Reference: Biol. Bull 182: 97-104. (February, 1992) The Divergence of Species-Specific Abalone Sperm Lysins is Promoted by Positive Darwinian Selection YOUN-HO LEE AND VICTOR D. VACQUIER Marine Biology Research Division 0202, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093 Abstract. Recognition by sperm lysin of the egg vitelline envelope may be one component in determining the spe-cies-specificity of fertilization in abalones. The amino acid sequences of lysin proteins of seven California abalone species were deduced from the cDNA sequences. This is the first extensive comparison of a gamete recognition protein from congeneric species. Each prelysin has a highly conserved signal peptide of 1 8 amino acids, followed by a mature sequence of 136-138 residues. Of 136 aligned positions, 68 have the same amino acid in all seven se-quences. The % identity relative to the red abalone lysin sequence is: white 90%, flat 83%, pinto 82%, pink 78%, black 71%, and green 65%. Hydropathy plots and a dis-tance tree of the seven lysins show that red, white, and flat lysins are more closely related to each other than to the lysins of the other four species. A hypervariable, spe-cies-specific, domain exists in all sequences between po-sitions 2-12. Amino acid replacements between any two lysins are mostly nonconservative. Analysis of the cDNA sequences shows the number of nonsynonymous substi-tutions (amino acid altering) exceeds the number of syn-onymous substitutions (silent) in 20 of the 21 pairwise comparisons of the seven sequences, indicating that pos-itive Darwinian selection must promote the divergence of lysin sequences. Introduction A striking feature of fertilization is the species specificity of sperm-egg interaction in mammals (O'Rand, 1988; Yanagimachi, 1988a, 1988b; Roldan and Yanagimachi, 1989) and invertebrates (Giudice, 1973; Summers and Received 25 July 1 99 1 ; accepted 1 1 October 1991. Abbreviations: Mr, relative molecular mass; VE, vitelline envelopes of abalone eggs. Hylander, 1975, 1976; Osanai and Kyozuka, 1982). Sperm-egg mixtures from the same species usually yield zygotes more efficiently than cross-species mixtures. Al-though cross-species hybrid zygotes can be obtained in mammals and invertebrates, the general observation is that much higher concentrations of sperm are needed in the insemination mixture to achieve fertilization. Blocks to cross-species fertilization can occur at four points in the process: induction of the sperm acrosome reaction by components of the egg surface, adhesion of sperm to the egg envelope, sperm penetration of the egg envelope, and fusion of sperm and egg cell membranes. In echinoderms, the greatest barrier to cross-species fertilization is the fail-ure of sperm to adhere to the egg vitelline envelope (Sum-mers and Hylander, 1975, 1976); in mammals it is the failure of sperm to adhere to and penetrate the egg zona pellucida (O'Rand, 1988; Yanagimachi, 1988a,b; Roldan and Yanagimachi, 1989). The divergence of gamete recognition proteins may be important in the establishment of barriers to cross-fertil-ization between populations. This may be an important factor in the speciation of marine invertebrates using ex-ternal fertilization. To learn how species-specific gamete recognition proteins have diverged during evolution, we have studied a protein from abalone sperm. Abalones are marine archeogastropods of the genus Haliotis. Approx-imately 70 extant species occur on coastlines of the world, eight of them on the Pacific Coast of North America. Although abalones are members of an ancient group of gastropods, the genus Haliolis is relatively recent, most fossils being from the Miocene (5-25 million years; Lind-berg, 1991). The abalone egg is contained within a glycoproteina-ceous vitelline envelope (VE) about 0.6 /im in thickness (Lewis et a/., 1982). The spermatozoon possesses a rela-tively enormous acrosome granule (Lewis et ai, 1980; 97
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