RICERCA ITALIANA     

Mitochondrial genomics in different groups of Metazoa: molecular and structural evolution and phylogenetic usefulness of the mitochondrial genome

Università degli Studi di Siena

Abstract

This collaborative project will be centered on the evolution of the mitochondrial genome (mtDNA), and on its usefulness as a phylogeographic and phylogenetic marker at different taxonomic levels in different taxa of Metazoa. The project represents the collaborative effort of four Research Units (R.U.), led by young Principal Investigators, but all of them characterized a remarkable tradition of research on the evolution of mtDNA, and by an outstanding record of publications in the field of molecular evolution and the phylogenetic significance of mitochondrial DNA.
From a methodological standpoint, the main effort will be dedicated to the sequencing of complete mitochondrial genomes in selected taxa of bivalves, hexapods, fishes and reptiles. The approach to whole-genome sequencing will be common to all R.U. and will be based on the application of Long-PCR and shotgun sequences. The potential usefulness of the Rolling Circle Amplification will also be tested. The presence of a common methodological strategy will allow extensive integration and collaboration between the four R.U. involved in the project, as well as the possibility of exploiting common sophisticated equipment. About 50 new complete mitochondrial genomes will be sequenced, annotated, described and submitted to GenBank, to make them available to the whole scientific community.
Selected taxa will be sampled at different taxonomic levels, according to the aim of the phylogeographic/phylogenetic analysis. Individuals from geographically isolated conspecific populations will be sampled in the reptile species Geochelone galapagoensis and Varanus komodoensis. These sequences will be used for assessing phylogeographic relationships in two endemic species from two archipelagos (Galapagos and Komodo) which have underwent extensive geological rearrangements over the past 5 myr. These data will also constitute a robust scientific background to support the planning of management strategies in these two endangered taxa. In the suborder Notothenioidei of endemic Antarctic and sub-Antarctic fishes, we will use mitochondrial genomics to infer phylogenetic relationships of a group of families whose evolution has been shaped by the occurrence of remarkable morphological and physiological changes over the past 25-30 myr, due to a long process of adaptation to increasingly extreme environmental conditions. Adaptation to the extreme Antarctic environment will also be studied at a molecular level by assessing the occurrence of directional selective pressures on specific domains of selected mitochondrial proteins, and by investigating the potential biochemical compensation for the loss of one mitochondrial gene. In hexapods, species from the basal apterygote taxa Protura, Collembola, Diplura, Microcoryphia and Zygentoma will be targeted. A detailed phylogenetic analysis will be performed in order to assess phylogenetic relationships within the order Collembola, and to investigate the placement of Protura, Collembola and Diplura in the phylogenetic tree of Pancrustacea. This analysis will also aim at testing whether the formal taxa Hexapoda and Crustacea, as traditionally defined, are monophyletic. Analyses will be performed exploiting the phylogenetic information contained in nucleotide and amino acid sequences, as well as the potential informativeness of gene order changes in mtDNA. The same phylogenetic approach will be used within the Bivalvia, to infer relationships among its major lineages. In this taxon, mtDNA data are particularly limited, and our study will contribute to extend the sampling and to provide a sufficient coverage of the biodiversity of Bivalvia. In addition, two species of Bivalvia (Tapes philippinarum and Musculista senhousia) will be used as model taxa to investigate the molecular mechanisms and the evolutionary significance of their aberrant mode of mitochondrial inheritance (Doubly Uniparental Inheritance). Cytological (fluorescence microscopy) and molecular (Real Time PCR and Suppression Subtractive Hybridization) methods will be used to assess the fate of sperm mitochondria after fertilization, the distribution of male- and female-derived mtDNA in different tissues and in individuals of different sex, and the functional significance of both genomes. This study will represent an important contribution to the understanding of the mechanisms of mitochondrial inheritance in all animals.
This project has the ambitious target of achieving outstanding results for unravelling the mechanisms of evolution of the mitochondrial genome, and of contributing to the solution of long-standing taxonomic and phylogenetic issues in several groups of Metazoa. We also aim at enforcing the level of collaboration and integration among four highly qualified Research Teams, and at stimulating the participation of young investigators (Graduate Students and Postdocs) to an articulated research project of considerable scientific value. <<<

Principal Investigator

Francesco Frati Università degli Studi di SIENA

Research Objectives

The outstanding scientific records of the Principal Investigators of each Research Units (see section 14 and the relative B forms) guarantees for the possibility to achieve the ambitious scientific targets set by this collaborative project. The common research target of this collaborative project is the sequencing of entire mitochondrial genomes in selected species from different taxonomic lineages of Metazoa. Entire mitochondrial genomes will represent the basic data for inferring evolutionary relationships at different taxonomic levels (phylogeography and phylogeny), for elucidating the molecular mechanisms of inheritance of mitochondrial DNA (mtDNA), and for unravelling processes of local adaptation mediated by relaxed and/or directional selection.
The collaborative project is based on a large sequencing effort, aiming at obtaining the complete sequence of about 50 mitochondrial genomes, for a ground total of almost 1 Gb of sequence information. These sequences will cover a broad taxonomic range (mollusks, hexapods, fishes and reptiles), and different taxonomic levels. Conspecific individuals will be sequenced in the reptile species Geochelone elephantopus and Varanus komodoensis, while species from different families will be sequenced in the teleost suborder Notothenioidei. In the Bivalvia (Mollusca) and the Hexapoda (Arthropoda), individuals from different families, representing different orders, will be sequenced. Such an extensive data set will allow the assessment of the significance of the mitochondrial genome as a phylogeographic and phylogenetic marker. In addition, a general target of the project is the assessment of various aspects of the molecular evolution of the mitochondrial genome at the nucleotide level, such as nucleotide composition, asymmetric compositional bias, heteroplasmy, rates of evolution, gene arrangement.
At the phylogeographic level, the performance of the mitochondrial genome will be evaluated in two insular systems, using as model species two taxa of endangered reptiles: the Galapagos giant tortoises and the Komodo dragon. These two species differentiated from the continental closest ancestor into different isolated populations, following the geological evolution of the Archipelago. They share a remarkable conservational importance, although on different paleogeographic settings. In fact, while colonization and dispersal history of giant tortoises across the Galapagos date back from 0.7 to 3 mya, vicariance due to eustatic changes in sea level affected gene genealogies of Komodo dragons during the last 140.000 years. Mitochondrial data will be used to infer the occurrence of historical events, such as colonization, isolation, population fragmentation and numerical reduction, by looking at the potential traces of these events on the genetic structure of populations, and by reconstructing phylogeographic networks. In addition, one major aim of this part of the project is to provide genetic data to support the implementation of management strategies, as well as the planning of correct practices of reintroduction where these are necessary.
In the suborder of endemic Antarctic fishes Notothenioidei, the aim of this project will be the reconstruction of phylogenetic relationships by comparing complete mitochondrial genomes of representatives of all families of these fishes. This study will provide genetic supported evidence for investigating the historical events that led to the colonization of the Antarctic ocean by this peculiar teleost lineage.
At a similar taxonomic level, but with a much broader world distribution, and a much more ancient evolutionary history, one target of the project will be the reconstruction of the phylogenetic relationships among the major families of the hexapod order Collembola. This cosmopolitan taxon of edaphic arthropods is organized in three suborders, Arthropleona, Symphypleona, and Neelipleona, whose phylogenetic relationships are not yet clear, and which will represent a specific target of this part of the project. The reconstruction of the phylogenetic relationships among collembolans have also the objective to assess whether the ancestor of these terrestrial species was itself terrestrial, or semi-aquatic.
On a broader taxonomic range, data collection will also focus on deeper phylogenetic relationships, such as those occurring within the Class Bivalvia and among the major lineages of Pancrustacea. Here, the aim of the project is to reconstruct phylogenetic relationships among the major lineages of Bivalvia, a group where such relationships are still debated, and for which mtDNA data are particularly scarce. In the Pancrustacea, we aim at assessing, by phylogenetic inference, the position of the basal hexapod orders Protura, Collembola and Diplura, and the reciprocal monophyly of the formal taxa Crustacea and Hexapoda, as traditionally defined, as well as providing a phylogenetic framework for interpreting crucial evolutionary steps, such as terrestrialization.
In these latter taxonomic contexts (Collembola, Bivalvia and Pancrustacea), useful phylogenetic information might also be retrieved from gene arrangement, and the identification of shared gene order changes across different taxa. In this effort, we also aim at providing real data to produce hypotheses over the mechanisms by which such changes occur.
At least two additional significant issues are addressed with this collaborative project, with noteworthy implications for the mechanisms of evolution and inheritance of the mitochondrial genome. One is the study of the unusual mechanism of inheritance of mitochondria found in many bivalves, which leads to the presence of different mitochondrial genomes within the same species (Doubly Uniparental Inheritance). One major aim of the project will be the elucidation of the cytological mechanisms leading to DUI, the fate of the mitochondria of both gametes, the nature of nucleus-mitochondrion interactions, the functional significance of the male-inherited mitochondrial DNA, and the genetic consequences of this peculiar mode of inheritance on the rates of evolution of the mitochondrial genome. The DUI system is likely to be a precious experimental system to elucidate the mechanisms of inheritance of mitochondria and the evolution of their genomes, since DUI is considered as a variation of the common matrilinear mechanism of inheritance of mitochondria.
The second major issue is represented by the importance of selection at the mtDNA level for the adaptation of notothenioid fishes to the Antarctic environment. One major aim of this project will be the assessment of the mechanisms by which the mitochondria of Antarctic fishes are able to compensate for the loss of the nad6 gene in the mitochondrial genome, and whether such a loss may be considered the result of relaxed selection over mtDNA gene content. At the same time, we also aim at determining whether the peculiar environment of the Antarctic ocean is able to exert other forms of directional mutational pressures, as preliminary data on accelerated rates of non-synonymous substitutions at specific functional domains in mtDNA-encoded proteins seem to suggest.
In this section dedicated to the “Aims of the Project”, we would also like to emphasize that a general target of this initiative is to promote the collaboration among four research teams, all led by comparatively young investigators, and composed by many motivated postdocs and PhD students for which this initiative may help in stimulating reciprocal interaction and improving their scientific training. <<<

First Results

As described in section 11, the present project is organized around eight main objectives. Two of them are typical phylogeographic studies, with special emphasis on island biogeography and conservational and management implications. Four objectives are typical phylogenetic studies, at different taxonomic levels (and age of the diversification events), in different taxa (Teleostei, Arthropoda and Bivalvia). One objective will deal with the mechanisms of inheritance of the mitochondrial genome in species of the Bivalvia showing the peculiar phenomenon of the Doubly Uniparental Inheritance of mtDNA. One objective will address the mechanisms and the consequences at the molecular level of the adaptation to the Antarctic extreme environment of a family of endemic teleosts.
In all instances, the principal interest of these research lines is represented by the advancement of the scientific knowledge on the different subjects, all of them being still highly controversial. But in the cases of the phylogeographic studies on the Galapagos giant tortoises, Geochelone elephantopus, and the Komodo dragon, Varanus komodoensis, their status of endangered species implies the likelihood that the data produced will represent a solid scientific background to support the planning of management strategies.
In the Galapagos giant tortoises, the comparison of the entire mitochondrial genomes will lead to draw an exhaustive picture of the relationships among the different subtaxa (races or subspecies) which have been described in 6 of the main islands of the Archipelago. This research will also allow the identification of the most important routes of colonization of the islands in relation with the palaeogeographic events that have shaped up the landscape of the Archipelago. The mtDNA data collected will also help in defining long-standing issues concerning the taxonomic status of the different forms, and the relationships between morphological and genetic differentiation. Of no lesser importance, the data collected in this project will also serve as a solid biological basis to support the planning of management strategies, especially in those island where the species has recently gone extinct, and where reintroduction practices must be executed using the original endemic subspecies.
Similar results are expected to be the outcome of the study on the phylogeography of the endemic Komodo dragon. Here, the evolutionary relationships between six different populations (from 5 different islands) will be described using the complete sequences of the mitochondrial genome, providing an important contribution to the issue of insular biogeography, in archipelagos of comparatively recent geological origin (less than 5 mya). These data will offer solid results concerning the ancestral lineage which originated the differentiation of Varanus komodoensis in the Komodo archipelago, the pattern of colonization of the islands (in an insular system which was geologically extensively modified during the glacial periods of the Pleistocene by remarkably eustatic changes of the sea level), and the influence of recent gene flow on the diversification of the populations. Our project will therefore provide a picture of the evolutionary history of this endangered species, with remarkable implications on the development of conservational strategies. Galapagos tortoises and Komodo dragons have different dispersal abilities and inhabit archipelagos characterized by different palaeogeographic patterns of formation. Therefore, this part of the project provides an opportunity to compare patterns of mitochodrial genome evolution under diverse spatial and temporal biogeographic scenarios.
In the suborder of endemic Antarctic and sub-Antarctic fishes Notothenioidei, we will provide a complete picture of the phylogenetic relationships of the taxon, which is confined to the cold Antarctic ocean or to nearby sub-Antarctic waters. This will represent the first exhaustive phylogenetic study of this suborder, which has been studied so far only on the basis of morphological characters or molecular data from single genes, which have provided contrasting results. We will therefore be able to obtain a solid phylogenetic scheme for investigating the evolution of this peculiar groups of families, that have differentiated in the past 25-30 mya after the Antarctic Ocean became separated from the remaining southern oceans. The assessment of a solid phylogenetic framework on which all evolutionary changes can be superimposed is particularly important for this group of fishes which underwent remarkable morphological, physiological, biochemical and molecular (see below) adaptations in order to survive in this extreme environment.
The sequencing of the complete mitochondrial genome will also serve as a data set to infer phylogenetic relationships among the major lineages (families) of the basal hexapod order Collembola. Here, with 8 species already sequenced, we will add the sequence of 9 additional species, from all three described suborders, bringing the total number of sequenced species for this order to 17, therefore assembling one of the taxonomically most diverse data set ever constructed for a single hexapod order. The phylogenetic analysis will provide a complete picture of the evolution of this 450-500 myr old lineage of terrestrial arthropods, with special emphasis on the relationships among the three suborders (Arthropleona, Symphypleona, Neelipleona), a test of their reciprocal monophyly, and the pattern of evolution of ecophysiological features such as the semi-aquatic habit found in some species.
In a broader phylogenetic context within the Arthropoda, a specific phylogenetic analysis will also address the question of hexapod monophyly, and the position of basal hexapod orders in the phylogenetic tree of the Pancrustacea. This objective will be achieved by sequencing additional species of apterygote species (9 species from Protura, Diplura, Microcoryphia and Zygentoma, in addition to the 9 species of Collembola), therefore assembling (with all taxa already available in GenBank) a large data set of pancrustacean species for which the sequence of the entire mitochondrial genome is available. This data set, with over 120 species and over 10 kb of sequence information, will represent the largest data set ever assembled for this subgroup of arthropods, and the resulting phylogenetic analysis will help shedding light on the evolutionary steps which occurred over 600 mya and which led to the diversification of the most successful taxon of animals in the Earth (the Insecta). The availability of such a phylogenetic scheme will provide a solid background information to interpret some of the most remarkable processes of evolution and adaptation, such as terrestrialization. The same data set will also help assessing the reciprocal monophyly of Crustacea and Hexapoda, an issue which is highly contentious among arthropod systematists. This analysis will also serve as a test of the performance of different methods of phylogenetic reconstruction, and of the recently developed matrices of amino acid substitution.
Mitochondrial genomics will also be used to approach the phylogenetic relationships of Bivalvia. For this Class of mollusks, a reasonably complete, although not exhaustive, data set will be assembled, sequencing the mitochondrial genome from at least 10 different species from different families. This will dramatically increase the number of complete mitochondrial sequences of Bivalvia available in GenBank, that are now only 13, and will constitute a sufficiently large data set to assess the phylogenetic relationships of the major families of the Bivalvia, using either nucleotide and amino acid sequence data, as well as the information contained in gene order changes.
The Bivalves will also represent the target taxon for another important aspect of our project, namely the issue of the Doubly Uniparental Inheritance, with obvious implications on the overall pattern of evolution and inheritance of the mitochondria genome. Here, our project is expected to contribute substantially to the description of the DUI in the species Tapes philippinarum and Musculista senhousia, whose mechanisms will be compared to those already described for Mytilus. By using in vivo systems for tracking mitochondrial movement, we will be able to describe, in both species, the fate of sperm mitochondrial after fertilization. Real Time PCR will be also used to quantify the relative abundance of M and F mtDNA in gametes, during embryonic development, in individuals of different sexes, and in different tissues of the same individual. In addition, the SSH technique will be useful to assess the molecular details of sex determination in these species, as well the interactions between nucleus and mitochondria leading to the suppression of the “sperm mitochondria elimination mechanism” that commonly produces matrilinear inheritance of mtDNA. This part of the project represents an absolute novelty in DUI studies and will likely contribute to unveil the molecular details of DUI. This will certainly be of remarkable importance for the general understanding of mitochondrial inheritance even in non-DUI species, since DUI is commonly considered just a variation of the matrilinear inheritance. This part of our study will also contribute to investigate the rate of occurrence of mitochondrial recombination, as this phenomenon might be particularly likely in tissues where highly differentiated mitochondrial genomes are present at the same time. Finally, as we will eventually provide molecular markers for the early sex-determination in T.philippinarum, a species of considerable commercial importance, the results of our study may bear potentially significant practical applications in the ability of recognizing the sex of the individuals before the development of the gonads.
The notothenioid fishes will also represent a model taxon for studying the molecular adaptation to the extreme environment of the Antarctic Ocean, as well as for the assessment of the consequences of such an adaptation on the genome of these fishes. Our study will lead to testing whether the peculiar environmental conditions are able to exert a directional mutational pressures on specific functional domains of selected mitochondrial genes, a test which will be done by studying the sequences of Antarctic and non-Antarctic species, and by comparing the rates of non-synonymous substitutions. In addition, the sequencing of the complete mitochondrial genome will provide a large data set to assess whether the loss of the nad6 gene, found in some mitochondrial genomes of Antarctic notothenioids, may be tolerated because of a relaxed selection pressure, or because of compensatory mechanisms at the biochemical level.
In total, our project will lead to the complete sequencing and description of about 50 new mitochondrial genomes, contributing in a significant manner to the study of the molecular evolution of the mitochondrial genome, its modes of inheritance, its utility as phylogeographic and phylogenetic marker, and the molecular consequences of adaptation to extreme environmental conditions on the evolution of this genome.
Finally, we consider as a significant result of our project the possibility offered to young researchers (PhD students and postdocs) to take part to an ambitious project of molecular evolution, to get acquainted with modern molecular techniques and bioinformatic tools, and to be involved in all different steps of the experimental procedures. This will eventually represent a significant improvement for their scientific training. <<<

Timescale

24 months

National and international background

The mitochondrial genome (mtDNA) is a closed circular molecule which exhibits an extraordinary degree of structural conservation across Metazoa. With very few exceptions, the mtDNA has a size ranging from 15 to 20 kb, and contains 37 genes (Boore 1999). Thirteen genes (PCGs) encode for subunits of enzymes involved in oxidative phosphorylation; there are three subunits (I-III) of the cytochrome c oxidase (cox1, cox2 and cox3), 7 subunits of the NADH dehydrogenase (nad1, nad2, nad3, nad4, nad4L, nad5, nad6), two subunits of the ATPase (atp6 and atp8), and the gene encoding for the cytochrome b (cytb). All proteins are used within the mitochondrion, in conjunction with additional subunits encoded in the nuclear genome and transported into the organelle. Twenty-two genes encode for their 22 corresponding tRNAs, one for each amino acid (trnX), with the exception of Leu and Ser which have two tRNAs each. Two genes (rrnS, rrnL) encode for the small and large subunits of ribosomal RNAs (12S and 16S), which participate in the assembling of the mitochondrial ribosomes. Their presence, and the complete array of tRNAs, allow the translation of mitochondrial genes to be carried out within the mitochondrion, making the organelle independent with respect to protein synthesis.
The genome has a very compact organization, with intergenic non-coding spacers being very short (few nucleotides), or absent: in certain circumstances, the coding sequences of tRNA genes overlap with the coding sequence of other genes. Normally, there is only a single large non-coding region, which is believed to contain the molecular signals for the beginning of the replication and the transcription of the genome (Saito et al. 2005), and is therefore called Control Region. In insects, the control region is unusually rich in A and T nucleotides (up to over 90%: Crozier &amp; Crozier 1993), and is therefore often referred to as the A+T-rich region.
In general, nucleotide composition is quite heterogeneous in mtDNAs, with insects and many arthropods exhibiting a remarkable nucleotide bias. This is most extreme in pterygote hexapods, with some hymenopterans characterized by values exceeding 80% (Crozier &amp; Crozier 1993). Another interesting issue is the asymmetric compositional bias observed between the two strands of the mtDNA. It has been shown (Hassanin et al. 2005) that the relative content in As and Ts of PCGs varies according to the strand where the genes are encoded. The frequency of T tends be higher (compared to the frequency of As) in both strands where the coding sequence is present, if compared to the complementary strand (Podsiadlowski et al. 2006; Carapelli et al. 2006). The same occurs for Gs vs. Cs. In addition, this T-bias tends to be less pronounced in the strand where most of the PCGs are encoded in the mtDNA of insects (J-strand), with respect to the other strand (N-strand). This indicates that other strand-specific evolutionary forces are present, in addition to the selective pressures due to the presence of the coding sequence of the gene, possibly linked to which of the two strands is transcribed first during the replication of the molecule. This phenomenon has interesting implications for the study of the selective pressures which regulate mtDNA molecular evolution, but it also has important implications when mitochondrial genes are used for phylogenetic inference. In fact, the translocation of a gene, with a strand change, would change the strand-specific selective pressures acting on it, differentiating this gene from its homologues located, in other taxa, on the original strand (Hassanin et al. 2005).
With gene content virtually conserved across all Metazoa, one peculiar feature of the mitochondrial genome is the order with which the 37 genes are arranged along the molecule. This is quite heterogeneous over a broad taxonomic range, but tends to be much more homogeneous within taxa, with little or no changes across PCGs within insects or within vertebrates (Boore 1999). Nevertheless, rearrangement “hot spots” have been reported in mtDNA, where gene translocations, especially of tRNA genes, seem to occur much more frequently (Dowton et al. 2003). The process by which gene translocations occur may be mediated by the duplication of large fragments of the mtDNA (encompassing several genes), followed by the quick loss of duplicated genes, a loss somehow driven by the tendency of mtDNA to economize its size. Two possible models have been proposed on the basis of the comparison of gene arrangement of mitochondrial genomes from different taxa, implying that the loss of the duplicated genes might be totally random (Boore 2000), or predetermined by molecular and structural features such as transcriptional polarity (Lavrov et al. 2002). Evidence supporting the occurrence of such a mechanism can be found in unusually large intergenic non-coding regions, sometimes bearing complete, or almost complete copies of duplicated tRNA genes (Campbell &amp; Barker 1999; Carapelli et al. 2006). Regardless of the process by which gene translocations occur, changes in gene arrangement my have important phylogenetic implications. Given their rarity, and the number of possible combinations, the likelihood that identical gene arrangements occur by random convergence is negligible, therefore assigning to any shared translocation a considerable phylogenetic value as a marker of shared evolutionary history (Boore et al. 1995). A keystone example is represented by the shared translocation of a tRNA gene in the arthropod mitochondrial genome, which is considered to be a rubust synapomorphy of all Pancrustacea (Boore et al. 1998).
The mitochondrial genome is also quite peculiar with respect to its mode of inheritance (Xu 2005). Being the mitochondria of the zygote derived from those of the oocyte, the mitochondrial genome of any organism is normally inherited only along the female line. This maternal mode of inheritance, associated with homoplasmy (all mtDNA molecules of all mitochondria of all cells of a single individual are identical), has been considered a dogma for many years, before evidence of heteroplasmy (Kann et al. 1998; Nardi et al. 2001) and paternal leakage (Bromham et al. 2003) were identified. A special case of heteroplasmy and paternal leakage is the so-called phenomenon of the Doubly Uniparental Inheritance (DUI) (Zouros et al. 1992), where two different mitochondrial variants are present (showing 10% to 30% nucleotide divergence: Passamonti et al. 2003; Mizi et al. 2005), which segregate in a different fashion in the two sexes, and in different tissues. While the maternal mtDNA (F) is inherited by both sexes, the paternal mtDNA (M) is inherited only by the male progeny. In the male progeny, which is effectively heteroplasmic, the two mtDNA haplotypes (M and F) have a non-homogeneous distribution, with mtDNA-F which is more abundant in somatic tissues, while mtDNA-M dominates in the gonads, and sperm cells appear to carry only mtDNA-M-bearing mitochondria. DUI is widely distributed among Bivalvia, where it has been demonstrated in species from different families (Mytilidae, Unionidae, Veneridae), leading to the suggestion that it evolved once in the whole taxon (Passamonti et al. 2003; Zouros 2000). The data collected so far indicate that DUI evolved because of a failure (or a change) of the egg recognition system of sperm mitochondria, and through the establishment of a special transmission pattern of mtDNA-M from males to their sons (Burzynski 2007). DUI also seems to be correlated with sex-determination in bivalves, implying functional differences between M and F mtDNAs (Kenchington et al. 2002), and making these species a unique model to study mitochondrial inheritance, as well as the evolution of the mitochondrial genome at the sequence level.
Since at least over fifteen years, the mitochondrial genome has become increasingly used as a phylogeographic and phylogenetic marker (Avise 1987; Caterino et al. 2000; Simon et al. 2006). The extensive application of mtDNA as such a marker begun when PCR and direct sequencing became easy-to-use tools for zoologists, allowing the production of sequence information in a relatively short time, and the inference of evolutionary relationships based on the comparison of nucleotide and amino acid sequences (Kocher et al. 1989; Simon et al. 1994). These efforts generated an extraordinary amount of sequence information, the development of universal primers which could be used across a wide range of taxa (Simon et al. 1994, 2006), and a considerable improvement of methods of phylogeographic and phylogenetic reconstruction (Simon et al. 2006). Although mitochondrial genes are thought to evolve more rapidly than single-copy nuclear genes (Lin &amp; Danforth 2004), the mitochondrial genome is indeed a mosaic of rapidly evolving sequences and slowly evolving ones, making it a suitable marker at different evolutionary levels. More recently, methodological improvements have made easier the sequencing of the complete mitochondrial genome (Boore et al. 2005), therefore allowing phylogenetic studies to be transferred at the whole-genome level. As a consequence of these sequencing efforts, a considerable number of complete mitochondrial genomes are now available in GenBank (over 130 in arthropods, over 500 in vertebrates). Several molecular features of the mitochondrial genome may become useful phylogeographic and phylogenetic markers: nucleotide and amino acid sequences of PCGs (Simon et al. 2006), as well as gene order data (Boore 1999), allowing the genome to be used at different taxonomic levels, from conspecific populations to the phylogeny of a Phylum.
As an example of this, the mitochondrial genome has been extensively studied in arthropods to infer phylogenetic relationships of their major lineages (myriapods, chelicerates, crustaceans and hexapods), leading to reconsider the evolutionary history of the whole Phylum. Analyses now based on the comparison of over 100 taxa, and over 10 kb of sequence information have resulted in the proposal of innovative hypotheses of relationships, such as the rejection of the Atelocerata which is replaced by the Pancrustacea (Telford &amp; Thomas 1995; Boore et al. 1998), the affinity between Chelicerata and Myriapoda (Hwang et al. 2001), and the reciprocal paraphyly of Crustacea and Hexapoda (Nardi et al. 2003; Cook et al. 2005; Carapelli et al. 2007).
At the intraspecific level, the mitochondrial genome is no less informative. The relative rapid rate of evolution of several genes, or the possibility to use hypervariable subsets of nucleotides, such as 3rd codon positions of PCGs or the control region, make mtDNA an ideal marker for reconstructing the history of populations over a time span of only a few thousands or a few millions of years (Caterino et al. 2000). This allows an accurate calibration of the molecular clock, and the assessment of the influence of paleogeographical events on the genetic structure of a species. A research field that may considerably profit by the application of mitochondrial genomics is island biogeography, especially in contexts where the geological age of the islands is relatively young, and the species are of considerable conservational value, therefore requiring a detailed assessment of the reciprocal relationships of isolated populations, with obvious implications on management strategies (Ciofi et al., 2007).
As mentioned earlier, one important contribution of mitochondrial genomics to phylogeny is exerted through the analysis of gene arrangement. A special case of this is represented by the rare examples where one gene is missing from the genome in a single evolutionary lineage. Such an event has been demonstrated in notothenioid fishes, endemic of the Antarctic ocean, which have lost the nad6 gene (Papetti et al. 2007). The rarity of such an event, coupled with the peculiar environmental context in which it evolved, stimulate interesting hypotheses on the mechanisms that these animals may have evolved to compensate for the gene loss, as well as on its possible adaptive value.
In conclusion, now that complete mitochondrial genomes may be sequenced relatively easily, and that methods of sequence analysis are constantly improving, mitochondrial genomics has developed an extraordinarily wide range of applications, from phylogeography to phylogeny, from the study of its modes of inheritance to the adaptive implications of the evolution of its genes and gene content.

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