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42 Asante-Owusu, R.N. et al. (1996) Heterodimerization between two classes of homeodomain proteins in the mushroom Coprinus cinereus brings together potential DNAbinding and activation domains. Gene 172, 25–31 43 Luo, Y.H. et al. (1994) Only one of the paired Schizophyllum commune Aα mating-type putative homeobox genes encodes a homeodomain essential for Aα regulated development. Mol. Gen. Genet. 244, 318–324

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44 Spit, A. et al. (1998) Heterodimerization targets a homeodomain protein complex to the nucleus. Proc. Natl. Acad. Sci. U. S. A. 95, 6228–6233 45 Kües, U. et al. (1994) A chimeric homeodomain protein causes self-compatibility and constitutive sexual development in the mushroom Coprinus cinereus. EMBO J. 13, 4054–4059 46 Hartmann, H. et al. (1996) The pheromone response factor coordinates filamentous growth and pathogenicity in Ustilago maydis. EMBO J. 15, 1632–1641

47 Urban, M., Kahmann, R. and Bolker, M. (1996) Identification of the pheromone response element in Ustilago maydis. Mol. Gen. Genet. 251, 31–37 48 Inada, K. et al. (2001) The clp1 gene of the mushroom Coprinus cinereus is essential for A-regulated sexual development. Genetics 157, 133–140 49 Murata, Y. et al. (1998) Molecular analysis of pcc1, a gene that leads to A-regulated sexual morphogenesis in Coprinus cinereus. Genetics 149, 1753–1761

Natural selection and the evolution of mtDNA-encoded peptides: evidence for intergenomic co-adaptation Pierre U. Blier, France Dufresne and Ronald S. Burton Mitochondrial DNA (mtDNA) variation is an important tool for the investigation of the population genetics of animal species. Recently, recognition of the role of mtDNA mutations in human disease has spurred increasing interest in the function and evolution of mtDNA and the 13 polypeptides it encodes. These proteins interact with a large number of peptides encoded in the nucleus to form the mitochondrial electron transport system (ETS). As the ETS is the primary energy generation system in aerobic metazoans, natural selection would be expected to favor mutations that enhance ETS function. Such mutations could occur in either the mitochondrial or nuclear genes encoding ETS proteins and would lead to positive intergenomic interactions, or co-adaptation. Direct evidence for intergenomic co-adaptation comes from functional studies of systems where nuclear–mitochondrial DNA combinations vary naturally or can be manipulated experimentally.

P.U. Blier F. Dufresne Dept de Biologie, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski, Quebec, Canada G5L 3A1. *e-mail: Pierre_Blier @uqar.qc.ca R.S. Burton Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-0202, USA.

Mitochondrial DNA has been used extensively in studies of population genetic structure and phylogenetic relationships among animals (but not plants1,2). Two properties of mtDNA make it particularly useful for such studies: high rates of nucleotide substitution compared with nuclear DNA (nuDNA)3,4, and maternal inheritance that is not subject to recombination5 (but also see Ref. 6). Although evolutionary and population genetic studies often assume that mtDNA undergoes NEUTRAL EVOLUTION (see Glossary) or nearly neutral evolution7,8, the important roles of all 13 mtDNAencoded peptides in cellular ATP production suggest that mtDNA variation could often have significant metabolic and fitness consequences. Indeed, the functional interactions between mitochondrial proteins encoded by mtDNA and nuDNA might result in strong selection for positive intergenomic interactions, or CO-ADAPTATION. One potential problem for the co-adaptation of the nuclear and mitochondrial genomes is the different substitution

rates of these genomes3,4. Peptides encoded by the mtDNA show a higher rate of substitution than their co-evolving, nuDNA-encoded counterparts, suggesting that co-adaptation will be driven by the mitochondrial genome. Two approaches can be employed when attempting to determine the role of natural selection on the evolution of mtDNA-encoded proteins. Either one can attempt to extract information from patterns of DNA sequence variation, or one can seek direct experimental evidence for functional differences among mtDNA variants. With regard to the former, evolutionary models provide testable predictions concerning the patterns of DNA sequence variation within and between species in the absence of selection (i.e. based on the neutral theory of molecular evolution). In fact, predictions derived from the neutral theory have been repeatedly rejected in analyses of animal mtDNA9–13. These studies reveal a striking pattern: there is a consistent excess of intraspecies amino acid polymorphism relative to interspecies amino acid divergence. Although some alternative explanations cannot be eliminated, a leading hypothesis is that the polymorphisms represent the accumulation of slightly deleterious mutations on the nonrecombining mtDNA molecule as GENETIC DRIFT prevents weak PURIFYING SELECTION from eliminating the weakly deleterious NONSYNONYMOUS SUBSTITUTIONS14. However, in the absence of recombination, such an underlying mechanism would be expected to affect all coding regions of the mtDNA equally15. Ballard16,17 obtained numerous complete mtDNA sequences from the four species in the Drosophila melanogaster group

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Glossary Co-adaptation: Selection by which harmoniously interacting genes accumulate in the gene pool of a population. Selective forces acting on one of the genes will also affect its interacting partners. Ectothermic: Organisms having a body temperature determined primarily by the temperature of the environment. Those organisms that maintain a body temperature independent of the temperature of the environment are endothermic. Introgression: The spread of genes of one species into the gene pool of another by hybridization and back-crossing. Neutral evolution: A neutral mutation is one that has no adaptive significance to the organism or is without a phenotypic effect. Such changes are therefore not subjected to selective forces. The theory of neutral evolution states that, at the molecular level, the vast majority of changes are neutral or nearly so and that evolution is primarily determined by random genetic drift, rather than by natural selection. Nonsynonymous substitutions (missense): A substitution that alters a codon and causes a change in amino acid residue. The number of substitutions per nonsynonymous site for any pair of sequences is Ka. Purifying selection (negative): A selection regime resulting in the removal of deleterious substitutions from the population. Random genetic drift: Random changes in a gene population over time. These fluctuations can lead to fixation or extinction of an allele without regard to their adaptive value. Sympatric: Population or taxa that occur together in the same geographical area. Synonymous substitutions (silent): A nucleotide substitution resulting in a codon specifying the same amino acid as before (e.g. AAA to AAG, both code for lysine). The number of substitutions per synonymous site for any pair of sequences is Ks.

(D. melanogaster, Drosophila simulans, Drosophila mauritania, Drosophila sechellia and Drosophila yakuba) and applied a sliding-window analysis to ascertain whether patterns of substitutions are uniform across the length of the mtDNA. The results show conclusively that different coding regions have different patterns of substitution, suggesting that different genes have experienced different selective pressures. For example, the ratio of the rate for SYNONYMOUS versus nonsynonymous substitution (Ks/Ka) is highest for COI, COII, COIII, ND3, cytochrome b, ND4L and ND1, lowest for ND2, ND5 and ND6 and intermediate for ATPase 8, ATPase 6 and ND4 (see Fig. 1 for summary of mitochondrial proteins). In addition, Drosophila simulans lineages have reduced polymorphism, a result consistent with the occurrence of ‘selective sweeps’ (where selection favoring a particular substitution leads to fixation of a single haplotype, thereby eliminating polymorphisms within the lineage). Interestingly, such purging of polymorphism could also be explained by genderspecific impairment of reproduction by the bacterial endosymbiont Wolbachia17. Although sequence analyses implicate a role for natural selection in mtDNA evolution – which is immediately apparent when considering the role of mtDNA mutations in human disease18,19 – they cannot typically predict the phenotypic effects of specific substitutions. Computational methods were recently employed to predict cytochrome c– cytochrome c oxidase interactions (because three-dimensional structures are known for both proteins)20, but even analysis of this pairwise interaction is an extreme simplification of the intracellular environment. Hence, assessing the physiological and fitness consequences of most http://tig.trends.com

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amino acid substitutions in mitochondrial proteins remains an empirical endeavor. Here we discuss a variety of approaches to assess the role of natural selection on the evolution of mtDNA-encoded proteins and the co-adaptation of mitochondrial and nuclear genomes (Table 1). Mitochondrial metabolism and the evolution of mtDNA-encoded peptides

In vertebrates, the mitochondrial genome contains 13 protein-coding genes, genes specifying two mitochondrial rRNAs and 22 mitochondria-specific tRNAs, and genes regulating transcription and replication (the D loop) (Fig. 1). The 13 peptides participate directly in electron transport and proton pumping (block III, Fig. 2) and ADP phosphorylation (block IV, Fig. 2). Because strict regulation of the ETS is required to prevent accumulation of harmful superoxide radicals, selective forces on mtDNA might involve not only the well-recognized problem of energy supply, but also the necessity for maintenance of appropriate cellular redox potentials21. Recent studies suggest that numerous proteins containing mtDNA-encoded peptides participate not only in translocation of protons and electrons, but also in the tight regulation of mitochondrial respiration (namely complexes I, III and IV; Refs 22–29). In assessing the functional significance of evolutionary changes in proteins encoded by the mtDNA, we can draw on the widely accepted principle that the rate of amino acid substitution within a protein is determined by the stringency of structural and functional constraints30. Examining hominoid primate COII, an mtDNA-encoded subunit of complex IV, Templeton11 found that amino acid substitutions in the cytosolic portion of the peptide were deleterious, whereas such substitutions in the transmembrane portion were neutral. From a phylogenetic analysis of the entire protein-coding portion of the mitochondrial genomes of 19 taxa, Naylor and Brown31 found a strong hydrophobicity constraint for sites encoding aliphatic residues. Although it would be simplistic to assume that all amino acid substitutions actually alter protein function, a broad array of protein structural and functional requirements dictate the range of acceptable amino acid substitutions. For protein-coding regions of the mammalian mitochondrial genome, nonsynonymous substitution rates vary greatly among genes, whereas synonymous rates are more uniform4. COX subunits are the most conserved, and NADH oxidoreductase (ND5 and ND6) and ATP synthase (ATP6 and ATP8) are more variable4. Similar patterns were observed in ECTOTHERMIC vertebrates32. The broad taxonomic generality of these patterns of amino acid substitution rates suggest that structural and/or functional constraints are gene specific rather than taxon

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Fig. 1. Function of mtDNA-encoded proteins in mitochondrial metabolism. (a) Mitochondrial DNA encodes 11 peptide constituents of the electron transport system (ETS) and two peptides of the ATPase complex. It also has genes specifying two mitochondrial rRNAs and 22 mitochondria-specific tRNAs, and genes regulating transcription and replication (D loop). (b) Seven mtDNA-encoded peptides (ND1–ND6) are subunits of NADH–ubiquinone oxidoreductase (complex I), which participates in the translocation of electrons from NADH to ubiquinone (UQ) and in pumping protons from the matrix to the intermembrane space. Of the 11 subunits comprising ubiquinol–cytochrome c oxidoreductase (complex III), only cytochrome b is encoded by mtDNA. Complex III catalyzes the oxidation of ubiquinol, the reduction of the cytochrome c and the proton translocation. The three largest subunits (COI, II and III) of cytochrome c oxidase (COX; complex IV) are also encoded in the mtDNA. COX is the last enzymatic complex of the ETS, catalyzing the transfer of electrons from reduced cytochrome c to molecular oxygen. Mammalian COX is formed of 13 subunits; the three mtDNA-encoded peptides form the functional core of the enzyme, containing the redox centres for electron transfer and proton pumping. Finally, ATPase6 and ATPase8 are mtDNA-encoded components of the ATP synthase complex (complex V), which translocates protons from the intermembrane space to the matrix and phosphorylates ADP. ATPase6 and ATPase8 are part of the transmembrane F0 subunit of ATP synthase, which translocates protons across the inner mitochondrial membrane. Green, the mtDNA-encoded subunits (ND1–ND6); yellow, nuclear DNA-encoded subunits of complex I and the ubiquinone; purple, the mtDNA-encoded subunit of complex III; orange, mtDNA-encoded peptides of complex IV; brown, subunits of complex V encoded by the mitochondrial genome; gray, the nuclear DNA-encoded peptides of complexes II, III, IV and V.

specific. For example, low substitution rates in the mitochondrial COX subunits suggest that there are tight constraints on amino acid composition across the peptides. Such constraints arise from interactions among the COX subunits themselves (encoded by either mtDNA or nuDNA), interactions with other ETS proteins and interactions with other structural components of the mitochondria (e.g. mitochondrial membranes). Such interactions might underlie observed correlations in the rates of amino acid substitutions in different mtDNA genes33. In this context, highly conserved mtDNA genes that encode peptides with the most constrained functions in mitochondrial metabolism (cytochrome b, COI, COII and COIII) might drive the evolution of many interacting peptides in the ETS (encoded by either mtDNA or nuDNA). Because the regulation of mitochondrial respiration is greatly affected by environmental conditions34,35, we would expect the kinetic properties of cytochrome c oxidase to show http://tig.trends.com

evolutionary adaptation to the thermal habitat of the organism. Also, we might expect a correlated evolutionary adaptation of cytochrome c to that same thermal environment. In fact, considering the broad range of environmental conditions where organisms occur, it is likely that at least part of the observed mtDNA divergence among populations and species is associated with enzyme adaptations to particular environments. Given the functional importance of proteins encoded by the mtDNA, selection for optimal catalytic capacity and regulatory efficiency of the ETS under diverse environmental conditions should lead to detectable patterns of adaptation. Co-adaptation of nuclear and mitochondrial genomes

The extensive and intimate associations between mtDNA-encoded proteins and nuclear-encoded proteins in the ETS are apparent in Fig. 1. As discussed above, to maintain physiological function, natural selection will continually favor evolutionary co-adaptation of interacting proteins encoded by the nuclear and mitochondrial genomes. Such a scenario could lead to environment-specific correlations of functional properties. For example, cytochrome c, encoded by a nuclear gene, transports electrons between complex III (cytochrome bc1) and complex IV (cytochrome c oxidase). This function requires cytochrome c to dock with these two protein complexes; electron transfer then involves formation of protein complexes guided by specific electrostatic interactions36. Hence, these proteins should be highly co-adapted. In fact, rates of amino acid replacement for the mtDNA-encoded COX subunit II (COII) and the COX substrate, cytochrome c, are highly correlated among mammalian lineages37. Furthermore, there is direct evidence that these correlations reflect a functional basis: Osheroff et al.38

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Table 1. Different methods to detect natural selection on mtDNA Methodology

Advantages

Disadvantages

Statistical analyses of mtDNA sequences

Powerful analytical tools to detect selection

Little power in detecting functional significance of specific mtDNA substitutions

Injection of foreign DNA into cells without mtDNA

Can study how highly divergent combinations of mtDNA and nuclear backgrounds interact together

Restricted to unnatural cell culture environments. Cannot assess whole organism effects

Laboratory crosses (population or species) to introduce mtDNA in a new nuclear background

Crosses can be performed under different environmental conditions to tease apart the relative roles of environmental and genetic factors in cytonuclear interactions

Difficult for species with long generation time and when species and/or populations are too divergent to produce viable hybrids

Natural introgressed populations

Study natural model systems

Limited by their natural occurrence

found that primate cytochrome c (encoded by nuDNA) showed a higher reaction rate with primate than with non-primate COX in in vitro assays. Although the in vitro assay approach provides a direct assessment of co-adaptation between ETS components, interpretation of such results in terms of their effects on total ETS activity might not be straightforward for two reasons: (1) relationships between enzyme activity at any one step of a pathway and total pathway flux is typically nonlinear, and (2) the extent to which assay conditions in vitro reflect in vivo activities is difficult to assess. Studies frequently suggest that individual ETS enzyme activities could be as much as fourfold more than required to sustain endogenous respiratory rates (Ref. 39, and references therein). However, Villani et al.39 recently found that reserve capacity for COX might be low in human cell lines. In any case, this problem suggests that alternative approaches, employing intact respiring cells, could be more fruitful in elucidating patterns of intergenomic co-adaptation. To address the issue of functional co-adaptation between mtDNA- and nuDNA-encoded proteins, investigations require analyses of cells or organisms containing control and experimental combinations of mtDNA and nuDNA. There are three potential sources for such materials: (1) direct manipulation of cells or embryos in culture (e.g. nuclear transplantation, repopulating mitochondria-less cell lines with foreign mitochondria, fusion of enucleated cells with mitochondria-less cells); (2) repeated back-crossing of animals in the laboratory, thereby INTROGRESSING foreign nuclear genes into a particular mtDNA background; (3) natural hybrids where unusual mtDNA–nuDNA combinations occur. Direct manipulation provides the greatest control over the genomic composition of the cells, but does not readily provide insight into the potential role of environmental factors as selective agents in mtDNA evolution. Repeated back-crossing allows analyses at both cellular and organismal levels, http://tig.trends.com

but genetic composition is less controlled (although the introgression scheme predicts that the entire nuclear genome can be replaced, strong selection favoring certain mtDNA–nuDNA combinations will perturb this). Finally, natural hybrids have the advantage that some inferences of environmental effects can be possible from the distribution of hybrids in nature, but such systems typically lack the control and replication of the former two approaches. Liepins and Hennen40 initiated direct manipulation approaches to understanding nuclear–mitochondrial DNA interactions. They transplanted single nuclei from Rana pipiens blastula cells into enucleated eggs of Rana palustris and found that normal COX activity required at least a haploid genomic complement derived from the same species as the mitochondrial genome. Over the past decade, techniques for repopulation of mitochondria-less cells with mitochondria from different sources have become important methodologies for studying mtDNA–nuDNA interactions. King and Attardi41 found evidence for cytonuclear interaction by repopulating human cell lines experimentally depleted of mitochondria and showing that COX activity varied with the source of exogenous mitochondria reintroduced to a common host cell line. Yamaoka et al.42 found that repopulation of mouse mtDNA-less cells with rat mtDNA restored mitochondrial translation but not respiratory function. Repopulation with mtDNA from a different mouse species restored both translation and respiration. These results suggest that co-adaptation of nuclear and mitochondrial proteins at the level of respiratory function is more stringent than that involved in mtDNA replication, transcription and translation. Together these studies indicate that COX function (or more inclusively, ETS function) is strongly dependent on co-adapted function of mitochondrial genes with nuclear genes (including, but not limited to, the nuDNA-encoded ETS-enzyme subunits and cytochrome c). It is important to note, however, that these studies typically address

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Fig. 2. Mitochondrial metabolism. Mitochondrial ATP production is accomplished by four interrelated sets (or functional blocks) of reactions occurring in different compartments within the organelle (for review see Ref. 27). The first functional block (metabolite supplier) includes the transport of the metabolites from the cytosol to the mitochondrial matrix and the integration of these metabolites into the citrate cycle. These metabolites enter the citrate cycle (block II) either as acetyl-CoA (mainly from pyruvate and fatty acids) or directly as citrate cycle intermediates. The citrate cycle enzymes decarboxylate and oxidize the metabolites through the reduction of NAD, resulting in the production of NADH. Electrons of NADH then pass through the third functional unit (the electron transport system or ETS) to molecular oxygen. Electron transport through the ETS induces the proton transfer from inside mitochondria to the intermembrane space. This process generates the proton motive force that can be used by the last functional block (ATP synthase and the ADP/ATP carrier, located on the inner membrane of mitochondria) to produce and transport ATP. The enzymes that compose the four functional units have to be closely integrated to ensure that the steady state flux of ATP can be adjusted and maintained according to the specific needs of the cell.

co-adaptation at the species level and do not take into account environmental differences among the species compared. Does such co-adaptation occur within genetically divergent, conspecific populations? What are the relative roles of the intrinsic genetic environment (i.e. the nuclear genetic background) and the extrinsic physical environment (e.g. ambient temperature regime) in the evolution of mtDNA-encoded proteins? Recent work suggests that studies of natural model systems could provide some insights. Nuclear and mitochondrial co-adaptation in Tigriopus californicus

The intertidal copepod Tigriopus californicus provides a valuable model system for the study of co-adaptation between nuclear and mitochondrial genomes because it has extremely high levels of interpopulation genetic divergence43. Levels of mtDNA sequence divergence among conspecific populations are among the highest intraspecific values yet reported; inferred amino acid divergence between populations at the COII locus is nearly 8% (Ref. 43). On the basis of the COII and partial COI sequences, over 20 diagnostic amino acid substitutions were found between T. californicus http://tig.trends.com

populations from Santa Cruz and Los Angeles. Can such highly divergent mitochondrial gene products function normally when placed in foreign nuclear genetic backgrounds, or has there been significant intergenomic co-adaptation? A direct method of testing for interactions between nuclear and cytoplasmic genetic elements (e.g. mitochondrial genes) employs laboratory crosses to substitute cytoplasm (including mitochondria) from one population onto the nuclear background of a second population. The method takes advantage of the differences in modes of inheritance of nuclear versus mitochondrial genomes: although nuclear DNA is inherited from both parents, mtDNA is typically maternally inherited. Hence, an F1 interpopulation hybrid between a female from population A and a male from population B will have a 50:50 mix of A and B nuclear genes, but only population A mtDNA. If the F1 organisms are then back-crossed to the paternal line (B), the resulting progeny will still have only A mitochondria, but will have 75% B nuclear genes. Continued back-crossing to the paternal population reduces the expected fraction of A nuclear genes by half each generation. Although the intended focus is the interaction between nuclear and mitochondrial genomes, it is also possible that other cytoplasmic factors (such as the Wolbachia infections observed in Drosophila17) can influence the physiology of the introgressed hybrids. Several studies have employed this type of approach to construct cytonuclear hybrids in Drosophila to test for intergenomic co-adaptation in fitness (e.g. Refs 44,45). The introgression approach can also be employed to study specific functional interactions. Edmands and Burton46 found a reproducible pattern of decreasing COX activity as mitochondria from a Los Angeles T. californicus population were moved to a purer and purer nuclear background from a distant Santa Cruz population, giving strong evidence for intergenomic co-adaptation. If co-adaptation within each population is a consequence of adaptation to local environmental conditions, one would expect a predicable pattern of hybrid breakdown; for example, northern populations would show stronger breakdown when crossed with southern populations than when crossed with another northern population. However, if co-adaptation arises during the course of stochastic population differentiation, hybrid breakdown would be population specific and unpredictable. Although not specifically designed to address this issue, the data in Edmands and Burton46 suggest that patterns of COX activity in introgressed lines are largely unpredictable and therefore favor the latter hypothesis. Although examination of COX activity in interpopulation hybrids is a direct functional approach to intergenomic co-adaptation, a number of caveats need to be considered. In addition to the usual issues of whether in vitro assays provide realistic

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assessment of functional differences between enzymes, an obvious problem concerns the fact that the assay of COX function required the use commercially available cytochrome c (derived from horse) as substrate46. Intergenomic co-adaptation might well involve the T. californicus cytochrome c gene, which is known to be differentiated among T. californicus populations47, but this possibility is not testable with the assay employed. Mitochondrial DNA introgression in natural populations of brook char

Acknowledgements Supported by an NSERC grant to P.U. Blier, an NSERC postdoctoral fellowship to F. Dufresne and a NSF grant to R.S. Burton.

Because mitochondrial metabolism is highly sensitive to environmental conditions, particularly temperature variations34,35, one can speculate that the mitochondrial genome of animals evolved in response to environmental or body temperature to maintain optimal function of mitochondria. To address this issue, we need to determine whether proteins encoded in different mitochondrial haplotypes have different functional properties and thermal sensitivities. But how can we dissect apart the effects of the mitochondrial and nuclear genomes on such functional properties? A population of brook char (Salvelinus fontinalis) from the north shore of the St Lawrence river has been found that possesses the mitochondrial genome of Arctic char (Salvelinus alpinus) exclusively, with no sign of nuclear introgression48. This introgression could be highly significant because Arctic char is a cold-adapted species, whereas brook char is more temperate. The introgressed population is found in the northern distribution range of brook char. A recent study on this population and a SYMPATRIC brook char population with no mitochondrial introgression revealed significant differences in the functional properties of mitochondria at different temperatures (H. Glémet, PhD thesis, Institut National de la Recherche Scientifique, Quebec, 1997), as well as higher activity of cytochrome c oxidase in mitochondria from introgressed population. Complete sequencing of the mitochondrial genome of Arctic and brook char (Doiron, S. et al., unpublished) revealed 47 amino acid substitutions between the species with only one

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in the cytochrome oxidase complex and 45 in the NADH oxidoreductase complex. Hence, the activity differences could be a result of (1) differential expression of introgressed mtDNA haplotypes; (2) differences in functional properties of enzymes encoded by the two different mtDNA haplotypes; (3) undetected differences in nuclear genomes, such as in the nuclear subunits of cytochrome oxidase; and (4) maternally inherited factors (sex chromosome, transposable element, endosymbionts, etc.). Distinguishing among these possibilities will require reciprocal introgression experiments, which could be done in captivity or through the injection of S. fontinalis mtDNA into S. alpinus eggs and the reciprocal. With all the above factors controlled, the effects of the DNA substitution on enzyme properties could be fully resolved. Injection of mtDNA of cold-adapted fish species in the eggs of warm-adapted fish would be especially insightful in examining the functional properties of mitochondrial haplotypes. Concluding remarks

Although statistical analyses of DNA sequences suggest that evolution of mitochondrial proteins is not always neutral, the functional significance of variation in these proteins has not been extensively explored. Such work requires biochemical and physiological studies of specific combinations of nuclear and mitochondrial genomes that can be produced experimentally using cell (or embryo) culture or controlled introgressive crosses in the lab. Available data suggest that the driving force behind evolutionary change is not always adaptation to the external environment. Subtle genetic changes introduced into a population’s gene pool by mutation face constant selection favoring the maintenance of functional interactions among proteins encoded by nuclear and mitochondrial genes. Hence, natural selection on mtDNA-encoded peptides simultaneously results both in adaptation to the environment and co-adaptation to the nuclear genome. Recent work suggests that both processes have profound effects on the evolution of mitochondrial proteins.

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Speciation This month’s special issue of Trends in Ecology and Evolution Guest Editor: N.H. Barton The mind of the species problem J. Hey Theory and speciation M. Turelli, N.H. Barton and J.A. Coyne The genetics of species differences A. Orr Chromosomal rearrangements and speciation L.H. Rieseberg Gene trees and species trees are not the same R.A Nichols

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Sexual selection and speciation T. Panhuis, R. Butlin, M. Zuk and T. Treganza Ecology and the origin of species D. Schluter Sympatric speciation in animals S. Via Phylogenetics and speciation T. Barraclough and S. Nee Scale and species numbers C. Godfray and J. Lawton Speciation in the fossil record M.J. Benton and P.N. Pearson