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This is to certify that the

dissertation entitled

Documenting the history of speciation in

Rhagoletis zephyria (Diptera: Tephritidae)

using molecular markers
presented by

Vesna Gavrilovic

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in Zoology

 

 

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a r professor

Date 05/1m2001

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6101 chlRC/DateDuopGS—MS

i_——— _—_____—————

DOCUMENTING THE HISTORY OF SPECIATION IN
Rhagoletis zephyrz'a (Diptera: Tephritidae)

USING MOLECULAR MARKERS
By

Vesna Gavrilovic

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Zoology and
Program in Ecology, Evolutionary Biology and Behavior

2001

ABSTRACT

DOCUMENTING THE HISTORY OF SPECIATION IN Rhagoletis zephyria
(Diptera: Tephritidae) USING MOLECULAR MARKERS
By

Vesna Gavrilovic

Rhagoletis pomonella (apple maggot) and R. zephyria (snowberry fly) belong to the R.
pomonella species group of the true fruit flies (Tephritidae) which are the center of a
long-lasting debate about modes of Speciation. Speciation in this group appears to have
been occurring sympatrically via host-plant shifts, in the absence of the geographic
barriers traditionally thought to be necessary for genetic divergence of populations and
establishment of reproductive isolation. The distribution of R. pomonella and R. zephyria
has been described as parapatric, while all the other species of the pomonella group are
broadly sympatric. In addition, while most of the species in the group show only allele
frequency differences for electrophoretic loci, there is a fixed difference at the Had locus
(hydroxyacid dehydrogenase) in R. zephyria (fixed for Hadl l l) and R. pomonella (two
other alleles - Had '00 and Had125 ). Therefore the relationship between these two species
provides a contrast to other species pairs in the group. This project investigates whether
R. zephyria has diverged by mechanisms similar to the ones that lead to divergence of
other taxa in the pomonella group. To address this question, the phylogenetic position of
R. zephyria within the group is studied using sequences of nuclear and mitochondrial

genes. Patterns of genetic variation in both apple maggot and snowberry flies throughout

the geographic ranges of both species are characterized not only using gene sequence
data, but also using amplified fragment length polymorphism (AF LP) patterns, in order to
infer polarity and time of the host shift that presumably occurred causing Speciation and
infer genetic mechanisms involved in species divergence. Rhagoletis pomonella appears
to represent a large and variable gene pool from which new species arise. The zone of
overlap of native geographic ranges of R. zephyria and R. pomonella is shown to be much
larger than previously described. Host shifi that led to divergence of R. zephyria from R.
pomonella and Speciation appears to have occurred from ancestral hawthorn to
snowberry. Speciation of R. zephyria appears to be very recent, as indicated by the
unresolved phylogeographic relationships, incomplete lineage sorting and lack of fixed
differences at all loci whose sequences were studied. The amount of genetic variation
found in R. zephyria was large, indicating that Speciation was not accompanied by
bottlenecks. Little geographic structuring was revealed by analyses of anonymous nuclear
loci, mitochondrial COI/COII gene and AF LP patterns. All analyses show that R.
zephyria appears to be a native inhabitant of eastern North America. Some AF LP
fragments were only amplified either in R. zephyria or R. pomonella. These fragments
can potentially be used as diagnostic markers for distinguishing between these two

species.

ACKNOWLEDGEMENTS

Numerous people have provided guidance, help and support in various ways
throughout the years I was working on this dissertation. Without them the project would
have been much weaker and the whole graduate school experience much less fun. Dr. Jim
Smith, my advisor, provided knowledgeable and professional guidance, support and
continuous understanding and a simple “Thanks” is just not enough to express how much
I appreciate it. It is difficult to find adequate words of gratitude to Dr. Guy Bush for his
continuous intellectual guidance and financial support. Thanks to Dr. Rich Lenski and
Dr. Jim Hancock for the time and effort they invested in serving on my Graduate
Committee.

Special thanks are due to present and past members of Smith and Bush or Bush
and Smith lab, who were always extremely supportive and provided technical help,
valuable discussions and suggestions: Dr. George Weiblen, Angela Roles, Joe Crossno,
Dr. Vanderlei Martins, Matt J aycox, Vince Borla, Chhaya Patel, Martha Smith Bressan
Caldas, Jessica Wolf, Elizabeth Kruszewski and Danielle Christman.

Amplification and sequencing of the nuclear alleles would not be possible without
Joe Roethele (University of Notre Dame) who constructed the genetic linkage map of
Rhagoletis pomonella, designed primers for the nuclear loci, helped tremendously in
cloning and sequencing, trained me to do sequencing myself, initiated valuable
discussions and always had friendly words of understanding and support. I would like to
thank Dr. Jeff F eder who kindly allowed me to use the primers designed in his laboratory

and do a large part of the work there. Help and support of members of his lab - Uwe

iv

Stolz, Dr. Bill Perry, Hattie Dambroski and Dr. Ken F ilchak are highly appreciated.
Thanks also to Nancy Perry for providing the warmth of home one winter when I worked
at Notre Dame.

For direct or indirect help with collecting the samples throughout the country I
would like to acknowledge Dr. Guy Bush, Dr. Jim Smith, Joe Crossno, Dr. Mark
Wiedrlechner (Iowa State University), Mike Klaus (Washington Department of
Agriculture), Dr. Diane Olsten (Utah State University), Charles Linn (Agriculture
Experimental Station, Geneva, NY), Dr. Ron Prokopy (University of Massachusetts,
Amherst) and Dr. Timothy Dickinson (Royal Ontario Museum, Toronto, ON). For help
with sequencing and AF LP GeneScanning thanks go to Sue Soltzfus, Therese Best,
Sherry Tjugum-Holland and Barbara Christian at the MSU DNA Sequencing Facility.

When data analyses seemed to be going to a dead end, Dr. Ron DeBry (University
of Cincinnati) and Dr. Susan Masta (San Francisco State University) provided advice that
helped me put things back on track. Their help is greatly appreciated.

Branislav Blagojevic was cheering for me from the very beginning, waiting
impatiently for a long time on the other side of the country, making sure that I did many
things in an unorthodox way, at least time—wise. Including the 3am phone calls which I
will surely miss, but gladly trade for conversations in person.

It would be very difficult to get through all the challenges without the support of
my family, even from across the ocean. Here I say thanks to Vladimir Gavrilovic, Ana
and Dragan Gavrilovic and J elena Todic.

Friends whose encouragement and sanity keeping are priceless include Sandra

Bencic, Dr. Danny Rozen, Jelena and Goran Poprzen, Natasa Nestorovic, Igor Pastirk,

Sanela Lampa Pastirk, Eva Maria Muecke, Alexandra Burch, Hilschers — Klara, Roman
and Ondrej, Silvia Fransen and Katarina Martic. And then also, thanks to Micaela
Szykman, Russ Van Horn and Ed Siuda for all the kind words and support.
Encouragement from Maxygen, Inc. that came from Dr. Lori Giver, Dr. Steve
delCardayre, Cristina Ivy, Troy Obrero, Walker Lutringer, Fred Gaume and many others
contributed greatly to my motivation to finish the project and finish it quickly, for which I

am very grateful to all of them and to Dr. Danny Rozen for helping me start that network.

vi

TABLE OF CONTENTS

LIST OF TABLES ............................................................................... viii V
LIST OF FIGURES ................................................................................ ix
KEY TO SYMBOLS AND ABBREVIATIONS .............................................. x
CHAPTER 1

INTRODUCTION .................................................................................. l
Speciation in the absence of geographic barriers ............................................... 1
Use of molecular markers in making inferences about Speciation ........................... 8
Rhagoletis pomonella group taxa, life history, host use and geography ................... 13
Specific objectives and hypotheses ............................................................. 20
CHAPTER 2

GEOGRAPHIC DISTRIBUTION OF R. zephyria .......................................... 23
Introduction ....................................................................................... 23
Materials and Methods ........................................................................... 27
Results ............................................................................................. 29
Discussion .......................................................................................... 39
CHAPTER 3

PHYLOGEOGRAPHIC RELATIONSHIPS OF R. pomonella AND R. zephyria. . ....44
Introduction ....................................................................................... 44
Materials and Methods ........................................................................... 47
Results ............................................................................................. 56
Discussion ......................................................................................... 90
CHAPTER 4

DIVERGENCE OF R. pomonella AND R. zephyria AS REVEALED BY
DIFFERENCES IN AF LP PATTERNS ....................................................... 99
Introduction ......................................................................................... 99
Materials and Methods .......................................................................... 102
Results ............................................................................................. 110
Discussion ........................................................................................ 1 19
CONCLUSIONS ................................................................................ 1 25
APPENDICES .................................................................................. 128
Appendix A: P220 sequence alignment ...................................................... 129
Appendix B: P2956 sequence alignment ..................................................... 146
Appendix C: P2480 sequence alignment ..................................................... 167
Appendix D: COI/COII sequence alignment ................................................ 180
REFERENCES ................................................................................... 201

vii

LIST OF TABLES

Table 1.1. Taxa belonging to the Rhagoletis pomonella species group and their host
plants .................................................................................................... 16

Table 2.1. Collection records for R. zephyria ..................................................... 34

Table 3.1. Populations of Rhagoletis zephyria and R. pomonella included in the
phylogeographic analyses ........................................................................... 49

Table 3.2. Oligonucleotide sequences used for PCR amplifications and sequencing. . . . . ...53

Table 3.3. Summary of the sample and loci analyzed in R. zephyria, R. pomonella and
other taxa of the genus Rhagoletis in this phylogeographic study .............................. 58

Table 3.4. Summary of the polymorphism at three anonymous nuclear loci and
mitochondrial COI/COII locus in R. pomonella, R. zephyria and other taxa of the genus
Rhagoletis ............................................................................................. 58

Table 3.5. Detection of natural selection at the four loci in R. pomonella, R. zephyria and
all other Rhagoletis taxa (pooled) using Tajima's (1989) and Fu & Li's (1993)

tests ..................................................................................................... 60
Table 3.6. HKA (Hudson-Kreitman-Aguadé) test for silent-site differences in four regions
between R. pomonella and R. zephyria ............................................................ 61
Table 3.7. Summary of divergence parameters between R. pomonella and R.

zephyria ................................................................................................ 62
Table 3.8. Summary of the parsimony analyses of the DNA sequences at three anonymous
nuclear loci and mitochondrial COI/COII locus .................................................. 63
Table 4.1 Populations of R. zephyria and R. pomonella sampled for the AF LP

analysis ................................................................................................ 103
Table 4.2. Cycling parameters used for selective amplification .............................. 108

Table 4.3 Estimated average heterozygosities and percentage of polymorphic loci in R.
zephyria and R. pomonella ......................................................................... 113

Table 4.4 Genetic distances (below diagonal) and geographic distances (in miles, above
diagonal) between R. zephyria and R. pomonella from different regions based on AF LP
data .................................................................................................... 118

viii

LIST OF FIGURES

Figure 1.1. Geographic distribution of the Rhagoletis pomonella group species ........... 17
Figure 2.1. Collection sites of R. zephyria ....................................................... 38
Figure 3.1. Genetic linkage map of Rhagoletis pomonella ..................................... 51
Figure 3.2. P220 neighbor-joining tree based on J ukes-Cantor distances ................... 64
Figure 3.3. P220 -— a random most parsimonius tree (of the 264500 equal MPRs). . . . . ....66
Figure 3.4. P220 - strict consensus of 264500 most parsimonious trees of length 262.
CI=0.523, RI=O.896, RC=0.468 .................................................................. 68
Figure 3.5. P2956 - neighbor-joining tree (J ukes-Cantor distances) ........................ 71
Figure 3.6. P2956 —- a random most parsimonious tree (of 285400 equal MPRs) ......... 73
Figure 3.7. P2956 - strict consensus of 285400 most parsimonious trees of length 309.
CI=O.628, RI=0.937, RC=0.588 .................................................................. 75
Figure 3.8. P2480 — neighbor-joining tree (J ukes-Cantor distances) ......................... 77
Figure 3.9. P2480 — a random most parsimonious tree (of 1920 MPRs) ..................... 79
Figure 3.10. P2480 - strict consensus of 1920 most parsimonious trees of length 582.
CI=O.308, RI=0.698, RC=O.214 .................................................................. 81
Figure 3.11. COI/COII — neighbor-joining tree (J ukes-Cantor distances) .................. 84
Figure 3.12. COI/COII — a random most parsimonious tree (of 89200 MPRs) ............ 86
Figure 3.13. COI/COII - strict consensus of 89200 most parsimonious trees of length 300.
CI=O.613, RI=0.780, RC=O.483 .................................................................. 88
Figure 4.1 Collecting sites for the populations sampled for AF LP analysis ............... 104
Figure 4.2. The principle of the AF LP DNA fingerprinting technique ..................... 107
Figure 4.3 Frequency distribution of number of fragments amplified from each

individual ............................................................................................ 1 12
Figure 4.4. Neighbor-joining tree based on the analysis of 255 AF LP loci ................ 1 14

ix

KEY TO SYMBOLS AND ABBREVIATIONS

aap (ptx) — Rhagoletis pomonella, Waxahatchie, Texas

has — R. basiola

bop (pga) - R. pomonella, Macon, Georgia

c — R. carnivora

cng — R. cingulata

com — R. completa

eL (electrom) — R. electromorpha

f — R. nr. mendax (flowering dogwood fly)

fa (nr. mendax a) - R. nr. mendax (flowering dogwood fly), adult
fp (nr.mendax p) - R. nr. mendax (flowering dogwood fly), pupa
fu — R. fausta

ind —— R. indiferens

jbt — R. juniperina

m — R. mendax

mel — R. mendax, East Lansing, Michigan

mga — R. mendax, Georgia

mmi - R. mendax, East Lansing, Michigan

mnj — R. mendax, Rutgers, New Jersey

mns - R. mendax, Nova Scotia

mon - R. mendax, Ontario

mOtis — R. mendax, Otis Lake, Michigan

mxH —- R. nr. pomonella, Mexico (highland)
mxhd — R. nr. pomonella, La Jolla, Mexico

me — R. nr. pomonella, Mexico (lowland)

NC (pmiNC) — R. nr. pomonella, Grant, Michigan
nr. mendax GA - R. nr. mendax (flowering dogwood fly), Georgia
nr. pom MX - R. nr. pomonella, Mexico

nwmx — R. pomonella, New Mexico

p - R. pomonella, Grant, Michigan

pco - - R. pomonella, Boulder, Colorado

pel — R. pomonella, East Lansing, Michigan

pga (bop) - R. pomonella, Macon, Georgia

pia - R. pomonella, Ames, Iowa

pil - R. pomonella, Riverwoods, Illinois

pma - R. pomonella, Amherst, Massachusetts
pmhbj - R. nr. pomonella, Louisiana (mayhaw fly)
pmi - R. pomonella, Grant, Michigan

pmiNC (NC) - R. pomonella, Grant, Michigan
pmn - R. pomonella, Staples, Minnesota

pne ‘- R. pomonella, Nebraska

pns - R. pomonella, Kentville, Nova Scotia

pny - R. pomonella, Geneva, New York

pon - R. pomonella, Toronto, Ontario

ppa - R. pomonella, Biglerville, Pennsylvania

xi

ptx (aap) - R. pomonella, Waxahatchie, Texas
put - R. pomonella, Wellsville, Utah

pwa - R. pomonella, St. Cloud Ranch, Washington
rpmx - R. nr. pomonella, Mexico City, Mexico
sparkga — R. nr. mendax, Georgia (sparkleberry fly)
str — R. striatella

sv — R. suavis

t — R. tabellaria, Washington

tzt — R. tabellaria, Ontario

zca — R. zephyria, Honeydew, California

200 — R. zephyria, Boulder, Colorado

zel — R. zephyria, East Lansing, Michigan

zid - — R. zephyria, Elmira, Idaho

zma — R. zephyria, Amherst, Massachusetts
zmio — R. zephyria, Mio, Michigan

zmn — R. zephyria, Hawby, Minnesota

zmt — R. zephyria, Swan Lake, Montana

zmtB — R. zephyria, Terry, Montana

znd — R. zephyria, Bismarck, North Dakota
zne — R. zephyria, Brady, Nebraska

zny — R. zephyria, Geneva, New York

zon — R. zephyria, Rice Lake, Ontario

zor — R. zephyria, Grants Pass, Oregon

xii

zpa — R. zephyria, College Park, Pennsylvania

zsb -— R. zephyria, Glen Haven (Sleeping Bear Dunes), Michigan
zsd — R. zephyria, Custer, South Dakota

zwa — R. zephyria, Dixie, Washington

zwi — R. zephyria, Waukesha, Wisconsin

zwy — R. zephyria, Moiser Gulch, Wyoming

In all trees, green branches represent R. zephyria, red R. pomonella, pink R. nr.
pomonella (Mexico), blue R. mendax and light-blue R. nr. mendax (flowering dogwood
fly). Squares next to R. zephyria labels and circles next to R. pomonella labels correspond
to geographic regions from which the samples were taken: red — Northeast, orange —
Great Lakes, green — Great Plains, blue — Rocky Mountains and Colorado Plateau, violet

(purple) — Northwest, and gray — South.

xiii

CHAPTER 1

INTRODUCTION

Tephritid fruit flies in the genus Rlzagoletis have been a model system for
studying sympatric Speciation via host-plant shifts with no geographic separation of
populations. Some of the species in this genus (suavis species group, Bush and Smith
1998) conform to the traditional view that new species arise when genetic differences
between populations accumulate as a result of geographic barriers to gene flow (Mayr
1963). However, species of the pomonella species group represent one of the examples of
incipient sympatric Speciation, not uncommon in phytophagous insects (Mitter et al.
1991, Guldemond and Mackenzie 1994, Emelianov et al. 1995) and some vertebrates
(Schliewen et al. 1994, Sturmbauer 1998, Hatfield and Schluter 1999). Rhagoletis
pomonella group species are also one of the best-understood examples where ecology has

played an important role in Speciation (Orr and Smith 1998).

SPECIATION IN THE ABSENCE OF GEOGRAPHIC BARRIERS

Species and Speciation are the center of the long-standing debate focused on
explaining how gene pools become split and isolated, giving rise to new species. Many
biologists still hold the view that this is possible only if physical barriers to gene flow
between populations exist, allowing accumulation of different mutations, different

selection pressures and stochastic processes to shape gene complexes within these

populations in such a way that, if they come into secondary contact, they will no longer
be able to produce viable and fertile offspring. If they accept the view that Speciation can
occur without physical barriers to gene flow, they often say it happens only rarely (e. g
Barraclough and Vogler 2000).

Parapatric divergence in a classic sense may be initiated in a small region at the
periphery of a widely distributed species. The new ecological “race” becomes adapted to
a new “niche” and, once established, spreads to occupy the range of the new habitat or
host (after Bush 1975). It is unlikely that a shift and adaptation to the new niche occurs
simultaneously along the entire ecotone. Recently, mosaic distribution of populations
throughout the species range has been referred as to parapetric distribution of populations
(for example, Gavrilets et al 2000). Models of Speciation under this definition of
parapatry assume that geographic variation exists between populations of a widely
distributed species and some gene flow occurs between neighboring populations. It is
intuitive that the geographic ranges of most species are much larger than the dispersal
distances of their individuals or gametes. Even though any particular mutation is a rare
event, the number of possible mutations is almost unlimited and therefore different
mutations are likely to accumulate in different populations of species throughout the
range. Different habitat conditions, both biotic and abiotic, are likely to exist in different
parts of the range, creating different selection regimes,which can contribute to
divergence of the populations despite some gene flow. The main questions here are how
fast reproductive isolation can develop and where does the first split occur (Gavrilets et al
2000). According to Mayr (1963), peripheral populations are more likely to diverge

because they are usually smaller, experience different selection pressures than the central

populations and are less affected by gene flow. Brown (1957), on the other hand, in his
concept of centrifugal Speciation, argues that the central populations represent the origin
of new species because they are often the source of new genetic variability. In a recently
developed model, Gavrilets et al (2000) have shown that both time until Speciation and
location of the first split depend on mutation rate, amount of genetic change required for
reproductive isolation and population size. One of the important implications of this
model is that it does not require strong divergent selection for rapid (few hundred to few
thousand generations) Speciation. Experimental study of premating reproductive
mechanisms in grasshoppers (Tregenza et al 2000) corroborated that the long periods of
allopatry are not necessary for Speciation.

At the opposite end to allopatric Speciation in the geographic continuum is
Speciation in sympatry. Key elements of sympatric Speciation are resource-based
disruptive selection, assortative mating and habitat (host) fidelity (Johnson et al 1996,

F utuyma 1997, Berlocher 1998, Schluter 1998). Habitat choice behavior is also attributed
the status of a key component in generating conditions under which reproductive
isolation between sympatric populations can evolve (Via et al 2000), because behavioral
changes often initiate the use of a new environment, allowing selection to operate on
morphological and physiological characters (F utuyma and Moreno 1988, Fry 1996).
Earlier models (Felsenstein 1981) involved non-habitat-choice assortative mating and
divergent selection. In these models, recombination easily separates alleles for assortative
mating from alleles for performance in two habitats, and little or no divergence is
possible. F elsenstein (1981) therefore concluded that sympatric Speciation is only likely

under highly restricted conditions. Models developed later (Rausher 1984) included loci

for habitat choice and demonstrated that habitat preference can develop easily even in
absence of fitness differences across environments. Therefore characters that influence
both specialized resource use (habitat preference) and assortative mating are particularly
important in evolution of ecological specialization and speciation. The significance of
ecological specialization in speciation has been widely recognized and documented for a
number of species (Bush 1994, Johnson et al 1996, Schluter 1996).

Experimental corroboration of this model comes from work of Via on pea aphids.
In several studies her group has shown that under experimental conditions two host races
of pea aphids have poor performance and fitness on alternative hosts simply because of
unwillingness to feed on that host, not because of physiological trade-off. Several other
studies have shown that trade-offs are not essential for ecological specialization (Fry
1996, Kawecki 1996, Whitlock 1996). Caillaud and Via (2000) suggest that the
physiological trade-off (toxicity of the alternative host) initially might have been the
selective force favoring host plant acceptance, but it no longer plays that role. In host
races of Rhagoletis pomonella, no evidence of larval feeding trade-off have been found
(Prokopy et a1 1988) — larvae of both races performed equally in hawthorn fruits, yet they
are highly specialized for different temporal resources (Feder et al 1997, F eder and
Filchak 1999, Filchak et al 1999).

Under the influence of natural selection, populations colonizing novel
environments can diverge very rapidly from the ancestral populations, which is a critical
early step in speciation (Coyne 1992, Travisano and Rainey 2000). Speciation by
ecological specialization is most rapid when assortative mating in different environments

is coupled with habitat preference (Johnson et al 1996). In the initial diversification phase

small amount of divergence occurs; this phase is followed by the quasi-equilibrium phase
during which little divergence occurs and completion phase when gene flow between
habitats stops quickly (in less than 1000 generations) and divergence is dramatic
(Johnson et al 1996). Fast speciation in sympatry can also occur when traits for habitat
choice, or traits correlated with them, are the direct target of disruptive selection
(Kondrashov and Kondrashov 1999). Reproductive isolation can develop in the presence
of limited gene flow via pleiotropy when divergent selection acts on multiple characters
(Rice and Hostert 1993). When the effect of selection is larger than gene flow, some
divergence will result; selection will then tend to further decrease gene flow, enabling
divergence of traits that initially were not diverging (Rice and Salt 1990).

It is usually not easy to estimate how fast reproductive isolation evolves,
especially in long-standing groups, since averaging rates over time obscures the
biologically important short-term evolution (Hendry and Kinnison 1999). The most
spectacular speciation rate has been estimated for cichlid fishes in lakes of Eastern Africa
(Danley et al 2000, Wilson et al 2000). Several factors contribute to rapid diversification
despite some gene flow in these lakes: philopatry, lack of dispersing larval stage, and
varying water levels which affects extinction and reestablishment of populations (Danley
et al 2000, Meyer 1993). A number of experimental studies with known timescales have
shown that reproductive isolation can evolve very rapidly - examples of rapid divergence
include limnetic and benthic forms of sticklebacks (Schluter 1996), evolution of
soapbeny bug populations on alternative hosts (Carroll et al 1997), morphological
differentiation of Anolis lizards introduced to Bahaman Islands (Losos et al 1997),

changes in life history traits and morphology in Neotropical guppies (Reznick et al 1997),

and morphological and genetic divergence between river and lake populations of salmon
(Hendry et al 2000). In all these cases, diverging populations were adapting to local
resources and genetic differences were small, especially in neutral markers, but isolation
developed in as little as 13 generations (Hendry et a1 2000). Accompanying
morphological and ecological changes were large enough to prevent cross mating, which
can further facilitate divergence and led to speciation in sympatry.

Ecological interactions, rather than ecological specialization, are the starting point
in models of sympatric speciation with an evolutionary branching approach (Doebeli and
Dieckmann 2000). These models are based on the theory of adaptive dynamics (Metz et
al 1992, Geritz et al 1998). In these models, ecological interactions such as resource
competition, predator-prey interactions or mutualism are the driving force of evolutionary
change and branching. A number of empirical examples provide evidence for sympatric
speciation under the evolutionary branching models. In sticklebacks (Schluter 1994,
Nagel and Schluter 1998), Anolis lizards (Losos et al 1998) and Darwin’s finches (Grant
et al 1985, Schluter 1988) branching is attributed to resource competition; however, it is
not clear that competition is an important factor in insect evolution. In orchid and orchid
bees coevolution and cospeciation are initiated by mutualistic interactions (Kiester et al
1984), whereas speciation in sub-Antarctic weevils illustrates the significance of
predator-prey interactions (Chown and Smith 1993).

Berlocher (1998) recognizes four stages in the process of sympatric speciation:
host races, species isolated by host fidelity, species with prezygotic and/or postzygotic
isolation unrelated to host fidelity, and totally isolated species. Taxa at all of the stages

are found within the Rhagoletis pomonella species group. Host races of R. pomonella

 

 

mate on fruits of different host plants and display no other pre- or postzygotic
reproductive isolation; however, gene flow between them is reduced by strong host
fidelity and selection. Genetic differences between the host races are small; in the case of
apple and hawthorn races of R. pomonella there are no unique allozyme alleles but
frequency differences between races are maintained by host choice behavior and
selection despite the 6% gene flow between them (Feder and Bush 1989). Species
isolated by host fidelity in the R. pomonella species group (R. pomonella and “flowering
dogwood fly”, Berlocher 1998) have larger allele frequency differences but still no
unique alleles have been identified. Hybridization between these species under laboratory
conditions results in fully viable and fertile offspring (Smith 1986). In nature, host choice
of these species is even more pronounced than in host races and this strong host fidelity
prevents them from interbreeding (Berlocher et al 1993). Species with prezygotic and/or
postzygotic reproductive isolation unrelated to host fidelity show non—fixed species-
specific allozyme alleles (private alleles) and morphological autapomorphies (Feder and
Bush 1989, Jenkins 1996). Low levels of gene flow are possible (Feder and Bush 1989),
but the assortative mating occurs under laboratory conditions, even in the absence of host
plants (Bierbaum and Bush 1990), and in nature where large host preference differences
prevent interbreeding (Feder and Bush 1989). Hybrids have reduced viability but are
fully fertile (Bierbaum and Bush 1990). Within the R. pomonella species group, this stage
is illustrated by R. mendax (blueberry maggot) and R. pomonella species pair (Berlocher
1998). Totally isolated species, such as R. cornivora and R. pomonella are separated by
large genetic distance and exhibit fixed allozyme differences at several loci (Berlocher et

al 1993), as well as morphological polymorphisms (Jenkins 1996).

 

USE OF MOLECULAR MARKERS IN MAKING INFERENCES ABOUT SPECIATION

Information about speciation can be inferred from analyses of the geographic
ranges of species, correlations between range overlaps and the degree of genetic
divergences, and by a biogeographic analyses of allele or haplotype genealogies
(Berlocher I998). Phylogeography brings an historical perspective to population genetic
phenomena such as population subdivision, genetic drift, gene flow and selection (Brown
et al. 1996), and allows estimates to be made about the order and polarity of ancestor-
descendant relationships and ecological niches (Berlocher 1998). Existing patterns of
allele relationships depend on the time since speciation and the mode of speciation.

Deep gene trees with major lineages separated by relatively large mutational
distances are usually observed when long-term, usually physical barriers to gene flow
separate the populations (Avise 2000). The observed genetic distances may arise as a
result of novel mutation accumulation in lineages after their geographic separation or as a
result of lineage sorting from a polymorphic ancestral gene pool; these two cases are not
mutually exclusive. However, the same results could occur following sympatric
divergence. Deep splits between distinct mtDNA lineages have been observed in
contiguous populations of black-backed jackal (Wayne et al 1990). The authors suggest
that this finding may be the result of secondary admixture of populations that were
separated in the past or retained distinct ancestral lineages despite high gene flow.

Recent sympatric speciation is reflected by alleles of ancestral species forming a
paraphyletic group, with alleles of derived species arising from within the ancestral clade

(Hanison 1991). Generally, if populations have been reproductively isolated for a

relatively short time, little differentiation is expected to be observed in neutral genes,
leading to unresolved relationships between the taxa, since parental genes are still being
shared among derived groups. The ancestral species is expected to be more
geographically structured than the descendant species, since populations of the ancestral
species have been adapting to local habitat conditions for a longer time (Brown et al
1996). For mitochondrial DNA, Nei gel and Avise (1986) have shown by computer
simulations that, following speciation, relationships among haplotypes go through phases
of poly-, para- and monophyly and that the time to reaching monophyly depends on
population sizes at and after speciation.

It should be noted that the general expectations about lineage sorting and
coalescence are only applicable to selectively neutral alleles/haplotypes. Different types
of natural selection will change the expectations in different ways. When directional
selection drives the frequency of an advantageous allele, it eliminates the variation at
linked sites by a selective sweep or hitchhiking effect (Stephan et al 1992). New variation
is recovered very slowly by accumulation of new neutral mutations in copies of
advantageous alleles. This shortens the coalescent time for the gene and linked loci,
depending on the strength of linkage (Li 1997).

Selection against deleterious mutations (background selection, Charlesworth et al
1993) also reduces neutral variation at linked sites, reducing the coalescent time at that
region as well. The effect of background selection is strongest when the mutation rate is
high and the recombination rate is low; it decreases rapidly with the increase in

recombination rate (Li 1997). Balancing selection, on the other hand, can slow down the

elimination (extinction) of alleles at a locus and linked sites, maintaining some lineages
over time scales much longer than expected and across speciation events (Clark 1997).

Polymorphisms can also be misleading in the estimation of patterns and time of
speciation when trans-species polymorphisms exist (Klein 1986). In such a case, the
divergence of allelic lineages occurrs before the divergence of species, so that
polymorphisms observed within species are older than the species itself, as illustrated by
the highly polymorphic human MHC loci (Klein et al 1993).

The type of molecular markers chosen for phylogeographic studies thus largely
influences the conclusions drawn from gene genealogies. Although the importance of the
marker choice is not questioned, the majority of the studies published are based on only
one type of molecular marker (for exceptions see Bemardi et al. 1993, Burton and Lee
1994, Hare and Avise 1998).

About 70% of all phylogeographic studies are based primarily or exclusively on
analyses of mitochondrial DNA (Avise 2000), which is a maternally inherited, haploid
marker. The mitochondrial genome is relatively small in size (16 kb in Drosophila) and
present in thousands of copies in each individual. mtDNA does not undergo
recombination, shows stable gene arrangement, ample polymorphism within species, and
often evolves faster than typical single-copy nuclear DNA (Avise 1994). Isolation of
haplotypes is relatively easy through PCR. Trees based on mitochondrial DNA sequences
reflect only a matrilineal portion of the evolutionary history (Maddison 1995), which
might be quite different from the history of nuclear genes.

Nuclear markers, depending on their nature (single-copy genes, repeated short

sequences, size polymorphisms) and position in the genome (in regions presumably under

10

 

 

 

 

selection vs. neutral), can reveal different patterns of variation and evolution. Nuclear
markers often used for studying genetic structure and relationships of populations and
species include allozymes, RFLPs (restriction fragment length polymorphism, reviewed
in Avise 1994), microsatelites (Weber and May 1989), VNTRs (variable number of
tandem repeats or minisatellites, Jeffreys et a1 1985) and AF LPs (amplified fragment
length polymorphism, Voss et a] 1995). These markers do not require DNA sequencing
and allow estimates of population genetic parameters such as allele frequencies, numbers

of alleles, heterozygosity, effective population size, Nem and Neil parameters, genetic

distances among populations, and F or 0 statistics (Wright 1965, Weir and Cockerham
1984)

The sequences of single-copy nuclear genes are more difficult to obtain and few
studies have used them for estimation of intra— or interspecific gene trees and
phylogeographic analyses (Hare and Avise 1998). In diploid organisms, if the individual
is heterozygous at a locus under study, PCR amplifies both alleles and it is important to
separate them and obtain both sequences. This is possible by cloning of the amplified
PCR products through a vector. However, misincorporation of nucleotides during the
previously performed PCR is possible (Keohavong and Thilly 1989). Some authors
ignore possible misincorporations in their analyses (Palumbi and Baker 1994, Sota and
Vogler 2001). Another approach to this problem involves sequencing multiple clones
from a single individual so that the sequences can be compared and allelic variants
distinguished (Bemardi et al 1993). Most phylogenetic information is recovered from
gene trees obtained from loci in regions with low recombination frequencies.

Recombination distorts the phylogenetic history of changes within and between lineages

ll

by producing homoplasy, which can erroneously cluster the genotypes and violate the
assumption that tree branches are non-reticulate (Avise 2000).

Estimates divergence times between species are based on the concept of a
“molecular clock” (Hillis et al 1996). This concept is based on the simplifying
assumptions that DNA sequences evolve at an approximately uniform rate over time in
all evolutionary lineages (Zuckerkandl and Pauling 1965) and that the rate of neutral
mutation equals the rate of evolution (Kimura 1968). For mtDNA it is estimated that 1-
2% sequence divergence accumulates over a million years (Brown et al 1979). However,
this clock has only been calibrated using insect and crustacean sequences (Brower 1994).
It is known that variation in rate of mtDNA evolution exists not only among taxa but also
among different genes (Simon et al 1994). Differences among taxa may stem from
different efficiencies of their DNA repair systems (Britten 1986), different generation
times (Laird et al 1969) or different metabolic rates (Martin and Palumbi 1993). More
accurate estimates of the rate of evolution are obtained if multiple loci are used and
averaged, since that reduces the variance (Takahata and Nei 1985). If the time since
lineage separation can be estimated from fossil evidence as well, than the clock can be
calibrated more accurately. Unfortunately, fossil data are not available for most taxa, so

the molecular clock can only be used for rough estimates.

12

 

 

RHAGOLETIS POMONELLA GROUP TAXA, LIFE HISTORY, HOST USE AND GEOGRAPHY

The Rhagoletis pomonella species group consists of 4 taxonomically described
species, at least two undescribed species (sparkleberry fly and flowering dogwood fly)
and several taxa of uncertain taxonomic status (host races, geographic races) (Table 1.1).
All Rhagoletis taxa are typical solitary parasites in that females search for host fruits for
their offspring, and oviposit only on acceptable fruits (Price 1977). R. pomonella group
species have specific hosts that belong to widely divergent plant families (Table 1.1). All
taxa within the R. pomonella species group have almost identical life cycles. They are
univoltine, with adults emerging over short periods of time in spring or summer.
Emergence of each species is synchronized with the beginning of the fruiting season of
their respective host. Adults feed on insect honeydew, bird feces, yeast and bacteria
found on leaf surfaces of host or non-host plants (Bush 1992). Females become sexually
mature 7 or 8 days after emergence, at which time they begin mating and ovipositing.
Mating occurs almost exclusively on the host fruit (Prokopy et al 1971). Females are
attracted to a suitable host fruit by chemical (Prokopy et al 1973, Frey et al 1998) and
visual cues (Moericke et al 1975), where they lay their eggs into the fruit by inserting
their ovipositor into the fruit. Oviposition is followed by marking of the fruit with
pheromones. That deters other females from ovipositing into the same fruit (Prokopy
1972). This probably reduces competition and perhaps enhances resource partitioning.
Each female typically lays one egg per fruit; however, multiple infestations have been
observed, particularly in large fruit such as apples (Filchak, pers. comm.; Gavrilovic and

Crossno, pers. observ.) and experimentally corroborated in R. zephyria (van Randen and

Roitberg 1996). Multiple ovipositions per single fruit were originally explained in
parasitoids by mistakes in oviposition (van Lenteren 1981), but more recently were
argued from a standpoint of adaptive superparasitism theory (van Alphen and Visser
1990). This theory states that when ecological or physiological conditions are
unfavorable (e.g. no uninfested fruit, high egg load) and there is positive probability that
an additional larva could survive feeding within the same fruit, females can increase their
reproductive fitness component by accepting previously marked (i.e. infested) host fi'uit.
Larvae feed and pass through 3 instars within the fruit over a 2-4 week period
(Bush 1966). Upon completion of larval development, which is synchronized with fruit
falling to the ground, larvae leave the fruits and pupate 3-5 cm in the soil, enter diapause
and overwinter in the pupal stage. The following summer adults emerge and complete the
life cycle in 20-30 days. Host fidelity in all species of the genus Rhagoletis is very high
and adults are known to disperse over large distances in search of favorable hosts when
local host fruit is not available (Bush, 1966). Many of the taxa in the pomonella species
group can be crossed in the laboratory (Bierbaum and Bush 1990); however, they rarely
interbreed in nature (F eder and Bush 1989). This indicates that host preferences and host
fidelity accompanied with mating on the fruits of the host plant serve as effective
premating reproductive isolation mechanisms between taxa utilizing different host plants.
Four species, two undescribed species close to R. mendax and two taxa of uncertain status
close to R. pomonella belong to the R. pomonella species group (Table 1.1). The status of
Mexican populations of R. pomonella is also uncertain — whether they represent
geographic race of R. pomonella or a separate species remain under investigation (Bush,

pers. communication; Feder et al, in prep.)

l4

Rhagoletis pomonella is native to eastern North America, from Nova Scotia and
Maine southward to central Florida and westward to northwestern Minnesota and eastern
Texas (Figure 1.1). Populations present in the Pacific Northwest and western slopes of
the Rocky Mountains are thought to be introduced (McPheron 1990). Ranges of all other
taxa, except for R. zephyria, are fully contained within the much broader range of R.
pomonella (see Bush and Smith 1998). Rhagoletis zephyria has been described as
occurring throughout western North America and is parapatric in Minnesota and
Manitoba with R. pomonella (Bush 1966). However, R. zephyria has recenty been reared
from native snowberries in Ontario (Smith, pers. comm), Michigan (Crossno, pers.
comm.) and southeastern Wisconsin (Gavrilovic, pers. observation, see also Chapter 2),
indicating that the zone of overlap is much broader (see Figure 1.1). Populations of R.
zephyria also exist in eastern North America, where they are thought to be introduced
from western North America with the host plant, Symphoricarpos albus var. Iaevigatus,

that is grown as an ornamental shrub (Feder et al 1999).

15

Table 1.1. Taxa belonging to the Rhagoletis pomonella species group and their host

 

 

 

 

 

 

 

 

 

plants.
TAXA HOST PLANTS FAMILY
hawthorn (Crataegus 15 spp.)
Rhagoletis pomonella . Rosaceae
_ apple (Malus pumzlla)
R. zephyria snowberry (Symphoricarpos 3 spp.) Caprifoliaceae
R mendax blueberry (Vaccinium 7 spp.) Ericaceae
' huckleberry (Gaylusaccia 3 spp.)
R. cornivora shrubby dogwoods (Cornus 2 spp.) Comaceae
ibifi’éi’éfyxfly) sparkleben‘y (Vaccim'um arboreum) Ericaceae
R. nr. mendax . .
(flowering dogwood fly) flowering dogwood (C ornus florzda) Comaceae
2:13:11) El; n):onella plums (Prunus 2 spp.) Rosaceae
211th 3:13;; 11a mayhaw (Crataegus 2 spp.) Rosaceae

 

16

 

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17

Except for R. cornivora, species of the pomonella group are morphologically
virtually indistinguishable. In the case of R. pomonella and R. zephyria, only males can
be distinguished on the basis of small difference in the shape of surstyli (Bush 1966,
Jenkins 1996). All R. pomonella group taxa show the same distinctive wing banding
pattern, that resembles the legs of jumping spider when observed from above.

Genetic variation at various levels and evolutionary relationships within the
pomonella group have been examined by allozymes (Berlocher and Bush 1982, Feder et
al. 1988, McPheron et al. 1988, McPheron 1990, Berlocher et al. 1993, Berlocher 1995,
Berlocher 2000) and mitochondrial DNA sequences (Smith and Bush 1997, Han and
McPheron 1997, McPheron and Han 1997). Most taxa of the group show only allozyme
allele fi'equency differences (Berlocher et al. 1993). Six diagnostic loci show constant
host-related frequency differences between apple and haw races of R. pomonella (Feder
et al 1988, McPheron et al 1988), as well as among other taxa within the group
(Berlocher et al 1993). Rhagoletis pomonella and R. mendax possess private alleles at
polymorphic loci aspartate—aminotransferase-l (Am-1'00 in R. pomonella), diaphorase-Z
(Did-2lOO in R. pomonella) and fumarase (F um15 8 in R. mendax), with other alleles at
these loci being shared among species. However, there is a nearly fixed allele difference
between apple maggot and snowberry fly at the hydroxyacid dehydrogenase (Had) locus
— R. zephyria is fixed for Had' ‘ ', while R. pomonella has two other alleles, Hadmo and
Had125 (Berlocher et al. 1993). It should be noted that Feder et al. (1999) found very low
frequencies of Hadloo in populations of R. zephyria from Michigan and central
Minnesota, and Hadl H in populations of R. pomonella from Washington, northem and

central Minnesota. The presence of these rare alleles is explained by low-level

l8

hybridization or occasional mistakes in oviposition. It should be noted again that R.
cornivora, the most genetically distant of the species within the R. pomonella group,
along with the fore-mentioned morphological differences, possesses 9 unique allozymes
alleles and appears to have diverged first from the remainder of the group (Berlocher et al
1993, Jenkins 1996).

Although much work has been done in order to characterize the genetic variability
of, and the relationships between, the species comprising the Rhagoletis pomonella group
(Berlocher et al. 1993, Smith and Bush 1997, Berlocher 2000), many questions remain
open, from the full resolution of phylogenetic relationships to the taxonomic status of
currently unplaced populations, and to testing the sympatric hypothesis of speciation. The
knowledge gained through extensive research and deployment of new molecular markers
raises unresolved questions, keeping the field vibrant and exciting for years to come. This
dissertation focuses on the placement of R. zephyria and its relationship to R. pomonella
(the putative ancestor for all the taxa in the R. pomonella species group), in order to infer
the mode of speciation that led to divergence of these two species. Speciation via host
shifts has been proposed for most of the broadly sympatric pomonella group taxa and it is
interesting to determine whether R. zephyria has diverged by a similar mechanism.
Rhagoletis zephyria is the only species in the group with distribution extending far
outside of the range of R. pomonella. It is also the only species in the pomonella group
with fixed allele difference at one of the allozyme loci (Had). That provides an important

contrast to all other species pairs in the group.

19

SPECIFIC OBJECTIVES AND HYPO’I‘HESES

Population genetic Studies based on allozymes indicate that R. pomonella is the
most genetically diverse (with highest number of alleles at any of the allozyme loci and
highest heterozygosity) and most highly structured (as inferred from F s, values) of the
pomonella group species (Berlocher et al. 1993, Berlocher 1995, Berlocher 2000). This
has led to a working hypothesis that the apple maggot fly represents a large and variable
ancestral gene pool from which new species of the pomonella group arise. Specific
hypotheses tested in this project are:

Hypothesis 1: Rhagoletis pomonella represents a large and variable gene pool,
containing ancestral polymorphisms, from which new species arise.

Hypothesis 2: Rhagoletis zephyria has diverged from R. pomonella more recently
than the blueberry maggot, R. mendax.

Hypothesis 3: The host shift that led to divergence of R. zephyria from R.
pomonella and speciation occurred from ancestral hawthorn host to snowberry.

Hypothesis 4: Rhagoletis zephyria in Eastem North America has been introduced
with the host plant (snowberry).

The extent of the range of overlap between R. pomonella and R. zephyria has
important implications for inferring mechanisms and geographic location of divergence
that led to speciation. Berlocher (1998) suggests that the range of overlap for these two
species, based on the previously described range of R. zephyria, is only about 15%
whereas mean range of overlap for all host-shifting species he examined was about 50%.

The size of overlap for apple maggot and snowberry flies is close to the mean range of

20

overlap found for non-host-shifters (12.4%), where speciation can be explained by
allopatric models (Berlocher 1998). To be able to address any hypotheses of speciation of
R. zephyria and its origin in eastern North America, I needed to obtain reliable
geographic distribution data (Chapter 2).

To test the hypotheses 1-3 I used a phylogeographic approach. Rhagoletis
pomonella and R. zephyria populations representative of the species geographic ranges
were surveyed for variation using molecular markers. In order to estimate levels of
genetic variation in R. pomonella and R. zephyria and compare them to other species of
the pomonella species group, as well as other species of Rhagoletis, I used DNA
sequences from three anonymous single-copy nuclear loci from three different linkage
groups in the R. pomonella genome (P220 from linkage group I, P2956 from linkage
group II and P2480 from linkage group III; Figure 3.1; Roethele et al. 1997), as well as
mitochondrial DNA (cytochrome oxidase subunits I and II withintergenic tRNALc"
coding region; COI/COII) (Chapter 3). The identities of the nuclear loci used here are
unknown. They display significant linkage disequilibrium with allozymes which display
host—specific frequency differences between host races of R. pomonella (Roethele et al.
1997). The degree of linkage to the allozyme loci (as determined by levels of
recombination, Roethele et al 1997) is different for these markers. Each of these loci
reflects the evolutionary history of the distinct part of the genome. This study thus relies
on a representative portion of a genome. As mentioned earlier, relationships between taxa
inferred from gene trees obtained using one gene can differ dramatically from those
obtained using a different gene or a different type of marker. Therefore gene trees may

not accurately correspond to species trees (Maddison 1995, Wang et al 1997). To

21

overcome this problem, I used not only sequences from different genes, but also
amplified fragment length polymorphism (AF LP) genotyping (Voss et al 1995). This
technique has recently been suggested for studying relationships between very closely
related and rapidly evolving Species (Albertson et al 1999), for studying population
structure and differentiation and estimating population genetics parameters (Reineke et al
1998). It generates a large quantity of information (polymorphic loci) by screening the
entire genome. I used two selective amplification primers to generate AF LP patterns for
individual flies from populations representative of the geographic ranges of R. pomonella
and R. zephyria and used these data to study phylogeographic relationships of the
populations of both species (Chapter 4).

If R. pomonella indeed represents a large, ancestral gene pool, it can be expected
that it will show a higher number of alleles, higher average nucleotide diversity, higher
heterozygosity, and higher F s, values across its geographic range than R. zephyria. It can
also be expected that R. pomonella is paraphyletic, while other species of the group are
expected to Show autapomorphic genetic differences, with alleles arising from pomonella
alleles. The polarity and time of the presumed host shift can also be inferred from allele
genealogies. If the data indicate that the divergence of R. zephyria from the ancestral
gene pool of R. pomonella was recent, then the polarity of host shift can be inferred to be
from ancestral hawthoms to snowberry. Cladograms and distance-based dendrograms
obtained from sequences of different genes and comparison of structure of R. zephyria
populations across the geographic range of the species and to the structure of R.
pomonella populations in the zone of overlap, should provide information on the origin of

R. zephyria in eastern North America, where one host plant species has been introduced.

22

CHAPTER 2

GEOGRAPHIC DISTRIBUTION OF R. ZEPHYRIA

INTRODUCTION

The Rhagoletis pomonella species group is one of the best-understood natural
model systems in which ecology is proposed to have played an important role in species
divergence (Orr and Smith 1998). Several closely related species in the R. pomonella
species group appear to have speciated in sympatry, with shifts to new hosts
accompanying all speciation events. Rhagoletis zephyria (snowberry fly) is a sister
species to R. pomonella (apple maggot). The geographic ranges of the R. pomonella
group taxa, except for R. zephyria, are completely contained within that of R. pomonella
(Bush 1966, F oote et al 1993). Speciation of R. zephyria, which infests fruits of
Symphoricarpos spp., is an interesting evolutionary question. Not only has R. zephyria
different geographic distribution (overlapping with R. pomonella and mostly allopatric to
other pomonella group taxa), but also this species is the only one of the R. pomonella
group taxa that displays fixed genetic differences with R. pomonella.

The working hypothesis about the origin of R. zephyria is that it has diverged and
speciated by a host shift. The shift must have had occurred in the zone of contact between
the ancestral and derived host, therefore in the zone of overlap of R. zephyria and its
ancestor. Another assumption is that the ancestral host is hawthorn, and that the ancestral
population was R. pomonella-like. It has been proposed that R. pomonella (apple maggot

fly) has the ability to shift from the original (ancestral) host to fruits of different host

23

plants and utilize them as a new resource, thus giving rise to new species (as implied in
Bush 1969). Host fidelity is an important factor in the speciation process, since adults of
all pomonella group taxa mate exclusively on or near their host fruits (Bush 1966). This
establishes and maintains premating reproductive isolation between the newly diverged
taxa. Although R. pomonella and R. zephyria can be crossed under laboratory conditions,
in nature that happens only rarely (Feder and Bush 1989, McPheron 1990, Smith et al
1993, Feder et al 1999).

To begin addressing the questions of how, when and where R. zephyria diverged
from an R. pomonella-like ancestor, it is important to establish the current distribution of
the two species. The geographic distribution of R. zephyria has not been well
characterized, and therefore it is not well known where and how large the zone of
parapatry is or has historically been. In addition, the host distribution of R. zephyria is
also uncertain. A precise map of the present geographic distribution of host and fly, as
well as evidence suggesting past distributions, are two of several variables that should be
known to document the history of speciation of any two taxa (White 1978). Many of the
other variables are already well documented for the R. pomonella species group taxa.
These include detailed morphological descriptions of taxa (Bush 1966, Foote et al 1993,
Jenkins 1996), ecological data on preferred habitats and the life history of the organisms
(Bush 1966, F oote et al 1993), behavioral information about possible ethological
isolation or habitat selection (Prokopy et al 1971, 1972, Prokopy and Bush 1972, 1973),
genetic descriptions based on cytological, biochemical and molecular data (Bush 1966,
Berlocher and Bush 1982, Feder et a1 1988, 1997, 1999, McPheron et al 1988, Berlocher

et al 1993, Berlocher 1995, 2000, McPheron and Han 1997, Smith and Bush 1997), data

24

on morphological and genetic geographic variation (McPheron 1990, F eder et al 1999),
experimental hybridization studies (Smith et al 1993) and information on the frequency
of hybridization in nature where the ranges overlap (McPheron 1990, Feder et al 1999).

R. zephyria infests fruits of Symphoricarpos spp. (Caprifoliaceae). The primary
host of R. zephyria in western North America is S. (1le var. laevz'gatus (S. rivularis). It
is a woody shrub, 1-2m tall, with dark-green leaves and small, pink, bell shaped flowers,
which give rise to pearl-like white berries in clusters. The fruit remains on the plants into
the late fall. The natural geographic distribution of this plant is limited to west of Rocky
Mountains (Jones 1940, US. Department of Agriculture, Forest Service 2001 online), but
following the Lewis and Clark expedition of 1804-05 this variety of snowberry was
introduced into the eastern part of North America and has escaped from cultivation over a
wide range (Bush 1966). R. zephyria infestations of the fruits of S. albus var. laevigatus
have been observed in eastern North America and have commonly been assumed to have
come along with the introduction of the host (F eder et al 1999).

On the east side of the Rocky Mountains and throughout the Great Plains, the
most common species of snowberry, and the primary host of R. zephyria, is S.
occidentalis, a small shrub up to 1m tall, with small pale pink flowers, which give rise to
round, dull greenish—white spongy fruits in clusters; the fruits quickly turn brown in early
fall. Symphoricarpos occidentalis has a very wide distribution, from British Columbia to
Ontario and south to Washington, Utah, New Mexico and Oklahoma (U .S. Department of
Agriculture, Forest Service 2001 online). It is most commonly found and is very
abundant in the prairies of the northern Great Plains. Although records of S. occidentalis

exist for Wisconsin, Illinois, Michigan, Ontario, Pennsylvania and New York (herbarium

25

data, Soper and Heimburger 1994, US. Department of Agriculture, Forest Service 2001
online), R. zephyria was not considered to be present in these states, except in association
with the cultivated S. albus var. laevigatus.

The third potential host species for snowberry flies is S. albus var. albus although
so far it has not been reported as a host of R. zephyria. S. albus var. albus is smaller in
size than S. albus var. laevigatus (up to 1m) with smaller flowers and fruits which grow
as solitary rather than in clusters. Symphoricarpos albus var. albus occurs in the eastern
United States and Canada, from eastern Quebec south to North Carolina and extends west
to the Rocky Mountains where it is only found on the eastern slopes (Jones 1940). This
variety readily hybridizes and is interfertile with S. albus var. leavigatus introduced to the
eastern North America (Smith, pers. comm.). This species of snowberry is only abundant
in the northern part of its range (US Department of Agriculture, Forest Service online),
but its presence throughout the entire region implies that the range of the three native
potential host plants of R. zephyria is more or less continuous throughout North America.

The geographic range of R. zephyria has been described as primarily western,
from southern British Columbia south to northern California, extending east to the
Mississippi River valley in Minnesota (Foote et a1 1993, Bush 1966). This implies that
the zone of parapatry between apple maggot and snowberry flies is relatively narrow,
since the native range of R. pomonella does not extend west of Minnesota (Figure 1.1,
after Bush 1966). Infestations by R. zephyria were previously reported for fruits of S.
albus var. laevigatus and S. occidentalis (Bush 1966). In order to determine precisely the
geographic range of the snowberry flies, fruits from all three potential hosts were

sampled throughout the ranges of these plants. The objectives were: i) to determine where

26

R. zephyria occurs; ii) whether all three potential hosts are infested and to what extent;
iii) whether infestations of the native hosts are limited to the west of the Mississippi
River valley; iv) where introduced hosts were infested; and v) how may the origin of R.
zephyria in the eastern North America be explained.

Adults of R. zephyria were reared from fruits of all three host plants as a result of
the extensive collection. The geographic range of R. zephyria extends south to South
Dakota and Nebraska; infestations of native hosts in Wisconsin, Michigan, Ontario and
New York are reported for the first time. The natural range of R. zephyria thus extends
east of the Mississippi River valley through the Great Lakes area. These findings have
important bearing on inferences about the speciation of R. zephyria, since they indicate
that the zone of overlap with R. pomonella or pomonella-like ancestor is or has been

much broader than previously thought.

MATERIALS AND METHODS

Field survey: Snowberry stands were located by searching herbarium records,
visiting sites with the prOper ecological characteristics but previously not known to be
inhabited by potential host plants, or from personal contacts. Fruits of S. albus var.
laevigatus , S. albus var. albus and S. occidentalis were hand-picked between the months
of August and October each year from 1993 to 2000 from localities in Massachusetts,
New York, Pennsylvania, Michigan, Wisconsin, Minnesota, North and South Dakota,

Nebraska, Colorado, Wyoming, Montana, Idaho, Washington, Oregon and California and

27

the province of Ontario (Table 2.1). Identifications of plant species were made in the
field. When uncertainty about the plant identity arose or when new sites were found,
plants were pressed and brought to the laboratory for identification.

Insect rearing: Fruits were collected into ZiplocTM bags containing a small
amount of vermiculite to prevent early rotting. Bags were stored in coolers in the field at
10-20°C until returned to the laboratory. All fruits were placed into plastic trays with
approx. 2cm layer of moist Grade 3 (medium) vermiculite and allowed to dry for 3-5
weeks at room temperature. During this time larvae completed their development, left the
fruits and pupated in trays. Fruits were removed and in most instances counted.
Vermiculite was sifted through a screen which retained the pupae, that were then counted
and infestation rates calculated as the number of pupae recovered divided by the number
of fruits. Pupae were placed in Petri plates (100x15mm) with a layer of approx. lem of
moist vermiculite. The plates were then placed in a 4°C refrigerator for 6-7 months to
simulate overwintering. Water was added to vermiculite monthly to keep the pupae
hydrated. After 6-7 months Petri plates were transferred to a controlled environmental
incubator (25°C, 14h light, 21°C, 10hr dark). Emerging adults were collected daily and
kept alive for 5-7 days on diet of 50% (w/v) yeast and 25% (w/v) sucrose.

Flies reared from Symphoricarpos spp. were assumed to be R. zephyria. Host fruit
remains one of the most reliable indications of Species identification. Since the host
fidelity in R. pomonella species group is very high and oviposition mistakes are rare
(Bush 1966, Bierbaum and Bush 1990) it is generally accepted that fly species can be

assigned based on fruits from which they were reared.

28

RESULTS

Host plants: Infestations of fruits were observed for all three host plants (S. albus
var. laevigatus, S. albus var. albus and S. occidentalis) between 1993 and 2000 (Table
2.1). Symphoricarpos albus var. laevigatus was sampled at 34 localities in its native
range west of the Rocky Mountains. It was found to be infested at 28 (82.4%) localities.
Infestations were also observed in Washington, Oregon, California, Idaho and Montana.
At three localities in California and three in Idaho fruits of S. albus var. Iaevigatus were
not infested (Table 2.1). Stands of introduced S. albus var. laevigatus were infested at all
localities east of the continental divide that were sampled. However, it should be noted
that uninfested S. albus var. laevigatus have been observed in Door County, WI (Bush,
pers. comm.) and at 17 localities in Nova Scotia (Crozier, pers. comm).

S. occidentalis was sampled at 26 localities both west of the Rocky Mountains
where its range overlaps with the range of S. albus var. laevigatus and in the Great Plains
where it is the predominant snowberry species. In all cases it was infested (Table 2.1 ).

S. albus var. albus was sampled at eight localities and observed at two more
where it was not sampled because permits to collect in National Parks and National
Monuments were not available. It was infested at six localities (75%) in New York,
Ontario, Michigan and Wisconsin. A low number of pupae were recovered from samples
from Ontario (2-18, Table 2.1). At several other localities S. albus var. albus was not
found to be infested, although R. zephyria was reared from the fruits of other hosts at

relatively close localities (Table 2.1).

29

Localities: Northwest (Washington, Oregon and California). Native S. albus var.
laevigatus was sampled at 11 localities in Washington, seven localities in Oregon and
seven localities in Northern California; S. occidentalis was sampled at one locality in
Washington. All S. albus var. laevigatus samples from Washington and Oregon were
found to be infested; R. zephyria was reared from four samples from California. At three
localities in California fruits were not infested with R. zephyria. Infestation rates ranged
from 0.8% at Olympia, WA to 62.9% at Dixie, WA, which was the highest infestation
rate observed for all samples (Table 2.1). S. occidentalis collected at Beacon Rock State
Park in the Columbia River Gorge was also infested with R. zephyria (infestation rate
30.0%, Table 2.1).

Rocky Mountains and Colorado Plateau (Idaho, western Montana, Wyoming and
Colorado). S. albus var. laevigatus was sampled at four localities in Idaho. A sample
from one locality sampled in 1995 (Elmira) was infested with R. zephyria. At three
localities sampled in 1997 no infestation was found (Table 2.1). In Montana, both S.
occidentalis and S. albus var. Iaevigatus were sampled at localities west of the
continental divide, where S. albus var. laevigatus is generally more common. Three S.
occidentalis and five S. albus var. laevigatus populations were sampled. All the samples
were found to be infested with R. zephyria. Most of the samples from Montana were
taken in 1994 and 1995 when the fruit number was not determined. All three potential
host species were sampled in Wyoming. S. albus var. laevigatus was sampled at a single
locality at high altitude (6700’, Glendo State Park). A large number of fruits was
collected (2689, Table 2.1), enabling detection of a very low rate infestation (0.04% -

only one pupa was recovered from the fruits). At lower altitudes, two samples of S.

30

occidentalis were found to be infested (1 .0-3.2%, Table 2.1). S. albus var. albus was
found growing at the Devil’s Tower National Monument where it was not collected.
Fruits did not look infested; however, this does not necessarily imply that they are not
infested, but only the inability to detect infestation by observation. At another site (Bear
Lodge campground), fruits of S. albus var. albus were relatively scarce and no pupae
were found. In Colorado, a small stand of native S. albus var. albus was found at Estes
Park. Fruits were sampled on several occasions (1996, 1997, 1999) but yielded no pupae.
Cultivated S. albus var. laevigatus growing as an ornamental plant on campus of the
University of Colorado in Boulder were sampled in early August of 1997, when an
infestation rate of 0.8% was observed (Table 2.1). In early September 1999, very large
berries were showing signs of heavier infestation; however, only 12 pupae were
recovered from 973 fruits (1.2%, Table 2.1)

Great Plains (eastern Montana, North Dakota, South Dakota, Nebraska,
Minnesota). S. occidentalis is a dominant host in eastern Montana. It is very common
across the broad areas of North and South Dakota and Minnesota where large stands are
frequently found and easily spotted along the roads. Three samples were taken from
Montana, four from North Dakota, three from South Dakota, two from Nebraska and five
from Minnesota. All samples yielded pupae; infestation rates ranged from 0.4% at Wasta,
SD to 4.2% at Rapid City, SD. Several large stands of S. occidentalis were observed in
Badlands National Park (Gavrilovic, pers. Obs.), but no collections were made in the
Park. A stand of S. albus var. albus was seen at Custer State Park, SD (Crossno, pers.

comm.) where no collections were made, also due to the lack of necessary permits.

31

Great Lakes (Wisconsin, Michigangnd Ontario). Herbarium records from the
University of Wisconsin, Madison indicated that S. occidentalis grows in Wisconsin.
However, most of the sites these records listed were converted to comfields (Gavrilovic,
pers. obs.). A stand of S. occidentalis found along the railroad tracks in Waukesha, about
15 mi west of Milwaukee, was heavily infested with R. zephyria (27.2%, Table 2.1). S.
occidentalis was also found near Mio, MI and pupae were recovered from the fruits.
Native stands of S. albus var. albus form the predominant ground cover at three sites in
Ontario where it was sampled in 1996 and 1998. All three samples were infested;
however, adults emerged only from samples collected at Glen Miller and Rice Lake
(Table 2.1). S. albus var. albus was also observed in Pinery Provincial Park (Gavrilovic
and Crossno, pers. obs.) but it was not collected. At Glen Arbor, MI (Sleeping Bear
Dunes National Lakeshore), plants found were either S. albus var. albus or its hybrid with
S. albus var. laevigatus. These fi'uits were also found to be infested (Table 2.1).
Infestations of S. albus var. laevigatus introduced to Michigan as horticultural plant were
previously known (Bush pers. comm., Smith pers. comm). This host plant was sampled
at five localities in Michigan and found to be infested at all of them (Table 2.1). The plant
is common in Lansing/East Lansing area and heavily infested. Lower numbers of pupae
were recovered fiom samples from Mio Dam and Harrisville (Table 2.1).

Northeast (Pennsylvania, New York and Massachusetts). Only introduced S. albus
var. laevigatus was found in Pennsylvania and Massachusetts, infested with R. zephyria
(Table 2.1 ), consistent with previous reports (Smith pers. comm., Bush pers. comm.) In
New York, two samples of S. albus var. laevigatus were found to be infested. The plants

at Wellesley Island were likely S. albus var. albus, however, the possibility that they were

32

hybrids with var. laevigatus cannot be excluded. This sample was also infested (Table
2.1), and was collected near the town of Alexandria Bay where infested S. albus var.

laevigatus was observed (Smith, pers. comm).

33

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38

DISCUSSION

Native snowberry species in the western part of North America (Washington,
Oregon, California, Idaho, Montana, Wyoming) and in the Great Plains (North Dakota
and Minnesota) were previously known to be host plants for R. zephyria (Bush 1966,
lFoote et al 1993). The natural range of the fly has been reported to extend east into
Minnesota and no further than the Mississippi River Valley. Findings of R. zehyria in
eastern North America have been attributed to human introduction, as the host, S. albus
var. Iaevigatus was introduced and cultivated as an ornamental plant (Feder et a1 1999).
Thus, the range of R. zephyria, including all introduced and native populations, was
thought to be discontinuous (Bush and Smith 1998). The main reason for this was the
assumption that the suitable host plants were not found in the Great Lakes area and
further east, into the northeast United States. Symphoricarpos albus var. albus, the native
snowberry species in the eastern North America, has never been reported infested with R.
zephyria.

In this field survey, stands of native snowberry species were located in a broad
geographic area covering the regions previously known as parts of the range of R.
zephyria and those where the fly has not been found although the hosts were known to
commonly occur (such as South Dakota and Nebraska), or where the only host available
for flies was thought to be the introduced S. albus var. laevigatus (Wisconsin, Michigan,
New York, Ontario). Here the observations of R. zephyria infesting native S. occidentalis
in Wisconsin and Michigan and S. albus var. albus in Michigan, Ontario and New York
are reported. These are the first reports of R. zephyria in Wisconsin and the first reports

of R. zephyria infesting native hosts east of Minnesota. The rearing of R. zephyria pupae

39

and adults from native hosts (S. occidentalis) in South Dakota and Nebraska is also
reported, and the findings of R. zephyria in Wyoming are extended into the eastern part
of the state. By these findings the known natural geographic range of R. zephyria is
extended.

Another species of the R. pomonella group, R. mendax (blueberry maggot), has
recently been found to infest native host plants in the Great Lakes area (Smith et al, in
review), a finding that questions the previously accepted view that the pest was
introduced with the cultivated host plants from the regions where it commonly occurs.
Similarly, this report of fly populations infesting native hosts raises a question of the
origin of R. zephyria in the eastern North America.

S. occidentalis is a native inhabitant of the Great Lakes area. This species is
typically found in prairies, which became established in this region after the Pleistocene
glaciers began receding (Thompson and Smith 1970, Catling and Catling 1993).
Herbarium records indicate that S. occidentalis was common in Wisconsin, Illinois,
Michigan, Ohio and New York as recently as 60 years ago, and distribution data from the
US Forest Service list this species from British Columbia to Ontario and as far south as
New Mexico and Oklahoma. Stands of S. occidentalis in Wisconsin and Michigan may
represent prairie remnants from the period after the Pleistocene glaciers began receding
and pupae and adults of R. zephyria were reared from both localities (Waukesha, WI and
near Mio, MI). Catling and Catling (1993) suggest that prairie remnants exist around
Lake Ontario as well, with S. albus var. albus being the predominant ground cover at
some areas, whereas Soper and Heimburger (1994) report S. occidentalis in Ontario, at

areas that correspond to Catling and Catling’s (1993) prairie remnants. Adults of R.

40

zephyria were reared from S. albus var. albus from two such sites (Glen Miller and Rice
Lake, ON). It is interesting to note that herbarium records from the University of
Michigan indicate that S. occidentalis was growing on the streets of Grand Rapids, MI
and in the Black River valley near Port Huron, MI about 100 years ago. An attempt was
made to locate native stands of either S. ocidentalis or S. albus var. albus in Ohio, but
information from the Department of Natural Resources indicated that the snowberries
were extirpated from Ohio relatively recently (Cusick, pers. comm.) All this suggests
that S. occidentalis was much more common in the Great Lakes area in the recent past,
indicating that the zone of overlap of R. zephyria and R. pomonella may have been much
wider than previously thought. This observation is important with respect to whether
populations of R. zephyria in eastern North America have been established by
importation of flies with the host plants or by spreading to the introduced host from local
native plants.

S. occidentalis appears to be the preferred host of R. zephyria. All the samples of
S. occidentalis were found to be infested (Table 2.1), whereas some samples of native S.
albus var. laevugatus were not (three samples from California and three from Idaho
yielded no pupae). Introduced S. albus var. laevigatus in Door County, WI, outside of the
native range of any snowberry species or variety, has never showed signs of infestations
(Bush, pers. comm.) Cultivated S. albus var. Iaevigatus in Nova Scotia is also not known
to be infested with R. zephyria (Crozier, pers. comm.) The same variety has been widely
introduced into Great Britain from the Pacific coast of North America, but no R. zephyria
associated with it has been observed (Gilbert 1995). Therefore it appears that infestations

of introduced S. albus var. laevigatus occur only where the native host is present as well.

41

This supports the hypothesis that R. zephyria may be a native inhabitant of eastern North
America.

AF LP analysis showed that R. zephyria reared from native host plants in New
York, Ontario, Michigan and Wisconsin possess AF LP fragments characteristic of R.
zephyria. Neighbor-joining analysis of AF LP patterns placed these individuals within the
R. zephyria cluster (Chapter 4). While mitochondrial and nuclear DNA sequences of flies
from these populations are very similar to other R. zephyria (Chapter 3), a lack of
geographic structuring does not allow me to distinguish between different scenarios about
the origin of snowberry flies in the east. However, the amount of variation observed
suggests that bottlenecks did not play a role in population divergence, which would be
expected if the populations in eastern North America were introduced and founded from
a small number of individuals. The possibility that these populations were established by
range expansion of R. zephyria west of the Mississippi River valley and colonization of
available hosts in the region cannot be eliminated. However, under such a scenario it
would be expected to see isolation by distance, which the data at this point do not
support.

The results of this field survey and rearing of R. zephyria from a previously
unrecorded host, S. albus var. albus, raise a question as to whether other species of
Symphoricarpos may serve as hosts for snowberry flies. It is important to note that where
S. albus var. albus was co-occurring or growing relatively close to S. albus var.
Iaevigatus or S. occidentalis, no infestations of S. albus var. albus were observed,
indicating that the other hosts are preferentially chosen. However, it may also indicate

that short dispersal distances and the patchiness of the suitable habitats may limit further

42

range expansion, even though the hosts may be present, as in the case of geometrid moths
[tame andersoni feeding on patchy distributed host plant, Dryas drummondii (Doak
2000). The relatively isolated, although physically close, stands of S. albus var. albus in
Colorado and Wyoming were not found to be infested (Table 2.1). The same was true for
stands of different species or varieties of Symphoricarpos observed or sampled in Utah,
Nevada and Arizona, where no signs of infestations were obvious and no flies were
reared from any of the samples (Bush, pers. comm., Smith, pers. comm.) This may
indicate that more or less geographically continuous presence of host plants is necessary
for dispersal of flies and range expansion.

The described findings of R. zephyria infesting native hosts in the Great Lakes
region may be similar to the observation of Smith et al (in review) of blueberry maggot
(R. mendax) infesting native Vaccinium corymbosum and V. angustifolium in the same
region. Similar to infestations of S. occidentalis, Vaccinium stamineum is reported to be
infested with R. mendax throughout its natural geographic range in eastern North
America, with V. corumbosum and V. angustifolium serving as “range extenders”. Even
so, the latter two hosts are not infested everywhere they grow, presumably because
abiotic factors render the habitat unsuitable for R. mendax (Smith et al, in review). The
same may be true for R. zephyria whose preferred host appears to be S. occidentalis, with
S. albus var. laevigatus and S. albus var. albus serving as “range extenders” respectively

to the west and east.

43

CHAPTER 3

PHYLOGEOGRAPHIC RELATIONSHIPS OF R. POMONELLA AND R. ZEPHYRIA

INTRODUCTION

The history of species divergence can be inferred from DNA sequence
polymorphism patterns at randomly selected genes (Kliman et al 2000) by comparing the
patterns between species, estimating DNA sequence divergence, inferring ancestral states
and reconstructing speciation events from the phylogenies. White (1978) stressed the
importance of describing genetic variation across the entire geographic ranges of two
sister taxa for documenting the history of their speciation. Previous DNA sequence
studies of phylogenetic relationships of taxa in the R. pomonella species group and
species of the genus Rhagoletis as a whole have sampled only a small number of
individuals (usually one) from each species (Smith and Bush 1997, McPheron and Han
1997, Han and McPheron 1997). This study represents the first effort to make inferences
about speciation of R. zephyria using a phylogeographic approach.

In this study I characterized genetic variation across the geographic ranges of
Rhagoletis zephyria and R. pomonella by analyzing DNA sequence divergence in
mitochondrial DNA and three anonymous nuclear loci. The general objective was to
examine biogeographic patterns within and between species at the DNA sequence level
and make inferences not only with respect to where the divergence of R. zephyria from R.
pomonella may have occurred, but also to the mode of speciation and subsequent species
divergence. Within this general framework, I addressed several specific questions: i)

what is the phylogenetic position of R. zephyria within the R. pomonella species group;

44

ii) how closely related are R. zephyria and R. pomonella; iii) is there geographic structure
within R. zephyria and/or R. pomonella and iv) are there any fixed differences between
the two species?

The phylogenetic position of R. zephyria within the R. pomonella group is
uncertain. Phylogenetic studies based on allozymes (Berlocher et a1 1993, Berlocher
2000) have placed R. zephyria at the base of the closely related R. pomonella group
species (with R. cornivora at the base of the entire group). However, the parsimony tree
in which R. zephyria was a sister to one of the R. pomonella clades was not a
significantly worse fit (Berlocher 2000). Studies based on mitochondrial DNA sequences
(l6S — McPheron and Han 1997, Han and McPheron 1997; COI/COII — Smith and Bush
1997) support the latter relationship.

Studies of genetic variation in multiple populations of R. pomonella so far have
all been based on allozymes (McPheron 1990, Berlocher 2000). Several populations of R.
zephyria were analyzed for allozyme and mitochondrial RFLP variation by Feder et al
(1999); their study focused on hybridization between R. pomonella and R. zephyria. No
large-scale geographic studies to date in either species have been based on DNA
sequence data.

R. pomonella and R. zephyria display a fixed difference at one of the six
diagnostic allozyme loci (Had) identified by F eder et al (1988) and McPheron et al
(1988). R. zephyria is fixed for Had I H while R. pomonella is polymorphic for two other
alleles, Had100 and Had'zs. Also, R. pomonella and R. zephyria display a fixed difference
in the shape of male claspers (surstyli) (Westcott 1989), and the lengths of female

ovipositors (however, the variation in the latter trait is large and ovipositor lengths

45

overlap, Bush 1966). No fixed differences in mitochondrial C011 and 168 DNA
sequences have been found (Smith and Bush 1997, McPheron and Han 1997).

As mentioned above (Chapter 1), relationships between taxa inferred from gene
trees obtained using one gene can differ dramatically from those obtained using a
different gene or a different type of marker. Therefore gene trees may not accurately
correspond to species trees (Maddison 1995, Wang et al 1997). Maddison (1997) remarks
that “phylogeny has a variance as well, represented by the diversity of trees of different
genes”. The use of multiple loci for estimating relationships between taxa permits one to
distinguish the forces that act on many genes (and ideally the entire genome) from those
affecting individual loci (Hudson et al 1987).

In this study I used DNA sequences of three anonymous single-copy nuclear
genes and a well-characterized mitochondrial COI/COII gene in order to minimize the
limitations of the single-gene approach. The nuclear loci represent different linkage
groups in the R. pomonella genome (Figure 3.1; Roethele et al 1997). While the identities
and function of the nuclear loci used here are unknown, they are each in significant
linkage disequilibrium with the allozyme loci that display host-specific frequency
differences between host races of R. pomonella (Roethele et a1. 1997). The degree of
linkage to the allozyme loci as determined by levels of recombination (Roethele et al
1997) is different for each of these markers and ranges between 1.8 and 7.50M.

The working hypothesis in this study is that the speciation of R. zephyria occurred
by a host shift of an R. pomonella-like ancestor from hawthoms to snowberries in a zone
of parapatry or sympatry in the upper Mississippi River valley of Minnesota. In studies

based on allozyme data (Berlocher and Bush 1982, Berlocher 2000) R. pomonella has

46

been shown to be the most genetically variable species of the group, with a large and
variable gene pool containing ancestral polymorphisms. In at least one case it has been
documented that R. pomonella has shifted from one host (hawthoms) to another (apples)
thus forming a new host race. This has led to a hypothesis that R. pomonella may be the
ancestor of other taxa in the group, which speciate by host shifts. It is further
hypothesized that the descendent R. zephyria spread west, and possibly east, on
snowberries. If there were no snowberries east of the Mississippi River valley prior to
this host shift, R. zephyria found in eastern North America today must have been
introduced with the infested host after the Lewis and Clark expedition (see Chapter 2).
One alternative hypothesis is that the host shift occurred at the Great Plains/Eastem
Woodland boundary, with subsequent dispersal of snowberry flies both east and west,
assuming that snowberries existed in eastern North America prior to the host shift (see

Chapter 2).

MATERIALS AND METHODS

Sample: Infested fruits of R. zephyria and R. pomonella host plants (snowberries,
hawthoms and apples) were collected during late summer and fall of 1994, 1995, 1997,
1998, 1999 and 2000 throughout the geographic ranges of both fly species (Table 3.1)
and brought to the laboratory, where the files were reared as described in Chapter 2.
Pupae of R. pomonella from Nova Scotia were obtained from R. Smith in 95% ethanol.

Adults were used for DNA extractions for all R. zephyria and most R. pomonella

47

samples; DNA was extracted from pupae of R. pomonella from Nova Scotia (ethanol
preserved), Massachusetts, New York and Washington State (samples collected in fall
2000) and Ontario (no adults emerged during two seasons). Individuals belonging to
other taxa included in the analyses were either reared in the laboratory of J. Feder at the
University of Notre Dame or obtained from S. Berlocher (University of Illinois at
Urbana-Champaign) or J. Smith. These flies were subjected to the same procedures as R.
zephyria and R. pomonella samples (see below) and sequences from them were obtained
in collaboration with J. Roethele (University of Notre Dame).

DNA extraction from R. zephyria and R. pomonella: Total genomic DNA was
isolated from individual flies using a protocol modified from Han and McPheron (1997).
Individual flies were homogenized in buffer (10 mM Tris, pH 8, 60 mM NaCl, 150 mM
sucrose, 10 mM EDTA, 0.5% (w/v) SDS, 0.1 mg/mL proteinase K) and crude
homogenates were incubated at 55°C for 30 minutes. Buffer containing 300 mM Tris, 150
mM sucrose, 100 mM EDTA and 0.75% (w/v) SDS was added to precipitate protein.
After incubation on ice for 10 minutes an equal volume of phenol was added and protein
and cell debris were removed by centrifugation (14,000 rpm, 5 min). Residual protein
contaminants were removed by subsequent phenolzchloroformzisoamyl alcohol (25:24: 1 ,
v/v/v) and chloroformzisoamyl alcohol (24:1, v/v) extraction followed by ethanol
precipitation at ~20°C overnight. DNA was resuspended in TE buffer (lOmM Tris, lmM

EDTA) containing lOOug/mL RNase A and stored at 4°C.

48

Table 3.1. Populations of Rhagoletis zephyria and R. pomonella included in the
phylogeographic analyses

 

 

 

 

 

332°” Locality Label
R. zflrhyria

Massachusetts Amherst ZMA
New York Geneva ZNY
Pennsylvania Penn State campus ZPA
Ontario Rice Lake ZON
Michigan Mio ZMIO
Michigan Sleeping Bear ZSB
Michigan East Lansing ZEL
Wisconsin Waukesha ZWI
Minnesota Hawby ZMN
North Dakota Bismarck ZND
South Dakota Custer SP ZSD
Nebraska Brady ZN E
Colorado Boulder ZCO
Wyoming Moiser Gulch ZWY
Montana Swan Lake ZMT
Montana Billings ZMTB
Idaho Elmira ZID
Washington Dixie ZWA
Oregon Grants Pass ZOR
California Honeydew ZCA
R. pomonella

Nova Scotia Kentville PNS
Massachusetts Amherst PMA
New York Geneva PNY
Pennsylvania Biglerville PPA
Ontario Toronto PON
Michigan E. Lansing PEL
Michigan Grant PMI
Georgia Macon PGA
Illinois Riverwoods PIL
Minnesota Staples PMN
Iowa Ames PIA
Nebraska ([80E,exit285) PNE
Colorado Boulder PCO
Texas Waxahatchie PTX
New Mexico (unknown) PNM
Utah Wellsville PUT
Washington St. Cloud PWA
Mexico Mexico City PMX
Mexico La Jolla MXHD

 

49

Choice of markers: Primer pair sequences for 10 anonymous nuclear loci located
in different linkage groups (Figure 3.1) within the genome, and showing different levels
of recombination with allozymes which show host-specific frequency differences in host
races of R. pomonella, were provided by J. Roethele and J. F eder (University of Notre
Dame). These loci (P181 and P220 from linkage group 1; P114 and P2956 from linkage
group 11; P7, P2156 and P2480 from linkage group III; P100 and P661 from linkage
group IV and P454 from linkage group V) were screened for variation and level of
phylogenetic signal. Based on the results of a preliminary study, P220, P2956 and P2480
were chosen for the phylogeographic study. P181, P2156 and P454 showed very little
variation; the phylogenetic signal from P661 was complicated by high recombination
rates within this gene; P114 provided less resolution than P2956; P7 was very difficult to

amplify and clone, and P100 did not amplify in R. zephyria.

50

8.7

20.0

7.0

3.3

3.3

2.5

0.4

4.2

4.9

11.1

6.2

- P3072
- P2160

~P2944

-P226
- P181

—P341

— Idh

—Ak

-P2648

— Pgm

 

1.7

4.6

02_

Iiialr4_

O

0

~Aat-2 2.0
LDia-2
—' 0.7

3.7

14.8

 

III I
- P667 -P2619
. . 6.3
" _. °. —Pe -2
"P138 8.3 p 12.0
"‘ P719 _ G'G'pdh
0.6 68
~P849
-Acon-2 ‘ 8 8 4
Anal 05 -P2156 -
0] ‘ P70
ilk -Had 3-2
P Adh-1 1 4 8,
M” 0.2 - 57
_P8 0.2 : 33a_.
' P114 1.4 i ‘4-1
1.2 ‘ P47
“P29261D'-P3082 «2
0.3 ' P2825 3.6
0.4 _ P33 5.6
‘ P3061
3
—P2473 L P22

 

51

 

 

V V
sex
chromosome
—P2963 _ Aat-1
— P2565 _ P309
— P1 147
—P1700
— P661
_ Gpi —P454
_ P369 -P535
-P2620
— P727 _ P9
— P6
_ P100 PP405
—P33 Dot
chromosome
Ac
P291 9
P249
P33b

Figure 3.1. Genetic linkage map of Rhagoletis pomonella. Map constructed by Roethele
et al. (1997) based on genetic recombination data obtained using allozyme and cDNA
markers. Underlined allozymes show allele frequency differences between the apple and
hawthorn host races of R. pomonella. cDNA markers are labeled with a “P”, followed by
a number; bold lines and marker labels indicate loci in gametic disequilibrium with an
adjacent allozyme locus. The loci used in this study are framed in red.

PCR amplification, cloning and sequencing for R. zephyria and R. pomonella:
Primers used for PCR amplifications of nuclear alleles were designed by J. Roethele and
are listed in Table 3.2. Reactions were performed in SOpL or 200pL volumes using
leibcoBRL PCR buffer, 6.25 mM MgC12, 1.25 mM of each of dATP, dCTP, dGTP and
dTTP, 10 pM of each primer, 5 units of Gibco Taq polymerase and luL of template DNA
(not quantified). The PCR cycling regime was: 95°C, 5 min; 35 cycles of 94°C 2 min,
52°C 1 min 30 sec, 72°C 2 min; 72°C 7 min. PCR products were initially gel-purified by
centrifugation through an Amicon Ultrafree-DA filter (Millipore, Bedford, MA) followed
by isopropanol precipitation for 2 hr at 4°C and cloned into the pCR2.l Dual Promoter
plasmid using a commercially available TA cloning kit (Invitrogen, Carlsbad, CA). After
overnight ligation at 14°C, chemically competent EFloL E. coli cells were transformed
and grown on LB medium plates containing 50 mg/mL ampicillin and 1.6 mg X-gal. PCR
products were also directly cloned without gel-purification, which yielded higher
numbers of putatively positive clones, so this technique was adopted for all subsequent
clonings. Putative positive clones were checked for the presence of inserted DNA by
direct PCR amplification from bacterial colonies, using the same primers and same
cycling regime as in the original PCR. Four positive clones from each individual fly were
purified (WizardS V Miniprep, Promega, Madison, WI) and sequenced. For about 30% of
the number of flies, sequencing was performed in both directions by cycle sequencing
with dye termination using universal primers M13 and M7 and the ALF automated
sequencer (Amersham-Pharmacia) at the University of Notre Dame. The rest of the
sample was sequenced unidirectionally, using universal primer M13 and ALF automated

sequencer at the University of Notre Dame, or specific T7 primers (Table 3.2) with the

52

BigDye termination (Applied Biosystems, Foster City, CA) and ABI377 sequencer at
Iowa State University or the University of Minnesota Some sequences were obtained by
sequencing purified PCR products from screening reactions (Qiaquick, Qiagen, Valencia,
CA). Several clones were sequenced at both University of Notre Dame and Iowa State
University to check for discrepancies. The results were consistent regardless of the
primers (universal or specific), template (purified plasmids or direct screening PCR
products) and sequencing method.

The mitochondrial COI/COII coding gene region was PCR amplified as a single
lkB fragment using the protocol of Smith and Bush (1997) and primers George (Cl-J-
2792, Bogdanowicz et al 1993) and Eva (TK-N-3722, Bogdanowicz et al 1993) (Table
3.2), with the modification that DNA was not quantified prior to amplification. PCR
products were purified (Qiaquick, Qiagen, Valencia, CA) and sequenced directly in both
directions using BigDye termination (Applied Biosystems, Foster City, CA) and ABI377

sequencer at Iowa State University.

Table 3.2. Oligonucleotide sequences used for PCR amplifications and sequencing

 

Primer Sequence

220T3 5’ CTG AAG TGG AAG ATG AAG AG 3’

220T7 5’ TTC GCG TAG TTA CAT ATT TAC 3’

2956T3 5’ CTG CGT TGC TGT TTT TGC 3’

2956T7 5’ CGC TAT TTA TTC CTG AAC ATA TTT TC 3’
2480T3 5’ GCC AAA GGG TAA TTT GTT TGA TAG 3’
2480T7 5’ TGC GAT TTG ACT TAT CTT AAT GG 3’
George 5’ ATA CCT CGA CGT TAT TCA GA 3’

Eva 5’ GAG ACC ATT ACT TGC TTT CAG TCA TCT 3’

 

 

53

Data analyses: Editing of nucleotide sequences was performed by using
Sequencher 3.1 (Gene Codes Corp. Ann Arbor, M1) or Mac Vector 6.0 (Oxford
Molecular Ltd., Oxford, UK). Sequences were aligned by eye using Se-Al v1.0 (Rambaut
1996 online) and compared to unpublished sequences of cDNA for each nuclear locus
(courtesy of J. Roethele) or published COl/COII sequences (Smith and Bush 1997).
Alignments (Appendices A-D) were exported as Nexus files for analyses of
polymorphism and phylogenetic relationships. GenBank accession numbers for the
sequences are being obtained at the time of the submission of this dissertation.

Measures of DNA polymorphism (number of haplotypes, haplotype diversity h
(estimate of heterozygosity), nucleotide diversity 7t and 0, and number of polymorphic
sites) were obtained using the program DnaSP ver. 3.50 (Rozas and Rozas 1999). This
program was also used to calculate values of Tajima’s D (Tajima 1989) as well as to
perform the Hudson-Kreitman-Aguadé test (HKA, Hudson et al 1987) of neutrality
(Kimura 1983). The average number of nucleotide substitutions per site between R.
pomonella and R. zephyria (ny) and the number of net nucleotide substitutions per site
between these two species (Da) were also estimated using DnaSP, as well as the
parameters that estimate gene flow (Nm).

Phylogenetic analyses were performed in PAUP* 4b (Swofford 1999) version 6.
For nuclear loci, analyses were performed on a PowerMacintosh G3, with 512MB RAM
memory and 400MB allocated to PAUP. For COI/COII, analyses were performed on a
PowerMacintosh 8600/200, with 64MB RAM and 48MB allocated to PAUP. For each
individual dataset, neighbor-joining and maximum parsimony analyses were done.

Neighbor-joining analysis was based on pairwise Jukes—Cantor distances (J ukes and

54

Cantor, 1969), with R. cingulata as outgroup for P220, R. basiola as outgroup for P2956,
R. tabellaria as outgroup for P2480 and R. suavis as outgroup for COI/COII. Identical
alleles (haplotypes) were then removed from the analysis in order to decrease the number
of possible rearrangements. Insertion/deletion events (indels) were hypothesized based on
the aligned sequences. For P220 and P2480 indels were scored as present or absent and
appended to the datasets (Siiltrnan et al 1995). For P2956, the majority of the indels were
overlapping so it was not possible to score them separately. For COI/COII there was only
one 5-bp deletion in one sequence and this indel was also not scored. Gaps were then
treated as missing data. In parsimony analyses, MP trees were first obtained using the
heuristic search option with simple sequence addition and TBR branch swapping. The
search was limited to saving 5,000 trees due to the large number of most parsimonious
trees. Multiple searches were than performed to search for the islands of shorter trees as
recommended by Maddison (1991) and using the method of Masta (2000) with 1000
replicate searches, each limited to saving 50 trees one step longer than the shortest trees
found. In all cases these searches found trees that were shorter than the ones obtained by
the initial simple sequence addition search. MPRs from these searches were then used as
starting trees for TBR branch swapping.

Images in this dissertation are presented in color. In all dendrograms and
phylograms presented here, green branches indicate R. zephyria, red R. pomonella, blue
R. mendax and light-blue R. nr. mendax (flowering dogwood fly). Squares to the right of
taxon label indicate R. zephyria, circles R. pomonella. Different colors of these symbols
correspond to geographic regions: red — Northeast, orange — Great Lakes, green — Great

Plains, blue — Rocky mountains, violet — Northwest and gray — South.

55

To assess the confidence estimates on the groups contained in the MP trees,
F elsenstein’s (1985) bootstrap approach was used with 500 replicates for both neighbor-
joining and MP trees. For parsimony bootstrapping, the fast stepwise sequence addition
option in PAUP* 4b6 was used. Branches with bootstrap confidence limits above 90 are
considered to be well supported, between 70 and 90 moderately well supported and

between 50 and 70 weakly supported.

RESULTS

Polymorphism. A summary of the number of sequences obtained at each locus is
presented in Table 3.3. DNA sequence variation is presented in Table 3.4. Two main
sampling strategies are usually found in phylogenetic studies based on DNA sequences
(F unk 1999). In one strategy, a small number of individuals (or only one) is sampled
from a large number of taxa and the phylogenies obtained from the sequence data are
usually well resolved and supported (Funk et al. 1995). An alternate strategy consists of
sampling several individuals from a number of geographic units, usually from the same
species, focusing on population genetic processes within species (Avise et al. 1987). Both
of these strategies have their limitations. When a small number of individuals per taxon is
sampled, intraspecific variation may not be detected; if sample sizes are large but limited
to one species inferences are also limited to that species. In this study, populations of R.
zephyria and R. pomonella were sampled across their geographic ranges and a small
number of alleles from each population was obtained for three nuclear and one

mitochondrial gene. Therefore the sampling for the phylogeographic analysis of the

56

divergence between R. zephyria and R. pomonella conforms to the “large number of taxa,
small number of individuals per taxon” strategy, since the populations of the two species
were the main objects (or taxa) of the study, with the goal to obtain information that can
provide insight on the process of speciation and divergence of R. zephyria.

The uneven number of sequences obtained from each taxon at each locus resulted
from unsuccessful amplifications or clonings or sequences that were too ambiguous at
multiple sites so that the calls could not be easily made. At P220 and P2956 haplotype
diversity in R. zephyria is slightly higher than in R. pomonella, whereas at P2480 and
COI/COII it is higher in R. pomonella. Summed over all nuclear loci, the weighted
average value of 0/bp was higher in R. zephyria (0.0652) than in R. pomonella (0.0513).
For mitochondrial COI/COII R. pomonella displays higher nucleotide polymorphism than
R. zephyria. The percentage of polymorphic sites was similar in the two species at P2956
and P2480 (7.7 and 20.5% in R. pomonella, 7.9 and 19.8% in R. zephyria). At P220,
21.9% of sites were polymorphic in R. zephyria, compared to 13.9% in R. pomonella. At
COl/COII, a low level of polymorphism was observed — only 3% of sites were
polymorphic in R. pomonella and 2.3% in R. zephyria. No fixed differences were

observed between R. pomonella and R. zephyria at any of the loci.

57

Table 3.3. Summary of the sample and loci analyzed in R. zephyria, R. pomonella and
other taxa of the genus Rhagoletis in this phylogeographic study.

 

 

Locus nZ np n0 Link. gr. Allozyme Length
P220 67 46 32 I Aat-2, Did-2 425
P2956 13 36 96 Il Acon-2, Mpi, Me 521
P2480 70 34 1 1 111 Had 439
COl/COII 25 23 32 mt n/a 1074

 

nz - number of R. zephyria sequences. np— number of R. pomonella sequences. n0 - number of sequences
obtained from other taxa. Link. gr. — linkage group (chromosome) where the locus is located in the R.
pomonella genome (Roethele et al 1997). Allozyme — allozyme locus to which the locus under study is
linked. Length — number of base pairs aligned.

Table 3.4. Summary of the polymorphism at three anonymous nuclear loci and
mitochondrial COI/COII locus in R. pomonella, R. zephyria and other taxa of the genus

Rhagoletis.

 

 

 

 

 

Locus Length Na] h Npoly n 0
porn 30 0.926 57 0.031 0.059
P220 425 zep 48 0.950 93 0.028 0.073
other 28 0.992 85 0.046 0.056
porn 22 0.943 40 0.020 0.021
P2956 521 zep 11 0.962 41 0.018 0.032
other 92 0.999 175 0.048 0.087
pom 32 0.996 90 0.070 0.080
P2480 439 zep 51 0.984 87 0.057 0.097
other 1 1 1.000 81 0.065 0.077
pom 13 0.874 32 0.009 0.020
COI/COII 1074 zep 11 0.800 25 0.006 0.014
other 22 0.970 139 0.041 0.059

 

pom — R. pomonella. zep — R. zephyria. Other — other taxa. N.. — number of different alleles (haplotypes).
NM, - number of polymorphic sites. h — haplotype diversity. 1: - nucleotide diversity. 0 - nucleotide

diversity, estimate of 4Ncu.

58

Tests of selective neutrality. Natural selection acting on different loci can change
expectations about lineage sorting and coalescence, so it is important to estimate whether
selection is acting on the loci under study. Selection can be detected using Tajima’s
parameter D based on the comparison of two different estimates of 0 which are strongly
correlated (Tajima 1989). If the loci are neutral, both estimates are expected to be the
same and D is expected to be 0. A values of D significantly different from 0 indicates that
natural selection acts on the locus. Using this method, selection was detected at P220 and
P2956 in R. zephyria and COI/COII in R. pomonella and R. zephyria (Table 3.5). An
alternative method (F u and Li, 1993) is based on weakly correlated estimates of 0. The
test statistic G was significant at P220 and COI/COII in R. pomonella and R. zephyria
and at P2480 in R. zephyria (Table 3.5). All estimates of D and G had negative values,
indicating an excess of low frequency polymorphisms and slightly deleterious alleles (Li

1997).

59

Table 3.5. Detection of natural selection at the four loci in R. pomonella, R. zephyria and
all other Rhagoletis taxa (pooled) using Tajima’s (1989) and F u and Li’s (1993) tests.

 

 

 

 

 

Locus Species Tajima’s D Fu & Li’s G
pom -1.648 -3.018*
P220 zep -2. 122* -4.704**
other -0.674 -0.221
pom - l .086 -1.966
P2956 zep -1.930* -2.153
other -1 .504 -1.731
porn -1.062 -1.673
P2480 zep -l .404 -3.377**
other -0.722 -0.367
porn -2.l72** -2.973*
COI/COII zep —2.219** -3.108**
other -1 . 190 -0.480

 

pom —- R. pomonella, zep — R. zephyria, other —- other Rhagoletis species. * - P<0.05, ** - P<0.01

Neutral mutation hypothesis predicts that the levels of within- and between-

species DNA variation should be positively correlated (Kimura and Ohta 1971). This

prediction can be tested by the Hudson-Kreitman-Aguadé (HKA) test which estimates

whether the levels of observed polymorphism and divergence are consistent across loci

(Hudson et al 1987). HKA test showed that levels of polymorphism and divergence

across the three regions were consistent within and between species and none of the loci

caused significant departure from neutrality in R. pomonella and R. zephyria (x2 non-

significant, Table 3.6). Pairwise comparison of P2956 and COI/COII was not possible

because none of the R. pomonella sequences at P2956 were obtained from the individuals

from which sequences of COl/COII were obtained.

60

Table 3.6. HKA (Hudson-Kreitman-Aguadé) test for silent-site differences in four
regions between R. pomonella and R. zephyria.

 

 

 

Region SL6 p Wd 12
obs exp obs exp

P220 38 38.20 16.27 16.07

vs.P2956 25 24.80 10.23 10.43 0.001

P220 33 33.89 9.64 8.75

vs. P2480 65 64.11 15.67 16.56 0.018

P220 37 38.09 9.31 8.22

vs.COII/COII 21 19.91 3.21 4.30 0.075

P2480 29 28.33 23.81 24.47

vs.P2956 8 8.67 8.15 7.49 0.012

P2480 66 66.95 15.21 14.26

vs. COI/COII 24 23.05 3.97 4.91 0.035

 

seg — number of segregating sites, pwd — average pairwise number of differences

Divergence between R. pomonella and R. zephyria. The average numbers of
nucleotide substitutions per site between R. pomonella and R. zephyria (ny) for each
locus are given in Table 3.7. The highest number of substitutions per site was observed at
P2480, whereas the lowest was in COI/COII. The number of net nucleotide substitutions
per site (D3) between the two species was highest at P220 (Table 3.7) and lowest at
COI/COII. Using these parameters and the Lynch and Crease (1990) method in DnaSP,
gene flow between the species was estimated (Nm, Table 3.7). Values of Nm at P2480
and COI/COII were high (Table 3.7). At these two loci natural selection was detected
using Fu and Li’s test (Table 3.5). The estimate of Nm was the lowest at P220 for which

Fu and Li’s G was also significant in both R. pomonella and R. zephyria (Table 3.5).

61

Table 3.7. Summary of divergence parameters between R. pomonella and R. zephyria.

 

Locus ny Da Nm
P220 0.0526 0.0233 0.31
P2956 0.0205 0.0056 0.66
P2480 0.0682 0.0039 3.97
COI/COII 0.0075 0.0002 15.24

 

 

Phylogenetic analyses. Neighbor-joining analyses based on J ukes-Cantor
distances and parsimony analyses were performed for each locus. The neighbor-joining
trees (Figures 3.2, 3.5, 3.8 and 3.11) illustrate the relative genetic distances between the
analyzed sequences. If the substitution rates at each locus were uniform (constant
molecular clock within and across loci), branch lengths of neighbor-joining trees would
be proportional to time since divergence. The constancy of the molecular clock at each
locus so far has not been demonstrated in Rhagoletis; at different loci branch lengths
differ considerably (Figures 3.2, 3.5, 3.8 and 3.11), which obscures estimates of times
since divergence. Generally, the amount of change in the R. pomonella species group was
found to be small (especially at COI/COII and P2956) which suggests recent divergence
ofthe taxa (Figures 3.2, 3.5, 3.8, 3.11).

A summary of the parsimony analyses is given in Table 3.8. At all loci the
number of informative characters used for parsimony analyses was low, and in some
cases (P220) lower than the number of individual sequences analyzed. This complicated
the analyses and resulted in thousands of equally parsimonious trees. Except for P2480,

consensus trees were poorly resolved and bootstrap support for clades was low (Figures

62

3.4, 3.7, 3.10 and 3.13). Consistency indices were also low, while retention indices were

relatively high (Table 3.8).

Table 3.8. Summary of the parsimony analyses of the DNA sequences at three
anonymous nuclear loci and mitochondrial COI/COII locus.

 

Locus n length n,- nMpR TL CI RI RC HI

P220 145 425 105 2645003 262 0.523 0.896 0.468 0.477
P2956 145 521 148 2854003 309 0.628 0.937 0.588 0.372
P2480 115 439 139 1920 582 0.308 0.698 0.214 0.692
COI/COII 80 1074 141 89200“ 300 0.613 0.780 0.483 0.387

n — number of sequences, n, -— number of informative characters, nMpR — number of equally parsimonious
trees obtained, TL — tree length, CI — consistency idex, RI — retention index, RC — rescaled consistency
index, HI — homoplasy index; 3 indicates that the search was terminated when the number of saved MPRs
exhausted the RAM before swapping on all trees.

Maximum parsimony analysis of P220 using 1000 replicate island searches
yielded 26500 equally parsimonious trees of length 262. Additional TBR swapping on
these trees yielded only trees of the same length (262) and exhausted the RAM after
saving 264,500 trees. A random MPR (most parsimonious reconstructions) is shown in
Figure 3.3. Strict consensus of the 264,500 trees is shown in Figure 3.4. Only 10 nodes
within the R. pomonella species group were supported and overall bootstrap support was

low.

63

Figure 3.2. P220 neighbor-joining tree based on Jukes-Cantor distances. Bootstrap values
greater than 50% are shown. Green branches represent R. zephyria, red R. pomonella,
blue R. mendax and light-blue R. nr. mendax (flowering dogwood fly). Squares next to
the taxon name symbolize R. zephyria, and circles symbolize R. pomonella. Colors of the
symbols represent geographic regions: red — Northeast, orange — Great Lakes, green —
Great Plains, blue — Rocky Mountains and Colorado Plateau, violet — Northwest and gray

— South. For taxon labels please refer to Key to symbols and abbreviations (p. xi).

64

 

El

 

 

 

_«A I
21141.3.
mom.
“‘2 .11
14 M» I
M3 o

 

 

 

 

 

—— 0.005 substitutions/site
Figure 3.2.

65

Figure 3.3. P220 - a random most parsimonius tree (of the 264500 equal MPRs). Green
branches represent R. zephyria, red R. pomonella, blue R. mendax and light-blue R. nr.
mendax (flowering dogwood fly). Squares next to the taxon name symbolize R. zephyria,
and circles symbolize R. pomonella. Colors of the symbols represent geographic regions:
red — Northeast, orange — Great Lakes, green — Great Plains, blue — Rocky Mountains and
Colorado Plateau, violet — Northwest and gray — South. For taxon labels please refer to

Key to symbols and abbreviations (p. xi)

66

Fun] I

ma.

Mu I

 

 

 

 

 

 

 

 

 

 

is:

 

is

 

«to
.‘OC'

 

l
i-

has.
a

4

 

 

 

 

 

 

Figure 3.3.

67

Figure 3.4. P220 - strict consensus of 264500 most parsimonious trees of length 262.
CI=0.523, RI=O.896, RC=0.468. Bootstrap values greater than 50% are shown above
branches. Green branches represent R. zephyria, red R. pomonella, blue R. mendax and
light-blue R. nr. mendax (flowering dogwood fly). Squares next to the taxon name
symbolize R. zephyria, and circles symbolize R. pomonella. Colors of the symbols
represent geographic regions: red — Northeast, orange — Great Lakes, green —- Great
Plains, blue — Rocky Mountains and Colorado Plateau, violet — Northwest and gray —

South. For taxon labels please refer to Key to symbols and abbreviations (p. xi).

68

u ...IIIII ltlnl I...

III; Ill-Ila.

nooooooc O

O

 

Figure 3.4.

69

P2956 was the only locus for which majority of the sequences obtained were from
the taxa outside of the R. pomonella group (Table 3.3, Figures 3.5, 3.6 and 3.7). Both
neighbor-joining and parsimony analyses at this locus suggest that R. zephyria groups
within the R. pomonella species group (Figures 3.5, 3.6 and 3.7), with the majority of the
groups weakly to moderately supported. Replicate island parsimony searches yielded
4550 trees of length 309, which were used as starting trees for TBR branch swapping.
The search was aborted after saving 285,400 trees and exhausting the RAM. A random
MPR (most parsimonious reconstruction) is shown in Figure 3. 6. A strict consensus of
the MPRs at P2956 (Figure 3.7) retains the R. pomonella species group clade with poorly
resolved relationships between the taxa and R. zephyria forming an unresolved group
within this clade, with the exception of a single R. zephyria from New York. Bootstrap
support for the clades was low to moderate (Figure 3.7).

Replicate island searches for the shortest parsimony trees at P2480 yielded 650
trees of length 582. After additional TBR swapping on these trees, 1920 trees of the same
length were found. A random tree representing the 1920 trees is shown in Figure 3.9, and
a strict consensus of these trees is shown in Figure 3.10. Although the resolution at this
locus was good (better than at other loci), consistency index and retention index had low

values (Table 3.8) and a low number of nodes was bootstrap supported (Figure 3.10).

70

Figure 3.5. P2956 — neighbor-joining tree (J ukes-Cantor distances). Bootstrap values
greater than 50% are shown. Green branches represent R. zephyria, red R. pomonella,
blue R. mendax and light-blue R. nr. mendax (flowering dogwood fly). Squares next to
the taxon name symbolize R. zephyria, and circles symbolize R. pomonella. Colors of the
symbols represent geographic regions: red -— Northeast, orange —— Great Lakes, green —
Great Plains, blue — Rocky Mountains and Colorado Plateau, violet - Northwest and gray

— South. For taxon labels please refer to Key to symbols and abbreviations (p. xi).

 

 

 

 

 

 

 

rs“
E
— 0.005 substitutions/site

Figure 3.5.

 

 

72

 

 

 

 

1“ £3:

 

 

 

Figure 3.6. P2956 — a random most parsimonious tree (of 285400 equal MPRs). Green
branches represent R. zephyria, red R. pomonella, blue R. mendax and light-blue R. nr.
mendax (flowering dogwood fly). Squares next to the taxon name symbolize R. zephyria,
and circles symbolize R. pomonella. Colors of the symbols represent geographic regions:
red — Northeast, orange — Great Lakes, green — Great Plains, blue — Rocky Mountains and
Colorado Plateau, violet — Northwest and gray — South. For taxon labels please refer to

Key to symbols and abbreviations (p. xi).

73

 

 

 

 

 

 

 

 

74

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3.7. P2956 - strict consensus of 285400 most parsimonious trees of length 309.
CI=0.628, RI=0.937, RC=0.5 88. Bootstrap values greater than 50% are shown above
branches. Green branches represent R. zephyria, red R. pomonella, blue R. mendax and
light—blue R. nr. mendax (flowering dogwood fly). Squares next to the taxon name
symbolize R. zephyria, and circles symbolize R. pomonella. Colors of the symbols
represent geographic regions: red — Northeast, orange — Great Lakes, green — Great
Plains, blue —- Rocky Mountains and Colorado Plateau, violet — Northwest and gray —

South. For taxon labels please refer to Key to symbols and abbreviations (p. xi).

75

 

Figure 3.7.

76

Figure 3.8. P2480 — neighbor-joining tree (J ukes-Cantor distances). Bootstrap values
greater than 50% are shown. Green branches represent R. zephyria, red R. pomonella,
blue R. mendax and light-blue R. nr. mendax (flowering dogwood fly). Squares next to
the taxon name symbolize R. zephyria, and circles symbolize R. pomonella. Colors of the
symbols represent geographic regions: red —— Northeast, orange — Great Lakes, green —
Great Plains, blue — Rocky Mountains and Colorado Plateau, violet — Northwest and gray

— South. For taxon labels please refer to Key to symbols and abbreviations (p. xi).

77

 

 

 

 

("LI I

 

”A O
14 NJ .

.1
7‘ $111.60 "In I

ciiia
5' 10.4

 

 

“.1

_ 0.005 substitutions/site

Figure 3.8.

78

Figure 3.9. P2480 — a random most parsimonious tree (of 1920 MPRs). Green branches
represent R. zephyria, red R. pomonella, blue R. mendax and light-blue R. nr. mendax
(flowering dogwood fly). Squares next to the taxon name symbolize R. zephyria, and
circles symbolize R. pomonella. Colors of the symbols represent geographic regions: red
— Northeast, orange — Great Lakes, green — Great Plains, blue — Rocky Mountains and
Colorado Plateau, violet — Northwest and gray — South. For taxon labels please refer to

Key to symbols and abbreviations (p. xi).

79

 

 

 

 

 

“.2
NJ

— 5 changes

Figure 3.9.

80

Figure 3.10. P2480 strict consensus of 1920 most parsimonious trees of length 582.
CI=0.308, RI=0.698, RC=0.214. Bootstrap values greater than 50% are shown above
branches. Green branches represent R. zephyria, red R. pomonella, blue R. mendax and
light-blue R. nr. mendax (flowering dogwood fly). Squares next to the taxon name
symbolize R. zephyria, and circles symbolize R. pomonella. Colors of the symbols
represent geographic regions: red — Northeast, orange — Great Lakes, green — Great
Plains, blue — Rocky Mountains and Colorado Plateau, violet — Northwest and gray —

South. For taxon labels please refer to Key to symbols and abbreviations (p. xi).

81

Figure 3.10.

 

 

 

 

 

 

 

 

 

 

 

 

ss-

tit

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sunrises...

 

 

 

5
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as:
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.—

 

 

 

 

 

 

82

     

s

Parsimony analysis of the COI/COII data using replicate island searches yielded
280 trees of length 300. Additional TBR branch swapping on these trees yielded 89,200
trees of the same length (Table 3.8) before exhausting RAM. A random MPR is shown in
Figure 3.12. A strict consensus of the MPRs did not resolve relationships among the taxa
of the R. pomonella species group (Figure 3.13) and the only clades that were bootstrap
supported were the clades consisting of the taxa outside of the R. pomonella species

group and R. carnivora.

83

Figure 3.11. COI/COII — neighbor-joining tree (J ukes-Cantor distances). Bootstrap values
greater than 50% are shown. Green branches represent R. zephyria, red R. pomonella,
blue R. mendax and light-blue R. nr. mendax (flowering dogwood fly). Squares next to
the taxon name symbolize R. zephyria, and circles symbolize R. pomonella. Colors of the
symbols represent geographic regions: red — Northeast, orange —- Great Lakes, green —
Great Plains, blue — Rocky Mountains and Colorado Plateau, violet — Northwest and gray

— South. For taxon labels please refer to Key to symbols and abbreviations (p. xi).

84

wane.
'Fpmz.
W11

“unit

b m2.
- MAO
an I

 

 

 

 

 

 

 

 

100

 

 

 

 

 

mum
«2.2

- 0.001 wbstltutlonslslte

Figure 3.11.

85

 

Figure 3.12. COI/COII -—- a random most parsimonious tree (of 89200 MPRs). Green
branches represent R. zephyria, red R. pomonella, blue R. mendax and light-blue R. nr.
mendax (flowering dogwood fly). Squares next to the taxon name symbolize R. zephyria,
and circles symbolize R. pomonella. Colors of the symbols represent geographic regions:
red — Northeast, orange — Great Lakes, green — Great Plains, blue -— Rocky Mountains and
Colorado Plateau, violet — Northwest and gray — South. For taxon labels please refer to

Key to symbols and abbreviations (p. xi).

86

was“.
«.1 I
MNA
MI
W
W1.
pic I
-—ptx1.20
“NLMH
—gm|2 I
W.
«mam. I
m I
not I
m-
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arm I
and I
m1.um1v.pd4l I I

 

 

 

 

 

 

 

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Figure 3.12.

I—ruu
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~61?
1m

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87

Figure 3.13. COI/COII - strict consensus of 89200 most parsimonious trees of length 300.
CI=0.613, RI=0.780, RC=0.483. Bootstrap values greater than 50% are shown above
branches. Green branches represent R. zephyria, red R. pomonella, blue R. mendax and
light-blue R. nr. mendax (flowering dogwood fly). Squares next to the taxon name
symbolize R. zephyria, and circles symbolize R. pomonella. Colors of the symbols
represent geographic regions: red — Northeast, orange — Great Lakes, green — Great
Plains, blue — Rocky Mountains and Colorado Plateau, violet —— Northwest and gray —

South. For taxon labels please refer to Key to symbols and abbreviations (p. xi).

88

anus“: O
“11.1 I
post: I
ammonia,“
"14.1

W2 '
pm I
anA
90102 I
m I
m1 I
not I
zone I
zma I
mmtt

rm.

man?

an“

man
won
me
meal I
“M6

 

 

MI
w.
9011.
HISI
[IMI
m1.
anae-

antr-
MI
anta-
anu-
MI
ml
m4-
marl
pma.
pm.
ala-

pcot I
m I
not I
not I
zmt I
an I
not I
put I
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zmtBr I
mMX
12110.1
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UM
doctmnt
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001111
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NM. 1
“2.2

 

Figure 3.13.

89

DISCUSSION

Phylogenetic position of R. zephyria. At all four loci analyzed here, both R.
pomonella and R. zephyria appear to be polyphyletic (Figures 3.2—2.13). Results obtained
from the different loci support the hypothesis that R. zephyria is a sister taxon to R.
pomonella and that it has diverged from the common ancestor more recently than R.
mendax. At all loci sequences of R. zephyria and R. pomonella are placed within the same
clades, and at P2956, P2480 and COI/COII alleles of R. mendax appear to have diverged
from the common ancestor with the present-day R. pomonella before alleles of R.
zephyria (Figures 3.7, 3.10 and 3.12). This is consistent with findings of Bush and Smith
(1997) and McPheron and Han (1997) based on mitochondrial DNA sequences.
Allozyme data (Berlocher et al 1993) suggested a different pattern and placement of R.
zephyria at the base of the closely related taxa of the R. pomonella species group (without
the more diverged R. cornivora). Recent analysis based on allozyme data agreed with the
previous one on basal placement of R. zephyria; however, this analysis included only two
populations of R. zephyria and it was suggested that the alternative placement (within the
group as a sister to R. pomonella) was not significantly poorer (Berlocher 2000).

At P220 most of the R. zephyria sequences were included in the same clade
(Figure 3.3) which also included R. pomonella from Michigan, Texas and Georgia, R. nr.
pomonella from La Jolla, MX, R. fausta and R. juniperina. Within this clade two smaller
clades of R. zephyria were observed. Within the larger of these two clades two R.
pomonella sequences were found, both from the Great Plains region. Other sequences of

R. zephyria or R. pomonella do not fall into the clades which reflect geographic regions.

90

A large R. zephyria clade is retained in the strict consensus with the same R. pomonella
sequences from Great Plains within the clade. A clade formed by R. tabellaria and R.
electromorpha, sister species from the R. tabellaria species group, was also retained in
the strict consensus tree (Figure 3.4). The remainder of the tree was poorly resolved
(Figure 3.4).

At P2956, species of the R. pomonella group formed a clade within which a single
R. striatella allele sequence was placed (Figure 3.6). R. zephyria formed a sister clade to
one of the R. pomonella clades, except for a single individual from New York (zny 3.4),
which was a sister taxon to another R. striatella and placed in a clade with R. suavis and
R. flavigenualz's which are distantly related to the R. pomonella species group. The clade
of R. pomonella species group (excluding some of the R. cornivora sequences which
were placed as a sister group to R. suavis and R. flavigenualis, Figure 3.7) was weakly
supported (bootstrap = 66). Moderate support was observed for the clade consisting of R.
pomonella group taxa without R. cornivora (bootstrap = 74). Clades consisting of the
taxa outside of the R. pomonella species group were weakly to moderately supported
(Figure 3.7).

The relationships among the R. pomonella species group taxa suggested by the
maximum parsimony analysis of the P2480 were consistent with the relationships
suggested by allozyme data (Berlocher et a1 1993). R. cornivora formed a sister clade to
the other R. pomonella group species (Figure 3.10). At the base of the more closely
related R. pomonella group taxa (without R. carnivora) was the clade formed by five R.
pomonella sequences. R. mendax and R. nr. mendax branched off from the remainder of

the taxa before branching of R. zephyria and R. pomonella. Sequences of R. zephyria and

91

R. pomonella (except for five in the basal clade) were placed in three distinct clades, none
of which corresponded to species. One of the three clades consisted mostly of R. zephyria
from the populations west of the Mississippi River Valley, with three R. pomonella
sequences (from Minnesota, Nebraska and Colorado) within this clade. Another large
clade consisted of R. pomonella and R. zephyria from both sides of the Mississippi, with
some structuring of R. zephyria into a predominantly western and predominantly eastern
group. R. pomonella sequences were placed with predominantly eastern R. zephyria
(except one R. pomonella from Michigan found in the “western” R. zephyria clade,
Figure 3.10). The third large clade contained mostly sequences of R. zephyria from the
Northeast and Great Lakes, with R. pomonella predominately from the Great Plains.

At COI/COII branch lengths within the R. pomonella species group were short,
which is consistent with the low levels of nucleotide polymorphism (Table 3.4) and low
sequence divergence between R. pomonella and R. zephyria (Table 3.7). A unique
haplotype was found in R. zephyria populations ranging from Massachusetts to Michigan.
Of all the eastern R. zephyria, only haplotypes found in Wisconsin and New York
populations was placed into the clade consisting of western R. zephyria and both eastern
and western R. pomonella (Figure 3.12). All mtDNA haplotypes observed in western R.
zephyria were either shared with or more closely related to R. pomonella.

The inability to better resolve the relationships among taxa in this study at three
out of four loci may stem from the very low number of informative characters and high
number of taxa (sequences) analyzed. High number of taxa also deflates consistency
indices, regardless of any change in information content (Kitching et a1 1998). It is

interesting to note that the only well-resolved locus in this study was P2480, which is

92

tightly linked to Had, the only allozyme locus that displays fixed difference between R.
pomonella and R. zephyria.

Some of the discrepancies between the gene trees presented here may be caused
by different forms of selection acting on the different genes. At P220, alleles from the
same heterozygous individuals were found in divergent clades, suggesting that balancing
selection may have played a role in Shaping the phylogenetic pattern observed at this
locus. Estimates of Tajima’s D and Fu and Li’s G at all loci were negative (Table 3.5).
The more robust Fu and Li’s statistic detected significant selection at P220 and COI/COII
in both R. pomonella and R. zephyria and at P2480 in R. zephyria (Table 3.5). This
finding was expected for the mitochondrial COI/COII gene region which codes for the
two subunits of cytochrome oxidase and has been shown to be conserved within the R.
pomonella species group (Smith and Bush 1997). Low nucleotide diversity in both
species and high similarity of mitochondrial COI/COII haplotypes indicate that this gene
region remains highly conserved under strong selection pressure that does not eliminate
only the mutations that maintain the functionality of the gene. Negative values of
Tajima’s D or F u and Li’s G reflect an excess of low frequency polymorphisms, which
can result from recent selection so that the variation is removed, or by a recent population
size expansion (Tajima 1989, Kliman et al 2000). The HKA test, on the other hand, did
not indicate a significant contribution of any of the loci to deviations from the neutral
mutation hypothesis (Table 3.6). Presence of selection also influences the estimates of
gene flow between R. pomonella and R. zephyria (Table 3.7). The indirect methods of
estimating gene flow (average number of migrants per generation, Nm) are based on the

parameters of divergence between the two species (average number of nucleotide

93

substitutions and number of net substitutions per site at each locus, Table 3.7) and the
assumption that the alleles at the loci under study are selectively neutral. If alleles are not
selectively neutral, and if selection favors the same allele everywhere (directional
selection), the rate of gene flow tends to be overestimated (Futuyma 1997), which can
explain the high Nm estimated for P2480 and COI/COII (Table 3.7). For these two loci
selection was detected by Fu and Li’s test (Table 3.5). Rate of gene flow can also be
overestimated if speciation has been recent and the two sister species have not had
enough time to differentiate. The high Nm at these two loci seems to support the
hypothesis of a recent speciation event. If selection favors different alleles in the two
Species, the rate of gene flow tends to be underestimated. Selection was detected at P220
by Fu and Li’s test (Table 3.5), however, Nm estimated for this locus was smaller (0.31)
than for the neutral P2956 (0.66).

Inferences about speciation from phylogeography. Overall phylogeographic
patterns observed seem to be consistent with recent speciation. If the speciation has
occurred within the past 10,000 years, unresolved relationships and shared ancestral
genes are expected to be found (Masta 2000). Host fidelity in Rhagoletis is high (Bush
1966) and hybridization between R. pomonella and R. zephyria occurs only rarely (Feder
et a1 1999). The phylogenetic patterns observed at all the loci, lack of fixed differences,
sharing of alleles and haplotypes between the two species and the placement of alleles
derived from geographically very distant populations can be explained by the recent
speciation and retention of ancestral polymorphism. Alternatively, these patterns could be
explained by interspecific gene flow. Other types of data are consistent with the recent

speciation hypothesis as well — R. pomonella and R. zephyria share alleles at all allozyme

94

loci except for Had (Berlocher and Bush 1982, Berlocher et al 1993, F eder et al 1999)
and are morphologically identical except for the shape of male genitalia (Bush 1966).
Low levels of genetic divergence are often found between ecologically differentiated
species which speciated by habitat shifts (sticklebacks — Schluter 1996, cichlid fishes —
Albertson et a1 1999, Danley et al 2000, damselflies — Brown et a1 2000) and the results
reported here are consistent with those findings. One possible alternative explanation for
sharing alleles and haplotypes between species would be if the recurrent mutations occur
in the two species independently (Kliman et a1 2000). However, it would be difficult to
explain the low levels of sequence divergence observed at all unlinked loci by this
phenomenon.

Generally, little geographic structuring was observed at any of the loci. When the
sequences of R. pomonella were found in predominantly R. zephyria clades (such as at
P220 and P2480), they tended to be from the populations of R. pomonella sampled in the
Great Plains (Minnesota, Nebraska, Iowa), indicating that the host shift from hawthoms
to snowberries and speciation of R. zephyria occurred in the Plains, in the zone where
Crataegus sp. and Symphoricarpos sp. were in contact when the glaciers receded after the
last glaciation period.

At P220 and P2480, some of the R. pomonella sequences were placed at the base
of the R. pomonella species group, supporting the hypothesis that the common ancestor
may have been R. pomonella-like. Based on the working hypothesis that the common
ancestor of R. pomonella and R. zephyria was R. pomonella-like and infested hawthorn

fruits (host of the present-day R. pomonella), the expectation was that R. pomonella

95

would show a higher number of alleles, higher average nucleotide diversity and higher
heterozygosity across its geographic range than R. zephyria.

Both species have high levels of polymorphism estimated by nucleotide
diversities (7t and 0) and haplotype diversity (h, also an indirect estimate of
heterozygosity) (Table 3.4). The levels of polymorphism observed in R. pomonella and R.
zephyria were much higher than in Drosophila simulans, D. mauritiana and D. sechellia
(Kliman et a1 2000). Only at COI/COII was the amount of variation low, as expected for
Rhagoletis mitochondrial genes (Smith and Bush 1997, Smith and Bush 2000). Over all
loci studied, R. zephyria had higher weighted average of 0/bp (0.0652) than R. pomonella
(0.0513), contrary to the expectation that R. pomonella should have higher diversity.
However, at some loci (P2480) sequences of R. pomonella were not obtained from the
entire geographic range, and generally numbers of sequences obtained were uneven at all
loci, which may have contributed to the unexpected finding that R. zephyria appears to be
more genetically variable than R. pomonella. Even so, it appears that the speciation of R.
zephyria was not accompanied by bottleneck.

It should be noted that high genetic diversity does not necessarily negate
population bottleneck at speciation - whether the bottleneck can be reflected by the
present genetic structure depends on how recent the speciation event is and how long the
bottleneck lasted relative to the effective population size (Eyre-Walkers 1998). Given the
lack of fixed differences between R. pomonella and R. zephyria at the loci studied here
and the similarity of their sequences reflected by the small numbers of phylogenetically
informative characters, shared alleles and haplotypes between individuals from different

species, relatively short branch lengths in neighbor-joining analyses and unresolved

96

phylogenetic relationships (Table 3.8, Figures 3.2-3.13, Smith and Bush 1997, McPheron
and Han 1997), the scenario in which the speciation occurred relatively recently seems
plausible. Therefore, it is unlikely that if the bottleneck played a role in speciation it
would not be reflected by the present structure.

It is difficult to infer the direction of population expansion from the phylogenies
at different loci (Figures 3.2-3.13), since little geographic structuring is revealed.
However, the better resolved P2480 MPRs suggest some structuring of R. zephyria,
supporting the hypothesis that dispersal to the west preceded the expansion to the east
(Figure 3.10). Of the three clades where R. zephyria is placed in the strict consensus of
1920 most parsimonious trees, the basal one consists mostly of alleles from populations
west of the Mississippi, indicating that these populations may have had more time to
adapt to local conditions and accumulate new mutations than the populations in the east.

Incomplete lineage sorting and the presence of both eastern and western alleles of
R. zephyria in the same clades at P2480 and P220 seems to support the hypothesis that R.
zephyria was not introduced to the Eastern North America, consistent with findings of
infestations of native hosts east of the Mississippi River and infestations of introduced
hosts only when the native hosts have been present (Chapter 2). If introduction had
occurred, populations in the east should have gone through a bottleneck, which would be
reflected in the MPRs by shorter branch lengths, similarity of the sequences in the east
and their placement together. This was not observed (Figures 3.2-3. 1 3). However, a
hypothesis that multiple introductions of R. zephyria from different areas in the west into

the east cannot be ruled out.

97

One possible way to obtain more information about the processes involved in
divergence of the two closely related species may be to assess genetic variation at many
more loci and more individuals, which I did using the amplified fragment length

polymorphism (AF LP) DNA fingerprinting (Chapter 4).

98

CHAPTER 4
DIVERGENCE OF R. POMONELLA AND R. zsrrmm AS REVEALED BY

DIFFERENCES IN AF LP PATTERNS

INTRODUCTION

Attempts to resolve phylogenetic relationships among taxa of the R. pomonella
species group and/or to determine phylogenetic position of R. zephyria so far have used
allozymes (Berlocher et al 1993, Feder et al 1999, Berlocher 2000), morphological
characters (Jenkins 1996), and sequences of mtDNA genes (Smith and Bush 1997,
McPheron and Han 1997) or nuclear loci (Chapter 3, Feder et al in prep). These studies
have shown that R. pomonella appears to be the most variable species of the group, which
has led to a working hypothesis that R. pomonella represents a large ancestral gene pool
from which new species have arisen. The lack of observed fixed differences between the
pomonella group species, except for the Hadl H allozyme allele in R. zephyria, and
similarity between mtDNA haplotypes (Smith and Bush 1997, McPheron and Han 1997)
indicates their recent divergence, with R. pomonella giving rise to new taxa faster than
alleles can become fixed. Since mitochondrial COI/COII is highly conserved and reflects
only a matrilineal fraction of the evolutionary history (Maddison 1995), nuclear gene
phylogenies for 3 different loci were constructed (Chapter 3). These phylogenies all
suggested different relationships between taxa and revealed little or no clear geographic
structuring of R. pomonella and R. zephyria populations. In addition, the results only
hinted to the origin of R. zephyria - where the host shift occurred, what the forces driving

divergence and speciation could have been, and whether the snowberry fly is a native

99

inhabitant of Eastern North America. Furthermore, attempts to identify diagnostic
markers that would be useful in determining whether specimens collected (trapped) in the
field as adults belong to one or another species have so far not been very successful.

The problem in the phylogeographic approach may be that an insufficient number
of sequences (individuals) or insufficient number of markers was used. It is not very
likely that the sampling strategy used for phylogeographic analyses provided insufficient
number of sequences, given the large number obtained from R. pomonella and R.
zephyria (Chapter 3). Kliman et al (2000) argue that repetitive sampling within species
tends to reveal true genealogical history, implying that the number of individuals can be
dramatically reduced if multiple loci are sampled. For rapidly evolving clades, Moran and
Komfield (1993) suggest that only a method that assays variation at a large number of
loci could overcome the effects of incomplete allele sorting in the time since species
divergence.

Recently, amplified fragment length polymorphism (AF LP, Vos et al 1995)
genotyping has been suggested as a method of choice for studying relationships between
closely related species (Albertson et al 1999), population structure and differentiation and
estimating population genetic parameters (Reineke et al 1998, Cardoso et al 2000). It is
widely used in characterizing variation in microbial (Rademaker 2000) and plant isolates
(Myashita et al 1999, Cho et al. 1996), as well as in mapping plant genomes (V oorips et
a1 1997, Brigneti et al. 1997, Vuylsteke et a1 1999, Castiglioni et a1 1999). It has less
frequently been applied to animal systems, probably because microsatellites are well
established for many species and have been extensively used as a method for studying

genetic variation between pOpulations (for example, Estoup et al 1996). However, a

100

number of recent studies indicate that AF LP fingerprinting is becoming more popular - it
has been employed in assessing parentage in birds (Quisteau et a1 1999), detecting
hybridization (Prowell et al unpublished, Nijman et al 1999) and for establishing linkage
maps (Liu et a1 1999).

By screening loci distributed throughout the entire genome, AF LP can be used to
generate a larger quantity of information than other markers such as allozymes, RAPDs,
RFLPs or microsatellites (Mueller and Wolfenberger 1999). Characters produced by
AF LP are highly replicable and provide good resolution of phylogenetic structure
(Albertson et a1 1999). A change in a restriction recognition site does not alter the size of
the fragment but removes it from the profile. The absence of a particular fragment can
also be the result of an insertion or deletion event between the cut sites, in which case the
size of the fragment is altered; it can also result from the failure to amplify some of the
fragments from unknown reasons. Therefore, one disadvantage of the AF LP technique is
that the characters behave as dominant markers, rather than co-dominant produced by
traditional RF LP. However, its advantages include that it does not require prior
knowledge of DNA sequences, detects variation over the entire genome and is highly
reproducible because it uses stringent reaction conditions (Vos et al 1995).

I used AF LP fingerprinting technique to compare intra- and interspecific variation
at sites distributed randomly throughout Rhagoletis genome among 55 R. pomonella
individuals from 13 populations and 59 R. zephyria individuals collected from 16
populations. These populations were chosen to represent the zone of overlap between the
two species and to cover the entire geographic range of R. zephyria. This study was

aimed at providing information about geographic structuring within R. pomonella and R.

101

zephyria and identifying relationships between their populations. This information is
crucial if we are to better understand the mechanisms of speciation of R. zephyria, infer
the location of the presumed host shift from ancestral hawthoms to snowberries and
explain the present-day geographic distribution of R. zephyria. In addition, potentially
useful characters that can be used to differentiate R. pomonella and R. zephyria genotypes
by a PCR-based technique were identified, eliminating the need for the fresh material

required for allozyme—based methods.

MATERIALS AND METHODS

Sample: Individual flies representing the zone of overlap of geographic ranges of
R. pomonella and R. zephyria and covering the entire range of R. zephyria were used for
the AF LP genotyping. I sampled at least 5 flies from each population (Figure 4.1). Not
all the samples amplified successfully (Table 4.1), so for calculation of genetic distances
and analyses of geographic structure samples were pooled according to geographic
regions as follows (Table 4.1): Northeast (Massachusetts, New York and Pennsylvania),
Great Lakes (Ontario, Michigan, Illinois and Wisconsin), Great Plains (Minnesota, Iowa,
Nebraska, North Dakota, South Dakota and Montana “B”), Rocky Mountains and
Colorado Plateau (Colorado, Montana, Utah and Idaho) and Pacific Northwest
(Washington, Oregon and California). Rhagoletis mendax individuals from four
populations (East Lansing, MI; Otis Lake, MI; Jasper Pulaski, 1N and Rutgers, NJ) were

also included in the neighbor-joining analysis (Table 4.1) in an attempt to infer the

102

polarity and order of host shifts, while two individuals of R. nr. pomonella sampled near

Mexico City, Mexico, were used as an outgroup.

Table 4.1 Populations of R. zephyria and R. pomonella sampled for the AF LP analysis.

 

 

 

 

 

2%?“ Locality Label n
R. zephyria

Massachusetts Amherst ZMA 3
New York Geneva ZNY 2
Pennsylvania PSU campus ZPA 3
Michigan East Lansing ZEL 4
Wisconsin Waukesha ZWI 5
Minnesota Hawby ZMN 5
North Dakota Bismarck ZND 5
South Dakota Custer SP ZSD 5
Colorado Boulder ZCO 3
Montana Swan Lake ZMT 3
Montana Melstone ZMTB 2
Idaho Elmira ZID 4
Washington Dixie ZWA 5
Oregon Grants Pass ZOR 7
California Honeydew ZCA 3
R. pomonella

Massachusetts Amherst PMA 5
New York Geneva PNY 2
Pennsylvania Biglerville PPA 3
Ontario Toronto PON 3
Michigan E. Lansing PMI 4
Illinois Riverwoods PIL 4
Minnesota Staples PMN 4
Iowa Ames PIA 5
Nebraska (180E,exit285) PNE 3
Colorado Boulder PCO 5
Utah Wellsville PUT 4
Washington St. Cloud PWA 4
Mexico Mexico City PMX 2

 

n - number of individual fingerprints used for the analysis

103

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104

DNA extraction: DNA was isolated fiom the individual flies following the
protocol of Han and McPheron (1997, see also Chapter 3). For AF LP genotyping, the
concentration of DNA in each sample was determined using TKOlOO fluorimeter
(Hoeffer Scientific Instruments, San Francisco, CA), since equal amounts need to be used
for each individual reaction.

AFLP genotyping: AF LP analysis was performed using a modified protocol
developed by PE Applied Biosystems (Foster City, CA) after Vos et al (1995) (Figure
4.2). Restriction and ligation of adaptors was carried out in a single 11 uL reaction for
each individual DNA sample. Approximately 400ng of total genomic DNA was digested
with two restriction enzymes: 1U of four-base recognition MseI and 5U of six-base
recognition EcoRI for 2.5 hours at 37°C in presence of IU of T4 DNA ligase and adaptor
pairs, short double-stranded sequences with one asymmetric end complementary to either
EcoRI or Msel end. Ligation of adaptor sequences modifies the restriction recognition
sequence so that restriction does not occur after the ligation. The restriction-ligation
samples were diluted to 200uL in TE buffer (20mM Tris-HCl, 0.1mM EDTA, pH 8.0) to
obtain the appropriate concentration for subsequent amplification reaction. Preselective
amplification reduces the number of fragments 16-fold by using the primers that match
recognition site, adaptor sequence and 3’C for M361 primer and 3’A for EcoRI primer
(Figure 4.2). Amplifications were performed in 20uL total reaction volume using 15 uL
of Amplification Core Mix (PE Applied Biosystems), l uL of preselective primer mix and
4uL of diluted restriction-ligation products. The cycling regime for preselective
amplification was 72°C 2min, 20 cycles of 94°C lsec, 56°C 3OSec, 72°C 2 min, followed

by an extension step of 30 min at 60°C, with all ramp times set to 0.01 on the PE9600

105

thermal cycler. PCR products from preselective amplification were diluted to 400uL with
TE buffer to obtain the appropriate concentration for selective amplification. Selective
amplification further reduces the number of fragments using the primers that match
restriction enzyme recognition site, adaptor sequence and three adjacent nucleotides
(Figure 4.2). EcoRI primers are labeled with fluorescent dye which enables the
visualization of fragments after the separation of amplified products on a DNA
sequencer. Each fragment for each primer combination, visualized as a band on the
denaturing gel, corresponds to a single AF LP locus. One Msel and one EcoRI primer
should be chosen according to the PE Applied Biosystems protocol; after initial screening
for polymorphism using 35 primer combinations, I chose the E-AGG/M-CTT and E-
ACT/M-CAG primer combinations. Selective amplification reactions were performed in
20uL volumes, using 15uL of Amplification Core Mix (PE Applied Biosystems), 1pL of
each primer and 3uL of diluted preselective amplification reaction products. Cycling
parameters for selective amplification are given in Table 4.2. Amplification products
were separated on 6% denaturing polyacrylamide gels for 3.5 hours using an ABI Prism
377 DNA sequencer with GS-500 ROX-labeled size standard. A subset of samples was
independently prepared twice and electrophoresed at both MSU and Iowa State
Sequencing Facilities to check for any possible discrepancies. The results were
consistent, so the rest of the sample was run at MSU. Sizes of fragments were determined
automatically by GeneScan 2.0.2 software (PE Applied Biosystems), by comparison to

the size standard.

106

V
AGenomic DNAcrfllntofmgmenlswiththerestricfionenzymesMselandEooRl
"::l_] if: EA H: "—
-.. 3 5. ___
B. Ligation of adaptors: 60081 is ‘and Msel is E
m

C. Genomic DNA fragments are modified.
Msel Msel

 

 

EOORI EOORI

    

   

 

...-

. Template preparation and ligation of AFLP adaptors

Prepared Template: Genomic DNA
Fragment. Modified with Adaptors

F1
F1

 

Preselective Primers:

 

- A : EcoRl adaptor + recognition site + A
or 15le adaptor + recognition site

Adaptors. Thermal
Core Mix Cycling C : Msel adaptor + recognition site + C

 

s, :3; C
' G

 

 

 

 

 

Preselective amplification of the prepared template
A Choose Selective AFLP Primers:
* - Axx }one of eight different fluorescent dye-labeled
* - Ax/Tx AFLP EcoRl Selective Amplification primers

1:1 Cxx - one of eight different AFLP Msel Selective
Amplification primers.

8. Run Selective Amplification:
T1

 

1:10
1 36 A

Primers. @ Thennal

Core Mix

Cycling

 

 

 

Selective amplification with fluorescent dyelabeled primers

Figure 4.2. The principle of the AF LP DNA fingerprinting technique. Total genomic DNA is
cut into fragments using two different restriction enzymes. At the same step, fragments are modified by
ligation of adaptors to prevent reannealing. Number of fragments is subsequently reduced by two PCR
amplifications (preselective and selective) with increasing specificity of primers. In selective amplification,
one of the primers is labeled with fluorescent dye, which enables visualization of fragments after their
resolution on a denaturing gel.

107

Table 4.2. Cycling parameters used for selective amplification

 

 

Hold Cycle Hold # of cycles
94°C 2 min 65°C 30 sec 72°C 2 min 1
94°C 1 sec 64°C 30 sec 72°C 2 min 1
94°C 1 sec 63°C 30 sec 72°C 2 min 1
94°C 1 sec 62°C 30 sec 72°C 2 min 1
94°C 1 sec 61°C 30 sec 72°C 2 min 1
94°C 1 sec 60°C 30 sec 72°C 2 min 1
94°C 1 sec 59°C 30 sec 72°C 2 min 1
94°C 1 sec 58°C 30 sec 72°C 2 min 1
94°C 1 sec 57°C 30 sec 72°C 2 min 1
94°C 1 sec 56°C 30 sec 72°C 2 min 23
60°C 30 min

4°C forever

 

108

Data analysis: AF LP fragments of in the size range of 200-450bp for primer
combination E-AGG/M-CTT and 150-450bp for primer combination E-ACT/M-CAG
were scored as present (1) or absent (0) for each individual. GeneScan automatically
assigns bands detected in each lane to a particular molecular weight. Because sizing by
the automated sequencer has a certain standard deviation, genotype patterns were
manually compared against each other and fragments were inferred to be the same if they
differed by less than lbp. Data were entered into a spreadsheet and subsequently
examined to pool the fragments that differed by lbp if no individuals displayed both
smaller and larger fragment. A frequency distribution of the number of fragments
obtained from each individual was plotted to check for deviations from normal. Five
percent of the samples with unusually high and 5% with unusually low numbers of
fragments were eliminated from the analysis. A high number of fragments has been
shown in some cases to be the result of incomplete digestion after which all digested and
partially digested fragments are amplified (PE Applied Biosystems 1996 protocol). A low
number of fragments resulted from unsuccessful amplifications with one of the primer
combinations. The dataset was then analyzed by neighbor-joining using PAUP*4b
(Swofford 1999) version 6 with mean character difference as the distance measure. The
relative strength of neighbor-joining clusters was evaluated by bootstrapping (F elsenstein
1985) based on 500 replicates. Characters supporting each cluster in the neighbor-joining
tree were determined by MacClade 4.0 (Maddison and Maddison 2000). Percentages of
polymorphic loci, heterozygosities, F5, values and genetic distances between geographic
regions were estimated using TFPGA (Tools For Population Genetic Analyses, Miller

1997). Percentage of polymorphic loci was calculated using both 99% and 95% criteria.

109

Since AF LP bands represent the dominant genotype at a locus, heterozygosities were
estimated based on a Taylor expansion of the estimate of frequencies of recessive alleles
(Lynch and Milligan 1994). F5. was calculated using Weir and Cockerham’s (1984)
method with jackknifing over loci to obtain variance estimates and bootstrapping with
1000 replicates to generate 95% confidence intervals. Genetic distances were calculated
as Nei’s (1972, 1978) distance measures. Approximate geographic distances were
estimated between the geographic centers of the populations sampled within the regions
and used in TFPGA to perform a Mantel test (Mantel 1967) with 10000 random
permutations to determine whether there is a correlation between unbiased Nei’s (1978)

genetic and geographic distances.

RESULTS

In a preliminary study I screened a total of 35 AF LP primer combinations for
amplification of polymorphic fragments in four R. zephyria and four R. pomonella
individuals, each from a different population. The majority of loci were polymorphic with
the polymorphism being shared between the two species. However, two primer pair
combinations (E-AGG/M-CTT and E-ACT/M-CAG) were identified that produced bands
unique to R. pomonella or R. zephyria. These primer pairs were used to generate AF LP
fingerprints for the entire sample. Fragments smaller than 200bp for primer combination
E-AGG/M-CTT and smaller than 150bp for primer combination E-ACT/M-CAG were

excluded from the analysis. The distribution of these fragments within each individual

110

genotype and between genotypes was too complex to score and interpret unambiguously.
Primer combination E-AGG/M-CTT amplified a total of 112 fragments between 200 and
450bp, with the average of 19 per individual; E-ACT/M-CAG amplified 143 fragments
between 150 and 450bp, with the average of 24 fragments per individual. Of these 255
AF LP loci, 239 (93.7%) were polymorphic across both species.

A frequency distribution of number of fragments amplified for each individual of
R. zephyria, R. pomonella and R. mendax is shown in Figure 4.3. Ninety percent of the
fingerprints obtained consisted of 29-62 fragments. Therefore all individuals for which
less than 29 or more than 62 fragments were amplified were eliminated from the analysis.

Average heterozygosities and the percentage of polymorphic loci within regions
for both species are given in Table 4.3. Levels of genetic variation, described by both
heterozygosities and % polymorphic loci were somewhat lower in R. pomonella than in
R. zephyria (Table 4.3). However, R. pomonella populations appear to be more
genetically structured than R. zephyria — the value of parameter 0 (which corresponds to
Fst) in R. pomonella is 0.106 (with a 95% confidence interval of 0072-0. 142), while in R.
zephyria it is 0.0615 (with a 95% confidence interval of 0.0440080). However, these
values were not significantly different (t=2.7510, df=110, P>0.05).

The neighbor-joining tree (Figure 4.4) shows that, in general, each of the three
species (R. pomonella, R. zephyria and R. mendax) clusters coherently. Rhagoletis
mendax appears as a sister group to R. pomonella, with R. zephyria forming a sister taxon
to them. The only R. zephyria that falls into the cluster with R. pomonella is a single
individual from South Dakota (ZSD16). Two individuals of R. zephyria (one from Idaho

— ZIDS, and one from Washington — ZWA8), as well as one bluebeny maggot fly from

111

New Jersey (MNJ 7), were placed outside of the main clusters in the neighbor-joining

analysis (Figure 4.4).

 

 

 

Number of samples

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109
115

Number of fragments

Figure 4.3 Frequency distribution of number of fragments amplified from each
individual. Samples with numbers of fragments between the dash lines were used in the
analysis.

112

Table 4.3 Estimated average heterozygosities and percentage of polymorphic loci in R.

zephyria and R. pomonella.

 

 

 

R. zephyria n H polyoo polyos Rpomonella n H polyog poly95
Northeast 8 0.066 25.098 25.098 Northeast 10 0.109 48.235 28.235
Great Lakes 9 0.108 45.098 45.098 Great Lakes 18 0.073 44.706 26.667
Gr. Plains 17 0.1 10 51.765 33.333 Gr. Plains 12 0.087 41.961 26.274
R°°ky . 10 0.110 45.098 23.529 R°°ky . 0.086 32.157 32.157
Mountains Mountains

Northwest 15 0.102 47.843 28.626 Northwest 4 0.080 25 .490 25.490
total 59 0.111 61.176 31.765 total 53 0.101 57.255 31.327

 

 

n — sample size, H — average heterozygosity, polyoo - % of polymorphic loci by the 99% criterion, pOIY95 -

% of polymorphic loci by the 95% criterion.

113

Figure 4.4. Neighbor-joining tree based on the analysis of 255 AF LP loci. Green
branches indicate R. zephyria, red R. pomonella and blue R. mendax. Colored squares
next to R. zephyria and circles next to R. pomonella sample labels indicate geographic
regions: red — Northeast, orange — Great Lakes, green — Great Plains, blue — Rocky
Mountains and purple — Northwest. For taxon labels please refer to Key to symbols and

abbreviations (p. xi). (Images in this dissertation are presented in color)

114

 

 

 

 

 

 

 

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115

Like in R. pomonella and R. mendax, groups within the R. zephyria cluster do not
correspond to regions, and genotypes from the same population are scattered throughout
the tree. Somewhat exceptional is the cluster of individuals from western populations at
the base of the large R. zephyria cluster and 3 snowberry flies from Massachusetts and
California each. In these cases genotypes from the same population cluster together, but
within groups that consist of individuals from different regions (plains, northwest and
northeast with Massachusetts flies and mainly plains with California flies).

Several characters define each cluster, and some of them are potentially useful for
discrimination between species. The fragment of 273bp amplified by the E-AGG/M-CTT
primer pair is present in all R. zephyria except the two individuals falling outside of the
main clusters and is found in no R. pomonella except one individual from Washington.
The same primer combination amplified a fragment of 316bp in all snowberry flies but
one from Idaho and one from South Dakota, both of which are not placed within the main
R. zephyria cluster. The 316bp fragment was amplified in only three R. pomonella and
two R. mendax. In 41 of the 59 snowberry flies primer pair E-ACT/M-CAG amplified a
fragment of 262bp, with frequency increasing from east to west. This fragment was
amplified in two apple maggots from Nebraska, two blueberry maggots from Michigan
(one from East Lansing, one from Otis Lake) and one from New Jersey (this individual
groups with two R. zephyria outside of the main clusters in the neighbor-joining tree,
Figure 4.4).

The primer combination E-ACT/M-CAG amplified a fragment of 150bp in most
R. zephyria and R. mendax, but appears to be lost in the R. pomonella lineage since it

amplified in only six R. pomonella from different populations. Fragment of 224bp

116

amplified by this primer pair was found in about 60% of snowberry flies, its frequency
being highest in the Plains, Northwest and Northeast, and lower in the Great Lakes and
Rocky Mountains populations. The same fragment was found only in one apple maggot
from Iowa. Similarly, fragment of 244bp was amplified in about 60% of R. zephyria, with
highest frequency in the Great Lakes and lowest in the Rocky Mountains. This fragment
was present in only three R. pomonella, one from Pennsylvania and two from
Washington. One of these apple maggots from Washington also had the 273bp fragment
amplified by E-AGG/M-CTT primers and present only in R. zephyria. The 305bp
fragment observed in all R. pomonella from Colorado and Utah (Rocky Mountains and
Colorado Plateau ecoregion) was rare in apple maggots from other regions and found in
snowberry flies from Wisconsin and North Dakota (four and one individuals,
respectively).

R. pomonella/R. mendax cluster is defined by fragments of 266 and 277bp
amplified by the E-AGG/M-CTT combination and 202bp amplified by the E-ACT/M-
CAG primer pair. All these characters are potentially useful for species identification.

Mantel test indicated that there is no significant correlation between genetic and
geographic distances. Coefficient of correlation between the two matrices in R. zephyria
was 0.369 and in R. pomonella 0.402, both of them were not significant, which was
consistent with the results of neighbor-joining analysis where no geographic structuring

was observed (Figure 4.4)

117

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DISCUSSION

The results obtained by the AF LP analysis of Rhagoletis pomonella and R.
zephyria demonstrated that this technique is powerful and promising for studying genetic
variation and relationships between the closely related species of Rhagoletis. The high
number of loci sampled at random and as a cross-section of the entire genome reflects
much higher variation and provides better resolution than sequences from any of the
individual nuclear or mitochondrial loci that have been examined thus far (Chapter 3).

Neighbor-joining analysis based on the AF LP loci revealed three well-defined
groups with almost perfect correlation of clusters with the three species (R. zephyria, R.
pomonella and R. mendax). The only exception was the placement of a single snowberry
fly from South Dakota within the R. pomonella cluster (Figure 4.4). This result is
consistent with the recent study of divergence of the pomonella group species based on
the allele frequencies at 29 allozyme loci (Berlocher 2000), where clustering of
populations corresponded to species. In that study R. zephyria was placed at the base of
the neighbor-joining tree, as a sister taxon to the cluster containing R. pomonella, R.
mendax and R. nr. mendax. AF LP analysis similarly places R. zephyria basally to the
pomonella/mendax group (Figure 4.4).

Branch lengths in the neighbor-joining tree indicate that the majority of genetic
variation is distributed within populations and species, suggesting that speciation
occurred without large population bottlenecks. This result is consistent with high allelic

and nucleotide diversity found within populations and species in studies of nuclear and

119

mitochondrial DNA sequence variation (Chapter 3, Feder et al in prep) and with
hypothesis that speciation occurred in sympatry.

Several characters define each cluster corresponding to species, although the
groups reflected by the neighbor-joining tree in Figure 4.4 do not have bootstrap support;
AF LP analysis thus for the first time provides a reliable PCR-based method for
distinguishing between the closely related species of the pomonella group. The primer
combinations used in this study amplified several fragments only in R. zephyria and very
rarely in R. pomonella or R. mendax. These fragments can theoretically be used as
diagnostic markers. Fragment of 273bp was successfully amplified by the primer
combination E-AGG/M-CTT in all R. zephyria except in two individuals falling outside
of the main cluster. In R. pomonella this fragment was only amplified in one individual
from Washington. This individual also possessed the fragment of 244bp amplified by the
primer combination E-ACT/M-CTT, present in 33 of the 59 R. zephyria and only two
other R. pomonella, one of which was from the same Washington population. This
suggests that very low-level hybridization with introgression of zephyria alleles into
pomonella may be occurring between apple maggot and snowberry flies in the West. R.
pomonella was introduced into the Northwest within the past 100 years (AliNiazee and
Brunner 1986, McPheron 1990); allozyme alleles characteristic for R. zephyria (Had1 I l)
have been found in low frequencies in flies reared from hawthoms and apples in
Washington (McPheron 1990) and Minnesota (F eder et a1 1999), suggesting male-
mediated gene flow in the direction from native into the introduced taxon’s gene pool. In
both these studies (McPheron 1990, Feder et a1 1999) multilocus genotypes, however, did

not indicate the presence of F. hybrids, suggesting extensive backcrossing to R.

120

pomonella. The placement of the potential hybrid individuals observed in this study
within the pomonella cluster (Figure 4.4) is consistent with this interpretation. Two
snowberry flies, one from Idaho and one from Washington, placed outside the main
zephyria cluster, both lack fragments characteristic for R. zephyria. The possibility that
this is an artifact produced by failure to amplify these fragments from these flies cannot
be ruled out. If there is some biological reason for the absence of zephyria-characteristic
fragments in these flies, their placement at the base of the neighbor-joining tree may
further indicate that the possible hybridization between apple maggot and snowberry flies
is rather rare. In cladistic analyses hybrid taxa tend to be placed as basal, causing
extensive homoplasy and reducing the resolution of relationships between other taxa
(McDade 1992).

To detect population structure in R. pomonella group large sample sizes are
needed (Berlocher 1995, 2000), as suggested by the allozyme studies (McPheron 1990,
Berlocher 2000). In studies of larger geographic scale (McPheron 1990, Feder et al 1999)
Fst values observed between the populations in native, eastern part of the R. pomonella
range, were low (0024-0032). Feder et al (1999) report the values of parameter 0 of
0.069 in R. zephyria and 0.073 in R. pomonella. Based on the analysis of 255 AF LP loci
and populations grouped in five regions, 0 in R. zephyria was 0.0615, while it was higher
in R. pomonella (0.1060). This finding is consistent with the hypothesis that R.
pomonella, as an ancestral species, is more geographically structured than the species that
have diverged from it. Contrary to the expectation based on the allozyme data (Berlocher
et a1 1993, Berlocher 2000, Feder et al 1999) that it should also have higher average

heterozygosity, AF LP data indicated somewhat higher value of this parameter in R.

121

zephyria (0.111 vs. 0.101 in R. pomonella). Lower average heterozygosity in
Northeastern R. zephyria is consistent with the hypothesis that snowberly fly was
introduced to this region from the west when the host plant, Symphoricarpos albus var.
laevigatus, was introduced as ornamental shrub. However, the possibility cannot be ruled
out that it reflects recent range expansion by colonization followed by genetic bottleneck
in this part of the species range.

Rhagoletis zephyria appears to have differentiated more from the present-day R.
pomonella than R. mendax (Figure 4.3). This result is consistent with morphological
(there is a slight difference in shape of male surstyli between apple maggot and
snowberry flies; Bush 1966, Jenkins 1996) and allozyme data (Hart111 is fixed in R.
zephyria and found only in very low frequencies in western R. pomonella - McPheron
1990; there are no fixed allele differences between R. pomonella and R. mendax). It may
suggest that the differences accumulated after the host shift and speciation in zone of
parapatry followed by the rapid range expansion to the west (Berlocher 1998), where the
flies filled new ecological niches and encountered different selection regimes. Grouping
of several flies from Northwest and Rocky Mountains at the base of the neighbor-joining
tree seems to indicate that these populations diverged most. Individuals from the Plains
region are present in every smaller cluster within R. zephyria and grouped with genotypes
from all other regions, which may indicate that speciation occurred somewhere in the
zone of parapatry in the Plains, as the hawthoms colonizing prairies alter the last
glaciation came into contact with snowberries, and highly genetically variable

subpopulations spread both east and west. Very low genetic distances between the Great

122

Lakes and all other regions except Northeast support the hypothesis that R. zephyria is
native to the Great Lakes area.

The only snowberry fly placed within the R. pomonella cluster (Figure 4.3) lacks
some of the fragments characteristic for R. zephyria and absent from R. pomonella.
However, it is difficult to assume that it shares pomonella genotype. This genotype may
indicate past hybridization with low frequency introgression of genes from R. pomonella
into the R. zephyria genome, or incomplete lineage sorting with retention of the ancestral
polymorphisms in very low frequencies in R. zephyria, or mistake in oviposition by R.
pomonella.

Data presented here do not seem to support the hypothesis of multiple speciation
events of R. zephyria from R. pomonella or pomonella-like ancestor. The observed
patterns do not allow distinguishing precisely between several scenarios of where the
speciation of snowberry flies has occurred and how the range has expanded. The ability
to further resolve relationships between populations of R. zephyria and R. pomonella
should provide better insight into the processes involved in their divergence. Lack of
geographic structuring and bootstrap support for the groups in neighbor-joining tree
indicates that even with this large number of markers we may still not have sufficient
data. To improve resolution and observe geographic structuring within species we may
need to sample even more AF LP loci using more primer combinations and obtain AF LP
fingerprints from more individuals from each population. Resolution, as well as the
support for particular groups, is expected to increase with increased number of loci
scored (Albertson et al 1999). It is also possible that resolution would improve with

decreasing the number of loci included in the analyses (Prowell et al in prep), since the

123

large number of similar loci may mask differentiation between populations and species.
This is reflected by the high levels of homoplasy, i.e. fragments present in some

individuals and some populations without any regular pattern.

124

CONCLUSIONS

Hypothesis 1: Rhagoletis pomonella represents a large and variable gene
pool, containing ancestral polymorphisms, from which new species arise.

Rhagoletis pomonella showed larger haplotype diversity than R. zephyria at one
of the three anonymous nuclear loci analyzed here (P2480) and at the mitochondrial locus
COI/COII. At all loci, nucleotide diversity 7: was higher in R. pomonella than in R.
zephyria. Surprisingly, however, the nucleotide diversity parameter 0, as well as
weighted 0/bp, was at all nuclear loci higher in R. zephyria, which may reflect the
influence of natural selection on polymorphism patterns in R. zephyria. Phylogenetic
analyses of two nuclear loci (P220 and P2480) placed some of the R. pomonella

sequences at the base of the tree, indicating that alleles of R. pomonella may be more

similar to ancestral.

Hypothesis 2: Rhagoletis zephyria has diverged from R. pomonella more
recently than blueberry maggot, R. mendax.

Phylogenetic analyses of anonymous nuclear loci and mitochondrial COI/COII
have indicated that R. mendax branched off from the ancestor of present-day R.
pomonella and R. zephyria before R. zephyria. Analysis of 255 AF LP loci, however,
showed that R. zephyria is more distant from R. pomonella than is R. mendax, contrary to
results of the analyses of individual loci and this hypothesis. New models of sympatric
speciation suggest that if species begin diverging in sympatry, the ranges of those that

speciated the latest should still be sympatric, whereas older species may be less sympatric

125

because of range changes over time (Berlocher, pers. comm.) The geographic range of R.
zephyria extends far outside of the range of R. pomonella, although the zone of overlap is
much larger than previously thought. The range of R. mendax is completely contained
within the range of R. pomonella, which may indicate that R. zephyria speciated before R.
mendax. Therefore, it is difficult to infer the timeframe and order of speciation events in
the R. pomonella species group. Nevertheless, polyphyly of R. zephyria and R.
pomonella, unresolved phylogenetic relationships at anonymous nuclear loci and
mitochondrial COI/COII analyzed in this study, shared alleles and haplotypes between
species, and lack of fixed differences indicate that speciation may have been recent and
could have happened in the past 10,000 years, after the glaciers of the last glaciation

receded and host plants (Crategus sp. and Symphoricarpos sp.) came into contact.

Hypothesis 3: The host shift that led to divergence of R. zephyria from R.
pomonella and speciation occurred from ancestral hawthorn host to snowberry.

If the ancestor of the present-day R. pomonella and R. zephyria was R. pomonella-
like, as suggested by the analyses of DNA sequences at different loci, then the host shift
occurred from the hawthorn host of the ancestor to snowberry. Placement of the R.
pomonella alleles sampled from populations from the Great Plains within the clades
consisting mainly of R. zephyria alleles seems to support the hypothesis that the host shift
may have occurred in the Plains. Little geographic structuring was revealed by the
analyses of DNA sequences and AF LP fingerprinting patterns, which makes inferences
about the direction of population expansion difficult. However, analyses of P2480

(neighbor-joining and parsimony analysis) and AF LP loci (neighbor-joining) indicate that

126

western populations of R. zephyria appear to be more divergent and placed as basal to the
rest of R. zephyria, suggesting that these populations may have been more isolated,

perhaps because they dispersed first.

Hypothesis 4: Rhagoletis zephyria in Eastern North America has been
introduced with the host plant (snowberry).

Evidence presented in this study supports the alternative to this hypothesis — that,
in fact, R. zephyria is a native inhabitant of eastern North America. Native hosts have
been found infested with R. zephyria in the Great Lakes region and in the Northeast, and
introduced Symphoricarpos albus var. Iaevigatus has been found infested only when the
native hosts were also available in relatively close proximity. Eastern populations of R.
zephyria showed no evidence of population bottleneck at any of the nuclear loci,
mitochondrial COI/COII or AF LPs, which might be expected if they were introduced
with the host plant and founded from a small number of individuals. Alleles of the eastern
R. zephyria were placed into same clades as the alleles of western R. zephyria without
much geographic structuring. If R. zephyria were introduced to eastern North America,
then it would be expected that alleles from eastern populations would group together and
lose to particular western populations from which they were introduced, which was not

the case in this study.

127

APPENDICES

128

APPENDIX A

P220 sequence alignment

129

.........................

....B.......... .......... ............. ..... ... .. . ............ 4. . . ..... . ....... .
....U.... .. ........... U.B . .. ... .... . ... .. ........... 4.. ..... . . H ............................
....U.... ...... .. . ..... U.B....... ............ . .... ....... . .4. . ... ..... .8 . .. . ....................
.. .B ..... . . ..4.4 ...... ....4... . 4. ..... 4 .....4 ....... l... U ii 4 . .Ummmo on mmmwmmmmmmmmmmwmmmm
....U ....... ....¢.¢ ..... U B..¢.... fl ........ .. fl ....... . 9.. I .... ...... . B..Ummmmmmmmmmmommmmmmmmwmwmm
....U...... . ..... . .. U B ......... . .......mmmmommmmmmmooooommmmmmoooommmmmmmwmo mommooommmmmmoooooooooo
...U... . .......... . U B ... . ........ . .. ............. i ............... B..wmmommmmmmmmwmmmmmwwwmmmmm
. ..B.. ....... . ....... . .. ...... . ........ ... .................. 4... . . ........ ..mwmoommmmmmmmmmmmmmmmmmmmm
....U... . .............. U.B .......... . ...... . . ............. . i .. .......... B...Uomommmmmwmmmmmmommmmmmmm
. .9 ........................................ ... .. . i . 4 ................ mmmmmmmoowmmmmmmmmmmmmmmmmmw
....B ......... . . ............ .. .. ................ . ........... i ................ mmwmmmmmmmmmwmmmmmmmmooooooo
....B. ..... ... ............. . ........ . . ...... .. . . ........ .-.. ...... . ...... .mommmo mmmmmmmmmmmmmmmmmmmwmm
....U...... ..4.4 ..... U B .4 ..... .4 ..... .. ...4.. ..4 ....... i . ...... 4.. B.mmmmmoo ooommmmmmmmmoooooooo
....B ..... ... ..4.4U ......... 4......4 ...... .....4... . E ii. . U11.B.4......U.momo oommmmmmmmmommmmwooo
.. .B... . ....4.4.. ........ 4. ....4 ....... . ..4. ............ ii ..... U..ii.4......U... oomooooommmmmmmwmmmmmm
....H ....... ....4... ......... 4 .... 4 ........... 4..... ..... . ..4.. ...U 4O4UBU.H4UU oomwmmmmmmmmmwmmommmmmmm
....B .......... .4.4 ...... .. 4......4 ........... 4 .......... 10 ii 4 U U9 4 .....UU mmmmmmmmmmmmmmmmmmwmmmm
....B... .. ...4.4.. ........ 4......4...........4..........iU.ii.....U..i..4. ..... U..mwmoeooommmmwmmmmmmmmmm
....B. ...... ....4.4... .. .. 4......4 ........ ...4...... .. .. 1.. . .......... .....Ummmmmmmmooommmmmmmoooooo
...B....... ...4.4 ..... U .. ......4 ....... .. .4 . ...... . ..... i .................. UU.mmmmmmmoooommmmmmmmwmmm
....U.. ............ . U B ..... .... . . ... .... ............ ..4... .. . ....... B..U....mmmo.mwmmmmmommmmmmmm
....U...................U.B............................ ..... ...i.......... ..... 9.......momooooooeoommmmmmmmm
. .U ................... U.H .................. . .......... . ...... 4.... ........... B.......mmmmmmmmmmmmmmmmwmmoe

..U... ...... . ......... U B ....... . ......... . . ... ......... .. 4 . .. .......... H eoomoommmmmooooooommmmmmmm

..U ................... U B ........ . ........ . ............. . . 4 .. .......... B .... .oooommmmmmmmmmmmmmmmm
. ..U ........ . .......... U B ...... . .......... .. ......... . .. 4 ............... B ...U.mmmmmmmmmmmmmmmmmmmmmm
....B ........................................... .. ........ . . 4 .................. ..U..wmmmmwmmmmmmmmmmmmmmm
. .U ...... . ............ U B .......... . ......... .. ........... . 4 ............... B oooommmmmmmwmmmmmmwmmmmmmmm
....U ................... U.B ....................... ... ....... . 4 ............... B Ummmmmmmmmmwmmoooommmmmmmm
....U ...... . ...........U.B ..... .... . ............ . .. ..... 4 . ............. B .....ooooommmmwmmmmommmmmmm
. ..U. . ............. .U.B .................... . .... . .... 4 .. U ......... B....... ooooooooomwmmmmmmmmmm
. ..U ....... .. ..... . ...U.B . ................... ... ....... 4 ...... . ........ B ..00.mmmmmmmmmmooooommmmmwm

....B.. .... ...... ....... . ... . ... ............... .. .. ..... 4 ...... . ......... mmmmmmmmmo mwmmwmmmmmmwmmmmmm

....B.... . ......................... . ................. . ........ 4 ................... ...mmmmoooooooooommmmmmmm

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4UUB4B4B4BU4UUBUBUiBBU4UUUUUlBBB49iUBHU88480040OBBH4UBUBUU4UB4UOiiUUUBBBU4UHUBUU8498999049984940499046OUUUBB
mbwmvmmaommbmmvmmaommbwmommaommbmmvmmaommbwmvmmdommbmmwmmflommbmmvmmaommbmmvmmaommbmmcmmaommbmmvmmaommbmmcmma
ooooooooommmmmmmmmmmmmmmmmmmwebbbbbhbhbmwmmmmwmmmmmmmmmmmmmvvvvvmvvvvmmmmmmmMMMNNNNNNNNNNHHHHHHaHHH
HHdHHHHHH

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130

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131

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132

. 9 ................... 0 .............. 04 .4 .................. 4 ............................................

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9 ........... 4 ....... 0 .............. 04 .4 .................. 4 ............................................

9 ........... 4 ....... 0 .............. 04 4 .................. 4 ............................................

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...0. . ... ........... 0.9 ........ . ....................... ... 4 ............... 9 ............................
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mbmmvmmaomwbmmvmmaommhwmvmmaommbmmvmmaommbmmvmmaommbmmvmmaommbmm«mmaommbmm«mmaommbmmvmmaommbmmIMNHommummvmma

ooooooooommmmmmmmmmmmmmmmmmmmbbbbbbbbbhmmmmmmmmmmmmmmmmmmmmvv¢vv«¢¢vvmmmmmmmmMMNNNNNNNNNNHHHaHHHHHa
HHHHHHHHH

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133

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII mIlflmoN

...BB...H.O ........ ...........O....... ..... . ..................... ... ......... ..... ..................... ..... N.HHMQ
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............ ................. . ................................................................. 4 ............ m.N0CQ
...BB...B.O ................... 0....B ........ . ..... U .................... . ....... . ............................ H.MGOQ
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...BB...B.U ...... . ............ U..... ......... .......... .............. ...... ........ . ........................ N.HMEQ
....B ............................................................................ B .......................... m.NMQQ
...BB...B.U....O .............. U... ............ ........ ......... ...0 ......................................... H.NMQQ
............................................................................................................ N.a0CN
... ......................................................................................................... H.H0CN
....................................... ..................................................................... H.HOHEN
BB 9 U ................... U. B 4 ........................................................ 4n ........... v.NMQN

.. ...... . ...... .. .................... . ......... 4 ........................ . ................................. H.HQQN
............................................................................................................ N.HMQN
. ................ . ................ 8.... . .................................................... 4 ............ N.NUCN
................................... B........................................................................ N.HUCN
.. . ........................ ..... ...................................................................... m.HUCN

B ............................................................................ B .......................... m.NUmN
B..B. .B ..................... U . F ................ B ................................................ . ...... v.HUmN
BB...B.O ................... O .................. . .......................................................... m.MHflQ
.HIIHIIIIH.U ................... U ............................................................................. H.MHHQ

. 99...“..0 . .............. O ..................... I ............................... I ....................... NoHHfiQ
PH. .9.0 ................... U ............................................................................. H.HHHQ
o.H:H.o.-.H..O . ................. O .................... o ................................... o .................... H.v0fla
.98. .8.0 ................... O .................... . ........................................................ m.mMHQ
...-I... .......... . .......... Uc. . . .................................................................... H.mm..flQ
BB. .H.O ................... O ............................................................................. m.¢CEQ
.PB. .B.O ................... O ........................................................................ . H.¢QEQ
.99...B.0 ................... O ................... . ..... . ................................................... m.NGEQ
.99. .B.0 ................... O ............................................................................. N.mHEQ

. orHrHI. ..Hyco ................... o ................. . ..... o ..................................................... H.mflEQ
.............................. m.U........................................................................... N.MH0N
. .. .......... . . ..................... .. ........ ... .. ..... . ...................... .. . . . ........... H.MHwN
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APPENDIX B

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166

APPENDIX C

P2480 sequence alignment

167

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179

APPENDIX D

COI/COII sequence alignment

180

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