Small Steps and Grand Leaps: A Study of Micro- and Macroevolutionary Processes

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UNIVERSITE LIBRE DE BRUXELLES FACULTE DES SCIENCES

DEPARTEMENT DE BIOLOGIE MOLECULAIRE LABORATOIRE DE GENETIQUE DE L’EVOLUTION

Small Steps and Grand Leaps:

A Study of Micro- and Macroevolutionary Processes

Thèse présentée en vue de l’obtention du titre de Docteur en Sciences

ATHANASIA C. TZIKA

DIRECTEUR DE THESE: PR. MICHEL C. MILINKOVITCH

ANNEE ACADEMIQUE 2007/2008

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“Seen in the light of evolution, biology is, perhaps, intellectually the most satisfying and inspiring science. Without that light it becomes a pile of sundry facts some of them interesting or curious but making no meaningful picture as a whole.”

"Nothing in Biology Makes Sense Except in the Light of Evolution", Theodosius Dobzhansky (1973)

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GENERAL INTRODUCTION ... 1

Historical background...1

The neo-Darwinian framework ...2

Macro-evolution and the Evo-Devo framework ...5

Evolutionary Leaps ...8

Genome evolution ...9

Transcriptome and Proteome ...10

PART I: POPULATION GENETICS, CONSERVATION AND PHYLOGENY INFERENCE... 12

Use of molecular data in conservation biology...12

The role of zoological and botanical institutions in conservation ...12

Chapter 1: Population structure of an endemic vulnerable species, the Jamaican boa (Epicrates subflavus)...14

Abstract...14

Introduction ...15

Materials and methods...16

Sample collection and species-specific molecular markers ...16

Clustering of individuals ...17

Network estimations ...18

Genetic diversity ...19

F - statistics...19

Phylogeny inferences...19

Results...20

Clustering of individuals using microsatellite data...20

Network estimation...21

Phylogeny inference ...23

Genetic diversity ...23

F - statistics...24

Discussion...25

Acknowledgements...27

Chapter 2: Molecular Genetic Analysis of a Captive-Breeding Program: The Vulnerable Endemic Jamaican Yellow Boa...28

Abstract...28

Introduction ...29

Methods ...30

Sample collection and DNA extraction ...30

Molecular markers ...30

Parental allocation and genetic variability ...31

Results...32

Discussion...37

Acknowledgements...39

Chapter 3: Using nuclear mitochondrial pseudogenes to investigate the phylogeny of cetaceans 40 Introduction ...41

Materials and Methods ...42

DNA extraction, amplification, cloning and sequencing ...42

Sequences analyses ...43

Results and Discussion ...43

Presence of multiple cytochrome b-like sequences in cetacean...43

A minimum of four ancient integrations of 2.9kb mtDNA into the nuclear genome...45

Chapter 4: Escaping the Mouse Trap; the Selection of New Evo-Devo Model Species...48

Abstract...48

Introduction: The earliest model species ...49

From maps to full-genome sequences and genetic engineering...50

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Identifying and understanding phenotypes...51

The search for evolutionary conservation (medicine-driven models) ...52

Understanding variation (Evo-Devo-driven models) ...53

Criteria for choosing new model species...54

Praise for a pragmatic approach and for Evo-Devo virtual zoos ...57

Acknowledgements...58

Chapter 5: A Pragmatic Approach for Selecting Evo-Devo Model Species in Amniotes ...59

Introduction ...60

Medicine-driven models (searching for evolutionary conservation) vs. Evo-Devo-driven models (understanding variation)...60

Criteria for choosing new model species...61

A multidisciplinary pragmatic optimization approach to the selection of model species ...63

Reptilia...63

Mammalia...67

Acknowledgements...72

PART III: FROM GENOMES TO PHENOTYPES: A BOTTOM-UP APPROACH... 73

Chapter 6: MANTiS; a phylogenetic framework for multi-species genome comparisons...75

Αbstract...75

Ιntroduction ...76

System and methods ...76

Characters mining ...76

Characters mapping ...79

Genome content ...80

Biological Processes and Molecular Functions...80

Gene expression ...81

Queries...82

The MANTIS views...84

A case study...85

Conclusions and availability ...87

Αcknowledgements...88

Chapter 7: Analyzing 35 metazoan full genomes in a phylogenetic framework...89

Abstract...89

Introduction ...90

Results And Discussion ...92

Data Mining...92

Character mapping ...92

Comparison to other databases...95

Re-analysis of previously investigated gene families...97

Biological Processes and Molecular Functions...98

Expression Patterns...101

Conclusions ...105

Methods ...105

Data Mining...105

Character mapping ...106

Re-analysis of previously investigated gene families and comparisons with other databases for the localization of gains/losses...106

Biological Processes and Molecular Functions...107

Gene Expression...107

PART IV: TRANSCRIPTOMES AND PROTEOMES – THE LINK BETWEEN GENOTYPE AND PHENOTYPE... 109

New technologies for ultra-fast sequencing ...109

Description of the 454 ultra-fast sequencing method ...110

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Assembly strategies ...110

Chapter 8: Between genotype and phenotype: an evolutionary analysis of full transcriptomes in vertebrates...112

Introduction ...113

Results...114

Proof of concept analyses ...114

Transcriptome sequencing...114

Contig assembly and BLAST searches for Crocodilus ...115

Protein sequencing ...116

Materials and Methods ...116

cDNA library construction ...116

cDNA analysis...117

Protein extraction, fractionation and sequencing...117

CONCLUSIONS AND PERSPECTIVES... 118

Population Genetics, Conservation And Phylogeny Inference ...118

From Genomes To Phenotypes: A Bottom-Up Approach...119

Utility of comparative genomic approaches in the Evo-Devo framework ...119

Evo-Devo and new model species ...124

Transcriptomes And Proteomes – The Link Between Genotype And Phenotype...126

SUMMARY... 127

BIBLIOGRAPHY... 128

APPENDIX: MICROSATELLITES ISOLATION BY ENRICHMENT... 142

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1 General Introduction

Historical background

The Origin of Species (1859) was a revolutionary book, yet its author, Charles Darwin (1809-82), could not have built his theory without the observations and ideas of his predecessors. Prior to the 17

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century, there were two widespread biogenesis beliefs among naturalists: (i) spontaneous generation, which held that living organisms are generated by decaying organic substances, e.g., that mice spontaneously appear in stored grain or maggots in meat, and (ii) transmutation, i.e., the ability of organisms to undergo massive transformation through time, either during the lifetime of an individual (like the metamorphosis of a butterfly), or from one generation to the next (e.g., the giraffe was thought to have arisen from a camel- leopard pairing). However, a great shift towards species fixism was observed mainly because of the work of Carolus Linnaeus (1707-78) and his contemporary naturalists.

The new theory was well supported by (i) experiments similar to the one of Francesco Redi (1626-97) and Lazzaro Spallanzani (1729-1799) rejecting spontaneous generation (figure 1) and (ii) an intense exchange program among horticultural gardens that demonstrated the reversibility of plant modifications due to climatic changes (e.g., a plant growing slower at a high-altitude and/or lower-temperature environment would change growth rate once transferred to more appropriate conditions and vise versa), against the common belief that these were actually transmutations.

Figure 1. (A) The naturalist of the 17

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century placed meat in an open jar and in two covered ones (one with gauze and the other with paper). He observed that no maggots appeared inside the covered containers where flies did not have access, although they did appear inside the open jar and on the gauze sealing where flies laid their eggs. (B) Lazzaro Spallanzani put broth into two flasks and boiled them. One flask was left open whereas the other was sealed up. Microorganisms developed only in the uncovered flask, demonstrating that microorganisms did not grow from the broth, but were in the air that entered the flask.

Species fixism has been important to the origin of evolutionary biology because it

set the stage for the construction of the Natural System (how one could have

constructed a coherent taxonomic system in which wheat could give rise to rye,