Positive Selection on an Acrosomal Sperm Protein M7 Lysin in Three Species of the Mussel Genus Mytilus
* Department of Biology, Duke University8wl, http://www.100md.com
{dagger} Department of Biological Sciences, University of Delaware8wl, http://www.100md.com
Abstract8wl, http://www.100md.com
Marine invertebrate sperm proteins are particularly interesting because they are characterized by positive selection and are likely to be involved in prezyogotic isolation and, thus, speciation. Here, we present the first survey of interspecific and intraspecific variation of a bivalve sperm protein among a group of species that regularly hybridize in nature. M7 lysin is found in sperm acrosomes of mussels and dissolves the egg vitelline coat, permitting fertilization. We sequenced multiple alleles of the mature protein-coding region of M7 lysin from allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). A significant McDonald-Kreitman test showed an excess of fixed amino acid replacing substitutions between species, consistent with positive selection. In addition, Kolmogorov-Smirnov tests showed significant heterogeneity in polymorphism to divergence ratios for both synonymous variation and combined synonymous and nonsynonymous variation within M. galloprovincialis. These results indicate that there has been adaptive evolution at M7 lysin and, furthermore, show that positive selection on sperm proteins can occur even when postzygotic reproductive isolation is incomplete.
Key Words: fertilization • gamete recognition • lysin • Mytilus • positive selection • sperm proteinjj3|j, 百拇医药
Introductionjj3|j, 百拇医药
Genes involved in sexual reproduction frequently show a strong signal of adaptive evolution at the molecular level (reviewed by ), and some of the most extreme examples are proteins expressed on the sperm surfaces of marine invertebrates. Despite the repeated action of positive selection on sperm protein genes, the underlying cause of selection is not understood (reviewed by and references therein). However, considering the evolutionary patterns of such genes across a wide array of taxa may help elucidate causes of selection.jj3|j, 百拇医药
Although there are several examples of positive selection among marine invertebrate sperm proteins, the exact pattern of selection is somewhat idiosyncratic. In general, marine invertebrates reproduce by spawning their gametes into the water column. Sperm and eggs are drawn together by chemoattraction, the sperm acrosomal vesicle opens, the egg and sperm bind, the sperm lyses the egg vitelline envelope, and the gametes fuse . Two sperm genes involved in the fertilization sequence have been extensively studied. Bindin is the gene causing sea urchin sperm to adhere to eggs. Positive selection in bindin (evidenced by a ratio of nonsynonymous to synonymous amino acid substitutions, dN/dS, between species exceeding 1) is restricted to a particular region of the gene, and considerable variation is observed both among and within species (. In contrast, positive selection in gastropod lysin (which lyses the egg vitelline envelope) is not restricted to one gene region. Many species pairs of abalone and teguline snails show substantially more nonsynonymous (dN) than synonymous (dS) substitutions across the entire gene, and within species, variation is nearly nonexistent . Other gastropod sperm genes with unknown functions show similar patterns to lysin (e.g., many amino acid changes between species but no variation within species ). The high ratios of dN/dS (>1) and the lack of intraspecific polymorphisms among gastropod sperm genes point to the action of selective sweeps that have rapidly fixed replacement substitutions within gastropod species and eliminated synonymous polymorphisms.
Studies to date have found positive selection on marine invertebrate sperm proteins among closely related species with overlapping geographic ranges (sympatry). Only sympatric groups of sea urchins show signatures of positive selection on bindin . In contrast, no evidence for positive selection has been found within genera characterized by allopatric species . In addition, the groups of gastropod mollusks (abalone and teguline snails) for which positive selection has been reported includes many sympatric species pairs..f, 百拇医药
Sympatry with closely related species also seems to be associated with increased sperm specificity. In sea urchins, allopatric species crosses require substantially less sperm for egg fertilization than crosses involving sympatric species pairs . Similarly, interspecific fertilization assays of abalone and snails show that sperm are much more efficient in fertilizing conspecific eggs than heterospecific eggs. The only direct evidence for a sperm protein's association with conspecific egg recognition comes from experimental crosses where sea urchin sperm were more likely to fertilize eggs with the same homozygous bindin genotype . Both sea urchins and abalone can be experimentally crossed to produce some viable offspring . However, the occurrence of hybrids in nature is rare, with hybrid frequencies estimated as 0.1% of Echinometra sea urchin populations and 0.37% of Haliotis abalone populations ).
Here, we focus on a group of closely related taxa that extensively hybridize in nature. Mussels in the Mytilus edulis (Mollusca: Bivalvia) species group (M. edulis, M. galloprovincialis, and M. trossulus) are common in the intertidal and shallow subtidal areas of the temperate Northern and Southern hemispheres . In the Northern Hemisphere, M. edulis is present in the North Atlantic on both the North American and the European coastlines. M. trossulus is present in the North Pacific, Atlantic Canada, and the Baltic Sea. M. galloprovincialis is native to the Mediterranean and Black Seas and western Europe, and it has been recently introduced to California and Asia . Some hybrid individuals have been found in every area where these species are sympatric: M. edulis and M. galloprovincialis in western Europe , M. edulis and M. trossulus in northern Europe and in Atlantic Canada), and M. trossulus and M. galloprovincialis in California and Asia ( but see ). In fact, hybridization is sufficiently frequent that hybrids comprise over 30% of the mussels in some regions of the major hybrid zones .
Despite extensive hybridization, these three species remain genetically distinct throughout much of their species range; therefore, some prezygotic or postzygotic processes must contribute to reproductive isolation. The mechanisms of fertilization in mussels have not been well studied, although recent laboratory crosses show that there is some degree of reduced fertilization success between M. edulis and M. galloprovincialis , suggesting that gamete recognition might play a role in isolating these species. In experimental crosses, F1 hybrid viability is also reduced for M. edulis x M. galloprovincialis and M. edulis x M. trossulus crosses, demonstrating some level of postzygotic isolation among the three species. However, F2 and backcross-type hybrid individuals are found in mussel hybrid zones, indicating that at least some F1 hybrids are fertile .+?/, http://www.100md.com
found three proteins in high concentration on the acrosomal surface of Mytilus sperm. One of these three proteins showed strong lysogenic activity, nonenzymatically dissolving the egg vitelline coat. This 20 kDa protein, which Takagi and colleagues named M7 lysin, also reinitiates the suspended meiosis by releasing the egg's first polar body. M7 lysin does not exhibit significant sequence similarity to lysins from abalone or snails, rather is classified by its function. Given the rapid rate of protein evolution observed for lysins in these gastropods, the lack of obvious similarity is not surprising and does not necessarily suggest independent origins of the different lysins.
Understanding how selection drives the evolution of sperm proteins and understanding the role of sperm proteins in speciation will be aided by studying such genes in diverse taxa, particularly among species with incomplete reproductive isolation. Consequently, we have sequenced multiple alleles of the mature protein coding region of M7 lysin in allopatric populations of M. edulis, M. galloprovincialis, and M. trossulus. By examining the patterns of polymorphism and divergence, we demonstrate that Mytilus M7 lysin has evolved in a manner consistent with positive selection, as with other marine invertebrate sperm proteins. This study provides a new phylogenetic perspective by demonstrating that positive selection of gamete recognition genes also occurs in bivalve mollusks. This is also the first evidence for positive selection on a marine invertebrate gamete recognition gene in a group characterized by extensive hybridization among species.|\0, http://www.100md.com
Methods|\0, http://www.100md.com
RNA Isolation and Sequencing
Mytilus edulis was collected from Woods Hole, Mass., and M. galloprovincialis was collected from Samos, Greece. These regions are known to have pure (nonhybrid) populations of each species. M. trossulus was collected from Penn Cove, Wash. Because imported M. galloprovincialis are cultivated in Washington alongside the native M. trossulus, the species identity of the M. trossulus individuals was confirmed using sequences of the mitochondrial COIII gene .[k*5[, 百拇医药
Total RNA was extracted from mantle tissue using Tri Reagent (Sigma). Fresh tissue was homogenized in 0.5 ml Tri Reagent and vortexed briefly after a 0.2 ml addition of choloroform. The tissue-Tri Reagent-choloroform mixture was centrifuged at 10,000 RPM for 15 min at 4°C. The top aqueous phase was pipetted into a fresh tube, and RNA was precipitated with 0.5 ml isopropanol. The RNA pellet was washed with 75% ethanol and dried at room temperature. The RNA pellet was resuspended in RNase-free water.[k*5[, 百拇医药
sequenced M7 lysin from one individual of M. galloprovincialis. To identify a part of the 3' untranslated region that was conserved among all three species and would thus be a suitable primer site, 3' RACE was used to amplify and sequence the 3' region from several individuals of M. edulis and M. trossulus. Total mRNA was reverse transcribed with SUPERSCRIPT RNase H Reverse Transcriptase (Life Technologies) using an oligo-dT adaptor (5'-AAG GAT CCG TCG ACA TCG ATA ATA CGA CAT TTT TTT TTT TTT TTT T-3'), and PCR was performed using the degenerate primer of Tagaki et al. (5'-GGA TTC AAY CAY GTN CAN GGN GAR AC-3') and a primer matching part of the adaptor sequence (5'-AAG GAT CCG TCG ACA TC-3'). A second, nested reaction was performed using a small amount of the first PCR product and the primers MEL-F6 (5'-AAC AAG GAT CAT GCA AAC -3') and a second primer matching the adaptor sequence (5'-GAC ATC GAT AAT ACG AC-3'), which amplified fragments from a number of M. edulis and M. trossulus individuals. These sequences of the 3' region, along with the Takagi et al. sequence of the 5' region, were used to design primers targeting the untranslated regions (UTRs) that would amplify the entire coding region of M7 lysin: MEL-F7, 5'-TGG AAG CCA AAT CTT TAC AT-3', and MEL-R5, 5'- GTC TTT AGT TCT TTA TTT TAT ATT GAC-3'. PCR was performed with Promega Taq DNA polymerase (Madison, Wis.) for 35 cycles of 20 s at 94°C, 30 s at 54°C, and 80 s at 72°C and amplified an approximately 770 bp fragment containing the entire 540 bp coding sequence for the mature peptide and 26 bp of the 5' UTR. The length of the amplified 3' UTR varied among alleles and was approximately 200 bp long.
PCR products were cloned (TA Cloning: Invitrogen). PCR with M13 primers was performed on individual colonies. These M13 PCR reactions were cleaned with a Qiaquick PCR Purification Kit (Qiagen) and then sequenced with M13 primers using the ABI PRISM BigDye Terminators ver. 2.5 Cycle Sequencing Kit. Sequencing reactions were visualized with an ABI 3700 DNA Analyzer. For each individual mussel, multiple colonies were sequenced in order to discriminate between polymorphism and polymerase error. Thus, two alleles were inferred for each individual, and each allele was represented by a minimum of three sequenced clones. Sequence chromatograms were visualized and edited using Sequencher ver. 4.1 software.yw#l, http://www.100md.com
Descriptive Statistics and Sequence Similarityyw#l, http://www.100md.com
Genetic variation within each of the three species was described using a number of measures. These measures do not test for selection but are useful for comparing M7 lysin to other genes. DNAsp ver. 2.5 ) was used to estimate species heterozygosity = 4Nµ, where N is the effective population size and µ is the neutral mutation rate) based on the number of segregating sites and the average number of pairwise differences . These two estimators of capture different elements of the frequency distribution of polymorphisms. Recombination rates were estimated by the methods of in the program SITES .
Although M7 lysin is one of the most abundant proteins in mussel sperm acrosomes, found no similarity between M7 and any other previously published sperm protein. Since their original description of M7, however, many more protein sequences have been deposited in public databases. To verify their results, we performed a protein BLAST search against the SwissProt and TrEMBL databases of proteins using the default BLOSUM matrix. In addition, we explicitly tried BLASTing the M7 lysin protein sequence against abalone lysin , teguline lysin (, and gastropod TMAP (, the other described sperm proteins for mollusks. Protein secondary structure was predicted for M. edulis using the PHDsec program implemented by PredictProtein for comparison to gastropod lysins. The charge and isoelectric static point of M7 lysin was determined by EMBL Isoelectric Point Service .a9tx!q', http://www.100md.com
Tests of Selectiona9tx!q', http://www.100md.com
We analyzed patterns of genetic variation within and among species in a number of ways to determine whether positive selection has shaped genetic variation at the mussel M7 lysin gene. The observed frequency spectra of polymorphisms for each species were compared with neutral expectations using Tajima's D statistic , as implemented in DNAsp. Heterogeneity in the polymorphism to divergence ratios across the gene was tested using the runs test, sliding G-test, and Kolmogorov-Smirnov test of . Because there was no a priori estimate of the whole-sequence recombination parameter, R, the program DNA Slider was run for values of R = 2, 4, 8, 16, etc., until P, the probability of obtaining the observed heterogeneity by chance, reached a peak and started to decline. For each value of R, 1,000 replicates were run; if the peak value of P was less than 0.10 for any test, 10,000 replicates were run. This was done for synonymous variation alone and for synonymous and amino acid replacement variation combined. One M. trossulus sequence (T765A) was used as the outgroup for testing heterogeneity within M. edulis and M. galloprovincialis, and one M. edulis sequence (E390B) was used as the outgroup for M. trossulus.
Relative frequencies of synonymous and amino acid replacement substitutions between species were also used to test for selection. McDonald-Kreitman tests compare the number of synonymous and nonsynonymous fixations (between species) with polymorphisms (within species). Standard McDonald-Kreitman tests were conducted on the whole data set. We also tested for an excess of radical amino acid fixations between species in the McDonald-Kreitman test framework using radical and conservative amino acid change definitions (charge, polarity, and polarity with volume) based on .ps:ou), 百拇医药
In addition, we looked at ratios of synonymous and nonsynonymous divergence along the three species branches, where a ratio of nonsynonymous to synonymous substitutions greater than unity is taken as evidence for positive selection. In MEGA 2.0 , the proportions of nonsynonymous substitutions per nonsynonymous site (dN) and synonymous substitutions per synonymous site (dS), based on the modified method, were calculated across the whole gene and for a 30-codon sliding window across the gene. Where dN was greater than dS, the significance of this difference was tested with Fisher's exact test . Yang and others have developed a series of nested ML tests that test for differences among dN/dS among codons in a gene and along branches of a tree . These methods were implemented with the CODEML program in PAML , based on a maximum likelihood tree generated in PAUP* (under an HKY with gamma model) .
Resultsn6#32, 百拇医药
Descriptive Statistics and Sequence Similarityn6#32, 百拇医药
Most of the sequenced fragments were M7 lysin, based on similarity to the sequence of ; a few M6 fragments from M. edulis were also sequenced but are not analyzed here (GenBank accession number ). No more than two distinct alleles were found per individual, consistent with M7 being a single copy gene. The sequenced fragments of M7 lysin included less than 26 bp of the 5' UTR, the entire 540-bp coding region, and 173 to 220 bp of the 3' UTR. The 3' UTR had a large amount of indel variation within and between species, making it difficult to align accurately; therefore only the coding region is analyzed here.n6#32, 百拇医药
It is unclear whether M7 lysin shares a common origin with gastropod lysins, as protein BLAST searches and analyses of protein structure failed to reveal any significant similarities between Mytilus M7 lysin and other proteins, including gastropod sperm proteins. Two {alpha}
-helices were predicted, at amino acid positions 77 to 86 and 97 to 109, and several short (<10 a.a.) ß-sheets were predicted interspersed along the protein. The two predicted {alpha}im;n, 百拇医药
-helices fall in the central region of the protein, a region containing {alpha}im;n, 百拇医药
-helices in abalone (based on crystallography: ). However, many of the other {alpha}im;n, 百拇医药
-helical regions in abalone lysin (and predicted for teguline lysin ) are not predicted for mussel lysin. M7 lysin is inferred to have a charge of –0.02 and an isoelectric point at 8.5, less than that for gastropods (10.0 to 11.6) ( Hellberg and Vacquier 1998). Although a common origin for gastropod and bivalve lysins cannot be rejected, there is no clear evidence to substantiate homology.im;n, 百拇医药
In all, we sequenced eight alleles from M. trossulus (GenBank accession numbers to ), 10 alleles from M. edulis (GenBank accession numbers to ), and six alleles from M. galloprovincialis (GenBank accession numbers to ). Although described their sequence from a Japanese mussel as being from an "M. edulis" individual, we include it with M. galloprovincialis because it is most similar to our Mediterranean M. galloprovincialis sequences and because introduced populations of M. galloprovincialis are the only "edulis-like" mussel in most of Japan (. Thus, our sample size for M. galloprovincialis includes seven alleles.
There was variation at M7 lysin both between and within species. Estimates of heterozygosity ({theta} ) were fairly similar across the three species . Allelic variation within species was characterized by recombination. The method described considerable recombination within M. edulis and M. trossulus , and our limited survey of within-species diversity found instances of all four possible recombinant gametes for M. galloprovincialis (positions 52 and 292; ) and for M. edulis (positions 252 and 510; ). A direct consequence of recombination is that there is no single genealogy for the alleles sampled. However, each species was defined by unique substitutions. There were 28 fixed differences (11 synonymous and 17 amino acid replacements) between the M. trossulus alleles and M. edulis and M. galloprovincialis alleles ( and ). M. edulis and M. galloprovincialis were each defined by one nonsynonymous substitution (sites 56 and 144; ). Thus, the species relationships inferred from lysin are the same as those inferred from mtDNA, where the M. trossulus species branch is the longest and there is less divergence between M. edulis and M. galloprovincialis .
fig.ommitted7?3vp, 百拇医药
Table 1 Allelic Diversity Within Mussel Species.7?3vp, 百拇医药
fig.ommitted7?3vp, 百拇医药
FIG. 1. Polymorphic nucleotide sites in the mature M7 lysin protein. Dots show identity to the consensus sequence. Species identities of alleles are described by the letters G, E, or T, where G is M. galloprovincialis, E is M. edulis, and T is M. trossulus, and A and B refer to two alleles within a single individual. For example, T776B is the B allele from M. trossulus individual 776. On the bottom row, S and N indicate whether polymorphisms are synonymous or nonsynonymous (e.g., causing amino acid substitutions). In two cases where there are three substitutions at the same site (positions 321 and 489), there is a synonymous polymorphism within M. edulis and a fixed nonsynonymous difference between M. trossulus and M. edulis/galloprovincialis.7?3vp, 百拇医药
fig.ommitted7?3vp, 百拇医药
FIG. 2. Alignment of M7 protein sequences from three mussel species. Dots show identity to the consensus sequence. Amino acid sites that are polymorphic within species are underlined. At two amino acid sites (positions 96 and 108, italicized) Mytilus trossulus is distinguished by two separate nucleotide substitutions, both of which are nonsynonymous. Substitutions at sites 56 and 144 are unique to M. edulis and M. galloprovincialis, respectively, and are shown in boldface
Tests of Selection28i5&, 百拇医药
Our tests of selection revealed an excess of fixed amino acid replacement differences between species, consistent with positive selection on the mussel lysin gene. A McDonald-Kreitman test of the entire data set was statistically significant (P < 0.0167) (, with nonsynonymous fixations exceeding synonymous fixations between species and synonymous polymorphisms exceeding nonsynonymous polymorphisms within species. McDonald-Kreitman tests based on ratios of conservative to radical amino acid substitutions did not show an excess of radical fixed differences. In fact, the proportion of fixed differences was greater for conservative changes than for radical changes considering charge, polarity, and polarity with volume change.28i5&, 百拇医药
fig.ommitted28i5&, 百拇医药
Table 2 McDonald-Kreitman Test of Selection Across All Three Species.28i5&, 百拇医药
While the McDonald-Krietman test demonstrated a pattern consistent with positive selection, the overall ratio of dN/dS was less than 1 (M. edulis vs. M. galloprovincialis: 0.25; M. edulis vs. M. trossulus: 0.48; M. galloprovincialis vs. M. trossulus: 0.48). A dN/dS less than 1 is expected if most replacement mutations are deleterious, which is likely to be the case for most protein coding genes. Sliding window analyses of dN/dS ratios located a region in the 3' end of the gene where dN/dS is greater than 1, but Fisher's exact test revealed that dN was not significantly greater than dS for any of the sliding window partitions. None of the maximum likelihood methods for identifying selection at specific sites or along specific branches yielded significant results. However, these methods assume no recombination and are not sensitive to changes along short branches such as those among closely related species .
Tajima's D statistic test of selection based on the frequency spectrum of polymorphisms did not show any strong deviations from neutrality ). The runs test, sliding G-test, and Kolmogorov-Smirnov test did not reveal significant heterogeneity in the polymorphism to divergence ratio within either M. edulis or M. trossulus. However, along the length of the M. galloprovincialis gene, the Kolmogorov-Smirnov test revealed significant heterogeneity, both for synonymous variation (P = 0.026) and for synonymous and nonsynonymous variation combined (P = 0.025), with the beginning of the sequence having a higher ratio of polymorphism to divergence than the latter portion .{ow%fu, 百拇医药
fig.ommitted{ow%fu, 百拇医药
FIG. 3. Sliding window plot of the polymorphism to divergence ratio in M. galloprovincialis, with M. trossulus used for divergence. For each window of 10 variable sites, the proportion of variable sites that are polymorphic (combined synonymous and nonsynonymous) was calculated. Each value is plotted at the midpoint of the window. G indicates the position of the nonsynonymous substitution defining the M. galloprovincialis lineage at bp position 430. Kolmogorov-Smirnov tests revealed significant heterogeneity in the polymorphism to divergence ratio both for synonymous variation (P = 0.026) and for combined synonymous and nonsynonymous variation (P = 0.025)
Discussion')ewn4(, 百拇医药
Positive Selection in Mussel Lysin')ewn4(, 百拇医药
In this first survey of molecular variation of a bivalve sperm protein, we find evidence for positive selection shaping patterns of genetic variation between closely related species that frequently hybridize in nature. The strongest evidence for positive selection comes from the significant (P < 0.0167) McDonald-Kreitman test. If we assume that ratios of replacement to synonymous polymorphism within species approximate a neutral distribution, then the McDonald-Krietman test points to an excess of fixed replacement substitutions between species. The proportion of adaptive substitutions can be estimated as {alpha}')ewn4(, 百拇医药
= 1-DSPN/DNPS , where DS and DN are the number of synonymous and nonsynonymous differences between species, and PS and PN are the number of synonymous and nonsynonymous polymorphisms. For this data set we estimate that 75% of the nonsynonymous differences between species are adaptive. Thus, the pattern of selection observed at mussel lysin confirms the emerging conclusion that marine invertebrate sperm proteins are frequently characterized by increased rates of amino acid substitutions.
Other studies have found dN/dS greater than 1 for gastropod lysins. However, many genes undergoing adaptive evolution are likely to have dN/dS less than 1, because the majority of nonsynonymous substitutions are probably deleterious even if a small fraction are adaptive. Here we find evidence for positive selection at a reproductive protein even though dN/dS is less than 1. This observation emphasizes the value of examining within-species polymorphism in addition to between-species divergence.|40^{q, 百拇医药
In addition to the significant McDonald-Kreitman test, significant heterogeneity in the polymorphism to divergence ratio along M7 lysin in M. galloprovincialis suggests that adaptive evolution has occurred. One nonsynonymous nucleotide substitution occurred in the M. galloprovincialis lineage after it split from M. edulis. This substitution is at amino acid site 144, near the 3' end of the coding sequence, which has a lower polymorphism/divergence ratio than the 5' end of the gene . Such a pattern is expected if a rapid, recent substitution at site 144 swept away the polymorphism in the 3' end of the gene, while, because of recombination, the 5' end of the gene was unaffected by the sweep. At least one intron of unknown length occurs in the lysin gene of Mytilus (Riginos, unpublished data), so it is not implausible that different parts of the coding region would have different evolutionary histories.
Comparisons of Mussel Lysin to Other Marine Invertebrate Sperm Proteinsm, 百拇医药
Like other lysins, Mytilus M7 lysin is found on the sperm acrosome and dissolves the egg vitelline envelope . However, its structure is not demonstrably similar to gastropod lysins, the only other molluscan lysins that have been described. Lysin proteins evolve quite rapidly, and so the lack of similarity is not surprising. For example, the rate of nonsynonymous substitutions per site per 109 years is estimated at 6 to 84 for abalone and 21 to 76 for snails . We estimate a rate of 6.5 for mussels, assuming a 3.5 million year divergence between M. trossulus and M. edulis/galloprovinicialis . Given these high substitution rates and a Cambrian gastropod-bivalve divergence (570 MYA; ), recognizable sequence similarity between gastropod and bivalve lysins is not necessarily expected.m, 百拇医药
All marine invertebrate sperm proteins studied to date show evidence of adaptive evolution. However, the patterns of intraspecific polymorphism and rates of divergence are substantially different. Gastropod sperm proteins have virtually no polymorphism and divergence is extremely rapid , suggesting that recent selective sweeps have removed any intraspecific variation. Sea urchin bindin genes, in constrast, are characterized by an excess of nonsynonymous variation between and within species , which may result from positive selection on new amino acid–changing mutations or positive assortative mating In mussels, we find a third pattern. As in gastropod lysins and urchin bindins, there is evidence that the majority of replacement substitutions between species are adaptive. However, unlike gastropod lysins, adaptive substitutions have not occurred so frequently that polymorphism has been swept away. Unlike urchin bindins, there is no significant evidence from the dN/dS ratio that amino acid polymorphisms are adaptive.
Clearly, more extensive surveys are needed to fully characterize the nature of intraspecific variation in Mytilus lysin. If lysin is directly involved in prezygotic isolation, then specific selection pressures may differ among populations, particularly those where hybridization does and does not occur. It will also be particularly interesting to examine this gene in populations of uncertain origin, such as those in the Baltic Sea and the Southern Hemisphere .l^a^.+8, 百拇医药
Given that all three Mytilus species surveyed here hybridize extensively in nature, our results imply that the action of selection on mussel lysin precedes the attainment of complete postzygotic reproductive isolation. Our results substantiate the emerging pattern that marine invertebrate sperm proteins are characterized by positive selection, particularly when ranges of closely related species overlap. It remains to be seen whether a common explanation underlies the general observation of adaptive evolution on genes involved in sexual reproduction.
Acknowledgements8gcp(1a, http://www.100md.com
We thank C. W. Cunningham, A. J. Abrams, B. Y. Kong, K. Zigler, and S. R. Palumbi for their assistance and comments throughout this project. Suggestions from three anonymous reviewers substantially improved this manuscript. This project was funded by the Duke University program in Molecular Evolution and Comparative Genomics, the PADI Foundation, and the National Science Foundation.8gcp(1a, http://www.100md.com
Literature Cited8gcp(1a, http://www.100md.com
Anisimova, M., J. P. Bielawski, and Z. Yang. 2001. Accuracy and power of the likelihood ratio test in detecting adaptive molecular evolution. Mol. Biol. Evol. 18:1585-1592.8gcp(1a, http://www.100md.com
Benton, M. J., ed. 1993. The fossil record 2. Chapman & Hall, London.8gcp(1a, http://www.100md.com
Biermann, C. H. 1998. The molecular evolution of sperm bindin in six species of sea urchin (Echinoida: Strongylocentrotidae). Mol. Biol. Evol. 15:1761-1771.8gcp(1a, http://www.100md.com
Bierne, N., P. David, P. Boudry, and F. Bonhomme. 2002. Assortative fertilization and selection at larval stage in the mussels Mytlius edulis and M. galloprovincialis. Evolution 56:292-298.
Comesaña, A. S., J. E. Toro, D. J. Innes, and R. J. Thompson. 1999. A molecular approach to the ecology of a mussel (Mytilis edulis-M. trossulus) hybrid zone on the east coast of Newfoundland, Canada. Mar. Biol. 133:213-221.(ik}5r, 百拇医药
Gardner, J. P. A. 1996. The Mytilus edulis complex in southwest England: effects of hybridization and introgression upon interlocus associations and morphometric variation. Mar. Biol. 125:385-399.(ik}5r, 百拇医药
Heath, D. D., P. D. Rawson, and T. J. Hilbish. 1996. PCR-based nuclear markers identify alien blue mussel (Mytilus spp.) genotypes on the west coast of Canada. Can. J. Fish. Aquat. Sci. 52:2621-2627.(ik}5r, 百拇医药
Hellberg, M. E., G. W. Moy, and V. D. Vacquier. 2000. Positive selection and propeptide repeats promote rapid interspecific divergence of a gastropod sperm protein. Mol. Biol. Evol. 17:458-466.(ik}5r, 百拇医药
Hellberg, M. E., and V. D. Vacquier. 1999. Rapid evolution of fertilization selectivity and lysin cDNA sequences in teguline gastropods. Mol. Biol. Evol. 16:839-848.(ik}5r, 百拇医药
Hey, J., and J. Wakeley. 1997. A coalescent estimator of the population recombination rate. Genetics 145:833-846.
Hilbish, T. J., E. Carson, J. Plante, L. Weaver, and M. Gilg. 2002. Distribution of Mytilus edulis, M. galloprovinicialis, and their hybrids in open-coast populations of mussels in southwestern England. Mar. Biol. 140:137-142.', 百拇医药
Hilbish, T. J, A. Mullinax, S. I. Dolven, A. Meyer, R. K. Koehn, and P.D. Rawson. 2000. Origin of the antitropical distribution pattern in marine mussels (Mytilus spp.): routes and timing of transequatorial migration. Mar. Biol. 136:69-77.', 百拇医药
Howard, D. J. 1999. Conspecific sperm and pollen precedence and speciation. Ann. Rev. Ecol. Syst. 30:109-132.', 百拇医药
Hudson, R. R., and N. L. Kaplan. 1985. Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111:147-164.', 百拇医药
Kresge, N., V. D. Vacquier, and D. C. Stout. 2001. The crystal structure of a fusagenic sperm protein reveals extreme surface properties. Biochemistry 40:5407-5413.', 百拇医药
Kumar, S., K. Tamura, I. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245.
Lee, Y.-H., T. Ota, and V. D. Vacquier. 1995. Positive selection is a general phenomenon in the evolution of abalone sperm lysin. Mol. Biol. Evol. 12:231-238.&\[i/|6, 百拇医药
Leighton, D. L., and C. A. Lewis. 1982. Experimental hybridization in abalones. Int. J. Inv. Repro. 5:273-282.&\[i/|6, 百拇医药
McDonald, J. H. 1998. Improved tests for heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence. Mol. Biol. Evol. 15:377-384.&\[i/|6, 百拇医药
McDonald, J. H., and R. K. Koehn. 1988. The mussels Mytilus galloprovincialis and M. trossulus on the Pacific coast of North America. Mar. Biol. 99:111-118.&\[i/|6, 百拇医药
McDonald, J. H., and M. Kreitman. 1991. Adaptive protein evolution at the ADH locus in Drosophila. Nature 351:652-654.&\[i/|6, 百拇医药
McDonald, J. H., R. Seed, and R. K. Koehn. 1991. Allozymes and morphometric characters of three species of Mytilus in the Northern and Southern hemispheres. Mar. Biol. 111:323-333.&\[i/|6, 百拇医药
Metz, E. C., G. Gómez-Gutiérrez, and V. D. Vacquier. 1998. Mitochondrial DNA and bindin gene sequence evolution among allopatric species of the sea urchin genus Arbacia. Mol. Biol. Evol. 15:185-195.
Metz, E. C., R. E. Kane, H. Yanagimachi, and S. R. Palumbi. 1994. Fertilization between closely related sea urchins is blocked by incompatibilites during sperm-egg attachment and early stages of fusion. Biol. Bull. 187:23-34.h\qo2/, http://www.100md.com
Metz, E. C., and S. R. Palumbi. 1996. Positive selection and sequence rearrangements generate extensive polymorphisms in the gamete recognition protein binding. Mol. Biol. Evol. 13:397-406.h\qo2/, http://www.100md.com
Metz, E. C., R. Robles-Sikisaka, and V. D. Vacquier. 1998. Nonsynonymous substitution in abalone sperm fertilization genes exceeds substitution in introns and mitochondrial DNA. Proc. Natl. Acad. Sci. USA 95:10676-10681.h\qo2/, http://www.100md.com
Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418-426.h\qo2/, http://www.100md.com
Nei, M., and W.-H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76:5269-5273.h\qo2/, http://www.100md.com
Nielsen, R., and Z. Yang. 1998. Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 148:929-936.
Owen, B., McLean, J. H., and R. J. Meyer. 1971. Hybridization in the eastern Pacific abalones (Haliotis). Bull. Los Angeles C. Mus. Nat. Hist. Sci. 9:1-37.7k]]()g, 百拇医药
Palumbi, S. R. 1999. All males are not created equal: fertility differences depend on gamete recognition polymorphisms in sea urchins. Proc. Natl. Acad. Sci. USA 96:12632-12637.7k]]()g, 百拇医药
Palumbi, S. R., and E. C. Metz. 1991. Strong reproductive isolation between closely related tropical sea urchins (genus Echinometra). Mol. Biol. Evol. 8:227-238.7k]]()g, 百拇医药
Rawson, P. D., V. Agrawal, and T. J. Hilbish. 1999. Hybridization between the blue mussels Mytilus galloprovincialis and M. trossulus along the Pacific coast of North America. Mar. Biol. 134:201-211.7k]]()g, 百拇医药
Rawson, P. D., and T. J. Hilbish. 1995. Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex. Mol. Biol. Evol. 12:893-901.7k]]()g, 百拇医药
Rawson, P. D., C. L. Secor, and T. J. Hilbish. 1996. The effects of natural hybridization on the regulation of doubly uniparental mtDNA inheritance in blue mussels (Mytilus spp.). Genetics 144:241-248.
Riginos, C., K. Sukhdeo, and C. W. Cunningham. 2002. Evidence for selection at multiple allozyme loci across a mussel hybrid zone. Mol. Biol. Evol. 19:347-351.!c, 百拇医药
Rost, B., and C. Sander. 1993. Prediction of protein secondary structure at better than 70% accuracy. J. Mol. Biol. 232:584-599.!c, 百拇医药
Rozas, J., and R. Rozas. 1999. DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174-175.!c, 百拇医药
Saavedra, C., D. T. Stewart, R. R. Stanwood, and E. Zouros. 1996. Species-specific segregation of gender-associated mitochondrial DNA types in an area where two mussel species (Mytilus edulis and M. trossulus) hybridize. Genetics 143:1359-1367.!c, 百拇医药
Shaw, A., D. E. McRee, V. D. Vacquier, and C. D. Stout. 1993. The crystal structure of lysin, a fertilization protein. Science 262:1864-1867.!c, 百拇医药
Skibinski, D. O. F., and J. A. Beardmore. 1979. A genetic study of intergradation between Mytilus edulis and M. galloprovincialis. Experientia 35:1442-1444.
Skurikhina, L. A., Y. F. Kartavtsev, A. Y. Chichvarkhin, and M. V. Pankova. 2001. Study of two species of mussels, Mytilus trossulus and Mytilus galloprovinicialis (Bivalvia, Mytilidae), and their hybrids in Peter the Great Bay of the Sea of Japan with the use of PCR markers. Russ. J. Gen. 37:1448-1451.0}, 百拇医药
Smith, N. G., and A. Eyre-Walker. 2002. Adaptive protein evolution in Drosophila. Nature 415:1022-1024.0}, 百拇医药
Suchanek, T. H., J. B. Geller, B. R. Kresier, and J. B. Mitton. 1997. Zoogeographic distributions of the sibling species Mytilus galloprovincialis and M. trossulus (Bivalvia: Mytilidae) and their hybrids in the North Pacific. Biol. Bull. 193:187-194.0}, 百拇医药
Swanson, W. J., and V. D. Vacquier. 1995. Extraordinary divergence and positive selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa. Proc. Natl. Acad. Sci. USA 92:4957-4961.0}, 百拇医药
Swanson, W. J., and V. D. Vacquier. 2002. The rapid evolution of reproductive proteins. Nature Rev. Gen. 3:137-144.0}, 百拇医药
Swofford, D. L. 1998. PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Mass.
Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585-595.y, http://www.100md.com
Takagi, T., A. Nakamura, R. Deguchi, and K. Kyozuka. 1994. Isolation, characterization, and primary structure of three major proteins obtained from Mytilus edulis sperm. J. Biochem. 116:598-605.y, http://www.100md.com
Vacquier, V. D. 1998. Evolution of gamete recognition proteins. Science 281:1995-1998.y, http://www.100md.com
Vacquier, V. D., K. R. Carner, and C. D. Stout. 1990. Species-specific sequences of abalone lysin, the sperm protein that creates a hole in the egg envelope. Proc. Natl. Acad. Sci. USA 87:5792-5796.y, http://www.100md.com
Vacquier, V. D., W. J. Swanson, and Y.-H. Lee. 1997. Positive Darwinian selection on two homologous fertilization proteins: what is the selective pressure driving their divergence? J. Mol. Evol. 44:S15-S22.y, http://www.100md.com
Väinölä, R., and M. M. Hvilsom. 1991. Genetic divergence and a hybrid zone between Baltic and North Sea Mytilus populations (Mytilidae: Mollusca). Biol. J. Linn. Soc. 43:127-148.
Vermeij, G. 1991. Anatomy of an invasion: the trans-Arctic interchange. Paleobiology 17:281-307.u*34t, 百拇医药
Watterson, G. A. 1975. On the number of segregating sites in genetical models without recombination. Theor. Pop. Bio. 7:256-276.u*34t, 百拇医药
Yang, Z. 1997. PAML: a program for phylogenetic analysis by maximum likelihood. CABIOS 13:555-556.u*34t, 百拇医药
Yang, Z., and R. Nielsen. 1998. Synonymous and nonsynonymous rate variation in nuclear genes of mammals. J. Mol. Evol. 46:409-418.u*34t, 百拇医药
Yang, Z., R. Nielsen, N. Goldman, and A.-M. K. Pedersen. 2000. Codon-substitution models for heterozygous selection pressure at amino acid sites. Genetics 155:431-449.u*34t, 百拇医药
Yang, Z., W. J. Swanson, and V. D. Vacquier. 2000. Maximum-likelihood analysis of molecular adaptation in abalone sperm lysin reveals variable selective pressures among lineages and sites. Mol. Biol. Evol. 17:1446-1455.u*34t, 百拇医药
Zhang, J. 2000. Rates of conservative and radical nonsynonymous nucleotide substitutions in mammalian nuclear genes. J. Mol. Evol. 50:56-68.u*34t, 百拇医药
Zhang, J., S. Kumar, and M. Nei. 1997. Small-sample tests of episodic adaptive evolution: a case study of primate lysozyme. Mol. Biol. Evol. 14:1335-1338.u*34t, 百拇医药
Zigler, K. S., and H. A. Lessios. 2003. Evolution of bindin in the pantropical sea urchin Tripneustes: comparisons to bindin of other genera. Mol. Biol. Evol. 20:220-231.u*34t, 百拇医药
Zouros, E., A. O. Ball, C. Saavedra, and K. R. Freeman. 1994a. Mitochondrial DNA inheritance. Nature. 368:817-818.u*34t, 百拇医药
Zouros, E., A. O. Ball, C. Saavedra, and K. R. Freeman. 1994b. An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus. Proc. Natl. Acad. Sci. USA 91:7463-7467.u*34t, 百拇医药
Accepted for publication October 9, 2002.(Cynthia Riginos and John H. McDonald)
{dagger} Department of Biological Sciences, University of Delaware8wl, http://www.100md.com
Abstract8wl, http://www.100md.com
Marine invertebrate sperm proteins are particularly interesting because they are characterized by positive selection and are likely to be involved in prezyogotic isolation and, thus, speciation. Here, we present the first survey of interspecific and intraspecific variation of a bivalve sperm protein among a group of species that regularly hybridize in nature. M7 lysin is found in sperm acrosomes of mussels and dissolves the egg vitelline coat, permitting fertilization. We sequenced multiple alleles of the mature protein-coding region of M7 lysin from allopatric populations of mussels in the Mytilus edulis species group (M. edulis, M. galloprovincialis, and M. trossulus). A significant McDonald-Kreitman test showed an excess of fixed amino acid replacing substitutions between species, consistent with positive selection. In addition, Kolmogorov-Smirnov tests showed significant heterogeneity in polymorphism to divergence ratios for both synonymous variation and combined synonymous and nonsynonymous variation within M. galloprovincialis. These results indicate that there has been adaptive evolution at M7 lysin and, furthermore, show that positive selection on sperm proteins can occur even when postzygotic reproductive isolation is incomplete.
Key Words: fertilization • gamete recognition • lysin • Mytilus • positive selection • sperm proteinjj3|j, 百拇医药
Introductionjj3|j, 百拇医药
Genes involved in sexual reproduction frequently show a strong signal of adaptive evolution at the molecular level (reviewed by ), and some of the most extreme examples are proteins expressed on the sperm surfaces of marine invertebrates. Despite the repeated action of positive selection on sperm protein genes, the underlying cause of selection is not understood (reviewed by and references therein). However, considering the evolutionary patterns of such genes across a wide array of taxa may help elucidate causes of selection.jj3|j, 百拇医药
Although there are several examples of positive selection among marine invertebrate sperm proteins, the exact pattern of selection is somewhat idiosyncratic. In general, marine invertebrates reproduce by spawning their gametes into the water column. Sperm and eggs are drawn together by chemoattraction, the sperm acrosomal vesicle opens, the egg and sperm bind, the sperm lyses the egg vitelline envelope, and the gametes fuse . Two sperm genes involved in the fertilization sequence have been extensively studied. Bindin is the gene causing sea urchin sperm to adhere to eggs. Positive selection in bindin (evidenced by a ratio of nonsynonymous to synonymous amino acid substitutions, dN/dS, between species exceeding 1) is restricted to a particular region of the gene, and considerable variation is observed both among and within species (. In contrast, positive selection in gastropod lysin (which lyses the egg vitelline envelope) is not restricted to one gene region. Many species pairs of abalone and teguline snails show substantially more nonsynonymous (dN) than synonymous (dS) substitutions across the entire gene, and within species, variation is nearly nonexistent . Other gastropod sperm genes with unknown functions show similar patterns to lysin (e.g., many amino acid changes between species but no variation within species ). The high ratios of dN/dS (>1) and the lack of intraspecific polymorphisms among gastropod sperm genes point to the action of selective sweeps that have rapidly fixed replacement substitutions within gastropod species and eliminated synonymous polymorphisms.
Studies to date have found positive selection on marine invertebrate sperm proteins among closely related species with overlapping geographic ranges (sympatry). Only sympatric groups of sea urchins show signatures of positive selection on bindin . In contrast, no evidence for positive selection has been found within genera characterized by allopatric species . In addition, the groups of gastropod mollusks (abalone and teguline snails) for which positive selection has been reported includes many sympatric species pairs..f, 百拇医药
Sympatry with closely related species also seems to be associated with increased sperm specificity. In sea urchins, allopatric species crosses require substantially less sperm for egg fertilization than crosses involving sympatric species pairs . Similarly, interspecific fertilization assays of abalone and snails show that sperm are much more efficient in fertilizing conspecific eggs than heterospecific eggs. The only direct evidence for a sperm protein's association with conspecific egg recognition comes from experimental crosses where sea urchin sperm were more likely to fertilize eggs with the same homozygous bindin genotype . Both sea urchins and abalone can be experimentally crossed to produce some viable offspring . However, the occurrence of hybrids in nature is rare, with hybrid frequencies estimated as 0.1% of Echinometra sea urchin populations and 0.37% of Haliotis abalone populations ).
Here, we focus on a group of closely related taxa that extensively hybridize in nature. Mussels in the Mytilus edulis (Mollusca: Bivalvia) species group (M. edulis, M. galloprovincialis, and M. trossulus) are common in the intertidal and shallow subtidal areas of the temperate Northern and Southern hemispheres . In the Northern Hemisphere, M. edulis is present in the North Atlantic on both the North American and the European coastlines. M. trossulus is present in the North Pacific, Atlantic Canada, and the Baltic Sea. M. galloprovincialis is native to the Mediterranean and Black Seas and western Europe, and it has been recently introduced to California and Asia . Some hybrid individuals have been found in every area where these species are sympatric: M. edulis and M. galloprovincialis in western Europe , M. edulis and M. trossulus in northern Europe and in Atlantic Canada), and M. trossulus and M. galloprovincialis in California and Asia ( but see ). In fact, hybridization is sufficiently frequent that hybrids comprise over 30% of the mussels in some regions of the major hybrid zones .
Despite extensive hybridization, these three species remain genetically distinct throughout much of their species range; therefore, some prezygotic or postzygotic processes must contribute to reproductive isolation. The mechanisms of fertilization in mussels have not been well studied, although recent laboratory crosses show that there is some degree of reduced fertilization success between M. edulis and M. galloprovincialis , suggesting that gamete recognition might play a role in isolating these species. In experimental crosses, F1 hybrid viability is also reduced for M. edulis x M. galloprovincialis and M. edulis x M. trossulus crosses, demonstrating some level of postzygotic isolation among the three species. However, F2 and backcross-type hybrid individuals are found in mussel hybrid zones, indicating that at least some F1 hybrids are fertile .+?/, http://www.100md.com
found three proteins in high concentration on the acrosomal surface of Mytilus sperm. One of these three proteins showed strong lysogenic activity, nonenzymatically dissolving the egg vitelline coat. This 20 kDa protein, which Takagi and colleagues named M7 lysin, also reinitiates the suspended meiosis by releasing the egg's first polar body. M7 lysin does not exhibit significant sequence similarity to lysins from abalone or snails, rather is classified by its function. Given the rapid rate of protein evolution observed for lysins in these gastropods, the lack of obvious similarity is not surprising and does not necessarily suggest independent origins of the different lysins.
Understanding how selection drives the evolution of sperm proteins and understanding the role of sperm proteins in speciation will be aided by studying such genes in diverse taxa, particularly among species with incomplete reproductive isolation. Consequently, we have sequenced multiple alleles of the mature protein coding region of M7 lysin in allopatric populations of M. edulis, M. galloprovincialis, and M. trossulus. By examining the patterns of polymorphism and divergence, we demonstrate that Mytilus M7 lysin has evolved in a manner consistent with positive selection, as with other marine invertebrate sperm proteins. This study provides a new phylogenetic perspective by demonstrating that positive selection of gamete recognition genes also occurs in bivalve mollusks. This is also the first evidence for positive selection on a marine invertebrate gamete recognition gene in a group characterized by extensive hybridization among species.|\0, http://www.100md.com
Methods|\0, http://www.100md.com
RNA Isolation and Sequencing
Mytilus edulis was collected from Woods Hole, Mass., and M. galloprovincialis was collected from Samos, Greece. These regions are known to have pure (nonhybrid) populations of each species. M. trossulus was collected from Penn Cove, Wash. Because imported M. galloprovincialis are cultivated in Washington alongside the native M. trossulus, the species identity of the M. trossulus individuals was confirmed using sequences of the mitochondrial COIII gene .[k*5[, 百拇医药
Total RNA was extracted from mantle tissue using Tri Reagent (Sigma). Fresh tissue was homogenized in 0.5 ml Tri Reagent and vortexed briefly after a 0.2 ml addition of choloroform. The tissue-Tri Reagent-choloroform mixture was centrifuged at 10,000 RPM for 15 min at 4°C. The top aqueous phase was pipetted into a fresh tube, and RNA was precipitated with 0.5 ml isopropanol. The RNA pellet was washed with 75% ethanol and dried at room temperature. The RNA pellet was resuspended in RNase-free water.[k*5[, 百拇医药
sequenced M7 lysin from one individual of M. galloprovincialis. To identify a part of the 3' untranslated region that was conserved among all three species and would thus be a suitable primer site, 3' RACE was used to amplify and sequence the 3' region from several individuals of M. edulis and M. trossulus. Total mRNA was reverse transcribed with SUPERSCRIPT RNase H Reverse Transcriptase (Life Technologies) using an oligo-dT adaptor (5'-AAG GAT CCG TCG ACA TCG ATA ATA CGA CAT TTT TTT TTT TTT TTT T-3'), and PCR was performed using the degenerate primer of Tagaki et al. (5'-GGA TTC AAY CAY GTN CAN GGN GAR AC-3') and a primer matching part of the adaptor sequence (5'-AAG GAT CCG TCG ACA TC-3'). A second, nested reaction was performed using a small amount of the first PCR product and the primers MEL-F6 (5'-AAC AAG GAT CAT GCA AAC -3') and a second primer matching the adaptor sequence (5'-GAC ATC GAT AAT ACG AC-3'), which amplified fragments from a number of M. edulis and M. trossulus individuals. These sequences of the 3' region, along with the Takagi et al. sequence of the 5' region, were used to design primers targeting the untranslated regions (UTRs) that would amplify the entire coding region of M7 lysin: MEL-F7, 5'-TGG AAG CCA AAT CTT TAC AT-3', and MEL-R5, 5'- GTC TTT AGT TCT TTA TTT TAT ATT GAC-3'. PCR was performed with Promega Taq DNA polymerase (Madison, Wis.) for 35 cycles of 20 s at 94°C, 30 s at 54°C, and 80 s at 72°C and amplified an approximately 770 bp fragment containing the entire 540 bp coding sequence for the mature peptide and 26 bp of the 5' UTR. The length of the amplified 3' UTR varied among alleles and was approximately 200 bp long.
PCR products were cloned (TA Cloning: Invitrogen). PCR with M13 primers was performed on individual colonies. These M13 PCR reactions were cleaned with a Qiaquick PCR Purification Kit (Qiagen) and then sequenced with M13 primers using the ABI PRISM BigDye Terminators ver. 2.5 Cycle Sequencing Kit. Sequencing reactions were visualized with an ABI 3700 DNA Analyzer. For each individual mussel, multiple colonies were sequenced in order to discriminate between polymorphism and polymerase error. Thus, two alleles were inferred for each individual, and each allele was represented by a minimum of three sequenced clones. Sequence chromatograms were visualized and edited using Sequencher ver. 4.1 software.yw#l, http://www.100md.com
Descriptive Statistics and Sequence Similarityyw#l, http://www.100md.com
Genetic variation within each of the three species was described using a number of measures. These measures do not test for selection but are useful for comparing M7 lysin to other genes. DNAsp ver. 2.5 ) was used to estimate species heterozygosity = 4Nµ, where N is the effective population size and µ is the neutral mutation rate) based on the number of segregating sites and the average number of pairwise differences . These two estimators of capture different elements of the frequency distribution of polymorphisms. Recombination rates were estimated by the methods of in the program SITES .
Although M7 lysin is one of the most abundant proteins in mussel sperm acrosomes, found no similarity between M7 and any other previously published sperm protein. Since their original description of M7, however, many more protein sequences have been deposited in public databases. To verify their results, we performed a protein BLAST search against the SwissProt and TrEMBL databases of proteins using the default BLOSUM matrix. In addition, we explicitly tried BLASTing the M7 lysin protein sequence against abalone lysin , teguline lysin (, and gastropod TMAP (, the other described sperm proteins for mollusks. Protein secondary structure was predicted for M. edulis using the PHDsec program implemented by PredictProtein for comparison to gastropod lysins. The charge and isoelectric static point of M7 lysin was determined by EMBL Isoelectric Point Service .a9tx!q', http://www.100md.com
Tests of Selectiona9tx!q', http://www.100md.com
We analyzed patterns of genetic variation within and among species in a number of ways to determine whether positive selection has shaped genetic variation at the mussel M7 lysin gene. The observed frequency spectra of polymorphisms for each species were compared with neutral expectations using Tajima's D statistic , as implemented in DNAsp. Heterogeneity in the polymorphism to divergence ratios across the gene was tested using the runs test, sliding G-test, and Kolmogorov-Smirnov test of . Because there was no a priori estimate of the whole-sequence recombination parameter, R, the program DNA Slider was run for values of R = 2, 4, 8, 16, etc., until P, the probability of obtaining the observed heterogeneity by chance, reached a peak and started to decline. For each value of R, 1,000 replicates were run; if the peak value of P was less than 0.10 for any test, 10,000 replicates were run. This was done for synonymous variation alone and for synonymous and amino acid replacement variation combined. One M. trossulus sequence (T765A) was used as the outgroup for testing heterogeneity within M. edulis and M. galloprovincialis, and one M. edulis sequence (E390B) was used as the outgroup for M. trossulus.
Relative frequencies of synonymous and amino acid replacement substitutions between species were also used to test for selection. McDonald-Kreitman tests compare the number of synonymous and nonsynonymous fixations (between species) with polymorphisms (within species). Standard McDonald-Kreitman tests were conducted on the whole data set. We also tested for an excess of radical amino acid fixations between species in the McDonald-Kreitman test framework using radical and conservative amino acid change definitions (charge, polarity, and polarity with volume) based on .ps:ou), 百拇医药
In addition, we looked at ratios of synonymous and nonsynonymous divergence along the three species branches, where a ratio of nonsynonymous to synonymous substitutions greater than unity is taken as evidence for positive selection. In MEGA 2.0 , the proportions of nonsynonymous substitutions per nonsynonymous site (dN) and synonymous substitutions per synonymous site (dS), based on the modified method, were calculated across the whole gene and for a 30-codon sliding window across the gene. Where dN was greater than dS, the significance of this difference was tested with Fisher's exact test . Yang and others have developed a series of nested ML tests that test for differences among dN/dS among codons in a gene and along branches of a tree . These methods were implemented with the CODEML program in PAML , based on a maximum likelihood tree generated in PAUP* (under an HKY with gamma model) .
Resultsn6#32, 百拇医药
Descriptive Statistics and Sequence Similarityn6#32, 百拇医药
Most of the sequenced fragments were M7 lysin, based on similarity to the sequence of ; a few M6 fragments from M. edulis were also sequenced but are not analyzed here (GenBank accession number ). No more than two distinct alleles were found per individual, consistent with M7 being a single copy gene. The sequenced fragments of M7 lysin included less than 26 bp of the 5' UTR, the entire 540-bp coding region, and 173 to 220 bp of the 3' UTR. The 3' UTR had a large amount of indel variation within and between species, making it difficult to align accurately; therefore only the coding region is analyzed here.n6#32, 百拇医药
It is unclear whether M7 lysin shares a common origin with gastropod lysins, as protein BLAST searches and analyses of protein structure failed to reveal any significant similarities between Mytilus M7 lysin and other proteins, including gastropod sperm proteins. Two {alpha}
-helices were predicted, at amino acid positions 77 to 86 and 97 to 109, and several short (<10 a.a.) ß-sheets were predicted interspersed along the protein. The two predicted {alpha}im;n, 百拇医药
-helices fall in the central region of the protein, a region containing {alpha}im;n, 百拇医药
-helices in abalone (based on crystallography: ). However, many of the other {alpha}im;n, 百拇医药
-helical regions in abalone lysin (and predicted for teguline lysin ) are not predicted for mussel lysin. M7 lysin is inferred to have a charge of –0.02 and an isoelectric point at 8.5, less than that for gastropods (10.0 to 11.6) ( Hellberg and Vacquier 1998). Although a common origin for gastropod and bivalve lysins cannot be rejected, there is no clear evidence to substantiate homology.im;n, 百拇医药
In all, we sequenced eight alleles from M. trossulus (GenBank accession numbers to ), 10 alleles from M. edulis (GenBank accession numbers to ), and six alleles from M. galloprovincialis (GenBank accession numbers to ). Although described their sequence from a Japanese mussel as being from an "M. edulis" individual, we include it with M. galloprovincialis because it is most similar to our Mediterranean M. galloprovincialis sequences and because introduced populations of M. galloprovincialis are the only "edulis-like" mussel in most of Japan (. Thus, our sample size for M. galloprovincialis includes seven alleles.
There was variation at M7 lysin both between and within species. Estimates of heterozygosity ({theta} ) were fairly similar across the three species . Allelic variation within species was characterized by recombination. The method described considerable recombination within M. edulis and M. trossulus , and our limited survey of within-species diversity found instances of all four possible recombinant gametes for M. galloprovincialis (positions 52 and 292; ) and for M. edulis (positions 252 and 510; ). A direct consequence of recombination is that there is no single genealogy for the alleles sampled. However, each species was defined by unique substitutions. There were 28 fixed differences (11 synonymous and 17 amino acid replacements) between the M. trossulus alleles and M. edulis and M. galloprovincialis alleles ( and ). M. edulis and M. galloprovincialis were each defined by one nonsynonymous substitution (sites 56 and 144; ). Thus, the species relationships inferred from lysin are the same as those inferred from mtDNA, where the M. trossulus species branch is the longest and there is less divergence between M. edulis and M. galloprovincialis .
fig.ommitted7?3vp, 百拇医药
Table 1 Allelic Diversity Within Mussel Species.7?3vp, 百拇医药
fig.ommitted7?3vp, 百拇医药
FIG. 1. Polymorphic nucleotide sites in the mature M7 lysin protein. Dots show identity to the consensus sequence. Species identities of alleles are described by the letters G, E, or T, where G is M. galloprovincialis, E is M. edulis, and T is M. trossulus, and A and B refer to two alleles within a single individual. For example, T776B is the B allele from M. trossulus individual 776. On the bottom row, S and N indicate whether polymorphisms are synonymous or nonsynonymous (e.g., causing amino acid substitutions). In two cases where there are three substitutions at the same site (positions 321 and 489), there is a synonymous polymorphism within M. edulis and a fixed nonsynonymous difference between M. trossulus and M. edulis/galloprovincialis.7?3vp, 百拇医药
fig.ommitted7?3vp, 百拇医药
FIG. 2. Alignment of M7 protein sequences from three mussel species. Dots show identity to the consensus sequence. Amino acid sites that are polymorphic within species are underlined. At two amino acid sites (positions 96 and 108, italicized) Mytilus trossulus is distinguished by two separate nucleotide substitutions, both of which are nonsynonymous. Substitutions at sites 56 and 144 are unique to M. edulis and M. galloprovincialis, respectively, and are shown in boldface
Tests of Selection28i5&, 百拇医药
Our tests of selection revealed an excess of fixed amino acid replacement differences between species, consistent with positive selection on the mussel lysin gene. A McDonald-Kreitman test of the entire data set was statistically significant (P < 0.0167) (, with nonsynonymous fixations exceeding synonymous fixations between species and synonymous polymorphisms exceeding nonsynonymous polymorphisms within species. McDonald-Kreitman tests based on ratios of conservative to radical amino acid substitutions did not show an excess of radical fixed differences. In fact, the proportion of fixed differences was greater for conservative changes than for radical changes considering charge, polarity, and polarity with volume change.28i5&, 百拇医药
fig.ommitted28i5&, 百拇医药
Table 2 McDonald-Kreitman Test of Selection Across All Three Species.28i5&, 百拇医药
While the McDonald-Krietman test demonstrated a pattern consistent with positive selection, the overall ratio of dN/dS was less than 1 (M. edulis vs. M. galloprovincialis: 0.25; M. edulis vs. M. trossulus: 0.48; M. galloprovincialis vs. M. trossulus: 0.48). A dN/dS less than 1 is expected if most replacement mutations are deleterious, which is likely to be the case for most protein coding genes. Sliding window analyses of dN/dS ratios located a region in the 3' end of the gene where dN/dS is greater than 1, but Fisher's exact test revealed that dN was not significantly greater than dS for any of the sliding window partitions. None of the maximum likelihood methods for identifying selection at specific sites or along specific branches yielded significant results. However, these methods assume no recombination and are not sensitive to changes along short branches such as those among closely related species .
Tajima's D statistic test of selection based on the frequency spectrum of polymorphisms did not show any strong deviations from neutrality ). The runs test, sliding G-test, and Kolmogorov-Smirnov test did not reveal significant heterogeneity in the polymorphism to divergence ratio within either M. edulis or M. trossulus. However, along the length of the M. galloprovincialis gene, the Kolmogorov-Smirnov test revealed significant heterogeneity, both for synonymous variation (P = 0.026) and for synonymous and nonsynonymous variation combined (P = 0.025), with the beginning of the sequence having a higher ratio of polymorphism to divergence than the latter portion .{ow%fu, 百拇医药
fig.ommitted{ow%fu, 百拇医药
FIG. 3. Sliding window plot of the polymorphism to divergence ratio in M. galloprovincialis, with M. trossulus used for divergence. For each window of 10 variable sites, the proportion of variable sites that are polymorphic (combined synonymous and nonsynonymous) was calculated. Each value is plotted at the midpoint of the window. G indicates the position of the nonsynonymous substitution defining the M. galloprovincialis lineage at bp position 430. Kolmogorov-Smirnov tests revealed significant heterogeneity in the polymorphism to divergence ratio both for synonymous variation (P = 0.026) and for combined synonymous and nonsynonymous variation (P = 0.025)
Discussion')ewn4(, 百拇医药
Positive Selection in Mussel Lysin')ewn4(, 百拇医药
In this first survey of molecular variation of a bivalve sperm protein, we find evidence for positive selection shaping patterns of genetic variation between closely related species that frequently hybridize in nature. The strongest evidence for positive selection comes from the significant (P < 0.0167) McDonald-Kreitman test. If we assume that ratios of replacement to synonymous polymorphism within species approximate a neutral distribution, then the McDonald-Krietman test points to an excess of fixed replacement substitutions between species. The proportion of adaptive substitutions can be estimated as {alpha}')ewn4(, 百拇医药
= 1-DSPN/DNPS , where DS and DN are the number of synonymous and nonsynonymous differences between species, and PS and PN are the number of synonymous and nonsynonymous polymorphisms. For this data set we estimate that 75% of the nonsynonymous differences between species are adaptive. Thus, the pattern of selection observed at mussel lysin confirms the emerging conclusion that marine invertebrate sperm proteins are frequently characterized by increased rates of amino acid substitutions.
Other studies have found dN/dS greater than 1 for gastropod lysins. However, many genes undergoing adaptive evolution are likely to have dN/dS less than 1, because the majority of nonsynonymous substitutions are probably deleterious even if a small fraction are adaptive. Here we find evidence for positive selection at a reproductive protein even though dN/dS is less than 1. This observation emphasizes the value of examining within-species polymorphism in addition to between-species divergence.|40^{q, 百拇医药
In addition to the significant McDonald-Kreitman test, significant heterogeneity in the polymorphism to divergence ratio along M7 lysin in M. galloprovincialis suggests that adaptive evolution has occurred. One nonsynonymous nucleotide substitution occurred in the M. galloprovincialis lineage after it split from M. edulis. This substitution is at amino acid site 144, near the 3' end of the coding sequence, which has a lower polymorphism/divergence ratio than the 5' end of the gene . Such a pattern is expected if a rapid, recent substitution at site 144 swept away the polymorphism in the 3' end of the gene, while, because of recombination, the 5' end of the gene was unaffected by the sweep. At least one intron of unknown length occurs in the lysin gene of Mytilus (Riginos, unpublished data), so it is not implausible that different parts of the coding region would have different evolutionary histories.
Comparisons of Mussel Lysin to Other Marine Invertebrate Sperm Proteinsm, 百拇医药
Like other lysins, Mytilus M7 lysin is found on the sperm acrosome and dissolves the egg vitelline envelope . However, its structure is not demonstrably similar to gastropod lysins, the only other molluscan lysins that have been described. Lysin proteins evolve quite rapidly, and so the lack of similarity is not surprising. For example, the rate of nonsynonymous substitutions per site per 109 years is estimated at 6 to 84 for abalone and 21 to 76 for snails . We estimate a rate of 6.5 for mussels, assuming a 3.5 million year divergence between M. trossulus and M. edulis/galloprovinicialis . Given these high substitution rates and a Cambrian gastropod-bivalve divergence (570 MYA; ), recognizable sequence similarity between gastropod and bivalve lysins is not necessarily expected.m, 百拇医药
All marine invertebrate sperm proteins studied to date show evidence of adaptive evolution. However, the patterns of intraspecific polymorphism and rates of divergence are substantially different. Gastropod sperm proteins have virtually no polymorphism and divergence is extremely rapid , suggesting that recent selective sweeps have removed any intraspecific variation. Sea urchin bindin genes, in constrast, are characterized by an excess of nonsynonymous variation between and within species , which may result from positive selection on new amino acid–changing mutations or positive assortative mating In mussels, we find a third pattern. As in gastropod lysins and urchin bindins, there is evidence that the majority of replacement substitutions between species are adaptive. However, unlike gastropod lysins, adaptive substitutions have not occurred so frequently that polymorphism has been swept away. Unlike urchin bindins, there is no significant evidence from the dN/dS ratio that amino acid polymorphisms are adaptive.
Clearly, more extensive surveys are needed to fully characterize the nature of intraspecific variation in Mytilus lysin. If lysin is directly involved in prezygotic isolation, then specific selection pressures may differ among populations, particularly those where hybridization does and does not occur. It will also be particularly interesting to examine this gene in populations of uncertain origin, such as those in the Baltic Sea and the Southern Hemisphere .l^a^.+8, 百拇医药
Given that all three Mytilus species surveyed here hybridize extensively in nature, our results imply that the action of selection on mussel lysin precedes the attainment of complete postzygotic reproductive isolation. Our results substantiate the emerging pattern that marine invertebrate sperm proteins are characterized by positive selection, particularly when ranges of closely related species overlap. It remains to be seen whether a common explanation underlies the general observation of adaptive evolution on genes involved in sexual reproduction.
Acknowledgements8gcp(1a, http://www.100md.com
We thank C. W. Cunningham, A. J. Abrams, B. Y. Kong, K. Zigler, and S. R. Palumbi for their assistance and comments throughout this project. Suggestions from three anonymous reviewers substantially improved this manuscript. This project was funded by the Duke University program in Molecular Evolution and Comparative Genomics, the PADI Foundation, and the National Science Foundation.8gcp(1a, http://www.100md.com
Literature Cited8gcp(1a, http://www.100md.com
Anisimova, M., J. P. Bielawski, and Z. Yang. 2001. Accuracy and power of the likelihood ratio test in detecting adaptive molecular evolution. Mol. Biol. Evol. 18:1585-1592.8gcp(1a, http://www.100md.com
Benton, M. J., ed. 1993. The fossil record 2. Chapman & Hall, London.8gcp(1a, http://www.100md.com
Biermann, C. H. 1998. The molecular evolution of sperm bindin in six species of sea urchin (Echinoida: Strongylocentrotidae). Mol. Biol. Evol. 15:1761-1771.8gcp(1a, http://www.100md.com
Bierne, N., P. David, P. Boudry, and F. Bonhomme. 2002. Assortative fertilization and selection at larval stage in the mussels Mytlius edulis and M. galloprovincialis. Evolution 56:292-298.
Comesaña, A. S., J. E. Toro, D. J. Innes, and R. J. Thompson. 1999. A molecular approach to the ecology of a mussel (Mytilis edulis-M. trossulus) hybrid zone on the east coast of Newfoundland, Canada. Mar. Biol. 133:213-221.(ik}5r, 百拇医药
Gardner, J. P. A. 1996. The Mytilus edulis complex in southwest England: effects of hybridization and introgression upon interlocus associations and morphometric variation. Mar. Biol. 125:385-399.(ik}5r, 百拇医药
Heath, D. D., P. D. Rawson, and T. J. Hilbish. 1996. PCR-based nuclear markers identify alien blue mussel (Mytilus spp.) genotypes on the west coast of Canada. Can. J. Fish. Aquat. Sci. 52:2621-2627.(ik}5r, 百拇医药
Hellberg, M. E., G. W. Moy, and V. D. Vacquier. 2000. Positive selection and propeptide repeats promote rapid interspecific divergence of a gastropod sperm protein. Mol. Biol. Evol. 17:458-466.(ik}5r, 百拇医药
Hellberg, M. E., and V. D. Vacquier. 1999. Rapid evolution of fertilization selectivity and lysin cDNA sequences in teguline gastropods. Mol. Biol. Evol. 16:839-848.(ik}5r, 百拇医药
Hey, J., and J. Wakeley. 1997. A coalescent estimator of the population recombination rate. Genetics 145:833-846.
Hilbish, T. J., E. Carson, J. Plante, L. Weaver, and M. Gilg. 2002. Distribution of Mytilus edulis, M. galloprovinicialis, and their hybrids in open-coast populations of mussels in southwestern England. Mar. Biol. 140:137-142.', 百拇医药
Hilbish, T. J, A. Mullinax, S. I. Dolven, A. Meyer, R. K. Koehn, and P.D. Rawson. 2000. Origin of the antitropical distribution pattern in marine mussels (Mytilus spp.): routes and timing of transequatorial migration. Mar. Biol. 136:69-77.', 百拇医药
Howard, D. J. 1999. Conspecific sperm and pollen precedence and speciation. Ann. Rev. Ecol. Syst. 30:109-132.', 百拇医药
Hudson, R. R., and N. L. Kaplan. 1985. Statistical properties of the number of recombination events in the history of a sample of DNA sequences. Genetics 111:147-164.', 百拇医药
Kresge, N., V. D. Vacquier, and D. C. Stout. 2001. The crystal structure of a fusagenic sperm protein reveals extreme surface properties. Biochemistry 40:5407-5413.', 百拇医药
Kumar, S., K. Tamura, I. Jakobsen, and M. Nei. 2001. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244-1245.
Lee, Y.-H., T. Ota, and V. D. Vacquier. 1995. Positive selection is a general phenomenon in the evolution of abalone sperm lysin. Mol. Biol. Evol. 12:231-238.&\[i/|6, 百拇医药
Leighton, D. L., and C. A. Lewis. 1982. Experimental hybridization in abalones. Int. J. Inv. Repro. 5:273-282.&\[i/|6, 百拇医药
McDonald, J. H. 1998. Improved tests for heterogeneity across a region of DNA sequence in the ratio of polymorphism to divergence. Mol. Biol. Evol. 15:377-384.&\[i/|6, 百拇医药
McDonald, J. H., and R. K. Koehn. 1988. The mussels Mytilus galloprovincialis and M. trossulus on the Pacific coast of North America. Mar. Biol. 99:111-118.&\[i/|6, 百拇医药
McDonald, J. H., and M. Kreitman. 1991. Adaptive protein evolution at the ADH locus in Drosophila. Nature 351:652-654.&\[i/|6, 百拇医药
McDonald, J. H., R. Seed, and R. K. Koehn. 1991. Allozymes and morphometric characters of three species of Mytilus in the Northern and Southern hemispheres. Mar. Biol. 111:323-333.&\[i/|6, 百拇医药
Metz, E. C., G. Gómez-Gutiérrez, and V. D. Vacquier. 1998. Mitochondrial DNA and bindin gene sequence evolution among allopatric species of the sea urchin genus Arbacia. Mol. Biol. Evol. 15:185-195.
Metz, E. C., R. E. Kane, H. Yanagimachi, and S. R. Palumbi. 1994. Fertilization between closely related sea urchins is blocked by incompatibilites during sperm-egg attachment and early stages of fusion. Biol. Bull. 187:23-34.h\qo2/, http://www.100md.com
Metz, E. C., and S. R. Palumbi. 1996. Positive selection and sequence rearrangements generate extensive polymorphisms in the gamete recognition protein binding. Mol. Biol. Evol. 13:397-406.h\qo2/, http://www.100md.com
Metz, E. C., R. Robles-Sikisaka, and V. D. Vacquier. 1998. Nonsynonymous substitution in abalone sperm fertilization genes exceeds substitution in introns and mitochondrial DNA. Proc. Natl. Acad. Sci. USA 95:10676-10681.h\qo2/, http://www.100md.com
Nei, M., and T. Gojobori. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3:418-426.h\qo2/, http://www.100md.com
Nei, M., and W.-H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76:5269-5273.h\qo2/, http://www.100md.com
Nielsen, R., and Z. Yang. 1998. Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 148:929-936.
Owen, B., McLean, J. H., and R. J. Meyer. 1971. Hybridization in the eastern Pacific abalones (Haliotis). Bull. Los Angeles C. Mus. Nat. Hist. Sci. 9:1-37.7k]]()g, 百拇医药
Palumbi, S. R. 1999. All males are not created equal: fertility differences depend on gamete recognition polymorphisms in sea urchins. Proc. Natl. Acad. Sci. USA 96:12632-12637.7k]]()g, 百拇医药
Palumbi, S. R., and E. C. Metz. 1991. Strong reproductive isolation between closely related tropical sea urchins (genus Echinometra). Mol. Biol. Evol. 8:227-238.7k]]()g, 百拇医药
Rawson, P. D., V. Agrawal, and T. J. Hilbish. 1999. Hybridization between the blue mussels Mytilus galloprovincialis and M. trossulus along the Pacific coast of North America. Mar. Biol. 134:201-211.7k]]()g, 百拇医药
Rawson, P. D., and T. J. Hilbish. 1995. Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex. Mol. Biol. Evol. 12:893-901.7k]]()g, 百拇医药
Rawson, P. D., C. L. Secor, and T. J. Hilbish. 1996. The effects of natural hybridization on the regulation of doubly uniparental mtDNA inheritance in blue mussels (Mytilus spp.). Genetics 144:241-248.
Riginos, C., K. Sukhdeo, and C. W. Cunningham. 2002. Evidence for selection at multiple allozyme loci across a mussel hybrid zone. Mol. Biol. Evol. 19:347-351.!c, 百拇医药
Rost, B., and C. Sander. 1993. Prediction of protein secondary structure at better than 70% accuracy. J. Mol. Biol. 232:584-599.!c, 百拇医药
Rozas, J., and R. Rozas. 1999. DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174-175.!c, 百拇医药
Saavedra, C., D. T. Stewart, R. R. Stanwood, and E. Zouros. 1996. Species-specific segregation of gender-associated mitochondrial DNA types in an area where two mussel species (Mytilus edulis and M. trossulus) hybridize. Genetics 143:1359-1367.!c, 百拇医药
Shaw, A., D. E. McRee, V. D. Vacquier, and C. D. Stout. 1993. The crystal structure of lysin, a fertilization protein. Science 262:1864-1867.!c, 百拇医药
Skibinski, D. O. F., and J. A. Beardmore. 1979. A genetic study of intergradation between Mytilus edulis and M. galloprovincialis. Experientia 35:1442-1444.
Skurikhina, L. A., Y. F. Kartavtsev, A. Y. Chichvarkhin, and M. V. Pankova. 2001. Study of two species of mussels, Mytilus trossulus and Mytilus galloprovinicialis (Bivalvia, Mytilidae), and their hybrids in Peter the Great Bay of the Sea of Japan with the use of PCR markers. Russ. J. Gen. 37:1448-1451.0}, 百拇医药
Smith, N. G., and A. Eyre-Walker. 2002. Adaptive protein evolution in Drosophila. Nature 415:1022-1024.0}, 百拇医药
Suchanek, T. H., J. B. Geller, B. R. Kresier, and J. B. Mitton. 1997. Zoogeographic distributions of the sibling species Mytilus galloprovincialis and M. trossulus (Bivalvia: Mytilidae) and their hybrids in the North Pacific. Biol. Bull. 193:187-194.0}, 百拇医药
Swanson, W. J., and V. D. Vacquier. 1995. Extraordinary divergence and positive selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa. Proc. Natl. Acad. Sci. USA 92:4957-4961.0}, 百拇医药
Swanson, W. J., and V. D. Vacquier. 2002. The rapid evolution of reproductive proteins. Nature Rev. Gen. 3:137-144.0}, 百拇医药
Swofford, D. L. 1998. PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Mass.
Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585-595.y, http://www.100md.com
Takagi, T., A. Nakamura, R. Deguchi, and K. Kyozuka. 1994. Isolation, characterization, and primary structure of three major proteins obtained from Mytilus edulis sperm. J. Biochem. 116:598-605.y, http://www.100md.com
Vacquier, V. D. 1998. Evolution of gamete recognition proteins. Science 281:1995-1998.y, http://www.100md.com
Vacquier, V. D., K. R. Carner, and C. D. Stout. 1990. Species-specific sequences of abalone lysin, the sperm protein that creates a hole in the egg envelope. Proc. Natl. Acad. Sci. USA 87:5792-5796.y, http://www.100md.com
Vacquier, V. D., W. J. Swanson, and Y.-H. Lee. 1997. Positive Darwinian selection on two homologous fertilization proteins: what is the selective pressure driving their divergence? J. Mol. Evol. 44:S15-S22.y, http://www.100md.com
Väinölä, R., and M. M. Hvilsom. 1991. Genetic divergence and a hybrid zone between Baltic and North Sea Mytilus populations (Mytilidae: Mollusca). Biol. J. Linn. Soc. 43:127-148.
Vermeij, G. 1991. Anatomy of an invasion: the trans-Arctic interchange. Paleobiology 17:281-307.u*34t, 百拇医药
Watterson, G. A. 1975. On the number of segregating sites in genetical models without recombination. Theor. Pop. Bio. 7:256-276.u*34t, 百拇医药
Yang, Z. 1997. PAML: a program for phylogenetic analysis by maximum likelihood. CABIOS 13:555-556.u*34t, 百拇医药
Yang, Z., and R. Nielsen. 1998. Synonymous and nonsynonymous rate variation in nuclear genes of mammals. J. Mol. Evol. 46:409-418.u*34t, 百拇医药
Yang, Z., R. Nielsen, N. Goldman, and A.-M. K. Pedersen. 2000. Codon-substitution models for heterozygous selection pressure at amino acid sites. Genetics 155:431-449.u*34t, 百拇医药
Yang, Z., W. J. Swanson, and V. D. Vacquier. 2000. Maximum-likelihood analysis of molecular adaptation in abalone sperm lysin reveals variable selective pressures among lineages and sites. Mol. Biol. Evol. 17:1446-1455.u*34t, 百拇医药
Zhang, J. 2000. Rates of conservative and radical nonsynonymous nucleotide substitutions in mammalian nuclear genes. J. Mol. Evol. 50:56-68.u*34t, 百拇医药
Zhang, J., S. Kumar, and M. Nei. 1997. Small-sample tests of episodic adaptive evolution: a case study of primate lysozyme. Mol. Biol. Evol. 14:1335-1338.u*34t, 百拇医药
Zigler, K. S., and H. A. Lessios. 2003. Evolution of bindin in the pantropical sea urchin Tripneustes: comparisons to bindin of other genera. Mol. Biol. Evol. 20:220-231.u*34t, 百拇医药
Zouros, E., A. O. Ball, C. Saavedra, and K. R. Freeman. 1994a. Mitochondrial DNA inheritance. Nature. 368:817-818.u*34t, 百拇医药
Zouros, E., A. O. Ball, C. Saavedra, and K. R. Freeman. 1994b. An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus. Proc. Natl. Acad. Sci. USA 91:7463-7467.u*34t, 百拇医药
Accepted for publication October 9, 2002.(Cynthia Riginos and John H. McDonald)