000000000000000000000000000000000
llllllllllllllll111111111111111000000000000000
1111111111111lllllllllllllllllloo0000000000000
ll11111111111111lllllllllllllllllllllll1111111
11111111111111]ll11lllllllllllllllllllllll1111
1111111111111111111llllllllllllllllllll1111111
1.111111111111111llllllllllllllllllll1111111111
1.111111111111111lllllllllllllllllllllll1111111
0000000000000000O000000000000001111111111111111
1110011100110111111111111111111000000000000000
0000000000000000000001110000000111111111111111
0000000000000000000000000000000111111111111111

1111111111111111111llllllllllllllllllllll11111
lllllllllllllllllllIllllllllllllllllllllllllll
000000000000000000000000000000011111111111111]
111111111111111111111111111111]11111111111111]
11111111111111.1111]..I.111111111111111111111111111
1111111111111111111111111111111000000000000000
1111111111111lllllllllllllllllloo0000000000000
11111111111111.1111111111111111111000000000000000
1111111111111111111111111111111000000000000000
1111111111111111111111111111111000000000000000
11111111111111.1111]111111111111000000000011111
111111111111111111111111111111111111111111111]
1111111111111111111111111lllllllllllllllllllll
ll11111111111ll1111111111111111000000000000000
1111111111111111llllllllllllllllllllll11111111

llllllllllllllll11111111111111111111111111.1111

 

 

64

Table 11 continued

650
700

1111
8543
5000

195
220
250
270
280
290
300
325
325
325
350
400
400
450

AAAAAAAAAAAAAAAAAAAAAAAAAA
GGGGGGGGGGGGGGGGGGGGAAAAA
GGGGGGGGGGGGGGGGGGGGGGGGG

 

000

abc

abcdabab

11111111111111111111111111111100000000000000000
l1.I.llllllllllllllllllllll111100000000000000000
0000000000000000000000000000000000000000000000
000000000000000000O000000000001111111111111111
0000000000000000000000000000001111111111111111

111111111111111111111111]lllllllllllllllllllll
.I.111111111111111111111111]lllllllllllllllllllll
l111111111111111111llllllllllllllllllll1111111
11.111111111111111lllllllllllllllllllll111111111
1111111111lllllllllllllllllllllll1111111111111
1l11111111111111111100000000000000000000000000
0000000000000000000000000000001111111111111111
.I.1111111111111111111111111111111011100111111110
11111111111111111111111111llllllllllllllllllll
.l.l111111111111111llllllllllllllllllllll1111111
111111000000000000O0000000000011111111111111111
l111111111ll1111111111111111110000000000000000
111001111111111111111.1111].llllllllllllllllllll
111001.I.l111111111llllllllllllllllllllll11111111
0000001000000000000000000000001111111111111111
0000001100000000000000000000001111111111111111
O00001100000000000000000000000111111111111.1111
0000000000000000000000000000001111111111111111
0010111111000001111100000000001111111111111111
1111101111111110000000000000001111111111111111
l11111llllllllllllllllllllll11111111111111.1111
1111111111llllllllllllllllll111111111111111111
000000000000000000000000000000111111111111.1111
111111111111.111111111111111llllllllllllllllllll

1100110000000000000000000000001111.1111].1111111

 

 

fi

Table l 1 continued

AAG650
AAG700
AAGl30
AAGI40
AAGISO
AGGlooS
AGGI95
AGG220
AGGZSO
AGGZ70
AGGZ80
AGGZ90
AGG300
AGG325
AGG325
AGG325
AGG350
AGG400
AGG400
AGG450
AGG450
AGG480
AGG480
AGG480
AGG480

 

abc

abcdabab

0000000000000000000000000000000000111111111111
0000000000000000000000000000000000111111111111
0000000000000000000000000000000000000000000000
111111111111lllllllllllllllllllllll000000000000
11111111llllllllllllllllllllll1111000000000000
111llllllllllllllllllllllllll11.111111111111111
llllllllllllllllllllllllllllllllllllllllllllll
111lllllllllllllllllllllllllllllllllllllllllll
11111111111111]1111111111111111.111111111111111
11111111111111111111.11111111111111111111111111111
0000000000000000000000000000000000111111111100
11111111111lllllllllllllllllllllllo00000000000
0000000000000000000000000000000001111111111111
11111111111111111111111111111111.111111111111111
111111111111111111111111llllllllllllllllllllll
111111111111111111111111111111111111000000000000
0000000000000000000000000000000000111111111.1111
1111111111111111111111111.111111111111111111.1111]
1111111111111111111111111.1111].1.111111111111111
11111111111.I.lllllllllllllllllllllloo0000000000
11111l111111111111111111111111.1111000000000000
1111111111111111111111111111111.1111111111111100
11111Ill111111111l111111111111111111000000000000
111111llllllllllllllllllllllll11111111111111.1100
llllllllllllllllllll1.1111111111111000000000000
111111111111lllllllllllllllllllllll11111111111
1111111111111.111111111111111111.111111111111111
11111.I.lllllllllllllllllllllll11111000000000000
llll1111111..11111111111111.111111111111111111.1111
11111llllllllllllllllllllllll1.1111000000000000

0000000000000000000000000000000000111111111111

 

 

66

Table 1 1 continued

650
700
130

1111
9854
5500

220
250
270
280
290
300
325
325
325
350
400
400
450

AAAAAAAAAAAAAAAAAAAAAAAAAA
GGGGGGGGGGGGGGGGGGGGGAAAAA
GGGGGGGGGGGGGGGGGGGGGGGGG

 

000

abc

abcdabab

11111111111111.1111].111111111000000000011111111
1111111111111111111111111111000000000011111111
0000000000000000000000000000000000000000000000
0000000000000000000000000000111111111100000000
0000000000000000000000000000111111111100000000
111111111111111111111.1111lllllllllllllllllllll
.I.11.111111111111111lllllllllllllllllll111111111
1.111111111111111111111lllllllllllllllllllll111
llllllllllllllllllllllllllllllllllllllllllllll
1111111111.111111111111111lllllllllllllllllllll
0000000011111111111111111111000000000011111111
0000000000000000000000000000111111111100000000
llllllllllllllllllllllllllllllllllllllllllllll
111111111111111111111.111111100000000001111111].
11111111111111.1111].111111lllllllllllllllllllll
0000000000000000000000000000111111111100000000
lllllllllllllllllll111111111000000000011111.1111
111111111111111111111111llllllllllllllllllllll
11111111111111.111qu111111lllllllllllllllllllll
0000000000000000000000000000111111111100000000
0000000000000000000000000000111111111100000000
0000000000000000000000000000111111111111111111
0000000000000000O00000000000111111111100000000
0000000000000000001111111lllllllllllllllllllll
0000000000000000001111111111111111111100000000
11111111111111.1111].111111111000000000011111111
Ill-11111111111111.1111llllllnnllll1.111111111111111
00000000ll111111111111111111111111111100000000
1111.I.1111111111111111111llllllllllllllllllllll
.00000OOOOBXKXYUOOOOOOOOAXXYUOOlllllli::}100000000

 

 

W

Table 11 continued

650
700

11111
98543
55000

220
250
270
280
290
300
325
325
325
350
400
400

AAAAAAAAAAAAAAAAAAAAAAAAA
GGGGGGGGGGGGGGGGGGGAAAAA
GGGGGGGGGGGGGGGGGGGGGGGG

 

000

abc

abcdabab

1.111111111110000000000000000000011111111110000
1111111111110000000000000000000011111111110000
0000000000000000000000000000000000000000000000
000O00O000001111111111111111111100000000001111
0000000000011111111111111111111100000000000000
1111111111111111111111111111111111111111111111
1111111111111111.1111111111111111111111111111111
11111111111111111111.11111111111100000000001111
11.1111111111111111.1111111111111100000000001111
11.11111111111111111111111111111100000000001111
1111111111110000000000000000000011111111110000
0000000000001111111111000000000000000000000000
111111111111111111111.1111111111111111111100000

1.111111111111111111111111111111100000000000000

11111111111100000000000000000000000000
00000000000011111111.111111111111000000
1.1111111111100000000000000000000111111
11111111111111111111111111111111000000
11111111111111111111111111111111000000
00000000000011111111111111111111000000
0000000000001111111111111111111.11000000
11111111111111111111111111111111000000
00000000000011111111111111111111000000
11111111111111.1111111111111111111000000
00000000000011111111111111111111000000
11111111111111111111111111111111111111
11111111111111111111111111111111111111
00000000000011111111110000000000111111
1.11111111111111111111111111111111111111
00000000000011111111111111111111000000
11.111111111100000000000000000000000000
1.1111111111111111111111111111111111111

1.1111111111100000000000000000000111111

 

WHH
WHH
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00001111
00001111
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00001111
00001111
00001111
00001111
00000000
00000000
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11111111
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11111111

11110000

man
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&

Table 11 continued

650
700

l
3
0

140

111
985
550

G

G

G

G

G

G

G
G220
G250
0270
G280
G290
G300
G325
G325
G325
G350
G400
G400
G450
G
G
G
G
G

 

000

abc

abcdabab

0000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000
1111111111111111111111111111111111111111111111
0000000000001110000001111100000111111111111111
1111111111111111111111111111111111111111111111
1111111111111111111111111111111111111111111111
1111111111111111111111111111111111111111111111
111111111111111111111111111111111111111111.1111
1111111111111111111111111111111111111111111111
0000000000000000000000000000000000000000000000
0111111000001100111111111111111111111111111111
0000001000000100111110000000000000000000000000
0000000000000000000000000000000000000000000000
111111111.11111111111111111111111111111111111111
11.1111111111111111111111111111111111111111111.1111
0000000000000000000000000000000000000000000000
11111111111111111111111111111111.111111111111111
11111111111111111111111111111111.111111111111111
1111111111100000111111111111111111111111111111
1111111111101100111111111111111111111111111.1111
0000000010110000000000000000000111111111111111
0000001000000000000000000000000111111111111111
0000000010010000000000000000000111111111111111
0000001010110000000000000000000.111111111111111
1.111111111111111111111111111111111111111111111
1.1111111111111111.111111111111111111111111111111
011111111111111111111111111111111111111111.1111
0111111111111111111110000011111000000000000000
1000000000001000000001111100000000000000000000
0000000000000000000000000000000111111111111111
1111111111111111111111111111111111111111111111

0000000000000000000000000000000000000000000000

 

612 4 IZWM523456123561 4 6mwwwm2345612 4 12346
mmmmmmmmmmmmmmwmummummmmmmwmmmmmwwmwmmmmmmmmmm

 

69

Table 11 continued

650
700
130
140
.150

ll
98
55

220
250
270
2000
290
300
325
325
325
350
400
400
450

AAAAAAAAAAAAAAAAAAAAAAAAAA
GGGGGGGGGGGGGGGGGGGGGAAAAA
GGGGGGGGGGGGGGGGGGGGGGGGG

 

0
0
0

abc

abcdabab

00000000000000111111000
00000000000000000000000
00000000000000000000000

lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
00000000000000000000000
lllllllllllllllllllllll
00000000000000000000000
00000000000000000000000

lllllllllllllllllllllll
lllllllllllllllllllllll
00000000000000000000000
lllllllllllllllllllllll
11111111111111111111111
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
00000111111111111111111
00000111111111111111111
00000000000000111000000
lllllllllllllllllllllll

00000000000000000000000

 

 

13456123 I. 3.1 3123
mmmmmflflmwgWEw_m_m_m_m_m_m_m_m_m__

m

 

Table 1 1 continued

llllllllllllllllllllllllllllllllllllll1
lllllllllllllllllllllllllllllllllllllll
000000000000000000000000000000000000000
000000000000000000000000000000000000000
11111llllllllllllllllllllllll1111111111
lllllllllllllllllllllllllllllllllllllll
1111111llllllllllllllllllllll1111111111
000000000000000000000000000000000000000
lllllllllllllllllllllllllllllllllllllll
000000000000000000000000000000000000000
000000000000000000000000000000000000000
000000000000000000000000000000111111111
1111111111llllllllllllllllllllo00000000
111ll111111llllllllllllllllll1111111111
1111111111111111lllllllllllllllllllllll
1111lllllllllllllllllllllllll1111111111
111111l11111111111lllllllllllllllllllll
1111111llllllllllllllllllllll1111111111
11111111111111111111lllllllllllllllllll
11111111111111111111lllllllllllllllllll
11111111111111111llllllllllllllllllllll
000000000000000000000000000000000000000
111ll11111111llllllllllllllllll11111111
000000000000000000000000000000111111111
1111111llllllllllllllllllllll1111111111

lllllllllllllllllllllllllllllllllllllll

 

QWA17 0

LLA]
LLA4
LLA6
LLAZS 0
LLA32 0
LLA37 0
LLA42 O
LLA49 0
LLAll 0
LLA19 0
CIAS
CIA10 O
CIAIS 0
CIA20 0
CIAZS 0
CIA30 0
CIA35 0
CIA40 0
CIA45 0
CIA50 O
SRA20 0
SRA30 0
SRA40 0
SRASO 0
SRA60 O
SRA35 0
SRA41
SRA43 0
SRASI
SRA53 0
QWA] 0
QWAIO 0

7l

 

 

Table 11 continued

lll1111111111111111111111111111111111111111111
ll11111lllllllllllllllllllll11111111111111.1111
000000000000000000000000000000011111111111.1111
000000000000000000000000000000011111111111.1111l
11.11111111111111111.1111111111111000000000000000
111111111111llllllllllllllllllloo0000000000000
111llllllllllllllllllllllllll1.1000000000000000
0000000000000000000000000000000111111111111111
llllllllllllllllllll11.111111111000000000000000
0000000000000000000000000000000.111111111111111
0000000000000000000000000000000.111111111111111
1.1111111111111111111111111111111.111111111111111
00000000000111111111111111111.11000000000000000
1.1111111111111111.11111111111111000000000000000
11111111111llllllllllllllllllllll1111111111111
10000000000O0000000001111111111.111111111111111
llllllllllllllllllll11111111111000000000000000
1.1111111111111111111111111111111000000000000000
1111111111111111111111111111111000000000000000
11111111111111.1111].111111111111001000000000000
11111111111111].1111l11111111111.111111111111111
0000000000000000000000000000000111111111111111
111111111111111111111111111111.11000000000000000
llllllllllllllllllll1111111111.1111111111111111
11111111111111].1111111111111110000000000000000

11111111111111]1111111111111111.111111111111111

 

72

 

 

 

Table 11 continued

 

llllllllllllllllllllllllllllllllllllllllllllll
llllllllllllllllllll11111111111111111111111111
llllllllllllllllllll11111111110000000000000000
.I.1111111111lllllllllllllllllllo000000000000000
00000000000000000000000000000011111111111.1111].
0000000000000000000000000000001111111111111111
0000000000000000000000000000001111111111111111
11111111111lllllllllllllllllllooo0000000000000
0000000000000000000000000000001111111111111111
llllllllllllllllllllll111111110000000000000000
ll1111l111llllllllllllllllllllooo0000000000000
1111111111111111111111111111100000000000000000
0000000000000000000000000000001111111111111111
0000000000000000000000000000001111111111111111

1111111111111111111111111111111111111111111111
llllllllllllllllllllllllllllllllllllllllllllll
0000000000000000000000000000001111111111111111
0000000000000000000000000000001111111111111111
0000000000000000000000000000000000000000111111
0000000000000000000000000000000000000000111111
llllllllllllllllllllllllllllllllllllllllllllll
111llllllllllllllllllll11111110000000000000000
0000000000000000000000000000001111111111111111
llllllllllllllllllllll111111100000000000111111
0000000000000000000000000000001111111111111111
llllllllllllllllllllllllllllllllllllllllllllll
111111111111.111111111111111111111111111111.1111
00000000000000000000000000000011111111111.1111]
1111111111lllllllllllllllllllllll1111111111111
llllllllllllllllllllll111111110000000000000000

llllllllllllllllllllll111111100000000000000000

 

930H113SBNHNM271 ll28HBMUB~A3HURM75HM$J$KwflWw.
mmmmmmm m mmmwwmmm mmmmmmmmmmmmmmmmmmm mmmmmmmmm

73

 

 

Table 11 continued

111111111111111111111111111.1ch11].1111111111111
llllllllllllllllll11111111111111.111111111111111
000000000000000000000000000000000011111111111]
0000000000000000000000000000000000111111111111
lllllllllllllllllll1.11111111111111000000000000
11111111lllllllllllllllllllllllll1000000000000
11111111111111111111111111111111.111000000000000
0000000000000000000000000000000000111111111111
l111111l111111111111111111111111111000000000000
0000000000000000000000000000000000111111111111
000000000000000000000000000000000011111111111].
0000000000000000000000000000000000111111111111
111111llllllllllllllllllllllll1111000000000000
111111llllllllllllllllllllllll1.111000000000000
1111111111111111111111111111111111111111111111
1111111111111111111111111111111llllllllllllllll
11111111ll11111111111111111111111.111000000000000
llllllllllllllllllllll111111111111000000000000
1111111111111111111llllllllllllllllllllllll100
111111ll11lllllllllllllllllllllll1111111111100
1111111111111111111llllllllllllllllllllllll111
0000000000000000000000000000000000111111111111
11111111111111111111111111111111111000000000000
111111111111110000000000llllllllllllllllllllll
11111111111111111111111111111111111000000000000

111111ll11111111111lllllllllllllllllllllllllll

111111ll111111111111111111111111].1111111111111
000000000000000000000000111111111111
000000

 

BRI-3l 0 O
BRI-35 0 0
3111-42 0 0
BRI-43 0 O
DCR-2 0 0
DCR-7 0 0
DCR-11 O O
DCR-14 0 0
DCR-19 0 O
DCR-21 O 0
DCR-22 O

DCR-23 0

DCR-24 0

DCR-25 0

74

 

 

Table 1 1 continued

1.11111111111111111111111111111.111111111111111111
1111111111111lllllllllllllllllllllll1111111111
1.111111111111111111111111111000000000011111111
llllllllllllllllllll111111110000000000111111111
0000000000000000000000000000111111111100000000
0000000000000000000000000000111111111100000000
0000000000000000000000000000111111111100000000
111111111111111.11111111111111000000000011111.1111
0000000000000000000000000000111111111100000000
1.111111111111111111111111111000000000011111111
llllllllllllllllllll11111111000000000011111111
1111111111llllllllllllllllllo000000000111111111
0000000000000000000000000000111111111100000000
0000000000000000000000000000111111111100000000
llllllllllllllllllll11111111111111111111111111
1111111111111lllllllllllllllllllllll1111111111
0000000000000000000000000000111111111100000000
0000000000000000000000000000111111111100000000
0000000000000000000000000000111111111100000000
0000000000000000000000000000111111111100000000
11.11111111111111111111111111111.11111111111111111
1.1111111111111111111111111110000000000111111.1111
0000000000000000000000000000111111111100000000
111llllllllllllllllllll11111111111111111111111
0000000000000000000000000000111111111100000000
11111111111111.1111lllllllllllllllllllllll1111111
llllllllllllllllllll111111111111111111111111111

 

75

 

 

 

Table 11 continued

1111111111111111111lllllllllllllllllllll1111111
l111111l11111111111l111111llllllllllllllllllll
1111111111110000000000000000000011111111110000
l111111111110000000000000000000011111111110000
00000000000011llllllllllllllllllo0000000001111
0000000000001lllllllllllllllllllo0000000001111
0000000000001111111111111111111100000000001111
111111111111000000000000000000001111111111000O
O00000000000111111111111111111110000000O001111
1111111111110000000000000000000011111111111111
1111111111110000000000000000000011111111111111
11111111111100000000000000000000111111111.1111].
000000000000llllllllllllllllllllo0000000000000
0000000000001111111111111111111100000000001111
11111111111111111111llllllllllllllllllll111111
111111111111111111ll111111llllllllllllllllllll
0000000000001111111111111111111100000000000000
O000000O0000llllllllllllllllllllo000000000000O
0000000O00001111111111111111111100000000001111
0000000000001111111111000000000000000000001111
l111111111111111111111111111.1111].1111111111111
1.11.1.1111.11i1.1110AUnuOAUnVOAUnUOAUnVOAUnV0Aunu0nU1i1.1111.11.l.11.0nunu0
0nunVOAUnVOAUnuOAUnul.1111.11.l.1111.1111.11.1.11.1.1nvOAUnVOAUnuOnunVOAUnVO
1111111l111111111111111111llllllllllllllllllll
0000000000001111111111111111111100000000001111
1111111111111llll111111111llllllllllllllllllll
l11111111111111111ll111111llllllllllllllllllll
1.111111111110000000000000000000011111111110000
1111111.I.1111.I.l11111111111111111111111111111111
0011111111110000000000111111111111111111110000

11111111111.10000000000000000000011111111110000
6nv 117.4.Dcu7.0 0.1«a1.4 m
Dnu

umnmnnnnn1579n
mm m m; wwwwmwwmwwmmwm

 

CflflN4
ON“M
KTA3
KTA6
KTA9
KTAl
KTAI
KTAl
KTAl
KTAl
KTAl
KTA2
FTAI
FTA4
FTA7
FTAS
FFAll
FFAIZ
“flql
SHEL3
SHEY7
SHE21
SHEZS
SHEfl?
[MRI
DERZ

76

 

 

Table 11 continued

11111111111ll1lllll111lllllllllllllllllllllll1
111111111111.1111111111111111].1.1111111111111111
0000000000000000000000000000000000000000000000
0000000000000000000000000000000000000000000000
111111111110000111111111111lllllllllllllllllll
1111111111100000000000000000000000000000000000
1111111110111011111111111111lllllllllllllllllll
0000000000000000000000000000000000000000000000
11111111100lll1111111111111lllllllllllllllllll
1000000000000000000000000000000000000000000000
1000000000000000000000000000000000000000000000
1111111111111111lll1111111111111111111111.1111]
0000000000000000000000000000000000000000000000
llllllllllllllllllllllllllllllllllllllllllllll
1111111111111111111111111111]1.1111111111111111
11111111111llllllllllllllllll11.111111111111111
0000000000011111000000000000000000000000011111
O000000000000000000000000000000000000000000000

1111111111111lll111111llllllllllllllllllllllll
1111111111111111111111llllllllllllllllllllllll
1111ll11111lll11111111llllllllllllllllllllllll
0000000000000000000000000000000000000000000000
0000000000000001000001111100000000000001101110
1111111111111111111111111111111111111111111111
llll1111111lll11111111llllllllllllllllllllllll
111l111111111l11111111llllllllllllllllllllllll
1111111111111l1111111111llllllllllllllllllllll
0000000000000000000000000000000000000000000000
l11111111111111111111111111lllllllllllllllllll
0000000000000000011001110000000000001000000000
0000000000000000000000000000000000000000000000

mmmmmmmmmmmw $mewwwwwmmmmmmmmmmmmwmmmm mwwmmmmm

 

77

 

 

Table 11 continued

lllllllllllllllllllllll
lllllllllllllllllllllll
00000000000000000000000
00000000000000000000000
lllllllllllllllllllllll
00000000000000000000000
lllllllllllllllllllllll
00000000000000000000000
lllllllllllllllllllllll
00000000000000000000000
00000000000000000000000
lllllllllllllllllllllll
00000000000000000000000
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
Ollllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
lllllllllllllllllllllll
00000000000000000000000
00000000000000000000000
lllllllllllllllllllllll
lllllllllllllllllllllll
llllllllllllllllllllll

11111111000000000000000
00000000000000000000000
lllllllllllllllllllllll
00000000000000000000000
00000000000000000000000

mmmmmmmmggmmwmmmmmwm

 

P_S_1_
El
PS3

78

 

S
. 1111111111
a 6.“ ATAG3750000000000000000000011111111111111111
Mfl.mp.ATAG47511111111111111.11111100000000000000000
um pdEACGGI800000000000000000000000000111110000000
. es
.N.» .A 66 1111111111111llllllllllllllllllllllll
fo C 330
S
mammu.BACGG4250000000000000000000011111000000000000
pl 1A 0000000000000000000000001111000000000
00 tCAAAC24O
. a 11 111111
c csAAAG475
.M«OubME.MACACIaJSllllll111.111.111.11.11100000000000000000
.MmmflACACl680000000000000000000011111111111111111
l .I.111111111111111111111.111111111111111
MMmmPACAC310
6 rr 0000000000000000000011111111111111111
mCCCIACAC37O
emwoneflACAC3750000000000000000000000000111110000011
«OlmllCACACSOOOOOOOOOOOOO00000000011111111111111100
m.smmwACAG4650000000000000000000011111111111111111
.1881
.101
zwnn
lwmmnm 529 7HM” % 41
12 1. 03721720
mmwmm 16w 4mmmmmlm3 1678wmmsmmMMMmmmmmmw13
o o t
.1?th L L LQQQQstsss H mm mmSSSSSAAAAA

101010111110010000111000010000111
79

101010111110010000111000010000111
10101011l110010000111000010000111

WFBB
WFBl7
WFB34

TCGC375111110000000000000000
TCGC450000000000000000000001
TCGC475111110000000000000000
TCCCIOOOO0000000000000000001
TCCCIIOllllll11111111111111]
TCCCISOOO0000000000000000001
TCCC160000000000000000000001
TCCCIOOOOOOOOOOOO000000000000
TCCC230111111.111111111111110
TCCCZSOOOOOO0000000000000001
TCCC30011llllllllllllllllllo
TCCC4251111111.111111111111111
TCCT95 llllllllllllllllllllo
TCCTIOOOOOOOO00000000000000].
TCCTIIOllllll11111111111111]
TCCT130000000000000000000001
TCCT175000000000000000000001
TCCTlooOll11111111111111.1111].
TCCT230000000000000000000001
TCCTZSOlllll11111111111111ll
TCCT27511111111111111111.1111
TCCT3101111111111111111.1111].
TCCT350111111111111111111111
ATAGI35111111111111111111110
A 111111111

Table 12 continue

1000101111111011101100101011110

HIW2

100010111111101110110010101111010
100010111111101110110010101111000
100010111111101110110010101111000
100010111111101110100010111111000
100010111111101110100010111111000
100010111111101110100010111111000
10001011ll11101110100010111111000
100010111111101110100010111111000
100010111111101110100010111111000
100010111111101110100010111111000

HIW8

HIW14

HIW2 1
SFA6

SF A7

SFAlO
SFA23
SF A37
ASP12
ASPZ l
ASP27
ASP32
ASPSO

WFBl

1000

l

000101111111011101000101111

1
100010111111101
100010111111101

000
000

10001011l111101110100010101111000
100010111111101110100010101111000
10001011ll11101110100010101111000
100010111111101110100010101111000
100010111111101110100010101111000

11
11

ll
11

1
1

1011
1011

101000
101000

WFB3
WFB8
WFBl7
WFB34

80

.w

.m

n

t

Table 12 co

TGCC2450
TGCCZSOI
TGCC2601
TGCC3100
TGCC3301
TGCC4251
TGCC4500
TCGC75 l
TCGC80 0
TCGC85 0
TCGC90 l
TCGCIIOO
TCGC1200
TCGC1300
TCGC1601
TCGCISOO
TCGCZOSO
TCGC2300
TCGC2350
TCGC2600
TCGC3001
TCGC3101
TCGC3200
TCGC3301

LLA]
LLA6

10110000010001001011011000

10110000010001001011011000

LLAZS

101100000100010010110110001100

LLA32
LLA49

1011000001000100101101100011001

01101000100010010110110001100110

ll

01101001100000000110110010100010
101101001100000000110110010100010

101101001100000000110110010100010
1011010011000000001101100101000

0000001

1
1
l
l
1
1
0

1001000001000000001101100001000
100100000100000000110110000100

HIS]

H156

0
0

01000
01000

0101001000011011010010111000010

100100000100000000110110000100

10010000010000000011011000

HIS7

H188

10010000010000000011011000

HISIO

HIW198

1

100
1

10010000110110100101110000

HIW2

1
1
1

00

10000110110100101110000100
0010000110110100101110000100

10000110110100101110000
0110001101100001011100001001

0110001101100001011100001001

010110110001101100001011100001001
010110110001101100001011100001001

00

ll

HIW8
HIW14
HIW2 l
SFA6
SFA7
SFAlO

1
1

110110001101100001011100001001
110110001101100001011100011001
110110001101100001011100011001
110110001101100001011100011001

00

00
010110110011101100001011100011001
010110110011101100001011100011001
01011011001110]100001011100011001
010110110011101100001011100011001
010110110011101100001011100011001

WFBB

WFB8

WFB17
WFB34

81

d
m
.m

n

t

Table 12 co

0000000000001111

TTGC47500000
TTGCSOOlllll
TTGC75011111
TTGC77511111
TGGC10511111

000

111
111
111
111

111

TGGC13500000000
TGGC15300000000
TGGC17000000000
TGCC75 00000000
TGCC10500000000

TGCC1070000

l10101101100000000101011111

111
111
111
111
111
000
111
000
111
00
00
00
00
00
00
00
00
11
11
00
11
11
00
11
00
11

11

00

100110101101100000000

111111111

1

l

1001101011011000000001010

11111

11111

00000
11000
00000
00000
00000
00000
00000
00000
00000
00000
11111
11111
00000
11111
11111
00000
11111
00000
11111
11111
00000
00000
11111

1652» 7mun1un 11 7
mmmmmWMMMMwmmmwmmm

111
111
110
110
000

111

10011010110110000000000
0011010110110000000000
1111011111111111001

1
1
1
0
0

1111111100110

1
1

1

1

11

1

l

1

11111100110001

111111110011000
l

1
1
11
11
00
11
11
11
11

00

00000000
100000

111111

10

l

10111001111111111110111001110

101110011111111111101110011

1111
1111
0000
0000
0000
1111
1111
0000
0111
1111
1111
1111
1111
1111
1111
1111
1111
1111
1111
1111
0000
1000

11111

0011111111111101100011

11111
11111

00000

0111001111111111110110001

O
0
0
0
0
l
l
1
0
0
0
0

1
1
1
l
l
l
l
1
1
1
1
l
l

1011100111000
0
0

l
1
1

1
1
1

01011100
1
1

00111111111111011100111000
00111111111111011100111000

100111111111111011100111000

101011100111111111111011100111000
101011100111111111111011100111000

1
1

1
1
1

10101
10101
10101

WFBl7

WFB34

82

TTGC14000000000000

TTGC14500000000000

TTGC17100000000000

TTGCISSOOOOOOOOOOO

TTGCZIOOOOOOOOOOOO

TTGC21500000000000

mm

Table 12 cont'

LLAZS
LLA49
QWB7
QWBIS
QWBZ4
QWBZ9
SFB]

LLA]
LLA6

00

ll
00
11
ll
00
ll
11
00
00
00

11
ll
11

ll

SFB32
SFB34

SFBll

100011011010
100011011010

11

SFB41
HISl

0000Ollllllllllllllllllll
1111100000000000000000000
0000011111111111111111111
1111100000000000000000000
1111100000000000000000000
0000011111111111111111111
11111111111111]1111111111
111111111111111111111111]
00000111111.111111111111111
0000Ollllllllllllllllllll
0000000000000000000011111
1111100000000000000000000
11111111111111]1111111111
1111100000000000000000000
1111100000000000000000000
0000Ollllllllllllllllllll
0000011111111111111111111
1111100000000000000000000
1111100000000000000000000
1111100000000000000000000
0000011111111111111111111
111111111111111111111111]
1.111111111111111111111111
1111lllllllllllllllllllll
1111100000000000000000000
1111100000000000000000000
11111111111111.11111111111
lllllllllllllllllllll1111
1111100000000000000000000
0000011111111111111111111
1111100000000000000000000

llllllllllllllllllllll111

1.111100000000000000000000

WFBI7
WFB34

HIS7
H1510
HIW198
HIW2
HIW8
HIW14
HIW2 1
SF A6
SFA7
SFAlO
SFA23
SFA37
ASPIZ
ASP2 l
ASP27
ASP32
ASPSO
WFBl
WFB3
WFBS

83

TAAG3050
TAAG3300
TAAG3350
TAAG4500
ATGGI750
ATGG3301
ATGG4251
ATGGGOOO
ATGG7001
ATAC1601
ATAC1701
ATAC1751
ATAC2251
ATAC2751
ATAC2901

TTGC117

TTGCIZO
TTGC1250
wTTGCIZooO
.mTTGCl351
HTTGC1381

t

Table 12 co

11101011010111111101100000
11101011010111111101100000

11000111101011010111111101100000
11000111101011010111111101100000
11000111101011010111111001100000
11000111101011010111111001100000
1100011110101101011111100110000

l

l

1

LLA]

LLA6

LLAZS
LLA32
LLA49
QWB4
QWB7

0
O
0
0
0

0
l
l

000
000

11
ll

0

0

0

0001111010110101111110011000

11000111101011010111111001100010
11000111101011010111111001100010
11000111101011010111111001100010
11000111101011010111111001100000
11000111101011010111111001100000
11000111101011010111111001100000
11000111101011010111111001100000
11000111101011010111111001100000
01111101011100101000010011111101
011l1101011100101000010011111101
011ll101011100101000010011111101

100110000
011ll101011100101000010011111101

100
100

QWBIS
QWBZ4
QWBZ9
SFB]
SFBll
SFB32
SFBM
SFB41
HISl
H186
HIS7
H158
H1810
HIW198

11101011100101000010011111101

101011100101000010011111101

ll

101011100101000010011111101
101011100101000010001111101
101011100101000010001111101
101011100101000010001111101

01011100101000010001111101

01111101011100101000010011111101
01111101011100101000010011111101
01111101011100101000010011111101
01111101011100101000010001111101
01111101011100101000010001ll1101

lll1101011100101000010001111101

011ll101011100101000010001111101

WFBl7
WFBB4

84

AGG450b
AGG450a
AGG480d
AGG480C
AGG480b
AGG4803
AGGSOO
ACT190 ab
ACT240
ACT270
ACT3503b
ACT375
ACT450
ACT500
TAAC218
TAAC220
TAAC228
TAAC230
TAAC231
TAAC240
TAAC245
TAACZSO
TAAC300
TAAGZOS
TAAGZIO
TAAGZZO
TAAGZZS
TAAG230
dTAAG265
WTAAGZ75
.MTAAG29O
TAAG300

11

Table 12 co

1010110001010001011110111111

1

 

00

1011000101000
1011000101000

0
0

101011000101000
0111101011000
0
0
0

l
1

1
1
1

111

1111

1111

1111

1
l
1
l

10 0

0 0

0 0
100010100010111111

1101011000101000

011010111101111

10
10

0
l

1 111111111
1010110000

1

l
0

l
l

01

001101011110111111101011000000
001101011110111111101011000000
001101011110111111101011000000

00000
00000

1
l

010
010

1
l

l
l

l
l

l
l
11001101011110111111111011000000

11001101011110111111111011000000

11001101011110111111111011000000

11001101011110111111111011000000
1

00110 011101111101
00110011101111111

11
ll
11
11
ll

HIW2

HIW8

HIW14
HIW2 l
SFA6

SFA7

SFA10
SFA23
SFA37
ASP12
ASPZ l
ASP27
ASP32
ASPSO

WFB]

1100000

0011010111101] 0101

1 l

00
00
00
00
10
11.
1.1
11
00
11
11
1.1
11
ll
11
ll

11

101
101

ll
11
11001101011110111111111011100000

11001101011110111111101011110000
11001101011110111111111011010100
11001101011110111111101011110111
11001101011110111111101011110010
11001101011110111111101011110000
l10011010111101]1111101011100000

1100110101
1100110101

WFBB
WFB8
WFB l7
WFB34

85

Table 12 continued

ACG225

ACG350b

ACG350
ACG400
ACG425
ACG450
ACG490
ACGSOO
AAGl70
AAGI95
AAG350
AAG450
AAG475
AAGSOO
AAG650
AAG700
AAGI30
AAG140
AAGISO
AGGlooS
AGGI95
AGGZZO
AGGZSO
AGGZ70
AGG280
AGGZ90
AGG300
AGG325

a

0
0
0
3b
8b
ab
3b

3b
3b
3.0
C

AGG325b

A0632.)
66350

3
3.0

A
A
A

GG400b
GG4008

110110010111111010000111110101111
110110010111111010000111110101111
110110010111111010000111110101111
110110010111111010000111110101111
110110010111111010000111110101111
110110010111111100000111111101111
110110010111111100000111111101111
110110000111111100000111111101111
110110000111111100000111111101111
110110010111111100000111111101111
110110010111111100000111111101111
110110000111111100000111111101111
110110010111111100000111111101111
110110010111111100000111111101111
110110010111111100000111111101111
110110110111111100000111111101111
110110110111111100000111111101111
110110110111111100000111111101111

LLA]

LLA6

LLAZS

LLA32

LLA49

QWB4
QWB7

QWBIS

QWBZ4

QWBZ9

SFBl

SFBll

8FB32

8FB34

SFB41
H181

H186
H187

110110010111111100000111111101111
110110010111111100000111111101111

H188

H1810

111011100111110001111101101011000

I-IIW198

111011100111110001111101101011000

HIW2

111011100111110001111101101011000

HIW8

HIW14

l11011100111110001111101101011000

111011100111110000001101100011000
111111101111110001111101101011000
111111101111110001111101101011000

111111101111110001111101101011100
111111101111110001111101101011000

HIW2 1
SFA6

SFA7

SFAlO
SFA23
SFA37
ASP 12
A8P2 1
ASP27
ASP32
ASPSO

WFB]

111111101111110001111101101011000
001111101111110001111101101011000
00111110111111000]111101101011000

111111101111110001111101101011000
111111101111110001111101101011000
111111101111110001111101101011000
lll111101111110001111101101011000

111011101111110001111101101011000

111011101111110001111101101011000
111011101111110001111101101011000
111011101111110001111101101011000

WFB3
WFBS
WFB17
WFB34

86

 

d
C

m
n

11

t.

Table 12 co

ACC90d
ACC90C
ACC90b
ACC90a
ACC100
ACC120 ab
ACC160
ACCZZSC
ACC225b
ACC2258
ACC3203b
ACC330
ACC475
ACGlooSC
ACGlooSb
ACGloosa
ACGZZO

11111000101110011
11111000101110011
11111000101110011
11111000101110011

LLA]
LLA6

LLAZS
LLA32

LLA49

11111000101110011
10110100101110011
10110100101110011

QWB4
QWB7

10110100101110011
10110100101110011

QWBlS

QWB24

10110100101110011
10111100101110011
10111100101110011
10111100101110011
10111100101110011

QWBZ9

SFBl

SFBll

SFB32

817334
SFB41
H181

10111100101110011
11111100101110011
11111100101110011
11111100101110011
11111100101110011
11111100101110011
01100111010001111
01100111010001111

H186

H187

H188

H1810

HIW198

HIW2

HIW8

01100111010001111
01100111010001111

HIW14

01100011010001111
01100111010001111
01100111010001111
01100111010001111
01100111010001111

HIW21

SFA6

SFA7

SFA10
SFA23
SFA37
ASP12
ASP2 l
ASP27
ASP32
ASP50

WFBl

01100111010001111
01100111010001111

01100111010001111
01100111010001111
01100111010001111
01100111010001111
01100111010001111

01100111010001111
01100111010001111
01100111010001111
01100111010001111

WFB3

WFB8

WFB17

WFB34

87

LITERATURE CITED

Abbott RJ. (1992). Plant invasions, interspecific hybridization and the evolution of new
plant taxa. Trends in Ecology & Evolution 7: 401-405.

Anderson, E. (1949). Introgressive Hybridization. Wiley, New York.

Anderson, N. O. and P. D. Ascher (1993). Male and Female Fertility of Loosestrife
(Lythrum) Cultivars. Journal of the American Society of Horticultural Science 118(6):
851-858.

Anderson, N. O. and P. D. Ascher (1994). Erosion of Style/Anther Length Integrity in
Introgressive Lythrum Hybrids. In “Pollen-Pistil Interactions and Pollen Tube Growth”
(03 Stephenson and T-h Kao, Eds), 269-272. American Society of Plant Physiologists,
Rockville, MD.

Angiolillo, 0., M. Mencuccini, and L. Baldoni (1999). Olive genetic diversity assessed
using amplifed fragment length polymorphisms. Theoretical and Applied Genetics 98:
41 1-421.

Arnold, ML. (1993). Iris nelsonii (Iridaceae): Origin and genetic composition of a
homoploid hybrid species. American Journal of Botany 80: 577 - 583.

Arnold, M.L., J.L. Hamrick and ED. Bennett (1990). Allozyme variation in Louisiana
irises: 0 test for introgression and hybrid speciation. Heredity 65: 297 - 306.

Arnold, M. L. J. J. Robinson, C. M. Buckner and B. D. Bennett (1992). Pollen dispersal
and interspecific gene flow in Louisiana irises. Heredity 68: 399 - 404.

Bai, G., M. Ayele, 1. Tefera, and l. T. Nguyen (1999). Amplified Fragment Length
Polymorphism analysis of Tef [Eragrostis tef (Zucc.) Trotter]. Crop Science 39: 819 -
824.

Batra, S. W. T., D. Schroeder, P. E. Boldt, and W. Mendl (1986). Insects associated with
purple loo sestrife (Lythrum salicaria L.) in Europe. Proceedings of the Entomology
Society Washington 88(4): 748-759.

Blackwell, W. 1. (1970). The Lythraceae of Ohio. Ohio Journal of Science 70: 346-
352.

Blossey, B. (1995). 0 Comparison of Various Approaches for Evaluating Potential
Biological Control Agents Using Insects on Lythrum salicaria. Biological Control 5:
113-122.

88

Cody, W. J. (1978). The status of Lythrum alatum (Lythraceae) in Canada. Canadian
Field Naturalist 92: 74-75.

Cutright, N. J. (1986). Regulation of purple loosestrife by states in the Midwest.
Proceedings of the North Central Weed Control Conference 41: 123-125.

Darwin, C. (1877). The different forms of flowers on plants of the same species.
London: Murray.

Dodd, 0. P. (1959). in Biogeography and Ecology in Australia, eds. Keast, 0/. R. L.
Crocker, and C. S, Christian. Monographiae Biologicae no. 8 (W. Junk, the Hague).

Ellstrand, N. C. and K. 0. Schierenbeck (2000). Hybridization as a stimulus for the
evolution of invasiveness in plants? Proceedings of the National Academy of Sciences
97(13): 7043-7050.

Ellstrand, N. C., R. Whitkus, and L. 1. Rieseberg (1996). Distribution of spontaneous
plant hybrids. Proceedings of the National Academy of Sciences 93: 5090-5093.

Ewel, J. J., D. J. O'Dowd, J. Bergelson, C. C. Daehler, C. M. Antonio, L. D. Gomez, D.
R. Gordon, R. J. Hobbs, 0. Holt, and K. R. Hopper (1999). Deliberate introductions of
species: research needs. BioScience 49: 619-630.

Fisher, R. 0. (1944). Allowance for double reduction in the calculation of genotypic
frequencies with polysomic inheritance. Annual Eugenics 11: 31.

Fisher, R. 0. (1949). The linkage problem in a tetrasomic wild plant, Lythrum salicaria.
Heredity Supplement p. 223.

Graham, 8. (1975). Taxonomy of the Lythraceae in the Southeastern United States.
SIDA 6(2): 80-103.

Heiser, C. B. (1947). Hybridization between the sunflower species Helianthus annus and
1. petiolaris. Evolution 1: 249 - 262.

Heiser, C. B. (1949). Study in the evolution of the sunflower species Helianthus annus
and 1. bolanderi. University of California Publications in Botany 23: 157 - 208.

Hill, M., 1. Witsenboer, M. Zabeau, P. Vos, R. Kesseli, and R. Michelmore (1996).
PCR-based fingerprinting using AFLP's as a tool for studying genetic relationships in
Lactuca ssp. Theoretical and Applied Genetics 93: 1202-1210.

Hufl‘aker, CB. and C. E. Kennett (1959). Journal of Range Management 12: 69-82.

Kok, L. T., T. J. McAvoy, R. 0. Malecki, S. D. I-Iight, J. J. Drea, and J. R. Coulson
(1992). Host Specificity Tests of Hylobius transversovittatus Goeze (Coleoptera:

89

 

Curculionidae), a Potential Biological Control Agent of Purple Loosestrife, Lythrum
salicaria L. (Lythraceae). Biological Control 2: 1-8.

Kok, L. T., T. J. McAvoy, R. 0. Malecki, S. D. Hight, J. J. Drea, and J. R. Coulson
(1992). Host Specificity Tests of Galerucella calmariensis (L.) and G. pusilla (Dufi)
(Coleoptera: Chrysomelidae), Potential Biological Control Agents of Purple Loosestrife,
Lythrum salicaria L. (Lythraceae). Biological Control 2: 282-290.

Krebs, S. L. and J. F. Hancock (1989). Tetrasomic inheritance of isoenzyme markers in
the highbush blueberry Vaccinium corymbosum L. Heredity 63: 11-18.

Lam, S. L. (1974). Origin and formation of unreduced gametes in the potato
[Chromosomes]. Heredity 65(3): 175-178.

Levin, D. 0. (1970). Assortative Pollination in Lythrum. American Journal of Botany
57(1): 1-5.

Levin, D. 0. (1983). Polyploidy and novelty in flowering plants. American Naturalist
122: 1.

Lindgren, C. J. and R. T. Clay (1993). Fertility of ‘Morden Pink’ Lythrum virgatum L.
Transplanted into Wild Stands of L. Salicaria L. in Manitoba. HortScience 28(9): 954.

Louis-Marie, P. (1944). La Salicaire dans le Quebec. Institut Agricole d’Oka. Prov. De
Quebec. 46 pp.

Lumaret, R. (1986). Doubled duplication of the structural gene for cytosolic
phosphoglucose isomerase in the Dactylis glomerata L. polyploid complex. Molecular
Biological Evolution 3: 499.

Mack, R. N. (1991). The Commercial Seed Trade: An Early Disperser of Weeds In the
United States. Economic Botany 45(2): 257 - 273.

Maughan, P. J ., M. 0. Saghai Maroof, G. R. Buss, and G. M. Huestis (1996). Amplified
fragment length polymorphism (AF LP) in soybean: species diversity, inheritance and
near-isogenic line analysis. Theoretical and Applied Genetics 93: 392-401.

Ottenbreit K. 0. and R. J. Staniforth (1994). Crossability of naturalized and cultivated
Lythrum taxa. Canadian Journal of Botany 72: 337-341.

Paul, S., F. N. Wachira, W. Powell, and R. Waugh (1997). Diversity and genetic

differentiation among populations of Indian and Kenyan tea (Camellia sinensis (L) O.
Kuntze) revealed by AFLP markers. Journal of Heredity 84: 34 -42.

90

Powell, W., M. Morgante, C. Andre, M. Hanafey, J. Vogel, S. Tingey, and 0. Rafalski
(1996). The comparison of RFLP, RAPD, AFLP and SSR (microsattelite) markers for
gerrnplasm analysis. Molecular Breeding 3: 225-238.

Pursh, F. (1814). Flora Americae Septentrionalis: or, a Systematic Arrangement and
Description of the Plants of North America. 2 Volumes. White, Cochrane and Co.,
London. Xxxvi, 751pp. + 24 t.

Qu, L., J. F. Hancock, and J. 1. Whallon (1998). Evolution in an Autopolyploid Group
Displaying Predominantly Bivalent Pairing at Meiosis: Genomic Similarity of Diploid
Vaccinium darrowi Camp and Autotetraploid V. corymbosum (Ericaceae). American
Journal of Botany 85(5): 698-703.

Rieseberg, L. l.(1995). The role of hybridization in evolution: old wine in new skins.
American Journal of Botany 82(7): 944 - 953.

Rieseberg, L. l, 1. Choi, R. Chan, and C. Spore (1993). Genomic map of a diploid
hybrid species. Heredity 70: 285 - 293.

Rieseberg, L. 1. and N. C. Ellstrand (1993). What can molecular and morphological

markers tell us about plant hybridization. Critical Reviews in Plant Science 12: 213 -
241.

Rieseberg, L. 1., D. E. Soltis, and J. D. Palmer (1988). 0 molecular examination of

introgression between Helianthus annus and 1. bolanderi (Compositae). Evolution 42:
227 - 238.

Rieseberg, L. l. and D. 0. Warner (1987). Electrophoretic evidence for hybridization
between T ragopogon mirus and T. miscellus (Composidae). Systematic Botany 12: 281
- 285.

Riley, 1. P. (1938). 0 character analysis of colonies of Iris hexagona var. giganticaerulea
and natural hybrids. American Journal of Botany 25: 727 - 738.

Roa, 0. C., M. M. Maya, M. C. Duque, J. Tohme, 0. C. Allem, and M. W. Bonierbale
(1997). AFLP analysis of relationships among cassava and other Manihot species.
Theoretical andApplied Genetics 95: 741-750.

Samuel, R. W. Pinsker and F. Ehrendorfer (1990). Allozyme polymorhism in diploid and
polyploid populations of Galium. Heredity 65:369-378.

Shore, J. S. (1991). Tetrasomic inheritance and isozyme variation in T urnera ulmifolia
vars. Elegans Urb. And intermedia Urb. (Tumeraceae). Heredity 66: 305.

91

Soltis, D. E. and L. 1. Reiseberg (1986). Autopolyploidy in Tolmiea menziesii
(Saxifragaceae): genetic insights from enzyme electrophoresis. American Journal of
Botany 73: 310.

Soltis, D. E. and P. S. Soltis (1989). Genetic consequences of autopolyploidy in T olmiea
(Saxifi'agaceae). Evolution 43: 586.

Soltis, D. E. and P. S. Soltis (1993). Molecular Data and the Dynamic Nature of
Polyploidy. Critical Reviews in Plant Sciences 12(3): 243 —- 273.

Soltis, D. E. and P. S. Soltis (1995). The dynamic nature of polyploid genomes.
Proceedings of the National Academy of Sciences USA 92: 8089 - 8091.

Soltis, D. E. and P. S. Soltis (2000). The role of genetic and genomic attributes in the

success of polyploids. Proceedings of the National Academy of Sciences USA 97: 7051
- 7057.

Stebbins, G. L. (1950). Variation and Evolution in Plants. Columbia University Press,
New York, 1950.

Stebbins, G.L. (1971). Chromosomal Evolution in Higher Plants. Arnold, London.

Strefeler, M. S., E. Danno, R. L. Becker, and E. J. Katovich (1996). Isozyme
characterization of genetic diversity in Minnesota populations of purple loosestrife,
Lythrum salicaria (Lythraceae) American Journal of Botany 83(3): 265-273.

Stuckey, R. L. (1980). Distributional History of Lythrum salicaria (Purple Loosestrife)
in North America. Bartonia 47: 3-20.

Tohme, J., O. D. Gonzales, 8. Beebe, M. C. Duque (1996). AFLP analysis of gene pools
of a wild bean core collection. Crop Sciences 36: 1375 - 1384.

Veronesi, F., 0. Mariani, and E. T. Bingham (1986). Unreduced gametes in diploid
Medicago and their importance in alfalfa breeding. Theoretical and Applied Genetics
72(1): 37-41.

Vos, R, R. Hogers, M. Bleeker, M. Reijans, T. van de Lee, M. Homes, 0. Frijters, J. Pot,
J. Peleman, M. Kuiper, and M. Zabeau (1995). AFLP: a new technique for DNA
fingerprinting Nucleic Acids Research 23: 4407-4414.

Vroh Bi, 1., 0. Maquet, J. P. Baudoin, P. du Jardin, J. M. Jacquemin, and G. Mergeai
(1999). Breeding for "low-gossypol seed and high-gossypol plants" in upland cotton.
Analysis of tri-species hybrids and backcross progenies using AFLPs and mapped
RFLPs. Theoretical and Applied Genetics 99: 1233 - 1244.

92

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