* Department of Biology, University of North Carolina, Chapel Hill
Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla
Correspondence: E-mail: willett4{at}email.unc.edu.
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Abstract |
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Key Words: mitochondrial DNA evolution ubiquinol-cytochrome c reductase cytochrome c1 rieske iron-sulfur protein cytochrome b Tigriopus californicus
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Introduction |
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In mammals, several lines of evidence suggest that the interactions involving the nuclear-encoded soluble cytochrome c (CYC) and cytochrome c oxidase (complex IV) have evolved rapidly in higher primates: (1) In vitro studies with heterospecific combinations of CYC and complex IV enzymes across these groups show reduced enzyme activity (Osheroff et al. 1983). Additionally, analyses of human/orangutan cell lines containing portions of both species nuclear genomes and orangutan mtDNA show reduced complex IV enzyme activity consistent with significant mtDNA/nuclear coadaptation (Barrientos et al. 2000). (2) Sequence analyses show that rates of amino acid substitution increase in simian primates in mtDNA-encoded subunits of complex IV (COI and COII) and in seven other nuclear-encoded subunits in concert with increases in substitution in CYC (Adkins, Honeycutt, and Disotell 1996; Wu et al. 2000; Schmidt, Goodman, and Grossman 2002). (3) At the structural level, rates of amino acid evolution are increased at interaction points of proteins for mtDNA-encoded proteins but not nuclear-encoded proteins (Schmidt et al. 2001). (4) Analyses of mtDNA-encoded cytochrome b (CYTB), nuclear-encoded cytochrome c1 (CYC1), and rieske iron-sulfur protein (RISP) from ubiquinol-cytochrome c reductase (complex III) also show accelerated substitution in humans/simian primates correlated with the changes in complex IV and CYC and may argue for extensive coadaptation of these two ETS complexes and CYC in higher primates (Andrews, Jermiin, and Easteal 1998; Grossman et al. 2001). Given the role of CYC as a carrier of electrons between complex III and complex IV, this coadaptation has an obvious potential functional basis, although the evolutionary forces driving this divergence in higher primates are not known.
Because of high levels of interpopulation genetic divergence, the intertidal copepod Tigriopus californicus presents an excellent model system for the study of evolutionary coadaptation between nuclear and mitochondrial genes. Allopatric populations of T. californicus along the western coast of North America show extreme divergence in mtDNA genes, sometimes over relatively short physical distances. Two mtDNA-encoded subunits of complex IV, COI, and COII, display divergences above 20% for nucleotide substitutions and up to 15% for amino acid divergence (Burton and Lee 1994; Burton 1998; Burton, Rawson, and Edmands 1999). The nuclear-encoded protein CYC also shows substantial amino acid divergence between populations, particularly given the generally high level of conservation of this protein (Rawson, Brazeau, and Burton 2000). Despite high interpopulation divergence, within-population polymorphism is generally low; the resulting high FST values suggest highly restricted gene flow. Clearly these populations have evolved independently for long periods of time and could be considered separate species under some species definitions (i.e., phylogenetic species concept); however, based on their reproductive compatibility, we will continue to refer to them as conspecific populations (also see Burton [1998]).
Several studies suggest that nuclear/mitochondrial coadaptation is evolving between genetically isolated T. californicus populations. Edmands and Burton (1999) used repeated backcrossing to study the effect of cytonuclear interactions on cytochrome c oxidase (complex IV) enzyme activity in hybrids. Reductions in complex IV activity in some interpopulation crosses were caused at least in part by nuclear/mitochondrial interactions. Rawson and Burton (2002) found direct evidence for functional coadaptation between CYC and complex IV. CYC derived from a San Diego (SD) population yielded significantly higher activity with SD populationderived complex IV than that derived from a Santa Cruz (SC) population, and reciprocally, SC populationderived CYC functioned best with SC populationderived complex IV. Willett and Burton (2001) found extensive differences in CYC genotypic viabilities in F2 hybrids from controlled crosses between populations (estimated from deviations from Mendelian ratios). CYC viability differences were observed in reciprocal crosses between specific populations, suggesting cytoplasmic effects consistent with the inference of nuclear/mitochondrial interactions.
The combination of extensive divergence in mtDNA-encoded genes in T. californicus and fitness/functional evidence for mitochondrial/nuclear coadaptation make this system a unique opportunity to examine molecular evolution of these interacting genes. In this paper, we will first examine the evolution of CYTB (the mtDNA-encoded subunit of complex III) and show that it is diverging rapidly between populations. Two nuclear-encoded subunits of complex III have functional domains containing redox centers, the rieske iron-sulfur protein (RISP), and cytochrome c1 (CYC1). We have obtained nuclear DNA sequences encoding these proteins from T. californicus and examine polymorphism within populations and divergence between populations. We then compare these genes, which function in the ETS, with other nuclear genes that have been obtained from T. californicus. Two major conclusions can be drawn from these results: (1) Proteins closely interacting with mtDNA-encoded proteins in the ETS can show evidence for selection for high rates of evolution (CYC), but not all appear to be evolving rapidly (CYC1 and RISP). (2) The mtDNA in T. californicus appears to have a much higher rate of substitution between populations than has the average nuclear gene for both synonymous and nonsynonymous changes.
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Materials and Methods |
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Identification and Sequencing of Nuclear-Encoded Proteins
We sought to characterize two nuclear-encoded genes from complex III of the ETS that contains the mtDNA-encoded protein CYTB. The T. californicus nuclear genes encoding the proteins CYC1 and RISP were initially identified using a 5' RACE (rapid amplification of cDNA ends) procedure on mRNA isolated from SD copepods. The kit Generacer (Invitrogen, Carlsbad, Calif.) was used with degenerate primers designed to match conserved regions of these two proteins (from alignments of human, Drosophila melanogaster, Saccharomyces cerevisiae, and Caenorhabditis elegans) to amplify from cDNA. Primer used for CYC1 was CYC1.rev (5'-GCNCCNCCNGGRAARTANGGRTT-3') and primer used for RISP was RISP.rev (5'-TARTGNSWNCCRTGRCANGGRCA-3') in conjunction with a Generacer kit primer. Resulting PCR products were gel purified, cloned, and sequenced. The complete mRNA was obtained with 3' RACE using T. californicusspecific primers designed from the new sequence.
Genomic DNA sequences were obtained from the SD population corresponding to the mRNA transcript for RISP and CYC1. PCR products were generated using species-specific primers and these products were sequenced directly. Polymorphism was inferred for sites when two clear peaks of equal intensity were observed in the chromatogram. Sequences for most of the coding region and intervening introns were obtained from multiple individuals from SD, SC, and AB individuals (some 5' end and 3' end codons were not sequenced). CYC sequences were published previously based on sequences obtained from clones (Rawson, Brazeau, and Burton 2000), and polymorphisms in these sequences were verified by examining directly sequenced products from same individuals as described above. Sequences were edited with the program Sequencer version 4.1 (Genecodes, Ann Arbor, Mich.), and alignments were made using ClustalX with some adjustments by hand in large indels in introns. DNAsp version 3.53 (Rozas and Rozas 1999) was used to calculate values, M-K tests, and numbers of fixed silent and replacement differences. PAUP* version 4.0b10 (Swofford 2001) was used to calculate amino acid identities between proteins and for phylogenetic analyses.
We also sought to characterize polymorphism and divergence in nuclear-encoded genes that are not a part of the ETS and not likely to interact closely with mtDNA-encoded proteins. Additional sequences of the gene glutamate dehydrogenase (GDH) were obtained from SD, SC, and AB population copepods. A complete genomic sequence of GDH from SD was recently obtained (Willett and Burton 2003) and a region of 1,320 bp, including a single intron of 71 bp, was sequenced from multiple individuals in each population. Sequences were obtained by direct sequencing from PCR products. Population samples from two other nuclear encoded proteins, 1-pyrroline-5-carboxylase synthase (P5CS) and
1-pyrroline-5-carboxylase reductase (P5CR), were previously published (Willett and Burton 2002). To verify heterozygous sites, sequencing traces were visually inspected for polymorphism as discussed above. Alignments of sequences are available as Supplementary Material online (http://www.mbe.oupjournals.org) and representative sequences from each population have been submitted to GenBank with accession numbers AY344446 to AY344470.
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Results |
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We obtained sequences of CYC1 and RISP from three T. californicus populations, SD, AB, and SC. The intron/exon structure of these genes was determined by comparing mRNA and genomic sequence for SD, and it is largely conserved between populations (fig. 2). A potential exception to this is intron 1 of CYC1 in the AB and SC populations, where the 3' intronending AG in SD has been changed to GG, and the next closest potential splice AG is 5 bp closer to the predicted start codon (this predicted boundary has not been verified by mRNA sequencing from SC and AB copepods). The four small introns in the two genes vary little in size between populations, whereas the two larger introns vary considerably (fig. 2). Based on the current annotations of the A. gambiea and D. melanogaster CYC1 and RISP genes, there appears to be little conservation in intron position among these two Diptera and T. californicus. However, unlike the other introns, the first intron in RISP does occur in approximately the same position in all three taxa (in the unalignable putative mitochondrial signaling peptide).
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Discussion |
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Few other studies have explicitly examined the average level of divergence at synonymous and nonsynonymous sites for comparisons of mtDNA and nuclear genes. Pesole et al. (1999) found 22-fold higher synonymous substitution in complete mtDNA from humans and chimps in comparison with divergence in 20 nuclear genes from both species. Nonsynonymous sites had a threefold higher rate of substitution in mtDNA for comparisons of the same proteins. In an examination of 13 complete animal mtDNA sequences, Lynch and Jarrell (1993) reported that rates of amino acid substitution may be somewhat lower in mitochondrial-encoded proteins than in nuclear-encoded proteins. Comparisons of COI, COII, and CYTB with nuclear genes for D. melanogaster and D. simulans show less than a two-fold increase in synonymous rates but greater than a three-fold decrease in nonsynonymous rates for the mtDNA-encoded genes (Ballard 2000b; Betancourt, Presgraves, and Swanson 2002; C. S. Willett, unpublished results). Both synonymous and nonsynonymous rates of substitution appear to be substantially higher in T. californicus, suggesting a much higher mutation rate for mtDNA than nuclear. Noncoding sequence in large introns is likely to be the least constrained sequence we have analyzed, and comparisons of divergences of noncoding and synonymous sites for the three genes with large introns analyzed (CYC1, RISP, and CYC) show a 1.5-fold to twofold higher rate for noncoding sites, suggesting some constraint on synonymous sites in coding regions of nuclear genes. For comparison of mutation rates between nuclear-encoded and mitochondrial-encoded genes, this constraint could inflate the difference if mitochondrial-encoded genes have less constrained synonymous sites (but only by about twofold).
Given the central importance of the mitochondrial-encoded subunits in the ETS, a high mutation rate of mtDNA in comparison with nuclear DNA in T. californicus can only explain a rapid rate of amino acid replacement if the fixed substitutions behave neutrally. An alternate explanation for increased amino acid divergence in mtDNA-encoded proteins could be environmental selection on the ETS. Is there evidence for selection for amino acid replacement acting on CYTB? A negative Tajima's D statistic or Fu and Li's D test could indicate recovery from a recent selective sweep. Polymorphism for CYTB has nonsignificant negative values for both of these measures in the SD population. Both SC and AB populations have positive Fu and Li's D test values and nonsignificant Tajima's D statistic values. These tests then provide no strong evidence for recent selective sweeps on mtDNA. MK tests can detect repeated fixations driven by selection, but comparisons of polymorphism and divergence across these three populations for CYTB do not show a pattern consistent with positive selection. In fact, for comparisons involving SC, an opposite deviation consistent with excess replacement polymorphism is seen. Therefore, examination of polymorphism and divergence in T. californicus populations lends little support for selection acting on CYTB to fix amino acid changes; instead, there is some evidence for excess slightly deleterious polymorphism, a pattern common in animal mtDNA.
If amino acid substitution in CYTB has not been driven by positive selection, could fixed differences be deleterious? Because of its ephemeral high intertidal habitat, T. californicus experiences dramatic fluctuations in population size (Burton 1997), which could facilitate the stochastic fixation of deleterious mutations. Extinction and recolonization in subdivided populations has been shown to increase the probability of fixation of deleterious mutations (Cherry 2003). Given the close physical and functional contacts of the mitochondrial-encoded and nuclear-encoded subunits of the ETS, compensatory amino acid change (via selection on nuclear-encoded subunits) is a potential consequence of accumulated deleterious substitutions. For two key subunits of complex III, RISP and CYC1, we find little evidence for selection for increased amino acid replacement in MK tests on these two proteins. This contrasts with apparently pervasive coadaptation occurring in primate complex IV proteins where amino acid substitution appears to be accelerated in many subunits of the complex (Adkins, Honeycutt, and Disotell 1996; Andrews, Jermiin, and Easteal 1998; Wu et al. 2000; Schmidt, Goodman, and Grossman 2002). Our finding shows that substantial change in CYTB can occur without numerous compensatory changes in RISP or CYC1. This could imply that the majority of the amino acid changes observed in CYTB between T. californicus populations are functionally neutral, or alternatively, most compensatory change occurs within the same subunit (for example within CYTB).
Mapping the positions of the amino acid changes from T. californicus populations in RISP and CYC1 on vertebrate complex III structures could also provide functional insights. Both differences from the SD sequence for CYC1 are found in the 1' helix, which is thought to be near the interaction site with soluble CYC (Iwata et al. 1998; Zhang et al. 1998) although they are not the specific interacting residues in yeast (Lange and Hunte 2002). They include adjacent sites 68 and 69 (based on the vertebrate CYC1 mature protein positions) and are Val to Ile in SC/AB, and Met to Ile in SC. In RISP, residue 24 (based on vertebrate RISP mature protein position) is in the matrix portion of the protein and is a Lys to Asn change in AB. At position 50 in the transmembrane helix, there is a change from Gly to Ala in SC/AB. None of these residues have been implicated in a specific intermolecular interaction in yeast or vertebrate complex III.
Sequence comparisons provide no evidence for selection acting on RISP or CYC1 in the evolutionary divergence of these two proteins between T. californicus populations; however, there is evidence for selection acting on the soluble CYC protein that transports electrons between complex III and complex IV. This evidence comes from MK tests that suggest an excess of amino acid fixation for comparisons involving the SC population (table 8). Divergences between T. californicus populations for CYC appear high based on the high level of conservation of CYC in animals, but a relative rate comparison with the CYC of prawn does not suggest a dramatic increase in the rate of CYC in the Tigriopus lineage in general (1.2-fold higher). CYTB in contrast shows a twofold to threefold increase along the Tigriopus lineage, suggesting that rates of amino acid substitution in CYC and in CYTB may not be evolving in concert over long periods of time. A more relevant comparison for CYC evolution may be with COII of complex IV rather than CYTB with which CYC has no direct interactions. Like CYTB, COII is highly divergent between T. californicus populations, and hydrophobic portions of this protein (involved in CYC binding) are unexpectedly divergent (Burton, Rawson, and Edmands 1999; Rawson and Burton unpublished results).
Functional and fitness data also suggest selective divergence of CYC between genetically isolated T. californicus populations. Rawson and Burton (2002) showed that the enzymatic activity of complex IV differed when using SD populationderived CYC versus SC populationderived CYC as substrate. Enzymatic activity was consistently higher when complex IV and CYC were derived from the same natural population. Large temperature/enzyme interaction effects were also detected. Other studies show that fitnesses (as determined by deviations from expected Mendelian ratios) of different CYC genotypes vary dramatically in F2 hybrids of AB, SC, and SD populations (Willett and Burton 2001). For AB/SC hybrids, dramatic influences of the rearing environment (including a different temperature regime) were observed; selection on CYC genotype was totally eliminated by a change from one environmental regime to another (Willett and Burton, 2003). The prevalence of environmental influences on CYC fitness and function suggest that local adaptation to temperature environment could be a selective force on CYC and influence its evolution. A model for molecular evolution in this system could involve a high mutation rate on mtDNA, leading to frequent fixation of neutral or slightly deleterious mtDNA mutations perhaps compensated by intramolecular changes. Coadaptation between mtDNA-encoded and nuclear-encoded proteins may then be driven by occasional bouts of adaptation to environmental conditions, leading to the evolution of specific components of the ETS complexes.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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