Multiple and widespread integration of mitochondrial DNA

sequences in the nuclear g n o m e s of geese

Branimir Gjetvaj

A thesis submitted to the Department of Biology

in conformity with the requirements for

the degree of Doctor of Philosophy

Queen's University

Kingston, Ontario, Canada

September, 1998

copyright O Brrinimir Gjetvaj, 1998

National Library Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographie Services seMces bibliographiques

395 W e l l i Street 395. rue Wellington OrtawaON K1AON4 OttawaON K1AûN4 canada canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/f&n, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts f?om it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

GJETVAJ, Branimir. Ph.D. Queen's University. Kingston. Ontario. Canada.

Septernber 1998. Multiple and widespread integration of mitochondrial DNA sequences in

the nuclear genomes of geese.

Supervising professor: Dr. Peter T. Boag

Sequences showing homology to a rnitochondrial DNA (rntDNA) fragment

containing the D-loop region (probe pmLSGûû3) have been found in the nuclear genomes

of five out of six goose species analyzed (Aves. Anserinae). In the Lesser Snow Goose

(Amer caenilescens caerulescens), there are about 3.000 - 5.000 copies of the 3.6 kb

insened element. The bulk of these sequences appear to result from a secondary

amplification, and are organized in a tandem m y in one location in the genome. Three out

of 233 ( 1.2%) of Lesser Snow Goose individuals are homozygously deficient for this

tandem array. This a m y was also identified in the genornes of the closely related Greater

Snow Goose (A. S. atlantica) and Ross Goose (A. rossii). The genomic DNA of these

three species did not contain sequences that would hybidize to two other mitochondrial

DNA probes containing ATPase subunit 6 (prnLSGOO 1 ) or cytochrome oxidase subunit 1

(pmLSG002). 1 found that White-fronted Goose nuclear DNA contains multiple fra, oments

that hybridized to d l three rnDNA probes. It appears that three separate re,' -ions

representing 60% of the goose mitochondrial genome have been insened in the nucleus of

this species through multiple and independent events. Brant and Canada Goose nuclear

genornes contain low-copy nurnber sequences that show homology to rntDNA probes

prnLSGûûI and pmLSG003. None of the mtDNA probes that I used hybridized to nuclear

sequences in the Emperor Goose.

1 present evidence thrit mtDNA-originating sequences are present in nuclear

genomes of various modern species of the subtarnily Anserinae. and suggest that these

sequences were tnnsferred and incorporated into the nuclear genomes on several occasions

along the evolution of goose lineaps. The implications for currently accepted goose

phylogeny are discussed. I funher show that Lesser Snow Goose populations spanning a

morph-ratio cline along the Gulf of Mexico coxst exhibit low genetic heterogeneity and high

levels of gene flow. Lack of barriers to gene flow in regard to rnorphological characters

and "species" recognition mechanisms may explain the presence of apparently similar

mtDNA-like sequences in Lesser / Greater. and Ross' Goose nuclear genomes. This study

supports the notion of promiscuity of mitochondrial and nuclear genomes in general. and of

frequent and continuous exchange of jenetic information between the mitochondrion and

the nucleus among nonpasserine birds.

This thesis is dedicated to the memory of rny father.

and to my rnother. for her courage

1 would like to thank Drs. Bradley White and Fred Cooke for taking me as a

graduate student. and for ail the help and suppon during the initial stages of this project.

Special thanks to Dr. Peter Boas for taking me under his wing when everybody else left.

and for overseeing this thesis to the end.

My gratitude goes to al1 graduate students and technicians at Queen's and McMaster

Universities who provided assistance. support. friendship and tons of laugh ter. I will

avoid to list al1 of them as 1 am afraid to forget any. Many thanks to Dr. Roger Doyle and

Doug Cook for help, and for allowing me to do some additional lab work at the Marine

Gene Probe Laboratory in Halifax. Doug was an exceHent teacher and 1 learned a lot from

him. Dr. Tim King was patient and supportive during the final (pûnic) stages of my thesis.

and for allowing me to analyze sorne of the data using faciiities at his laboratory in West

Virginia.

1 am grateful to Dr. Tom Quinn for providing Snow Goose mDNA and single-

locus DNA probes. and to the following people for providing the samples or helping with

the sample collection: R. Helm. T. Quinn, L. Lougheed, H. Bell, D. Rechard, G.

Linscombe, R. Bisbee, J. Crabtree. D. and H. Lobtres, H. McKay, M.Cronen, A. Reed.

and G. Gauthier.

Many thanks to Dr. Marianne Douglas for cawing up, and beating me with the

Thesis Stick. I never befieved thrit a piece of driftwood can hurt so much.

And tïnally. thanks to my mom for being patient. supponive. and for living through

a wu- without her younger son.

Abs tract

Dedication

Acknowledgments

Table of Contents

List of Tables

List of Figures

List of Abbreviations

General Introduction

Chapter 1 : Mitochondrial genes in the nucleus

1.1 Generd features of animal mitochondrial DNA

1.2 Transfer of mitochondrial genomes to the nucleus

Provision of a genetic element within the donor genome

Transfer and integration of genetic information

Activation and regulation of the tnnsferred sequences

Loss of mitochondrial gene copy after it becomes redundant

1.3 Mechanisms of transfer and integration of mtDNA sequences

1.4 Gene content of inserted rntDNA sequences

1.5 Sequence divergence between mtDNA and nuclear copies

1.6 Implications for population and evolutionary studies

Chapter 2: Materials and methods

Samples

DNA extractions

Restriction digests. electrophoresis. and hybridiziitions

Mitochondnal DNA sequencing and sequence database search

Chapter 3: Mitochondnal DNA in nuclear genomes of geese

3.1 Identification of mtDNA sequences in the Lesser Snow Goose genome

3.2 Copy numbrr of the 3.6 kb element in the Lesser Snow Goox genome

3.3 Phylogenetic distribution

3.4 Gene content of Lesser Snow Goose mtDNA probes

I V

v

vi --.

V l l l

i x

X

Chapter 4: Genetic structure of Lesser Snow Goose populations dong

a colour-morph cline

4.1 Introduction

4.3 Materials and methods

Samples

DNA extractions. restriction digests, hybtidizations and data analysis

4.3 Results

Scoring of RFLP polymorphisms. and levels of DNA variation

Testing for Hardy-Weinberg equilibrîum. and linkage disequil ibrium

Allele frequency distribution. and measures of genetic differentiation

4.4 Discussion

Chapter 5: Discussion

5.1 mtDNA sequences integrated in the nuclear genomes of geese

5.2 Goose phylogeny and possible evolutionary origins of the transposed

mtDNA sequences

5.3 Content of transposed mtDNA sequences

5.4 Repeated character of the 3.6 kb nucDNA fragment and its presence

in natural populations

Summary and Conclusions

References

Vita

vii

LIST OF TABLES

Table Page

1 Overview of the presence of mtDNA sequences in nuclear genomes 13 of multicellular animals

2 Gene content of Lesser Snow Goose mtDNA probes 45

3 Lesser Snow Goose sampling locations and number of saraples 52

4 Restriction fragment length polyrnorphism identified by 6 DNA probes 56

5 List of loci with significant deviation frorn Hardy-Weinberg equilibrium 60

6 Cornparison of allele frequencies and test of population differentiation 63

7 Cornparison of allele frequencies and test of colour morph differentiation 65

8 Exact test of population differentiatîon, and pairwise FST values 67

Figure

Southem blot of Lesser Snow Goose DNA probed with a mtDNA probe pmLSG003. showing repetitive character of the inserted fra, ornent

Screening for the presence of nuclear homolog in the Snow Goose

Southem blot with a minisatellite-like banding pattern

Screening for the presence of nuclear homolog in six species of geesr

Southem blot of Brant Goose DNA digested with several restriction enzymes and hybridized with pmLSG003

Sequence alignment of clone pmLSGûû 1 with chicken and duck mtDNA

Sequence alignment of clone pmLSGûû2 with chicken and duck mtDNA

Origin of three Lesser Snow Goose mtDNA probes

Map with sampling locations

Restriction fragment length pot ymorphism in Lesser Snow Geese detected with DNA probe DQSG- 1

Allele frequencies of 8 loci across populations

Mode1 of the possible evolutionary origins of mtDNA-like sequences in nuclear genornes of geese

Page

32

34

36

38

40

43

44

46

53

58

62

76

The abbreviations used in this thesis folIow those listed in the Proceedings of the National Academy of Sciences U.S.A. 84. vi-vii. with these additions:

bp

kb

mtDNA

nucDNA

RFLP

rRNA

SDS

tRNA

base pair

kilobase pair

rnitochondrial DNA

nuclear DNA

restriction fragment length polymorphisrn

ribosomal RNA

sodium dodecyl sulfate

transfer RNA

The compact and economical animal mitochondrial genome (Attardi 1985) is the

result of a gradua1 transfer of genetic information from organelle to the nucleus. a process

impl ied in the endosymbiont hypothesis (Margulis 1970; Gray 19%). A massive transfer

of proto-organellar genes streamlined the genome of a prokaryotic endosymbiont that

invaded an ancient eukaryotic cell. However, the recent transfer of rnitochondrial DNA

sequences (mtDNA) and their integrrition into nuclear genomes has k e n reported for an

increasing number of modem plant (Blanchard and Schmidt 1995). and animal taxa

including crustaceans (Schneider-Broussand and Nigel 1997). insects (Gellissen et al.

1983: Sunucks and Hales 1996). sea urchins (Jacobs et al. 1983). and various vertebrates

(Sorenson and Fleischer 1996: Lopez et al. 1997; Kidd and Friesen 1998; Zischler et al

1998: and references in Zhang and Hewitt 1996). Sequence analysis of various

mitochondrial genes and corresponding nuclear copies suggest that. in a few organisms.

such transposition events have occurred on multiple occasions (Fukuda et al. 1985: Nugent

and Palmer 199 1 ; Hu and Thi tly 1994. 1995; Collura and Stewart 1995: Sorenson and

Fleischer 1996; Sunnucks and Hales 1996). Hence, the mechanism responsible for the

streamlining of the mitochondrial genome may still remain intact. and the transfer of DNA

sequences from mitochondrion to the nucleus may not only be a historical event but also an

ongoing process.

Partial or complete sequences of at lem eight of the thineen rnitochondrial protein-

coding genes. both ribosomal RNA genes, and the control region have been inserted in

animal nuclear genomes (reviewed in Zhang and Hewitt 1996). Nuclex copies may be

inserted in apparently rmdom locations with low-copy numbers of integated fragments

(Jacobs et al. 1983; Smith et al. 1992). dispersed throughout the nuclear genome

(Gellissen ct al. 1983; Zullo et al. 1991 : Fukuda et al. 1985; Kamimura et al. 1989). or

mriy form more than 300 - 600 kb of localized. tandemiy repeated arnys (Lopez et al.

1994). In locusts. aphids. birds. rats. and hominoids the rntDNA-like sequences are

present as multiple copies (Gellissen et al. 1983; Zullo et al. 1991 : Hu and Thilly 1994:

Collun and Stewart 1995; van der Kuyl et al. 1995: Sunnucks and Hales 1996: this

study). Different molecular mechanisms were inferred in different inteeration events

(Blanchard and Schmidt 1996). More extensive studies on mechanisms of transfer need to

be conducted.

Detection of organisms where transfer and integntion of mitochondrial DNA

sequences into the nucleus took place, provides an opportunity to study mode and relative

rates of evolution of mitochondrial and nuclear DNA. In organisrns where sequence data

are available. different patterns of sequence evolution between the originating mitochondrial

genes and their nuclear copies were observed. Mitochondrial sequences insened in the

nucleus evolve at a nearf y equivaient (and presumabl y selectivel y neutral) "pseudogene"

rate and are often referred to as "nuclear fossils" (Zischler et al. 1995; Perna and Kocher

1996). while the original mitochondrial genes evolve at different rates depending on the

constraints of their function (Lopez et al. 1997). Better understanding of the relative rates

of evolution of mtDNA (and corresponding nuclear DNA) has a paramount importance for

the appropriate use of such sequences in evolutionary studies (Hoeh et al. 1995; Lopez et

al. 1997).

The presence of mitochondrial sequences in the nuclear environment could prove

to be an obstacle when mitochondrial DNA is used for phylogenetic and population biology

studies (Smith et al. 1992: Quinn 1992: Zhang and Hrwitt 1996). This is panicularly

relevant to studies that use the Polymerase Chain Reaction (PCR) amplification of target

DNA sequences wi th "universal" or degenerate pri mers ( Kocher et al. 1 989). Paralogous.

nuclear-encoded mtDNA pseudogenes could potentially be amplified by PCR primers

drsigned on sequences from related taxa. thus produc ing erroneous resul ts in ph y logenet ic

or population analyses (Colluri et al. 1996: Zhang and Hewitt 1996). Clear knowledge on

the presence and phylogenetic distribution of nuclear homologs is thus necessa-.

The rnajority of studies dealing with mtDNA transpositions have concentrated on a

single species. However. nuclear insertions involving the same mtDNA regions can be

observed in related taxa. Widespread phylogenetic presence of transposed mtDNA

sequences has only recently k e n documented in birds (Arctander 1995: Sorenson and

Fleischer 1996: Kidd and Friesen 199%). and a variety of other taxa (Hu and Thilly 1994:

Lopez et al. 1994. 1996; Collura and Stewart 1995; Sunnucks and Hales 1996; Zischler

et al. 1998). Furthemore. only a few studies have paid attention to the Ievel of variation in

nuclear inserts on a population level (Zischler et al. 1995; Sorenson and Fleischer 1996).

Mitochondrial DNA transpositions which appear to occur frequently and have broad

phylogenetic distribution allow a study of molecular mechanisms of gene duplication.

transfer and integration of exogenous DNA, and in addition provide an insight into the

evolutionary history of the mitochondrial genorne over long evolutionary timescales (Hu

and Thilly 1994).

The detection of mtDNA-like sequences in the nuclear genome of Lesser Snow

Goose (Quinn and White 1987) raised a question of whether such sequences are present in

other, ctosely related species. A distribution of the mtDNA inserts that are localized and

presumably reptitive. lead to a hypothesis that the nuclear integrations occumd recently in

this species, and that the mtDNA integrations would not have widespread occurrence in

other species of geese. In this study 1 will: a) test for the presence of rntDNA sequences in

nuclear genomes of six species of geese (Aves. subfamily Ansennae) (the species analyzed

in this study were selected to include representatives of two genera of the subfamily

Anserinas: genus Branta (Canada Goose and Brant). and genus Anser (Lesser and Greater

Snow. Ross'. Emperor. and White-fronted Goose)): b) I will show that in the Lesser

Snow Goose. 3.6 kb nucDNA fragments containing mitochondrial inserts occur aï ri

homogeneous. tandemly repeated amy. as digestion with either md III, Barn HI, or Ava

I alorte recover identical sizcd fragments; c) I will show that the tandem anay of 3.6 kb

inserts has not yet reached fixation in Lesser Snow Goose populations. as scrrrning for the

presence of the inserted element reveaied that 1.2% of individuals are homozygously

deficient for the array; and d) based on the presence / absence of rntDNA integrations and

results of hybridization experimen~s. 1 will discuss the implication of this study on the

presently accepted phylogeny of Anserinae.

The Lesser Snow Goose is a dimorphic taxon. with gray ("Blue") and white

("Snow") plumage colour phases. Gray and white plumage foms of Lesser Snow Geese

were considered separate species as late as 1960s. Cooke and coworkers ( 1988) suggested

that the clinal distribution of the two colour phases dong the Gulf of Mexico coast was

formed due to a secondary contact and interbreeding after a Ions period of isolation in

allopatry . The extent of the inter "species" transfer of integrated mtDNA-Iike sequences

(i.e. between Ross' and Lesser / Greater Snow Geese) can be inferred from a study of

levels of genetic differentiation and gene flow between taxa that were recently considered as

separate species (Le. "Blue" and "Snow" colour morphs of Snow Geese). In this thesis. 1

will analyze population genetic structure of the colour-morph cline along the Gulf of

Mexico coast to uncover the extent of genetic differentiation between the two colour

morphs, and to determine the extent of gene flow between the populations dong the cline.

This study will document the presence of multiple integrations of separate mtDNA

sequences in nuclear genomes of geese. and show rhat mtDNA transpositions in birds are

probably much more common and widespread than previously thought. I will provide data

on the character, frequency and extent of the mtDNA insertions among a selected group of

Anserinae, which could have implications for the use of nuclear homologs in avian

evolutionary studies (Quinn 1992: Arctander 1995; Sorenson and Fleischer 1996: Kidd

and Friesen 1998).

CHAPTER 1: MITOCHONDRIAL GENES IN THE NUCLEUS

In this chapter I will present an overview of relevant literature regardin% transfer of

animal mitochondrial DNA (mtDNA) sequences to the nucleus. I will briefly outline the

general features of animal mtDNA. and review published research on the mechanisms of

gene transfer between mitochondrion and nucleus. proposed mechanisms of insertion. gene

content of tnnsferred rntDNA fragments in animals. and levels of sequence divergence

between the mitochondrial and corresponding nuclear sequences.

1.1 General features of animal mitochondrial DNA

Mitochondria are energy-producing celluIar organelles that contain their own

Denomes. with up to 10.000 copies of the organelle genome present per cell. The size of 3

the mitochondrial genome in rnulticellular animals (rnetazoa) varies by a factor of three.

from 13.8 kb (Okimoto et al. 1992) to 43 kb (La Roche et al. 1990). Usually, this size

difference is due to variation in the non-coding portion of the molecule (reviewed in

Harrison 1989. and Wolstenholme 1992), with occasional large-scale duplications of

coding and non-coding portions of the genome (Moritz and Brown 1987; Hyman et al.

1988; Wallis 1987; Zevering et al. 199 1 ; Gjetvaj et al. 1992; Fuller and Zouros 1993;

Hyman and Beck-Azevedo 1996: Kumazawa et al. 1996). There appears to be no

correlation between mtDNA size and taxonomic relationship. The mitochondrial genomes

are present as circular molecules within the organelle cornpartment. with the exception of

cnidarians which possess one or two linear mtDNA molecules of 8 kb to 18 kb (Warrior

and Gall 1985: Bridge et al. 1992).

In contmt to the restricted size variation in animal mtDNA. plant mitochondrial

gcnonies Vary in s i x from 15.8 kb in the green alga Chlamvdomon~s reinhardtii (Gray and

Boer 1988) to over 367 kb in flowering plants. which feature many aspects of size-relaxed

nuclear genomes (Unseld et al. 1997). The large size of higher plant mtDNA c m be

accounted for by the presence of numerous introns. long open reading frames. gene

duplications. remnants of transposons of nuclear ongin and other imported foreign DNA

sequences. Fungal mtDNAs also exhibit large size variation. from 17.3 kb to 10 1 kb

(Paquin and Lang 1996; reviewed in Gray 1992). The mtDNA gene content in piants and

fungi is more conserved than the size variation,

Human. mouse, and cow mitochondrial genomes were the first to be completely

sequenced and analyzed for gene content (Anderson et al. 198 I . 1982; Bibb et al. 198 1 ).

Animal rntDNA molecules sequenced to date are found to be compact and efficient in

genome organizaiion. Most of the mtDNA molecules contain the same set of 37 genes: two

gertes for the RNA components of the mitochondrial ribosomes (ssu-rRNA and lsu-rRNA).

32 transfer RNAs (tRNAs), and 13 genes for enzymes or components of enzymes involved

in oxidative phosphorylation; cytochrome c oxidase (CO) subunits 1. II, and III. ATPase

subunits 6 and 8, respiratory chain NADH dehydrogenase components 1 through 6 and l L .

and cytochrome b. In addition. animal mtDNA molecules contain between 125 bp to

approximately 8000 bp of DNA sequence that Iack genes but inciude the molecule's ongin

of replication and DNA tnnscript promoters (Clayton 1992). The notable exceptions for

this conserved gene content are nematodes Caenorhabditis ele~ans, Ascaris suum, md

Meloidogme iavanica, and the bivalve Mytilus edulis (Wolstenholme et al. 1987: Okimoto

et al. 199 1. 1992; Hoffmann et al. 1992) which Iack the ATPase subunit 8. and the

octocoral Sarco~hvton elaucum which contains a gene for a homolog of MutS. a

component of bacterial MutSLH mismatch repair pathway (Pont-Kingdon et al. 1995.

I998). In Mvtilus edulis, there are 23 tRNA genes (Hoffmann et al. 1992). The additional

tRNA gene appears to splcify methionine. making mtDNA in this species unique in having

two ~ R N A M " genes. An Australian marsupial. the wallaroo Macroous robustus (Janke et

al. 1997). Iacks the completr sequrnce for the lysine tRNA sene. An alternative ~ R N A ~ ~ S

was not found within the wüllaroo mitochondriti. The mitochondrial genomcs in organisms

close to the base of the animal evolutionary tree are also the most unusual; the sea anemone

mtDNA (Metridium senile. Wolstenholme 1992) contains only two (RNA gnes which

specify tryptophan and formyl-methionine. Analysis of partial sequences of two other

cnidarian mitochondrial grnomes has also revealed the presence of only two (RNA genes.

Import of cytoplasmic tRNA molecules into mitochondrion must take place in these

organisms. in order for mitochondrial protein synthesis to proceed. Furthemore, the CO 1

and NADH 5 genes of sea anemone each contain a group 1 intron. These are the only

known introns in any animal mitochondrial genome.

In contrast to the gene content uniformity, the relative gene arrangement varies

widely in animals belonging to different phyla. Mitochondrial genomes appear to undergo

rearrangements on a time scale appropriate for resolving distant phy logenet ic relationshi ps.

and cornparisons of the relative order of mitochondrial genes has been used for evaluating

relationships among distantly related groups of organisms (Brown 1985; Jacobs et al.

1988b: Sankoff et al. t 992; Smith et al. 1993; Boore and Brown 1994; Boore et al.

1995; Kumazawa and Nishida 1995). The inferred mtDNA gene rearrangements include

both g n e transpositions and inversions. Perhaps the most stnking feature in animal

mtDNA is the abundance of tRNA transpositions (e-g. Wolstenholme et al. 1987:

Cantatore et al. 1988; PZibo et al. 1991). The proximity of tRNA genes to mtDNA gene

duplications in species where such mutations were observed, their apparent enhanced

mobility relative to other rnitochondrial genes. and their potential to form similar secondary

structures. suggest the possi bil ity that they might facil itate mt-genome rearran_oemen ts

(Cantatore et al. 1987). Transfer RNA genes have k e n implicated as serving aî insertion

sites for duplicated genes within the mtDNA molecule (Kumazawa et al. 1996). as well as

targeei sites for insertion of prokaryotic gnetic elements into a host genome (Reiter et al.

1989).

Mitochondrial gene order is usually conserved among animals belonging to the

sümc phylum. In anhropods. the mitochondrial gene order between Droso~hilo (Clary and

Wolstenholme 1985; Garesse 1988). Apis (Crozier and Crozier 1993). locust (McCracken

et al. 1987). and other organisms with known partial mtDNA sequences. differ on1 y by

Iimited tRNA gene transpositions. Among echinodems there is only one inversion

differentiating sea urchins frorn starfish (Himeno et al. 1987; Jacobs et al. 1988a;

Cantatore et al. 1989: Smith et al. 1993). Mitochondrial genes are arranged in the same

relative order in most vertebrates (Anderson et al. 198 1. 1982; Roe et al. 1985: Desjardins

and Morais 1890: Tzeng et al. 1992), although minor rearrangements are found in sea

lamprey . Petromvzon marinus (Lee and Kocher 1 995). the Amencan buIl frog. Rana

catesbeiana (Yoneyama 1987). several reptiles (Kurnazawa and Nishida 1995: Quinn and

Mindel1 1996; Macey et al. 1997), birds (Desjardins et al. 1990; Desjardins and Morais

1990, 199 1 ; Quinn and Wilson 1993: Wenink et al. 1994), and marsupials (Paiibo et al.

199 1 ; Janke et al. 1994). In avian mtDNA, NADH dehydrogenase subunit 6 and ~ R N A ~ I u

are positioned immediately to the 5' end of the D-loop region. whereas in other vertebrates

cytochrome b gene occupies this position. This change in avian gene order may have

resulted from a duplication within the mtDNA during the evolution of higher vertebrates

(Desjardins and Morais 1990: Quinn and Wilson 1993).

Each mitochondrial genome contains a non-coding control region (called a

displacement or D-loop in venebrates) essential for the initiation of replication and

transcription. Replication is unidirectional around the molecule and highly asymmetrical.

In venebrates. replication of one strand (heavy or H-strand) is initiated at a specific location

in the control region and proceeds for two-thirds of the distance around the molecule before

the synthesis of the second (light or L-srrand) is initiated in a noncoding sequence within a

clustcr of tRNA genes. well distiinced from the D-loop. In Drosophila mtDNA. synthesis

of the first strand can be alrnosr complete before synthesis of the complementary strand is

initiaied. Animal rntDNAs are vcry compact and contain few if any sequences brtween

genes. such as in the nematode C. eleoans mtDNA (Okimoto et al. 1992) where there are

only 38 Ïntergenic nucleotidrs (but see McKnight and Shaffer 1997).

With the exception of sea anemone mtDNA (Wolstenholme 1992). genes are not

split by introns, whereas introns were found in both fungal and plant rnitochondrial

genomes. The consequence of this feature of animal mtDNA molecules is that there are

alrnost no spaces for regions that woilld initiate and control DNA transcription. Genes are

transcribed from both strands of the mtDNA molecule in a symmetrical fashion. originating

within the control region (Clayton 1992). However. there is a strong bias in gene

transcription. In most animal mDNAs studied to date, the majority of genes are encoded

on the heavy DNA strand. Extrernes are documented in blue mussels (Hoffmann et al.

1992), and annelid earthworm Lumbricus terrestris (Boore and Brown 1995) where al1 of

the 37 genes present are transcribed from the same DNA strand. Individual gene transcripts

are precisety cleaved from primary polycistronic transcripts of mtDNA strands by

processing at the tRNA sequences that flank virtually every gene, and have few if any

upstream and downstrearn non-translated nucleotides (Rossmanith et al. 1995). In some

instances, gene transcripts end in U or UA that are adenylated to provide complete

translation termination codons. An alternative RNA editing strategy must be invoked for

posttranscriptional extension of many overlapping tRNA and protein-coding genes. since

the primary transcripts of at least one of the overlapping genes will have incomplete

sequence (Yokobori and P a b o 1997). Animal mitochondrial RNA polymerase may have a

dual role in transcription and replication (Schinkel and Tabak 1989). RNA polymerases in

animal and fungal mitochondria are nuclear - encoded and struchirally related to

bacteriophage T3 and T7 RNA polymerases. but not to eubacterial or eukaryotic nuclear

RNA pol ymerüses (Schinkel and Tabak 198%. It appears that early in mi tochondrial

evolution. a phage - type polymense was recruited. allowing loss of the ori_oinal RNA

pol y merase inherited from the protobacterial ancestor. The on1 y exception in the animal

kingdom is the heterotrophic flagellate Reclinomonas americana (Lang et al. 1997) where

gent. transcription appears to be eubacterial - like. since its genome encodes al1 four

components of a eubacterial - type RNA pol ymerase. Mitochondrial genomes do not

encode al1 of the proteins needed for organellar function: a majority of the proteins

functioning in the mitochondria are encoded by the nuclear genome. synthesizrd in the

cytoplasm and imported into the organelle (Hartl and Neupert 1990). Protein sorting and

import into mitochondria is nuclear - encoded as welI.

Animal mitochondrial genomes exhibit a vanety of geenetic novelties. Instead of 32

tRNAs, mitochondrial genetic systems utilize only 22 tRNAs An exception is a marsupial.

wallaroo (Janke et al. 1997) where the mitochondrial genome lacks a ~ R N A L Y ~ gene.

Although RNA import into mitochondria of higher vertebntes is still debated (Yoshionari et

al. 1994). Janke and coworkers ( 1997) proposed that the translational function in marsupial

mitochondria is achieved by the import of a nuclear encoded ~RNALY? Tarrasov et al.

( 1995a. 1995b) describe the import mechanisms for cytoplasmic lysine-tRNA in yeast. and

Hauser and Schneider ( 1995) provided data on the frequent irnport of tRNA molecules into

Trypanosoma mitochondria. The ability to use only a limited number of tRNA molecules is

achieved by the tàct that mitochondrial tRNAs are capable of reading eithrr two or four

synonymous codons. Also. there are at least six unonhodox translation initiation codons

used in transcription of mitochondrial protein genes. including a11 ATN codons. GTG. and

TTG. Some of the tRNA molecules have unusual secondary structure (Wolstenholme

1 992; WoIstenholme et ai. 1 987). In many organisms. the mitochondrial genetic code

differs from the "universal code" and different organisms use different variants of this

code. For example. in vertebrate mitochondria (Anderson et al. 1 98 1. 1982; Roe et al.

198% Desjardins and Morais 1990; Tzeng et al. 1992). UGA codes for the amino acid

tryptophan instead of a stop codon. and AGA and AGG are read as stop signals rather than

arginine, as in the universal genetic code. The codes AGA and AGG specify serine in

nematodes (Okimoto et al. 1992). platyhelminths (Garey and Wolstenholme 1989).

mollusks (Hoffmann et al. 1992: Boore and Brown 1994: Hatzoglou et al. 1995). starfish

(Hirneno et al. 1987). and seri urchin mtDNA (Jacobs et al. 1 9 8 8 ~ Cantatore et al. 1989).

In starfish and sea urchins. AUA codes for isoleucine as in the universril code. but

methionine in most mitochondd systems. In sea urchins AAA codes for asparagine rather

than lysine. and the stop codons UAA or UAG are used as amino acid encoding triplets in

1 I out of 13 protein-coding genes.

During invertebrate evolution. each lineage rnay have developed its own

mechanism of mtDNA replication and transcription. and of RNA processin_o and

translation. The serine-specific capacity of AGA and AGG codons may have ken retained

in ail inveriebnte mtDNAs. The apparent absence of AGG codons from Drosophila

mtDNA (Clary and Wolstenholrne 1985: Garesse 1988) may be a function of the extremely

low use of G in the third positions of codons. In vertebrate mtDNA. codons AGA and

AGG are either not used, or they signal translation termination. This switch in specificity

could have taken place either just before or following the development of venebrates (Garey

and Wolstenholme 1989).

1.2 Transfer of mitochondrial genomes to the nucleus

The exceptionally compact and economical animal rnitochondrial genome (Attardi

1985) is the result of a gradua1 transfer of genetic information from organelle to the

nucleus. a process implied in the endosymbiont hypothesis (Margulis 1970; Gray 1992).

Molecular evidence has now confirmed that mitochondria originated from an eubacterial

ancestor. most likel y from the alpha-subdivision of purple bacteria (Proteobacteria). A

massive transfer of proto-orgünellar genes streamlined the genome of an endosymbiont that

invaded the ancient eukaryotic cells, causing a gradua1 transfer of coding functions from

rnitochondria to the nucleus. Tnnsfer of genetic information between the mitochondrion

and nucleus was not confined to the early stage of organismal evolution: recrnt transfer of

mtDNA sequences and integrütion in nuclear genomes bas k e n reponed for an increasing

nurnber of modem taxa. including fungi (van der Boogaan et al. 1987: Wright and

Cummings 1983; Farrelly and Butow 1983: Thorsnsss and Fox 1990. 1993). plants

(Kemble et al. 1983), and a variety of invertebrates and vertebrates (references listed in

Table 1 ). The largest amounr of data regarding the tnnsfer of mtDNA sequences to the

nucleus has been docurnented for humans (Hu and Thilly 1994; Nomiyama et al. 1984.

1985; Kamimurri et al. 1989; Srivre-Train et al. 1992: Shay and Werbin 1992: van der

Kuyl et al. 1995. and references listed in Table 1 ).

Tnnsfer of senetic material does not on1 y occur between rnitochondrion and the

nucleus, but also between chloroplast and nuclear genornes (reviewed in Blanchard and

Schmidt 1995) as well as between chloropIast and rnitochondrial genomes (Stem and

Lonsdale 1982: Lonsdale et al. 1983; Timmins and Scott 1983: Fejes et al. 1988; Wintz

et al. 1988: Lonergan and Gray 1994). An interesting case of mitochondrial gene transfer

was documented in sea urchin mtDNA. where a transfer tRNA gene with a specificity for

one group of leucine codons lost its tRNA function and becarne part of a protein-codin:

NADH gene (Cantatore et al. 1987). Sequence analysis of various mitochondrial genes and

corresponding nuclear pseudogenes suggest that, in a few organisms. gene trrinsfer events

have occurred on multiple occasions (Lewin 1983; Fukuda et al. 1985: Nugent and Palmer

199 1 ; Hu and Thilly 1994. 1995; Collura and Stewart 1995: Sorenson and Fleischer

1996: Sunnucks and Hales 1996). and that the mechanism responsible for the streamlining

of the mitochondrial genome sti11 rernains intact.

A minimal mechanism for the transfer of genetic material from mitochondrion to

the nucleus can be throretically divided into several evolutionary steps (Obar and Green

1985): gene duplication and provision of mitochondrial genetic material for the transfer.

transfer of the genetic information. integration of the trmsferred sequence. activation and

regulation of the integrriteci element. and tinally selection and loss of the donor

(rnitochondrial) sequence.

Table I : Published reports on the presence of mitochondrial DNA sequences in nuclear

genomes of multicellular animals. When more than one species of a particular

genus contains nuclear copies of mtDNA. only the genus name is listed. Data on

size of transposed mtDNA sequences refer to the size of characterized sequrnce

inserted in the nuclear genome. Missing or unspecified data for the copy number

of inserts and / or frequency of transposition events are marked with a question

mark. For primates, only the most recent publications per author are given. A full

!kt of references is provided in the text.

Organ i sm size of copy number gene content References

insert (kb) [integrations]

Invertebrates - Stone crabs:

Menippe adina 0.56 1 16s rRNA Schneider-Broussard and

M. mercenaria - [single'!] Neige1 1 997.

- Locusts:

Locusta 3 -9 several 12s and 16s Gellissen et al. 1983.

mi~ratoria hundred rRNAs Gellissen and Michaelis

[ ? j 1987.

Shistocerca > 1.0 multiple 12s rRNA, Zhang and Hewitt 1996b

gre gari a [single?] control region

- Aphids:

Sitobion 0.8 multiple COI. COU. Sunnucks and Hales

[multiple] t R N A ~ ~ u 1996.

- Sea urchins:

S. purpuratus - 2.8 low 16s rRNA. Jacobs et al. 1983.

[single'?] COI Jacobs and Grimes 1986.

Vertebrates

- Birds:

Cephus sp. < 1.0 9 portion of Kidd and Friesen 1998.

[single] D-loop. NADH 6

Table 1 . - cont. size of copy number gene content Re ferences

insen (kb) [integrations]

Vertebra tes

Anser 0.18 3 D-loop Quinn 1993.

caerdescens [ ? 1 Aythva sp. O -4 ? D-loop Sorenson and Fleischer

Netta sp. [single] f 996.

Scvtalopus sp. 0.32 ? cyt b Arctander 1995.

M~ornis senilis [single]

- Rodents:

Rattus 0.5 repetitive 12s and 16s Hadler et al. 1983.

norveoicus [single] rRNAs. D-loop Zullo et al. 199 1.

Chroeomvs 0.36 3 cyt b Smith et al. 1992.

ielskii -- [ ? l - Cats:

Felis sp. 7.95 38-76 . I2Sand16S Lopez et al. 1994. 1996.

repetitive rRNAs. NADH 1

[single] NADH 2, COI.

COII, D-loop,

R N As

- Primates:

Homo sa~iens 0.2 to 3.0 from a few 12s and 16s Tsuzuki et al. 1983.

to 1OOO rRNAs, tRNAs, Fukuda et al, 1985.

[single / CO 1-111. D-loop, Karnimura et al. 1989.

multiple] NADH 2, 4. 4L. Shay et al. 199 1 : Collura

NADH 5, cyt b et al. 1995: Hu and

Thilly 1995; van der

Kuyl et al. 1995:

Zischler et al. 1995.

GoriIlri oorih. 0.4 to 3.0 < 10 12 rRNA. Collura et al. 1995.

Hvlobates agilis [sinsle / cyt b. D-loop van der Kuyl et al. 1995.

Pan sp.. Pongo - multiple] Zischler et al. 1998.

Provision of a genetic element within the donor genome

Extra sequences avaitable for the transfer can be obtained either from the redundant

mtDNA molecules present within a cell. and / or from copies of duplicated genes and non-

coding sequences. Each ceIl can contain up to 600 mitochondria in its cytoplasrn.

Furthemore. each organelle itself harbors a number of mitochondrial genomes. providing

an additional pool of DNA moiecules. A number of rntDNA sequence duplications.

comprising of either complete gene. or mtDNA gene segment duplications. have been

documented in animals, providing redundant mtDNA sequences available for transfer

(Moritz and Brown 1996. 1987; Hyman et al. 1988: Wallis 1987: Zevering et al. 199 1 :

Hyman and Beck-Azevedo 1996: Kumazawa et al. 1996). Deletion of a large portion of

the mt-genome prier to transfer does not have to be deleterious; the organism in question

can still possess intact mitochondria in a heteroplasmic state (Boursot et al. 1987)-

Transfer and integration of genetic information

At least one copy of the donor genetic element is moved and integrated into the

recipient genome. Molecular evidence indicates that gene transfers are principally

unidirectional from plastids or mitochondria to the nucleus. Thorsness and Fox ( 1990)

calculated that in yeast, DNA moves from mitochondria to the nucleus at a surprisingly high

frequency of 2 x 10-5 per ceIl per generation. The movement of genetic materiûl in the

opposite direction was calculated to occur in at least 100.000 times lower frequency. The

most massive trünsfer of genetic elements so far recorded occurs between flowering plant

chloroplasts and their mitochondria (reviewed in Hanson and Folkerts 1992).

Transfer of mitochondrial genrs into the nucleus cm be distinguished as

"primordial" and "recent" (Gillham 1994). The former cIass of transfers occurred soon

after the original endosymbiotic event. and before the diversification of mitochondrion-

containing lineages. It is chancterized by a massive loss of genetic content by a proto-

mitochondrion. A good example of such a primitive mitochondrial genome is found in the

freshwater protozoan Reclinomonas amencana (Lang et al. 1997). This organism's

mitochondrion contains 97 genes in 69 kb of mtDNA sequence. This is the largest

collection of genes so far identified in any animal mtDNA. but much less than could be

found in their free-living eubacterial progenitor. Hence. Reclinomonaî foms a "missing

link" between extant. derived mitochondria and their relatively unchanged bacterial

progenitors. The latter class of gene tramfers took place in specific mitochondrial lineages.

Examples for such transfers can be found in plants (Nugent and Palmer 199 1 ), humans

(Hu and Thilly 1994), and birds (Sorenson and Fleischer 1996).

Several proposed mechanisms of transfer and integration of mtDNA sequences

will be presented in more detail in the following section of this chapter.

Activation and regulation of the transferred sequences

The newIy acquired genetic element is transcribed. and if it encodes a protein,

uanslated by the nuclear genome. The recipient genome (nucleus) fine-runes the regulation

of the newly acquired gene, synthesis of its product and transfer of each product to its

functional site. This is the most difficult step of successful gene transfer. as there are

several obstacles with respect to the correct regulation and expression of genes at their new

sites: the difference in codon usage between the two cell compartments have to be

eliminated. and the product of the now nuclear gene must be correctly targeted to the

mitochondrial cornpanment (in the case of proteins this is most çommonly achieved by

a single peptide). The duplication of a sea urchin tRNA sene (Cantatore et al. 1987)

followed by an anticodon change. provides an example of the type of mutationrit

adjusiments and shifts in the genetic code which could have taken place during evolution of

transferred mtDNA sequences. An adjacent and correct open reading frame is necessary for

the nucleur copy to be functional und active. In two crises of mtDNA trrinsfers in human

carcinoma cells (Shay et al. 199 1 ; Savre-Train et al. 1992). the partial transferred genes

were transcnbed together with the adjoining nuclear sequences. and their transcription

products could potentially be translated when complemented with tRNAs expressed from

normal mitochondrial genomes. As there is little additional data available on nuclear

sequences surrounding mitochondrial DNA inserts in animals. I will presrnt here data for

higher plants. In two documented cases of successful transfer of rnitochondrial sequences

in cowpea and soybean. for which sequences upstream of the protein coding reg ions were

obtained. the appropriate open reading frames were indeed observed (Nu, oent and Palmer

1991 : Covello and Gray 1992). In both species of legumes this open reading frame is

separated by an intron from the sequences homologous to the CO II gene. which are likely

to be descendants of the same gene transfer. The presence of an intron sequence possibly

allows for a more relaxed insenion of foreign DNA fragments. a process reminiscent of the

hypothesis of exon shuffiing (Nusent and Palmer 199 1 ). Nevenheless. a blunt connection

of an open reading frame and tnnsferred mtDNA sequences is possible and has k e n

documented in the evening primrose (Oenothera. Grohmann et al. 1992). Here. the open

reading frame is directly connected to the gene for the ribosomal protein S 12 (rps 12). The

transit sequence necessary to target this protein to the mitochondrion in encoded by a 5'-

extension of the open reading frame.

Simple insertion in the vicinity of a potentially correct regulatory environment does

not necessady preclude the automatic activation and regulation of inserted mtDNA

sequences. Rather. an assembly of functional mitochondrial genes in the nucleus seem to

be created during evolution. at least in the case of CO II gene in cowpea (Nugent and

Palmer 199 1 ). In this species. a nuclear copy of the active mitochondrial gene is not

expressed. and thus rnay require an adaptation of the nuclear environmenc for its full

functional expression.

Loss of rnitochondrial gene copy after it becomes redundant

Once the nuclear copy assumes full control of the genetic element and its product.

the mitochondrial copy becomes redundant, and can be inrictivated by a regulatory or

functional Irsion. An example for this stage of gene transfer can be seen in the structural

gene for ATPase subunit 9 which is present in the mitochondrial genome of fungi (Borst

and Grivrll 1978: Gray 1992). However. the ATPase 9 is transcribed from a nuclear copy

in Neurospra and Aspereillus (van der Boogaart et al. 1982). whereas the mitochondrial

copy of this g n e remains silent. In animals, the ATPase 9 g n e is present only as a nuclear

copy. A second example involves the CO II gene of plant mitochondria. In soybean. a

perfectly intact copy of this gene is present. although the protein is synthesized from a

nuclear copy (Covello and Gray 1992).

Loss of the mitochondrial gene is not an active process. Rather. it can involve

partial and sequential loss of fragments of transferred gene. before the last remnant of the

gene is eliminated from the mitochondrion. Flowering plants harbour a functional gene for

ribosomal protein S 12 (rps 12) in their mitochondria. The cornplete gene is present in the

nucleus of the evening primrose Oenothera (Grohrnann et al. 1992). while about two thirds

of the coding region has k e n deleted from the mitochondrial copy. Such partial losses and

remngements of non-essential coding sequences can be facilitated by formation of

dupticated regions and creations of pseudogenes within the mitochondrial genome. In the

animal world. probably the best examples for creation of redundant and fractionated

mtDNA sequences are docuniented in lizrirds (Zevering et al. 199 1 ). and nematodes

(Hynian and Beck-Azevedo 1996).

The final stage of gene transfer from mitochondria to the nucleus is achieved when

the redundünt copy is coinpletely eliminüted frorn the mitochondrion. This stage has

appürently k e n reached for rnost of the mitochondrial proteins which are now encoded in

the nuclear genornc. An ençcptionülly illuminating exümple of gradua! transfer of

mitochondrial genes to the nucleus is documented for the CO II gene of plant mitochondr-ia.

This gene seems to be tnnsferred to the nucleus during the course of evolution in legumes

(Nugent and Palmer 199 1 ). Both mitochondnal and nuclear CO II genes are present in pea.

soybean. and comrnon bean. The mitochondrial gene is expressed in pea. while the nuclear

gene is expressed in soybean and bean. Only the nuclear gene is present and expressed in

cowpea and mugbean. Among molluscs. a gene for ATPase subunit 8 is present in

completely sequenced mtDNA of two gastropods. Albinaria coerulea and Cepea nemoralis.

and a chiton Katharina tunicata (Hatzoglou et al. 1995; Terret et al. 1996: Boore and

Brown 1994). whereas it is absent from the mtDNA of a bivalve Mytilus edulis (Hoffmann

et al. 1992).

1.3 Mechanisms of transfer and integrstion of mtDNA sequences

Nurnerous studies dealing with the transfer and incorporation of oqanellar

sequences into the nucleus failed to reveal a unifying mechanism or common DNA

sequence which may be directly involved in the process. The most difficult obstacle for

transfer of mtDNA sequences is the passage of highly charged nucleic acids through the

organelle membranes. Nevenheless. limited transport of nucleic acid across the membranes

is stiil possible. Since not al1 tRNAs required for translation are encoded by the

mitochondnal DNA, and some of the tRNA found within mitochondrion are encoded by

nuclear genes. it seems that transport mechanisms for small nucleic acids through

mitochondnal membranes do exist (see Nagley 1989: Yoshionari et al. 1994: Akashi et al.

1997). Furthermore. mitochondrial genetic information can be transferred to the nucleus

via RNA intermediates through the process of reverse transcription. Such a mechanism

was suggested for transfer of organelle sequences in plants (Schuster and Brennicke 1 987:

Nugent and Palmer 1991; Covrllo and Gray 1992; Grohmann et al. 1992).

There are other opportunities in the normal course of the life of cells when nucleic

acids could migrate from one cornpartment to another without encountering boundaries

imposed by membranes. Those include aging. when the systems that remove damaged

mitochondria became inefficient (S hay and Werbin 1992). Insertion of DNA fragments

into the nuclear genome could take place by the same mechanisms that facilitate the

incorporation of other exogenous DNA. Previous studies (Richter 1988: Richter et al.

1988) have suggested that peroxidative damage. both natural and through intake with food

and environmental exposure. could lead to fragmentation of mtDNA. They propose that

due to massive oxidative damage. combined with the inefficient DNA repair in

mitochondria, large amounts of DNA fragments are present in mitochondria in vivo. Some

of these fragments may escape from the organelle and integrate into the nuclear DNA. One

of the rntDNA fragments that integrated into the human nucleus harboun a mutation

suspected to have occurred prior to integration (Collura and Stewart 1995). kistensen and

Pryz ( 1986) even documented the presence of intact mitochondrial DNA in cell nuclei.

Inserted mtDNA sequences would have the highest chance of fixation if the

insertion event occurred during germ-line development or during rapid cell division in early

embryologica1 growth. A defective or panially degraded mitochondnon could be inserted

in the chromosomes of an anaphase blastomere that is one of the germ-line progenitors,

thus increasing the probability of fixation and transmission into the next generation.

Kobayashi et al. ( 1 993) detemined the presence and active role of mi tochondrial large

subunit ribosomal RNA (Isu-rRNA) as a cytoplasmic factor in the formation of pole cells

durins earl y em bryogenesis. They suggest that the Isu-rRNA molecules are transponed out

of the mitochondnon and into the cytoplitsm only during the limited time of gem-line

establishment in early ernbryonic drveloprnent. and that they are subsequently removed.

Such extrineous mtDNA sequcnces could have been i ntegrated in the chromosomes.

Indeed. nuclear integrcitions of rnitochondriül ribosomal RNA genes have ken documented

in humans (Tsuzuki et al. 1983: Nomiyamü ct al. 1985: Kamimura et al. 1989).

Studies presented to date display great variability of sequences adjacent to the

mitochondrial insem. suggesting a nndom mode of integntion (Blanchard and Schmidt

1996). There seems to be no unifying mechanism or common DNA sequence that may be

directly involved in the transposition and integration of mtDNA fragments. Furthemore.

the nuclear genomic environments where the integntion occurred show no similarity to

each other. Reported hotspots for integration are mostl y noncoding reg ions that incl ude

introns. intergenic regions, repetitive sequences. direct repeats. and transposable elements.

It seems plausible that transposed mtDNA sequences are associated with noncoding regions

or repetitive sequences, as insertion into singlecopy genes would probably be fatal to the

gene and the organism. Kato et al. (1980) found that repetitive sequences are preferred

sites for the integration of exogenous DNA. This is the most frequently reported pattern of

mtDNA sequence integrations (Gellissen et al. 1983: Jacobs et al. 1983: Tsuzuki et al.

1983; Nomiyama et al. 1984; Fukuda et al. 1985: Wakasugi et al. 1985). If inserted in

the vicinity of regions with high frequency of recombination. mtDNA sequences are

potentially prone to fragmentation and local remngements (Farrely and Butow 1983:

Jacobs et al. 1983).

Zischler and coworkers (1995) determined the presence of a 6 bp direct repeat at

the ends of integrated mtDNA fragments in humans. Individuals that did not possess the

nuclear copies also Iacked this particular 6 bp repeat. The ends of insertions in that

particular human nuclear integration. coincide approxirnately with the ends of 7s DNA (a

third DNA strand that makes the triple-stranded D-loop portion of the mitochondrial control

region). Transfer and integration of the same portion of the control rezion has been

described i n rats (Zullo et al. 199 1 ). Zullo and coworkers ( 199 1 ) determined that inserted

D-loop onginating fragments in the rat genome are flanked by 88% identical aquences.

and were associated with LINE-like repetitive elements. In at lest two other cases. long

interspersed repetitive elrments (LINEs) or cryptic retroviral sequences werr CO-isolated

with the nuclear homologs of mitochondrial genes (Tsuzuki et al. 1993: Wakasugi et al.

1985). Not al1 repetitive elements are presumed to be involved in the original mobilization

and integntion of mitochondrial sequences. In sea urchins (Jacobs and Grimes 1986). and

cats (Lopez et al. 1994. 1996), the location of the repeat elements suggest that they may

have ken involved in the post-insertion rearrangements that resulted in duplications and

local transpositions.

Nuclear in tegrations may resu f t in mtDNA sequences k i n g dispersed throughout

the nuclear genome (Gellissen et al. 1983: Zullo et al. 1991 ) or in localized. tandemly

repeated arrays (Lopez et al. 1994: this study ). Fukuda et al. ( 1985) inferred 10 to 13

copies of each of four mtDNA regions to be dispersed in human chromosomes (however.

see Kamimum et al. 19831. In contrat, there are only a limited number of integrated

nuclear copies present in sea urchins and akodontine rodents (Jacobs et al. 1983: Smith et

al. 1992).

1.4 Gene content of inserted rntDNA sequences

Mitochondrial DNA sequences integrated in nuclear genornes of higher organisms

appear to contain both ribosomal RNA genes. at least eight of thirteen protein-coding

genes. and a control region (Table 1). No particular region of the rnitochondrial genome,

with the exception of the control region, seem to be preferentially integrated in the nuclear

genorne. Initial studies that reponed the presence of mtDNA sequences in the nucleus

utilized a variety of cloning and hybridization techniques to detect such sequences

(Gellissen et al. 1983; Jacobs et al. 1953: Zullo et al. 1991). With the increased use of the

polymrrase-chain reaction (PCR) in population and evolutionary biology (Saiki et al. 1986:

Arnheim et al. 1990). the majority of rrcent studies used this rnethod to amplify target

mtDNA sequenccs. The higher frequency of detection of nuclear inserts through accidental

PCR amplificürions of püralogous genes could be biased by the use of specific PCR

primcrs (mostly to amplify D-hop. cytochrome b. or rRNA genes). More detailed and

extensive analysis. such as the sequence database analysis of plant. yeast and human DNA

attempted by Blanchard and Schmidt ( 1 996). will be required to determine whether there

are any trends in preferential gene tnnsfen between the mitochondrion and the nucleus.

Furthemore. future studies will have to address the question of whether there is a tendency

for organelle genes to be tnnsferred and inserted into specific sites within the host genome.

In plant chloroplast (Cheung and Scott 1989) and human mitochondrial gene transfers

(Kamimura et al. 1989). separate organelle sequences were found to be incorporated

together and present as a contiguous unit within the nuclear DNA.

1.5 Sequence divergence between mtDNA and nuclear copies

Variable lengths of inserted rntDNA sequences have k e n reported. ranging from

less than 200 bp (overview in Table 1). to 300-60 kb of a tandemly repeated 7.95 kb

fragment in cats (Lopez et al. 1994. 1996). The level of sequence similarity between the

original mtDNA fragment and the nuclear copy also varies to a great extent. In the Iocust

Shistocerca (Zhang and Hewitt 1996) the nuclear copies are highly conserved in sequence

and length. reflecting a recent integration event. On the other side of the spectrum. nuclear

rntDNA-like sequences in guillemots (Kidd and Friesen 1998) show only 508 sequence

similarity to their mitochondrial homologs. MtDNA integration events in sea urchins

(Jacobs and Grimes 1986). or primates (Fukuda et al. 1985) can be as old as 30 million

years or more. In multiple integrations of the same sequence of human mitochondrial 16s

rRNA gene (Hu and Thilly 1994). the level of sequence divergence between mtDNA and

corresponding nuclear pseudogene ranges from 62.4% to 93.2%.

The existence of paralogous sequences in the nucleus (sequences related by gene

duplication), raises the possibility of estimating evolutionary patterns between nuclear

copies and functional rntDNA (Sunnucks and Hales 1996; Arctander 1995; Lopez et al.

1997). Where sequencing data are availliblr. patterns of nucleotidr substitutions reveal that

the majority of differences between originating and transferred mtDNA fragments are due to

the continuing evolution of mtDNA sequences. Given the evidence rhat mtDNA fragments

would most likely be insened into untranscribed regions (Shay and Werbin 1992:

Blanchard and Schmidt 1996). the inserted mtDNA sequences are expected to evolve at

rates similar to nuclear pseudogenes. Mitochondrid gene sequences inserted in the nucleus

of cats (Lopcz et al. 1997) do evolve at a nearly equivalent (and presurnably selectively

neutral) pseudogene rate, while the original mitochondrial genes evolve at different rates

depending on the constraints of their function. Mitochondrial fragments exhibit a higher

rate of sequence heterogeneity. compared to their nuclear counterparts: 14.4 tirnes higher in

human control region integrations (Zischler et al. 1998). and 5.7 times higher for

cytochrome b in the bird Scvtalo~us (Arctander 1995). The higher rate of sequence

substitutions and length variations has provoked several explanations, including: ( 1 ) that

mitochondrial DNA gamma-plymerase may be more error prone than the nuclear DNA

polymerase, (2) that oxidative damage may be more likely in the mitochondrion due to the

abundance of free radicals. (3) that mitochondria may have defective mismatch-repair

mechanisms (a homologue of bactend mutS mismatch-repair mechanism gene in the

octocoral mtDNA is thought to be acquired by lateral transfer; Pont-Kingdon et al. 1998),

and (4) that expanded codon recognition in mitochondria might lead to relaxed selection

(Wallace 1982; Brown et al. 1979. 1982; Clayton 1992).

Differences in modes of evolution between functional rnitochondrial gene copies

and nuclear pseudognes include generall y higher substitution rates in mitochondrial copies

(with the exception of CO 1 gene in cats: Lopez et al. 1997); higher transition to

transvcrsion rates of substitutions in mitochondria: and higher frequency of stop codons

and frarneshift mutütions in open readi ng frames of nuclear inserts. Mitochondrial

sequrnces ülso rxhibit a skewrd distribution of substitutions according to the codon

posiiion. with third position substitutions being predominant in protein-coding genes.

Transiiions at thc third position of rnitochondrial codons evolve at least 39 times faster than

the corresponding positions in the nuclear inse- (Arctander 1995) whereas nuclear inserts

tend to have a more even distribution of substitutions. Nuclear copies of mitochondrial

genes are assurned to be nonfunctional because of differences between mtDNA and the

nucleus in their genetic code. and the observed presence of insertions or deletions causing

frameshift mutations (Smith et al. 1992: Lopez et al. 1994: Collura and Stewart 1995:

Sunnucks and Hales 1996).

1.6 Implication for population and evolutionary studies

As mentioned in the previous section. the presence of mtDNA-like sequences in

the nuclear genome could cause a problem in population and evolutionary studies (reviewed

in Zhang and Hewitt 1996a). particularly when preparations from total cellular DNA are

subject to PCR amplifications with universal prirnen (Kocher et al. 1989). Because recent

nuclear integrations retain high sequence identity to corresponding mtDNA fragments.

nuclear inserts could potentially be amplified by PCR primers designed on scquences from

related taxa. Although the presence of aberrant stop codons and frameshift mutations

would indicate inadvertent PCR amplification of nuclear pseudogenes. it will be more

difficult to identify transferred copies of non-coding regions such as the D-loop. often used

in evolut ionary analyses (Sorenson and Fleischer 1 996).

Various procedures to overcome the problem of CO-amplification of nuclear insens

have k e n suggested. These include the design of specific. discriminating oligonucleotide

primers. use of several different overlapping primer pairs to amplify the sequence of

interest. PCR amplification of different mtDNA regions. and the use of enriched or purified

DNA. In birds, rntDNA crin be extracted from mitochondria-rich cells rather than blood.

because avian red blood cells are nuclerited and contain few mitochondria (Quinn 1992:

Arctander 1995: Sorenson and Fleischer 1996). Collurri and coworkers ( 1996) su,, *=est an

clegünt method by which the PCR products of original mtDNA genes and nuclear

pseudogenes can be distinguished. Because the mitochondrial genome is tnnscnbed and

processed into polyadenylated mRNAs (Anderson et al. 198 1 : Clayton l992), reverse

trmscriptasecoupled PCR can be used to amplify the functional mitochondrial version of

the coding sequence. This method was shown to clearly distinguish DNA sequences

obtained from mitochondrial cytochrornr b gene and its nuclear homologue in primates

(Collura et al. 1996).

This concludes a summary of the relevant literaiure regarding tnnsfer of animal

mtDNA sequences to the nucleus. The work addressed by this thesis involves an analysis

of transferred segments of mtDNA in six species of geese (Aves. Anserinae).

CHAPTER 2: MATERIALS AND METHODS

Samples

Two hundred and fony three individuals of Lesser Snow Goose (Anser

caerulescens caemlescens) were screened for the presence of nuclear copies of mtDNA

sequences. One hundred and sixty one onginated from hunter-killed birds or from blood

samples taken dunng bird-banding drives organized by local authonties at wintering

grounds in Texas and Louisiana. U.S.A. Sixty seven samples were from the breeding

colony near Churchill. Manitoba, Canada. and 15 represented a breeding population from

the Wrangel Island. Russian Commonwealth. The blood samples from Churchill and liver

tissue samples from Wrangel Island used in this study were from the collection of Lesser

Snow Goose samples stored at Queen's University. Kingston. Canada. and used

previously (Quinn 1988). Sixteen blood samples of Greater Snow Goose (A. c. atlantica)

were collected at the breeding colony on Bylot Island. Northwest Temtories. Canada.

Ross' Goose (A. rossii, n = 12) blood and heart tissue samples were collected from adult

hunter-killed birds on wintering grounds in Texas and Louisiana. Emperor Goose (A.

canacica, n = 4) blood samples were from Old Chevak. Alaska. U.S.A. White-fronted

Goose (A. albifrons frontalis) blood samples (n = 4) were from Koyukuk, Alaska. whereas

hean tissue samples (n = 15) were collected during the winter months in Texas and

Louisiana. Twelve heart tissue sarnples of Canada Goose (Branta canadensis ssp.) were

col lectsd from hunter-ki lIed birds on wintering grounds in Texas and Louisirinri.

Subspecies designations wrre not recorded for thesr specimens. Ten blood san~ples of

Brant Goose (B. bemicla bemicla) were obtained from captive birds. Worplesdon. Surrey.

United Kingdom. All samples werc collected and / or irnportcd into Canada with

appropriate permits.

DNA extractions

Total cellular DNA was extracted either by using Nucleic Acids Extractor model

340A (ABI). or rnanually. The procedure was according to a method described by Seutin

et al. ( 199 1 ) with minor modifications. Briefly. between 50 pl to 150 pl of blood samples

were treated with 1 x AB1 lysis buffer ( in total volume of 3.5 ml) at 37OC for 24 to 48 hrs.

with occasional inversion of tubes. The Proteinase K digestion (62.5 U in 0.5 mI of 20

mM Tris-HCI, pH 8.0) proceeded at 37OC for 48 hrs. Heart tissue samples were

pulverized in liquid nitrogen, mixed with 3.5 ml of l x AB1 lysis buffer and kept at 370C

between 72 to 96 hrs. The Proteinase K step was the same as with blood sampIes. The

RNAse treatment step. suggested by Seutin et al. ( 199 1 ) was omitted in this protocol.

After the manual phenol / chloroform and chloroform extractions. the DNA was precipitated

in 1/10 final volume of 3 M sodium acetate and an equal volume of isopropanol. spooled on

sealed g l a s tips. rinsed in 70% ethanol. and redissolved in Ix TNEz ( 10 miM Tris-HCI

pH 7.8. 10 mM NaCI, and 2 mM EDTA).

Restriction digests, electrophoresis, and hybridizations

Restriction digests of 2 - 5 pg of DNA per sample were camied out for a minimum

of 2 hrs, under conditions recommended by the manufacturer (Gibco - BRL. Gaitherburg,

MD). Restriction fragments were electrophoretically separated at 2.2 V 1 cm for 16 - 18

hrs. in 0.8% to 1.5% agarose gels in 1 x TAE buffer (40 rnM Tris-acetate. and 1 mM

disodium-EDTA. pH 7.8). and transferred to Immobilon-N membranes (Mill ipore Corp..

Bedford. MA) by Southern btotting (Maniatis et al. 1982). Pulsed field gel electrophoresis

separation was at 14.3 V / cm for 1 6 hrs. in 1 -5% agarose gels in 0 . 5 ~ TBE buffer (45 mM

Tris-borate. and I mM disodium-EDTA. pH 7.8).

Mitoçhondritil DNA probes pmLSGûû 1. pmLSGOO2. and prnLSG003 (5.5 kb.

1.95 kb. and 1.3 kb &d I I I Lesser Snow Goose mtDNA fragments respectively. inserted

into the plasmid vector pUC8). and senomic probes DQSG 1. DQSGI, DQSG3. DQSGS.

DQSG6. and DQSG8 (Quinn and White 1987) were labeled by primer extension

(Pharmacia oligolabelling kit) with [a1pha-3~~] dCTP. according to Feinberg and

Vogelstein ( 1983). Membranes were prehybridized for 2 hours at 65OC in a sealable bag

containing prehybridization buffer. prepared with 6 x SSC ( 1 x SSC = 0.15M NaCI.

0.01 5M sodium citrate pH 7.0). 0.5% SDS, 5x Denhardt's solution (5x Denhardt's =

O. 1 C/c bovine semm albumin. 0 .18 Ficoll. O. 1 % polyvinylpyrrolidone). and 100 p@mi

denatured salmon spem DNA. Hybridizations were carried out overnight at 65oC. Two

washes of 10 min were can-ied out at room temperature in 1 8 SDS. 2 x SSC. followed by

two washes of 30 min at 650C in O. 1% SDS. 0.5 x SSC. The membranes were exposed to

X-ray film (Dupont. Cronex) at -700C for various tirnes.

Mitochondrial DNA sequencing and sequence database search

MAX efficiency DH5 alpha competent cells (Gibco - BRL. Gaithersburg. MD)

were transformed according to manufacturer's instructions. and plated on standard colour

screening plates (Maniatis et al. 1982). Plasmid DNA was isolated by a rapid mini-prep

procedure: 1.9 ml of bacterial culture was spun for 10 - 15 sec. at maximum speed in an

Eppendorf centrifuge. The pellet was cornpletely resuspended in 180 pl of STET buffer

( 8 9 sucrose. 5% Triton X IOO. 50 m M EDTA. 50 mM Tris-HCI pH 8.0). Twenty pl of IO

mgml lysozyme was added to each tube and mixed. After incubation at roorn temperature

for 75 sec.. samples were kept in boiling watrr for 75 sec. Cell dsbris was removrd by

centrifugation for I O min. The supematant wÿii collrcted and mixcd with an equal volume

of ice-cold isopropanol. chiiled at - 7 0 T for 1 O min. and spun in an Eppendcrof centrifuge

at maximum sperd for 10 min. Alcohol waï removed and the rcmaining pellet was

resuspendcd in 100 pl of TE buffer ( 10 m M Tris-HCI pH 8.0. I mM EDT A). The

nucleotidr sequencc of Lesser Snow Goose mtDNA probes pmLSGOO 1 and pmLSGOO2

wzs determined by the dideoxynucleotide chain termination method of Sanger et al. ( 1977).

The T7 sequencing kit with M 13 universal primers (Phmacia. Piscataway. NJ) w m used

for nucleotide sequencing. After electrophoresis in 8% polyacrylamide wedge gels. the

obtained DNA sequences were visualized by autoradiography. The rntDNA sequencing

was completed at the Marine Gene Probe Laboratory . Dalhousie University. Halifax. N.S..

DNA sequence databases were accessed through computer resources at the Aquatic

Ecology Lab. BRD-USGS, Kearneysville, WV. The GenBank databases (National Center

for Biotechnology Information. National Institutes of Helath, Bethesda, MD) were

searched using algori thrns in the BLASTN search program (Altschul et al ., 1997). Partial

DNA sequences of probes pmLSGûû 1 ( 138 bp long) and pmLSGOO2 (23 1 bp long) were

used sequentially to search sequence databases. The gene content of rntDNA probes was

detemined by cornparison with mtDNA sequences deposited in the GenBank. and by

cornparison to published sequences of chicken (Desjardins and Morais 1990). duck

(Rarnirez et al. 1993). and human (Anderson et al. 198 1) mtDNA. Sequence alignment

was performed using software package Sequencher 3.0 (Gene Codes Corporation. Ann

Arbor. MI). and manually. Numbers of transitions and transversions. percent sequence

divergence and translation changes were analyzed using MEGA version 1 .O 1 (Kumar et aI.

1993).

In this chapter I will present evidence of the occurrence of mtDNA sequenccs in

nuclear genomes of six species of geese: Lesser and Greater Snow. Ross'. White-fronted.

Emperor, Canada, and Bnnt Goose. 1 will describe experiments leading to identification

and characterization of a 3.6 kb DNA fragment containing rntDNA-li ke sequences insened

in the Lesser Snow Goose nuclear genorne. assess the copy number of the inserted

element, and determine the phylogenetic distribution of mtDNA inserts in the six species

under study .

3.1 Identification of mtDNA sequences in the Lesser Snow Goose genorne

Southern blots of Lesser Snow Goose genornic DNA digested with m d III and

hybridized with the mtDNA probe pmLSG003 reveal two distinctive fragments: a 5.5 kb

fragment of mitochondrial origin, and a 3.6 kt, fragment of nuclear origin (Quinn and White

1987). Confirmation that the 3.6 kb fragment represented the mtDNA sequences insened

in the nuclear genome of Snow Geese corne from a differential amount of hybrîdization

when the source of DNA is blood or tissue (Quinn and White 1987), and from sequence

data (Quinn 1992).

Prominent nuclear DNA (nucDNA) bands of approximately 3.6 kb were found

when DNA was digested with Hind III. & 1. and BamH 1. Additional fragments in

digests with these three enzymes were 7.1 kb and 10.8 kb long. suggesting tandemly

organized arrangement of the 3.6 kb sequence. To test this hy pothesis. 1 prepared a series

of incomplete digests with dec~as ing amounts of md III. The autoradiograph in Figure I

shows a ladder of bands consisting of monorner units (3.6 kb). dimer units (7.2 kb). trimer

units ( 10.8 kb) etc., with a smalIer proportion of the 3.6 kb monomers following the

rcduction in concentrations of the restriction enzyme.

Figure 1 . Southem blot of Lesser Snow Goose DNA probed with a mitochondrial DNA

probe pmLSGOO3. Two pg of DNA extncted from blcmd of the same individual

were digested with various amounts of & ~ d III at 370C for one hour. and

separated on a 0.7% horizontal agarose gel. The sarnple in lane I was treated with

an excess of enzyme: IO units per pg. The sarnple in lane 2 was treated with one

unit of m d III: each subsequent sarnple (lanes 3 to 8) was treated with one haIf

the enzyme used for the previous simple. In Iane M is the size marker. a mix of

lambda phage DNA digested with Pvu II and EcoR 1 / h d III (double digest).

The increased proportion of dimen and tnmers of the 3.6 kb nucDNA fragment.

apparent in lane 3 and lane 4 of Figure 1. is a result of a reduction in cleaved Hind III

recognition sites within the array. Undigested genomic DNA also hybridized to the probe

and appears as large molecular weight fragments on the autoradiognm (lanes 5 to 8 in

Figure 1 ). These resuits suppon the hyporhesis that the majority of mtDNA sequences

transferred and inserted in the nuclear genome of Lesser Snow Goose are associated with

sequences arranged in a tandem array.

The presence of additional bands that do not follow the ladder-type pattern

described above (e.g lanes 8 and 12 to 15 in Figure 2) indicate that a portion of the inserted

mtDNA sequences may not be confined to the tandem amy of 3.6 kb monomers. These

individuals showed the same banding pattern even after cutting the DNA with excess of

Hind III. The presence of non-ladder type banding patterns was detected on Southern blots - of the same individuals after digestion of genomic DNA with Ava I and BamH 1. Severn1

Lesser Snow Goose DNA samples were digested with K D ~ 1 (an enzyme with recognition

sites not present in the 3.6 kb element), and the fragments were separated by pulsed field

electrophoresis (data not shown). Probe pmLSG003 hybridized to high molecular weight

fragments with the fragment sizes and intensity of hybridization varying both within and

arnong indi viduals.

During the course of this study. 1 screened 243 Lesser Snow Geese for the

presence of a prominent 3.6 kb nucDNA band. and found 3 individuals ( 1 -2%)

homozygously deficient for this array (e.g. lane I I in Figure 2). 1 treated genomic DNA

extracted from these individuals with Ava 1 and BarnH 1. and determined the concordant

absence of the 3.6 kb nucDNA fragment. However. autoradiographs of Southern blots

with DNA from these three birds still contained a high molecular weight frqmcnt that

would hybridize to pmLSGûû3 (see Ime 1 I in Figure 2).

Figure 2. Screening for the presence of mtDNA-like sequences in the nuclear genome of

Lesser Snow Goose. Two pg of DNA from 15 individuals were digested with

Hind III. The Southem blot was hybridized with pmLSGûû3. The individual in -

lane 1 1 is lacking the 3.6 kb nucDNA fragment (with the mitochondria -

originating sequences). The 5.5 kb fragment is of mitochondnal origin. In lane M

is a size rnarker. lambda DNA digested with &d III.

A complex minisatellite-like banding pattern was observed when the Snow Goose

DNA was digested with restriction enzyme III and hybridized with pmLSGûû3 (Figure

3). Multiple DNA fragments. ranging in size from about 2.5 kb to more than 10 kb. can be

seen in sevenl individuals chosen at random from a natural population. Individual geese

that did not have the 3.6 kb nuclex insen also lacked the minisatellite-like frügmçnts in &

III digests (lanes I and 2 in Figure 3). The 1.7 kb and 1.8 kb fragments seen in those two

lanes could represent DNA of mitochondrial origin (the rntDNA probe pmLSG003 is 5.5

kb long). To test whether the observed banding pattem follows the Mendelian mode of

segregation, a Southem blot containing DNA from a family of two parents and six goslings

was prepared and hybridized with pmLSG003. Due to low quality of the gosling DNA. 1

was not able to obtain results consistent with a Mendelian mode of inheritance (data not

shown). Funher experiments will be needed to complrte this test.

MtDNA probes prnLSG00 1 and prnLSG002 did not hybtidize to any sequenccs in

the nuclear genorne of Lesser Snow Goose (data not shown).

3.2 Copy number of the 3.6 kb element in the Lesser Snow Goose genorne

To assess the number of copies of the 3.6 kb m d III nucDNA fragments in

Lesser Snow Goose, a concentration senes of total DNA and a concentration series of

pmLSG003 was applied to the same Immobilon-N membrane by dot blotting using a dot

blot apparatus (Gibco - B K ) . The dot blot procedure followed the GeneScreen Plus

membrane protocol (Du Pont. Boston. MA). Hybridizntion with pmLSG003 and medium

stringency washes were under conditions desct-ihed in the Methods section. From

compiirisons of the relative intensity of hybridization of mtDNA probe to the srries of

oenomic DNA concentrations. and relative intensity of hybridization of the pmLSG003 to s

the probe concentration series. 1 estimated that the nucDNA fragrnsn ts are present in iit lemt

3 . 0 0 - 5.000 copies.

Figure 3. Southern blot showing a minisatellite - like banding pattern in the Snow Goose.

Four pg of DNA from 6 individuals were digested with & III and separated on a

0.8% agarose gel. The Southem blot was hybridized with mtDNA probe

prnLSG003. Individuals in lanes 1 and 2 are lacking the 3.6 kb repeated element

containing the inserted mtDNA-like sequences, and do not show the minisatellite

pattern. Fragment sizes in kb pairs indicafed length of the size rnarker in lane M.

Considering that the avian nuclear genome contains approximately 2 x IO9 base pairs. the

Lesser Snow Goose genome contains a minimum of 0.5% mtDNA-onginating sequences.

3.3 Phylogenetic distribution

The existence of Lesser Snow Goose individuals that do not possess the

characteristic array of 3.6 kb nucDNA fragments. as weI1 a.. the fact that the majority of

mtDNA insens are localized within a defmed cluster of repeats, suggests a relatively recent

transposition and / or amplification event. Consequently. one would expect to see mtDNA

sequences inserted in nuclear genomes of Lesser Snow Goose and possibly in its close

relatives (i. e. Greater and Ross' Goose). but not in the more distantly related species ( i . e.

Brant and Canada Goose). To detemine the phylogenetic distribution of rntDNA

transposition events. I analyzed DNA sarnples from sevenl species of the subfamily

Anserinae.

From the results of experiments where Southern blots with various goose species

DNA was hybridized to mtDNA probe pmLSGûû3.I detennined the presence of a

prominent 3.6 kb m d III nucDNA fragment in Greater Snow and Ross' Goose genomes

(Figure 4). Similar to the results of hybridization expenments with Lesser Snow Goose

DNA. both Greater Snow and Ross' Goose exhibited the presence of DNA fragments in

addition to the 3.6 kb nucDNA fragment. that would hybridize to pmLSG003. Southern

blots with E-&d III. BamH 1 and Ava I digested genomic DNA showed little DNA banding

pattern differences among the three taxa (data not shown). As with the Lesser Snow

Goose. probes pmLSGûû 1 and pniLSGûû2 did not hybridize to any sequenccs in the

nuclear genomes of Greater Snow or Ross' Goose.

Figure 4. Screening for the presence of rntDNA sequences integrated in the nuclear

senornes of six goose species covered in this study. AI1 DNA samples were

digested with Hind III. The Southem blot was hybridized with pmLSG003.

Lanes 1.2. and 3 are DNA samples from three individual Lesser Snow Geese.

and lanes 6 and 7 are DNA samples from two individual White-fronted Geese.

The DNA samples were extracted from blood (lanes 1. 2.4. 5.6. 8. and 10). or

tissue (lanes 3. 7. afid 9)-

Southern blots of probe pmLSG003 hybridized to White-fronted Goose nuclear

DNA revealed more complex DNA fragment patterns (lanes 6 and 7 in Figure 4). It seems

that the mtDNA sequences were insened in several locations in the White-fronted Goose

nuclear genome. without going through extensive Iocnlized amplification seen in the Lesser

Snow Goose. Southem blots of DNA from White-fronted Goose hybridized with

pmLSGOO I and pmLSGOO2 indicated the presence of additional mtDNA sequences

incorporated in the nuclear genome of this species.

Ail three mtDNA probes faiIed to hybridize with any fragments in the nuclear

genome of the Emperor Goose. Even after extended exposure tirnes. and analysis of

digests with several informative restriction enzymes (such as that presented in Figure 5). al1

bands present on the autoradiographs were of mitochondria1 origin.

Probe prnLSG003 hybridized to 3.9 kb and 1.65 kb md III fragments of Brant

mtDNA (Figure 4 and Figure 5). 1 obsewed two additional fragments of 2.5 kb and 2.15

kb md III that showed reiatively weak hybridization. It is possible that these two novel

Hind III fragments represent the mtDNA sequences integrated in the Brant nuclear genome. -

The 5.5 kb mtDNA probe pmLSGûû3 hybridized to a 1.9 kb BamH 1 fragment. as well as

to two large molecular weight fragments of approximately 10.5 kb and 13 kb (Figure 5).

Mitochondrial DNA probe prnLSGOO 1 hybridized to 5.0 kb. 3.7 kb, and 1.4 kb a d III

fragments, as well as 12.5 kb. 9.6 kb. 9.1 kb and 2.3 kb BamH 1 DNA fragments in the

Brant Goose genome.

Southern blots of probe pmLSG003 hybridized to DNA from Canada Goose

reveal strong hybridization to the 5.5 kb md III fragment of mitochondrial ongin. ris well

as weak hybridization to 1 1.8 kb. 8.5 kb and 3.1 kb U d I I I fragments (data not shown).

Probe pmLSGOO 1 hybridized to the 1.9 kb &d I I I mtDNA fragment. aï well as to 8.2 kb.

5 . 1 kt, and 3.8 kb t-&d I I I fragments (data not shown).

Figure 5. Southem blot demonstrating restriction fragment patterns of Brant DNA

extracted from blood, digested with various enzymes. and hybridized with mtDNA

probe prnLSG003. In lane M is the lambda DNA size standard mixture. selected

fragment sizes are indicated on nght.

3.4 Gene content of Lesser Snow Goose mtDNA probes

aments To determine the content of mtDNA clones t h a hybndized to DNA fra,

inserted in nuclear gnomes of geese covered in this study. I sequenced two clones:

pmLSGOOl and pmLSGûû1. The sequence of the third clone (pmLSGûû3) was published

previously (Quinn 1992; Quinn and Wilson 1993). and determined to contain partial

NADH 5 and I ZS ribosomal genes. cytochrome b. NADH 6 and the control region (D -

loop). 1 had consistent problems with obtaining sequencing products with the universal

M 13 forward sequencing primer. Thus, the partial sequences of clone prnLSGOO 1 ( 1 38

bp. Figure 6), and pmLSGOO2 (23 1 bp. Figure 7) were obtained by using onl y the M 1 3

reverse primer.

The gene content of both clones was determined on the basis of results of

sequence similarity searches of GenBank databases (Table 2). Clone pmLSGûû I was

determined to contain regions with high sequence similarity to the chicken and duck

ATPase 6 gene. Thus 1 believe that the 3' end of this clone contains a portion of the

ATPase 6 gene. which is approximately 680 bp long in chicken and duck mtDNA. After

the initial BLAST alignment search. sequence of the Snow Goose clone prnLSGOO 1 was

aligned to known chicken and duck mtDNA (Figure 6). There are 24 nucleotide

substitutions and 6 insertions / deletions within the 138 bp of goose and chicken mtDNA

sequences. Transitions (A-G. T-C: n= 16) outnumber the transversions (n=8). The

majority of point mutations are silent. causing only 9 amino acid changes. Snow Goose

and duck sequence cornparison reveals 13 substitutions (transitions : trünsvcrsions = 14 :

9). and 5 nucléotide insertions / deletions.

A seltrch of the GsnBank sequence database reveliled that the Snow Goose clone

pmLSGûû7 contains sequrnce with high similnrity to duck. chicken and humün

rnitochondrial CO 1 genr (Table 2 and Figure 7). DNA sequence cornparison between the

Snow Goose clone (23 1 bp in Icngth) and chicken mtDNA rcvelils 40 nuckotide

substitutions (transitions : transversions = 23 : 17) and 15 mutations causing length

changes. Cornparison to the corresponding duck mtDNA repion clone (only 7 1 1 bp of

sequence compared) reveals 28 nucleotide substitutions (transitions : transversions = 18 :

10). The extent of length mutations in the analyzed region of COI gene is more

pronounced. causing several changes in the amino acid composition.

Figure 8 depicts a map of the chicken mitochondrial gene order (Desjardins and

Morais 1990). and relative position of the three DNA probes used in this study. Avise and

coworkers ( 1992) published a map of restriction sites in the Snow Goose mtDNA.

However, I was not able to further map the position of mtDNA probes used in this study

( U d III fragments) and infer the gene content for DNA probes pmLSG01 and

prnLSG002.

Figure 6. Sequence cornparison of the clone pmLSGûû I (Lesser Snow Goose partial

ATPase 6 gene). to the corresponding sequences in chicken (Desjardins and

Morais 1990), and duck mtDNA (Rmirez et al. 1993). Deletions in DNA

sequences are indicated by dahes. The nucleotide position numbering refers to

the chicken mtDNA sequence. Variable amino acids are indicated in bold face.

pmLSGOOl - ATPase 6

Gly Ile Pro Leu I l e Leu Pro Ser Leu Leu Leu Pro Ala Leu Leu Leu

chicken: GGA ATC CCT CTA ATC CTC CCA TCA CTC CTT CTT CCA GCC CTC CTA CTT Leu Phe

goose: GGC ATC CCT -TA ATC CTA CTA TC- CTA CTC TTC CCA GCC CTA CTA -TC Val Leu Phe

duck: GTC ATC CC- CTG ATC CTA CTA TCC CTG CTT CTT CCA GCC CTA TTG TTC

Pro Ser Pro Gly Açn A r g Trp Ile Asn Asn Arg Leu Ser Thr Ile Gln

chicken: CCA TCA CCA GGA AAC CGA TGG ATC AAC AAC CGC CTC TCC ACC ATC CAA Ser V a l LYS

goose: CCG TC- CCA AGC AAC CGA TGA GTC AAC AAC CGC CTC TCT ACT ATC AAA duck: CCA TCC CCA GGC AAC CGA TGA ATC AAC AAC C G . CTA TCC ACC ATC CAA

Leu Trp Phe T h r His Leu I l e Thr Lys G l n Leu Met Thr Pro

chicken: CTC TGA TTC ACC CAC CTA ATC ACA AAA CAA CTA ATA ACC CCC Leu Ile Ala Thr

goose: CA- TGA CTC ATT CAC CTA AT- ACA AAA CAA CTA ATA GCC ACT Leu Leu f le

duck: CTG TGA CTC CTA CAC CTA ATC ACA AAA CAA CTA ATA ATC CCA

Figure 7. Alignment of 23 1 bp nucleotide sequence of clone pmLSGOO? (Lesser Snow

Goose partial CO 1 gene). with corresponding sequences in the chicken

(Desjardins and Morais I990), and duck mtDNA (Ramirez et al. 1993). Only 21 1

bp of duck COI gene was available for the cornparison. The nucleotide position

nurnbering refers to the chicken mtDNA. "N" denotes uncertain nucleotide.

pmLGS002 - COI

chicken: GCC TCA TCT ACC GTA GAA GCT GGG GCC GGC ACA GGA TGG ACA GTT TAC goose: GCC TCA TCC ACT GTA GAA GCT GGC GCC GGC ACA GGC TGA ACT GTC TAC duc k : GGC GCT GGT ACA GGT TGA ACC GTA TAC

chicken: CCC CCT TTA GCC GGC AAC CTA GCC CAC GCT GGC GCA TCA GTA GAC CTA goose: CCT CCC CTA GCA GGC AAC CTC GCC CAC GCC GGA GCT TCA GTA GAC CTG duck: CCA CCT CTA GCA GGC AAC CTA GCC CAC GCC GGA GCC TCA GTG GAC CTG

chicken: GCC ATC TT- TCA -TT -AC TTA GCA GGT GTT TCC TCC ATT CTA GGA GCC goose: GCT ATC TTC TCA CTC CAC TTA GCC GGT ATC TCC TCC AT- CTT GGG GC- duck: GCT ATC TTC TCA CTT CAC CTG GCC GGT GTC TCC TCC ATC CTC GGA GCC

chicken: ATC AAC TTT ATC ACT ACC ATC ATC AAC ATA AAA CCC CCC GCA CTG TCA goose: ATC A-C TTC AT- AC- ACA GCC ATC AAC ATA AA- CCC C-- GCA CTC TCA duck: ATN AAC TTC ATT ACC ACA GCC ATC AAC ATA AAA CCC CCN GAA CTC TNA

chicken: CAA TAC CAA ACA CCC CTA TT- -CG TA- TGA TCC GTC -CT goose: CAA TAC AAC CCA CTA TTG CTG ACG TAC TAA TAC GCA TCT duck: CAA TAC CAA ACN CCA CTN TT- -CG T-C TGA TCA GTC -CT

Table 2: Regions of sequence similarity between Lesser Snow Goose mtDNA clones and

mtDNA of chicken (Desjardins and Morais 1990). human mtDNA (Anderson et al.

198 1 ). and partial sequence of duck rntDNA (Ramirez et al. 1993: Mindel1 et al.

1997). as revealed after the BLAST sequence search. Accession number is for

DNA sequences registered with the GenBank database. position is according to the

author's numbering in the sequencing file.

Clone Accession Position S ize % Origin of segment

number (bp) identity

pmLSGW 1 X52392 9345 - 9416 72 84 chicken ATPase 6

(138 bp) L22476 1204- 1282 79 83 duck ATPase 6

pmLSG002 U83787 1-114 114 87 duckCOi

(23 1 bp) X52392 6988 - 7090 1 03 83 chicken CO I

V00662 6244 - 6383 140 76 humanCo1

Figure 8. Origin and relative position of Lesser Snow Goose mtDNA probes prnLSGûûl

(containing a ponion of ATPase 6 gene). prnLSGOO2 ( containing a ponion of CO

1 gene), and pmLSG003 (containing cytochromr b. NADH 6. and D-loop). The

relative position of the three probes is overlaid over a map of the chicken

mitochondrial genome (adapted from Desjardins and Morais 1990).

CHAPTER 4: GENETIC STRUCTURE OF LESSER SNOW GOOSE POPULATIONS

ALONG A COLOUR-MORPH CLINE

From the data presented in Chapter 3. it is possible to propose that a portion of the

mtDNA molecule (that hybridized to prnLSGûû3) was inserted in the nuclear genome of an

ancestral gwse pnor to the sepantion of Ross' and Lesser / Greater Snow Goose lineages

(for discussion see Chapter 5, section 5.2). An alternative hypothesis is chat the rntDNA-

like sequences that hybridized to pmLSG003 were distributed in dl three "taxi' dirough

occasional hybridization and cross-taxon gene transfer. As late as the 1960s. "blue" and

white plumage forrns of Lesser Snow Geese were considered as separate species on the

basis of plumage polymorphism. Cooke and coworkers (1988) have proposed that the

"blue" and white phase geese were allopatric as recently as the 1920s. and that recent

sympatry and gene fiow have resulted in merging of the two colour morphs. with formation

of a colour-deliniated cline in the central portion of their range. The extent of the inter

"species" transfer of integrated mDNA-like sequences (Le. Ross' - Lesser / Greater Snow

Goose) can be inferred from a study of sene flow between the two "taxa" that were until

recently considered as separated species (Le. "Blue" and "Snow" phases of Snow Geese).

In this chapter 1 will present results of a population genetic study in the Laser Snow

Geese. by concentrating on the analysis of genetic structure of the colour-morph cline dong

the Gulf of Mexico coast. 1 will analyze variation in the restriction-frigment-length-

polymorphism (RFLP) detectable with single-locus DNA probes (Quinn 1985; Quinn and

White I987b). and asses the lsvel of genetic differentiation and gene flow among the mid-

continental group of Lesser Snow Goose populations.

4.1 Introduction

The Snow Goose is a North American species present in two allopatric subspecies:

the Lesser Snow Goose (Anser caerulescens caerulescens) and the Greater Snow Goose

(A. g. atlantica). The subspecies are rnainly differentiated by size. with a considerable

overlap in rnorphometric characters (Cooke et al. 1995). Distribution of Lesser Snow

Goose nesting colonies ranges from Wrangel Island in northeastem Russia, through the

Canadian Arcric, to Greenland. The majority of Greater Snow Goose nesting colonies are

located on Bylot, ElIesmere. and Baffin Islands, with migration routes and wintering

grounds along the Atlantic flyway and north-east coast of the United States. Pair formation

in geese occurs on wintering grounds or during migration in early spring (Cooke et al.

1995). At that time the genetic structure of breeding populations is king formed. Some

Lesser Snow Geese from the Hudson Bay / Foxe Basin nesting area winter along the

Atlantic coast. Consequently, gene exchange may occur between the Lesser and Greater

Snow Geese. The central group of Lesser Snow Geese populations winter along the Gulf

of Mexico coast of Louisiana. Texas and Mexico. Pairs formed in this region fly to their

nesting areas in the Hudson Bay - Foxe Basin region. with some birds nesting in the Queen

Maud Gulf and Central Arctic region. The Pacific and southwestern group of populations

(referred to as "western" populations) winter from southern British Colurnbia to northem

Mexico. and inland to New Mexico. Birds from Wrangel Island, Yukon and the western

Northwest Temtories use the same wintering grounds. Band recovery data of birds from

the central group of populations which nest around Hudson Bay and Foxe Basin and winter

dong the Gulf coast. and the westem. predominantly white populations that winter dong

the Pticitk coaït. show that thcre is limited exchange of animals between the groups

(Dzuhin 1974).

The Lesser Snow Goose is a dimorphic taxon. with gray ("Blue") and white

("Snow") plumage colour phrises. The two colour morphs were previously considered to

be distinct species (Anser hvperborea and Anser caemlescens). This dichromatism is

controlled by a single gene or a tightly linked group of genes. in which the blue allele is

incompletely dominant over the white (Cooke and Mirsky 1971: Rattray and Cooke 1984).

The two colour phases are not randomly distnbuted in the central wintering area. but show

clinal distribution dong the Gulf of Mexico coast. The blue ( p y ) phase predominates in

the east pan of the range. while white birds predominate in the West. There is a steep

change in coior phase ratios close to the Louisiana and Texas borders (Cooke et al. 1988).

Nesting colonies in the Hudson Bay and Foxe Basin that will be covered in this study.

show a distribution of colour phases reflecting the one dong the Gulf coa t (Cooke et al.

1975; Francis and Cooke 1992). A small number of white geese from the western

breeding group (birds nesting on Wrangel Island) were also included in this study.

There is a substantial amount of knowledge about Lesser Snow Goose ecology.

behaviour. and migration routes (Cooke 1987; Cooke et al. 1995). Historical evidence

shows that the two colour morphs had alrnost allopatric distribution until the third decade of

the 20th century. A recent change in phase ratio distribution has k e n noticed (Cooke et al.

1988). with the blue-phase birds increasing in relative numbers in the predominantly white.

western breeding range. and white-phase birds showing a corresponding increase in

numbers in blue. eastem colonies. Differential migration of birds from the same colony is

still detectable. with blue geese tending to overwinter more easterly than the white birds

. (Cooke et al. 1975: Francis and Cooke 1992). An increase in the availability of winter

food due to changed agricultural practices in the coastal marshes of Texas and Louisiana in

the late 1910s. and incre~sed numben of nrwly established National Wildlife Refuges in

the United States at the beginning of the 20th century. may be the reasons for rnodified

migration routes and changes in distribution between the two colour phases. The rate of

change in colour phases is so rapid that Cooke and coworkers ( 1988) suggestrd that the

rnixing of the morphs could have occurred as recently as 10 generations ago.

Recent secondary contact of formerly allopatric forms after a p e n d of isolation is

one of the explanations suggested for the observed clinal distribution of colour morphs in

the Lesser Snow Goose (Geramita et al. 1982; Cooke et al. 1988). Birds of the two colour

morphs are assurned to have ken geneticaliy isolated in allopatry. and one thus expects to

observe allrle frequency differences between the populations containing various

proportions of "blue" and white birds. Such differences could have accumulated in

formerly allopatric populations through drift and / or selection, and although the two

morphs now interbreed freely. some residual genetic differences are expected to be seen.

In the Lesser Snow Goose most of the allozyme loci studied show low levels of

polymorphisrn under standard electrophoretic conditions (Bargiello et. al 1977; Cooke et

al. 1988). rendering the protein electrophoresis of little use in a study of genetic

differentiation within groups of closely related populations. Development of single-locus

nuclear DNA probes that detect restriction fragment length polymorphism (RFLP) in the

Lesser Snow Goose (Quinn 1988: Quinn and White 1987b), allowed for more sensitive

analysis of genetic varîation in the natural populations of this subspecies. 1 decided to use a

subset of these DNA probes in my study. After this study was initiated, Quinn ( 1992) and

Avise et al. ( 1992) used mtDNA sequence and RFLP analysis for the genetic study of

Lesser Snow Goose populations, and detected genetic substructuring of the populations

ricross the breeding range.

In this study. 1 will test whether thrre are residual genetic differences between the

populations forming a "hybrid zone" dong the Gulf of Mexico coast. 1 will determine the

level of diffcrentiation betwecn the populations comprising the cline, and obtain an estimate

of p n ç tlow along the cline. This will estüblish the rate of introgression from "blue" geese

into '-white" gecse populations. and provide a measure of intensity of gene tlow through the

zone of secondary contact. This study will concentrüte on the central group of Snow

Goose populations. and will differ from the previous population genetic studies in this

species by analyzing samples collected on wintering grounds where pair formation occurs

and the genetic structure of nestinz groups is detemined.

4.2 Materials and Methods

Samples

Blood samples were collected dunng reguiar bird-banding drives iit the Sabine

National Wildlife Refuge. Cameron. LA, from December 1990 to January 199 1. One to

three ml of blood was drawn from the brachial vein and immediately injected into a 5 ml

vacutainer containing lysis buffer (Perkio Elmer - Applied Biosysterns ( A M ) Division.

Fosier City. CA). The sample tubes were inverted and kept chilled until long-tem storrige

at -2BC. Heart tissue samples were collected during the same time period. from birds

brought by hunters to check-in or cleaning stations. Locations and nurnber of samples are

listed in Table 3. About 2 - 5 g of tissue was cut into small pieces and stored chilled in the

AB1 lysis buffer or pickling solution (Seutin et al. 199 1 ). After delivery to the laboratory at

Queen's University. tissue samples were kept for long-term storage at -20°C. Fifteen liver

samples representing a breeding population from Wrangel Island. Russian Commonwealth

(7 1 N. 180" E) were from the Lesser Snow Goose tissue collection stored at Queen's

University. Kingston. Canada. and used previously (Quinn 1988).

DNA extractions, restriction digests, hybridizations and data analysis

Total cellular DNA was extracted as described in the '-MateriiiIs and Methods"

chapter (Chapter 21. Restriction digests. rlectrophoresis and hybridizations were

performrd as described in Chapter 2. Six Lesser Snow Goose single-locus DNA probes

were used in this study: DQSG- 1 . DQSG-2. DQSG-3. DQSG-S. DQSG-6. and DQSG-8

(Table 4).

Table 3: Sampling locations and number of samples used for the Lesser Snow Goose

population study. Samples from Eagle Lake and Ganvood are in geognphical

proximity. and for the purpose of this study considered to represent one locality.

Due to a small sample size. samples from Freeport were pooled together with the

samples from Eagle Lake and Garwood.

Location Number of Colour Tissue type % sam~les momh

Eagle Lake. Texas

(29"33' N 96'20'

Garwood, Texas

(29'28' N 96'2 1 '

Freeport. Texas (29'03' N 95' 15'

22 Snow heart

7 Blue hem

37 Snow hem

4 Blue heart

6 Snow hem

14.5

Cameron. Louisiana (29'47' N 93" 19' W)

Lake Arthur. Louisiana (29'57' N 92'52' W)

Louisa (New Iberia), Louisiana (29'35' N 9 1°50' W)

Wrangel Island. Russirin

Commonwealth (Quinn. 1988)

77 -- Snow blood

19 Blue blood 46.3

8 Snow heart

20 Blue hem 71-4

2 Snow heart

46 Blue hem 95.8

15 Snow liver

o. 0

Figure 9. Top panel: colour-phase ratios of Lesser Snow Geese on wîntering grounds

dong the Gulf of Mexico Coast (adapted from Cooke et al. 1995)

Bottom panel: sampiing locations for the Lesser Snow Goose population study

(A= Eagle lake. Garwod and Freeport, Texas: B= Cameron. Louisiana: C=

Lake Arthur, Louisiana; D= Louisa, Louisianri).

The numbers of alleles present and observed heterozygosity were calculated for

each population. lit each locus. Basic information on the data set (allele frequencies.

observed and expected number of homozygotes and heterozygotes). and conformity to

Hardy-Wein brrg equilibrium were obtained by ushg GENEPOP software package version

3.0 (Raymond and Rousset 1995). This program calculates the expected and observed

number of homozygotes and heterozygotes and tests the significance of the deviation from

Hardy-Weinberg equilibrium using either the complete enurneration method for loci with

less than 5 alleles. or the Markov chah method proposed by Guo and Thompson ( 1992).

Cooke and coworkers ( 1988) detected heterozygote deficiency for allozyme data in a

population study of the Lesser Snow Goose. Consequently. 1 tested for the alternative

hypothesis H 1 ="heterozygote deficiency" (Rousset and Raymond 1995). rather than using

the exact Hardy-Weinberg test (Weir 1996).

Tests of heterogeneity between populations and colour morphs were perfomed

with the GENEPOP program. which tests for independence between 1 populations and k

alleles as described in Raymond and Rousset (1995b). This test is analogous to the

Fisher's exact test on a 2x2 contingency table extended to a 1 x contingency table. AH

potential states of the contingency table are expIored with a modified Markov chain

approach (Guo and Thompson 1992). where the probability of observing a table less or

equally likrly than the observed sample configuration under the nuIl hypothesis tested. H,:

"the allele distribution is identical across populations". As with the test of deviation from

Hardy-Weinberg equilibriurn. a standard emor of an unbiased estimate of probability value

is provided with each calculrition.

A test for genotype linkage disequilibrium was performed on al1 loci with the

GENEPOP software packqe. A contingency table with the number of genotypes at locus

n and genotypes at locus n+l. for al1 pairs of loci in each population. is creatrd and -

subjrcted to a Fisher exact test for each contingency table using a modified iMarkov chah

method (Guo and Thornpson 1992). The hypothesis tested is Ho: " genotypes at one locus

are independent from genotypes at the other locus".

A multilocus estimate of the effective nurnber of migrants (h. where N is the

local population size and a is the proportion of migrants) was calculated using the private

allele method of Slatkin ( 1985). based on Wright's ( 193 1 ) islnnd mode1 of migration. The

estimate of number of migrants was corrected for sample size (Banon and Slatkin 1986).

AI1 calcuIations were perforrned using GENEPOP.

4.3 Results

Scoring of RFLP polymorphism, and levels of DNA variation

Six single-locus DNA probes developed by Quinn (1988) and Quinn and White

( 1987b) were used to m e s s the levels of poiymorphism in Lesser Snow Goose

populations: DGSG- 1. DGSG-2, DQSG-3. DQSG-5. DQSG-6. and DQSG-8 (Table 4).

In nearly a11 of the cases, the detected RFLP pattern cm be interpreted as base substitutions

in the enzyme recognition site. For some probe 1 enzyme combinations. polymorphism

was caused by length variation in one of the fragments (Figure 10).

Only fragments of relatively high hybndization intensity were included in the

allelic descriptions and analysis. Some of the probe /enzyme combinations had to be

excluded due to the Iück of consistent hybridization results with DNA fragments smaller

than 1 kb. Allele designations were as suggested by Quinn (1988) and Quinn and White

( 1987b). and represent ü region of DNA detrcted with particular probe / enzyme

combinations rather than change in a single recognition site. For most of the probes.

complete al lele designations were possi blc. However. for somr probe 1 enzyme

combinations the source of variant band. or band combination was not obvious. These

were marked as "xx k b fragment absent" (ser Tablc 4, DQSG-8 I MspI. allele CI-5).

Table 4: Restriction fragment length polymorphism identified by 6 DNA probes. Allele

designation follows Quinn (1988) and Quinn and White (i987b). with addition of

new alleles detected in this study. Alleles at locus DQSG-5 1 HindIII - BI and BIII

were not scored as some fragments were small and difficult to score with certainty

needed for a population study .

Probe Enzyme 2N Allele Fragment sizes (kb) Frequency name (W

DQSG- 1

DQSG- I

DQSG-2

DQSG-3

CI- 1

CI-2

CI-3 C 1-4

CI-5

CI-6

DI- 1

DI-2

DI-3

DI-4

DI-5

DI- 1

DI-2

DI-3

CI- I

CI-2

CI-3

C 1-4

CI-5

C 1-6

Table 4. (cont.)

Probe Enzyme 2N Allele Fragment sizes (kb) Frequency (W

DQSG-5 W d I I I 354 BII- 1 2.4. BI. BIII fragments * BII-2 2.6, BI, BI11 fragments

BII-3 2.3. BI, BIII fragments

DQSG-8 W d I I I 350 BI- l 3.8. 2.3, 1.6. 1.3. 1 .O, BI1 fragm. 86.6

B K 3.8. 3.5. 1.6. 1.3. 1.0. BI1 f r q m . 12.8

BI-3 3.8. 2.4, 1.6, 1.3, 1.0. BI1 frrtgm. 0.6

BII- 1 4.8, BI fragments 90.3

BI12 2.9, 1.9.BIfragrnents 9.1

BII-3 2.55. 2.25, BI fragments 0.6

DQSG-8 MSPI 340 CI-1 13.6

CI-? 7.6, 6.0

CI-3 1 1 .O. 2.5

C I 4 9 . 1 , 4 . 5

CI-5 7.6. 6.0 absent

* DQSG-5 HindIII - BI and BIII fragments are: 3.4. 2.8. 2.45, 1.5. 1.24. 1.13. I.ZI kb

Figure 10. Example of detection of restriction fragment length poly morphisms in Lrsser

Snow Geese involving single restriction endonuclease recognition site and length

variation in one of the fragments. 32~-labeled DNA probe DQSG- 1 was

hybridized to Southem blot of MSDI digested genomic DNA in five individuals.

Genotype designations are shown above each lane. In lane M is the size marker.

human adenovims-2 DNA digested with Aval and BamHI (double digest).

Fragment sizes in kilobase-pairs are indicated on right.

Additional alleles uncovered in this study are listed in Table 4. As the IeveI of

RFLP variation in females did not differ significantly from the variation in males

(probability values for loci across al1 populations were 0.799 > P > 0.088). 1 pooled the

sexes to avoid further reduction of sample sizes. The allele frequencies pooled across

populations are presentrd in Table 4. Al1 of the loci except DQSG- 1 / Ta1 were

polymorphic under the 95% rule. The 95% frequency de finition of a polymorphic gene is

arbitrary (Hm1 and Clark 1989). and defines a polymorphic gene as a gene at which the

most common allele has a frequency of less than 0.95.

Testing for Hardy-Weinberg equilibrium, and linkage disequilibrium

Since most tests of genetic substructuring make the assumption that the genetic

markers king considered are in Hardy-Weinberg equilibrium. and that the genotype data at

each locus are independent from genotype data at the other loci. 1 conductsd tests of

conformity to Hardy-Weinberg equilibrium. and linkage disequilibrium for al1 loci across

al1 populations. Problems of low genotype and allele counts in the analysis were avoided

by using the GENEPOP software package that performs specific calculations by using a

Markov chain randomization method (Guo and Thompson 1992). No significant

deviaiions from Hardy-Weinberg equilibrium were detected. except in the Lake Arthur

population (at loci DQSG-2 / -1. and DQSG-8 / MspI). Louisa population (loci DQSG-5

/ &dIII-B. and DQSG-5 / EcoRI). and Wrangel Island population (locus DQGS-8 /

~VspIi. To test for the presrnce of significant association between pairs of loci. a painvise

linkagr disequilibrium test bctween al1 pairs of loci was conducted with the GENEPOP

software package. There wüs no evidence of linkage disequilibria between any pair of loci.

so the gnotype daia can be regardcd aï independent for each locus (data not shown).

Tiible 5: List of loci that showed signîficant deviation from Hardy-Weinberg equilibriurn.

The degree of deviation of genotype frequencies from the equilibrium is descnbed

by the index D where D= (Ho-H,) / H,. Ho= observed heterozygosity . md H,=

expected heterozygosi ty . A deficienc y of heterozygotes against expectations is

indicated by a negative value of D. The P value indicated represents the

probability of being wrong when rejecting the alternative hypothesis (Le. H i =

"heterozygote deficit"). Standard error of the estimate of probability value for the

locus DQSG-6 / EcoRI (Louisa. LA) was 0.0026 (Markov chain rnethod).

Population LOCUS N N A D P

Lake Arthur. LA DQSG-2 / -1 - ?5 3 - 0.39 1 0.007

DQSG-8 / MSDI 23 4 - O. 143 0.022

Louisa. LA DQSG-5 / HindIlI 48 3 - 0.975 0.044

DQSG-6 / b R J 46 5 - 0.353 0.024

Wrangel Island DQSG-8 / Mspi I I 3 - 0.322 0.006

N= sarnple size. NA= number of different alleles (allele types)

Allele frequency distribution, and measures of genetic differentiation

Allele frequencies per locus. pooled in respect to the population origin and colour

morph. are presented in Table 1. Since no overall differences in allelc frequencies were

found betwren male and fernale samples (combined set of loci excluding DQSG- I / 3 1 :

Fisher exact test: Chi'= 14.490, d.f-= 16. P= 0.5990). data for the two sexes were pooled

in al1 subsequent analyses. Table 6 presents allele frequencies of the three most common

aileles for al1 loci in samples from the Gulf coast and Wrangel Island. and results of the test

of population differentiation. 1 was not able to detect any significant differences between

populations at any of the 8 loci . The most common allele at DQSG-1 / -1 had a

frequency of p > 0.960 in al1 of the Gulf coast populations and data for this locus were

excluded from population analyses. The allele frequencies at this locus in the Wrangel

Lsland population were p= 0.889. q= O. 1 I I (ZN= 18). Differences in allrle frequencies at

this locus were not significant among populations (P= 0.3 137, S.E.= 0.010). The overall

differentiation of allrle frequencies between populations was not signiticantly different from

non-randorn distribution (Fisher exact test. ch iZ= 15.386. d.f.= 16. P= 0.5236).

I also conducted analyses to see whether there are any significant differences in

allele frequencies between blue and white birds. For this comparison. 1 pooled the data for

sampIes from al1 five populations. There were no significant di fferences in allele

frequencies between the two colour morphs when data were analyzed per locus (Table 7).

or for üII loci cornbincd (Fisher exact test. c h i 2 = 10.030. d.f.= 16. p= 0.7102). Due to the

Iück of overdll differenccs betwren colour rnorphs and small samplrs sizes per plumage

morph in some populiitions. I did not conduct analyses of allele frequency distributions of

the two colour inorphs w ithin and betwren individual populiit ions.

Figure I 1 . Top panel: percentage of Lesser Snow Geese of g'blue" colour morph in each of

the five sampling locations. Bottom panel: allele frequencies for the most cornmon

allele at 8 gene loci from Wrangel Island and four locations along the Gulf of

Mexico Coast. Locus designations have k e n shortened for ciarity: DQSG- 1 /

MspI = 1 -Msp. DQSG-8 I HindiII allele BI = 8-Hind A. etc.

O 1 -Msp O 2-Taq A 3-MSP @ 5 - Hind B + 6 - ECO % 8 - Hind A

8 - Hind B 8-MSP

Table 6: Cornpaison of allele frequencies for 8 loci across 5 populations and the estimüte

of the P-value of the probability test for population differentiation (Ho= "no

differentiation"). "S.E." is the standard error of the estimate of probability value.

"7N" is the number of scored alleles. "p'.. "q". and "r" are frequencies of the three

rnost common alleles. Locus designations have been shortened for clzirity.

1-Msp

Wrangel Isl.

Eagle Lake

Carneron

Lake Arthur

Louisa

2-Taq

Wrangel Isl.

Eagie Lake

Cameron

Lake Arthur

Louisa

3-Msp

Wrangel Isl.

Eagle Lake

Cameron

Lake Arthur

Louisa

5-Hind B

WrangeI I d .

Eagle Lake

Cameron

Lake Arthur

Louisa

9 r P + S.E.

0.719 1 0.0 12 1

0.227 0.091

0,150 0.150

O. 167 O. 128

O. 159 O. 136

0.114 0.114

Table 6 (cont.)

6-Eco

Wrangel Isi.

Eagle Lake

Cameron

Lake Arthur

Louisa

8-Hind A

Wrangel Isl.

Eagle Lake

Cameron

Lake Arthur

Louisa

8-Hind B Wrangel Isl.

Eagle Lake

Cameron

Lake Arthur

Louisa

8-Msp

Wrangel [ S I .

Eagle Lake

Cameron

Lake Arthur

Lou i sa

0.1 15

0.230

0.244

0.200

0.228

O. 136

O. 130

O. 100

0.096

O. 167

0.182

o. 1 10

O. 100

0.058

0.063

0.173

o. 122

O. 162

O. 1 Oc)

0.100

-+ S.E.

Table 7: Analyses of allele frequency distribution at 8 loci for two colour rnorphs.

Population data were pooled. See legnd to Table 6 for symbol description.

2N P 9 r P 2 S.E.

1-Msp

Snow

Blue

2-Taq

Snow

Blue

3-Msp

Snow

Blue

5-Hind B Snow

Blue

6-Eco

Snow

Blue

8-Hind A

Snow

Blue

8-Hind B Snow

Blue

8-Msp

Snow

Blue

A second test of population differentiation was conducted with the Arlequin

software package (Schneider et al. 1997). Sirnilar to the analysis with GENEPOP. this

proprn utilizes a Markov chain approach for the exact test of population differentiation. as

described in Raymond and Rousset ( 1995b). and Goudet et al. ( 1996). No sionificant t

di fferences in allele frequencies were detected among populations (P= 0.9007. S.E.=

0.0543 üfter ZOOûû iterations). Results of the analysis between all pairs of populations are

presented in Table 8.

Population genetic structure of Lesser Snow Goose populations along the Gulf of

Mexico coast was: funher investigated through the analysis of variance of gene frequencies

(F-statistics) following Weir and Cockerham ( 1984). A hierarchical analysis of variance

partitions the total variance into components due to differences within individuals. among

individuals within subpopulations. and among subpopulations. The senetic structure of

populations can be inferred by the measurement of the arnong-population componeni of

oenetic variance (FsT): populations (or species) with little gene flow among strongly 2

differentiated subpopulations have FST values which approach a value of 1.0. Genetically

homogeneous species (populations) with extensive gene flow have FST values approaching

0.0. Table 8 lists pairwise FST values calculated for the five Lesser Snow Goose

populations covered in this study .

3.4 Discussion

On the bais of RFLP analysis using 6 single-locus DNA probes. it was possible

to detect a wsak trend in distribution of üllele frequencies across the geopphic range of the

central goup of wintering populations in Lesser Snow Goose (Figure I 1 ). Cooke and

coworkers ( 1988) collected extensive histot-ical evidence showing that the "blue" and white

colour phases of Lesser Snow Geese were separated in allopatry for an extensive amount of

tirne. and thtit they carne in secondary contact with ii consequent exchange of genes between

Table 8: Panel A: Exact test of population differentiation between al1 pairs of populations.

Probability values are listed below the diagonal. and standard error values of

probability estimate are listed above the diagonal. Null hypothesis rested H,,: "no

differentiation among populations". Panel B: Population pairwise FST values.

A: exact test of population differentiation

-

Eagle Lake Carneron Lake Arthur Louisa Wrangel Isl.

Eagle Lake 0.0237 0.0092 0.0000 0.06 17

Cameron 0.90 1 9 0.0263 0.0 173 0.05 17

Lake Arthur 0.9740 0.69 12 0.0290 0.0280

Louisa 1 .O000 0.9082 0.7689 0.0535

Wrangel [si. 0.6035 0.6 152 0.7892 0.538 1

13: pairwise genetic distances (FST values)

EagleLake Cameron LakeArthur Lou isa

Eagle Lake

Cameron 0.0033

Lake Arthur 0.0097 0.0 1 32

Louisri 0.0054 0.0035 0.0 142

Wrangel M. 0.0 182 0.0044 0.0445 0.0 123

populations as recently as 70 years ago. They suggested that formerly allopatric

populations of the two colour phases could have accumulated and retained genetic

differences through drift and 1 or selection. Indeed. such differences were detected by the

analysis of allozyme polymorphisms (Cooke et al. 1988). The RFLP analysis of genomic

DNA (Quinn 1 988) revealed significant di fferences between western and central group of

populations, but not within the central group.

With the data collected and presented in this study. I was not able to detect

di fferentiation between the populations along the Gulf of Mexico coast. Samples from

populations collected on wintering grounds were considered in this study to represent true

genetic units. as at that time the pair formation and genetic structure of individual breeding

oroups is determined. Tests of population differentiation at each locus. and over al1 loci. b

failed to detemine significant differences among populations. The only significant

difference was detected in painvise comparisons at locus DQSG-8 I MspI for population

pairs: Wrangel Island and Eagle Lake (P= 0.0339. S.E.= 0.0033). Wrangel Island and

Lake Arthur (P= 0.047 1. S.E.= 0.0035). and Wrangel Island and Louisa (P= 0.0344.

S.E.= 0.0038). Differences between Wrangel Island and Cameron were non significant

(P> O. 1 ). The two most common aileles at this locus in the Wrangel Island population have

frequrncies of p= 0.636. q= 0.273, whereas in the other four populations the frequency of

the two most common alleles at this locus are p > 0.8 13. q< 0.162. Allele frequencies at

locus DQSG- 1 1-1 are not listed in Table 6: this locus is monomorphic (under the 95%

rule) in tour populations (p > 0.960). and polymorphic in the Wrangel Island population

(p= 0.889. q= O. 1 1 1 ). However. the allele frequency differences at this locus did not

producr a signitïcant rrsult after the exact test of population differentiation (P= 0.3 137.

S.E.= 0.0 12 1 ). As mentioned in the introduction. there is very limited documented

exchanse of animal?; betwern the western group of Lesser Snow Goose populations

(Wrangel Island) and the central group (al1 the other populations). allowing for a possible

developrnent of hcterogeneity in allele frequencies.

1 was not able to detect significant differences in the overall cornparison of the two

colour rnorphs. Subsequent cornparison of colour phases by individual population along

the colour-morph cline was not conducted. as this would further reduce the sample size and

complicate the analysis. Analysis of FST values revealed lack of population differentiation

at these loci as well. Overall. FST values were rather low. indicating low levels of

population heterogeneity and high gene flow. Cooke and coworkers ( 1988) used 6

polymorphic allozyme loci on blue and white birds from LaPerouse Bay colony (central

group of populations) and obtained an FST value of 0.0033 with the same calculation

method. Quinn ( 1988) obtained comparable results from the same colony with a set of 16

single-locus DNA probes (FST = 0.0039).

Departure of genotypic frequencies from Hardy-Weinberg expectations. showing a

deficit of heterozygotes, occurred at four loci (Table 5). Considering that only the Lake

Anhur population showed a deficit of heterozygotes at two loci. with other loci being at

equilibrium, it is likely that they are a statistical artifact of multiple cornpansons rather than

a result of evolutionary forces acting on this particular population (Le. selection against the

heterozygote, inbreeding due to a smail population size. non-random mating. or the

Wahlund effect due to population substructuring).

Estimates of the number of migrants using the private allele mode1 (Slatkin 1985:

Barton and Slatkin 1985) obtained the Nm value (after correction for sample size) of 6.59

individuais per generation. This value is rather high compared to other vertebrate species

(Hart1 and Clark 1989) indicatine high b e l s of gene flow and introgression of genes along

the cline. Pair formation of Lesser Snow Geese that nest in Hudson Bay and Foxe Brisin

occurs on the wintering grounds in Texas and Louisiana. If pair formation tiikes place

between birds of different nestins colonies. the male returns to the fernrile's natal colony.

The resulting gene tlow betwsen nesting colonies could be extensive as a result of influx of

new genes brought in by mignting males. Occasional movement of fsmales brtween

colonies occurs as well. further increasing the rimount of gene exchange between the

breeding groups (Genmita and Cooke 1982). There are no bamers for gene exchange

between blue and white birds either: hybridization of the two plumage forms seems to take

place without reduced reproductive fitness in hybrids (Findlay et al. 1985). However. a

rapid approach to equilibrium of colour ratios in both breeding and wintering populations.

and consequent rapid gnetic homogenization of populations. is not expected to occur.

This is due to ii strong positive assortative mating in this species. and the clinal wintering

distribution. Lesser Snow geese choose their mates according to the colour of their parents

and soblings (Cooke et al. 1976: Geramita et al. 1983). and mixed pairs are only 40% as

common as expected under random mating. Birds of different colour phase tend to

concentrate at opposite ends of the wintering range. and consequently have increased

probability of finding a homotypic partner. Nevertheless. some error in choosing a colour-

matching mate does occur. A female may choose her mate based on some character other

than plumage coloration. or mate choice may be infiuenced by different av~lability of the

two morphs in mixrd wintering flocks. In addition. egg dumping can result in birds

choosing the "wrong" colour if some of their sibs are of such colour. Although positive

assortative mating with respect to plumage polymorphism will restnct dispersa1 of the

colourdetermining gene along the cline. it should not influence the introgression of

unlinked loci (Barton 1983). As a consequence. the Lesser Snow Goose populations dong

the colour-morph cline could be more homogeneous in their genetic structure than it is

possible to deduct from the distri but ion of the colour character itself.

Sînce the inception of this study. Avise and coworkers ( 1992) analysed Lesser

Snow Goose population structure with RFLPs in mitochondrial DNA and found no

significant differences ümong populations. Quinn ( 1992) analysed DNA sequences of the

rapidly evolving control region of the mtDNA. and determined a more intricate substmcture

of Lssser Snow Goose populations than any previous study. He described reticulate

existüncc of iwo distinct mtDNA clades present in the entire Lesser Snow Goose range.

without detectable correlation to colour or location. Only one of the mtDNA clades showed

geographic substructuring. correlated with the West (Wrangel Island) - east division

(Hudson Bay I Foxe Basin colonies). The presence of two distinct clades was not evident

in my data. probably due to the fact that 1 concentnted on collecting samples from a central

area of the subspecies range. and used nuclear DNA markers. Mitochondrial DNA markers

(particulary the analysis of mtDNA sequences) often provide more information about

population sustnicture. This is due to a higher rate of mtDNA sequence rvolution

compared to the nuclear DNA (Brown et al. 1979, 1982). as well aï the fact that mtDNA is

matemally inherited and is more sensitive in detecting genetic substructure in species with

female natal phylopatry such as the Snow Goose. Any further study into the gnetic

structure of Lesser Snow Goose colour-morph cline will include the analysis of npidly

evolving regions of the mi tochondrial genome.

The presence of mitochondrial DNA-like sequences has frequently been

documented in nuclear genomes of higher orgmisms (reviewed in Chapter 1 ; Table 1 ).

Such sequences were detected with various techniques. such as DNA blotting and

hybridizations (Gellissen et al. 1983: Jacobs et al. 1983; Zullo et al. 199 1) . or accidental

PCR amplification (Smith et al. 1992: Quinn 1992: Collura and Stewart 1995; Zhang and

Hewitt 1996a). Transposed mtDNA fragments have been shown to have nuclear DNA

locations by probing Southem blots of differently purified DNA fractions, in situ

hybridizations of chromosomes, and sequencing through sites of insertions (Lopez et al.

1994 used al1 three methods). in this thesis. I demonstrated the existence of mtDNA-like

sequences in the nuclex genomes of five species of geese (Aves. Ansennae) through

hybridizations of Southem blots with mitochondrial DNA probes.

5.1 rntDNA sequences integrated in the nuclear genomes of geese

in Chapter 3 1 have shown that the genomic DNA of Lesser Snow Goose after

digestion with &d III, BamH I or Ava 1 yields a 3.6 kb DNA fragment, which hybridizes

to a 5.5 kb mtDNA probe (pmLSG003). The 3.6 kb fragments represent mtDNA

sequences inserted in the Snow Goose nuclear genome (Quinn and White 1987; Quinn

1992). and are arranged in tandem arrays. The same mtDNA probe detects a minisatellite

type of banding pattern on Sourhrrn blots with Y- III digested Snow Goose DNA.

Preliminary screrning of DNA sarnples chosen randomly from a natural population

determined low levels of shrired bands rimong the individuals. The observed pattern was

very similar to DDNA tïngerprinting profiles developed by Jeffreys and coworkers ( 1990).

and could represent subtle variation within the mÿy of repeats. The vast majority of

integrÿted mtDNA sequences forrning the tandem array of 3.6 kb repeats contain a

recognition sequence for m d III. BamH 1 and Ava 1. Some repeat units. but not all. also

have an additionai recognition sequence for Hae III. Variation in band position on a

fingerprint profile is caused by differences in length of DNA fragments drtected. due to the

variation in the number of repeats between the III recognition sites. DNA

fingerprinting techniques have been successfully used in pedigree studies of birds (Burke

1989), and the probe pmLSG003 could potentially be used in sirnilar studies at least in

Snow Geese.

Al1 of the individuals of Greater Snow and Ross' Geese screened for the presence

of nucDNA sequences with homology to the mitochondrial genome. possess the prominent

3.6 kb nuclear fragment as detected by the rntDNA probe pmLSG003. Similar to

observations already described for the Lesser Snow Goose. both Greater Snow and Ross'

Geese were determined to contain integrated rntDNA sequences that are not confined to the

tandem array of 3.6 kb monomers. 1 was not able to detect any species-specific bands

indicating sequences with mitochondrial inserts that would hybridize to prnLSG003.

Rather. al1 of the observed variation in the banding patterns was due to the variation within

the particular taxon.

In addition to DNA fragments of mitochondrial origin, ail three mtDNA probes

(pmLSGOO 1. prnLSGûû2. and prnLSGûû3) hybridized to DNA fragments of nuclear

origin on Southem blots containing White-fronted Goose DNA. Confirmation that the

additional bands represent nucDNA sequences cornes from a differential amount of

hybridization when the source of DNA was blood or tissue. A larger portion of the

mitochondrial gnome was incorporated in the White-fronted Goose nuclear genome. as the

oments seen three DNA probes represrnt about 609 of goose mtDNA. The number of fra,

on Southem blots that hybridized to the mtDNA probes suggest more than rt single

integration of mtDNA sequencrs. as well üs distribution in several locations of the White-

fronted Goose grnonie. Multiple integrations of mtDNA sequences hlis been documented

previously in a varicty of organisms: sea urchins (Jacobs et al. 1983 ). aphids (Sunnucks

and Hales 1996). birds (Arctander 1995; Sorenson and Fleischer 1996), rats (Zullo et al.

199 1 ). cats (Lopez et al. 1994. 1996). and primates (Fukuda et al. 1985: Collura and

Stewart 1995; van der Kuyl et al. 1995).

Probes pmLSGûû1 and pmLSG003 hybridized to Southem blots with DNA from

Brant and Canada Goose revealed fragments of mitochondrial origin, as well as additional

DNA fragments. Digests with several restriction enzymes (Figure 5) revealed fragments

whose total length adds up to more than 20 kb. which is greater than the length of the

mtDNA molecule itself. This seemingly contradictory observation might be explained by

the presence of mtDNA variation within individuals (heteroplasmy), intra-rnitochondrial

gene duplications. or that additional fragments represent nuclear copies of transposed

mtDNA sequences. Previous mtDNA studies in Brant and Canada Goose report rare

instances of heteroplasmy (Shields and Wilson 1987: van Wagner and Baker 1990). In

this study, extra fragments observed on autoradiographs were of the same size and present

in al1 screened individuais (data not shown). Brant and Canada Goose mitochondrial

pnomes are about 16.7 kb long (Shields and Wilson 1987) and although some mtDNA

length variation has k e n reported in birds (Avise and Zink 1988; Edwards and Wilson

1990). none was extensive enough to account for the additional mtDNA sequences reported

in this study. The most likely explanation for the presence of extra bands seen on

autoradiographs with DNA from Brant and Canada Goose is that they reveal nuclear-

encoded mtDNA sequences.

None of the three DNA probes used in this study detect mtDNA sequences

incorporated in the nuclear genome of Emprror Goose.

5.2 Goose phylogeny and possible evolutionary origins of the transposed

mtDNA sequences

1 propose that a portion of the mtDNA rnolecule (that hybridized to pmLSG003).

was inserted in the nuclear genome of an ancestral goose pnor to the separation of Ross'

and Lesser / Greater Snow Goose lineages (event "A" in Figure 12). Ross' Geese breed in

central arctic Canada with breeding pairs sometimes found in the sarne colonies as the

more numerous Lesser Snow Geese. Blue colour phases of Ross' Geese have been

observed. but they are extremely rare (McLandress and McLandress 1979). Although the

Snow and Ross' Goose interbreed and produce fertile offspring (McLandress and

McLandress 1979) they are considered as separate species. Previous studies determined

low levels of genetic differentiation between these two taxa using both mtDNA (Shields and

Wilson 1987: Avise et al. 1992). and allozyrne markers (Patton and Avise 1985). Both

Snow and Ross' Geese populations exhibit the presence of two distinct mtDNA clades

detected by RFLP analysis (Avise et al. 1992). The presence of two mtDNA clades in

Lesser Snow Goose populations was confirmed with the analysis of D-loop sequences

(Quinn 1992). Quinn ( 1992) determined the nucleotide sequence for a portion of the D-

loop region in the Lesser Snow Goose mitochondrial genome. as well as the partial

sequence of the nuclear homolog. and concluded that the rntDNA homolog onginated

before the divergence of the two mitochondrial DNA clades.

The age of nuclear integrations can be estirnated in at least two ways. The level of

divergence between the nuclrar and mitochondrial copies can be used to infer the date of the

transfer event by considering the diffrrencc in the rate ofevolution of rnitochondrial senes

and corresponding nuclear pseudogenrs (Quinn 1992). However. since nuclear copies

may be in different chromosonilil positions and experience v~stly divergent rates of nuclear

DNA evolution (Vawter and Brown 1986). the estimate of absolute age of pseudogenes by

their overall sequrnce similnrity to mtDNA sequences is not accurate.

Figure 12. Model of the possible evolutionary origins of mtDNA-like sequences in nuclear

genomes of goose species covered in this study. Phy logenetic relatedness is based

on RFLP analysis of mtDNA (modified after Shields and Wilson 1987, and Quinn

et al. 199 1 ). The phylogenetic position of the Emperor Goose (Anser canagica) is

ambiguous. Letters indicate possible time of transfer and integration of mtDNA

sequences dong the evolutionary pathway of goose lineages (see Discussion).

The species are Ross' Goose (A. rossii). Lesser and Greater Snow Goose (A.

caemlescens caerulescens and A. c. atlantica), White-fronted Goose (A. albifrons

frontalis). Canada Goose (Branta canadensis), and Brant Goose (B. bemicla).

ROSS' Goose

LesserIGreater Snow

Emperor Goose White-fronted Goose

Canada Goose

Brant Goose

Mitochondrial DNA fragments transferred before the divergence of two species

will usually be present in nuclear pnomes of both species. With an independent species

phylogeny based on morphological. molecular and fossil data. the approximate date of the

transfer event can be inferred frorn the phylogenetic distribution of mtDNA inserts. The

divergence time of Lesser Snow and Ross' Goose is estimated to be approxirnately 0.5 x

10"ears ago. and between these two taxa and White-fronted Goose approximately 1 -3 x

106 years ago (Shields and Wilson 1987: Avise et al. 19%). This is the time window for

possible integration of mtDNA sequences that show sequence similarity with pmLSG003.

The hypothesis of very recent speciation of the two taxa, with retention of the original

polymorphism in mtDNA and allozyme profiles. is supported by results presented here.

An alternative explanation is that occasionaI hybridization among Lesser / Greater Snow

and Ross' Geese. cross-taxon tnnsfer. and subsequent homogenization of the array

accounts for the lack of substantial differences in sequence variation arnong the rntDNA

inserts. From the results presented in Chapter 4, as weli as from results of allozyme

(Cooke et al. 1988) and mtDNA data analysis (Avise et al. 1992, Quinn 1992). i t can be

concluded that there is a low level of genetic differentiation and high level of p n e flow

between the "Blue" and "Snow" Lesser Snow Geese. two phenotypic forms that were as

late as the 1960s considered as separate species (Cooke and Cooch 1968). Two subspecies

of Snow Geese: the Lesser Snow and Greater Snow, are mainly differentiated by size.

with a considerable overlap in size measuremenn so the subspecific division has little utility

(Cooke et ai. 1995). Ross' Geese occasionaily form mixed pairs with Snow Geese. and

their offspring produce fertile offspring so these taxa are close genetically (Cooke et al.

1995). Nevertheless. genetic exchange ixtween the "taxa" has to be extensive in order to

overturn the effect of conçened svolution of repetitive arrays. where one would expect to

see higher homogeneity within the species than between arrays taken from different

species. This was not obvious from data presented in this study.

The Emperor Goose is a monomorphic species. occuning oniy in a dark (gray)

colour phase. Breeding and wintering grounds of this species. along the western toast of

Alaska. do not overlap with the corresponding areas of Ross' and Snow Geese. The

phylogenetic relatedness of the Emperor Goose to the Lesser / Greater Snow. Ross'

Goose, and White-fronted Goose is controversial. W ith the data presented here. 1 suggest

that the Emperor Goose is not as closely related to the Ross' and Snow Geese as

considered by some authors. Taxonomists disagree whether the Anser group of Arctic

nesting geese should be split into two genera. Anser and Chen. Lesser / Greater Snow.

Ross', and Emperor Geese are considered by some authors to be closely related and to

belong to a separate genus Chen (The Amencan Omithologists' Union 1983. and

Supplement of July 1985). Hybrids amon$ the Chen group of species as well as between

Chen species and White-fronted Goose have been reponed in nature. with many crosses - producing fertile offspring (Gray 1958). Based on the currently accepted phylogeny of the

Anser / Chen species. 1 suggest that the ancestor of Emperor Goose had separated from the

Snow and Ross' Goose lineage relatively early in the evoiution of Arctic geese. before the

incorporation of mtDNA sequences found in these two taxa (Figure 12). Insertions of a

larger segment of the mtDNA molecule (that hybridized to prnLSGOO 1. pmLSGOO2. and

pmLSG003) must have occurred subsequent to the sepantion of the White-fronted Goose

lineage (event "B" in Figure 12). Events "C" and "D' in Figure 12 mark proposed separare

insertions of a smaller portion of mtDNA molecule (detectable with pmLSGOO 1 and

prnLSGûû3) in the Brant and Canada Goose lineages.

5.3 Content of transposed rntDNA sequences

Mitochondrial srquences in nuclear genornes of higher orpnisms appear to

contain both ribosomal RNA genes. at least eight of thineen protein-coding genes. and the

control region (Table Il . Rrpetitive ürriiys of trinsposcd mtDNA srquences in Laser

Snow. Greater Snow and Ross' Goose are derived from a 3.6 kb monomer, which

hybridized to the mtDNA probe pmLSG003. This probe contains genes for cytochrome b.

NADH subunit 6. the D - loop region. and portions of 16s rRNA and NADH 5 genes

(Quinn 1993: Quinn and Wilson 1993). 1 detected the presence of sequences homologous

to al1 three mtDNA probes in the nuclear genome of White-fronted Goose. Through partial

sequencing of prnLSGûû 1 1 detemined that this probe contains sequences with high

similarity to the chicken ATPase subunit 6 (nucleotide position 9339 to 9423 in Desjardins

and Morais 1990), as well as to the sarne gene in the duck mitochondrial genome

(nucleotide position 1204 to 1282 in Ramirez et al. 1993). The mtDNA probe prnLSGûû7

contains sequences corresponding to the chicken cytochrome oxidase subunit 1 (nucleotide

position 6988 to 7090 in Desjardins and Morais 1990). It seems that there were several

transposition events involving a larger portion of the rntDNA molecule. with insertions

taking place in dispersed reg ions of the White-fronted Goose nuclear genome.

Many studies report that transposed mtDNA sequences are surrounded by

repetitive elernents (Gellissen et al. 1983: Jacobs et al. 1983; Tsuzuki et al. 1983: Fukuda

et al. 1985: Zullo et al. 199 l ) , or even insertion of K D ~ 1 repetitive elements into the

mtDNA-originating sequences (Nomiyama et al. 1984; Wakasugi et ai. 1985).

Mitochondrial DNA-iike sequences in Lesser & Greater Snow Goose. Ross' Goose and

White-fronted Goose could be associated with repetitive elements, which may have

facilitated the amplification (and dispersal) of mtDNA homologs seen in these three species.

Brant and Canada Goose nuclear genornes contain sequences which hybridized to

oments on rntDNA probes pmLSGûûI and pmLSGûû3. I was not able to detect any fra,

Southern blots thrit would indicrtte the presence of tandem arrays containing rntDNA

insens. Ail of the mtDNA-likr sequences are present as a singlecopy or low-copy-number

Oments. f rd,

5.4 Repeated character of the 3.6 kb nucDNA fragment and its presence in

natural populations

Results of this study suggest that the mtDNA sequences of Lesser Snow Goose

were inserted within the nuclear genome of this species and went through periodic tandem

amplification. 1 have shown that the insened mitochondrial sequences are arnngd in

tandem arrays containing 3000 - 5000 copies. and represent at least 0.5% of the Lesser

Snow Goose nuclex genome. 1 was not able to determine whether there were multiple

transpositions of mtDNA sequences in Lesser Snow Goose with subsequent amplification

of one of the inserts. or whether the ancient insertion went through sevenl sets of

rearrangements and duplications. On the basis of DNA sequence data Quinn ( 1992)

suggested that there could be one or more nuclear homologs in the Lesser Snow Goose that

were very recently derived from the mitochondrion. Nevertheless. the majority of these

sequences appear to be organized in a tandem array in one location in the genome. with little

sequence polymorphism among the elements of the array. Secondary duplication after the

integration hzis been suggested for rntDNA homologs in rats. primates and cats (Zullo et al.

199 1; Collura and Stewart 1995: Lopez et al. 1994. 1997). Lopez and coworkers ( 1994)

proposed a mode! of mtDNA transposition and sequence amplitlcation based on analogous

recornbination and extrachromosomal amplification of petite mutations in yeast. In shon.

the model proposes deletion and circularization of a segment of mitochondrial genome

(possibly mediated by in trachromosomal recom bination). which becomes amplified

extrachromosomally and persist ris an episome. Subsequent chromosomal integrrition

positions the amplified rntDNA kgment within the nucleus. This model is highly

speculative and it is not clertr whether the ciit mtDNA inserts were amplified

extrzichromosomally or after the chromosornril integration took effect.

A small portion of the Lesscr Snow Goose population ( 1.28) is homozyaously

deficient for the array of 3.6 kb elements that rire charxtcrized by the presence of I&d III.

Ava 1 and BamH 1 recognition sites. With techniques used in this study (Southem blottin: - and hybridizations) it was not possible to determine whether individuals are heterozygous

or homozygous for the insertion and thus determine the exact number of chromosomes

containing mtDNA sequences. It is possible to speculate that a portion of the Lesser Snow

Goose population is comprised of individuals in which the inserted mtDNA sequences did

not undergo secondary amplification of the 3.6 kb nucDNA fragment. However. due to a

slow rate of genomic turnover mechanisms relative to the rate of chromosome assortment in

a sexually reproducing population. one would expect that most individuals in a given

generation have a sirni lar copy nurnber of the new mutation (Dover 1982). 1 suspect the

nuclear copies that hybridized to prnLSGo3 are not widely dispersed throughout the

chromosomes of Lesser Snow Goose and. considering the relative1 y smal l copy-number of

elements. potentially prone to elimination. Thus, there could be an interaction between

non-phenotypic and phsnotypic selection (in terms of Sapienza and Doolitle 198 1 )

determining prevalence of repeti tive sequences containing mtDNA homologs in this

species. DNA samples from three individuals that do not possess the homologs were from

juvenile birds collected at the breeding colony near Churchill. Manitoba. From bird-

banding records, we know that those birds were not from the sarne nest, and there is a Iow

probability of them king close relatives. In individuals from the breeding colony near

Churchill. Manitoba, the only sample where we collected blood or tissue from juvenile and

adult birds. it appears that the proportion of juvenile birds without the 3.6 kb nucDNA

element (3 out of 25) is greater than the proportion of adult birds without the element (O /

12) (Fisher exact test. P = 0.W8. d.f. = 1 ). Is it possible that deletion of the array caused

disruption of grnes activaied in the later stage of goslin_e developrnent. producing difference

in fitness for those individulils*? Wu and coworkers ( 1989) deterrnined fitness differences

in Drosophila üssociatsd with the deletion of a satellite DNA or functional regions close to

the satellite nmy. Similar studies in Seese would require detection of phenot ypic characters

that cm trace individuals with deletion mutations. At the moment we are not aware of such

characters.

As mentioned in Chapter 1. the presence of rnitochondrial sequences in the nuclear

environment could prove to be an obstacle when mitochondrial DNA is used for population

biology studies (Smith et al. 1992: Quinn 1992: Collun et al. 1996: and references in

Zhang and Hewitt 1996). particularly when PCR amplification is used to obtain sufficient

quantities of DNA for analysis. However. inserted rntDNA fragments in nuclear genomes

of closely related species provide an opponunity to use such sequences as an outgroup in

phylogenetic analyses. In an elegant example. Zischler et al. ( 1995) used a nuclear copy of

the human D-toop region to resolve a controversy surrounding the origin of modem

humans. Previous to their study. the chimpanzee D-loop has been used as an outgroup

sequence despite the fact that the chimpanzee sequence is too divergent to make a good

outgroup for human D-loop phylogeny. By using the nuclear copy of D-loop transferred

after the divergence of the hurnan and chimpanzee lineages. Zischler and coworkers were

able to root the human phylogenetic tree with higher confidence. Multiple insens of

mtDNA-like sequences in nuclear genomes of geese may provide the most appropriate

outgroup sequences in phylogenetic studies of this group of species.

Due to a reduced rate of sequence evolution in nuclear pseudogenes. the ancestral

state of mtDNA genes can be inferred. Nuclear copies of mtDNA sequences are often

referred to as "nuclear fossils" (Zischler et al. 1995: Perna and Kocher 1996). Multiple

copies of nuclear insens that have arisen separately during differenr stages of genome

evolution (Hu and Thilly 1994) could potentially fom a nearly continuous historical record

of the mtDNA sequence evolution. In this study. 1 address the possibility for existence of a

similür '-evolutionary trail" of insrned mtDNA fragments (see ülso Quinn 1997. for D-loop

integrations in Lesser Snow Goose). Funher sequence analysis of mtDNA-like sequences

in Lesser / Greüter Snow. Ross' and White-fronted Geese could reveül whether multiple.

independent intrgration events took place. Nevertheless. one hüs to be üware that due to

the uni parental inheritance of mtDNA and rapid lineage soning. cenain mitochondrial

lineages could become extinct after the nuclear integntions. Consequently. mtDNA

sequences and corresponding nuclear copies observed today rnay not always share a direct

comrnon ancestor.

SUMMARY AND CONCLUSIONS

This study raises several possibilities for funher research into the coevolution of

mitochondrial and nuclear genomes. with the emphasis on mechanisms of transposition.

integration and gene duplication. Transposed mtDNA sequences in geese provide an

opponunity to study direcrly paralogous (intracellular) duplication events and relative rates

of sequence evolution in mtDNA and its corresponding nuclear pseudogenes. prirnarily

due to abundant transpositions present in several closely related species. Such studies have

recently gained a lot of attention (Arctander 1995; Perna and Kocher 1996: Sorenson and

Fleischer 1996; Lopez et al. 1997; Kidd and Friesen 1998: Zischler et al. 1998). This

study also provides data on the frequency and extent of rntDNA insertions among different

avian taxa. which has direct implication for the use of nuclear hornologs as outgroup

sequences in population and phylogenetic studies (Quinn 1992: Collura and Stewart 1995:

Sorenson and Fleischer 1996). Due to a slower rate of nucleotide substitution and reduced

sequence length heterogeneity. nuclear copies of mitochondrial DNA have the advantage in

detemining the ancestral mitochondrial States more accurately (Lopez et al. 1997: Zischler

et al. 1998). The mostcommon ancestral sequence deducted frorn nuclear insens present

in different species will be more reliable than an ancestral sequence denved from fast

evolving mtDNA. This will allow for tracing the history of sequence evolution in fast

evolving mtDNA sequences over long evolutionary tirnescales (Pema and Kocher 1996).

Results presented in Chapter 4. as well as the population analysis of allozyme

(Cooke et al. 1988). nuclear RFLP (Quinn 1988). and mtDNA markers (Quinn 1992:

Avise et al. 1992). reveal a lack of extensive heterogeneity in genetic structure of mid-

continent Lesser Snow Goose populations. If the two coiour phases indeed rvolved in

allopatry. they did not develop substantial between-population genetic differrnces.

Altematively. low spatial hrterogctneity in modem-day Lesser Snow Goose populations

could be a consequence of postgliicial range tluctuations. repopulations. and extensive gene

flow between the breeding groups. Lack of barriers to gene flow in regard to

rnorphological characters and "species" recognition mechanisms may support the

hypothesis of a rapid inter-species transfer of novel characters. which would explain the

presence of apparently similar mtDNA-like sequences in Lesser / Greater. and Ross' Goose

nuclear genomes.

Future research of mtDNA-like sequence integrations in nuclear genomes of geese

rnay concentrate on isolation of the site of integration from a chromosome lacking the insert

in Lesser Snow Goose. This would involve sequencing of regions fianking the inserts.

particularly to compare individuals with and without the rntDNA inserts. The results of

such experiments would allow identification of integration target sites and sequence

changes during the integration events. Primers flanking the point of insertion could be

designed. facilitating PCR-based screening for the presence of nuclear inserts with

homology to mtDNA probe prnLSGûû3 in related taxa. This would serve to confirm the

existence of nuclear inserts in Greater Snow, Ross' and White-fronted Geese. and

potentially Brant and Canada Goose. With in situ hybridizations of mtDNA probes to

cytological preparations of Lesser Snow Goose, and possibly White-fronted Goose

chromosomes (Gall and Pardue 1969) the chromosomal locations of integrated mtDNA

fragments could be determined. Results of in situ hybridization experiments would be

particularly important in detecting the distribution of homologs in White-fronted Goose.

where multiple copies of mtDNA-like fragments appear to be present in the nuclear

genome.

In rems of potential for future research. of particular interest are the mechanisms

of concerted evolution in the 3.6 kb inserted element found in the nuclear genornes of

Lesser and Greater Snow. and Ross' Goose, The observations presented in this study may

suppon the idea that some mutations in repeated pseudogene fimilies spread rapidly within

populritions through molecular mechanisms of turnover within the genome ("molecular

drive". Dover 1982). in addition to the forces of natural selection and genetic drift.

REFERENCES

Akashi. K.. Hirayama. J.. Takenaka. M.. Yarnaoka. S.. Suyama, Y.. Fukuzawa. H., and Ohyama. K. 1997. Accumulation of nuclear-encoded ~ R N A T ~ ~ ~ ~ ~ in mitochondria of the l ivewon Marchantia polvmomha. Biochim Biophys. Acta 1350: 262-266.

Altschul. S.F.. Madden. T.L.. Schiiffer, A.A.. Zhang. J.. Zhang. 2.. Miller. W.. and Lipman. D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3407.

American Ornithologists* Union. 1983. Checklist of North American birds. 6th ed. American Omithologists' Union. Washington. DC

Anderson. S.. Bankier. A.T.. Barrell. B.G.. deBruijn, M.H.L.. Coulson. A.R.. Drouin. J.. Eperon. I.C.. Nierlich. D.P., Roe. B.A., Sanger. F., Schreier. P.H.. Smith. A.J.H.. Staden. R.. and Young, I.G. 198 1. Sequence and organization of the human mitochondrial genome. Nature 290: 457-465.

Anderson. S.. deBruin. M.H.L., Coulson. A.R.. Eperon. I.C.. Sanger. F.. and Young. I.G. 1982. Complete sequence of bovine mitochondrial DNA: conserved features of the mammalian mitochondrial genome. J. Mol. Biol. 156: 683-7 1 7.

Arctander. P. 1995. Cornparison of a mitochondrial gene and a corresponding nuclear pseudogene. Proc. R. Soc. Lond. B 262: 1 3- 19.

Arnheim. N.. White. T.. and Rainey. W.E. 1990. Application of PCR: or_panismül and population biology. BioScience 40: 174- 182.

Attardi. G. 1985. Animal mitochondrial DNA: an extreme example of genetic economy. Int. Rev. Cytol. 93: 93-145.

Avise. J.C., Alisauskas, R.T.. Nelson, W.S.. and Ankney, C.D. 1992. Matriarcha] population genetic structure in an avian species with fernale natal philopatry. Evolution 46: 1084- 1096.

Avise. J-C.. and Zink. R.M. 1988. Molecular genetic divergence between avian sibling species: King and Clapper rails. Long-billed and Short-bilied dowitchers. Boat- tailed and Great-tailed grackles. and Tufted and Black-crested ti tmice. Auk 105: 5 16-528.

Bargiello. T.A.. Grossfield. J.. Steele. R.W.. and Cooke. F. 1977. Isoenzyme status and genetic variability of semm esterases in the Lesser Snow Goose. Amer caerulescens caerulescens. Biochem. Genet. 15: 74 1-763.

Barton. N.H. 1983. Multilocus clines. Evolution 37: 454-47 1 .

Barton. N.H.. and Slatkin. M. 1986. A quasi-equilibnum theory of the distribution of rare alleles in a subdivided population. Heredity 56: 409-4 1 5 .

Bibb. M.J.. van Etten. R.A.. Wright. C.T.. Walberj. M.W.. and Clayton. D.A. 1981. Sequence and gene organization of mousr mitochondrial DNA. Crll 26: 167- 180.

Blanchard, J.L.. and Schmidt G.W. 1995. Pervasive migration of organellar DNA to the nucleus in plants. J. Mol. Evol. 41: 397-406.

Blanchard. I.L.. and Schmidt, G.W. 1996. Mitochondrial DNA migration events in yeast and humans: integntion by a common end-joining mechanism and alternative perspecives on nucleotide substitution patterns. Mol. Biol. Evol. 13: 5 37-548.

Boore. J.L.. and Brown. W.M. 1994. Mitochondrial genomes and the phylogeny of mollusks. Nautilus lOS(Supp1. 2): 6 1-78.

Boore. J.L.. and Brown, W.M. 1995. Complete sequence of the mitochondrial DNA of the annelid worm Lumbricus terrestris. Genetics 141: 305-3 19.

Boore. J.L., Collins, T.M., Stanton, D., Daehler, L.L., and Brown, W.M. 1995. Deducting the pattern of arthropod phylogen y from mitochondrial DNA rearrangements. Nature 376: 163- 1 65.

Borst. P., and Grivell. L. 1978. The rnitochondrïal genome of yeast. Cell 15: 705-733.

Boursot. P.. Yonekawa H., and Bonhomme, F. 1987. Heteroplasmy in mice with deletion of a large coding region of mitochondrial DNA. Mol. Biol. Evol. 4: 46- 55.

Bridge, D.. Cunningham, C.W., Schierwater, B., Desalle. R.. and Buss, L.W. 1992. Class-level relationships in the phylum Cnidaria: evidence from rnitochondrial oenome structure. Proc. Natl. Acad. Sci. USA 89: 8750-8753. tz

Brown. W.M.. George Jr.. M.. and Wilson. A.C. 1979. Rapid evolution of animal mitochondrial DNA. Proc. Natl. Acad. Sci. USA 76: 1967- 197 1.

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

Brown. W.M. 1985. The mitochondnal genome of animals. Pp. 95- 130. b: MacIntyre. R.J. (ed.), Molecular cvolutionary genetics, Plenum, New York.

Burke, T. 1989. DNA fingerprinting and other rnethods for the study of mating success. Trends Ecol. Evol. 4: 139- 144.

Cantritore. P.. Gadaleta. M.N.. Roberti, M., Saccone, C., and Wilson. A.C. 1987. Duplication and rernoulding of tRNA genes during the evolutinary rearrangemenr of mitochondrial genomes. Nature 329: 853-855.

Cantritore. P., Roberti, M., Rainaldi, G., Saccone, C.. and Gadaleta, M.N. 1988. Clustering of tRNA genes in Paracentrotus lividus mitochondrial DNA. Curr. Genet. 13: 91-96.

Cantritore, P., Roberti. M., Rainaldi, G., Gadaleta. M.N., and Saccone. C. 1989. The complete nucleotide sequence. gene organization, and genetic code of the mitochondrial genorne of Paracentrotus lividus. J. Biol. Chem. 264: 10965- 10975.

Cheung. W.Y.. and Scott. N.S. 1989. A contiguous sequence in spinach nuclear DNA is homologous to 3 separated sequences in chloroplast DNA. Theor. Appl. Genet. 77: 625-633.

Clary. D.O.. and Wolstenholme. D.R. 1985. The mitochondrial molecule of Dros~hilri yakuba: nucleotide sequence. genome organization. and genetic code. J. Mol. Evol. 22: 3 2 - 3 7 1 .

Clayton. D.A. 1992. Transcription and replication of animal rnitochondrial DNA. Int. Rev. Cytol. 131: 217-232.

Collura. R-V.. and Stewan C.-B. 1995. Insertions and duplication of rntDNA in the nuclear genornes of Old World monkeys and hominoids. Nature 378: 448489.

Collura. R-V.. Auerbach M.R.. and Stewan C.-B. 1996. A quick. rapid method that can differentiate expressed mitochondrial genes from their nuclear pseudogenes. Curr. Biol. 6: 1337- 1339.

Cooke. F. 1987. Lesser Snow Goose: a long t e m population study. Pp. 107-432. b: Cooke. F.. and Buckley. P.A. (eds.), Avian genetics. Academic Press. London.

Cooke. F.. and Cooch. F.G. 1968. The genetics of the polymorphism in the goose Anser caerulescens. Evolution 22: 289-300.

Cooke. F., and Mirsky. P.J. 1972. A genetic analysis of Lesser Snow Goose families. Auk 89: 863-87 I .

Cooke. F.. Finney, G.H.. and Rockwell. R.F. 1976. Assonative rnating in Lesser Snow Geese. Behav. Genet. 6: 137-140.

Cooke. F.. MacInnes. CD.. and Prevet, 1.P. 1975. Gene flow between breeding populations of Lesser Snow Geese. Auk 92: 493-510.

Cooke. F., Parkin. D.T.. and Rockwell. R.F. 1988. Evidence of former allopatry of the two color phases of Lesser Snow Geese (Chen caerulsecens caemlsecens). Auk 105: 367-479.

Cook. F.. Rockwell. R.F.. and Lank. D.B. 1995. The Snow Goose of Laperouse Bay: natural selection in the wild. Oxford Univ. Press.. New York.

Covello. P.S.. and Gray. M.W. 19%. Silent mitochondrial and active nuclear genes for subunit 2 of cytochrome c oxidase (u II) in soybean: Evidence for RNA- mediated gene transfer. EMBO J . 11: 38 15-3820.

Crozier. R.H.. and Crozier. Y.C. 1993. The mitochondrial genome of the hrneybee Apis mellifera: cornplste sequencr and genome organization. Genetics 133: 97- 1 17.

Desjardins. P.. and Morais. R. 1990. Sequence and gene organization of the chicken mitochondrial genome: a novel gene order in higher vertebrates. J. Mol. Biol. 212: 599-634.

Desjardins. P.. and Morais. R. 199 1 . Nucleotide sequence and evolution of coding and noncoding regions of a quail mitochondrial genome. J . Mol. Evol. 32: 153- 16 1 .

Desjardins. P.. Ramirez. V.. and Morais, R. 1990. Genr organization of the Pecking duck mitochondrial genome. Curr. Genet. 17: 5 15-5 18.

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

Dzubin. A. 1974. Snow and blue goose distribution in the Mississippi and Central Flyways: compendium of results of bird recovery analyses. Memo Canadian Wildlife Service. prepared for Central and Mississippi Flyway Councils. June 1974. Univ. of Saskatchewan. Saskatoon.

Edwards. SV.. and Wilson, A.C. 1990. Phylogenetically informative length pol y morphism and seqence varïability in mitochondrial DNA of Australian songbirds (Pomatostomus). Genetics 126: 695-7 1 1.

Farrelly. F.. and Butow, R. 1983. Rearranged mitochondrial genes in the yeast nuclear genome. Nature 301: 296-30 1.

Feinberg, A.P.. and Vogelstein. B. 1983. A technique for radiolabelling DNA restriction fragments to high specific activity. Anal. Biochem. 136: 6- 13.

Fejes. E.. Masters. %.S., McCarty. D.M.. and Hauswinh. W.W. 1988. Sequence and transcriptional andysis of a chloroplast insen in the mitochondrial jeorne of & mays. Curr. Genet. 13: 509-515.

Findlay. C.S., Rockwell. R.F.. Smith. J.A.. and Cooke. F. 1985. Life history studies of the Lesser Snow Goose (Anser caerulescens caerulescens). VI. Plumage polymorphism. assortative mating and fitness. Evolution 39: 904-9 14.

Francis. C.M.. and Cooke, F. 1992. Migration routes and recovery rates of lesser snow geese from southwestern Hudson Bay. J. Wildl. Manage. 56: 279-286.

Fukuda M.. Wakasugi S.. Tsuzuki T.. Nomiyama K.. and Shimada K. 1985. Mitochondnal DNA-like sequences in the human nuclear genome: characterization and implications in the evolution of mitochondrial DNA. J. Mol. Biol. 186: 257- 266.

Fuller. KM.. and Zouros. E. 1993. Dispersed discrete length polymorphism of mitochondnal DNA in the scallop Placomcten mage1 lanicus (Grnelin). Curr. Genet. 23: 365-369.

Gall. J.G.. and M.L. Pardue. 1969. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. USA 63: 378-383.

Garesse. R. 1988. Droso~hiia melanognster mitochondrial DNA: gene orgünization and cvolutionary considerations. Genetics 118: 649-663.

Garey. 1.R.. and Wolstenholme. D.R. 1989. Platyhelrninth rnitochondrial DNA: evidence for early evolutonary origin of the tRNAscrA~~ that contains a dihydrouridine a m replacement loop. and of serine-specifying AGA and AGG codons. J. Mol. Evol. 28: 374-387.

Gellissen, G.. Bradfield J.Y.. White B.N., and Wyatt G.R. 1983. Mitochondrial DNA sequences in the nuclear genorne of a locust. Nature 301: 63 1-634.

Gellissen. G.. and Michaelis. G. 1987. Gene transfer: mitochondria to nucleus. Ann. NY Acad. Sci. 503: 39 1-401.

Geramita J.M.. and Cooke. F- 1982. Evidence that fidelity to natal breeding colony is not absolute in female snow geese. C m J. Zool. 60: 205 1-2056.

Geramita. LM.. Cooke. F.. and Rockwell. R.F. 1982. Assortative mating and gene flow in the Lesser Snow Goose: a modelling approach. Theor. Popul. Biol. 22: 177- 203.

Gillham. N.W. 1994. Organelle genes and genomes. Oxford Univ. Press.. New York.

Gjetvaj. B., Cook, D.L. and Zouros, E. 1992. Repeated sequences and large scale size variation of mitochondrial DNA: a common feature among scailops (Bivalvia: Pectinidae). Mol. Biol. Evol. 9: 106- 134.

Goudet, J. , Raymond. M.. de Meeüs. T.. and Rousset. F. 1996. Testing differentiation in diploid populations. Genetics 144: 1933- 1940.

Gray. A.P. 19%. Bird hybrids. A checklist with bibliography. Technical Comm. No. 1 3. Commonwealth Agricult. Bureaux. Bucks, Great Britriin.

Gray. M.W. 1992. The endosymbiont hypothesis revised. Int. Rev. Cytol. 141: 233- 357.

Gray. M.W.. and Boer, P.H. 1988. Organization and expression of algal (Chlarnvdomonas reinhrrrdtii) mitochondrial DNA. Phil. Trans. R. Soc. Lond. B 319: 135- 147.

Grohmann. L.. Brennicke. A.. and Schuster. W. 1992. The mitochondnl gene encoding ribosomal protein S 12 has k e n translocated to the nuclear genome in Oenothera. Nucl. Acids Res. 20: 564 1-5646.

Guo. S.W.. and Thompson. E.A. 1992. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics 48: 36 1-372.

Hadler. H.I.. Dirnitrijevic. B.. and Mahalingam. R. 1983. Mitochondrial DNA and nuciear DNA frorn normal rat liver have a common sequence. Proc. Natl. Acad. Sci. USA 80: 649545499.

Hancon. M.R.. and Folkerts. O.F. 1992. Structure and function of the highcr plant mitochondrial genome. Int. Rev. Cytol. 141: 129- 172.

Hani. D.L.. and Clark. A.G. 1989. Principles of population generics. 2nd sd. Sinauer Assoc.. Sunderland. MA.

Hartl. F.-U.. and Neupert. W. I W O . Protein soninp to mitochondria: evolutionary conservations of folding and ÿssernbly. Science 247: 930-938.

Hatzoglou, E., Rodakis. G.C.. and Lecanidou, R. 1995. Cornplete sequence and gene organization of the rnitochondrial genome of the land snail Albinaria coerulea. Genetics 140: 1353- 1366.

Himeno. H.. Masaki. H.. Kawai. T.. Ohta. T.. Kurnagai. 1.. Miura. K.-(.. and Watanabc. K. 1987. Unusual genetic codes and novel gene structure for tRNAserA~y in starfish mitochondrial DNA. Gene 56: 2 19-230.

Hoeh. W.R.. Stewart. D.T.. Sutherland, B.W., and Zouros, E. 1995. Cytochrome g Oxidase sequence cornparisons suggests an unusually high rate of mitochondrial DNA evolution in Mvtilus (Mollusca: Bivalvia). Mol. BioI. Evol, 12: 31 8-42 1.

Hoffmann, R.J., Boore, J.L.. and Brown, W.M. 1992. A novel mitochondrial genome organization for the blue mussel. Mvtilus edul is. Genetics 131: 397-4 1 2.

Hu, G., and Thilly, W.G. 1994. Evolutionary trail of the mitochondrial jenome as based on human 16s rDNA pseudogenes. Gene 147: 197-204.

Hu. G.. and Thilly, W. G. 1995. Multi-copy nuclear pseudogenes of mitochondrial DNA reveal recent acute genetic changes in the hurnan genome. Curr. Genet. 28: 4 10- 4 14.

Hyrnan. B.C.. and Beck-Azevedo, I.L. 1 996. Similar evolutionary patterning among repeated and single copy nematode mitochondrial genes. Mol. Biol. Evol. 13: 12 1 - 232.

Hyman. B.C., Beck, J.L.. and Weiss, K.C. 1988. Sequence amplification and gene remangement in parasitic nematode mitochondrial DNA. Genetics 120: 707-7 12.

Jacobs, H.T., Posakony, LW.. Grula. J.W., Roberts, J.W., Xin, LH. . Britten. R.J.. and Davidson. E.H. 1983. Mitochondrial DNA sequences in the nuclear genome of Strongvlocentrotus mupuratus. J. Mol. Biol. 165: 609-632.

Jacobs, H.T.. and Grimes. B. 1986. Complete nucleotide sequence of the nuclear pseudogenes for cytochrome oxidase subunit 1 and the large rnitochondrial ribosomal RNA in the sea urchin Strono_ylocentrotus our6uratus. J. Mol. Biol. 187: 509-527.

Jacobs. H.T.. Elliott, D.J.. Math. V.B.. and Farquharson, A. 1988a. Nuclotide sequence and gene organization of sea urchin mitochondrial DNA. J. Mol. Biol. 202: 185- 2 17.

Jacobs. H.T., Balfe. P.. Cohen. B.L.. Farquharson. A.. and Comito, L. l988b. Ph1 yogenetic implications of genome remangement and sequrnce evolution in echinoderm mitochondrial DNA. Pp. 12 1 - 137. : Paul. C.R.C.. and Smith. A.B. (eds.), Echinoderm phylogeny and evolutionary biology. Clarendon Press. Oxford.

Janke. A.. Xu, X.. and Arn~son. U. 1997. The complete mitochondrial genome of the wallaroo (Macropus robustus) and the phylogenetic relationship among Monotremata, Marsupialia. and Eutheria. Proc. Natl. Acad. Sci. 94: 1 276- 1 28 t .

Jeffreys. A.1.. Neumann. R.. and Wilson. V. 1990. Repeat unit sequence variation in minisatellites: a novel source of DNA polyrnorphism for studying variation and mutation by single molecule analysis. Cell 60: 473485.

Kato. S.. Anderson. R.A.. and Camenni-Otero. R.D. 1986. Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L ce11 senorne. Mol. Cell. Biol. 6: 1787-1795.

Kamimura. N.. Ishii. S.. Liandong. M.. and Shay. J.W. 1989. Three separate mitochondrial DNA sequences are contiguous in human gznomic DNA. I. Mol. Biol. 210: 703-707.

Kemble. R.J.. Mans. R.J.. Gabay-Laughnan. S.. and Laughnan. J.R. 1983. Sequences homologous to episomal mitochondrial DNAs in the maize nuclear pnorne. Nature 304: 744-747.

Kidd. M.G.. and Friesen. V.L. 1998. Sequence variation in the Guillemot (Alcidae: Cepphus) mitochondrial DNA region and its nuclear homolog. Mol. Biol. Evol. 15: 61 -70.

Kocher. T.D.. Thomas. W.K.. Meyer. A.. Edwards. S.V.. Paabo. S.. Villablanca. F.X.. and Wilson. A C . 1989. Dynarnics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86: 6 196-6200.

Kristensen, T.. and Prydz. H. 1986. The presence of intact rnitochondrial DNA in HeLa ce11 nuclei. Nucleic Acids Res. 14: 2597-2609.

Kurnar. S.. Tamura. K.. and Nei. M. 1993. MEGA: molecular evolutionary genetics analysis. v. 1 .O 1. Penn. Stace Univ.. University Park. PA.

Kumazava, Y.. and Nishida. M. 1995. Variations in mitochondrial tRNA gene organization of reptiIes as phylogenetic markers. Mol. Biol. Evol. 12: 759-772.

Kumazawa. Y.. Ota. H., Nishida. M., and Ozawa. T. 1996. Gene rearrangements in snake rnitochondrial genornes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster. Mol. Biol. Evol. 13: 1 242- 1254.

Lang. B.F.. Burger. G.. O'Keily. C.J.. Cedergren. R.. Golding. C.B.. Lemieux, C.. Sankoff. D.. Tunnel. M.. and Gray. M.W. 1997. An ancestral mitochondrial DNA resembling a eubacterial genorne in miniature. Nature 387: 493-497.

La Roche. I.M.. Snyder. M.. Cook. D.I.. and Zouros. E. 1990. Molecular charlurterization of a repeat element causing large-scale size variation in the mitochondrial DNA of the sea scallop Placopecten maoellanicus. Mol. B iol. Evol. 7: 35-64.

Lee. W-J.. and Kocher. T.D. 1995. Complete xquence of a seü lamprey (Petromvzon marinus) mitochondrial genorne: eiirly est;iblishment of the vertebnte genome organization. Genetics 139: 873-887.

Lewin. R. 1983. Promiscuous DNA leaps al1 barriers. Science 219: 478479.

Lonergan, KM., Gray. M. W. 1 994. The ri bosomal RNA gene region in Acanthamoeba castellanii mitochondrial DNA. A case study of evolutionary transfer of introns between mitochondria and plastids? J. Mol. Biol. 239: 476499.

Lonsdale. D.M., Hodge. T.P.. Howe, Cl, and Stem, D.B. 1983. Maize mitochondrial DNA contains a sequence homologous to the ribulose- 1 .5-bisphosphate carboxylase large subuni t gene of chloroplast DNA. Cell34: 1 OO7- 10 14.

Lopez, J.V., Yuhki N.. Masuda R., Modi W.. and O'Brien S.J. 1994. Numt, a recent transfer and tandem amplification of mitochondrial DNA to the nuclear genome of the domestic cat. J. Mol. Evol. 39: 174- 190.

Lopez, J.V.. Cevario, S., and O'Brien, S.J. 1996. Complete nucleotide sequences of the dornestic cat (Felis catus) mitochondrial pnome and a transposed mtDNA tandem repeat (Numt) in the nuclear genome. Genomics 33: 229-246.

Lopez. J.V., Culver. M.. Stephens. J-C.. Johnson, W.E.. and O'Brien. S.J. 1997. Rates of nuclear and cytoplasmic mitochondrial DNA sequence divergence in mamrnals. Mol. Biol. Evol. 14: 277-286.

Macey, J.R., Larson, A.. Ananjeva. N.B.. Fang. Z.. and Papenfuss, T.J. 1997. TWO novel gene orders and the role of Iight-strand replication in remangement of the vertebrate mitochondrial genome. Mol. Biol. Evol. 13: 9 1 - 1 04.

Maniatis. T.. Fritsch. E.F.. and Sambrook. 1. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. New York.

Margulis, L. 1970. Origin of eukaryotic cells. Yale Univ. Press.. New Haven. Conn.

McCracken, A., Uhlenbusch, 1.. and Geilissen, G. 1987. Structure of the cloned Locusta migratoria rnitochondrial genome: restriction mapping and sequences of its ND-1 (URF-1) gene. Curr. Genet. 11: 625-630.

McKnight, M.L.. and Shaffer, H.B. 1997. Large. rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander farnily Ambystomatidae (Amphibia: Caudata). Mol. Biol. Evol. 14: 1 167- 1 176.

McLandress, M.R., and McLandress. 1. 1979. Blue-phase Ross' Geese and other blue- phase geese in Western North Arnerica. Auk 96: 544-550.

Mindell. D.P.. Sorenson. M.S.. Huddleston, C.J.. Miranda. H.C.. Knight. A.. Sawchuk. S.J., and Yuri. T. 1997. Phylogenetic relationships among and within selected avian orders based on mitochondrial DNA. Pp. 2 1 1-245. In: Mindel 1. D.F. (ed. ). Avian molecular rvolution and systemaiics. Academic Press. New York.

Moritz. C.. and Brown. W.M. 1986. Tandem duplications of D-loop and ribosomal RNA sequences in lizard mi tochondrial DNA. Science 233: 1425- 1 427.

Moritz. C., and Brown, W.M. 1987. Tandem duplications in animal mitochondrial DNAs: variation in incidence and gene content amon; lizards. Proc. Natl. Acad. Sci. USA 84: 7 183-7 187.

Nagley. P. 1989. Trafficking in small mitochondrial RNA molecules. Trends Genet. 5: 67-69.

Nomiyama. H.. Tsuzuki. T.. Wakasugi. S., Fukuda. M.. and Shimada, K. 1984. Interruption of a hurnan nuclear sequence homologous to mitochondrial DNA by a member of the K y 1 1.8 kb family. Nucleic Acids Res. 12: 52254234.

Nomiyama. H., Fukuda, M.. Wakasugi. S.. Tsuzuki. T.. and Shimada, K. 1985. Molecular structures of rnitochondriül DN A-li ke sequences in human nuclear DNA. Nucleic Acids Res. 13: l a 9 - 1658.

Nugent. J.M.. and Palmer. J.D. 199 1. RNA-mediated transfer of the gene cox II from the mitochondrion to the nucleus during flowering plant evolution. Cell 66: 47348 1.

Obar. R.. and Green, J. 1985. Molecular archaeoiogy of the mitochondriai genome. J. Mol. Evol. 22: 243-25 1.

Okimoto. R., Chamberin, H.M.. Macfarlane, J.L.. and Wolstenholme, D.R. 1 99 1. Repeated sequence sets in mitochondrid DNA molecules of root knot nematodes (~eloido~vne): nucleotide sequences. genome location and potential for host race identification. Nucleic Acids Res. 19: 16 19- 1626.

Okimoto. R.. Macfarlane, J.L., Clary, D.O., and Wolstenholme. D.R. 1992. The mitochondrial genomes of two nematodes, Caenorhabditis eleoans and Ascaris suurn. Genetics 130: 47 1-498.

Piiabo. S.. Thomas. W.K.. Whitfield, KM.. Kumazawa. Y.. and Wilson, A.C. 199 1. Rearrangements of mitochondrial transfer RNA genes in rnarsupials. J. Mol. Evol. 33: 426-430.

Paquin. B.. and Lang, B.F. 1996. The mitochondrial DNA of Allomvces macrogvnus: the complete genomic sequence from an ancestral fungus. J. Mol. Biol. 255: 688- 701.

Patton. J-C.. and Avise. J-C. 1985. Evolutionary genetics of birds IV. Rates of protein divergence in waterfowl ( Anatidae). Genetica 68: 1 29- 143.

Perna. N.T., and Kocher. T.D. 1996. Mitochondrial DNA: molecular fossils in the nucleus. Curr. Biol. 6: 125- 129.

Pont-Kingdon. G.A.. Oknda. N.A.. Macfarlane. J.L.. Beagley. C.T.. Wolstenholme. D.R.. Cavalier-Smith. T., and Clark-Wrilker. G.D. 1995. A coral mitochondrial mutS gene. Nriture 375: 109- 1 1 1 .

Pont-Kingdon, G.A.. Okridri, N A . . Macfarlane. J.L.. Beagley. C.T., Watkins-Sims, CD., Cavalier-Smith, T.. Clark-Walker, G.D.. and Wolstenholme, D.R. 1998. Mitochondrial DNA of the coral Sarcophvton glaucum contains a gene for a homologue of bricterial MutS: a possible case of gene transfer from the nucleus to mitochondrion. J. Mol. Evol. 46: 4 19-33 1.

Quinn. T.W. 1988. DNA sequence variation in the Lesser Snow Goose, Anser caerulescens c;icrulrscrns. Ph. D. thesis. Queen's University. Kingston.

Quinn. T.W. 1992. The genetic legacy of mother goose: phylogenetic patterns of lesser snow goose Chen caemlescens caerulescens matemal lineages. Mol. Ecol. 1: 105- 117.

Quinn. T.W., and Mindell. D.P. 1996. Mitochondrial gene order adjacent to the control region in crocodile. turtle. and tuatara. Mol. Phyl. Evol. 5: 3 4 - 3 5 1 .

Quinn. T.W.. and White. B.N. 1987. Analysis of DNA sequence variation. Pp. 163- 198 in: Cooke. F.. and Buckley. P.A. (eds.). Avian genetics. Academic Press. London.

Quinn. T.W.. and White. B.N. 1987b. Identification of restriction-fragment-length polymorphisms in genomic DNA of the Lesser Snow Goose CAnser caerulescens caerulescensJ Mol. Biol. Evol. 4: 126- 143.

Quinn, T.W., and Wilson. A C 1993. Sequence evolution in and around the mitochondnal control region in birds. J. Mol. Evol. 37: 417-425.

Quinn. T.W., Shields, G.F., and Wilson, A C 199 1 . Affinities of the Hawaiian Goose based on two types of mitochondnal DNA data. Auk 108: 585-593.

Ramirez, V.. Savoie, P.. and Morais, R. 1993. Molecuiar characterization and evolutîon of a duck rnitochondrial genome. J. Mol. Evol. 37: 296-3 10.

Rattray. A.B., and Cooke, F. 1984. Genetic modelling: an analyis of a colour polymorphism in the Snow Goose. 2001. J. Lin. Soc. 80: 437-444.

Raymond. M., and Rousset, F. 1995. GENEPOP: population genetics software for exact test and ecumenism. I. Heredity 86: 248-249.

Raymond. M.. and Rousset. F. 1995b. An exact test for population differentiation. Evolution 49: 1280- 1283.

Reiter, W.-D., Palrn, P.. and Yeats, S. 1989. Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elernents. ~ u d . Acids es. 17: 1907- 1914.

Richter. C. 1988. Do rnitochondrkl DNA fragments promote cancer and aging'? FEBS Letters 241: 1-5.

Richter. C.. Park. J-W.. and Ames B.N. 1988. Normal oxidative damage to mitochondrial and nuclex DNA is extensive. Proc. NAtl. Acad. Sci. USA 85: 6465-6467.

Roe. B.A.. Ma. D.-P.. Wilson. R.K.. and Wong. J.F.-H. 1985. The complete nucleotide sequence of the Xeno~us laevis mitochondrial genome. J. Biol. Chem. 260: 9759- 9774.

Rossmanith. W., Tullo. A., Potuschak. T., Karwan, R., and Sbisa. E. 1995. Human mitochondrial tRNA processing. I. Biol. Chem. 270: 12885- 1289 1 .

Rousset. F., and Raymond. M. 1995. Testi ng heterozygote excess and deficicncy . Genetics 140: 1413- 1419.

Saiki. R.K.. Bugawan. T.L.. Hom. G.T.. Mullis. K.. and Erlich. H.A. 1986. Analysis of enzymatically amplified beta-globin and HLA-DQalpha DNA with ailele-specific oligounucleotide probes. Nature 324: 163- 166.

Sankoff. D.. Leduc. G.. Antoine. N.. Paquin. B.. Lang. B.F.. and Cedergren. R. 1992. Gene order cornparisons for phylogenetic inferernce: evolution of the mitochondrial genome. Proc. NatI. Acad. Sci. USA 89: 6575-6579.

Sapienza. C.. and Doolitle W .F. 1 98 1 . Genes are things you have whether you want them or not. Cold Spring Harbor Syrnp. Quant. Biol. 45: 177- 182.

Savre-Train, 1.. Piatyszek. M.A.. and Shay. J.W. 1992. Transcription of deleted mitochondral DNA in human colon adenocarcinorna cells. Hum. MOI. Genet. 1: 203-204.

Schinkel. A.H., and Tabak. H.F. 1989. Mitochondrial RNA polyrnerase: dual role in transcription and replication. Trends. Genet. 5: 149- 154.

Schneider. S.. Kueffer. L M . . Roessli. D.. and Excoffier. L. 1997. Arlequin ver. 1.1: a software for population genetic data analysis. Genetics and Biornetry Labontory. Univ. of Geneva, Switzerland.

Schneider-Broussard, R., and Neigel. J.E. 1997. A large-subunit mitochondrial ribosomal DNA sequence translocated to the nuclear genorne of two Stone cnbs (Menippe). Mol. Biol. Evol. 14: 1 56- 165.

Schuster. W.. and Brennicke. A. 1987. Plastid. nuclear and reverse transcriptase sequences in the mitochondnal genome of Oenothera: is genetic information transferred between organelles via RNA'? EMBO J. 6: 2857-2863.

Seutin. G.. White. B.N.. and Boag, P.T. 199 1. Preservation of avian blood and tissue samples for DNA analyses. Can. J. 2001. 69: 82-90.

Shay. J.W., and Werbin, H. 1992. New evidence for the insertion of mitochondrial DNA into the human genome: significance for cancer and aging. Mutat. Res. 275: 227- 7 7 <

Shay. J.W.. Baba. T.. Zhan. Q.. Kamimura. N.. and Cuthben. J.A. 1991. HeLaTG cells have mitochondrial DNA inserted into the c - m ~ c oncogene. Oncogene 6: 1869- 1874.

Shields. G.F.. and Wilson. AC. 1987. Calibrrition of mitochondrial DNA evolution in geese. J. Mol. Evol. 24: 2 12-2 17.

Slatkin. M. 1985. Rare alleles ~LS indicators of genr tlow. Evolution 39: 53-65.

Smith. M.F.. Thomas. W.K.. and Patton J.L. 1992. Mitochondrial DNA-like sequences in the nuclear genome of an akodontine rodent. Mol. Biol. Evol. 9: 204-2 15.

Smith. M.J.. Arndt. A.. Gorski. S.. and Fajber. E. 1993. The phylogeny of echinoderm cl~sses bÿsed on mitochondrial genome arrangements. J. Mol. Evol. 36: 545-554.

Sorenson. M.D.. and Fleischer, R.C. 1996. Multiple independent transpositions of mitochondrial DNA control region sequences to the nucleus. Proc. Natl. Acad. Sci. USA 93: 15239-15243.

Stern, D.B.. and Lonsdale, D.M. 1982. Mitochondrial and chloroplast genomes of maize have a 12-kilobase DNA sequence in common. Nature 299: 698-702.

Sunnucks, P.. and Hales. D.F. 1 996. Numerous transposed sequences of mi tochondrial cytochrome oxidase 1-11 in aphids of the genus Sitobion (Hemipterx Aphididae). Mol. Biol. Evol. 13: 5 10-524.

Terrett. J.A.. Miles, S., and Thomas, R.H. 1996. Compfete DNA sequence of the rnitochondrial genome of Cepea nemonlis (Gastropoda: Puhonata). J. Mol. Evol. 42: 160- 168.

Thorsness, P.E., and Fox. T.D. 1990. Escape of DNA from mitochondria to the nucleus in Saccharomvces cerevisiae. Nature 346: 376-379.

Thorsness, P.E., and Fox, T.D. 1993. Nuciear mutations in Saccharornvces cerevisiae that affect the escape of DNA from mitochondria to the nucleus. Genetics 134: 2 1 - 28,

Timmins, J.M., and Scott. N.S. 1983. Sequence homology between spinach nuclear and chloroplast genomes. Nature 305: 65-67.

Tsuzuki, T., Norniyama. H.. Maeda, S.. Setoyarna. C.. and Shimada. K. 1983. Presence of mitochondrial-DNA-like sequences in the human nuclear D N A Gene 25: 223- 229.

Tzeng, C-S.. Hui, C-F.. Shen. S-C.. and Huang, P.C. 1992. The complete nucleotide sequence of the Crossostoma lacustre mitochondrial genorne: conservation and variation among vertebrates. Nucleic Ac ids Res. 20: 4853-4858.

Unseld, M., Marienfeld, J.R.. Brandt. P., and Brennicke. A. 1997. The rnitochondrial genome of Arabidopsis thaliana contains 57 genes in 366,924 nucleotides. Nature Genet. 15: 57-6 1.

Vawter. L., and Brown. W.M. 1986. Nuclear and mitochondrial DNA cornparisons reveal extreme rate variation in the rnolecular clock. Science 234: 194- 196.

van Wagner. C.E., and Baker. A.J. 1990. Association between mitochondrial DNA and morphological evolution in Canada Geese. J. Mol. Evol. 31: 373-382.

van den Boogaan. P.. SamalIo. J.. and Agsteribbe. E. 1982. Similar grnes for a mitochondrial ATPasc subunit in the nuclear and mitochondrial genomes of Neuros~ora crissa. Nature 298: 187- 189.

van der Kuyl, A.C., Kuiken. C.L.. Dekker. J.T.. Perizonius. W.R.K.. and Goudsmit. J . 1995. Nuclear counterpans of the cytoplasrnic mitochondriall ZS ribosomal RNA gene: a problern of ancient DNA and molecular phylogenies. J. Mol. Evol. 40: 652-657.

Wakasugi, S.. Nomiyama. H., Fukuda, M.. Tsuzuki. T.. and Shimada, K. 1985. Insertion of a long K D ~ 1 family rnernber within a mitochondrial-DNA-like sequence present in the human nuclear genorne. Gene 36: 28 1-288.

Wallace. D.C. 1992. Mitochondrial genetics: a pandigm for aging and degenentive disease'! Science 256: 628-632.

Wallis. G.P. 1987. Mitochondrial DNA insertion poly morphism and g e m line heteroplasmy in the Triturus cristatus cornplex. Heredity 58: 229-238.

Warrior, R.. and Gall. 1. 1985. The mitochondrial DNA of Hvdra attenuata and Hvdra littoralis consist of two linear molecules. Arch. Sci. Geneve 38: 439-445.

Weir, B.S. 1996. Population data analysis II: methods for discrete population genetic data. S inauer Assoc., Sunderland, MA.

Weir. B.S.. and Cockerham. C.C. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38: 1358- 1370.

Wenink. P.W.. Baker. A.J.. and Tilanus. M.G.J. 1994. Mitochondrial control region sequences in two shorebird species. the tumstone and the dunlin. and their utility in population genetic studies. Mol. B iol. Evol. 11: 22-3 1.

Wintz. H.. Grienenberger, J.M.. Weil, J.H.. and Lonsdale, D.M. 1988. Location and nucleotide sequence of two tRNA genes and a tRNA pseudo-gene in the maize mitochondrial genome: evidence for the transcription of a chloroplast gene in mitochondria. Curr. Genet. 13: 247-254.

Wu. C.-I., True. J.R., and Johnson, N. 1989. Fitness reduction associated with the deletion of a satellite DNA array. Nature 341: 248-25 1.

Wolstenholme, D.R. 1992. Animal rnitochondrial DNA: structure and evolution. Int, Rev. Cytoi. 141: 173-2 16.

Wolstenholme. D.A.. Macfarlane, J.L.. Okimoto. R.. Clary. D.O.. and Wahleithner. J.A. 1987. Biuare tRNAs inferred from DNA sequences of mitochondrial genomes of nematode worms. Proc. Natl. Acad. Sci. USA 84: 1324- 1328.

Wright. R.M.. and Cummings. D.J. 1983. Integration of rnitochondrial gene sequences within the nuclear genomr during senescence in a fungus. Nature 302: 86-88.

Wright. S. 193 1 . Evolution in Mendelian populations. Genetics 16: 97- 159.

Yokobori. S-i.. and Paiibo. S. 1997. Polyadenylation creates the discrirninator nucleotide of chicken mitochondrial ~ R N A T Y ~ . J. Mol. BioI. 265: 95-99.

Yoneyama. Y. 1987. The nucleotide sequrncrs of the heavy and light strand replication origins of the Rana catesbeiana mitochondriril grnome. Nippon Ida Daioaky Zasshi 54: 429-440.

Yoshionari. S.. Koike. T.. Yokogawa. T.. Nishitawa. K.. Ueda. T.. Miura. K-i.. and Watanabc. K. 1994. Existence of nuclear-encoded SS-rRNA in bovine mitochondria. FEBS Lett. 338: 1 37- 142.

Zevering. C.E.. Moritz, C.. Heideman. A.. and Sturm. R.A. 199 1 . Parallel origins of duplications and the formation of pseudogenes in mitochondrial DNA from parthenogenic lizards (Heterodonta binoei: Gekkonidüe). J. Mol. Evol. 33: 13 1 - 44 1 .

Zhang. D.X.. and Hewitt .G.M. 1996(a). Nuclear integrations: challenges for rnitochondrial DNA markers. Trends Ecol. Evol. 11: 247-35 1.

Zhang, D.X.. and Hewit. G.iM. 1996(b). Highly conserved nuclear copies of the mitochondrial control region in the desen locust Schistocerca sreoriria: some implications for population studies. Mol. Ecol. 5: 795-300.

Zischler, H.. Geisert, H., Vonhaeseler, A., and Paabo. S. 1995. A nuclear "fossil" of the mitochondrial D-loop and the origin of modem humans. Nature 378: 489-492.

Zischler. H., Geisert, H., and Castresana. J . 1998. A hominoid-specific nuclear insertion of the mitochondrial D-loop: implications for reconstructing ancestral mitochondrial sequences. Mol. Biol. Evol. 15: 463-469.

Zullo, S.. Sieu, L.C.. Slightom, J.L.. HadIer. H.I.. and Eisenstadt. J.M. 199 1 . Mitochondrial D-loop sequences are integrated in the rat nuclear genorne. J . Mol. Biol. 221: 1223- 1235.

IMAGE EVALUATION TEST TARGET (QA-3)

APPLIED INlclGE . lnc 1653 East Main Street - -. - Rochester. NY 14609 USA -- -- - - Phone: i l W48~-03OO -- -- - - F a 7361288-5989