Cytochrome c Oxidase Sequence Comparisons Suggest an Unusually ...

Letter to the Editor Cytochrome c Oxidase Sequence Comparisons Suggest an Unusually Mitochondrial DNA Evolution in Mytilrcs (Mollusca: Bivalvia)

High Rate of

Walter R. Hoeh,* Donald T. Stewart,* Brent W. Sutherland,* and Elefherios Zouros*+ *Department of Biology, Dalhousie University; and j-Department of Biology, University of Crete and Institute of Marine Biology of Crete

Blue mussels of the genus Mytilus have an unusual mode of mitochondrial DNA (mtDNA) transmission: males receive mtDNA from both parents and transmit their paternal mtDNA to their sons; females receive mtDNA only from their mother (Skibinski, Gallagher, and Beynon 1994~2, 1994b; Zouros et al. 1994~2, 1994b). This mode of mtDNA transmission has been termed “doubly uniparental inheritance” (DUI; Zouros et al. 1994a). It is known from the work of Hoffmann, Boore, and Brown (1992) and Boore and Brown (1994) that the gene order of Mytilus mtDNA is much more different from that of vertebrates, insects, or the chiton Katharina (itself a mollusk) than these other mtDNA gene orders are from each other. Here we report another exceptional feature of the Mytilus mtDNA genome, specifically, an accelerated rate of sequence divergence. Based on a comparison of nucleotide and amino acid sequences, we demonstrate that the Mytilus mtDNA is the most divergent metazoan mtDNA known today and that it evolves at a higher rate than is typical for animals. We suggest that the fast rate of mtDNA evolution in Mytilus is causally related to DUI. Nucleotide sequences of both the “female” (F) and “male” (M) mitotypes of Mytilus edulis for cytochrome c oxidase subunits I (COI, 660 bp) and III (COIII, 762 bp) were obtained as described elsewhere (Folmer et al. 1994; Stewart et al. 1995). Ten additional sequences representing major metazoan lineages were either obtained from Genbank or communicated to us. Estimates of sequence divergence and the construction of neighborjoining trees were carried out using MEGA version 1.02 (Kumar, Tamura, and Nei 1993) and were based on the combined COI/COIII data set. Pairwise proportional distances for first and second nucleotide positions and for amino acids are shown in table 1. Given the taxonomic distance of the compared taxa, nucleotide substitutions at third positions were saturated and were excluded from nucleotide distance calculations. Key words: Mytilus, doubly uniparental lecular evolution, phylogeny.

inheritance,

rates of mo-

Address for correspondence and reprints: W. R. Hoeh, Department of Zoology, Miami University, Oxford, Ohio 45056. E-mail: RHOEH@ AC.DAL.CA. Mol.

Biol. Evol. 13(2):418421. 1996 0 1996 by the Society for Molecular Biology

418

and Evolution.

ISSN: 0737-4038

The neighbor-joining tree (fig. 1) obtained from the nucleotide data of table 1 has two unusual features. The first is that Ascaris is unexpectedly grouped with Myti Zus. The grouping of Ascaris with Mytilus is unexpectec on the basis of previous studies of metazoan phylogen: (e.g., Eernisse, Albert, and Anderson 1992; Conwa; Morris 1993; Philippe, Chenuil, and Adoutte 1994). Thi grouping is almost certainly due to the long branch at traction phenomenon (e.g., Kuhner and Felsensteil 1994). High rates of sequence evolution have previousl! been proposed as an explanation for seemingly anoma lous phylogenetic placements of nematodes (e.g., Ced ergren et al. 1988; Philippe, Chenuil, and Adoutte 1994) The other relationships on the tree are consistent wit1 traditional views of metazoan phylogeny and do no change when Ascaris is removed from the analysis. The second and most important feature of the tree is that the Mytilus sequences were the most divergent The long branch length of the Mytilus lineage was ob served regardless of which option in MEGA for esti mating distances was used. We have used Drosophila a, a reference in relative rate tests between Mytilus and the two other mollusks, the chiton Kutharina and the snai Albinaria. The difference in nucleotide distances be tween Mytilus and Drosophila and Albinaria and Dro sophilu was KMMD- K,, = 0.123 + 0.016, and tha between Mytilus and Drosophila and Katharina ant Drosophila K,, - KKD = 0.155 2 0.015, both signif icantly different from zero at the 1% level. This resul was independent of whether the F or the M sequence o Mytilus was used. Similar results were obtained if amine acid distances were used or if Drosophila was replacec with another outgroup species. Clearly, Mytilus has ex perienced an accelerated rate of mtDNA sequence di vergence compared to the other molluscan taxa. Comparison of nucleotide or amino acid pairwist distances of Mytilus with those of Ascaris (bold in tabh 1) shows that Mytilus was marginally but consistently more divergent than Ascaris. Specifically, in all nine comparisons the Ascaris distances were smaller than tht F or M type of Mytilus, with the exception of the M nucleotide sequence in the comparison involving Home and the F amino acid sequence in the comparison involving Parucentrotus. In no case, however, was the Ascm-is distance significantly smaller than the corresponding Mytilus distance. It is interesting that this higher

Letter to the Editor

419

Table 1 Pair-wise Proportional Distances from Nucleotides at First and Second Codon Positions (Above Diagonal) and from Inferred Amino Acids (Below Diagonal) in the Combined COYCOIII Data Set (SE)

Myths

edulis F . . . . . . .

Myths

edulis M . . . . . .

Ascaris suum Albinaria

.. .......

turrita

.... ...

Katharina tunicata

.....

Drosophila

yakuba

... ..

Anopheles

gambiae

....

Artemia franciscana

....

Paracentrotus lividus Xenopus laevis

...

. .......

Gallus gallus

.........

Homo sapiens

.........

Mytilus edulis F

Mytilus edulis M

-

0.068 (0.009) -

0.073 (0.013) 0.552 (0.025) 0.487 (0.025) 0.465 (0.025) 0.470 (0.025) 0.470 (0.025) 0.496 (0.025) 0.487 (0.025) 0.479 (0.025) 0.479 (0.025) 0.492 (0.025)

0.560 (0.025) 0.484 (0.025) 0.465 (0.025) 0.467 (0.025) 0.477 (0.025) 0.489 (0.025) 0.501 (0.025) 0.487 (0.025) 0.477 (0.025) 0.496 (0.025)

Ascaris suum 0.384 (0.017) 0.394 (0.017) 0.440 (0.025) 0.455 (0.025) 0.443 (0.025) 0.457 (0.025) 0.467 (0.025) 0.492 (0.025) 0.457 (0.025) 0.450 (0.025) 0.472 (0.025)

Albinaria turrita

Katharina tunicata

Drosophila yakuba

Anopheles gambiae

Artemia franciscana

Paracentrotus lividus

0.347 (0.016) 0.343 (0.016) 0.308 (0.016) -

0.320 (0.016) 0.324 (0.016) 0.305 (0.016) 0.233 (0.014) -

0.337 (0.016) 0.341 (0.016) 0.316 (0.016) 0.214 (0.014) 0.182 (0.013) -

0.344 (0.016) 0.345 (0.016) 0.324 (0.016) 0.220 (0.014) 0.194 (0.013) 0.063 (0.008) -

0.362 (0.016) 0.353 (0.016) 0.341 (0.016) 0.267 (0.015) 0.226 (0.014) 0.171 (0.013) 0.166 (0.013) -

0.339 (0.016) 0.347 (0.016) 0.338 (0.016) 0.253 (0.015) 0.219 (0.014) 0.202 (0.014) 0.203 (0.014) 0.210 (0.014) -

0.311 (0.023) 0.302 (0.023) 0.309 (0.023) 0.350 (0.024) 0.358 (0.024) 0.331 (0.023) 0.338 (0.023) 0.329 (0.023)

0.277 (0.022) 0.287 (0.022) 0.302 (0.023) 0.336 (0.023) 0.263 (0.022) 0.280 (0.022) 0.290 (0.022)

0.093 (0.014) 0.241 (0.021) 0.324 (0.023) 0.268 (0.022) 0.280 (0.022) 0.285 (0.022)

0.236 (0.021) 0.329 (0.023) 0.25 1 (0.021) 0.270 (0.022) 0.273 (0.022)

0.346 (0.024) 0.311 (0.023) 0.304 (0.023) 0.311 (0.023)

0.294 (0.023) 0.304 (0.023) 0.321 (0.023)

Xenopus laevis

Gallus gallus

Homo sapiens

0.349 (0.016) 0.345 (0.016) 0.321 (0.016) 0.265 (0.015) 0.205 (0.014) 0.199 (0.014) 0.191 (0.013) 0.219 (0.014) 0.189 (0.013) -

0.351 (0.016) 0.352 (0.016) 0.333 (0.016) 0.270 (0.015) 0.225 (0.014) 0.218 (0.014) 0.213 (0.014) 0.213 (0.014) 0.196 (0.013) 0.101 (0.010) -

0.357 (0.016) 0.353 (0.016) 0.354 (0.016) 0.272 (0.015) 0.234 (0.014) 0.214 (0.014) 0.211 (0.014) 0.218 (0.014) 0.204 (0.014) 0.113 (0.011) 0.107 (0.010) -

0.141 (0.017) 0.153 (0.018)

0.180 (0.019)

Nom.-Distances involvingMytilus andAscaris are in bold.

degree of Mytilus divergence also applies to the molluscan species (Albinaria and Katharina). Because nematodes are traditionally thought to represent a more basal metazoan lineage than do mollusks (e.g., Eernisse, Al-

0.034

MJtitw

M

Mytitus F

FIG. 1.-Neighbor-joining consensus tree (1,000 replicates) showing the long branches of Mytilus and Ascaris. Pairwise distances between taxa were based on the proportion of nucleotide differences at first and second codon positions for the combined COIKOIII mtDNA data set. Bracketed numbers indicate the proportion of bootstrap support for a given node. Open numbers indicate branch lengths (in numbers of substitutions/nucleotide site). Taxa represented include: blue mussel Mytilus edulis (female and male lineages), nematode Ascaris suum, snail Albinaria turrita, chiton Katharina tunicata, fruit fly Drosophila yakuba, mosquito Anopheles gambiae, brine shrimp Artemia franciscana, urchin Paracentrotus lividis, frog Xenopus laevis, chicken Gallus gallus, and human Homo sapiens.

bert, and Anderson 1992), we contend that the Mytilus sequences are more divergent than the Ascaris sequence. From dates of appearance of taxa in the fossil record and estimates of patristic distances from the neighbor-joining tree (after excluding Ascaris because of its anomalous position), it is possible to calculate approximate rates of sequence divergence for each of the major lineages represented here. To produce a conservative estimate for the rate of evolution of Mytilus mtDNA we have used the upper estimate of the time of divergence for the lineage leading to Mytilus and the lower estimates for the arthropod and deuterostome lineages leading to the species used in this analysis. Assuming 600 million years (Myr) as the time of the divergence of Mytilus from gastropods, the Mytilus lineage has diverged at a rate of 3.67 X 1O-4 substitutions/nucleotide site/Myr (based on p-distance). The corresponding rate is 2.24 X lop4 for humans (based on 250 Myr since divergence from chicken) and 2.00 X 10e4 for Drosophila (based on 355 Myr since divergence from Artemia). Using the Kimura 2-parameter model to generate distances, the rate of substitution estimated for Mytilus (5.23 X 10e4 substitutions/nucleotide site/Myr) is more than double that for humans (2.48 X 1O-4 substitutions/

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nucleotide site/Myr) or Drosophila (2.31 X 10e4 substitutions/nucleotide site/Myr). The same pattern holds for estimates of uncorrected or corrected amino acid substitution rates. We conclude that both relative and absolute rate tests indicate that the Mytilus mtDNA lineage has diverged at a faster rate than is typical for animal mtDNA and that this accounts for the higher amount of differentiation we see in this molecule. The rapid mtDNA evolution in nematodes was previously noted for both the small and large rRNA gene sequences (Okimito, Macfarlane, and Wolstenholme 1994). Earlier, Cedergren et al. (1988) made the same observation for nematode nuclear rRNA gene sequences and attributed it to high mutation rate. Higher mutation rates resulting from shorter generation times or higher metabolic rates are commonly invoked to explain differences in rates of molecular evolution (e.g., Martin, Naylor, and Palumbi 1992; Martin and Palumbi 1993; Stewart and Baker 1994). It is difficult to evaluate the impact of these factors on the long-term rate of evolution of mtDNA given that life-history parameters may have changed considerably during the species’ evolutionary history (Crozier, Crozier, and MacKinley 1989). With this caveat in mind, Mytilus does not appear to be remarkable with regard to either metabolic rate or generation time in comparison to related extant forms. In a comparative study of molluscan metabolic rates, Bayne and Newall (1983) demonstrated higher rates in grazing gastropods (e.g., Littorina) than in sessile suspension feeders, such as mussels, scallops, oysters, and the sessile gastropod Crepidula. It is more difficult to assess the possibility that the high rate of evolution in Mytilus is due to a higher number of DNA replications in the germ line of Mytilus than in the other taxa represented in fig. 1. This force is responsible, at least in part, for the greater divergence between Y chromosome genes than X chromosome genes in primates, where the number of germ-cell divisions per generation is greater in males than in females (e.g., Shimmen, Chang, and Li 1993). Mytilus is an external spawner capable of producing millions of gametes in any given spawning season (Seed and Suchanek 1992). We do not know how this translates into number of germ cell divisions and how, in turn, the number of cell divisions correlates with the number of mtDNA replications. It is doubtful that the number of mtDNA replications per real time unit could account for the relationships among evolution rates shown in fig. 1, which include not only the exceptional high rates of the two Mytilus lines (and of Ascaris), but also the relatively similar rates for taxa that differ greatly in generation times and gametic outputs. As an alternative explanation we propose that, as a result of doubly uniparental inheritance, mussel mtDNA is under more relaxed selection than mtDNA molecules

with conventional uniparental transmission. In malt mussels, the M type is the predominant (if not the ex. elusive) form of mtDNA present in sperm. In contrast the M type is in the minority in all somatic tissues 01 males (Stewart et al. 1995; B. Sutherland, unpublishec data). Females (and their eggs) contain almost exclu, sively the F type. The physical division of the M and I genomes into sperm and eggs (Skibinski, Gallagher, ant Beynon 1994b; Zouros et al. 1994b) and the apparently negligible role of the M type in somatic tissues may impose different functional constraints (and selective pressures) on these distinct mtDNA lineages. If the sun total of selection pressure on each lineage is reducec because of this “division of labor,” it could result in thf accelerated evolution of ikfytilus mtDNA. It follow: from this hypothesis that when the two lineages of A4y. tilus are compared, the M lineage must be found tc evolve faster and to contain more intraspecific variability than the F lineage. Skibinski, Gallagher, and Beynor (1994b), Rawson and Hilbish (1995), and Stewart et al (1995) have independently demonstrated a faster rate ol evolution for the M lineage using different parts of the Mytilus mtDNA genome (COIII/ND2, 16s rRNA, ant COIII, respectively). Stewart et al. (1995) comparec CO111 substitution rates for synonymous, Ks, and nonsynonymous, KA, sites for the M and F lineages in M. edulis and M. trossulus and found that the ratio of silenl to replacement substitution rates was more than six times greater in the F lineage (Ks/K, = 110.8) than in the M lineage (Ks/K, = 16.9). They also observed thal there was a greater diversity of male types than female types in the natural population they studied. Both ot these observations are consistent with the hypothesis 01 relaxed selection on the M lineage. DUI has now been reported in the freshwater mussel Pyganodon (formerly Anodonta, see Hoeh [ 19901: grandis (Liu, Mitton, and Wu 1995), whose lineage must have been separated from that of Mytilus by more than 400 Myr. From RFLP comparisons, Liu et al. alsc observed that the M mtDNA lineage of P. grandis evolves faster than the F lineage and attributed the difference to relaxed selection on the M lineage. The presence of DUI in the distantly related Mytilus and Pyganodon lineages opens the possibility for independent tests of the hypothesis we propose here, i.e., that relaxed selection leads to faster evolution of the M lineage intraspecifically and to faster evolution of both lineages with respect to the mtDNA of related taxa that have retained the conventional uniparental mode of transmission. Acknowledgments We thank A. Ball and R. Singh for assistance with primer design and for help sequencing the CO111 gene

Letter to the Editor

in Mytilus. We also thank Dr. G. Rodakis for kindly providing the Albinaria sequences prior to their publication. This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to E.Z. and by the Research Development Fund (Dalhousie University) to W.R.H. and D.T.S. W.R.H. and D.T.S. were provided with postdoctoral fellowships by NSERC.

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Accepted

September

reviewing 11, 1995

editor