ALMA MATER STUDIORUM – UNIVERSITÀ DI BOLOGNA DOTTORATO DI RICERCA in BIODIVERSITA’ ED EVOLUZIONE Ciclo XXI° Settore/i scientifico disciplinari di afferenza: BIO/05 TITOLO TESI ”Mitochondrial Genomics: structure, inheritance and phylogenetic utility of the Mitochondrial genome in Bivalvia and Insecta” Presentata da: Dr. Andrea Ricci Coordinatore Dottorato Relatore Prof. Giovanni Cristofolini Dr. Marco Passamonti Esame finale anno 2009 1. FOREWORD ........................................................................................................................................................2 2. INTRODUCTION................................................................................................................................................2 2.1 THE MITOCHONDRIAL DNA ..................................................................................................................2 2.2 ANIMAL MITOCHONDRIAL DNA: DISTINCTIVE FEATURES........................................................4 2.2.1 gene content ............................................................................................................................................6 2.2.2 genome architecture ...............................................................................................................................8 2.2.3 The asymmetric distribution of genes...................................................................................................9 2.3 THE MITOCHONDRIAL GENOME IN BIVALVE MOLLUSKS .......................................................11 2.4 BIVALVIA AND DOUBLY UNIPARENTAL INHERITANCE (DUI)................................................13 2.4.1 DUI and sex-ratio .................................................................................................................................15 2.4.2 DUI, selection and molecular evolution of gender-mitochondrial genomes....................................17 2.4.3 “Masculinization” or “role reversal-event” and its implications for Mitochondrial dna recombination ................................................................................................................................................18 2.4.4 DUI distribution and phylogeny of paternal and maternal genomes. ...............................................21 2.4.5 The mytilid Musculista senhousia: an unusual case of gender-associated mitochondrial dna heteroplasmy. .................................................................................................................................................23 2.5 THE MITOCHONDRIAL GENOME IN INSECTA................................................................................25 2.5.1 The paleozoic diversification of the insect orders and the “ORTHOPTEROIDS PROBLEM”.....29 3 RESULTS AND DISCUSSION.........................................................................................................................30 3.1 MOLLUSCA BIVALVIA...........................................................................................................................30 3.1.1 Overall gene order................................................................................................................................30 3.1.2 Nucleotide composition .......................................................................................................................31 3.1.3 Ribosomal and transfer RNA genes....................................................................................................32 3.1.4 Gene junctions ......................................................................................................................................32 3.1.5 Gene content .........................................................................................................................................33 3.1.6 Comparative sequence analysis...........................................................................................................35 3.1.7 Codon usage..........................................................................................................................................36 3.1.8 Large Unassigned region (LUR) and sex-linked mt-DNA transmission..........................................38 3.2 PHASMATODEA .......................................................................................................................................40 3.2.1 Results...................................................................................................................................................40 3.2.2 Phylogenetic analyses and effect of data manipulation .....................................................................41 3.2.3 Discussion.............................................................................................................................................43 3.2.3.1 Methodological effects of various approaches to phylogenetic reconstruction........................45 4. Materials and methods........................................................................................................................................46 4.1 SECTION-1: MOLLUSCA BIVALVIA ...................................................................................................46 4.1.1 Sample collection .................................................................................................................................46 4.1.2 PCR amplifications, sequencing and annotation................................................................................46 4.1.3 Annotation, alignment and phylogenetic analyses.............................................................................47 4.2 SECTION-2: PHASMATODEA ................................................................................................................48 4.2.1 Sample collection .................................................................................................................................48 4.2.2 PCR amplifications and sequencing....................................................................................................48 4.2.3 Annotation, alignment and phylogenetic analyses.............................................................................50 4.2.4 Phylogenetic analyses and data partitioning.......................................................................................50 5. REFERENCES ...................................................................................................................................................52 6. TABLES..............................................................................................................................................................78 7. FIGURES ............................................................................................................................................................90 8. APPENDIX...................................................................................................................................................... 100 1 1. FOREWORD This PhD Thesis is the result of my research activity in the last three years. My main research interest was centered on the evolution of mitochondrial genome (mtDNA), and on its usefulness as a phylogeographic and phylogenetic marker at different taxonomic levels in different taxa of Metazoa. From a methodological standpoint, my main effort was dedicated to the sequencing of complete mitochondrial genomes, and the approach to whole-genome sequencing was based on the application of Long-PCR and shotgun sequences. Moreover, this research project is a part of a bigger sequencing project of mtDNAs in many different Metazoans’ taxa, and I mostly dedicated myself to sequence and analyze mtDNAs in selected taxa of bivalves and hexapods (Insecta). Sequences of bivalve mtDNAs are particularly limited, and my study contributed to extend the sampling. Moreover, I used the bivalve Musculista senhousia as model taxon to investigate the molecular mechanisms and the evolutionary significance of their aberrant mode of mitochondrial inheritance (Doubly Uniparental Inheritance, see below). In Insects, I focused my attention on the Genus Bacillus (Insecta Phasmida). A detailed phylogenetic analysis was performed in order to assess phylogenetic relationships within the genus, and to investigate the placement of Phasmida in the phylogenetic tree of Insecta. The main goal of this part of my study was to add to the taxonomic coverage of sequenced mtDNAs in basal insects, which were only partially analyzed. 2. INTRODUCTION 2.1 THE MITOCHONDRIAL DNA Mitochondria are known for their “cellular power plants” role, that is the production of ATP through oxidative phosphorylation. Such a role is unquestionably of great interest, but many other important features and functions are coming to light as the numerous researches proceed. As a matter of fact, there are strong evidences about an involvement of mitochondria in many biological processes of the eukaryotic cell life. Mitochondria have a central role in the control of cellular cycle (in particular cell growth and death; see McBride et al., 2006 for a review) and are responsible for several aspects in cell signaling, fertilization, development, differentiation, ageing, apoptosis and even sex determination, just to name some (Lenaz, 1998; Werren and Beukeboom, 1998; Wang, 2001; Brookes et al., 2002; Van Blerkom and Davis, 2007). Moreover, mitochondria are implicated 2 in human diseases such as miopathy, Kearns-Sayre syndrome,