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This is to certify that the

dissertation entitled

CHARACTERIZATION OF THE RIBOSOMAL DNA -
-OF THE GENUS RHAGOLETIS
(DIPTERA: TEPHRITIDAE) .

 

presented by

-YUE MING

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in ENTOMOLOGY

 

/.7i/m/v

/ Major %—\

 

27 OCT. 1995

 

MSU i: an Affirmative Action/Equal Opportunity Institution 0- 12771

 

 

 

 

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LIBRARY
Michigan State
University

 

 

 

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TO AVOID FINES rotum on or baton dot. duo.

DATE DUE DATE‘ DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I JI I |

MSU In An Nflmativo Action/Emil Opportunity Inctltwon
WM!

 

CHARACTERIZATION OF THE RIBOSOMAL DNA
OF THE GENUS RHA GOLETIS
(DIPI'ERA: TEPI-IRITIDAE)

By

Yue Ming

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

DOCTOR OF PHILOSOPHY

Deparunent of Entomology
1995

[130'

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ABSTRACT

CHARACTERIZATION OF THE RIBOSOMAL DNA
OF THE GENUS RHAGOLEHS
(DIP'TERA: TEPHRITIDAE)

By

Yue Ming

Although the Nearctic species of Rhagoletis have been well studied with respect to
morphology and allozymes, there are still unanswered questions regarding the
differentiation of sibling species, placement of certain species, and relationships among the
existing species groups. In order to gain new information on these subjects, I have
characterized the Rhagolea's ribosomal DNA, especially the non-coding spacers such as the
internal transcribed spacers (ITS).

I have presented the ITS sequences of four North American sibling species of the
R. cingulata species group. The inter—specific variation in this group is not significantly
higher than the intra-specific variation. Consequently, the ITS sequences are of limited
application for inferring phylogeny of the members in this group. However, several
molecular markers in the ITS sequences have been described which can be potentially
useful for differentiating some of the sibling species in this group. A few highly conserved
secondary structure elements in the ITS regions have also been described and compared
with those in Drosophila.

The ITS sequences have been obtained for eight additional Rhagoletis species,
including pomonella, carnivora, contpleta, juniperina, fausta, electromorpha, basiola, and

sm'atella, and a phylogenetic analysis was performed This study indicated that R.

comivora belongs to the pomonella group; R. juniper-ind was removed from the tabellaria
group and may be more closely related to the pomonella group; close relatives of the
cingulata group are more likely closely aligned with the suavis group rather than the
pomonella group; R. fausta may be related with the tabellan'a group; R. basiola and R.
striatella ITS sequences are highly divergent from other species analyzed, indicating that
Rhagoletis may not be monophyletic.

A genomic library for R. pomonella was constructed and several rDNA clones
identified. Furthermore, the region containing the intergenic spacer and extemal
transcribed spacer of rDNA from two R. cingulata flies of different host plants was PCR
amplified and cloned. The regions were partially sequenced and found to be significantly
divergent between the two R. cingulata of different host plants. Whether this observed
divergence is related to the different fly host origins is an inu'iguing question worth further

investigation.

© IneMing 1995
All Rights Reserved

DEDICATION

To the memory of my father, brother and nephew

 

 

 

St

Critic

allow
HOusi
my far
Mrs_ K
mOre C
CDCOuI-a
COUrsa.
Bedojan

ACKNOWLEDGEMENTS

I would like to thank my major professors Drs. Guy L. Bush and Frederick W.
Stehr for their effort throughout all the different stages of my development as a scientist and
person while at Michigan State University (MSU). Special thanks also to my Doctoral
committee consisting of Drs. Alexander S. Raikhel, J. Mark Scriber and Donald 0. Su'aney
for their patience, encouragement and support. I thank Dr. Hugh M. Robertson for
providing the PCR primers, and Drs. Stewart H. Berlocher, Randy Cooper, Ranjan Gupta,
James Nugent, Jerry A. Payne, Adam Peters, Ron J. Prokopy, Gary J. Steck, George C.
Steyskal, William J. Turner and John Wilterding for collecting the specimens used in this
Dissertation. Special thanks to Dr. James J. Smith for helping me getting started in Dr.
Bush's laboratory and for his technical assistance. I thank John Jenkins and Judith Sirota
for their friendship over the years and for their helpful suggestions and specimen
collections. I thank Dr. David R. Engelke from the University of Michigan (UM) for
critically reading the secondary structure part in Chapter H and Dr. John P. Langmore for
allowing me to use his laboratory computers at UM. I would also like to thank the Family
Housing Community Center at UM for providing the computer and study facilities. I thank
my family in China and my in-laws in Pullman, WA, especially my parents—in-law Mr. and
Mrs. Krikor O. Bedoyan, for their support and encouragement that made my life at MSU
more enjoyable. Very Special thanks to my husband Jirair K Bedoyan for his support,
encouragement and assistance throughout all this time. His love and faith kept me on
course. Last and most importantly, I thank God for giving me a healthy baby, Sarah M.

Bedoyan, otherwise it would have been impossible for me to complete my Ph.D. program
at MSU.

LIST
LIST

 

CHM

‘ II.

SPEC
‘ PHi’I

111.
Tim

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. ix

LIST OF FIGURES ................................................................................. x

CHAPTERS

I. INTRODUCTION .......................................................................... 1
Bibliography for Chapter I ...................................................... 24

II. rDNA INTERNAL TRANSCRIBED SPACERS 1 AND 2 OF R. CINGULATA
SPECIES GROUP: SEQUENCE, CONSTRAINED SECONDARY STRUCTURES AND
PHYLOGENETIC IMPLICATIONS

Introduction ....................................................................... 31
Materials and Methods ........................................................... 35
Results ............................................................................. 39
Discussion ......................................................................... 67
Bibliography for Chapter II ..................................................... 71

III. PHYLOGENETIC IMPLICATIONS FROM ANALYSIS OF INTERNAL
TRANSCRIBED SPACER REGIONS IN THE rDNA OF TEN RHAGOLEHS SPECIES

Introduction ....................................................................... 76
Materials and Methods ........................................................... 78
Results ............................................................................. 82
Discussion ....................................................................... 107
Bibliography for Chapter III ................................................... 117

 

 

 

IDEN

SPAC]

VI

 

IV. CONSTRUCTION OF AN R. POMONELLA GEN OMIC LIBRARY FOR AND
IDENTIFICATION OF THE COMPLETE rDNA REPEATING UNIT

Introduction ...................................................................... 120
Materials and Methods .......................................................... 124
Results and Discussion ......................................................... 132
Bibliography for Chapter IV ................................................... 143

V. PARTIAL CHARACTERIZATION OF THE EXTERNAL TRANSCRIBED
SPACER REGIONS OF RHAGOLETIS CINGULATA

Introduction ...................................................................... 145
Material and Methods ........................................................... 147
Results and Discussion ......................................................... 149
Bibliography for Chapter V .................................................... 159
VI. CONCLUSIONS ........................................................................ 161

 

 

 

Tab}

Tabl

Tabl:

Table

Table

Table C

Table 7

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

LIST OF TABLES

Collection Sites and Host Plants of Rhagoletis Species Used in This Study ..... 36

A-T Content and Length of the Analyzed Rhagoletis ITS Sequences ............. 41

Average Percent Nucleotide Substitutions (Nucleotide Substitutions per 100

Nucleotide Sites) in the ITS sequences for the Rhagoletis cingulata Species

Group and R. pamonella in Pairwise Comparisons ................................. 50

Potential Molecular Markers in ITS] for Distinguishing Members of the

R. cingulata Species Group. ........................................................... 55
Collection Sites and Host Plants of the Rhagoletis Species Used ................. 79
A-T Content and Length of the Rhagoletis ITS Sequences Analde ............. 89

Average Percent Nucleotide Substitutions (Nucleotide Substitutions per 100
Nucleotide Sites) for the Rhagoletis IT 81 and ITSZ Sequence Pairwise

Comparisons ............................................................................. 92

Transition to Transversion Ratios for the Rhagoletis ITSl and partial IT 82

Sequences ................................................................................ 93

 

Fi g1

 

Figure

Figurg‘

Figure 6

Flam 7

 

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figme 5.

Figure 6.

Figure 7.

Figure 8.

LIST OF FIGURES

PCR amplification products from Rhagoletis ITSZ using primers 108F
and 52R ................................................................................ 42

ITSl sequences and alignment for Rhagoletis cingulata species group and
R. pomonella .......................................................................... 44

ITSZ sequences and alignment for Rhagoletis cingulata species group and
R. pomonella .......................................................................... 47

Phylogeny inferred from the combined IT 81 and ITS2 sequences from
Rhagaletis cingulata species group and R. pomonella ............................ 56

Typical computer generated secondary-structure models for Rhagolen's lTSl
(A and B) and ITSZ (C and D) ...................................................... 59

Sequences and alignment of specific structural domains found in the Rhagoletis
ITS sequences ......................................................................... 61

Secondary-structure models for specific domains in the Rhagoletis ITS
sequences .............................................................................. 63
Rhagoletis ITSl sequences and alignment .......................................... 83

Fi‘

Fl!

5

Fig

 

Figt

Flam

 

Flgur;

figure

58’er 12

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

Figure 16.

Figure 17.

Figure 18.

Rhagoletis ITS2 sequences and alignment .......................................... 9S

Phylogenetic analyses of Rhagoletis using the ITSI sequence alignment 101

Phylogenetic analyses of Rhagoletis using the ITS2 sequence alignments 103

Phylogenetic analyses of Rhagoletis using the combined IT 81 and IT 82

sequences ............................................................................. 105

Overall map of the Drosophila melanogaster rDNA repeat unit ................ 121

Schematic outline of the procedure for generating a Rhagoletis pomonella

genomic DNA library and screening for the rDNA repeat unit ................. 125

Detailed outline of several specific steps in Figure 14 .......................... 127

Screening the Rhagoletis pamonella genomic DNA library with Dm238
pro be .................................................................................. l 3 3

Southem blot analysis of the restriction digestion products of DNA purified
from R. pomonella rDNA clones .................................................. 133

Products of the PCR amplification reaction of the IT 81 and ITS2 regions of

Rhagoletis pomonella ............................................................... 137

 

 

 

 

Figure 1'

Figure 2(

Figural]

Figure 22

 

 

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Nucleotide sequences of the ITSI and ITSZ from Rhagoletis pamonella and
pairwise comparison with their counterparts from Drosophila melanogaster

.......................................................................................... 139
Designing the appropriate primers for PCR amplification of the IGS/ETS
regions of Rhagoletis ................................................................ 150
Products of PCR amplification of the IGS/ET S region of Rhagoletis
cingulata from sour cherry .......................................................... 152
Alignment of the sequenced regions from the two Rhagoletis cingulata

clones RCI-A and RC3 with homologous regions from Drosophila
melanogaster ......................................................................... 155

 

 

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CHAPTER I
INTRODUCTION

The genus Rhagolen‘s Loew currently has more than 60 described species, and is
widely disu'ibuted over the Palearctic (Rohdendorf 1961; Kandybina 197 2), Nearctic (Bush
1966; Berlocher and Bush 1982) and Neotropical regions (Foote 1981; Frias and Martines
1991). Because larvae of Rhagoletis feed in a wide variety of developing fruits, many
Rhagolea's species are serious pests of fruits such as apples, cherries, blueberries, walnuts
and tomatoes (Bush 1966). Many species in Rhagoletis have the ability to rapidly shift to
new hosts, including introduced cultivated plants (Boller and Prokopy 1976).
Morphologically, populations on the old and new hosts are often hard to distinguish. The
rapid shifting of host plants conuibutes to the difficulty of controlling these pests because
the wild hosts function as a reservoir for pest populations year after year. The presence of
newly established host-associated populations and sympatric sibling species in Rhagoletis
has also made the genus a model system for studying sympauic speciation (Bush 1969;
1974; 1975; 1992; 1994).

Over the last century, an extensive literature on the biology, ecology and control of
certain Rhagoletis species, especially those in North America, has accumulated. A brief
overview of a few outstanding features of the biology of Rhagoletis is presented in this
chapter as they not only play important roles on the evolution of the flies themselves, but
also offer clues that may be used to interpret the status of some taxa covered in my
dissertation. A second section stresses the issues concerning host shifts in Rhagoletis—
especially host race formation in two unique species groups, the pomonella and cingulata

Species groups. A third section presents the current taxonomy of North American

 

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2
Rhagoletis and discusses existing problems regarding the status of certain taxa in this

genus. It is followed with an overview of the organization of the remaining parts of my

dissertation.

Biology of Rhagoletis

Eggs and larvae

Rhagoletis eggs hatch within about a week after being laid underneath fruit skins
(Prick et al. 1954). First instar larvae usually mine directly to the interior of the fruit within
24 hours after hatching (Frick et al. 1954), probably avoiding parasites as a result (Bush
1992 and references within). Larvae usually confine their feeding to the same fruit in
which the eggs are laid and complete their development in about 8 to 40 days, depending
on the Rhagaletis species and growth conditions (Ries 1935; Frick et a1. 1954; Boller and
Prokopy 1976). Fruit quality, such as sugar content and acidity, can dramatically influence
larval growth rate and survival; larval mortality has been reported to be 100% in some
varieties and species of apple and haws (Dean and Chapman 1973; Bush et al. 1989). In
addition, temperature can also significantly affect larval development rate. In R.
indtfi'erens, for example, larval development takes about 10 days at 85°F vs. 35 days at
60°F; larval development ceases at 55°F and death occurs when larvae are exposed to 28°F
for 4 hr (Prick et al. 1954). Mature larvae leave the fruit usually after fruit drops to the
ground and burrow into the soil under the host tree. They pupate within a few days 4 - 10
cm under the ground (Boller and Prokopy 1976).

Pupae and Diapause

Although some Neotropical Rhagoletis are facultatively multivoltine—R. tomazis
has at least five to six generations per year in Chile (Frias et al. 1991)—most temperate
Rhagoletis are univoltine and their pupae undergo a winter diapause. Fly deve10pment

continues when spring temperature and moisture increases. However, in some cases,

 

 

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3

small portion of pupae can remain in diapause in the soil for 2 to 5 winters before
completing development (Boller and Prokopy 197 6). Such delay in diapause termination
provides a pupal reservoir and insures that the population will survive in case of
catasuophe, such as the failure of their host plants to fruit (Boller and ProkOpy 1976).

Small fractions of R. pomonella (lllingsworth 1912) and R. indifierens (Frick et al.
1954) populations, without undergoing pupa diapause, may complete development directly
after a few weeks of pupation, resulting in a new generation of adults. However, the
second adult generations are usually unable to oviposit before low temperatures anive in
the Fall. Diapause induction, as in other insects, is regulated by photOperiod and
temperature (Prokopy 1968). Populations of R. pomonella adapted to apples and
hawthoms respond differently to those diapause regulating factors (Prokopy 1968; Feder et
al. 1993). Post-diapause regulation in Rhagoletis shows high correlation with thermal
units accumulated over a deve10pmental threshold temperature (Reissig‘ et aL 1979; Feder et
al. 1993). Post-diapause eclosion time of different host-associated populations in R.

pomonella is significantly different and genetically programmed (Smith 1988a).

Eclosion and Adult Feeding

After over-wintering below the ground, Rhagoletis adults emerge from their puparia
at specific times, in most cases, during the spring and summer. Adult emergence occurs
primarily in the moming, probably stimulated by the rising morning temperature (Balduf
1959). After bursting the puparium, adult flies propel through the soil by contraction and
elongation of the body and ptilinum in order to reach the ground surface (Christenson and
Foote 1960). On average, females emerge a few days earlier than males; however, at peak
emergence the sex ratio reaches equilibrium (Boller and Prokopy 197 6). Synchronization
of adult emergence with the fruit maturation of host plants has been demonsuated in many
Rhagoletis species and has been proven an important trait in differentiating host races in R.

cerasi (Boiler and Bush 1974). Within two hours of emergence, most flies are capable of

flight 11
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flight and feeding, spending little time on the ground before taking off (Boiler and Prokopy

197 6).

Rhagoletis adults feed on many different kinds of food, such as insect honeydew,
yeast, bacteria and fungal spores. Nectar and plant liquid exuding from glandular
structure, wounds and oviposition stings are probably additional food sources (Boller and
Prokopy 1976). Bird droppings are also natural protein source for Rhagoletis (Prokopy et
aL 1993). Bird droppings treated with antibiotics were significantly less attractive than
untreated ones, indicating that bacteria may be involved in generating attractive volatile(s)
(Prokopy et al. 1993). Carbohydrate obtained in the form of leachate by extensively
'grazing' on surface of host foliage can sustain fly longevity (Hendrichs et al. 1993a).
Rhagoletis flies engorged with a great volume of dilute food, have been observed to
exu'ude orally droplets of liquid crop contents ("bubbling") followed by subsequent re-
ingestion (Hendrichs et al. 1992; 1993b). Through the bubbling behavior, flies eliminate
excess water by evaporation to concentrate nutrients suspended in dilute solution while
foraging for other resources (Hendrichs et al. 1992; 1993b). Both sexes of R. pomonella
require carbohydrates, certain vitamins and amino acids for gonadal maturation (Bush
1992), which in most Rhagoletis species occurs within two weeks of emergence (Boller
and Prokopy 1976). _

Search for food in Rhagoletis is not restricted to the larval host plant but, in some
instances, to various types of neighboring vegetation. Under normal crop conditions,
movement associated with feeding is nondispersive, rarely taking individuals far from their
host plants (Maxwell and Parsons 1968; Neilson 1971). Although R. cerasi is capable of
flying several kilometers, most dispersive flights observed in Rhagoletis were influenced
more by the availability of suitable fruit for oviposition than the search for food (Boller and
Prokopy 1976).

Foragiflé

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Foraging for Mates and Oviposition Sites

During sexual maturation, both male and female Rhagoletis adults congregate on
their larval host plants, where courtship, mating and oviposition occur (Bush 1969;
Prokopy et al 1971). Flies search for host trees through visual cues such as foliage color,
tree shape and tree size (Moericke et al. 1975), as well as olfactory cues from susceptible
host fruit (Prokopy et al 197 3). Flies appear to have difficulty locating trees at a distance of
more than 1.6 m (Roitberg et al. 1982). Although model size, shape and color have
profound effects on a fly's response to various models (Prokopy 1973a; 197 3b; 197 3c;
Green et al. 1994), these visual cues are not host plant specific. Olfactory and contact
chemical cues, however, play a more important role in the final detection of a correct host.
The odors emanating from ripening host fnrits provide specific attractants used by the flies
to identify their correct host fruits (Prokopy et al. 1973). For example, a number of
straight-chain esters (e.g., butyl hexanoate) isolated from exu'acts of volatile produced by
host fruits of R. pomonella elicit highly selective behavioral responses by R. pomonella,
suggesting that this fly is narrowly adapted to respond to specific compounds emanating
from its hosts (Averill et al. 1988; Green et al. 1994).

Once on the host plant. flies detect the fruit on the basis of shape, contrast-color
against the background and size (Boller and Prokopy 1976). In R. pomonella, if fruit
visual stimulus is strong (e.g., red color), chemical stimuli such as synthetic apple volatile
blend, do not increase the probability of finding fruit or fruit models; however, as the
visual stimuli became progressively weaker (red to green to clear), fnrit odor (irrespective
of concentration) appears to aid flies during the fruit-finding process (Aluja and Prokopy
1993). Female flies inspect the fnrit for oviposition on the basis of its size, surface
structure, and stage of ripeness (Boller and Prokopy 1976) using chemical stimuli, such as
contact and volatile stimulation with various chemicals associated with host fruits, received
by ovipositor sensilla provide the fly with information about host suitability and/or quality

(ijar et aL 1989). Female flies oviposit more often and remain longer on trees harboring

high vsj
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6
high vs low densities of fnrit clusters (Roitberg et a1. 1982), and will leave their host trees

within a short time if they discover no fruit (Roitberg et al. 1982). The intertree distance
also influences the foraging behavior of R. pomonella in the field. Flies generally invest
less search time on a tree when neighboring trees are nearby than when farther away,

apparently reducing travel costs (Roitberg and ProkOpy 1982).

Mating and Oviposition

Mating in Rhagoletis occurs almost exclusively near or on host fruit; the host plant
thus acts as an important site for courtship and mating (Bush 1969; Prokopy et al. 1971),
as well as for larval development. Visual cues such as body coloration and wing pattern
are important in courtship and species recognition, especially those from the R. suavis
group, whose members have suikingly different color patterns but infest mainly the same
plant genus Juglans (walnuts) (Bush 1966; Yokoyama and Miller 1994). These visual
cues, however, are effective only at close range and they can not be considered as
important reproductive isolating mechanisms in sibling species groups such as the
pamonella and cingulata groups. Sibling species in the R pomonella group, for instance,
have almost always shifted to new hosts in the course of speciation. They therefore meet
and mate on different hosts (Bush 1969; 1974). Even though different species meet one
another occasionally visual cues seem not completely prevent them from attempting to
courtship as different species in copula have been observed in nature (Prokopy and Bush
1973a). Furthermore, wing patterns and body coloration of most pomonella and cingulata
group species are not distinguishable in almost all cases (Bush 1966). Males of several
Rhagoletis species (mendax, cingulata, tabellarr'a, pomanella and camivora) also are
apparently unable to distinguish between the sexes and mount other males as often as
females (Prokopy and Bush 1973a; Smith and Prokopy 1982; Smith .1984; 1985a; 1985b).

Male Rhagoletis are highly territorial. For example, male walnut flies guard egg-

laying punctures on host walnut to increase access to females and defend these sites from

cum.
on t
(Prc
the 1

1P1?“
Smit

fema

husk: (La

 

7
conspecific and heterospecific males (Papaj 1994). The visual stimulus of a moving female

on the same or nearby fruit elicits attention and initiates courtship by waiting males
(Prokopy et al. 1971) which usually involves wing waving, posturing and ‘pawing’ with
the prothoracic legs (Biggs 1972; Prokopy and Bush 1973a). In walnut flies, mating
generally takes place as female initiates oviposition (Papaj 1994). Male Rhagoletis
approach the female either directly or obliquely from the rear (Smith and Prokopy 1982;
Smith 1984; 1985a; 1985b). If the female is receptive the male is allowed to mount onto
females' abdomen usually by a jump or short flight (Prokopy and Bush 197 3a).

Male R. pomonella secrete a pheromone that is assumed to function primarily as an
aphrodisiac which he waffs to female as he waves his wings. This pheromone is active
only over short distances in nature (Prokopy 1975). So far, no long distance sex
attractants in Rhagoletis has been observed. Because attracrion is short range, adults of
both sexes must first find the correct host plant and locate fnrit before they can meet the
opposite sex. Therefore, host selection and mate recognition are directly conelated. The
restriction of mating to the fruit of a specific host plant thus serves as an important
precopulatory reproductive isolation in several species (Bush 1966; Prokopy and Bush
1973a; Feder et al. 1994) and has important implications in sympatric host race formation
of these flies which will be discussed later.

Female flies of most Rhagoletis Species lay only one eg at a time, usually in nearly
ripe rather than immature fruit (Messina et al. 1991). However, walnut infesting flies, R.
suavis (Loew) and R. completa for example, lay eggs in batches (Boyce 1934; Ries 1935).
The walnut husk flies readily use sting holes made by conspecifics as oviposition Sites
(Ries 1935; Lalonde and Mangel 1994) and this probably accounts for the fact that a
hundred or more eggs are not uncommon in a single puncture (Ries 1935).
Superparasitism is probably a viable strategy because husks have sufficient food for more
than one fly offSpring and are difficult to parasitize initially due to the toughness of the
husks (Lalonde and Mangel 1994). Also, the walnut husk contains very high levels of

juglone-
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leasr init
1992). l
parasitisr
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8
juglone—a very toxic substance. By feeding gregariously, larvae may receive more

protective benefit in detoxifying this substance (Bush, personal communication).

After oviposition, females of several Rhagaletis species deposit oviposition
detening pheromone (ODP) on the fruit surface to reduce intraspecific larval competition at
least initially (Crnjar and Prokopy 1982; Averill and Prokopy 1989; Aluja and Boller
1992). However, the benefit of host marking may be offset by increased risk of egg
parasitism by wasps (Roitberg and Lalonde 1991). Receptor cells sensitive to exu'acts of
the ODP have been identified in the tarsal D-sensilla of some Rhagoletis species (ijar and
Prokopy 1982; Stadler et al. 1994), and isomers and derivatives of ODP for R cerasi have
been synthesized and bioassayed (Aluja and Boller 1992; Stadler et al. 1994). Application
of such synthetic ODP in an experimental cherry orchard caused a tenfold reduction in fruit
infestation suggesting the pheromone may potentially be useful as a fruit fly management
tool (Aluja and Boller 1992; Stader et al. 1994).

Since Rhagolen’s females select host fruits for oviposition and larvae have no choice
in which host fruit they develop, successful foraging for oviposition sites may be more
closely related to genetic fitness than is the successful foraging for food by other animals
whose young may move between and select resources to which they are best adapted. This
is supported by evidence which suggests that phenotypic differences in host response
pattern between hawthorn and apple origin flies of R. pomanella have an underlying
genetic basis (Prokopy et al. 1988; Feder et al. 1994).

Longevity

Average adult longevity in nature, although not yet established accurately, ranges
from 2 to 6 weeks depending on the species (Boller and Prokopy 1976). Longevity is
usually greater in cool weather; with light, humidity and food availability also effecting
adult longevity (Boller and Prok0py 1976; Hendrichs et al. 1993a).

E
disCOV'el'.‘
differenc:
Rhagolen
1969: 19‘.
described
1983; Ber
on Rosacr
Snow; the
Comaoeac‘
recognizer
(Smith 19:
Marshall (f.
Ufldtscfibt
1993) and
1993). Sp

 

new host f
group are 1
ma DOW r

Simpalric

 

 

 

Hybridizn’
by Earlier r
Stitches (Bo

   

Mariam;

9
Host Shifts in Rhagoletis

Extensive study of Rhagoletis biology over the past thirty years has led to the
discovery of abundant sympatric sibling species which have little or no morphological
differences. Speciation in species groups consisting mostly of sibling species in
Rhagoletis has always been accompanied or preceded by a shift to a new host plant (Bush
1969; 1992; 1994). The R. pomonella species group, for example, consists of four
described and at least two undescribed sibling species (Bush 1966; Berlocher and Bush
1982; Berlocher et al. 1993; Bush, personal comm); these are the apple maggot or haw fly
on Rosaceae, R. pomonella (Walsh); the snowberry fly on Caprifoliaceae, R. zephyria
Snow; the blueberry fly on Ericaceae, R. mendex Curran; and the shrubby dogwood fly on
Comaceae, R. carnivora Bush. Recently two additional undescribed species have been
recognized, the flowering dogwood fly whose larvae feed in the fruit of Cornusflorida L.
(Smith 1988b; Berlocher et al. 1993) and the sparkleberry fly on Vaccinium arboreum
Marshall (Ericaceae) (Payne and Berlocher 1995). There also appears to be other
undescribed species, such as those southem populations infesting wild plums (Bush 1966;
1992) and the spring population on mayhaw in eastern Texas (Berlocher and Enquist
1993). Speciation in the pomonella group has been accompanied by a shift to a radically
new host family in almost every case (Bush 1969). However, members of the pomonella
group are difficult to distinguish morphologically (Bush 1966; Westscott 1982), and many
taxa now recognized as distinct species were originally considered as host races or
sympatric subspecies by earlier authors (Bush 1966; Diehl and Prokopy 1986).
Hybridization, oviposition-choice, ecological, and comparative serology studies canied out
by earlier researchers (reviewed in Bush 1966; 1969) as well as more recent allozyme
studies (Berlocher and Bush 1982; Feder, et al. 1989; Berlocher et al. 1993),
electroantennogram studies on host odor recognition (Frey and Bush 1990), natural
hybridization studies (Feder and Bush 1989a; Smith et al. 1993) and host associated
behavioral differences (Bierbaum and Bush 1988) strongly support the view that the three

gym pal
isolatet
occurs ;
iflnnes
pomone
Westcot
species .
and Bus
by’ondiz

pomonel.

populatic
Kaneshin
does not 2
hybridizar
discmsed
than is nor

DOVCI [ECO

 

1 O
sympatric eastern forms, R. pomonella, R. mendax, R. cornivora are reproductively

isolated from one another and represent distinct sibling species. Rhagoletis zephyria which
occurs primarily in the western United States and is sympau‘ic only with R. pomonella in
Minnesota (Bush 1966; Westcott 1982; McPheron 1990a; 1990b) differs slightly from R.
pomonella in surstyli configuration, wing band ratio and ovipositor length (Bush 1966;
Westcott 1982). Although R. zephyria is the most divergent morphologically of the four
species (Bush 1969), it is the most closely related on the basis of allozyme data (Berlocher
and Bush 1982; Berlocher et al. 1993). It is not surprising that a. low level of interspecific
hybridization between these two sibling species has been reported in areas where R.
pomonella has recently been introduced into western North America (McPheron 1990a;
1990b). Such interspecific hybridization has been also noted between sympatric
populations of Drosophila heteroneura and D. silvesm's species in Hawaii (Carson and
Kaneshiro 1989). Although F1 and F2 progeny are produced; interspecific hybridization
does not appear to result in the loss of species identity. The role and outcome of
hybridization between closely related animal species is an intriguing problem. As
discussed by Bush (l992),'interspecific hybridization may be more widespread in insects
than is now realized, and in parasite insects a low level of hybridization may give rise to
novel recombinant genotypes that facilitate the colonization of a new host.

Besides the above mentioned host plants, R. pomonella-like flies also infest other
plants such as native plums (Prunus spp.), sour cherries (P. cerasus L.), pears (Pyrus
communus L.), rose hips (Rosa rugosa Thumb.) and apricots (P. armeniaca L.) (Bush
1992). In addition, R. pomonella has also been reared from chokecherry (P. virginiana L.)
(although rarely), sweet cherry (P. avium L.), mahaleb cherry (P. mahaleb L.), ornamental
hawthorn (Crataegus monogyna Jacquin and C. mollis Scheele), river hawthorn (C.
douglassr' Lindley), crabapple (Malus spp.), pyracantha (Pyracantha coccinea Roemer), and
quince (Cydonia oblonga Miller) (Allred and Jorgensen 1993; and references within).

Some of these host associated Rhagoletis populations, such as those southern populations

 

 

 

 

associate
recently e
Giush 19'

O
which wa
hawthorn
Feder and
on the apt
original h;

Ht
suggesting
flies. Wit]
significant
Substantial
Observed, i
llelJFOduce
fidelity, as
hmhoms
and Bitsh (
elecmph}.

preferenm

 

ability to n
1992). n.
Worm.

Ecl
”0mm;

batmOm t

 

1 1
associated with wild plums, may represent undescribed species and others appear to be

recently established host races, for example, those infesting sour cherry and rose hips
(Bush 1966; 1992).

Of particular interest in the pomonella group is a new host race of R. pomonella
which was established on introduced apples approximately 150 years ago from the original
hawthorn (Crataegus Sp.) infesting form (Bush 1966; 1969; 1974; 1975; Bush et al. 1989;
Feder and Bush 1989b; Bush 1992). Over the years, extensive study has been carried out
on the apple race of R. pamonella and it is found that the apple race is distinct from the
original hawthom race in several characteristics:

Host Preferences — Prokopy et al. (1988) have provided behavioral evidence
suggesting significant differences in host response pattem between apple and hawthorn
flies. With respect to choice of fruit for oviposition, female apple flies chose apples
significantly more often than did hawthorn flies. Similarly, male apple flies tend to stay
substantially longer on apples than male hawthorn flies. Feder et al. (1993; 1994) have
observed, in the field mark-release-capture experiments, that R. pomonella tend to
reproduce on the same host species in which larvae of the flies developed. This host
fidelity, as a premating barrier between sympatric R. pomonella populations on apples and
hawthoms, restricts gene flow to about 6% per generation (Feder et al. 1993; 1994). Frey
and Bush (1990) also noticed a difference between the two host races in
electrophysiological response to host odors, further supporting the conclusion that host
preference is genetically-based although prior experience of adult R. pomonella effects their
ability to find host fruit (Prokopy et al. 1994) and on their host preference behavior (Bush
1992). The two races of R. pamonella also show different learning ability to reject novel
fruit species (Bush 1992; and references within).

Eclosion Time — It has been demonstrated that apple flies are genetically
programmed to develop faster and emerge sooner after diapause is terminated than
hawthorn flies (Smith 1988a; McPheron et aL 1988a; Feder etal. 1993). This difference in

l 2
emergence times between the races corresponds to the difference in fruit maturation

between their apple and hawthorn hosts. This allochronic separation of the races accounts
for part of the isolation of these two host races (Feder et al. 1993). This divergence of R
pamonella in eclosion time which is heritable (Smith 1988a) may substantially resuict gene
flow among different host-associated populations and thereby conuibute significantly to the
initial divergence of new R. pomonella host races.

Allozyme Frequency — Several studies have demonstrated allozyme frequency
differences between the apple and hawthorn p0pulations (Feder et al. 1988; 1989; 1990a;
1990b; McPheron et al. 1988a). Allele frequency divergence is possibly linked to other
loci involved with adaptation to apple and hawthorn, such as eclosion time, host fidelity
and response to host odors (Bush 1992).

This evidence of genetically based difference between the population of R.
pomonella associated with apple and haws is now sufficient to support the view that they
represent genetically distinct host races (for host race criteria see Bush 1992). Because the
two host races differ from each other in several biologically significant ways as the
divergence is due to adaptations to different host plants, Bush (1969, 1974, 1992) has
proposed that such adaptation might eventually lead to complete reproductive isolation
without geographical isolation. To account for the rapid host race formation of R.
pomonella and for the evolution of other sibling species in the pomonella group, Bush
(1969; 1974; 1975) developed a model of sympatric host race formation based on genetic
changes in host preference and host-based larval survival genes. The proposed host
preference speciation (I-IPS) model suggested that recombination between new alleles of a
host preference gene (also called habitat preference or host selection gene) and host-based
larval survival gene (also called habitat-based fitness gene) will produce new genotypes that
can colonize new host plants. Furthermore, gene flow reduction between the newly
established and parental populations could be enhanced by other factors such as allochronic

isolation on unrelated plants with different fruiting times (emergence patterns), conditioning

(associatl
(Bush, lS
simulatio
a third. ur
developm
conditions
1989). M
sympatric
based asso
simulation
genetically
linkage dis
fitness (fit)
andfir loci ;
P1012383 is Cl
conditions c

generations.

 

 

1 3
(associative learning by induction), disruptive selection and semigeographic isolation

(Bush, 1969, 1974 and 1975). The HPS model has later been supported by computer
simulation (Diehl and Bush 1989), in which two unlinked loci influencing larval fitness and
a third, unlinked locus involving habitat preference. Progress toward speciation (i.e.,
development of reproductive isolation) is likely to occur under a broad range of biological
conditions when assortative mating is coupled with habitat preference (Diehl and Bush
1989). More recently, Johnson et al. (1995) have developed a multi-locus model for
sympauic speciation in which habitat preference, habitat-based fitness and non-habitat
based assortative mating genes are considered simultaneously. Using computer
simulations, they demonstrate how, in organisms that mate within a preferred habitat,
genetically based host preference initiates the process of sympatric speciation leading to
linkage disequilibrium between the assortative mating gene (asm) loci and host-based
fitness (fit) loci in diploid populations. Completion of linkage disequilibrium of the asm
and fit loci yields no further interbreeding (gene flow), which implies the speciation 1
process is complete. This process can occur sympatrically under a wide variety of
conditions of selection pressure and gene penetrance, and may take less than 1000
generations.

In the case of R. pomonella, colonization on apples resulted in an escape from most
parasites. In Washington State, for example, the average level of parasitism of R.
pomonella on hawthorn (C. monogyna ) is up to 90%; while no parasitoids emerged from a
total of 4385 pupae reared from apple (Gut and Brunner 1994).

As Bush (1975) suggested, species groups having the potential for shifting to a
new host plant might have substantially higher level of genetic polymorphism, especially at
those loci involved with host adaptation. Indeed, R. pomonella has pronounced population
heterogeneity in allozyme frequency (McPherOn et al. 1988b; Feder et al. 1990a; Feder et
al. 1990b; Feder and Bush 1989b; Berlocher and McPheron, unpublished data). Such a
great population differentiation in R pomonella may be related to its extreme flexibility in

diapause
capacity I
times (lab
Jorge set
Il
Rhagolen'.
infonnatio
genetic her
have subst
Still. howe‘
possible w:
established
distribution
repeated est

(Berlocher .'

be [3111631 01

“Terms.

 

[Elite PHI/m

Mutated Ch;

l 4
diapause strategy and eclosion phenologies (Feder et al. 1993), resulting in its great

capacity to adapt to many different native hawthorn species with a wide range of fruiting
times (late April to early November in Texas, Berlocher and Enquist 1993; Allred and
Jorgensen 1993).

The process of host shifts is the core of the model of sympatric speciation in
Rhagoletis pr0posed by Bush in 1969. Over the years, an effort has been made to obtain
information on various behavioral and ecological aspects of these flies and to examine the
genetic basis of host selection and genetic differences for allozyme frequencies. The results
have substantially clarified many points and placed the model on a firmer basis. There are
still, however, some details which need to be established. Berlocher (1989) discussed a
possible way in which a host race could arise in R. pomonella. If the apple race was
established from a single colonization event and spread gradually through the apple
distribution, a genetically homogeneous population should form, otherwise, independently
repeated establishment on apples would result in several genetically distinct subpopulations
(Berlocher 1989).

Relevance to Speciation in the R cingulara group

The pattern of sympatric speciation occurring in R. pomonella species group may
be typical of many host-specific insects. The model proposed for R. pomonella group
could, in principle, be applied to the members of R. cingulata group equally well. The R.
cingulata species group consists of four native North American species: R. cingulata, R.
indifi'erens, R osmanthi and R. chionanthi (Bush, 1966) and one sub-tropical species, R.
turpiniae, described recently from Mexico, infesting two species of Turpinia
(Staphyleaceae) (Hernandez-Ortiz 1993).

Rhagolea's cingulata and R. indrfierence originally infested the fruits of different
native Prunus (Rosaceae) and now both have established themselves on inu'oduced

cultivated cherries (P. avium and P. cerasus). The two species appear to be allopauically

isolater
North I
needed
Hugo/r
05mm
species 2
morpholt
difficult
even as b
\h
Species su
cherry), P
and P. salt
1966), In .
and fruits l
native host
introduced 1
to 5,000 fee
Normally; rt
1975). even
Occasional]

Zone with n

 

 

l 5
isolated from one another in the eastern (R. cingulata) and western parts (R indijj‘erens) of

North America (Blanc and Keifer 1955; Bush 1969), although further investigation is
needed to verify the situation in the central plain states where cultivated cherries are grown.
Rhagoleris chionanthi and R osmanthi have been reared from Species of Chionanthus and
Osmanthus (Oleaceae) respectively in southeastern USA, where the two olive-infesting
Species are sympatric with the eastern cherry fly, R. cingulara (Bush 1966). The minimal
morphological differences among the four North American species make it extremely
difficult, if not impossible, to differentiate between them. They had been generally treated
even as host races or subspecies before Bush's 1966 revision.

Western cherry fruit fly, R indifierens has been reported to infest several Prunus
species such as its principal host, P. emarginata (Dougl.) D. Dietr. (wild pin or bitter
cherry), P. virginiana L. var. demissa (Nutt) Torr., P. subcordata Benth (Pacific plum)
and P. salicina Lind]. (inu'oduced Japanese plum) (reviewed in Frick et al. 1954 and Bush
1966). In California, the native bitter cherry grows at higher altitudes (3,500 to 9,000 feet)
and fruits late in the Summer and Fall (Bush 1975). R. indifierens usually infests the
native host in August (Bush 1975). Cultivated chenies, P. avium and P. cerasus,
introduced to Califomia 100-150 years ago, are grown mainly at relatively low altitudes (O
to 5,000 feet) and fruit much earlier than the native bitter chen'y (Bush 1969; 1975).
Normally, the cultivated cherries in California are not infested by R indijj'erens (Bush
1975), even when they are completely surrounded by the native bitter cherry (Bush 1969).
Occasionally, however, late maturing cultivated cherries, growing in the altitudinal overlap
zone with the wild bitter cherry, become infested (Bush 1969; 1975). These newly
formed, but highly localized, populations are often periodically eliminated by the California
Department of Agriculture (Bush .1969; 1975). Usually the same infested area is free from
attack of this fly the following year (Bush, 1975). This approach has effectively prevented
permanent establishment of R indifi'erens on commercial chenies in California (Bush

1969; 1975).

altitt'
space
estab.
popul
the ne
of tint
emerg
to low:

two po

popular
1969; 1'

(margin
Other on
1975; lo
Cherries a
(Bush 19
Si
110511133“
Wanna).
(Sitter the
the 53mm
SPines, the
Wise 0n the

mftefOre. h;

l 6
Since the majority of domestic chenies in California are allochronically and

altitudinally semi-isolated from the wild bitter cherry, there is only a narrow window in
space and time when a successful host Shift can occur (Bush 1975). If permanent
establishment of the fly population on the cultivated cherries were to be allowed, the
population would probably become permanently established on California chenies. Within
the newly established population, individual flies emerging earlier would have an advantage
of finding a greater abundance of oviposition sites. Selection would favor individuals with
emergence time shifted to an earlier date and the newly established population would spread
to lower altitudes where cultivated chenies are more abundant (Bush 1975). Eventually
two populations with different emergence times and host preferences would evolve.

In Oregon and Washington, R indrflerens apparently established permanent
population on introduced domestic cherries (P. avium and P. cerasus L.) (Bush 1966;
1969; 1975). There appear to be two races coexisting in these areas, one on native P.
emarginara at high altitude whose fruits mature from late July to early September, and the
other on domestic chenies at low altitudes during late May and early July (Bush, 1969;
1975; Jones et al. 1991). The two indrferens populations from native host and cultivated
chenies are almost completely allochronically isolated from one another north of California
(Bush 1969; 1975).

Similar differences in emergence patterns have been observed between different
host-associated populations of R. cingulata. In addition to its native host, black cherry (P.
seroa'na), R cingulata also now infests inuoduced cultivated cherries such as P. avium
(sweet cherry), P. cerasus (sour cherry) and occasionally P. mahaleb (Mahaleb cherry) in
the eastern United States. Since cultivated cherries mature earlier than native Prunus
species, the majority of fruit in commercial orchards is semi-allochronically isolated from
those on the wild host. The fly populations on cultivated chenies and wild black chenies,

therefore, have different emergence times and if following the pattern of divergence in R.

porno

races.

than:

R chit

beard)

Idevtlv

alternat
suggest
overlap}
Osmantr
possibly
Species 5
Species.

0mm
diverged
CSIablishc
same H101
050mm
hire lnCt‘t
he IOITna'
formmn
“‘0 host ;
hinted b:
beCame ge
Shim O

“Patric,

1 7
pomonella possibly different host preferences, Showing some evidence of isolation as host

races.

Allochronic isolation is even more pronounced between R osmanthi and R.
chionanthr‘, both infest native olives (Oleaceae) in southeastern United States (Bush 1966).
R chionanthi infests the fruit of Chionanthus virginicus (the fringe-nee or old man’s
beard) in the summer, while R. osmanthi attacks the fruit of Osmanthus americanus
(devilwood) during midwinter (Bush 1966; 1969; 1975). Bush (1969) proposed three
alternative explanations for the origin of the two olive infesting species. One explanation
suggests that the host plants Chionanthus and Osmanthus may have considerably
overlapped in fruiting time and both were infested by one Rhagoletis species. Later.
Osmanthus shifted its fnriting time to cooler winter months in response to climatic changes,
possibly occurring during the Pleistocene. During the process, the original olive infesting
species split into two distinct, allochronically isolated races that eventually evolved into two
species. Bush's second explanation suggests that a new host race may have established on
Osmanthus from original Chionanthus-infesting population after the two host plants
diverged in fruiting time. To me it seems also possible that a host race could become
established when the two host plants had fruiting times broadly overlapping, following the
same model for apple race formation in R. pomonella. Later, the fruiting time shift of
Osmanthus in response to climatic changes, resulting in fly emergence time shifting, would
have increased isolation of the host race from the parental population, eventually leading to
the formation of two distinct, allochronically isolated species through sympatric host race
formation. The third explanation proposed by Bush involves geographic isolation of the
two host plants. Originally, the two host plants may have fruited at the same time and were
infested by one olive-infesting species. Later, each host plant with its fly population
became geographically isolated. Meanwhile, fnritin g tiine of one host plant may have
shifted. Once geographical contact was reestablished, the two fly populations, although
sympatric, may have become allochronically isolated from each other. A fourth possibility

is th
Chic

5P5“

group
broad
ciu'ort

Uniter

accom;
Changes
Species
below
unaluit
darted
DNA re
ClInsular

Wits

1 8
is that the Chionanthus population arose from a sympatric cingulata then a few adults of the

Chiananthus population emerged during late Fall or Winter and established the Osmanthus
species.

Therefore, the cingulata species group is similar in many ways to the pomonella
group. It consists of sibling species with minimal morphological difference but with a
broad range of host plants. The two olive-infesting species(R osmanthi and R.
chionanthr) and the cherry-infesting species (R; cingulata) are sympatric in the southeastern
United States. Although slight differences in morphological characters do occur which
distinguish the three species these characters are not consistently cleaneut. Also R
cingulata and R indrfierens are host specific on different but closely related native Prunus
species, and have independently established populations on inu'oduced sweet and sour
cherries. These host associated populations Show evidence of isolation as host races.

Speciation in the cingulata group, as in the pomonella group, has apparently been
accompanied by a shift to a new host plant. Before the kinds and numbers of genetic
changes that promote, accompany and follow the colonization of the members of cingulara
species group of a new host can be established, an accurate means of distinguishing
between host races or even closely related species is required. In the absence of
unequivocal distinguishing morphological traits altemative means of identification must be
devised. As a Step towards resolving this problem, I have employed specific genomic
DNA regions as molecular markers to resolve species and racial boundaries within the R.
cingulata species group and explore the relationships of this group with other Rhagoletis
species (see Chapter II).

Cunent Taxonomy of North American Rhagoletis
The North American Rhagoletis species were most recently monographed by Bush
(1966), with 21 species segregated into seven species groups (pomonella, cingulata,
tabellaria, suavr's, ribicala, stn'atella and alternata) and one Species , R. fausta , unplaced.

BUSh t
genital
analyst
(1982)
electro;
conserr
p0ll10nr
morpht
Solanat

the clad

l 9
Bush classified the species groups mainly on the basis of structural similarities of the

genitalia, chaetotaxy, wing venation, and karyotype. Since then the only phylogenetic
analysis on North American Rhagoletis was the one conducted by Berlocher and Bush
(1982), based on electrophoretic data. The main areas of agreement between the
electrophoretic analysis and the conventional classification (Bush 1966) are the
conservation of the suavis and cingulata groups and, in 2 out of 3 cladistic trees, the
pomonella species groups. In addition, the species possessing the most ancestral
morphological characteristics, such as R striatella, a pest of husk tomatoes (Physalis sp.,
Solanaceae), and R. basiola, infesting fmit of Rosa (Rosaceae), branch from the base of
the cladistic trees generated from electrophoretic data.

Despite the above congruence there are some areas of disagreement between the
above mentioned Studies. For example, R juniperina, which infests Juniperus
(Cupressaceae), is removed from the tabellaria group and placed with cingulata group in
Berlocher and Bush (1982). The tabellaria group conventionally consists of 4 early
described species: tabellaria. juniperina, persimilis, and ebbettsi (Bush 1966), plus a
recently described species, R electrommpha Berlocher (Berlocher 1984). Members of the
tabellarr'a group share similarities in genitalia, wing pattern and body coloration (Bush
1966). Host plants of persimilis and ebbettsi are unknown. R. tabellaria is a wide ranging
species infesting two Camus species (Comaceae), C. stolonifera and C. amomum, in
eastern North America, and C. stolonr'fera in the north central and western North America
(Bush, personal comm.). In the west a race or undescribed species is known infesting
Vaccinium (Ericaceae) in western North America (Bush 1966; Bush, personal comm.). R.
electromarpha Berlocher, the most recently described tabellan'a-like species, infests two
different Camus Species—C. drummondi and C. racemosa in Illinois (Berlocher 1980;
Berlocher 1984). Morphologically, R. electromorpha is almost identical to tabellaria. Also
the presence of gland-like tubular sac at the end of phallotheca, at the junction with

aedeagus, relates R electromorpha most closely with R tabellaria with considerable

confid
rubella
ditiere.
put the

andinr

that R r
and Bu:
Howevt
species:
placeme
know wl
support I
Ringo/er
Oedicare
Hon-eve;
MUN 0r
nontoort
Another n
is the plao
Howetem
Phcement
TherefOre, 1
[0 fHillier in
In at
mulls. such

“nitrite.

2 O
confidence. In contrast, the morphology of juniperina is sufficiently different from R.

tabellaria to suggest their relatively distant relationship. However, the great morphological
difference between juniperina and the four North American members of the cingulata group
put their close relationship in doubt. The Status of R. juniperina, therefore, is debatable
and in need of careful consideration.

Also, allozyme studies (Berlocher and Bush 1982; Berlocher et al. 1993) indicate
that R cornivora may notbelong to the pomonella group. A recent mtDNA Study (Smith
and Bush, in preparation) also places R cornivora outside of the pomonella group.
However, the close morphological affinities between R. comivora and the rest of the three
species in the pomonella group made earlier Rhagoletis researchers hardly doubt the
placement of R comivora in the pomonella group. Therefore, it would be interesting to
know whether any DNA data, such as ribosomal DNA (rDNA) sequence analysis, will
support the morphological implication regarding the relationship of R. cornivora to other
Rhagoletis species. In addition, R. striatella, the tomato husk fly, is placed with genera
Oedicarena and Zonosemata in Berlocher and Bush (1982) rather than within Rhagoletis.
However, the placement of striatella with Zonosemata is not surprising as Bush (1966)
pointed out, R striatella shares similarity with Zonosemata in karyotype, number of lower
fronto-orbital bristles, certain characteristics of male genitalia and host plant relationship.
Another new insight gained from the electrophoretic analysis of Berlocher and Bush (1982)
is the placement of previously unplaced species, R. fausta, with the suavis group.
However, the recent mtDNA data (Smith and Bush, in preparation) does not support this
placement. Instead, R. fausta forms a clade with R. juniperina according to mtDNA data.
Therefore, the placement of R. fausta is cunently still not completely resolved and subject
to further investigation.

In addition to the uncertainty of placement of some species in certain species
groups, such as R. juniperina, carnivara, striatella and fausta, the phylogenetic relationship

among different species groups are not completely resolved either. For example, closest

relati‘
electr
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demon

1989).

Ringo/t
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and unit
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2 1
relatives of the cingulata group in North America is the pomonella group according to

electrophoretic studies (Berlocher and Bush 1982; Berlocher et al. 1993), while based on
mtDNA data (Smith and Bush, in preparation) the cingulata group is more closely related to
the suavis group.

In summary, although the species of Rhagoletis, especially those from the Nearctic
region, have been well studied and segregated into a number of species groups, there are
still several unanswered questions regarding placement of certain species and relationships
among the existing species groups. Furthermore, monophyly of the genus has not been
demonstrated and its relationships to other Carpomyina are poorly understood (N orrborn
1989).

Dissertation Objectives

My research has two objectives 1) to estimate the phylogeny of North American
Rhagoletis species and 2) to explore the genetic variation among the sibling species of the
cingulata species group. The following ten species are analyzed in this study: R. cingulata
and indzflerens are representing the cingulata group; R. pomonella and comivora
representing the pomonella group with carnivora's placement in this group deeming further
verification using rDNA data; R. completa represents the well-defmed suavis group; R.
electmmorpha and juniperina are from the tabellaria group with juniperina ’s relationship
with the rest of the tabellaria group questionable; R. fausta was unplaced anywhere (Bush,
1966) or its placement is in disagreement from two previous independent molecular studies
(Berlocher and Bush 1982; Smith and Bush, in preparation); and R. basiola which
possesses some of the most ancient morphological characters and is used as outgroup for
my rDNA phylogenetic analysis. The placement of R. striatella in the genus Rhagoletis has
been questionable and will be further tested in this study using rDNA spacers.

I use internal transcribed spacers to explore the following problems: 1) What

phylogeny do the rDN A spacer sequence data support? 2) Is the phylogenetic implication

from
mort
whici
cingu
mole:
the di
comp:
useful
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identified a
Chapter II]
mung the z
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2 2
from the rDNA data congruent with any existing systematic of the genus based on

morphology and allozyme data? 3) Are there any molecular markers in the rDNA spacers
which can be used to differentiate sibling species of the morphologically indistinguishable
cingulata complex? and if so, what phylogenetic relationship can be inferred from those
molecular markers? 4) Are there any genetic polymorphism in the rDN A spacers among
the different host-populations of R. cingulata and if so, how high is the level of variation
compared to interspecific variation in R. cingulata group. In addition, I evaluate the
usefulness of the secondary structure of internal transcribed spacers (ITS) in inferring
phylogeny. Investigation on other rDNA spacer regions such as the external transcribed
spacer (ETS) has also been conducted (Chapters IV and V) and the preliminary results from
such investigation may form basis for future studies to gain insight into the phylogeny of

morphologically indistinguishable species complex.

Organization of the Dissertation

The next four chapters are presented in scientific format, with each Chapter
subdivided into an introduction, material and methods, results, discussion, and
bibliography sections. In some cases the result and discussion sections are combined into
one section. Chapter V1 is a concluding summary.

Chapter II presents complete sequences and several constrained secondary structure
elements of the rDNA ITS regions of the four North American sibling species of the
cingulata group. In addition, molecular markers to differentiate those sibling species are
identified and phylogenetic implications from those molecular markers are discussed.
Chapter III makes use of the rDN A ITS regions to establish phylogenetic relationship
among the ten representative Rhagolen's species noted above. The phylogenetic
implications from the ITS regions are compared with those from earlier studies based on
morphology and other molecular data. Chapter IV describes the construction of a R.

pomonella genomic DNA library and identifies several clones which represent different

3?]

inu

2 3
segments of the complete rDNA repeat unit. The research described in Chapter IV was

performed at the beginning of my Ph. D program before the PCR technology was widely
applied in molecular biology. Chapter V represents a partial characterization of the rDNA
ETS region of two R. cingulata flies, one from native black cherry and the other from

introduced sour cherry.

BIBLIOGRAPHY FOR CHAPTER I

Allred, D. l
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24

Bibliography for Chapter I

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66:5 1-59.

 

Berlochc
differr
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No. 8.

Biggs. I. I
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fruit fly

Boller. E.
Europe;
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Bullet. E. 1
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Bol'ce. A l
8:363-5'

Bush. G. L.
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25

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26

Carson, H. L., and K. Y. Kaneshiro. 1989. Natural hybridization between the sympatric
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ijar, R., A. Angioy, P. Pietra, J. G. Stoffolano Jr., A. Liscia, and I. T. Barbarossa.
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fidelity is an effective premating barrier between sympatric races of the apple maggot fly.
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27

Feder, J. L., T. A. Hunt, and G. L. Bush. 1993. The effects of climate, host plant
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28

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29

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Smith, D. C. 1985b. General activity and reproductive behavior of Rhagoletis tabellaria
(Diptera: Tephritidae) flies in nature. J. Kansas Entomol. Soc. 58:737-739.

 

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30

Smith, D. C. 1988a. Heritable divergence of Rhagoletis pomonella host races by seasonal
asynchrony. Nature 336:66-67.

Smith, D. C. 1988b. Reproductive differences between Rhagoletis (Diptera: Tephritidae)
fruit parasites of Camus amomum and C. flarida (Comaceae). J. New York Entomol.
Soc. 96:327-331.

Smith, D. C., and R. J. Prokopy. 1982. Mating behavior of Rhagoletis mendax (Diptera:
Tephritidae) flies in nature. Ann. Entomol. Soc. Am. 75:388-392.

Smith, D. C., S. A. Lyons, and S. H. Berlocher. 1993. Production and electrophoretic
verification of F-l hybrids between the sibling species Rhagoletis pomonella and R.
cornivora. Entomol. Exp. Appl. 69:209-213.

Stadler, B., B. Ernst, J. Hurter, and E. Boller. 1994. Tarsal contact chemoreceptor for
the host marking pheromone of the cherry fruit fly, Rhagoletis cerasi: responses to
natural and synthetic compounds. Physiol. Entomol. 19:139-151.

Westcott, R. L. 1982. Differentiating adults of apple maggot, Rhagoletis pomonella
(Walsh) from snowberry maggot, R. zephyria Snow (Diptera: Tephritidae) in Oregon.
Pan-Pacif. Entomol. 58:25-30.

Yokoyama. V. Y., and G. T. Miller. 1994. Walnut husk fly (Diptera: Tephritidae) pest-
free and preovipositional periods and adult emergence for stone fnrits exported to New
Zealand. J. Econ. Entomol. 87:747-751.

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CHAPTER II
rDNA INTERNAL TRANSCRIBED SPACERS 1 AND 2 OF THE RHAGOLETYS
CINGULATA SPECIES GROUP: SEQUENCE, CON STRAINED SECONDARY
STRUCTURES, AND PHYLOGENETIC IMPLICATIONS

Introduction

The genus Rhagoletis (Diptera: Tephritidae) is composed of several economically
and biologically important species groups. Speciation in some of these species groups
have been accompanied by colonizing new host plants. The R. pomonella Species group,
for example, has emerged as a model system for studying host race formation and
sympatric speciation (Bush 1993). The apple and hawthorn races of R. pomonella are
morphologically indistinguishable, but biologically they show several genetically-based
character differences (see chapter 1). Host shifts to introduced plants have also occurred in
other, less well studied, North American Rhagoletis species, but their biological status
deems further investigation. Of particular interest are members of the Rhagoletis cingulata
species group which have undergone rapid host shifts but are also difficult to distinguish
using morphological characters alone. .

The R. cingulata species group consists of four native North American species: R.
cingulata, R. indrfl'erens, R. osmanthi and R. chionanthi (Bush, 1966) and one sub-
tropical species, R. turpiniae, described recently from Mexico, infesting two species of
Turpinia (Staphyleaceae) (I-Ienrandez-Ortiz 1993). The four North American sibling
species show little or no morphological difference, but have a wide range of host plants.
R. cingulata and R. indrflerens are serious cherry pests in the eastern and western United
States, respectively. These two species originally infest different native wild chenies

3 1

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(Prunus spp.), and now both have established themselves on introduced cultivated cherries

such as sweet chenies and sour chenies. For each of the two species, populations on the
old native host and introduced cherries appear to be semi-allochronically isolated due to
different emergence times which are synchronous with the maturation of their different
respective host fruit. The two host-associated populations of R. indifierens, on native pin
cherries and introduced commercial cherries are also semi- geographically isolated from
each other because cultivated chenies are usually grown at considerably lower altitude than
the wild chen'y in the western United States. Therefore, two host races, with different
emergence patterns and different host associations, exist for each of the cherry fly species.
The other two sibling species in the cingulata group, R osmanthi and R. chionanthi, infest
native olives (Oleaceae) in southeastern United States where they are sympatric with R.
cingulata (Bush 1966). R. chionanthi infests the fnrits of the fringe-tree, Chionanthus
virginicus, which fruits in the summer; while the larvae of R. osmanthi are found in
devilwood, Osmanthus americanus, which fruits during midwinter (Bush 1966; 1969;
1975). Bush (1969) proposed, as in the case of the formation of the apple race of R.
pomonella, host races adapted to the two olive species could have established themselves
when the two host plants had broadly overlapping fruiting times . When the fnriting time
of Osmamhus shifted, probably in response to climatic changes, the pattern of fly
emergence time also shifted. As a consequence, isolation of the host race from the parental
population would have been increased, eventually leading to the formation of two distinct,
allochronically isolated species through sympatric host race formation specialized on
different host plants.

The cingulata species group is, therefore, similar in many ways to the pomonella
group. It consists of sibling species infesting various plants but with minimal overlapping
morphological differences. The morphological characters that distinguish two olive-
infesting species (R. osmanthi and R. chionanthr) and the cherry-infesting species (R.

cingulata) which are sympatric in the southeastern United States are thus not clear-cut. The

 

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allopatric R. cingulata and R. tridr’fi’erens which utilize as hosts different native Prunus

species, have also established populations on introduced sweet and sour chenies that show
some evidence of isolation as host races. Speciation in the cingulata group, as in the
pomonella group, has probably been accompanied by a shift to a new host plant. Before
the kinds and numbers of genetic changes that promote, accompany and follow the
colonization of host plants by the members of cingulata species group can be established,
an accurate means of distinguishing closely related species or even host‘races is required.
In the absence of unequivocal distinguishing morphological traits alternative means of
identification must be devised. As a step towards resolving this problem, I have employed
nuclear rDNA internal transcribed spacers (ITS) as molecular markers to investigate species
and racial boundaries within the R. cingulata species group and explore the relationships of
this group with other Rhagoletis species.

Sebcting the sequence to be analyzed is probably the most important decision to be
made in designing a DNA analysis in phylogenetic studies because the level of sequence
variation should be sufficient to display enough variation but not too much that there is
substantial homoplasy of nucleotide substitution. Nuclear rDNA is unique in the sense that
it has both highly variable and conserved regions, providing information across a broad '
phylogenetic spectrum (Hillis and Davis 1986). In eukaryotes, rDNA is composed of
tandemly repeated transcriptional units separated from each other by intergenic spacers.
The entire unit is transcribed by RNA polymerase I as a single 458 precursor molecule,
which is then processed to yield mature 188, 5.88 and 288 rRNAs (Hadjiolov 1985;
Sonnet-Webb and Tower 1986). The highly conserved coding regions (188, 5.88 and
288) and relatively fast-evolving spacers allow investigation of both distantly and closely
related taxa. In addition, the highly conserved coding regions flanking the spacers make
rDNA an excellent system for PCR amplification and analysis. For instance, the internal
transcribed spacers (ITSl and 1182) are located between the well-conserved coding regions

188 and 5.88, and 5.88 and 288, respectively. Even though the sequence of Rhagoletis

fort

3 4
rDNA is not known, PCR primers can be designed based on either highly conserved

sequences or known sequences of closely related taxa. Because of the above mentioned
features of rDNA, the rDNA spacers have recently become an attractive source of
phylogenetic characters for differentiating populations (Nazar et al. 1991; Bakker et al.
1992; Kooistra et al. 1992; O'Donnell 1992; Gardes and Bruns 1993; Fritz et al. 1994;
Volger and DeSalle 1994) and for phylogenetic analysis (Lee and Taylor 1991; Baldwin
1992; Pleyte et al. 1992; Wesson et al. 1992; Wingfield et al. 1994). Because of the
relatively rapid rate at which new mutants are fixed in rDNA spacers, these regions may
distinguish closely related species that otherwise show little genetic divergence (Brown et
al. 1972; Furlong and Maden 1983; Tautz et al. 1987; Porter and Collins 1991). In
addition, ITSI and IT 82 RNAs in yeast Saccharomyces cerevisiae, have been shown to
function independently and are important for the processing of the pre-rRNA to the mature
forms (Musters et al. 1990; van der Sande et al. 1992).

A secondary-structure model for S. cerevisiae 1182, based on chemical and
enzymatic probing, has been proposed (Y eh and Lee 1990). The ITS regions have a high
propensity of forming secondary structures in several other organisms as well
(Kupriianova et aL 1989). Some of these conserved potential secondary structures in ITS
are presumed to be functionally important. Therefore, within the ITS sequences several
regions may be relatively constrained and not free-evolving as in the case of Drosophila
(Schlotterer et al. 1994). Having those conserved secondary structures as partial alignment
guides will increase the accuracy of aligning homologous regions rather. than only similar
regions which might be a consequence either of common ancestry or of chance (Olsen and
Woese 1993). Because it is very important to compare aligned homologous regions in
phylogenetic study, secondary structure analysis has become essential when we extract
phylogenetic information from rDNA sequences (Wesson et al. 1992; Schlotterer et al.
1994).

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My immediate goals in this study are to 1) obtain suitable primers and PCR reaction

conditions for amplifying, for the first time, the ITS regions of rDNA in the genus
Rhagoletis; 2) determine the sequences of ITS regions of the 4 North American sibling
species in the cingulata group and those of R. pomonella for comparison; 3) examine the
level of ITS sequence polymorphism among different individual flies of each species, as
well as among different host-associated p0pulations of R cingulata; 4) establish molecular
characters which can be used for distinguishing the sibling species in the cingulata group
and evaluate the usefulness of the ITS sequences in the phylogenetic analysis of those
closely related species and/or host-associated population in this group; and 5) identify
constrained potential secondary-structure elements in ITS of Rhagoletis using an analysis
based on the principle of positional covariance in addition to the computer-based minimum
free energy method. Some conserved secondary-structure elements will be compared with
those from Drosophila. I also infer a phylogenetic relationship and investigate the
systematic status of taxa in the cingulata group. The suitable PCR primers and reaction
conditions determined in this study will'be employed in future phylogenetic analysis of an
expanded number of taxa in the genus Rhagoletis, especially those whose placement is
uncertain or in question as mentioned in Chapter I. The level of intra- and inter-specific
variation discovered here will help evaluate the usefulness of the ITS regions in future
phylogenetic analysis of other taxa in the genus Rhagoletis. Identification and further
characterization of nucleotide changes within specific secondary structural elements may
provide additional insight to the mode of evolutionary divergence of certain Rhagoletis

species and functional significance of the ITS during processing of precursor rRNA.

Materials and Methods
Biological Material
All species were collected during 1988-90 from various locations and host plants in

the United States of America (Table 1). Larvae emerged from field infested fruit and were

Tab!

36

Table 1. Collection Sites and Host Plants of Rhagoletis Species Used in This Study

 

 

Spades Sample Sex Host Plant (Common Name) Location (USA)

R. cingulata RC1 M Pmnus avium (sweet chary) Traverse City, MI
R. cingulata RC2 F Prunus avium (sweet cherry) Traverse City, MI
R. cingulata RC 3 M Prunus cerasus (sour cherry) Hart, MI

R. cingulata RC4 F Prunus cerasus (sour cherry) Hart, MI

R. cingulata RC5-12 M Prunus serotina (black cherry) Roselake, MI

R. cingulata RCS-Il F Pmnus serotina (black cherry) Roselake, MI

R. chionanthi RKl M Chionanthus virginicus (fringe-tree) Perry, GA

R. chionanrhr' RK2 F Chionanthus virginicus (fringe-tree) Perry, GA

R. osmanthi ROI M Osmanthus americanus (wild tea-olive) Alligator Lake, FL
R. osmanthi R02 F Osmanthus amen'canus (wild tea-olive) Alligator Lake, FL
R. indrfi'erens R11 M Prunus cerasus (sour cherry) Pullman, WA

R. indifl’erens R12 M Pnarus cerasus (sour cherry) Pullman, WA

R. Werens R13 F Prunus cerasus (sour cherry) Pullman, WA

R. pomonella RPl M Crataegus spp. (hawthorn) E. Lansing, MI

R. pomonella RP2 M Craraegus spp. (hawthorn) E. Lansing, MI

R. pomonella RP3 ND Mains pumula (apple) . Door Co., WI

R. comivora RCol M Camus amomum (dogwood berries) E. Lansing, MI

R. juniperina RJ 1 M Juniperus virginiana (E red cedar) Dixon Springs, IL
R. fausta RFl M Prunus cerasus (sour cherry) Fish Creek, WI

 

Note.—ND=NotDetermined

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3 7
allowed to pupate in fine, moist vermiculite. Pupae were sifted from the vermiculite and

stored at 4° C for at least 5 months. Pupae were then removed from the cold and held at
22° C under a 15 hr light and 9 hr dark cycle to terminate diapause. Within 2-7 days after
emergence most adult flies were frozen at -70° C for subsequent genomic DNA isolation.

Specimens from each collection were pinned for species identification.

DNA Isolation and Amplification

Total genomic DNA was isolated from individual flies as described by Procunier
and Smith (1993). The ITSl region was amplified using primer
1406F 5’CC'ITTGTACACACCGCCCGT (matching the 3' end of 188) and primer
35R 5'AGCI'RGCTGCGTI‘C’ITCATCGA (matching the 5' end of 5.88). The IT82
region alone was amplified using primer 108F 5'GAACATCGACHHKTYGAACGCA
(matching the 3' end of 5.88) and primer 52R 5'GT1‘AG’I'ITC‘1TITCCI‘CC8CT
(matching the 5' end of 28S). Amplification of the ITSI and IT82 regions as a combined
region on one DNA fragment was performed for R. indrfierens, using the 1975F and 52R
primers. Amplification by the polymerase chain reaction (PCR) was canied out in 25 IJJ
(final volume) containing 10 mM Tris-HCl, pH 8.5, 50 mM KCl, 3 mM MgC12, 375 11M
of each dNTP, 0.1-0.4 11M primer 1975F (or 108F) and 0.104 M primer 35R (or 52R),
and 1.25-2.50 units of Ampli Taq DNA polymerase (Perkin Elmer
Cetus) with 5-20 ng genomic DNA. Amplification parameters were 92° C for 3 min 10
sec; 30 cycles each at 92° C for 15 sec, 65° C for 15 sec and 72° C for 2 min; and 72° C for
6 min 10 sec. Amplified DNA was subjected to electrophoresis on a 1.0 % agarose gel and
visualized with ethidium bromide. Bands containing the DNA of interest were excised
from the gel and the DNA purified using the Prep-A-Gene DNA purification matrix (Bio-

Rad), according to the manufacturer's instructions.

Cloning a

1'1
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3 8
Cloning and Sequencing

The TA Cloning kit (Invitrogen) was used in a one-step cloning strategy for the
direct insertion of the purified PCR products into a plasmid vector, followed by
transformation into competent cells. Plasmid vector and competent cells were supplied by
the manufacturer. Plasmid DNA was purified from individual clones using the Magic-Prep
DNA purification kit (Promega), following the manufacturer's instructions. One clone was
randomly picked from each fly and, in general, several flies were sequenced for each
species. DNA sequencing was performed according to the chain-termination method of
Sanger et al. (1977), and using the Sequenase Version 2.0 DNA sequencing kit (USB) and
35S-dATP (Amersharn). The same primers used in the amplification reactions were used
to determine the DNA sequence in both directions. Once a stretch of DNA was sequenced
additional primers were employed to complete the sequencing of the ITS regions from the
different species: Primers 35R-GB 27 5'ACC(CT)AAACATTITCAAGT(CT)GCG was
used for the ITS] regions; and primer 108F-GB25
5'A(AT)(AG)(AG)AATC(AT)(CT')AGTAT1‘CCC was used for the ITSZ regions.

DNA Sequence, Structure and Phylogenetic Analysis

The PILEUP and FOLDRNA programs in the GCG package of the University of
Wisconsin Genetics Computer Group (UWGCG package, version 8.0) were used for
alignment and secondary-structure calculations, respectively. Alignments were done first
with the computer (gap weight = 3.00 and gap length weight = 0.20) and then manually
adjusted. Estimates of the percent nucleotide substitutions per nucleotide site between each
pair of taxa and their standard errors were determined by the methods of Juke and Cantor
(1969) and Tamura (1992) using Molecular Evolutionary Genetics Analysis (MEGA)
software version 1.01. All gap sites were removed from the subset data during pairwise
comparisons. Because several flies from each species were sequenced, which resulted in

several percent substitution estimates for pairs of the same species, a weighted average of

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3 9
the data points was calculated taking into account the standard error values for each pair of

taxa by the following equation:

2(xi/O’i2)
l1 = 2
2(1/Ci )

 

where xi is the Jukes-Cantor estimate for each pairwise comparison and 0'; its standard
error estimate with p. the average number of substitutions per nucleotide site. The standard
error of the weighted average value was determined by taking the square root of the
reciprocal of the denominator in the above equation. Subsequently, the average percent
nucleotide substitution (number of nucleotide substitutions per 100 nucleotide sites) was
obtained by multiplying 11 and its standard error value by 100. Phylogenetic analysis was
accomplished by the maximum parsimony method in which all uninforrnative characters
were ignored using the programs in PAUP version 3.1 by D. L. Swofford (University of
Illinois, Champaign, IL). Uninforrnative characters were ignored and gaps were
considered as missing data. Taxas RC2 with its ITSl sequence and R13 with its IT 82
sequence were excluded in the PAUP analysis because their corresponding IT82 and ITSl
sequences, respectively, were not determined in this study (see Table 2) and equivalent
taxas are required when combining informative characters from both ITS sequences in a
PAUP analysis. The exhaustive search option was employed to find the most
parsimonious tree(s). The three R. pomonella flies were taken as outgroup and made a

monOphyletic sister group to ingroup (i.e., rooted).

Results
DNA Amplification _
The PCR amplification of the ITSI and IT82 or a combined region containing the

two spacers was successful. Most of the amplification products were visualized as a single

 

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4 0
sharp band on agarose gels as in Figure 1. Some of them do not even need further

purification and can be directly used for cloning.

Sequence Analysis

The complete ITSI and IT82 sequences of the 4 sibling species in the cingulata
group and of R. pomonella are presented in Figures 2 and 3, respectively. In addition to
the ITS sequences presented in Figures 2 and 3, I also sequenced approximately 50 to 200
bp of rDNA coding regions. The boundaries between the ITS and the coding regions were
defined by comparing the Rhagoletis sequences with published Drosophila melanogaster
sequences (T autz et al. 1988). For ITSI, of the 182 nt sequenced within the 3’ of the 188
(using 1406F), I found 2 insertion/deletions and one substitution; of the 54 nt sequenced
within the 5’ end of 5.88 (using 35R) no variation was found. Similarly, for IT 82, of the
99 nt sequenced within the 3’ end of 5.88 (using 108F) only 2 substitutions were
observed; of the 80 nt sequenced within the 5’ end of 288 (using 52R) no variation was
observed.

The sequences of the different members in the cingulata species group are highly
conserved with very few nucleotide changes. However, there are considerable
insertion/deletion differences in both ITSI and IT82 between the R. cingulata species
group and R. pomonella. Furthermore, the region between nucleotide positions 206-300 in
ITSI appears to be quite variable between the three individual flies of R. pomonella, two of
which are from hawthorns and the third from apples.

The average percent nucleotide substitutions (nucleotide substitutions per 100
nucleotide sites) for the ITS sequences were calculated based on alignments in Figures 2
and 3, and are presented in Table 3. The results from the pairwise comparisons were, in
most cases, identical between two different statistical approaches; Jukes and Cantor (1969)
and Tamura (1992). The Tamura (1992) approach compensates for biases in

transition/transversion rates and G+C content in addition to compensating for multiple hits

Tal

41

Table 2. A-T Content and Length of the Analyzed Rhagoletis ITS Sequences

 

 

 

ITSI ITSZ

Species Sample A-T Length A-T length

Content (nt) Content (nt)

(%) (%)

R. cingulata RC 1 79.8 660 82.7 555
R. cingulata RC2 79.8 657 ND ND
R. cingulata RC3 79.9 662 82.7 555
R. cingulata RC4 79.7 661 82.7 554
R. cingulata RC5-Il&2 80.1 652 82.9 556
R. chionanthi RKl 80.1 658 82.6 553
R. chionanthi RK2 80.1 659 82.8 554
R. osmanthi ROI 80.0 657 82.4 553
R. asmanthi R02 80.0 657 82.7 554
R. indiflerens R11 79.7 659 82.7 555
R. indifierens R12 79.7 660 82.8 557
R. indifi’erens R13 ND ND 82.8 557
R. pomonella RPl 80.4 684 82.0 471
R. pomonella RP2 79.2 653 81.7 471
R. pomonella RP3 80.1 674 81.7 475
R. comivara RCol 80.7 652 81.2 482
R. juniperina RJl 81.0 683 82.7 557
R. fausta RFl 80.0 624 8 1.7 527

 

Note.— ND = Not Determined

42

Figure 1. PCR amplification products from Rhagoletis 1182 using primers 108F and 52R

PCR products were analyzed on a 0.8% agarose gel by electrophoresis. The 123
bp DNA ladder (lanes M), PstI (lane M') and HindIII (lane M") digests of A

DNA were used as molecular size markers. Lanes 1-9 conespond to pomonella,
completa, electromorpha, comivora, striatella, fausta, basiola, indrferens and
cingulata. The genomic DNA used in this analysis were prepared from male flies
except for R. cingulata.

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43

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Figure 1

 

44

Figure 2. IT 81 sequences and alignment for Rhagoletis cingulata species group and R.
pomonella. Identical nucleotides are denoted by dashes, and gaps are denoted by
dots. Phylogenetically informative characters are denoted by asterisks.
Conserved computer generated secondary structure domains are boxed, whereby
the loop regions of the stem-loop structures (see Fig. 5) are separately boxed and

labeled. The stem regions are indicated. The labeling of the strucnrral domains

corresponds to the numbering in Fig. 5.

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47

Figure 3. 1182 sequences and alignment for Rhagoletis cingulata species group and R.

pomonella. Notations same as in legend of Figure 2.

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48

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3

70 180
13‘: 110 120 130 140 150 . 160 1 . 190 200
RTCCATIAiihTIATRGCAAAAAAAGAAATRRRTRARATTTCTTCQAATCC..TCTIARTGARRTCTCTTRTARAAAAGAATCTCAGTATTCCCTTRAGA
..... -o m * --- — -——-
--.. ......................... Tc--- D- ...............
...... " .---’c'-"-----c I --------------------------"-"--
-- ---------------- c -------------------------------------------
210 220 230 240 250 260 270 280 290 300
0 mg n- —> 1M . .
CRTTRTTTGRRTTRATATTTRAATRATATATATGAGGAGCAASCTCTAGCATAAR’RTTCA ATTCTAGARTTCCCTCTA.TRGATAT
--- ------------------------------- .4 ..... qr- ----------------------------
......................................... d—ooccdboc-ncuncc-cuc---.-a--—:-------
-------------------------------------------- GCCOD-1L----------------—----.----——-
----------------------------------------------- --—----tl----—--—----—--—--—-—-.--—----
--. .---c -------------------------------------------------------------- A ------ T--A----
--. c ---------------------------------------------- A ------ run-u—
, --. c -------------------------------------------- a ------ run-«-
310 330 340 350 360 370 380 390 400
06-12” it- o c . '
ATTTATRTTRRTRTCTGGRTIATCTTCQTTTTTTCTGAATCAACAAAARATAAAARTRTRAARTGTRTTRRTRTRAAAGTTAT .................
..... --------------------—-- A----GA---- .... U I IOOOIC'OODO
..... -....-C‘---‘..-...-"-. T..- n- Colt-0.00.0000:
-------------------- - T------------.................
----‘ ..................... -- ----- T ............ O .............
----- ------- ---- C-----..-—--—.................
...... b-----------------------------------------T-----I c--’--0 one. Ion-coo...-
""‘P -------------------- G -------------------- T ----- G. ......................
-------------------------- G-- T-----..-----.................
c ............ “------c-nooucu-s-ccococouscocoscoocoooososcouousooo--'ATATATATATATATAn
C ----- Cb ------ C- ........................... .... ............... ATATATATRTRTRTNTT
c '4’--’---C’ - v o s u I o o n o - I o s o c sssssss o I I I o I o o o ........... ---ATATATATATATATA07

 

l
D

1‘

I I)
la.

:1..

51’.

Fll-‘lt

tho

1'8

re:

3 (c

 

RC1
RC3
RC4
RC5
R11
R12
R13
RRI

R01
R02
R91
RP2
RP3

49

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

410 420 430 440 450 460 470 480 490 500
U D O I . D C . C O O I
.......................ATTRRTAAAAARCGGGATG.ARAGR.TCTTTTTTTTT.CTRATIAGRTRRRR..TTTCRRGRAG.....TATTTCTT
............................ 0 on Aooooc----’---
:::::::::::::::::::::::-------------------: ..... : ..... .. .. ...... .........
coco-coco... aoosocooo- ................. A ..... a ”””” I 0- ADO... ........
0000.00.00 ooooooooooooooo A ...... o 0. A0... ---------
ooooooo ....-c0......con-------------------A "' a CO al.-oo--------
soon-ooo-o-soo-c-soooco ................... A ..... c ----------- 0 00 A00... .......
Ollooooooooooclooclonnl ”--A .... o no Pic-co. .......
loco-ocoooooooo-oaogo .................... A ..... o .... T 00 3‘00... --------
......CIOCCOIOOIOIICQ ..................... A ---------- . .. A.-I.C ........
...-oooloooooooooooooco ................. A .... c- I to ‘l‘ll... ........
Mnnfllnflh.émm """"""""""""" A"'"GT """"""" co ..... -c-- ----- cocoon-.0000000'oo .......
M"“flhnflAsAmm ................ A""6T ......... coo ..... -c ....... 000.000.00.000... ........
Moose-oooosTAoAmm ................. A---"GT ......... a ....... .c ....... n ......... ATATAT ........
510 520 530 540 g 550 560 570 580 590 600
0
TGRRRAARCTRRRRRARTRTRRRRATTATRTATGATTTTATAACACTTTARTCACTATTRTRAARRRRTTTTTRTTATTTTTCTCTTTRRRTRTTTTBTT
.............................. 7
I------::::I22222222222231: .... A '2:
----c ............ on. --------------------------------------------- oooo-ooc-ocooooo ...... o ..... ....--
——--c---——--—---..... ...................................................... o ............. ’---UUCI--s
----C------------.... ...................................... o ...... 0.0.0.... ooooooooooooo ----....--.
609
ARARTTGTR

Figure 3 (cont’d)

a).

hxmth.

50

Table 3. Average Percent Nucleotide Substitutions (Nucleotide Substitutions per 100
Nucleotide Sites) in the ITS sequences for the Rhagoletis cingulata Species Group and R.
pomonella in Pairwise Comparisons

 

 

 

nsr 1 2 3 4 5
1 cin 0.18 i 0.07
(10)
2. ind 0.21 :1: 0.06 0.35
(10) (l)
3. chi 0.40 i 0.08 0.52 i 0.15 0.00
(10) (4) (1)
4. osm 0.40 i 0.08 0.52 :1: 0.15 0.35 i 0.12 0.00
(10) (4) (4) (1)
5. porn 5.77 :1: 0.27 5.88 r 0.42 5.70 i 0.42 5.32 :1: 0.40 0.20 i- 0.11
(15) (6) (6) (6) (3)
H32
1 cin 0.27 :1: 0.11
(6)
2. ind 0.27 i 0.09 0.24 i 0.17
(12) (3)
3. chi 0.24 :1.- 0.12 0.24 :1: 0.17 0.00
(8) (6) (l)
4. osm 0.24 :1: 0.12 0.24 as 0.17 0.00 0.00
(8) (6) (4) (l)
5. porn 3.33 :1: 0.26 3.29 :t 0.30 3.21 :1: 0.36 3.21 i 0.36 0.24 i 0.17
(12) (9) (6) (6) (3)

 

Note.—cin = R. cingulata; ind = R. indrfl'erens; chi = R chionanthi; osm = R. osmanthi;
and porn = R. pomonella. Number in the brackets represents the number of pairs analyzed.
All gap sites were removed from the subset data before the pairwise comparisons.

 

as in the
from the
l
0.07% it
the diffe
values id
observec
represen
1994). f
substitua
represen
1
DNA arr
Empemt
range frt‘
about O'l
Because
and hlg‘
the Error
after 30 t
that as a
plasmid ,
Whirled
“0 differ.
"Emilie.

“mile 11‘
f

 

5 1
as in the Jukes and Cantor (1969) approach. Therefore, here I only present the results

from the Jukes and Cantor (1969) approach.

Among the different R. cingulata flies the level of genetic variation was 0.18 i
0.07% in ITSI and 0.27 :1: 0.11% in IT 82. Similarly, the level of genetic variation among
the different R. pomonella flies was 0.20 i 0.11 in ITSI and 0.24 i 0.17 in 1182. The
values for the level of within-species variation in the ITS sequences are comparable to those
observed in a recent study of five different isofemale Drasaphila melanagaster lines,
representing five different individuals from different countries (Schlotterer and Tautz
1994). From Table I of Schlotterer and Tautz (1994) I calculated the percent nucleotide
substitutions of the ITS sequences between isofemales lines (i.e., intra—specific assay
representing different individuals) to be 0.10 i 0.07.

The fidelity of Taq polymerase is highly dependent on the conditions used during
DNA amplification — especially dNTP and Mg“2 concentrations, and annealing
temperature (Gelfand and White 1990). The average nucleotide mutation rate per cycle can
range from 1.7 x 10'4 (at 1.5 mM each dNTP, 10 mM MgC12 and 37° C annealing) to
about 0.5 x 10'5 (at 200 11M each dNTP, 1.5 mM MgC12 and 54 to 55° C annealing).
Because the conditions used for amplification in this study are at the lower end for dNTP
and Mg“2 concentrations as well as higherannealing temperature, I conservatively estimate
the error frequency from DNA amplification to be 53 mutation per length of ITSI or ITSZ
after 30 cycles. This is within the limits of deviation found in Table 3. It is noteworthy
that, as a control, when the D. melanagaster ITS sequences were amplified by PCR from
plasmid po238 (which contains the complete D. melanogaster rDNA repeat unit;
provided by Dr. G. A. Dover) and sequenced using the conditions in this study, I detected
no difference from published results (Tautz et a1. 1988; Schlotterer et al. 1994). Significant
DNA slippage-induced length variation is observed when simple repeating sequences are
amplified by various DNA polymerases in vitra (Schlotterer and T autz 1992). Variations at

simple repeat loci (e.g., length expansions) have also been observed in viva and appear to

 

arise ft:
1994: at

 

length V
u'rro art
or SpecieI
be the pl
DeSallel
|
nucleon
those in
hetero gt

large 1e.r

Species .

the intra
Val'laiior

inthell

 

 

5 2
arise from DNA slippage synthesis by DNA polymerases as well (see Tautz and Schlotterer

1994; and references within). Therefore, it seems possible that some of the observed
length variation in the simple repeats found in ITS of Rhagoletis may be attributed to the in
vitra amplification step before cloning and not an inherent variation among the populations
or species studied. Other researchers have also attributed this type of sequence variation to
be the product of slippage events in other ITS sequences (Wesson et al. 1992; Vogler and
DeSalle 1994). This is partially the reason why gaps were excluded in the calculation of
nucleotide substitution rates. However, other length expansions in simple repeats, such as
those in R. pamanella (206-300 in 1'18] and 384-423 in 1182), may reflect inherent
heterogeneity within R pomonella because I do not observe a random distribution of such
large length expansions of such di— and tri-nucleotide motifs (e. g., TA or TAA) among
species of the cingulata group.

The inter-specific variation in the cingulata group was not significantly higher than
the intra-specific polymorphism, indicating that the ITS regions do not display sufficient
variation distinguishing closely-related members of this group. However, the divergence
in the ITS sequences between the cingulata group and R. pomonella were significantly high
(about 5.8% vs. 0.2% intra-specific variation in ITSl for R. pomonella). Therefore, the
ITS regions may provide information on the phylogeny of taxa from different species
groups of the genus Rhagoletis rather than on relationships among members of the
cingulata group.

The length and A-T content of the ITS regions for the R. cingulata species group
and R pomonella are shown in Table 2. On average the ITSI sequences of the R.
cingulata group were about 100 nt longer than the H82 sequences (658 i 3 nt for ITSI vs.
555 i 1 nt for 1182), while in R. pomonella ITSI is about 200 nt longer than IT82. In
addition, ITSl of R pomonella showed considerable length heterogeneity ranging from
653 to 684 nucleotides among three flies, implying the potential usefulness for future
genetic study on host-associated populations of R pamanella species. The ITS of the

 

Drosop}
with the
Imuscrip
rellt‘ated

u: A-T

(hlll'ltOv

 

 

5 3
cingulata species group had a very high A-T content, with 79.9 i 0.2% (average i sample

standard deviation) in ITSI and 82.7 i 0.2% in ITSZ. The high A-T content found for the
Rhagoletis ITS sequences is comparable to that of Drasaphila (approx. 75% in ITSI and
79% in IT 82 from Schlotterer et a1. 1994) and Cicindela beetles (approx. 79% in ITSI
from Volger and DeSalle 1994), but much higher than is found in Aedes mosquitoes
(approx. 42% in ITSI and 47% in ITSZ from Wesson et al. 1992). The A-T content of
Rhagoletis ITS exceeds that found for noncoding DNA in Drasaphila, which is about 60%
(Moriyama and Hartl 1993), but is less than the 96% A-T content found in the 4,601 bp A
+ T region of D. melanogaster mitochondrial DNA (Lewis et al. 1994). The significance of
high A-T content found in the ITS sequences is not clear but in the case of Rhagoletis, it
may have some relation to a recent observation that R. pomonella rDN A clusters are located
at the periphery of fibrillar centers in the nucleolus (Procunier and Smith 1993) or may be
related to the organization of rDNA gene clusters into heterchromatin as suggested for
Drasaphila by Schlotterer et al (1994). High A—T content DNA is known to be associated
with the nuclear matrix (or nuclear scaffold), which may affect the processes of
transcription and replication (van Holde 1989). Drasaphila histone gene clusters, tandemly
repeated about 100-fold, were found to be periodically attached to type 1 nuclear scaffold
via A-T rich sequences lying in the spacers between histone H1 and histone H3
(Mirkovitch et al. 1984). ‘

Molecular Markers and Phylogeny

Thirty-four and 15 informative characters were obtained from 1T 81 and IT 82,
respectively (asterisks, Figures 2 and 3). Forty-four out of the total 49 characters group
the cingulata species group as a separate cluster distinct from R pomonella. The remaining
5 characters (#s 115, 137 and 175 in I'T81;#s 350 and 371 in IT82) were used to
determine relationships among the members of the cingulata species group. Because of the

limited number of informative characters, a phylogenetic analysis on the combined ITSI

 

 

and
two
flies
I'Eifi
two
4A

Fig:

and.

 

5 4
and IT82 data was performed. Two most parsimonious trees were found (Figure 4). The

two trees were basically the same except for the outcome of the two R. asmanthi individual
flies (R01 and R02). Both trees have same length 50, consistency index (CI) 1.00 and
retention index (RI) 1.00. Both a strict and 50% majority-rule consensus analyses of the
two most parsimonious trees gave me a tree with the same topology as the tree in Figure
4A. On the other hand, the semi-strict consensus tree had the same topology as the tree in
Frgure 4B. The trees indicate that R. asmanthi is the most ancestral species in this group
and R cingulata forms a derived clade with R indifi'erens. It should be noted that the trees
were based on a very limited number of characters and all phylogenetic implications from
those trees are tentative and subject to further investigation. In ITSI there are three
positions (#s 115, 137 and 175) potentially useful as molecular markers for distinguishing
different members of the cingulata species group (Table 4). Nucleotide composition at
these three positions were compared with several other Rhagoletis species which are
covered in more details in the following Chapter. Position 115 is composed of the residue
C in the two cherry-infesting species (cingulata and indrfierens, total 7 flies), but T in the
two olive-infesting species (asmanthi and chiananthi, total 4 flies), as well as in 4 other
Rhagoletis species (pamanella, camivara, juniperina and fausta, total 6 flies). At position
# 175, R. chiananthi is the only Rhagoletis species studied which has residue G, while the
other 7 Rhagoletis species presented here, including the remaining three members of the
cingulara group, have C at this position. Position #175, together with position # 137
which is A in chiananthi and G in asmanthi, possibly can be used to distinguish the two
olive-infesting species from one another. These three informative positions in IT 81 lie
within a very well aligned region, adding confidence to their potential use as molecular
markers. In addition the three positions are tightly packed in a relative short fragment (only
60 nts from #115 to 175) and the sequence can be easily obtained in a one-step sequencing
reaction or in an automatic sequencer. Between the two cherry infesting species (R.

cingulata and R. indrfierens) there was also a characteristic nucleotide position (position

 

55

Table 4. Potential Molecular Markers in ITSI for Distinguishing Members of the R.
cingulata Species Group

 

R. cingulata species group Other Rhagoletis species

 

Position cin(5) ind (2) chi (2) osm (2) porn (3) cor (1) jun (1) fau (1)

 

115
137
175

n>n
>0
>-1

new
o-r
>-r
>-r
:-

 

Note.—cin = R cingulata; ind = R. indrjferens; chi = R chiananthi; osm = R. asmanthi;
porn = R pamanella; cor = R. camivara; jun = R. juniperr'na; and fan = R. fausta. The
numbers in brackets represent the number of flies sequenced to determine the type of
nucleotide at the specified position. The nucleotide composition at the equivalent positions

for R camivara, R juniperina and R fausta ITSI sequences were taken from data in
Chapter III.

   

56

 

A
Figure 4. Phylogeny infened from the combined ITSI and 1182 sequences from
Rhagoletis cingulata species group and R. pomonella. Two most parsimonious _
trees were obtained using PAUP. All uninfonnative characters were ignored and
gaps in the alignments were treated as missing data. The number of character-
state changes along each branch are indicated.
B
4

figure 4

 

 

Figure 4

57

 

RC3
RC4
RC1
RC5
RIl
R12
RKl
RK2
R01
R02
RP1
RP3
RP2

RC3
RC4
RC1
RC5
R11
RI2
RKl
RK2
R01
R02
RP1
RP3
RP2

 

 

 

#3711

subsut

Secont

llSlS

 

5 8
#371 in IT82; Figure 3) which distinguishes between them. However, this position needs

further verification because it seems dimorphic in R. chiananthi, although this is transition

substitution rather than transversion.

Secondary Structure Analysis:
ITSI Secondary Structures

Several secondary structural domains in ITSI were determined using FOLDRNA
and presented in Figure 2. The computer generated secondary structure models for the
cingulata species group are generally similar and represented by that of R. asmanthi in
Figure 5A. For comparison. the secondary structure of R. pomonella is also shown
(Figure 5B). The nomenclature used to describe the structural elements of Rhagoletis ITS
structure models was as follows: The frrst two symbols refer to either ITSI (11) or 1182
(12), R stands for Rhagoletis and the last symbol refers to the number of the major stem-
loop structure from 5’ to 3' of the ITS sequences (Figures 2, 3 and 5). In the ITSI two of
the structural elements (IlR4 and IlR5) were found to be highly conserved among all the
studied Rhagoletis species, including 4 non-cingulata group species (Figures 6 and 7).
Furthermore, the IlR4 structural element was associated with several compensatory
changes between the cingulata species group and the other Rhagoletis species studied. The
FOLDRNA assigned the base-pair A354:U380 for the cingulata species group and this
same pair covaries with U354:A380 in R. pamanella and R. carnivara suggesting that a
stem region may indeed exist (Fig. 7A). In addition, two compensatory
deletions/'msertions were found; A350:U383 and U349:G384 base pairs in the cingulata
species group are absent in R. pomonella (Fig. 7A, boxed nucleotides). The non-canonical
U:G pair (found in 11R4 by FOLDRNA) is common among the 16S rRNA of (eu)bacteria
and other RNA helices (Gutell et al 1994; and references within). Although the IlR4
stem-loop structure for R. pamanella is outlined according to FOLDRNA calculations in

Figure 2, it is more likely that the loop region for this species is the same as

59

Ill;
1411’!

 

Figure 5. Typical computer generated secondary-structure models for Rhagoletis ITSI (A

at
and B) and ITS2 (C and D). Panels A and C correspond to R. asmanthi, while
Panels B and D correspond to R pomonella. Several of the domains indicated in
Figs. 2 and 3 are boxed and labeled. Free energy values determined by the
FOLDRNA program in kcal/mol are indicted for each structure.

 

 

 

 

 

 

60

         

v.31 .huuoam
unannoaon .Q

Q

 

 

m.mHHr .hnuoun
1:23-35— .2

 

m 2.5mm

wmuH

23: My» 1

 

 

m.wou: .huuonm
33230 . 2

U

 

 

      

Snag- :Guoum
.z

 

r?”

61

Figure 6. Sequences and alignment of specific structural domains found in the Rhagalea's
ITS sequences. The numbers in brackets show the number of flies sequenced.
The proposed 100p regions for the secondary structural domains are boxed and
labeled (see Figs. 2, 3 and 7). The sequences in Panels A, B and C correspond

A
to domains 11R4, IlR5 and 12R4, respectively, and in two cases (Panels A and
It. cin
B) sequences adjacent to the domains (as defined in Figs. 2, 3 and 7) are shown. 2 :21: I
Asterisks in Panel C correspond to identical nucleotides found at those positions :I :2,
1. car
among 8 Drasaphr'la species (see text). Gaps and identities are denoted by dots : 1run
- an
and dashes, respectively. Nucleotide positions are numbered by following the
B
numbering system of Figs. 2 and 3. Furthermore, to remain consistent with the
numbering system in Figs 2 and 7, the nucleotide positions in Panel A :3 if;
r. em
correspond only to cingulata species group and R pomonella (i.e., gaps are not 1 on
- po-
numbered). R stands for A or G residues. R. cin = R. cingulata, R. ind = R. :3 :3;
l. f
indifierens, R. chi = R. chiananthi, R. osm = R. asmanthi, R. pom = R. an
pomonella, R. cor = R. camivara, R. jun = R. juniperina and R. fau = R. C
fausta.
R. cm
R. m
L cm
1. 0..
2. PC.
1. Co,
1. Jun
1.

fan

 

figure 15

R.
R.
R.
R.
R.
R.
R.
R.

R.
R.

R.
R.
R.
R.
R.

tau

tau

(5)
(2)
(3)
(3)
(3)
(1)
(1)
(1)

(5)
(2)
(3)
(3)
(3)
(1)
(1)
(1)

(4)
(3)
(3)
(3)
(3)
(1)
(1)
(1)

Figure 6

662

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

340 350 360 370 380 390

. . . 11114 hoop. . .
mononucxnuux . oncxun . cccuuuc um: PIUQECCCJ‘llfil - . AUGMUUUGUGAAUU
---.- ....... 3---- . ------ . ----------- .4. ............ . . ...............
---------------- . -‘---- . -------r----b------------ . . ---------------
---------------- .------o--‘---‘ ----r------------oo-"------------
................ ---u--o ‘ - 9-‘-----R-..-..--'---------
---------------- c---u--c--------------------—--rt-.c- . . .-----------
........... 6---..-G-u‘-o a- 9‘--AC--------“--
---------- A’---o00"‘C’oo-----‘"---‘----‘--‘---Goooo00.-----‘-----
‘30 440 ‘50 ‘60 ‘70 480
o o 0 1185 L009 0 o
MUNRUAGUUGURCUCRUURUUURG URUCGRUU CUMGAURRGUUMUUUGU
------------------ G—----- _---—-—-------—--———

250 260 270 280 290

. - :2!“ Loop- - .

.00 0.00.0000.0.00.00.00.0000000 C... .0. 0..

 

Figure

 

 

63

Figure 7. Secondary-structure models for specific domains in the Rhagoletis ITS
sequences. Pr0posed canonical (W atson-Crick) base pairs are connected by
lines, and non-canonical U:G pairs are connected by filled dots. Nucleotide
positions are marked with a tick mark and numbered every 10th position; the first
and last positions for each structural element are numbered. Thick arrows and
symbols with double arrowheads represent nucleotide or nucleotide pair
replacements at those positions. Nucleotides associated with thick arrowheads
denote additions at the specified position among specific Rhagoletis species
(nucleotides not circled) and Drasaphila species (circled nucleotides). Panel A
represents the secondary structure model for domain IlR4 in ITSI for the R
cingulata species group. Nucleotides in bold are invariant among all the
Rhagoletis species surveyed in this study. Several nucleotide or nucleotide pair
replacements (symbols with double arrowheads) or additions (thick arrowhead)
are shown for R pomonella and R carnivara only (see Fig. 6A and text). The
boxed nucleotide pairs are absent among R. pomonella. Panel B represents the
secondary structure model for domain IlR5 in 1151 (see Figs. 5A and 513).
Nucleotides in bold are invariant among all the Rhagoletis species surveyed in
this study. Panel C represents the secondary structure model for a partial region
of domain 12R4 in IT82 (see Figs. 3, 5C and 5D). Nucleotides in bold are
invariant among all the Rhagoletis species surveyed in this study and among 8
other species of Drasaphila (see text) and correspond to those with asterisks in
Figure 6. Circled nucleotides or nucleotide pairs correspond to those found
among specific Drasaphila species (see text). The boxed nucleotide replacement
found for Rhagoletis was absent in the Drasaphila species.

 

64

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“H.012:
:16
D D
41p
onmtt
are
e p
p 4
4

6 5
that for the R. cingulata species group (i.e.UAAUG) because of the above mentioned

compensatory changes. Apart from the three compensatory changes, there was only one C
to T transition (from the cingulata group to R. pomonella) found at position 372 (Figures
6A and 7A), which is a relatively less significant position regarding secondary structure
because of its location at a bulge (i.e., non base-pairing region; Figure 7A). R. cornivora
sequence for the IlR4 region appears to diverge more than that of R. pomonella; for
example, three insertions of C residues after positions 350, 356 and 381 (Figures 6A and
7A) are noted — again, at or near bulges. Actually, the insertion of C residues may even
enhance the stem structure because additional non-canonical base pairs can be potentially
formed (Figure 7A, the C residues with arrowheads). In two other Rhagoletis species
(iuniperina and fausta), the potential loop motif in IlR4 was still preserved, however, the
flanking region, especially those forming the lower part of the stem structure show more
divergence in these two Species (Figure 6A). The above observations in IlR4 are
compatible with the electrophoretic results which indicated that the closest relatives of the
cingulata group are the members of the pomonella group which consists of several sibling
species such as R. pomonella, R. mendax, R. zephyria and R. carnivora (Berlocher and
Bush 1982; Berlocher et a] 1993).

The IlRS structural element, like the IlR4, was also highly conserved among the 8
Rhagoletis species surveyed here (Figures 6B and 7B). The extensive numbers of
canonical base pairs along the pr0posed stem (Fig. 7B) and the common non-canonical
U451:G47 2 pair (determined by FOLDRNA; Gutell et al. 1994) leaves almost no doubt
that the proposed secondary structure indeed exists. Actually the MRS appears even more
constrained than the IlR4 because there is no sequence variation in this region of more than
50 nucleotides among all the 8 Rhagoletis species. except the C- residue was replaced with
G in R. fausra at position 448 (Figure 6B), which does not significantly alter the secondary

structure because of its location at a bulge (Figure 7B).

6 6
IT82 Secondary Structures

Several secondary structural domains in IT82, determined using FOLDRNA, are
shown in Figure 3. The computer generated secondary structure models for R. osmanthi
and R. pomonella are shown in Figures 5C and 5D, respectively. The structural domains
at the 5' end of ITS2, including the potential stem-loop structures 12Rl, 12R2, and 12R4,
appear to be highly conserved between the cingulata species group and R. pomonella
(Figures 3, 5C and 5D). The loop of the 12R4 structural element (AUUGAU) and the
remaining sequence from position 245 to 290 was actually conserved even among several
other Rhagoletis species (Figures 6C and 7C). The FOLDRNA did not pair positions 258
and 277 in I2R4, however, a non-canonical pair of C258:U277 (C:C in fausta) can
possibly exist because Y:Y (i.e., pyrimidine: pyrimidine) non-canonical pairing, although
rare, have been observed in various helices (Gutell et al. 1994). In R pomonella and R.
cornivora the G at position 286 was replaced by A and this change may not affect the
outcome of the secondary structure because this position forms at a bulge in the pr0posed
model (Figures 6C and 7C).

A secondary structural element, described as D3 in Drosophila by Schlotterer et al.
(1994), appears to be equivalent to the 12R4 of Rhagoletis in this study (Figures 6C and
7C). The sequences between positions 215-234 and 250-269 in ITS2 of D. melanogaster
(nucleotide positions numbers are as in Fig. 18 of Schlotterer et a1. 1994) matches almost
perfectly with sequences between positions 245-264 and 271:290 in ITS2 of Rhagoletis
(Figure 6C, letters with asterisks; Figure 7C, nucleotides in bold), except for minor
transition and insertion/deletions. There are also compensatory changes in the 12R4
between Rhagoletis and Drosophila. The computer predicted base-pairing GZ48:C287 in
Rhagoletis was replaced by A:U (218:266) in 6 Drosophila species (sechelia, simulans,
mauritiana, melanogaster, arena and yalacba, position numbers for Drosophila as in Figure

1B of Schlotterer et al. 1994). However, in D. pseudobscura and D. virilis, which

6 7
diverged much earlier from the above mentioned six Drosophila species, the same pairing

position remains G:C as in the Rhagoletis species surveyed here (Figure 7C).

Discussion

Although the primers were designed based on coding sequences of distant relatives
of Rhagoletis, such as Drosophila, the PCR amplification of Rhagoletis rDNA spacers
were successful. The PCR reaction conditions determined in this study yielded the desired
products. The same primers and reaction conditions will be useful guides for future
application of the rDNA spacers in Rhagoletis studies.

The ITS regions are thought of as being universal fast-evolving genomic DNA
regions suitable for resolving closely related species that otherwise show little genetic
divergence (Brown et al. 1972; Furlong and Maden 1983; Tautz et al. 1987; Porter and
Collins 1991). In the case of Rhagoletis, however, the ITS regions seem to display a
limited amount of genetic variation and only a few informative characters were available for
inferring a phylogenetic relationship among the closely related sibling species in the
cingulata group. Some of the observed sequence variations could be attributed to in vitro
slippage synthesis events in simple repeat motifs. Such Variations, however, generate
phylogenetically uninfonnative (autapomorphic and homoplastic) characters, which do not
affect the results of phylogenetic analysis. Other observed slippage mutations in simple
repeats may have an in viva basis, created probably in regions of low selective (structural
and/or functional) constraints. This interpretation has been suggested by other researchers
as well (T autz and Schlotterer 1994; and references within). A few key nucleotide
positions, however, have been described in ITSI that could be useful to distinguish some
of the morphologically indistinguishable species in this group. By designing primers from
the conserved regions of I'I‘Sl adjacent to positions 115 and 137, and taking advantage of
current PCR and sequencing technology, one could in a short time differentiate between the

cherry and olive infestin g species of insects. Moreover, the considerable divergence

6 8
discovered between the cingulara group and R pomonella in this study (Figures 2 and 3;

Table III) indicates that the ITS sequences may provide valuable information in the
phylogenetic study of taxa from different species groups of the genus Rhagoletis.
Although a secondary-structure model of minimum free energy can be produced
with the FOLDRNA, the real secondary structure may, on the basis of base-pairing and
other constraints, have a different free energy value (Zucker and Stiegler 1981; Gutell
1993; Gutell et al. 1994; and references within). For the past decade, a comparative
approach based on the concept of positional covariance has been applied to elucidate the
Escherichia coli 168 and 238 rRNA higher-order structure and identify functionally
important elements in these molecular structures (for review see Gutell et al. 1994). In this
study, using the FOLDRNA program and some principles of comparative analysis based
on compensatory nucleotide changes, a number of constrained secondary structural
elements in ITS of several Rhagoletis species have been described. One of them, namely
12R4, is highly conserved between Rhagoletis and Drosophila indicating the possible
functional importance of this stem-loop structure given the estimated divergence time
between the families Tephritidae and Drosophilidae which ranges from 77 MYR (million
years ago; Kwiatowski et al. 1994) to 90 MYR (Collier and MacIntyre 1977) and even to
123 MYR (Beverley and Wilson 1984). The Drosophila radiation is estimated to be
between 40 to 62 MYA (Beverley and Wilson 1984; Spicer 1988; Kwiatowski et al. 1994),
at least 15 MYR after the divergence of Tephritidae and DmSOphilidae. Phylogenetic
studies of Drosophila based on the ITS sequence comparisons (Schlotterer et al. 1994)
implies that D. psuedoobscura and D. virilis , both with G:C at a specific pairing position
in 12R4, diverged much earlier than the 6 Drosophila species with A:U at the same paring
position. This evolutionary divergence suggests that the G:C pair in the secondary
structural element of IT82 (2481287 in Figure 7C) among the 8 known Rhagoletis species
and the two older Drosophila species is a primitive condition, which later may have mutated

to A:U among the 6 Drosophila species sometime during their common evolutionary

6 9
history. However, convergent evolution by independent substitution events (A:U to G:C)

having occurred in Drosophila and Rhagoletis can also be envisioned and can not be
completely ruled out.

Among the ITSI structural elements investigated, the proposed IlR5 stem-loop
structure was highly conserved among all the 8 Rhagoletis species presented in Figures 6B
and 7B, while another such element, IlR4, demonstrated high conservation only among
the cingulata species group and diverges considerably beyond this Species group (Figures
6A and 7 A). This observation suggests the existence of differential levels of constraint
throughout the length of ITS l, presumably due to its functional or higher order structural
role(s). Furthermore, the variation in the IlR4 suggests that the cingulata species group
may be more closely related to the members in the pomonella group rather than to R
juniper-inn and R. fausta, as indicated by electrophoretic data (Berlocher and Bush 1982;
Berlocher et a1 1993).

To obtain further information on the phylogeny of the cingulata group, a faster
evolving DNA may be more satisfactory, such as those coding for alcohol dehydrogenase
which show considerable divergence among sibling species of Drosophila (Bodmer and
Ashbumer 1984) and other non-coding rDNA spacers, i.e., external transcribed spacers
and intergenic spacers (see chapter IV and V)

In conclusion, I have presented the complete ITS sequences of the 4 members of
the cingulata species group as well as that of R. pomonella. The Rhagoletis ITS sequences
are highly A-T rich. I found low levels of interspecific ITS variation in the cingulata
species group, implying that ITS sequences are of limited application in phylogenetic
analysis of host—associated populations and/or closely related sibling species. A few
molecular markers have been described and can be potentially useful for distinguishing the
olive infesting species (R. osmanthi and R. chionanthi) from the two cherry flies and for
differentiating between the two olive infesting flies. The high sequence divergence found

between the cingulata group and R. pomonella indicates that the ITS regions can provide

7 0
better resolution for studying phylogenetic relationship of taxa from different Rhagoletis

species groups. The overall computer generated secondary structure model of the ITS was
presented for the cingulata species group and R. pomonella. Several highly conserved
secondary structural elements were determined by the FOLDRNA and comparative
analysis. One such element (I2R4) seems to be conserved even among two distant
families, Tephritidae and Dros0philidae, which may have diverged in the mid to late
Cretaceous period (65 to 130 million years ago). The conserved ITS secondary structural
elements should be a valuable guide for accurate alignment of taxa from different species
groups of Rhagoletis. The mode of ITS evolutionary divergence should also be useful in
future investigations of the structure, function and processing of precursor rRNA.
Furthermore, future studies on the ITS of other Rhagoletis species will allow us to
elucidate the degree of functional and structural constraints on the ITS sequences in
Rhagoletis.

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71

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75

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CHAPTER III
PHYLOGENETIC IMPLICATIONS FROM ANALYSIS OF INTERNAL
TRANSCRIBED SPACER REGIONS IN THE rDNA
OF TEN RHAGOLET IS SPECIES

Introduction

Nearctic Rhagoletis were comprehensively reviewed by Bush (1966), with 21
species segregated into seven species groups (pomonella, cingulata, tabellaria, suavis,
ribicola, striatella and alter-tiara). Bush’s revision was based mainly on morphology and
karyotype analysis. Since then attention has been given to the mode of host race formation
and sympatric speciation in one of the Rhagoletis species groups (i.e. the pomonella
group). Currently, the only phylogenetic analyses of this group are those based on
allozyme studies (Berlocher and Bush 1982; Berlocher et al 1993), and mtDNA COII
(Smith and Bush, unpublished data) and morphological characters (Jenkins, in
preparation). The results from the allozyme studies have some congruence with the
conventional classification of Bush (1966), such as the conservation of the suavis,
cingulata, and to some extent, pomonella species groups. However, there are a few
discrepancies between the previous studies .— even among the biochemical and molecular
studies (i.e., the allozyme and mitochondrial DNA studies). To date, the placement of
several species in this genus, such as R. juniperina, cornivora, striatella, fausta and
basiola, is still uncertain. In addition, the phylogenetic relationships among different
species groups are yet to be completely resolved. For example, the close relatives of the

cingulata group in North America may either be the pomonella group, according to

76

7 7
electrophoretic studies (Berlocher and Bush 1982; Berlocher et al. 1993), or the suavis

group, according to mtDNA COII data (Smith and Bush, unpublished data).

Although the Rhagoletis species of the Nearctic region have been well documented
and segregated into a number of species groups, the phylogeny of Rhagoletis in the
Nearctic region is not well established as there are still several unanswered questions
regarding placement of certain species and relationships among the existing species groups.
Furthermore, monophyly of the genus has not been demonstrated and its relationship to the
closely related genus Carpomyina is poorly understood (Norrbom 1989). Since the genus
Rhagoletis contains many economically important pest species and some of Rhagoletis
species groups are believed to speciate sympatrically through host shifting, a reliable
phylogenetic framework is not only required for understanding the evolutionary history of
this genus and testing evolutionary theories such as speciation, but also required for
providing accurate information for appropriate control of those pest species, some of which
show little or no morphological differences. Therefore, in order to get further insight into
the phylogeny of this group, I have sequenced the internal transcribed spacers (IT 81 and
ITS2) of the nuclear rDNA of 7 additional Rhagoletis species.

The taxa covered in this ITS sequence analysis are the following nine species: R.
cingulata and indifierens represent the cingidata group; R. pomonella and comivora
conventionally belong to the pomonella group, but comivora‘s placement in this group is
questionable and needs verification; R. completa represents the well-delimited suavis
group; R. electromorpha and juniperina are placed in the tabellaria group with juniperina ’3
relationship with the rest of the tabellaria group yet to be resolved; R. fausta is unplaced
(Bush, 1966) or its placement in previous independent studies (Berlocher and Bush 1982;
Smith and Bush, unpublished data) does not agree; and R. basiola which appears to
possess some of the most primitive morphological characters, is used as outgroup in this

phylogenetic analysis.

7 8
The results of this study show both congruence and disagreement with earlier

studies. In addition, there are some points which are not recovered in previous analyses.
The ITS sequence analyses strongly indicate that l) R. cornivora belongs to the R
pomonella group, 2) R. juniperina is removed from the R. tabellaria group and may be
more closely related to the R. pomonella group, 3) a possible relationship between R.
fausta and the R. tabellaria group is indicated, 4) the close relatives of the R. cingulata
species group are more likely the members of the R. suavis group, and 5) R. basiola is
indeed quite distinct from the other Rhagoletis species.

In addition to the above phylogenetic implications, the usefulness of the ITS
regions in phylogenetic studies and its feasibility in this particular study are discussed.
Some sequence features characteristic of Rhagoletis rDNA ITS are compared with
published ITS information from other insects.

Materials and Methods

Biological Material

All species were collected during 1988-90 from various locations and host plants in
the United States of America (Table 5). larvae emerged from field infested fruit and were
allowed to pupate in fine vermiculite. Pupae were sifted from the vermiculite and stored at
4° C for at least 5 months. Pupae were then removed from the cold and held at 22° C under
a 15 hr light and 9 hr dark cycle to terminate diapause. Within 2-7 days after emergence
most adult flies were frozen at -70° C for subsequent genomic DNA isolation. Specimens

from each collection were pinned for species identification.

DNA Isolation and Amplification

Total genomic DNA was isolated from individual male flies as described by
Procunier and Smith (1993). The ITSI region was amplified using primer 1975F
STAACAAGGT'ITCCGTAGGT‘G (matching the 3' end of 18S) and primer 35R

79

Table 5. Collection Sites and Host Plants of the Rhagoletis Species Used

 

 

Species Location Host Plant (Common Name)

1. R. cingulara Hart, MI Prunus cerasus (sour cherry)

2. R. indifl'erens Pullman, WA Prunus cerasus (sour cherry)

3. R. completa Grand Junction, CO Juglans nigra (black walnut)

4. R. juniperina Dixon Springs, IL Juniperus virginiana (E. red cedar)
5. R. fausta Fish Creek, WI Prunus cerasus (sour cherry)

6. R. electromorpha Meridian Twshp., MI Cornusfoemina

7. R. pomonella E. Lansing, MI Crataegus mollis (hawthorn)

8. R. cornivora E. Lansing, MI Cornus amomum (dogwood berries)
9. R. basiola Montrose, CO Rosa spp.

 

8 0
5'AGCTRGCTGCGTI‘CTTCATCGA (matching the 5' end of 5.88). The IT 82 region

was amplified using primer 108F 5'GAACATCGACHHKTYGAACGCA (matching the 3'
end of 5.88) and primer 52R S'GTTAGTITCTI'ITCCTCCSCT (matching the 5' end of
288). Amplification of the ITSI and IT82 regions as a combined region on one DNA
fragment was performed for R basiola, R. completa, R. fausta, R. juniperina, R.
cingulata, and R. indifi‘erens, using the 1975F and 52R primers. The IT 81 and IT82
regions from R. cornivora and R pomonella were amplified as separate pieces but from the
same individual fly. From R. electromorpha, I was able to amplify only the ITS2 region.
Amplification conditions were as described in Chapter II. Amplified DNA was subjected to
electrophoresis on a 1.0% agarose gel and visualized with ethidium bromide. Bands
containing the DNA were excised from the gel and the DNA purified using the Prep-A-

Gene DNA purification matrix (Bio-Rad), according to the manufacturer's instructions.

Cloning and Sequencing

The TA Cloning kit (Invitrogen) was used in a one-step cloning strategy for the
direct insertion of the purified PCR products (see earlier sections) into a plasmid vector,
followed by transformation into competent cells. Plasmid vector and competent cells were
supplied by the manufacturer. Plasmid DNA was purified from individual clones using the
Magic-Prep DNA purification kit (Promega), following the manufacturer's instructions.
DNA sequencing was performed according to the chain-termination method of Sanger et al.
(1977), and using the Sequenase Version 2.0 DNA sequencing kit (USB) and 3SS-dATP
(Amersham). The same primers used in the amplification reactions were employed to
determine the DNA sequence in both directions. Once a certain stretch of DNA was
sequenced additional primers were employed to complete the sequencing of the whole
specific region from the different species: Primers 35R-GB27
5'ACC(CT')AAACAT'ITI‘CAAGT(CT)GCG (for all the species) and 1975F-GB28
S'AAATAAGCCAAACAAAGGAG (for basiola alone) were used for the ITSI regions;

8 l
and primer 108F-GB25 5'A(AT)(AG)(AG)AATC(AT)(CT)AGTATTCCC was used for

the ITS2 regions for all the species. The ITS sequences of R. cingulata, R. indifi'erens and
R. pomonella were taken from an extensive study of intraspecific polymorphism’s in the

ITS region (RC4, R11 and RP1 respectively in Chapter 1).

DNA Sequence and Phylogenetic Analysis

The PILEUP program in the GCG package of the University of Wisconsin
Genetics Computer Group (UWGCG package, version 7.0) was used to align the ITS
sequences. Alignments were done first with the computer (gap weight = 3.00 and gap
length weight = 0.20). They were then manually adjusted taking into account, in some
cases, secondary structural constraints as determined using the FOLDRNA program in
GCG and/or by a comparative analysis approach of looking for compensatory substitutions
between taxa (Chapter D.

Because several secondary structural and/or potential functional elements may
constrain Rhagoletis ITSl and IT82 sequence evolution differently in different parts of
these molecules, two different approaches were used for infening a Rhagoletis phylogeny
for the nine species surveyed in this study. One approach is based on the analysis of
pairwise-distance measures and another on the analysis of discrete molecular characters.

Phylogenetic analyses by parsimony methods were carried out using the programs
in PAUP version 3.1.1 (Swofford 1993). The exhaustive search option was used to
generate the most parsimonious tree(s). In addition, throughout these analyses, all
uninfonnative characters were ignored and gaps were treated as missing data. Bootstrap
50% majority-rule consensus trees using the branch and bound option in PAUP were
constructed from 500 replicates (using seed number 1). The percentage of times that a
group of taxa appeared as a clade in the bootstrapped parsimony trees, the bootstrap

confidence limits (BCL), were indicated at the internal nodes of the trees.

8 2
Phylogenetic analyses by the distance matrix methods were performed using

programs in the Molecular Evolutionary Genetics Analysis (MEGA) version 1.01.
Estimates of the average percent nucleotide substitutions (number of nucleotide
substitutions per 100 nucleotide sites) between each pair of taxa were determined using the
method of Kirnura (1980) and the percent sampling standard deviations (SD) calculated.
All gap and missing information sites were removed in the pairwise comparisons. The
Kirnura matrix of distances was used to produce phenograms by the neighbor-joining
method (Saitou and Nei 1987) using MEGA. As with the parsimony approach mentioned
above, bootstrapped distance trees were generated from 100 replications (with random seed
number 1), and the BCL in each tree were indicated. With the latter bootstrap analyses,
gaps were removed only in the pairwise comparisons. In calculating transition to
transversion ratios using MEGA all gap sites were removed from the subset data.
Calculations were performed by first employing the Kimura two-parameter distance option
in MEGA.

Results

ITSI Sequence Analysis

The ITSI sequences of 7 Rhagoletis species are presented in Figure 8A At least
three clones for each of these species were obtained but for some species only one clone
was sequenced due to cost and time constraints. The remaining clones were preserved at
-70° C for future examination of within-individual variation. Similarly, three ITSI clones
for both R. basiola and R. striatella were obtained and one clone for each species was
sequenced. However, their sequences are too diverged to align with the other Rhagoletis
species (Figure 8B). Because of the inability to PCR amplify the IT S1 region of R.
electromorpha, the IT S1 sequence for this species was not included in this study.

The length of the ITSI regions studied here ranged from 625 to 922 hp, with
basiola about 250bp longer than the other surveyed species (Table 6). The ITSI of

83

Figure 8. Rhagoletis IT 81 sequences and alignment. A) The complete ITSI sequences of

 

 

the species surveyed except for the longer R. basiola sequences (Table 6).
Phylogenetically informative characters are denoted by asterisks. Identities are
denoted by dashes, and gaps are denoted by dots. B) The complete R. basiola
sequence compared with the partial sequence of R. sm'atella. The alignment was
done using GAP in GCG with the gap weight = 1.00 and gap weight length =
0.10. The complete length of R. striatella remains to be determined. bas = R.

basiola, str = R. striatella, and ND = not determined.

 

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str

Figure 8B

51

48

101

70

151

84

201

113

251

153

301

200

348

246

377

296

412

451

495

542

582

87

TTGTGTTCCTATCCGTAAAAAAAAATATATAATATAATACAATATTATAT

IIII III IIIIIII IIIII | | IIIIIIIII III IIIII
TTGTATTCTTATCCGTGAAAAAGCA.AGGAAATATAATA.TATA.TATAT

TACATGTTTGAACAAAAAAATAAAAATTATTCTGGTTCACGTCCCGCCAG
II II I IIIIIIIII IIIII
TATATAT....ACAAAAAAAAAAAAA ........................

AGTAATTATATGCTTGTCTTTAAGATTAAGCCACGCCATATGTAATGCCT

II II I II III
..I .................. AAAAT ....... GGACA....AAAT....

CATGACAATACATCTAACGTATTGTGGATTTGCATACATTGGAAAATACC
III I III II III II I III IIII II
..TGA....AAATCGAA.GTA ...... ATCT..ATA ..... TAAAACAC.

AATAAAAAAAGAACAAAAAACAAAAATTTTGGTTTTGTACTTTTCATTCA
III IIIIII II I IIIII IIIIII IIIIIII III
.ATATAAAAAG ......... CATATATTTTCTTTTTGTTCTTTTCACTCA

ATTTCTGTAAAAACATTTGTTTTTGACATAACTTTGAGTGTTTTTCTTTT

I I III I III III I IIIIII IIIIIIIIIIIII III
AATCTTGT...TATATTGATTTATAACATAATATTGAGTGTTTTTCCTTT
TTTCTTT...ACATACATTGAAAAAGACAAAATCAAATGATATGTTTGAA
II: III I II IIII IIIII I II II ||II| |I|

'I'I'NG'I'I'I'I‘CGAAA'ICCA'I‘I‘AAAAAA . . . C A'I‘GC CATGTG . TA'I‘G‘I‘GTGAC
AATAA....GCCAA ....... aCAAAGGAGAAA.TATC ......... GTT
I|I|| II | I III IIIII I II III
AATAAATTTGCATATATCTTTAAGAAGAAGAAACTTTCTTCTTGTGTGTT

C....TT....ACATAAAATACAATATA.TATATGTATT..AGAGA....

I II I III I III III I III
CCTCTTTG (ND) AAATAGGAAATGCATTAAAAAGATGCA

........ ACGAAAATTAT.TTCTCACAGTACTAATTGTAATTAGTA..C

III : III I I I II : III IIIII I:I
GGGTGCTCGCGAGNGCTATATGCAAAAAGGNATAAAAATAATTTGNATTT

AA..GAAGTGTGCATGC.AAAAATTATAATTGTAAACAA...AATGAATG
II IIII IIII II I IIII II III II I III I
AATTGAAGGGTGCCGGCTGAGCCCTATA..TGCAAAAAACCTACAGAAGG

AAACGAATGTATATTTTTGTA...TAGCCACACTATTATGAAATTTTNAA

I I III I IIII II II I IIII :III II :II
ATAACAATTATTGTTTTCCTATCCTAAGAAAACTAAGGCRAAAATTCGAA

T...ATGTGGAAAATAT.CA ...... TATTTTCGCACATTATGCTTGTCT
I II I IIII II II I I II I IIIII
TTGCAT.TAAAAAAAATGCAGGGTGCTGGCTGAGCCCTATATGC ......

CAAAGATTAAGCCACG.AATATGTAATGCTTCATCACAATATATGTAAAT

IIII I II I I IIIIII III I II II III II II
CAAA....AGGCAAAGAAATATGCAATAAATtAT....AT.TATTTATAT

100

69

150

83

200

112

250

152

300

199

347

245

376

295

411

450

ND

494

541

581

630

ND

88

631 GTATTGTGAATTTGCATACG.TTGAAAAAAC ....... AACCTTTAAACA 672

I II I III I I I III II IIII IIIIIII
ND GCATATTTAATAACAAAAAGATTCTGTCCACTTTTGTTAACCCTTAAACA ND

673 TATATAGTTGTACTCAATTATTTAGTAGCGATACTAAATGCTAAGTTGAT 722

IIIIIIIIIIIIIII IIIIIIIII I I IIIIIII IIIIII II
ND TATATAGTTGTACTC.ATTATTTAGATTTGTTTCTAAATGATAAGTTAAT ND

723 TTCTTTACATTAACGTGTGTT .................. TTCTTTTTTTT 754
I IIIIIIIIIIIIIIII II I II I | I
ND TAGTTTACATTAACGTCTATTTATTAAAANCGGTGTACATACTATGTAAT ND

755 AAA.AAGAAAA ......... TACTGTTTTTGTAGAC.TAAGCCATACGCA 793
III IIIIIII IIIIIIIIIIIII II IIII ||I IIII
ND AAAGAAGAAAAACTAATGATTACTGTTTTTGTAAACTTAAQACAtGCGCA ND

794 AC..TTCAAAATGTTTGGGTTTAAAATaATAATTTATTGAAGGAATTTTA 841

II IIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIII II
ND ACTTTTGAAAATGTTTGGGTTTWAAATTATAATTTATTGAAAGA ...... ND

842 TTTATATTACAAATATTTCTAATTTACTCTTTCAATAAATAANAA....A 887
I II I IIIIIIIIIIIII:|I I
ND .......GAGAA...............TTTTTCAATAAATAAAAATCAGA ND

888 AATGA.CATACAAATTAA ....... AAAAAAAAAATTATCA 920

I III IIIIII | || IIIII IIIII IIII
ND AGTGACCATACATGTAAAGAGATGTAAAAAgAAAATGATCA ND

Figure 8B (cont’d)

89

Table 6. A-T Content and Length of the Rhagoletis ITS Sequences Analyzed

 

 

 

 

ITSI IT82

Species A-T Content length A-T Content Length

(%) (nt) (%) (nt)
1. R. cingulata 79.8 662 82.7 557
2. R. indifi‘erens 79.7 660 82.7 558
3. R. completa 80.4 634 81.2 541
4. R. juniperina 81.0 684 82.7 557
5. R. fausta 80.0 625 81.7 527
6. R. electromorpha ND ND 81.0 501
7. R. pomonella 80.5 686 82.0 478
8. R. cornivora 80.7 653 81.2 482
9. R. basiola 75.3 922 - 76.6 642

‘_

Note.— ND = Not Determined

9 O
R. fausta had two major insertions of approximately 30-40 bp long (positions 15-53 and

747-780 in Figure 8A). The first insertion had long stretches of A and T residues while the
second insertion had long stretches of T immediately followed by (AT)5 and then by
(ACAT)5 repeats. R fausta also had one major deletion of approximately 80 bp long,
shared almost completely with R comivora (positions 263-335 in Figure 8A) and partially
with R. pomonella (positions 300 -333 in Figure 8A). The sequence differences of the
ITS l were generally distributed evenly from 5' to 3’. However, a number of relatively
conserved regions were found, such as positions 500-563. The A-T content of the ITS]
regions were high (about 80%; Table 6). The high AT concentration was partially
contributed by long stretches of A and T such as regions around positions 140 (A)7-10, 224
(T)9,388 (T)12 and 857 (A)lo. Simple direct repeats involving A and T residues were also
common in the ITSI. For instance, the AAT motif was repeated 5-6 times around position
340 in R. pomonella and R. comivora, and AT motif occurred approximately 14 times

Within positions 620-650 of R. comivora. A number of other, less frequent motifs were

also detected (e.g., TGTA and CATA).

The average number of nucleotide substitrrtions per 100 nucleotide sites for
Pairwise comparisons and transition to transversion ratios were calculated based on the
alignment in Figure 8 using three different statistical approaches (i.e., the Jukes and Cantor
1969, Kimura 1980 and Tamura 1992 approaches). Because the results from these three
Statistical approaches were not significantly different, only the values from the Kimura
(1980) approach are shown in Tables 7 and 8. In comparison with the intraspecific
nucleotide substitution rates obtained in Chapter II (0.00 to 0.35 average percent nucleotide
Sllbstitutions), the interspecific nucleotide substitutions are substantially higher (2.99 to
7-88) except for the two sibling species from the cingulara group (i.e., R. cingulata and R.
i"difi'erens). Because generally only one clone was sequenced for each surveyed species,
the level of intraspecific variation in rrsr could not be determined. However, the

int—l‘aspecific variation of R. cingulara, calculated from 10 pairs, was 0.18 :1: 0.07 average

9 1
percent nucleotide substitutions (see Chapter II) and the values for several other Rhagoletis

species, including 3 R. pomonella individuals, were similarly low (see Chapter II).
Therefore, it is reasonable to assume that the intraspecific variations for those unknown
Rhagoletis species are also low and negligible when comparing interspecific substitution
rates for phylogenetic analysis. The transition to transversion ratios for the pairwise
sequence comparisons are presented in Table 8. These ratios range from 0.94 to 3.04,
with transitions alone contributing 100% of the substitutions observed between R.
cingulata and R. indifierens. In principle, a transition to transversion ratio of 0.5 is the
expected rate, if there were no bias at all, because there are twice as many ways that a
transversion (AtoT,AtoC,GtoTanthoC)can occurthanatransition (AtoGandT
to C). Some of the transition to transversion ratios found in this study are relatively higher

than those in Drosophila (Schloterer et aL 1994).

TI‘S 2 Sequence Analysis
The complete IT82 sequences of 9 Rhagoletis species are presented in Figure 9.
Two additional taxas (R. electromorpha and R. basiola) have been included in comparison
With the ITSI sequence analysis in the earlier section (Figure 8). The length of the ITS2
Varies from 478 to 642 bp (Table 6). Among the aligned sequences R. basiola was 84-164
bp longer than the other surveyed species, in part due to the long insertion/deletion found
[36an positions 487 to 572. Generally, the IT82 was about 100-300bp shorter than the
reSpective I'TSI sequences. Like in ITSI, the ITS2 region had a high A-T content (81.3 i
1 -9% A-T content; Table 6). Long stretches (3 to 11 bp) of A or T residues were
Predominant in the ITS2 sequences; for example, the (A)4 sequence occurred at least six
tfines in R. cingulara IT82. Long stretches of TA simple repeats were also common, such
as the (TA)9 around position 380 in R. basiola. The 5' half of the ITS2 was relatively
cotlserved among the surveyed Rhagoletis species. Actually one region (249-295) was

found to be highly conserved even between Rhagoletis and Drosophila, suggesting this

92

Table 7. Average Percent Nucleotide Substitutions (Nucleotide Substitutions per 100
Nucleotide Sites) for the Rhagoletis ITSI and ITSZ Sequence Pairwise Comparisons

 

Average Percent Nucleotide Substitution, Calculated by the Kimura
Method (1980), for IT 81 (above Diagonal) and partial IT S2 (below the
Diagonal) for Pairwise Analysis (gaps excluded only in pairwise
comparisons)

 

I

1. cin - 0.30 5.22 5.12 7.35 ND 6.76 6.35 ND
2. ind 0.31 - 5.22 4.79 7.37 ND 6.76 6.55 ND
3. com 2.24 1.91 - 6.14 7.11 ND 7.09 6.79 ND
4- jun 1.59 1.27 1.27 - 6.67 ND 5.51 5.91 ND
5. fau 5.21 4.88 4.89 3.88 - ND 7.88 7.79 ND
6. ele 3.22 2.89 2.90 2.23 3.89 - ND ND ND
7- pom 3.55 3.21 3.20 2.24 5.89 3.88 - 2.99 ND
3. cor 3.23 2.90 2.89 2.24 5.57 3.88 1.59 - ND
9. has 8.03 7.64 7.62 6.81 9.20 8.00 8.34 9.13 -

I

N0te.— cin = R. cingulata; ind = R. indifi'erens; com = R. completa; jun = R. juniperina;
fau = R. fausta; ele = R. electromorpha; pom = R. pomonella; cor = R. cornivora; and
has = R. basiola. ND = not determined.

93

Table 8. Transition to Transversion Ratios for the Rhagoletis ITSI and partial IT82
Sequences

 

Transition/Transversion Ratios, Calculated by the Kimura Method (1980),
for ITSI (above Diagonal) and partial IT 82 (below the Diagonal) Where

 

 

All Gaps Are Excluded

1 2 3 4 5 6 7 8 9
1. cin - NS 2.76 1.48 1.20 ND 2.75 1.98 ND
2. ind NS - 2.76 1.36 1.29 ND 2.88 2.09 ND
3. com 1.35 1.01 - 0.94 0.67 ND 1.74 1.25 ND
4. jun 4.06 3.03 0.00 - 0.47 ND 1.31 0.94 ND
5. fau 1.02 0.87 0.29 0.37 - ND 1.25 0.96 ND
6. ele 1.53 1.27 0.28 0.40 0.57 — ND ND ND
7. porn 3.59 3.06 0.60 1.01 0.67 0.84 - 3.04 ND
8. cor 4.11 3.59 0.81 1.35 0.79 1.02 NS - ND
9. bas 1.51 1.38 0.76 0.92 0.80 0.70 1.13 1.23 -

I

N0te.— The numbers corresponding to the different taxa are as in Tables 5-7. NS =
not shown; corresponds to cases where transitions = 100% and transversions = 0%.
= not determined.

9 4
region may be functionally important (for details see Chapter II). In contrast, the 3' half of

the IT82 region showed considerable sequence divergence making this region rather

difficult to align unambiguously, especially the region beyond about position 340.

The average percent nucleotide substitutions and transition to transversion ratios
were calculated according to the alignment in Figure 9 and presented in Table 7 and Table
8, respectively. Since the region beyond the position 340 was difficult to align
unambiguously this region was excluded in the above calculations. Except for the
cingulata-inditferens pair, the interspecific percent nucleotide substitution rates (1.27-9.20)
were significantly higher than the intraspecific variations observed for R. cingulata, R.
indifferens and R. pomonella (00.24 percent nucleotide substitutions; see chapter 11). The
transition to transversion ratios varied from 0.00 to 4.11, with transition alone contributing
100% of the substitutions in the cingulata-indijj‘erens and pomonella-comivora pairs of
taxa, The ratios in the ITSZ were lower, in most-cases, than those measured for ITSI; the

ratios for most pairs among completa, juniperina, fausta and electromorpha were lower

than 0.5.

Phylogenetic Analyses

A total of 32 informative characters were obtained from the ITSI (asterisks in
Figure 8A) and 19 from the IT S2 (asterisks and crosses in Figure 9) aligned sequence data.
Only one most parsimonious tree for the IT 81 sequence data was found with R. fausta
taken as the outgroup (Figure 10A). The tree is of length 40 with consistency index (CI)
0.925 and retention index (RI) 0.919. Bootstrap analysis using PAUP gave 100% support
fOI‘ the branches of R. cingulata and R. indifierens, and R. pomonella and R. comivora;
88% support for the clade of R. cingulata-R.indijferens and R. completa; and 61% support
f0? the clade of R. pomonella-R comivora and R. juniperina (Figure IOB). The same tree
Was obtained using the NJ distance approach in MEGA. The bootstrap showed, again.

1 00% support for the R. cingulata and R. indifi'erens branch, 98% support for the

95

 

Figure 9. Rhagoletis IT82 sequences and alignment. A) The complete IT82 sequences of
the species surveyed except for R. striatella. Phylogenetically informative
characters are denoted by asterisks and crosses. Notations same as in legend of
Figure 8. B) The R. basiola sequence compared with the sequence of R.

striatella. The alignment was performed as in Figure 8B. Notations are as in
Figure 8.

 

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II III I IIII IIIIIIIIII II II IIIII IIII
TCAAAAAAAAAAAAAAGAATCATAGTATTCCCGAAAAACGTCAACGTTAT

TTGAAT ..... TAACATTTAAATAAATATATATGTGAAGAGGAATGTCTA

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TTGCATTTATATACAATATATATATGTATATATGAAAAGAGGAATGTCTA

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IIIIIIII II II IIIIIIIIIIIIIIIIIIII III IIIIII
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R. pomonella and R. comivora branch, 57% support for the clade of R. cingulata-

R. indifl'erens and R. completa and 54% support for the clade of R. pomonella-R. comivora
and R. juniperina (Figure 10C).

For the ITSZ, since the 3' half was so highly divergent and difficult to align with
confidence, I chose only the informative characters between positions 1 to 340 (11 out of
the total 19 PAUP informative characters; asterisks in Figure 9) in the search of a most
parsimonious tree rooted—with R. basiola as the outgroup. An exhaustive search gave only
one most parsimonious tree with length 13, CI = 0.923 and R1 = 0.909 (Figure 11A). Of
the 8 excluded character sites (crosses in Figure 9), three of them grouped R. cingulata and
R. indifi’erens together, one grouped R. pomonella with R. comivora, and one grouped R.
cingulata, R. indifl’erens and R. completa together. However, almost all of them had
significant number of gaps and their reliability was dependent on the sequence alignment.
Bootstrap analysis using PAUP of the ITSZ sequence data (up to position 336) showed
97% support for the R. cingulata and R. indz'fierens branch, 100% support for the R.
pomonella and R. comivora branch, and 73% support for the R. fausta and R.
electromarpha branch (Figure 11B); placements of R. juniperina and R. completa were
unresolved. A similar bootstrap analysis in PAUP but using instead the whole IT82
sequence showed 100% support for the R. cingulata and R. indifferens branch, 100%
support for the R. pomonella and R. comivora branch, and 61% support for the R. fausta
and R. electromarpha branch (Figure 11C). Again, R completa and R. juniperina
remained unresolved. A similar tree, but with higher resolution, was obtained from a NJ
approach using IT82 sequences up to position 340 (Figure 11D). The clade of R. fausta
and R. electromorpha became a sister group to the other taxa which had R. juniperina
forming a tritomy with two other clades. The first clade included an earlier branched R.
completa and the derived cingulata-indifl’erens pair, while the second had R. pomonella and
R. camivora. A bootstrap test of the NJ tree showed 95% support for the R. cingulata and

R. indijferens branch, 96% Support for the R. pomonella and R. comivora branch, and

101

Figure 10. Phylogenetic analyses of Rhagoletis using the ITSI sequence alignment. a)
This single most parsimonious tree (consistency index (CI) = 0.925 and

retention index (RI) = 0.919) was obtained for the ITS] sequence alignment

 

(Figure 8A) using the exhaustive search option in PAUP. All uninfonnative
characters were ignored and gaps in the alignment were treated as missing data.
The number of character-state changes along each branch are indicated. b) 50%
majority-rule bootstrap consensus tree (using the branch and bound option in
PAUP) after 100 replications with random seed number set at 1. All
uninfonnative characters and gaps were treated as in part a. The bootstrap
confidence limits (BCLs) are indicated. c) Bootstrap distance tree analysis on
the ITSI sequence alignment (Figure 8A) using the Kimura (1980) matrix of
distances and the neighbor-joining (NJ ; Saitou and Nei 1987 ) tree generating
options in MEGA (see Material and Methods) with 100 replications and random
seed number set at l. BCls are indicated. The scale corresponds to 0.06

average percent nucleotide substitutions.

102

 

0 s
12 1 c1n
3 —‘_—_ ind
a com

 

 

 

 

 

 

 

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fau

 

 

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um

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l (BCHD
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Scale: each - is about equal to 0.06 percent nucleotide substitutions

Figure 10

103

Figure 11. Phylogenetic analyses of Rhagoletis using the l'I‘SZ sequence alignment. a)
This single most parsimonious tree (CI = 0.923 and R1 = 0.909) was obtained
for the IT82 sequence alignment (Figure 9A) using the exhaustive search option
in PAUP. All characters beyond position 340 (Figure 9A) were excluded in

 

this analysis. Other parameters were as indicated in the legend for Figure 10A.
The number of character-state changes along each branch are indicated. b) 50%
majority—rule bootstrap consensus tree (from PAUP) with the parameters set as

in the legend for Figure 103. All characters beyond position 340 (Figure 9A)

 

were excluded in this analysis. BCLs are indicated. c) Same as in part b except
the complete IT82 sequences were used in this analysis. d) Bootstrap distance
tree analysis on the IT82 sequence alignment (Figure 9A) as indicated in Figure

10C. The scale corresponds to 0.07 average percent nucleotide substitutions.

104

cin
ind
com
jun
pom.
cor
fau
ele

bas

 

:B ’ cin
Bootstrap ind
com
jun
pom
cor
fau
ele
bas

    
    

ein
ind
com.
jun
fau
ele
pom.
cor
bas

Bootstrap

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Scale: each - is spproxinetely equel to 0.07 percent nucleotide substitutions

meell

105

Figure 12. Phylogenetic analyses of Rhagoletis using the combined ITSI and ITSZ
sequences. a) This single most parsimonious tree (CI = 0.925 and R1 = 0.917)
was obtained after combining the phylogenetically informative characters from
the PAUP analyses in Figures 10A and 11A (ie. asterisks in Figures 8A and
9A) and then performing an exhaustive search. Gaps were treated as missing
data. The number of character-state changes along each branch are indicated.
b) 50% majority-rule bootstrap consensus tree of the combined ITSI and ITS2
data set (as in part a) with the parameters set as in the legend for Figure IOB.
BCLs are indicated. c) Bootstrap distance tree analysis on the combined ITSI
and IT82 sequences as indicated in Figure 10C. However, only positions 1-
340 of the IT82 alignment (Figure 9A) were included in this analysis. The

scale corresponds to 0.05 average percent nucleotide substitutions.

106

 

 

 

 

, 15 [_ 2 gin
3 2 1nd
L

 

 

 

 

 

B.

Bootstrap f 80 I 99 { gig

52 com
53 jun

100 pom

cor
fau
ele
bas

 

 

 

 

 

 

 

 

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70+ ----------------------------------- I
+ """ I +-- ind

 

Scale: each - is about equal to 0.05 percent nucleotide substitutions

Figure 12

1 0 7
73% support for the R. fausta and R. electronwrpha branch (Figure 11D). R. completa

was grouped with the cingulata-indtfierens pair 71% of the time, and the placement of R.
juniperina, again, remained unresolved.

By combining informative characters from IT S1 (32) and IT82 (11, only those
found between positions 1 and 340 of ITSZ; Figure 9A), I found one most parsimonious
tree of length 53, CI = 0.925, HI = 0.075 and R1 = 0.917 (Figure 12A). In this PAUP
analysis of nine taxa, the absent electrommpha and basiola lTSl sequences were treated as
missing data (i.e., by dots). To check the effect of this treatment on the tree topology I
repeated the PAUP analysis of the combined ITS sequence data, but with the IT82 of
electromamha and basiola excluded (i.e., 7 taxa analyzed). One most parsimonious tree
with the same topology was obtained, but with a shorter tree length (data not shown).
Bootstrap analysis of the combined sequence data showed 99% support for the R. cingulata
and R. indifierens branch, 100% support for the R. pomonella and R. comivora branch,
61% support for the R. fausta and R. electromarpha branch (Figure 12B). R. completta
was grouped with the cingulara-indrfierens pair 80% of the time and R. juniperina was
grouped with the pomonella-comivora pair 53% of the time. Similarly, a bootstrap test of
NJ distance tree from the combined 11‘ S1 and l'I‘S2 (again, only positions 1-340)
sequences showed 100% support for the R. cingulara and R. indrjferens branch, and 100%
support for the R. pomonella and R. comivora branch (Figure 12C; electromarpha and
basiola sequences were excluded from the analysis because of their ITSI absent in the
alignment). R. campletta was grouped with the cingulata-indzflerens pair 70% of the time

and R. juniperina was grouped with the pomonella-comivora pair 45% of the time.

Discussion
ITS Sequence Divergence
Generally, the values for the average substitution rates are much less than the

saturation level (around 40%) found between the Drosophila melanogaster species group

l O 8
and D. pseudoobscura (or D. virilis; Schlotterer et al. 1994). This observation is consistent

with the generally high transition to transversion ratios observed for the pairwise sequence
comparisons (Table 8) when compared with those from the Drosophila study (Schlotterer et
al. 1994). This may imply that the evolutionary history of the Rhagoletis species surveyed
in this study is not as long as those of the Drosophila species. However, the observed
differences in substitution rates and transition/transversion ratios may also reflect the fact
that the rates of nucleotide changes are faster in Drosophila because they have many more
generations per year than Rhagoletis. In general, gene sequences of closely related taxa
will differ predominantly in transitions because, these occur at a far greater rate than do
transversions (Quicke 1993). A decrease in the transition to transversion ratio is expected
when the period of divergence increases (Brown et al. 1982). In the large subunit
mitochondrial rDNA of Hawaiian Drosophila, transitions account for 90% of the number of
total substitutions between taxa that had been separated for 51 Myr but account for only
40% of the differences between taxa separated for 210 Myr (DeSalle et. al 1987). For
cingulata-indifi’erens and pomonella-comivora species pairs, 100% of the total substitutions
were transitions suggesting that the species diverged relatively recently (Table 8).

However, nucleotide substitution rates and transition to transversion ratios are not always
correlated well. For example, in the completa/juniperina pair of taxa, the transition to
transversion ratios in the IT S2 were very low, while the corresponding substitution rates
are not very high.

The significance of the high A-T content in the ITS sequences of Rhagoletis is not
clear but may have some relation to a recent observation that R. pomonella rDNA clusters
are located at the periphery of fibrillar centers in the nucleolus (Procunier and Smith 1993;
also see Chapter II). The high A-T content found for the ITS sequences of Rhagoletis is
not generally typical for eukaryotic organisms although high AT contents have been also
observed in Drosophila (T autz et al. 1988; Schlotterer at aL 1994) and in Cicindela beetles
(V olger and DeSalle 1994). In several Aedes mosquito species, only 350% A-T content

1 0 9
were reported for the ITSZ (Wesson et al. 1992). The IT S sequences in vertebrates and

plants have an A-T content as low as 20-30% and 30-50%, respectively (Torres et al. 1990;
Yokota et al. 1989). The decreased AT content in higher animals has been proposed to be
associated with the shift from cold-blooded to warm-blooded vertebrates (Bemardi et al.
1988). The low AT content in hematophagous mosquitoes may be linked to their feeding
on warm-blooded animals (Wesson et al. 1992).

The Feasibility of Using the ITS for Rhagoletis Phylogenetic Analysis

The rDNA ITS sequences have become attractive markers for phylogenetic studies.
Variation in these regions has been analyzed using restriction fragment length
polymorphism (RFLP) (Hillis and Dixon 1991) and more recently, also using PCR and
sequencing technologies. The rDNA array in animal species typically consists of several
hundred tandemly repeated copies. The sequence diversity among those copies within a
single individual (i.e., intra—individual variation) and within a population (i.e., inter-
individual variation) can exist. With current techniques, it is impossible to determine the
sequence for each of the hundreds repeating units and to obtain the total amount of genetic
diversity within a population or even within a single individual. As a consequence the
existence of undetected polymorphism among repeat units within a single individual or
within a population can complicate the use of the rDNA in phylogenetic analysis.
However, those tandem repeats are generally homogenized to produce a uniform sequence
in all repeating units of a given species through several mechanisms, such as unequal
crossing over and gene conversion, (i.e. concerted evolution; Dover 1982). Empirical
evidence of the homogenization of tandem repeats through gene conversion has been
provided by studying parthenogenetic lizards (Hillis et al. 1991). Probably because of this
general assumption of concerted evolution, some studies on the rDN A do not pay much
attention to intraindividual and intraspecific variation although Williams et al. (1988)

warned that homogenization of the tandem repeats within a species may take a relatively

l 10
long period of evolutionary time. However, substantial variation within certain Drosophila

species (Williams et a1 . 1987) have been noticed and a great amount of polymorphism in
the IT 82 sequence has been reported in Aedes mosquitoes with highest intraindividual
variation 1.52% and intraspecific variation 2.55% (Wesson et al. 1992). High intra-
individual variation has also been described in a Cicindela beetle ITSI sequence (V olger
and DeSalle 1994). Those finding further support that homogenization of the rDNA
repeats indeed requires long periods of evolutionary time and thus it becomes necessary to
access the intraspecific variation in the ITS before it is used for phylogenetic analysis.
Since events of unequal crossing over, gene conversion and interchromosomal reciprocal
exchange are possible mechanisms responsible for homogenization of tandem repeats of
rDNA the location of rDNA arrays on chromosomes.(i.e. multiple arrays on different
chromosomal loci or different chromosomes) in a given species may affect the sequence
uniforrning process. In most Anopheles mosquitoes the rDNA is localized on the X
chromosome while the rDNA arrays of Aedes can occur at least at two loci on different
chromosomes (Park and Fallon 1990; Fritz et al. 1994; Kumar and Rai 1990; Wesson et al.
1992). This may partially account for the disparity in the degree of intraindividual and
intraspecific ITS variation between Aedes and Anopheles, several species in the later genus
shows little or no such variation in their ITS sequences (Porter and Collins 1991; Fritz et
al. 1994). D. melanogaster is known to have rDNA loci on both the X and Y
chromosomes and different rDNA sequences on the two chromosomes have been detected
(Y agura et al. 1979), with the X-linked rDNA arrays probably under selective constraints
(Williams et al. 1987). Using in situ hybridization, Procunier and Smith (1993)
determined that the rDNA in Rhagoletis pomonella is located on two homologues of
chromosome number one, which may be sex-linked with the X chromosome containing a
larger block of rDNA. Therefore, high levels of inua-individuallintraspecific variation. in
Rhagoletis ITS sequences should not be surprising. The investigation of intraspecific
polymorphism carried out in the previous chapter became necessary before the ITS

l l 1
sequence is used in the phylogenetic analysis of the taxa covered in this chapter. Compared

with the interspecific variation among the concerned taxa in this study (most of them
representing different species groups) the intraspecific variation found in the previous
chapter was significantly low, indicating that intraspecific variation can be regarded as
negligible in phylogenetic analysis of taxa at the species group level, but not among sibling
species of some species groups, such as the cingulata group.

Phylogenetic Implications from the ITS sequences

The lack of sufficient morphological characters between species groups of
Rhagoletis has made it difficult to establish a reliable phylogenetic framework for this
genus. Allozyme analyses of certain species in the genus Rhagoletis have provided insight
into the phylogeny of this genus. However, a good consensus between the results from
morphology and allozyme data is still far from being reached and some conflict appears to
exist between the biochemical and molecular data (i.e. allozyme vs. mtDNA) with regard to
the placement of certain species in the phylogeny of this genus. The results from my ITS
study are in agreement with one or another of the earlier systematic studies on the genus
Rhagoletis (Bush 1966; Berlocher and Bush 1982; Smith and Bush, unpublished data).
There are also conflicts, with the previous studies, with regard to the placement of certain
species. First, the ITS data support the view that R. comivora belongs to the pomonella
group. This placement is consistent with all the analyses performed in this study, which
includes the PAUP and NJ analyses of both IT 81 and IT S2 as separate as well as
combined regions. The BCL values for all the analyses were consistently high, usually
about 100%. The closeness of R. comivora and R. pomonella is in agreement with their
affinity in their morphology. Rhagoletis comivora, morphologically, resembles R.
pomonella so much that it had been treated as a sympatric subspecies of R. pomonella until
Bush (1966) recognized it as a distinct species. The unique karyotype and several features

in wing pattern unambiguously place R. comivora in the pomonella group (Bush 1966).

1 12
Although R. pomonella/R. comivora hybridization are unlikely to occur in nature, a few

hybrids between these two species were obtained in the laboratory and confirmed as
hybrids with electrophorectic analysis (Smith et al., 1992). However, this placement is
contradictory to the results of mtDNA analysis, which places R. comivora far away from
the pomonella group, and groups R. comivora with R. juniperina or with the tabellaria
group (Smith and Bush, unpublished data). Electmphoretic study (Berlocher and Bush
1982) indicates that the genetic distance between R comivora and the other members of the
pomonella species group is about 9 times greater than the distance among the other
members of this group themselves. Therefore, R. comivora appears to have diverged
much earlier than its sibling species during the evolution of this group. Although UPGMA
clustering analysis of Nei standard distance (Berlocher and Bush 1982) places R. comivora
outside the pomonella group (Berlocher and Bush 1982), that analysis did not group R.
comivora with R. juniperina as in the mtDNA analysis. Although the ITS regions of other
sibling species in the pomonella group, such as R. mendax, R zyphyria and a pomonella-
like species on the flowering dogwood in Florida, were not analyzed in this study, the
species of this group are predicted to form a unambiguous clade if their ITS sequences are
available because R. comivora -— the most genetically divergent based on allozyme and
mtDNA data — is shown here to be closely related to R pomonella. Because of the
important role of the pomonella group in the theory of sympatric speciation (via host
shifting) and because this group also contains major economic pests, such as R. pomonella
on apples and R. mendax on bluebenies, accurate relationship among the members of this
group is of particular interest both practically and theoretically. The nucleotide substitution
rates between the R. pomonella and R. comivora pairs of taxa were 2.99 in ITSI and 1.59
in ITSZ while the highest rates among members of the R. cingulata species group were
only 0.52 in ITSI and 0.73 in ITSZ. It is clear that the genetic distance in the

pomonella/comivora species pair is higher than that among sibling species in the cingulata

1 13
group. However. R. pomonella and the other pomonella group species are almost as

closely related as cingulata group species.

Rhagoletis completa, the walnut husk fly, represents the suavis group in which
speciation is believed to be allopatric (Bush 1975), unlike that for the R. pomonella and R.
cingulata species groups. The close relatives of this group has been difficult to determine
based on morphological characters (Bush 1966). There are contradictory molecular data
regarding the placement of this group in the genus Rhagoletis. This study, unlike the result
from the allozyme study (Berlocher and Bush 1982) but in support of the mtDNA analysis
(Smith and Bush, unpublished data), indicates that the suavis group is more closely related
to the cingulata group than to the pomonella group. However, bootstrap values (BCL)
supporting the closeness of these two groups are not consistent and vary from 36%
(mtDNA analysis) to 88% (lTSZ PAUP bootstrap in this study). Analysis of the ITS
sequences of other sibling species in the suavis group, such as R. juglandis, R. boycei and
R. zoqui, may eventually provide more information on the relationship of this group with
other Rhagoletis species groups.

Rhagoletis electromorpha is a representative of the tabellan’a species group which
conventionally also includes R. juniperina and two other species (Bush 1966). In this
study, however, R. juniperina is far removed from the tabellaria group, which is congruent
with allozyme and mtDNA analyses. Morphologically, R. juniperina is probably the most
divergent among the other members of the tabellaria group. It is becoming clearer that R.
juniperina may indeed not belong to the tabellaria group. However, to verify this point, the
ITS of other members of the tabellaria group could be analyzed. Based on allozyme
analysis, R. juniperina is more closely related to the cingulata group; but the ITS data
indicates R. juniperina is either more closely related to the pomonella group (Figure 10) or
its placement is unresolved (Figure 11). The closeness of R. juniperina to the pomonella
group has also been noted in the mtDNA protein parsimony cladograrn (Smith and Bush,
unpublished data). Consequently, the ambiguous placement of R. juniperina is still in need

l 14
of a more extended ITS and mtDNA sequence analysis of this genus and some reanalysis

of morphological and other biochemical data sets.

Morphologically, R fausta, which is a serious cherry pest. did not show enough
affinity with any North American Rhagoletis species groups and remained unplaced (Bush
1966). Allozyme data (Berlocher and Bush 1982) on the other hand consistently placed
this species with the suavis group. The ITS sequence of R. fausra does not support this
placement but finds it more closely related to R. electromarpha, which is a representative of
the tabellaria group, than to the campleta, which belongs to the suavis group. This
interpretation is consistent with mtDN A analysis which has several different placements for
R. fausta, none of which supports the placement of R. fausta with the suavis group either
(Smith and Bush, unpublished data). However, the placement of R. fausta with R.
electromarhpa in this study is very tentative because it is based on IT82 sequence alone,
showing low BCL. Therefore, the placement of R. fausta remains in doubt. To better
resolve this question other members of the suavis group, such as juglandis, suavis and
boycei, as well as several other species from the rabellaria group should be sampled and a
comprehensive analysis of their ITS sequences performed.

Rhagoletis basiola and R. striatella have undergone extensive sequence divergence
in the ITS regions. Except for the ITS2 of R. basiola, the ITS sequences for these two
species, especially R. striatella, are very difficult to confidently align with Other Rhagoletis
species surveyed here. Their large genetic distance in the ITS sequences from other
Rhagoletis species are also reflected in their possessing of several primitive morphological
characters (Bush 1966) and in their divergent allozyme frequencies (Berlocher and Bush
1982). The striking difference in allozyme frequency between R. sm'atella and other
Rhagoletis has even led to the placement of this species outside of the genus Rhagoletis
(Berlocher and Bush 1982). In mtDNA COII nucleotide parsimony cladograrns (Smith and
Bush, unpublished data), R. striatella formed a polytomy with non-Rhagoletis outgroups

such as Zonosemata electa and Oedicarena laufi'ons. R. basiola belongs to the R. alternata

l 15
species group which mainly consists of Eurasian species and are considered to be distinct

genus by some authors (Bush 1966). It is not surprising that the monophyly of the genus
Rhagoletis as currently defined has been questioned by several workers (Norrbom 1989;
Foote et al. 1993; Smith and Bush, unpublished data; and Jenkins, personal
communication). To further test the monophyly of the genus Rhagoletis using ITS
sequences, some close relatives of Rhagoletis, such as Z electa and 0. latifrons, need to be
analyzed and compared with the respective Rhagoletis species.

In summary, phylogenetic implications from the ITS sequence study are partially in
agreement with some of the previous studies on Rhagoletis phylogeny, and partially
contradictory to the previous results. The results from this study indicate that R. comivora
belongs to the pomonella group, further confirming the placement based on morphology
and karyotype Bush (1966), but contradictory to the mtDNA study (Smith and Bush,
unpublished data); R. juniperina is removed from the tabellaria group, in support of
allozyme and mtDNA analyses, and this species may more closely relate to the pomonella
group rather than the cingulata group, which is in agreement with the mtDN A (Smith and
Bush, unpublished data), but not with allozyme analysis; close relatives of the cingulata
group are more likely the members of the suavis group rather than those in the pomonella
group, which is congruent with the mtDNA study, but not with the allozyme results; R.
fausta may relate to the tabellaria group, which was not implied by previous studies; R.
basiola and R. sm’atella are most divergent in the ITS sequences, correlating with their
possession of several ancient morphological characters (Bush, 1966) and their basal
branching in the analyses of mtDNA and allozyme frequency, further supporting the idea
that Rhagoletis, as currently defined, may not be monophyletic. This is the first study of
rDNA ITS sequences of the genus Rhagoletis. Their application in phylogenetic study of
Rhagoletis has provided some insight into the relationship between certain taxa from
different species groups of this genus. The study of ITS should be expanded in the genus

Rhagoletis which should provide a new avenue for the understanding of the evolutionary

1 16
history of this genus and providing a reliable phylogenetic framework to test some

important evolutionary theories such as sympatric speciation through host shifting.

BIBLIOGRAPHY FOR CHAPTER III

117

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Berlocher, S. H., and G. L. Bush. 1982. An electrophoretic analysis of Rhagoletis
(Diptera: Tephritidae) phylogeny. Systematic Zoology 31:136-155.

Berlocher, S. H., B. A. McPheron, J. L. Feder, and G. L. Bush. 1993. Genetic
differentiation at allozyme loci in the Rhagoletis pomonella (Diptera: Tephritidae) species
complex. Ann. Entomol. Soc. Am. 86:716-727.

Berlocher, S. H., B. A. McPheron, J. L. Feder, and G. L. Bush. 1993. Genetic
differentiation xx allozyme loci in the Rhagoletis pomonella (Diptera: Tephritidae)
species complex. Ann. Ent. Soc. Amer. 86: 1-1 1.

Bemardi, G., D. Mouchiroud, C. Gautier, and G. Bemardi. 1988. Compositional

patterns in vertebrate genomes: conservation and change in evolution. J. Mol. Evol.
28:7-18. *

Brown, W. M., E. M. Prager, A. Wang, and A. C. Wilson. 1982. Mitochondrial DNA
sequences of primates: Tempo and mode of evolution. J. Mol. Evol. 18:225-239.

Bush, G. L. 1966. The taxonomy, cytology, and evolution of the genus Rhagoletis in
North America (Diptera: Tephritidae). Bull. Mus. Comp. Zoo]. 134: 431-562.

Bush, G. L. 19693. Mating behavior, host specificity, and the ecological significance of
sibling species in frugivorous flies of the genus Rhagoletis (Diptera: tephritidae). Amer.
Nat. 103: 669-672.

Bush, G. L. 1969b. Sympatric host race formation and speciation in frugivorous flies of
the genus Rhagoletis (Diptera: Tephritidae). Evolution 23:237-251.

Bush, G. L. 1975. Sympatric speciation in phytophagous parasitic insects. Pp. 187-205
in P. W. Price, ed. Evolutionary strategies of parasitic insects and mites. Plenum
Publishing Corp., New York.

Bush, G. L. 1993. Host race formation and sympatric speciation in Rhagoletis fruit flies
(Diptera: Tephritidae). Psyche 99: 335-357.

Bush, G. L. 1994. Sympatric speciation in animals: new wine in old bottles. Trends
Ecol. Evol. 9:285-288.

De Salle, R., T. Freedman, E. M. Prager, and A. C. Wilson. 1987. Tempo and mode of
sequence evolution in mitochondrial DNA of Hawaiian Drosophila. J. Mol. Evol. 26:
157-164.

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Diehl, S. R., and G. L. Bush. 1984. An evolutionary and applied perspective of insect
biotypes. Annu. Rev. Entomol. 29:471-504.

Dover, G. 1982. Molecular drive: a cohesive mode of species evolution. Nature
299: l 1 1-1 17.

Dover, G. A. 1989. Linkage disequilibrium and molecular drive in the rDNA gene family.
Genetics 122: 249-252.

Feder, J. L., and G. L. Bush. 1991. Genetic variation among apple and hawthorn host
races of Rhagoletis pomonella (Diptera: Tephritidae) across an ecological transition zone.
Entomol. Exp. Appl. 59:249-265.

Fritz, G. N., J. Conn, A. Cockbum, and J. Seawright. 1994. Sequence analysis of the
ribosomal DNA internal transcribed spacer 2 from p0pulations of Anopheles nuneztovari
(Diptera: Culicidae). Mol. Biol. Evol. 11:406-416.

Hillis, D. M., and M. T. Dixon. 1991. Ribosomal DNA: molecular evolution and
phylogenetic inference. Q. Rev. Biol. 66:411-453.

Hillis, D. M., C. Moritz, C. A. Porter, and R. J. Baker. 1991. Evidence for biased gene
conversion in concerted evolution of ribosomal DNA. Science 251:308-310.

Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules. Pp. 21-132 in H.
N. Munro, ed. Mammalian protein metabolism. Academic Press, New York.

Kimura, M. 1980. A simple method for estimating evolutionary rate of base substitutions
through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 111-120.

Norrbom, A. L. 1989. The status of Urophora acuticomis and U. sabroskyi (Diptera:
Tephritidae). Entomol. News 100:59-66.

Park, Y. J ., and A. M. Fallon. 1990. Mosquito ribosomal RNA genes: characterization of
gene structure and evidence for changes in copy number during development. Insect
Biochem. 20: 1-1 1.

Porter, C. H., and F. H. Collins. 1991. Species-diagnostic differences in a ribosomal
DNA internal transcribed spacer from the sibling species Anopheles freebomi and
Anopheles hermsi (Diptera: Culicidae). Am. J. Trop. Med. Hyg. 45:271-279.

Procunier, W. S., and J. J. Smith. 1993. Localization of ribosomal DNA in Rhagoletis
pomonella (Diptera: Tephritidae) by in situ hybridization. Insect Mol. Biol. 2: 163-174.

Quicke, D. L. J. 1993. Principles and techniques of contemporary taxonomy. Chapman
and Hall, New York.

Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for
reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406-425.

Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-
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119

Schlotterer, C., M. Hauser, A. von Haeseler, and D. Tautz. 1994. Comparative
evolutionary analysis of rDNA ITS regions in Drosophila. Mol. Biol. Evol. 11:513-
522.

Swofford, D. L. 1993. PAUP: phylogenetic analysis using parsimony, version 3.1.1.
University of Illinois, Champaign.

Tamura, K. 1992. Estimation of the number of nucleotide substitutions when there are
strong transition-transversion and G+C content biases. Mol. Biol. Evol. 9:678-687.

Tautz, D., J. M. Hancock, D. A. Webb, C. Tautz, and G. A. Dover. 1988. Complete
sequence of the rDNA genes of Drosophila melanogaster. Mol. Biol. Evol. 5: 366-376.

Torres, R A., M. Ganal, and V. Hemleben. 1990. GC balance in the internal transcribed
spacers ITSI and IT82 of nuclear ribosomal RNA genes. J. Mol. Evol. 8:669-686.

Vogler, A. P., and R. DeSalle. 1994. Evolution and phylogenetic information content of
the ITS-l region in the tiger beetle Cicindela dorsalis. Mol. Biol. Evol. 112393-405.

Wesson, D. M., C. H. Porter, and F. H. Collins. 1992. Sequence and secondary
structure comparisons of ITS rDNA in mosquitoes (Diptera: Culicidae). Mol.
Phylogenet. Evol. 1:253-269.

Williams, S. C., G. Fumier, E. Fuog, and C. Strobeck. 1987. Evolution of the
ribosomal DNA spacers of Drosophila melanogaster: different patterns of variation on X
and Y chromosomes. Genetics 116:225-232.

Williams, S. C., R. W. DeBry, and J. L. Feder. 1988. A commentary on the use of
ribosomal DNA in systematic studies. Syst. 2001. 37:60-62.

Yagura, T., M. Yagura, and M. Murarnatsu. 1979. Drosophila melanogaster has different
ribosomal RNA sequences on X and Y chromosomes. J. Mol. Biol. 133:533-547.

Yokota, Y., T. Kawata, Y. Iida, A. Kato, and S. Tanifuji. 1989. Nucleotide sequences of
the 5.88 rRNA gene and internal transcribed spacer regions in carrot and broad bean
ribosomal DNA. J. Mol. Evol. 29:294-301.

CHAPTER IV
CONSTRUCTION OF AN RHAGOZETYS POMONEIJA GENOMIC LIBRARY AND
IDENTIFICATION OF THE COMPLETE rRN A REPEATING UNIT

Introduction

Higher eukaryotic nuclear rDNA is composed of tandemly repeated transcriptional
units separated from each other by the non-transcribed intergenic spacer regions (IGS).
Each transcriptional unit contains the genes for the 188, 5.88 and 288 ribosomal RNA
(rRNA) and the external and internal transcribed spacers (ETS and ITS, respectively;
reviewed in Gerbi 1985; see Figure 13). The entire rDNA unit is transcribed by RNA
polymerase I as a single precursor molecule, which is then processed to yield mature 188,
5.88 and 28S rRNAs (Hadjiolov 1985; Sollner-Webb and Tower 1986). The
uanscriptional promoter of rDNA is located in the IGS/ETS boundary region and upstream
in the IGS there are also multicopies of promoter-like sequences called transcription
enhancers. Sequence comparison between species shows that the rDNA spacers including
IGS and ETS diverge much faster than the gene coding regions (reviewed in Long and
Dawid 1980). Further evidence that the transcription initiation region (i.e, IGS/ETS
boundary) undergoes rapid evolutionary change comes from the fact that the RNA
polymerase I of a particular species usually transcribes rDNA from that species only and
fails to recognize the rDNA transcription initiation region of another species in in vivo and
in vitro transcription assays among mammals, insects, and protozoans (for reviews, see
Arnheirn 1983; Dover and Flavell 1984). Therefore, sequence conservation of enhancer
regions in the IGS and initiation recognition site in the ETS within a given species may be
functionally important and the sequence divergence in these regions may be expected to be

I 2 0

121

Figure 13. Overall map of the Drosophila melanogaster rDNA repeat unit. Adapted from
studies described by Tautz et al. (1987 and 1988). Abbreviations: IGS,

 

intergenic spacer; ETS, external transcribed spacer; ITS, internal transcribed

 

spacer; rRNA coding regions (188, 5.88 and 288); E, EcoRI; B, Bng; H,
HindIII; Ha, HaeIII. Only 1 HaeIII is shown here. DNA probes used in this
study: 1) from D. melanogaster, 11.7 kb EcoRI fragment from plasmid
pDM238; and 2) from D. virilis, approx. 5 kb HindIII/HindIII fragment which
includes IGS and ETS, DvH25. The forward orientation, direction of the
primary transcript, is shown as reference for the primers used in the
experiments using the polymerase chain reaction (PCR). The scale is indicated
by the bar.

 

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species specific. Furthermore, Williams et a1. (1985) found that individual flies of

Drosophila mercatorum from different geographic regions have characteristic length of the
IGS, which range from about 4kb to more than 6.5kb. Those length variations may serve
as markers for determining the geographic origin of individuals. In light of these findings,
the IGS/ET S is a promising region for phylogenetic analysis of closely related species, in
particular those species (or host races) that are morphologically indistinguishable, such as
those often encountered in the genus Rhagoletis.

Although an increasing body of literature on rDNA has been accumulated for
Drosophila, Aedes, Glossina and Xenopus (Furlong et al. 1983; Gerbi 1985; Cross and
Dover 1987a; Cross and Dover 1987b; Gale and Crampton 1989), the rDNA of
agriculturally important insects in the genus Rhagoletis has not been characterized. To
evaluate the potential use of Rhagoletis-rDN A in studies on speciation and phylogenetic
reconstruction, I carried out a preliminary characterization on the organization of R.
pomonella rDNA. I first constructed a genomic DNA library from R. pomonella , then
took advantage of the highly conserved 188 and 288 genes using the complete repeating
rDNA unit of Drosophila melanogaster (provided by Dr. G. Dover, see Figure 13) as a
probe to identify R. pomonella rDNA clones. In this study, for the first time, I report
several positive rDNA clones for R. pomonella, which together cover all of the rDNA
coding regions and the various spacers. In addition, the R. pomonella ITSI and ITSZ
regions were sequenced and compared with those from D. melanogaster. The R.
pomonella rDNA clones from this study will form the basis for a more detailed study of the
sequence organization of the rDN A repeating unit for the genus Rhagoletis in the future.
This is of particular interest since the IGS and ET S are potentially useful regions for
phylogenetic study of closely related species or even host races in the genus Rhagoletis.
Phylogeny inferred from the IGS/ET S will allow us to compare the phylogeny obtained
from the ITS regions.

1 2 4
Materials and Methods

Biological Material

Rhagoletis pomonella larvae were collected in late summer from the fruit of Malus
pumula (domestic apple; Door Co., WI) by Jeff Feder and allowed to pupate in the
laboratory. Upon emergence the following spring flieswere stored at -70°C since 1984 for
rDNA analysis.

Genomic DNA Isolation and Sau3AI Partial Digestion

R. pomonella genomic library construction is briefly outlined in Figure 14. Total
genomic DNA was isolated from 50 flies as described by Procunier and Smith (1993).
Twenty ug of the genomic DNA was partially digested with 0.29 U of Sau3AI (GIBCO-
BRL) in 500 pl (reaction volume) containing 10 mM Tris-HCI, pH 7.5, 100 mM NaCl and
7 mM MgC12 at 37°C for 30 min. The reaction products were extracted twice with an equal
volume of phenol/chloroform/isoamyl alcohol (PCI; 25:24: 1) and once with an equal
volume of chloroformfrsoamyl alcohol (CI; 24:1). The partially digested DNA was
precipitated with NH40Ac/ethanol and then dissolved in TE buffer (10 mM Tris-HCI, pH
7.5, and 1 mM EDTA). The Sau3AI digestion conditions were optimized to generate
genomic DNA fragments ranging in size between 6 and 16 kb. About 10 ug of the
Sau3AI-treated genomic DNA was used in a partial fill-in reaction of the first two
nucleotides of the Sau3AI site (Figure 15A). The 25 pl final reaction volume contained 50
mM Tris-HCI, pH 7.2, 10 mM MgSO4, 0.1 mM dithiothreitol, 12.5 [lg acetylated BSA
(Promega), 1 mM dATP, 1 mM dGTP and 3 U of the Klenow enzyme (GIBCO-BRL).
The partial fill-in reaction was carried out at 37°C for 30 min. The reaction products were
extracted with PCI and CI as before, precipitated with ethanol and then dissolved in 2 pl of
TE buffer.

 

125

Figure 14. Schematic outline of the procedure for generating a Rhagoletis pomonella
genomic DNA library and screening for the rDNA repeat unit.

126

Isolation of R. pomonella Genomic DNA

Partial Digestion with SauBAI

Partial Pill-In Reaction
using Klenow, dATP and dGTP

Ligation to LambdaGEM-ll XhoI Half-Site Arms

In Vitro Packaging into Bacteriophage 7t

Transduction into E. coli

Plaque Lifting and Screening with mass Probe

Figure 14

127

 

Figure 15. Detailed outline of several specific steps in Figure 14. Panel a represents the
Sau3AI partial-digestion reaction of Rhagoletis pomonella genomic DNA and
the subsequent partial fill-in reaction with dATP and dGTP. Panel b presents
details of the LambdaGEM-ll Xhol half-site arms purchased from Promega
and the cloning product. The two SalI restriction sites are about 100 bp away
from the )0101half-sites. Bold letters indicate the nucleotides that were added
during the partial fill-in reactions. The figure is not drawn to scale.

 

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1 2 9

Cloning into Bacteriophage h Half-Site Arms and Packaging

The partial frll-in reaction products were cloned into LarnbdaGEM-ll XhoI half-site
arms (Promega; Cat No. B1960; Figure 15B) using T4 DNA ligase (GIBCO-BRL)
according to the manufacturer's (Promega) instructions. Control cloning reactions without
insert and with a test insert provided by the manufacturer were also performed. The
ligation reactions were packaged using the Packagene in vitro packaging system (Promega;
Cat. No. K315) according to the manufacturer's instructions. Packaged h phage titers
were checked on Luria-Bertaini (LB) plates using Esherichia coli LE392 (Promega),
according to the manufacturer's instructions, and were found to range between 1.2 x 104 -

2.8 x 105 pfu/ml. As expected, controls without inserts showed no plaques on LB plates.

DNA Probe Generation and Genomic Library Screening

The 11.7 kb Dm238 EcoRI insert (Figure 13) from plasmid pDM238 (provided by
Dr. G. Dover) was prepared as described in Procunier and Smith (1993). Purified Dm238
EcoRI insert (200 ng) was labeled with digoxygenin-dUTP (Boehringer Mannheim; Cat.
N 0. 1093088) by random-priming using a commercially available kit (Boehringer
Mannheim) according to the manufacturer's specifications. In addition to the Dm238
EcoRI fragment, the approximately 5 kb HindIII/HindlII fragment of D. virilis rDNA
repeat unit, DvH25 (T autz et al. 1987), was also used to generate digoxygenin-labelled
probes. Labelled probes were precipitated from an 80 it] reaction mixture by adding 10 ul
of 3M NaOAc, pH 5.0, 1 111 of 20 mg tRNA per ml (Boehringer Mannheim; Cat. No.
109495) and 250 pl 100% ethanol. After centrifugation, the pellets were washed with 70%
ethanol, air dried and dissolved in 100 u] TE containing 0.1% SDS. The probes (about 1-2
ng/ul) were stored at 4°C until use.

Bacteriophage l» plaques were immobilized onto Gene Screen nylon membranes
(DuPont Inc.; Cat No. NEF-983) as described elsewhere (Sambrook et al. 1989).
Membranes were prehybridized in buffer A (2X SSC [0.15 M NaCl, 20 mM sodium

1 3 O
citrate, pH 7.0], 0.5% [w/v] casein [Sigma; Cat. No. C5890], 0.1% [w/v] N-lauroyl

sarcosine and 0.2% [w/v] SDS) at 68°C for at least 2 hr. Hybridization was performed
overnight at 68°C with fresh buffer A containing digoxygenin-labelled Dm238 probe
(approx. 3 ng labelled Dm238 per ml buffer A). Filters were washed twice at room
temperature with 2X SSC/0. 1% SDS for 10 min each and once at 68°C with 0.2X
SSC/0.1% SDS for 15 min. Hybridizcd DNA was detected with an anti-dioxygenin
alkaline phosphatase conjugate (50 mU/ml) and LumiPhos substrate of the Genius system
(Boehringer Mannheim) using the protocol outlined in the Boehringer Mannheim Technical
Bulletin for LumiPhos 530 (900264R3/10M; Jan 1991) substituting casein for the blocking
reagent. Membranes were then exposed to X-OMAT film (Kodak) for 1 min to 2 hr for

visualization.

Characterization of Recombinant Phage by Gel Electrophoresis and Southern Hybridization
After identifying several positive plaques, the h bacteriophages corresponding to
those plaques were purified and the phage DNA was isolated using protocols described by
Sarnbrook et al. (1989). Isolated phage DNA was digested with various restriction
enzymes and the products analyzed by agarose gel electrophoresis according to common
procedures described by Ausubel et al. (1987). After electrophoresis, the gel was stained
with 0.5 rig/ml ethidium bromide and visualized using a transilluminator. The gel was also
photographed using a red filter with Polaroid positive/negative films (type 55). DNA in the
gel was then transferred by capillary action to Gene Screen nylon membranes (DuPont Inc.;
Cat No. NEF-983) and Southern hybridization was performed using the labelled Dm238

and DvH25 probes (see earlier section), as described elsewhere (Sambrook et al. 1989).

PCR Amplification and DNA Sequencing
The ITSI region was amplified using primers 1406F
S'CCT'ITGTACACACCGCCCGT (matching the 3' end of 18S) and 35R-GB14

1 3 l
5'AGCTRGCTGCGTTCTTCATCGA (matching the 5' end of 5.88). The ITS2 region

was amplified using primers 108F 5'GAACATCGACHHKTYGAACGCA (matching the
3' end of 5.88) and 52R 5'GT'I‘AGTI'TC'I'I'I'I‘CC'I‘CCSCT (matching the 5' end of 288).
The letters F and R refer to the forward and reverse orientation of the primers, respectively,
with respect to complete rDNA repeat unit (Figure 13). Amplification by the polymerase
chain reaction (PCR) was carried out in 25 pl (final volume) containing 10 mM Tris-HCI,
pH 8.5, 50 mM KCl, 3 mM MgC12, 375 pM of each dNTP, 0.1-0.4 pM primer 1406F for
ITSI (or 108F for ITSZ) and 0.1-0.4 pM primer 35R-GB14 for ITSI (or 52R for IT82),
and 1.25-2.50 units of Ampli Taq DNA polymerase (Perkin Elmer Cetus). Amplification
parameters were 920C for 3 min 10 sec; 35 cycles each with 920C for 15 sec, 650C for 15
sec and 720C for 1 min 10 sec; and 720C for 6 min 10 sec. Amplified DNA was subjected
to electrophoresis on 081.0% agarose gels and visualized with ethidium bromide. Bands
containing the DNA were excised from the gel and the DNA purified using the Prep-A-
Gene DNA purification matrix (Bio-Rad), according to the manufacturer's instructions.
The TA Cloning kit (Invitrogen) was used in a one-step cloning strategy for the
direct insertion of the purified PCR products into a plasmid vector, followed by
transformation into competent cells. Plasmid vector and competent cells were supplied by
the manufacturer. Plasmid DNA was purified from individual clones using the Magic-Prep
DNA purification kit (Promega), following the manufacturer's instructions. DNA
sequencing was performed according to the chain-termination method of Sanger et al.
(1977), and using the Sequenase Version 2.0 DNA sequencing kit (USB) and 35S-dATP
(Amersham). The same primers as in the amplification reactions (see earlier sections) were
used to determine the DNA sequence in both directions. Once a certain stretch of DNA was
sequenced other primers were employed to complete the sequencing of the IT S1 and IT 82
legions; primers 35R-GB27 5'ACC(CT)AAACATI'ITCAAGT(CT)GCG and 108F-GB25
5'A(AT')(AG)(AG)AATC(AT)(CDAGTATTCCC were used for the ITSI and IT82

regions, respectively.

1 3 2
Computer Analyses

A number of programs, including FETCH, GAP, MAPSORT, SEQED, and
STRINGSEARCH, in the GCG package of the University of Wisconsin Genetics
Computer Group (UWGCG package, version 8.0) were used in sequence comparison of
ITSI and IT82 between R. pomonella and D. melanogaster. The sequence of D.
melanogaster rDNA repeating unit, from clone po238, was found using Stringsearch
under the locus name DROGRAB and accession numbers M21017 and M29800. Bases 1
to 7232 and 7206 to 12026 were described in Tautz et al. (1988) and Tautz et a1. (1987),
respectively. In the D. melanogaster DROGRAB locus, the actual region between the 288
and 188 subunits of rDNA, which includes the IGS and ETS regions, subtends between
bases 7206 to 11729.

Results and Discussion

Genomic DNA Library Analysis

A high number of positive recombinant bacteriophage plaques were identified after
plaque-lifting and probing membranes with Dm238 (Figure 16A shows only one of the
several membranes). After collecting the well-separated plaques and repeating the
transduction and screening protocols, almost all of the plaques on the plates turned out to
be positive recombinants when probed with Dm238 (Figure 16B). The well-separated
plaques from the second screening were picked and named RpA, RpB, RpC, and RpD
(Figure 16B only shows the filter for clone RpD). Several plaques from the first screening
with relatively low or no signals when probed with Dm238 were also selected (Figure
16C). Some of these latter plagues did indeed have genomic inserts, but non-rDN A , and
were used as negative controls in subsequent experiments (see below). Surprisingly,
plaques containing the control test insert (provided by Promega as negative control) also
showed positive signals with Dm238 (data not shown). Upon correspondence with

researchers at Promega, it was confirmed that their test insert contained significant amounts

Figure 16.

 

Frgure 17.

 

 

133

Screening of the Rhagoletis pomonella genomic DNA library with Dm238
probe. In panel A arrows indicate several positive recombinant plaques
(positive for nuclear rDN A). Smaller spots represent non-rDN A recombinant
and/or nonrecombinant (background) plaques. One such spot was used as a
negative control. On the LB plates all plaques appeared of equal size. Panel b
represents clone RpD after the second screening protocol. Panel c represents

the negative control plaque from the first screening in Panel a.

Southern blot analysis of the restriction digestion products of DNA purified
from R. pomonella rDNA clones. Panels a and b represent blots hybridized
with DvH25 and Dm238, respectively. lanes 1 (1'), 2 (2'), 3 (3') and 4 (4')
correspond to DNA from clones RpA, RpB, RpC and RpD, respectively. DNA
in lanes 1, 2, 3 and 4 from Panel A was digested with SalI. DNA in lanes 1',
2', 3' andi4' from Panels A and B was digested with BglII. T and N stand for
test insert (from Promega) and negative control (see Material and Methods, and

Results sections), respectively.

9.4
6.6

2.3

Figures 16(top) and 17(bottom)

134

.3.
. t *'
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... .
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'- ' ' "

DvH25

2.3

Dm238

1 3 5
of rDNA from mice. To further characterize the R. pomonella rDNA clones, purified DNA

from the positive recombinant plaques was digested with Sall. This releases the complete
DNA insert flanked on each side with about 100 bp of A DNA (see Figure 15B for the Sall
sites). The choice for using Sall restriction enzyme to release the insert was made in part
because a search, using MAPSORT in GCG, for Sall site(s) in the complete Drosophila
rDNA repeat unit showed no such site; consequently, I reasoned that Rhagoletis rDNA
repeat units may also lack Sall site(s). Separation of the digested products by gel
electrophoresis and Southern hybridization using DvH25 probe (also provided by Dr. G.
Dover; Figure 13) showed one major band from clones RpA, RpB, RpC and RpD with the
fragment size varying between 8 and 12 kb among the various clones (Figure 17A lanes 1-
4). Another minor band was found in clones RpB, RpC and RpD in around the
compression region of the gel (about 23 kb). This latter band is most likely due to
incomplete digestion with Sall because the Sau3AI partial digestion products of the R.
pomonella genomic DNA ranged in size between 6 and 15 kb and, consequently, any Sall
site in the R. pomonella rDNA repeat should only lead to shorter DNA fragments and not
as large as 2023 kb. When the same membranes were stripped and probed with Dm238
(longer than DvH25) only the same Sall fragments were observed as using DvH25 and no
additional fragments were detected indicating that indeed Sall released the inserts as a
whole fragment in the above positive clones (Figure 17A only shows results using
DvH25). The size of the insert in clone RpD was comparable to the size of the complete D.
melanogaster rDNA repeat unit, which is about 11.7 kb (Coen et al. 1982). Further
digestion of the clones with 33111 and hybridization of membranes with DvH25 and
Dm238 probes (Figure 17; Panels A and B) showed DNA fragments comparable in size to
the 4.6 kb BglII/Bglll fiagment of D. melanogaster rDNA which contains ITSI and I'I‘S2.
In all cases, the negative control which contained a DNA insert (when gels were visualized
by ethidium bromide; data not shown) did not hybridize with either of the mentioned two

probes (Figure 17; Panels A and B).

1 3 6
ITS Sequence Analysis

The ITSI and IT 81 fragments, PCR amplified from one of the positive recombinant
phage DNA (RpD) using primers matching the highly conserved regions of the 188, 5.88,
and 288 genes are shown in Figure 18. There is one major DNA band of approx. 1 kb for
ITSI of R. pomonella and a similar size DNA fragment was also amplified for the ITSI
region of Drosophila (from Dm238 DNA) as a positive control (Figure 18A). Similarly,
one major band of approx. 680 bp was detected for the ITS2 region of the same clone of R.
pomonella and one band of approx. 550 bp from Drosophila (Figure 18B). The major
PCR bands from both R. pomonella and D. melanogaster were purified (using Prep-A-
Gene), cloned into the pCR II vector (from Invitrogen) and then sequenced. The sequence
comparison of ITS for the two species were carried out using the GAP sequence alignment
program in the GCG package and presented in Figure 19. The length of the ITSI and ITS2
sequences from R. pomonella clone RpD were 680 and 475 bp, respectively. The
identities of the ITS sequences were verified by sequencing about 100 nucleotides for the
adjacent highly conserved coding regions (188, 5.88 and 288) since these regions are
almost identical between Rhagoletis and Drosophila (T autz et al. 1988). R. pomonella ITSI
and ITS2 sequences were 46 bp shorter and 90 bp longer than the respective D.
melanogaster ITS sequences (Tautz et al. 1988). The A-T content of the R. pomonella
ITSI and IT82 sequences were 80.1 and 81.7%, respectively; slightly higher than those of
the D. melanogaster (73.1 and 80.0%, respectively; Tautz et al. 1988; Schlotterer et al.
1994). According to the ITSI and IT82 sequence alignments shown in Figure 19, there is
substantial divergence between R. pomonella and D. melanogaster. including several
insertion/deletion differences. However, a number of highly conserved regions are also
observed between the two along the length of the spacers (for example, the underlined
sequence in Figure 19B). The latter point is especially obvious for IT82, indicating that

some of these regions may be of structural/functional significance.

137

Figure 18. Products of the PCR amplification reaction of the ITSI and IT82 regions of
Rhagoletis pomonella. Panels a and b correspond to ITSI and IT82,
respectively. Lanes 1 and 2 in both panels represent products of PCR
amplification reactions using Dm238 DNA and purified recombinant
bacteriphage 7t DNA from clone RpD, respectively. Lanes with M represent the
123 bp DNA ladder as molecular size markers.

 

H1214

 

138

 

Figure 18

139

Figure 19. Nucleotide sequences of the ITSI and IT82 from Rhagoletis pomonella and
pairwise comparison with their counterparts from Drosophila melanogaster.
The GAP sequence alignment program in the GCG package was used to
perform this pairwise comparison. GAP penalty parameters (factors) were set
at: gap weight = 1.00; gap length weight = 0.10. Bars represent nucleotide
identities and gaps are represented by dots. Panels a and b are for ITSI and
ITS2, respectively. The underlined region in Panel b represents part of a
constrained structural element conserved between Rhagoletis and Drosophila,
and discussed in Chapter II. R. pom = R. pomonella and D. mel = D.
melanogaster.

11.9001
D.rne11
40
51
82
100
116
150
162
200
210
250
258
299
308
348
352
395
399
439
432
489
481
534
527
583
575
630
617
677
667

719

Figure 19A

140

TTGTGT....TCCTATCCGT..AAAAATAT ..... ATTATATRTATATCT
IIII I IIII IIII I IIIIII IIIIII I III
TTGTATAATATCCTTACCGTTAATAAATATTTGTflATTATACAAATAAAA
CATATATATAATAATAATTAATATATATATAT.ATT ....... ATAAAAA
II II I IIIII IIIIII I II III II IIII
ACAATTTACCAAAATAA.AAATATAACAAAATGATTCCATGGAATCAAAA

...AAGAAAAAAATA....GAAAA....CTATATTT.TATAGAT....TA
II I IIIIII III I I IIIII IIIII I I
GTTAAAATCAAAATAAAACGAAGATGGGTTTTATTTATATAGTTAGTGTG

GAAGATATAAAGCACAACATATACTTATAAC...TCTAGT.TTTCTTTTT
I I II I II I I II IIII IIII I I I II I
GGGCTTGGCAACCTCATAAAAAGATTTTAACATTTCTAATGTATGTTGTG

GTTCTTTTCATCCAATTTGTAAAATAA..ACTTTTTTTGTTGTTATTATT
I III I I I II II II I III I I II II
CGTATTTCTGGCGAGTACTTACAACAACGGCGTTTCCTATAAAAATAATG

GTTAAAATATATACATATAATATATATA..TATATAATACATTCTTTAAA
II II II I II II I I I I I I I I I II II
TTTCGAACATGAAAATCGAAGAAACAAAATTCGAAAGTGGA.AGTCGAAT

CAAATTTCAATGTTTTTCTTTTTTTTCTTTTCATTTACTTACCTTTGTAA
IIII I III III I I I I IIII I II I I III
CAAAATAAAATAATTTCGAATGTGTGGTAATCATCGAAATAAGTGT.TAA

TGCATTAGG ...... GCAAAAAAATTTGTGAATTTGCATACATTGAAAAT
I I I II II II III IIIIII I III II
TATAATTGGTAGATATTAACTAATTTTTAAAATTTGTGTGTATT...TAT
GAAAATGATGAAAC.CTTTAAAACATATATAGTTGTACTCATTATTTA..
I II I II I II I IIII II IIII I III III
TACTAT....ACACGCGTTCCGA.ATATGTA.TTGTTCATCTTAGTTATG

.GTAT.CGAT.TCTAA ......... ATG..ATAAGTTAATTTGT...TCG
I II II I IIII II IIII I IIII I I
GGCATACGTTGGCTRATGCAACAACCTGAAATAAACAATGTTGTACCTGG

CATTAACGCGTATTTTCTTTTCTGCTTAATTAATAATATTATAT.ACACG
III I I I I IIII I II III I III I III
CATCCATCAGGTTAATGTTTTAT ..... ATRAATTGCAGTATGTGTCACC

CATATATAGAGTA....GTATTCAAAAATAAAAAGAAGGAAAATACGGTT
II I IIII I II III I I I I I I III I I
CA.AAATAGCAAACCCCATAACCAACCAGATTATTRTGATACATAATGCT

TTTGTG.GACTAAGACA.TGCGCAACTTGGAAATGTTTGGGTTTAAAATT
I I II IIIIIIIII I IIIIII I I III III II II I

TATATGAAACTAAGACATTTCGCAACATTTA...TTTTAGGTATATAAAT
ATRATTTGTTGAAAGAATCATCTTATAT ........ TATTTTATATTTCA
I IIII IIIII IIII I IIIII II I IIIII
A.CATTTATTGAAGGAAT..TGATATATGCCAGTAAAATGGTGTATTTTT
AATTTACTCTTTCAATAAATAAAGAAGAATGACATACAAATGAAAAAATA
IIII IIIIIIIIIII III I I IIIIII II IIIIII
AATT...TCTTTCAATAA..AAACATAATTGACAT...TATATAAAAATG
AAAAATAAATTATC 680

II IIII

AATTATAA ...... 726

39

SO

81

99

115
149
161
199
209
249
257
298
307
347
351
394
398
438
431
488
480
533
526
582
574
629
616
676
666

718

R. pornl

D. mel 1

Figure 19B

51

37
101

76
151
111
201
148
251
181
293
231
343
272
393
314
442

355

141

AGACTAT‘GCTMATMGTPGCTTAWAGTAMATMGAGAATPCA
IIIIIIIIIIII IIIIIIIIIIII IIII I I
AGACTATGCTM'I'I‘MG’I‘I‘GCTTAT .......... AAA? . . . . T'I'I‘TATA

AGCATATGGTATAT'PA'I'I‘GGA‘I'I‘P’I'I‘GT’I'I'I‘ATA'I‘T'I‘ATATAMATTTM
IIIIIIIIIIIIIIIIIIII III IIII II III I
AGCATATGGTATA’I‘TAT'I‘GG ........... ATAMTATMTM'I'I‘T’I‘TA

TCCATMTGNMTAGWGAMTMATAMAN‘PCTI‘GMATCC
IIIIIII II IIIIII IIIIII II III III
'I'I‘CA'I‘AATAT’I' ....... WTAMWCATT ....... AT . C

TCTCAATGAMTC‘I'I‘ATAAAAMGM’I‘C'I'CAGTAT'I‘CCCTTMGAC‘I'I'CA
IIIII III I IIIIIIII I IIII III
TCACATT’I‘GAA'I‘GT . . GMACGAAGAGAAATATT .......... 'I'TC .

50
36
100
75
150
110
200

147

GCANAWWMTAMWTAATATATATGAW 2 SO

IIIIII IIIIIIIII III IIIIIIIII
..T'I'I'P'I‘CAA ....... TCAMTAATA ...... CW

W-TATHAHATA
IIIIIIIIIII lIlIII IIIIIIIII III IIIIII II

WANMTGATP

TATTTATA'I'I‘AATATCCGGA'I‘AATG'I'I‘GA’I'I'I‘T'I'I‘GCGTATATATATATA
III II III IIIIIII III I III I
CGGAAATAGAAAAATCT'I‘GGT'I‘ATG’I'I‘A'I‘I‘ATTC’I‘I‘CGT ......... TG

TATATATMTAATTATTTPAT‘PAATAMAMCGGGATGAMAGGW
I IIIII I IIIIIIIIII I I III I
GT'I‘CG'I'I‘AAAAA . . . . TGGATAAATMAMCT‘I‘I‘GCATACMGMT . . . .

'I'P'I'I‘CTMTACGATMAATA'I'I‘ . TCMGAM’I‘A'I‘A‘I‘TA’I'I‘TC‘I'I‘TGAACA
IIIII IIIIIIIIIIIII IIII IIII I II
..... TAATA . . . .AMATG'I'I‘ATAACGAA'I'I‘TAATTAAATGT'P‘I'I‘ATCA

MGTWTTATATA'I'I‘TMAATAATA 47 5
III III IIIIII II IIII
T'I'ATATATAAAGAA'I'I‘TAT . . . GGCAAGA’I‘AAAG 3 8 S

180
292
230
342
271
392
313
441

354

l 4 2
Future Direction

The construction of a genomic library for R. pomonella has been successful. The
primary characterization of the positive clones indicate that the inserts are indeed rDN A
fragments. The ITS sequences from one of the clones further confirmed the presence of
rDNA in that positive clone. The large insert size of the clones suggests that it is very
possible that the complete repeating rDN A unit has been obtained, at least in some of the
clones if not all. However, since this is only a preliminary investigation of the rDNA of R.
pomonella, more detailed characterization needs to be done in order to determine which
clone has which part of the rDNA. Accordingly, the desirable clones may be further
subcloned into plasmid vectors, allowing one to sequence the IGS/ETS boundary region,
together with the whole ETS , with a primer matching the 5' end of 18S. The sequence
information obtained for the transcription initiation region, together with other available
sequence information such as those of Drosophila and Glossina for the same region, will
help to design the primers for PCR amplifying the ETS region of Rhagoletis flies and
further conduct phylogenetic analysis of closely related species or host races in the genus

Rhagoletis.

BIBLIOGRAPHY FOR CHAPTER IV

Cl

C1

Pu

Ga

Ge

Ha

Lt

 

 

143

Bibliography for Chapter IV

Amheirn, N. 1983. Concerted evolution of multigene families. Pp. 38-61 in M. Nei and
R. K. Koehn, eds. Evolution of genes and proteins. Sinauer, Sunderland, Mass.

Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and
K. Struhl. 1987. Current protocols in molecular biology. John Wiley and Sons, New
York.

 

Coen, B., T. Strachan, and G. Dover. 1982. Dynamics of concerted evolution of
ribosomal DNA and histone gene families in the melanogaster species subgroup of
Drosophila. J. Mol. Biol. 158217-35.

Cross, N. C. P., and G. A. Dover. 1987a. A novel arrangement of sequence elements
surrounding the rDNA promoter and its spacer duplications in tsetse species. J. Mol.
Biol. 195:63-74.

Cross, N. C. P., and G. A. Dover. 1987b. Tsetse fly rDNA: an analysis of structure and
sequence. Nucleic Acids Res. 15:15-30.

Furlong, J. C., and B. E. H. Maden. 1983. Patterns of major divergence between internal
transcribed spacers of ribosomal DNA in Xenopus borealis and Xenopus laevis, and of
minimal divergence within ribosomal coding regions. EMBO J. 2:443-448.

Gale, K., and J. Crarnpton. 1989. The ribosomal genes of the mosquito, Aedes aegypti.
Eur. J. Biochem. 185:311-317.

Gerbi, S. A. 1985. Evolution of ribosomal DNA. Pp. 419-517 in R. J. MacIntyre, ed.
Molecular evolutionary genetics. Plenum Press, New York.

Hadjiolov, A. A. 1985. The nucleolus and ribosome biogenesis. Springer Verlag, New
York.

Long, E. O., and I. B. Dawid. 1980. Annu. Rev. Biochem. 49: 727-764.

Procunier, W. S., and J. J. Smith. 1993. Localization of ribosomal DNA in Rhagoletis
pomonella (Diptera: Tephritidae) by in situ hybridization. Insect Mol. Biol. 2: 163-174.

Sarnbrook, J., E. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual,
2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-
terrninating inhibitors. Proc. Natl. Acad. Sci. USA 12:5463-5467.

144

Schlotterer, C., M. Hauser, A. von Haeseler, and D. Tautz. 1994. Comparative
evolutionary analysis of rDNA ITS regions in Drosophila. Mol. Biol. Evol. 11:513-
522.

Sollner-Webb, B., and J. Tower. 1986. Transcription of cloned eukaryotic ribosomal
RNA genes. Annu. Rev. Biochem. 55:801-830.

Tautz, D., C. Tautz, D. Webb, and G. A. Dover. 1987 . Evolutionary divergence of
promoters and spacers in the rDNA family of four Drosophila Species: implications for
molecular coevolution in multigene families. J. Mol. Biol. 195:525-542.

Tautz, D., J. M. Hancock, D. A. Webb, C. Tautz, and G. A. Dover. 1988. Complete
sequence of the rDNA genes of Drosophila melanogaster. Mol. Biol. Evol. 5: 366-376.

Dover, G. A., and R. B. Flavell. 1984. Molecular coevolution: DNA divergence and the
maintenance of function. Cell 38:622-623.

Williams, S. M., R. DeSalle, and C. Strobeck. 1985. Homogenization of geographical
variants at the nontranscribed spacer of rDNA in Drosophila mercarorum. Mol. Biol.
Evol. 2:338-346.

CHAPTER V
PARTIAL CHARACTERIZATION OF THE EXTERNAL TRANSCRIBED
SPACER REGIONS OF RHAGOLETIS CINGULATA

Introduction

Insect sibling species are difficult to distinguish morphologically. This is even
more often the case with the genus Rhagoletis since many species have the ability to rapidly
shift hosts from native wild plants to introduced cultivated crops. As a consequence,
serious pest species with little or no morphological difference can arise in a relatively short
period of time under sympatric conditions. Taxonomist and systematists are often
challenged to seek alternative markers rather than morphological characters to detect and
differentiate such species and to infer their phylogenetic relationship. Accurate
identification of such sibling species is essential for appropriate management of these pest
species. Furthermore, the correct assessment of genetic differences among the sibling
species could be useful in our understanding of their evolutionary history, which in itself is
necessary for testing various evolutionary theories regarding speciation.

In the past few years, as a candidate of alternatives to morphological method,
nuclear rDNA spacers, especially the ITS sequences, have been applied to differentiate
morphologically-indistinguishable species (Porter and Collins 1991) or subspecies (V olger
and DeSalle 1994) and infer phylogenetic relationship among closely related species (Pleyte
et al. 1992; Wesson et al. 1992; Schlotterer et al. 1994). In chapter 11, I carried out a
similar study on the R. cingulara species group, in which there are few morphological
differences in the four North American species. However, except for a few potentially

useful molecular markers to differentiate some of the species, the ITS sequences seem not

145

l 4 6
to display sufficient interspecific variations to confidently infer a phylogenetic relationship

for this group. Moreover, between the two cherry-infesting species (R. cingulata and R
indifferens) only one potential molecular marker (position 371 in ITS2) existed in the
whole ITS regions. Since several other species groups in Rhagoletis, such as the
pomonella and tabellarr‘a groups. also consist of a number of morphologically hard-to-
distinguish sibling species (Chapter I), alternative methods need to be devised to
distinguish them accurately.

In order to find a suitable gene marker for distinguishing sibling species and/or host
races in Rhagoletis and for infening the phylogenetic relationship of closely related species
in this group, I explored the same non-coding regions of rDNA as in Chapter IV, namely
the ETS and IGS (see Figure 13 in Chapter IV), but using a different approach. Because
the IGS/ET S region is flanked by highly conserved rDNA coding regions, I took advantage
of published sequences of distantly related organisms to design primers for PCR
amplification of this highly variable region in Rhagoletis. Previous characterization of the
IGS/ET S region in eukaryotic organisms were exclusively performed by the long drawn
out procedures involving genomic library construction and screening. For the purpose of
identifying species and phylogenetic analysis, which usually requires the estimation of
intra- and inter-individual variations based on a large sample size of the same species, a
simpler technique using PCR to obtain the DNA fragment(s) of interest must be developed.

The objectives of this study were 1) to design suitable primers, according to
published sequences from other organism, which matches the 3’ end of 28S and 5’ end of
18S of Rhagoletis rDNA (i.e., flanking the IGS/ET S region); 2) to optimize conditions for
amplifying the rDNA spacers of interest in Rhagoletis; 3) to clone the PCR products; 4)
partially sequence the clones from both ends and verify that the appropriate region was
obtained by comparing with published sequences of other organisms; and 5) to assess the

value of the ETS region for phylogenetic reconstruction and species recognition.

l 4 7
I obtained two types of clones from PCR amplification products of R. cingulata

flies on different host plants; approx. 2.6kb fragment from a fly on sour cherry and 3.9kb
fragment from a fly on wild black cherry. The two types of clones were partially
sequenced from each end and both types of clones appear to have the ends matching the 3’
end of 28S and 5’ end of 18S, indicating that both types of clones may contain the IGS and
ETS of R. cingulata. However, the sequences of the two type clones are surprisingly
diverged, even at some regions within 3' end of the 28S gene. Whether the observed

divergence is related to their different host origins remains to be determined.

Materials and Methods

Biological Material

R. cingulata larvae were collected from two different field infested host plants,
Prunus cerasus (sour cherry; from Hart, MI) and Prunus serorina (black cherry; from
Roselake MI) during 1988-90. Larvae were allowed to pupate in fine moist vermiculite.
Pupae were sifted from the vermiculite and subsequently stored at 4°C. After at least 5
months, pupae were removed from the cold and held at 22°C, under a 15:9 hr lightzdark
cycle to terminate diapause. Within 2-7 days after emergence most adult flies were frozen
at -70°C for subsequent genomic DNA isolation. Specimens from each collection were

pinned for species identification.

DNA Isolation and Amplification

Total genomic DNA was isolated from a single female R. cingulata fly reared from
black cherries and a pool of 30 R cingulara flies reared from sour chenies respectively as
described by Procunier and Smith (1993). In addition, po238 (provided by Dr. G. A.
Dover), which contains the complete D. melanogaster rDNA repeat unit, was used as a
positive control for the PCR amplification reactions and also as a test for the accuracy of the

sequencing procedures in this study. The region between the 283 and 18S subunits of

l 4 8
rDNA, which includes the IGS and ETS sequences, was amplified using the following

primers:

GB16 5'GT'ITAGACCGTCGTGAGACAGGTTA (matching the 3' end of 28S),
GB4 5'AGACATGCATGGCTTAATCTTI‘GAG (matching the 5' end of 18S), or
GB4' 5'ACAAGCATATAACTACTGGCAGGAT (matching the 5' end of 18S).

The sequences for the primers GB4 and GB 16 were chosen after aligning the 18S
and 288 regions of the several different organisms (see section below; Figures. 2 and 3a)
and searching for highly conserved regions in the 5' end of 18S and 3' end‘of 288 genes,
respectively. Amplification by the polymerase chain reaction (PCR) was carried out in 25
pl (final volume) containing 33.5 mM Tris-HCI, pH 8.8, 8.3 mM NH4SO4, 3.35 mM
MgC12, 3.35 M EDTA, 15.0 M mercaptoethanol, 10% DMSO (Fluka), 160 pg/ml BSA
(Boehringer Mannheim), 1.25 mM of each dNTP, 0.1-0.4 p.M primer GBl6 and 0.1-0.4
p.M primer GB4, and 2.0 units of Ampli Taq DNA polymerase (Perkin Elmer Cetus).
Amplification parameters were 950C for 7 min; 35 cycles each with 940C for 1 min, 60°C
for 1 min and 650C for 8 min; and 650C for 15 min. Amplified DNA was subjected to
electrOphoresis on a 0.8% agarose gel and visualized with ethidium bromide. Bands
containing the DNA were excised from the gel and the DNA purified using the Prep-A-

Gene DNA purification matrix (Bio-Rad), according to the manufacturer's instructions.

Cloning and Sequencing

The TA Cloning kit (Invitrogen) was used in a one-step cloning strategy for the
direct insertion of the purified PCR products (see earlier sections) into a plasmid vector,
followed by transformation into competent cells. Plasmid vector and competent cells were
supplied by the manufacturer. Clones were randomly selected and stored as frozen stock at
-80°C. Plasmid DNA was purified from individual clones using the Magic-Prep DNA
purification kit (Promega), following the manufacturer's instructions. DNA sequencing

was performed according to the chain-termination method of Sanger et al. (1977), and

l 4 9
using the Sequenase Version 2.0 DNA sequencing kit (USB) and 358-dATP (Amersham).

Vector primers NB7 5'AATACGACI‘CACTATAG and NB 11
5'GTCATAGCTG I'I'I CCI‘ G were employed to determine the DNA sequence.

Computer Software and Analyses

Several programs, such as FETCH, LINEUP, PILEUP, MAPSORT, SEQED and
STRINGSEARCH, in the GCG software package of the University of Wisconsin Genetics
Computer Group (UWGCG package, version 8.0) were used in this study. The 288, 188,
IGS and ETS regions of rDNA from Aedes albopicrus (mosquito; mqsmagn.gb_in),
Daphnia pulex (water flea; daprrnaspa:gb_in), Drosophila melanogaster (fruit fly;
pdm238.gb_in), Glossina morsitans (tsetse fly; gmednal.gb_in); Lytechinus variegatus
(sea urchin); Rattus norvegicus (rat; mrgm4a.em_ro), Saccharomyces cerevisiae (yeast);
Tenebrio molitor (beetle; trnrn183.gb_in); and Xenopus laevis (frog; xlrn01.gb_ov) were
found using STRINGSEARCH. Alignments were performed using first LINEUP and
then PILEUP, with gap weight set at 1.00 and gap weight length set at 0.10. Restriction
enzyme sites on the sequences were located using MAPSORT.

Results and Discussion

At the start of this study, two PCR primers, named GB4 and GB 16, were designed
according to the highly conserved 18S (5'end) and 288 (3' end) regions of several distantly
related organisms such as frog and water flea (Figure 20; Materials and Methods). Because
the negative control with GB4 alone gave nonspecific PCR products with 1 DNA (data not
shown), another primer, named GB4', adjacent to GB4 was also designed and used
concurrently. With the genomic DNA of pooled R. cingulara from sour cherry, a major
band of approx. 2.6 kb and a slow migrating minor band were observed after amplification
with GB4' and GB16 (Figure 21). Control amplification reactions using each primer

separately showed faint low molecular weight bands (Figure 21; lanes 1 and 2) the origin

 

 

150

Figure 20. Designing the appropriate primers for PCR amplification of the IGS/ETS
regions of Rhagoletis. Panel a shows a map of the region in question. Refer to
legend of Figure 13 (Chapter IV) for notations and abbreviations. The partially
sequenced regions of the two R cingulara clones (RC1-A and RC3) are
indicated by the solid line; regions not sequenced yet are denoted by the dashed
line. Panel b shows the alignment of a 3’ end region of the 28S gene from
various organisms, including the two R. cingulara clones from this study.
Panel c shows the alignment of the 5’ end of the 18S gene from various
organisms, including the two R. cingulara clones from this study. Nucleotide
positions are numbered according to the start of coding region of the respective

genes of Drosophila melanogaster. The PCR primers are underlined

151

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152

Figure 21. Products of PCR amplification of the IGS/ETS region of Rhagoletis cingulata

 

from sour cherry. Lanes 1 and 2 represent products from amplification using
only GB4’ and GB16 primers, respectively. Lane 3 represents the major PCR
product (approx. 2.6 kb band) obtained after amplification using GB4’ and
GB16 together. lanes M and M’ represent the HindIII-Pstl double digest of A
DNA and 123 bp DNA ladder as molecular size markers.

 

H123HM’

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1 Hill It.

 

Figure 21

l 5 4
of which may be the result of the amplification condition used in this study. Using GB4

and GB16, one major band of approx. 3.9 kb was obtained for the single fly DNA
preparation (R. cingulata from black cherry); in addition, using the same two primers, a
major band of approximately 5 kb was obtained from po238 (data not shown).

After preparative scale amplifications, purification of the major DNA bands and
cloning, seven clones from the single fly DNA preparation, named RC1-A through RC1-
G, two clones from the pooled R. cingulata DNA preparation, named RC2 and RC3, and
one clone from Pdm238, named Dml2, were randomly selected. The seven clones from
the single fly DNA preparation were partially sequenced from both ends using the vector
primers (see Materials and Methods). More than 200 nts were sequenced from both ends
of each clone. Two of the seven clones (RC1-A and -B) had identical sequences (only the
sequence of RC1-A is presented in Figure 22). Each of the other five clones, however,
had identical sequences at both of their ends (the sequence matched the 5' end of 18S
corresponding with GB4; data not shown). The latter results were surprising and may be
explained as follows: 1) they may be an artifact generated as a result of the amplification
step or 2) they may reflect the presence of high numbers of genetic inversions within the
rDNA sequences, in particular within regions including the 5' end of 18S. To my
knowledge, there appears to be no precedent for such high numbers of genetic inversions
within rDNA sequences in other organisms; however, this possibility cannot be ruled out at
this time. More detailed characterization and investigation was necessary to resolve this
perplexing set of results and deemed, at the present time, to be beyond the scope of this
study.

One of the two clones from the pooled DNA preparation (RC3) and the Dm 12 from
PDm 238 were also partially sequenced from both ends. The Dm 12 sequences were
identical with published data for Pdm238 (Tautz et al. 1987; 1988). The sequences from
RC1-A and RC3 were aligned, using PILEUP, with the relevant D. melanogaster regions
(Figure 22; Panels A and B). Several patches of well conserved regions with

 

 

155

Figure 22. Alignment of the sequenced regions from the two Rhagoletis cingulata clones
RC1-A and RC3 with homologous regions from Drosophila melanogaster.
Alignments were performed using PILEUP with gap weight = 1.00 and gap
weight length = 0.10. Panel a shows the sequences downstream from the I
GB16 primer (underlined sequence) position (i.e., the 3’ end of the 28S. Panel
b shows the sequences upstream from the GB4 and GB4’ primer (underlined
sequences) positions (i.e., the 5’ end of the 188). Conserved nucleotides and
gaps are denoted by dashes and dots, respectively. Unidentified nucleotides are
denoted by a question mark. Arrows denote the start and end nucleotides of the

188 and 288 coding regions, respectively.

 

 

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RC1-A
RC3

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RCl-A
RC3

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RC1-A
RC3

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RC1-A
RC3

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RC3

Duel
RC1-A
RC3

Duel
RC1-A
RC3

Duel
RC1-A
R03

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RC1-A
RC3

Duel
RC1-A
RC3

156

3' end of 288

 

 

 

 

10 20 3° 6° 50 6° 70 80 90 100
. . . . O U . C . .
- ----- -- ----c--- . . .G--A---A-C-TA-m-T-A--GAA-T -------------- .---'r'rc--'r----
--- - - '--'G--- . . .m--------A-T--T—A-m-T -------------- o r-rm-T'A"
110 120 130 1‘0 150 160 170 180 190 200
CCAAJGGCK.CIIIICTTa11cumGOGI‘C‘GTGGTITGICGCTIoCGTCCCTTGGAJ1I1GCCTGALCaCCTC1IAGGTCGTITCCGTGCTGCACTGCA
n-r---'r-moc-oc---c---'r--ac--A--c-cc--A----'r-A----c-A---A---c -------------- -ccA-A---'r----<:A-- .....
n-rn-‘r-mooc-cu-cr-«ncc- -cc- —ooc--A----c-A--'r-c---- c m-A----A----A-- .....
210 220 230 260 250 260 270 280 290 300
AIGAaIAAIIAGGGaCIATT1UCIa1c1IaGaCTTCTIAACCAITTIAAcTT1IIIITT1ICT1TIaIIACCACAASGGATGTGATGCCAATGT‘A111C
o o e e .-.”-e c e o om-e o o c e o .C"o"'m«'-o o o o 0"” o e 0-4-0-A.G--0 0 0A--. 0 o’T'fl'fl"”C'mm"o - -AT
o e c . .M’C‘m'4'. o o e e e .C"'C'm’. e e o ...-.6 o .“G‘C’C'G'TM’- I o--m-o o o o .--“-m.G-. e---
310 320 330 360 350 360 370 380 390 400
TIACAaIGTaAIlTGGGAGCAJCTTCGIICACCTCATGCCGCGCTIGTTiCAIItIAAAGCAT1aa11ISliCA‘flG‘Cifl‘GCCT’GA‘JCAATTCTIA
--. .G. e--.fl.-
--. om---.fl--
61° 62° ‘30 660 650 660 ‘70 690 490
ACCACTTTTCTIACAGGCAAGGTCTTaTIAG1GGTTCAGCAGCTGCCATiCTCCGATCCACTCAAGCTTIECCTTTGCTTGITGATTCCR

Ind of 288-1

E'I'S/S' end of 188

10 20 30 60 SO 60 70 90 90 100
GCGCTCGGTTTTI1GTTIJITIJTICCAGAGAGTTITITGAAAAGAGATAAAT11TIAATTTITCASCAAGATGCAAATCATTTmACTTWJITTTCGTTK
............. ----O. ....--...-..-C-............'.----....... ...-.----. 0
"T:w- -' IIIIIII 00-0-00-00.----oooo"c-c--m.G---ocoo-oo--A---ocoe ooooo --G-'
110 120 130 140 150 160 170 180 190 200
AACAIIRNTJn3UuuUIERIKIafl1CAAAAflTTITcTI3CTTUGAAAIaAAA1GATIITTTIGAA1GIAASIIaTGTIIIIITIAAGACAAAATTATAG
....-GCC-.-CA--T‘-A‘.............----.-A----- ....... ......C‘-'-TT‘GCC'--CG--.-'C--CC---- ............
C-rccccc-A-T-'GTch-G~-....CCC-C-CC’TC-A-GG----CCCC-...CC-CGC--T..G-CC--TG-CC-GGCGCC'- ..............
210 220 230 260 250 260 270 290 290 300
AAAIaISII1ICAAaII11GTI1CIIC11c1TGT1IaIITGC1IAAACAAGTIGIAITTIAAAITOGGIASICGAAS‘ICGHGTGCTIT’SIAAARTCGC
o e e e - .‘C"G‘. e o e I. o o’GA"’M“—CA-C‘j-". o--m'- o e u o .-----T.c on O’T‘-.fl-o-‘-c----e--e e e n o a n u e -- . n "'
..CC-CGGr-Tbo-.......o.°-C?GC-'C’CC??---TCAP-G--~-C-CA-C'C-...CG.CT‘-T‘T-.CC~C-G’CCA- ......... G-A--A
310 320 330 360 350 360 370 380 390 400
CGTIJTCGIITUGNTTEIT.o..TTT1ISIAAIITITTTIAAASTTTTICCCAAAGGCAAAAINTTGIATTICIITCAA:..TIATIIIAAAAAATCC‘A
o .----. e o e o 0 .---fl. 0 e 0---. e e o""’ e - .‘C-m'C---. I o o e u a oA‘T‘G--m"m.T-- e o o--. o em-‘---m-’cm- - o .
a-ccu'r- .c ----- m-acm-x-c-c-uc- . -Acc--c- . . . . . -A-T- -c--ooocc-c-crtc-ccc-c-c---ccocc-ccc-c . --c-c-c- .
61° 62° 630 660 450 660 ‘70 680 490 500
T1I3IIIJIAAGTUGAAII1CTI1IIaIIT1IJITTGCTTIT1TCAATTCAAAAAAIISGIATGAAAEIJGIAAAG..AAAACATTITTCTGGTTCATCC
os........---'1"....-.....o.......‘--m-""-...--..-T"C-'-.....C-‘C"C“m‘oea"T-G'm ...........
. . . .--c-c-c-ac-cu:Gc-Gsc-. . . . . . . .-GA-M-c-G-oc-c---Gc---- . . .c--cc--c-A'r'r-c--'r're'r'rc-r-c-n -------- __ --- -
510 520 530 540 I
- - - ° Start of 188
TGCCIGTIGT1IJImGCTTCTCTCAAAGRTTIAGCCAEGCATGTCT

 

 

Figure 22

1 5 7
D. melanogaster were observed immediately downstream of the GB 16 primer, such as

positions 26-42, 71-90 and 160-178 (Figure 22A). Actually these three patches were even
conserved among other organisms such as Aedes albopictus, Xenopus laevis, Daphnia
pulex and Ram norvegicus (Figure 20A; and other data not shown). Other well
conserved patches were observed immediately upstream of the GB4 and GB4’ primers
(positions 490-521 for RC1-A and 489-496 for RC3; Figure 22B). The T to C transition
detected at position 511 was also observed among other organisms (Figure 20C position
25). Therefore, the conservation observed at both ends of the inserts from clones RC l-A
and RC3, clearly indicates that at least one of the two clones, if not both, contains the
complete R. cingulata IGS and ETS regions.

Surprisingly, however, beyond the highly conserved regions mentioned above, the
partially sequenced regions of the two R cingulara clones appear to be considerably
diverged -- even the 3’ end of the 288 gene (Figure 22; Panels A and B). This was
unexpected given that these sequences are from the same species although from different
host plants. Compared with their sequence divergence in the ITS regions (about 0.2
percent nucleotide substitutions), the level of genetic variation between the two different
host-associated flies of R. cingulata seemed to be excessively high in those partially
sequenced regions. However, I detected considerable variation in the 3’ end of the 28S
(downstream of GB 16) among other organisms whose sequences were from the GCG
database (e. g., Drosophila, Aedes, Xenopus, Daphnia, Rartus; data not shown). Indeed,
the presence of variable regions at the 3’ end of the 28S gene (also called expansion
segments) has been reported for humans and Drosophila (Gonzalez et al. 1990; Linares et
al. 1991).

Expansion segments are absent in prokaryotic rRNA genes but are known to vary
considerably in length and sequence between different eukaryotic organisms (Linares et al.
1991). This fact has prompted the use of expansion segments for the resolution of

phylogenetic relationships between closely related species (Gonzalez et al. 1990). It is also

1 5 8
noteworthy that the two R. cingulara clones were different in length; RC l-A and RC3 were

about 3.9kb and 2.6kb long, respectively. Length variation in the IGS is probably due to
different number of subrepeats in IGS. Large amount of length variation in several IGS
clones from D. melanogaster have been reported and the variations arose from varying
numbers of an internal repeat of about 250 bp long (Coen et al. 1982; Sirneone et al. 1982).
Similar length differences were also noted for D. mercarorum; individual flies from
different geographic regions have characteristic length for the IGS which range from 4.0 kb
to 6.5 kb (Williams et al. 1985). Williams et al. (1985) proposed that those length
difference patterns may serve as markers for determining the geographic origin of
individuals. In addition, a Y chromosome-linked length variant in the IGS is also present
in D. mercatorum (Williams et al. 1985). Therefore, it is not yet clear whether the
heterogeneity in sequence and length between these two R cingulara clones is an artifact or
a reflection of inherent variation between host-associated populations.

The R. pomonella clones described in Chapter IV may help resolve this puzzle since
some of the clones, as mentioned in Chapter IV, very likely have the complete rDNA
repeating unit of R. pomonella . Upon subcloning the DNA fragments which contain the
appropriate R. pomonella IGS-ET S region, a direct comparison of this region with
homologous regions from the RC1-A and/or RC3 could be undertaken. Therefore, in the
future, the 3’ end of 288 as well as the IGS/ETS boundary region could be useful regions
for the resolution of the phylogenetic relationships between the host-associated populations

of R. cingulata.

BIBLIOGRAPHY FOR CHAPTER V

159

Bibliography for Chapter V

Coen, E., T. Strachan, and G. Dover. 1982. Dynamics of concerted evolution of
ribosomal DNA and histone gene families in the melanogaster species subgroup of
Drosophila. J. Mol. Biol. 158:17-35.

Gonzalez, 1. L., C. Chambers, J. L. Gorski, D. Stambolian, R. D. Schmickel, and J. E.
Sylvester. 1990. Sequence and structure correlation of human ribosomal transcribed
spacers. J. Mol. Biol. 212127-35.

Linares, A. R., J. M. Hancock, and G. A. Dover. 1991. Secondary structure constraints
on the evolution of Drosophila 28S ribosomal RNA expansion segments. J. Mol. Biol.
219:381-390.

Pleyte, K A., S. D. Duncan, and R. B. Philips. 1992. Evolutionary relationships of the
salmonid fish genus Salvelinus inferred from DNA sequences of the first internal
transcribed spacer (ITSI) of ribosomal DNA. Mol. Phylogenet. Evol. 1:223—230.

Porter, C. H., and F. H. Collins. 1991. Species-diagnostic differences in a ribosomal
DNA internal transcribed spacer from the sibling species Anopheles freeborni and
Anopheles hermsi (Diptera: Culicidae). Am. J. Trop. Med. Hyg. 45:271-279.

Procunier, W. S., and J. J. Smith. 1993. Localization of ribosomal DNA in Rhagoletis
pomonella (Diptera: Tephritidae) by in situ hybridization. Insect Mol. Biol. 2: 163-174.

Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-
terrninating inhibitors. Proc. Natl. Acad. Sci. USA 12:5463-5467.

Schlotterer, C., M. Hauser, A. von Haeseler, and D. Tautz. 1994. Comparative
evolutionary analysis of rDNA ITS regions in Drosophila. Mol. Biol. Evol. 11:513-
522.

Sirneone, A., A. de Falco, G. Mancino, and E. Boncinelli. 1982. Sequence organization
of the ribosomal spacer of D. melanogaster. Nucleic Acids Res. 10:8263-8272.

Tautz, D., C. Tautz, D. Webb, and G. A. Dover. 1987. Evolutionary divergence of
promoters and spacers in the rDNA family of four Drosophila species: implications for
molecular coevolution in multigene families. J. Mol. Biol. 195:525-542.

Tautz, D., J. M. Hancock, D. A. Webb, C. Tautz, and G. A. Dover. 1988. Complete
sequences of the rRNA genes of Drosophila melanogaster. Mol. Biol. Evol. 5:366-376.

Vogler, A. P., and R. DeSalle. 1994. Evolution and phylogenetic information content of
the ITS-1 region in the tiger beetle Cicindela dorsalis. Mol. Biol. Evol. 11:393-405.

160

Wesson, D. M., C. H. Porter, and F. H. Collins. 1992. Sequence and secondary
structure comparisons of ITS rDNA in mosquitoes (Diptera: Culicidae). Mol.
Phylogenet. Evol. 1:253-269.

Williams, S. M., R. DeSalle, and C. Strobeck. 1985. Homogenization of geographical
variants at the nontranscribed spacer of rDNA in Drosophila mercatorum. Mol. Biol.

Evol. 2:338-346.

. F'V-T'J-lz'. .‘It ua-FI I r' -

CHAPTER VI
CONCLUSIONS

In this dissertation, I have presented the complete ITS sequences of the 4 members
of the cingulara species group as well as that of R pomonella. The Rhagoletis ITS
sequences are highly A-T rich like those from Drosophila and beetles. I found low levels
of interspecific ITS variation in the cingulara species group, implying that ITS sequences
are of limited application in phylogenetic analysis of host-associated populations and/or
closely related sibling species. Between the two cherry infesting species (R cingulata and
R indifi’erens) there is only one potential nucleotide position that could differentiate them.
However, a few molecular markers have been described, which can be potentially useful
for distinguishing between the two olive infesting species themselves (R. osmandri and R.
chionanthr) and differentiate them from the two cherry flies species. The high sequence
divergence found between the cingulata group and R pomonella enabled me to use the ITS
regions for analyzing the phylogenetic relationship of taxa from different Rhagoletis species
groups. The overall computer generated secondary-structure models of the ITS sequences
were presented for the cingulara species group and R pomonella. Several highly
conserved secondary-structural elements were determined by FOLDRNA and comparative
analysis. One such element (12R4) seems to be conserved even among two distant
families, Tephritidae and Drosophilidae, which diverged in the Cretaceous period between
65 to 130 million years ago implying an important functional role for this structural-element
in the processing of precursor rRNA. The conserved ITS secondary-structural elements

have been helpful guide for increasing the accuracy of aligning regions based on homology

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rather than similarity which might be a consequence either of common ancestry or of

chance.

In addition, I have sequenced the ITS regions of 7 other Rhagoletis species, which
include comivora, completa, juniperina, fausta, electromorpha, basiola, and striatella.
Phylogenetic implications from this study are in partial agreement with some of the
previous studies on Rhagoletis phylogeny which use other methods, and partially
contradictory to these previous results. This study indicates that R. comivora belongs to
the pomonella group, supporting the placement based on morphology and karyotype
similarities but is not in agreement with the mtDNA analysis; R. juniperina was removed
from the rabellaria group, in support of allozyme and mtDNA analyses, and this species
may be more closely related to the pomonella group rather than the cingulata group. This
result is in agreement with the mtDNA data but not with the allozyme analysis. With
respect to the relatives of the cingulara group members of the suavis group rather than those
in the pomonella group appear closest, which is congruent with the mtDNA study, but not
with the allozyme results; R fausta may be related with the tabellaria group, which had
been left unplaced in previous studies; R. basiola and R striatella are most divergent in the
ITS sequences, correlating with their possession of several ancient morphological
characters and their basal branching in the analyses of mtDNA and allozyme data and
further supporting the idea that Rhagoletis may not be monophyletic. The application of
ITS sequences in the phylogenetic study of Rhagoletis has provided some insight into the
relationship between certain taxa from different species groups of this genus.

This is the first study of rDNA ITS sequences of the genus Rhagoletis. The study
of the ITS should be expanded in the genus Rhagoletis which could Open a new avenue for
the understanding of the evolutionary history of this genus and providing a reliable
phylogenetic framework to test some important evolutionary theories of speciation through
host shifting. In addition, the mode of ITS evolutionary divergence should be useful in

future investigations of the structure, function and processing of precursor rRNA. Future

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studies on the ITS of other Rhagoletis species will allow one to elucidate the degree of

functional and structural constraints on the ITS sequences in Rhagoletis.

Besides the studies on the ITS regions, I have also conducted preliminary
investigations on other noncoding regions of Rhagoletis rDNA. The construction of a
genomic library for R pomonella has been successful. The primary characterization of the
positive rDNA clones were substantiated by sequencing the ITS regions in these clones.
The large insert size of the clones suggests that it is very possible that the complete
repeating rDNA unit has been obtained, at lease in some of the clones if not all. However,
since this is only a preliminary investigation of the rDNA of R. pomonella, more detailed
characterization needs to be done in order to determine which clone has which part of the
rDNA. Accordingly, the desirable clones may be further subcloned into plasmid vectors,
allowing one to sequence the IGS/ET S boundary region, together with the whole ETS ,
with a primer matching the 5' end of 18S. The sequence information obtained for the
transcription initiation region, together with other available sequence information such as
those of Drosophila and Glossina for the same region, will help in the design of primers for
PCR amplification of the ETS region of Rhagoletis flies, and further aid in the phylogenetic
analysis of closely related species and host races in the genus Rhagoletis.

I also attempted to PCR amplify the IGS/ET S region from two R. cingulata flies of
different host plants. Two types of clones were obtained; one with an approx. 2.6kb insert
from the fly on sour cherry and the other with an approx. 3.9kb insert from’the fly on wild
black cherry. The two types of clones were partially sequenced from each end and both
types of clones appear to have the ends matching the 3’ end of 28S and 5’ end of 18S,
indicating that both types of clones may contain the IGS and ETS of R. cingulara.
However, the sequences of the two type clones are surprisingly diverged, even at some
regions within 3' end of the 28S gene. Whether the observed divergence is related to their

different host origins remains to be determined.

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