*Department of Molecular Biology and Genetics, Cornell University;
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For example, the sea urchin sperm protein bindin shows high levels of sequence polymorphism and an excess of amino acid replacements compared to silent substitutions (Metz and Palumbi 1996
). Recently, it was shown that bindin sequence polymorphism has consequences relating to fertilization success (Palumbi 1999
). The fastest evolving protein in Drosophila is the male reproductive protein Acp26Aa (Herndon and Wolfner 1995
; Schmid and Tautz 1997
; Tsaur and Wu 1997
; Aguadé 1998
). Analysis of sequence divergence between closely related species has demonstrated that the evolution of Acp26Aa is driven by positive Darwinian selection, but the selective pressure remains a mystery (Tsaur and Wu 1997
). Additionally, the female receptor for Acp26Aa remains unknown. The only system in which the evolution of both cognate male and female reproductive proteins has been investigated is that of the abalone (genus Haliotis; Swanson and Vacquier 1998
).
The abalone system is also the best characterized system for understanding the molecular and evolutionary basis for species-specific fertilization (Swanson et al. 1998
). Abalones are large marine archeogastropods with external fertilization. Seven species coexist off the west coast of North America; many have overlapping breeding seasons and habitats. Despite the potential for hybridization, the species remain distinct, with hybrids only rarely being found in the wild (Owen, McLean, and Meyer 1971
). The basis for maintaining distinct species could be in part attributed to species-specific fertilization, which can be quantitatively demonstrated in the laboratory (Leighton and Lewis 1982
).
Following the release of gametes, the events of abalone fertilization which can exhibit species specificity include chemotaxis of sperm to the egg, induction of the sperm acrosome reaction, dissolution of the egg vitelline envelope (VE), and binding and fusion of the two gametes (Vacquier and Lee 1993
; Swanson and Vacquier 1997
; Vacquier 1998
). The dissolution of the VE has been extensively studied in the abalone and has been demonstrated to exhibit species specificity (Vacquier, Carner, and Stout 1990
). Dissolution of the VE is mediated by the sperm protein lysin, which nonenzymatically creates a hole in the VE by stereospecifically competing for hydrogen bonds and hydrophobic interactions among the fibers comprising the VE, leading to the unraveling of the fibers and the creation of a hole in the VE (Lewis, Talbot, and Vacquier 1982
). The vitelline envelope receptor for lysin (VERL) is a fibrous molecule of 1,000 kDa containing approximately 28 repeats of 153 amino acids (Swanson and Vacquier 1997, 1998
). In-solution binding kinetics demonstrate that lysin and VERL interact with high affinity (EC50 10 nM). Furthermore, lysin-VERL binding shows positive cooperativity and the same species-specificity as does lysin mediated VE dissolution, indicating that the specificity resides in the two isolated molecules (Swanson and Vacquier 1997
).
Lysin is extremely divergent between closely related species (Lee, Ota, and Vacquier 1995
) and is among the fastest evolving metazoan proteins yet discovered (Metz, Robles-Sikisaka, and Vacquier 1998
). Analysis of the number of nonsynonymous substitutions per nonsynonymous site (dN) compared with that of synonymous substitutions per synonymous site (dS) shows that dN exceeds dS by as much as fourfold in pairwise comparisons of species (Lee and Vacquier 1992
; Lee, Ota, and Vacquier 1995
). Lysin is monomorphic in California red abalone, indicating that red abalone lysin evolution proceeds by a series of selective sweeps (Metz, Robles-Sikisaka, and Vacquier 1998
). Amino acid replacements are scattered over the entire sequence, but the N- and C-termini are especially divergent between species. Site-directed mutagenesis shows that the species specificity can be attributed, in part, to the N- and C-termini (Lyon and Vacquier 1999
).
To gain insights into the selective forces driving the rapid divergence of lysin, the evolution of VERL was previously investigated by sequencing VERL repeats between species of abalone (Swanson and Vacquier 1998
). VERL repeats were shown to be more similar within a species than between species. This process of homogenization of repeats has been termed concerted evolution and occurs by unequal crossing over and gene conversion (Elder and Turner 1995
). In contrast to lysin from these same species (Lee and Vacquier 1992
), the VERL repeats did not show signs of positive Darwinian selection based on dN/dS ratios. It was hypothesized that the redundant nature of the VERL molecule reduced the functional constraint of each VERL repeat, leading to relaxed selection on repeats (Swanson and Vacquier 1998
).
To investigate the evolutionary forces affecting the divergence of VERL, a polymorphism survey of the VERL locus was conducted. To insure comparison of orthologous regions of the repetitive VERL molecule, we identified the C-terminal VERL repeat (the last repeat) and a novel nonrepetitive portion of the protein. After obtaining polymorphism sequence data for both pink abalone (Haliotis corrugata) and red abalone (Haliotis rufescens) species, additional tests of neutrality were performed. Some of these tests may be more powerful at detecting certain types of positive Darwinian selection than interspecific dN/dS ratios calculated over the entire molecule (Kreitman and Akashi 1995
; Aquadro 1997
).
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Polymorphism PCR
DNA was prepared as described in Metz, Robles-Sikisaka, and Vacquier (1998)
. Briefly, one peripheral epipodial tentacle was clipped from each living abalone, washed in 20 mM Tris (pH 7.6) and 20 mM EDTA, and homogenized in 300 µl of the same solution containing 10% (vol/vol) chelating resin (Sigma) in a 1.5-ml tube. The tube was boiled for 5 min, vortexed, and centrifuged. PCR amplifications for the last VERL repeat and a portion of C-terminal nonrepeat region were carried out with primers vbend2 (CCAGAGCAAACTGATCGACTG) and mendR (CCATGATACTCCTATGTGCAG) in a 50-µl reaction containing 50 pmol of each primer, 1 µl Taq polymerase, 1 x Taq polymerase buffer (Promega), 1.5 mM MgCl2, 0.2 mM of each dNTP, and 1 µl of template DNA. Thermal cycle settings were 94°C for 30 s, 50°C for 30 s, and 72°C for 90 s for 36 cycles. PCR products were purified and sequenced directly. The sequences presented here are available in GenBank under accession numbers AF250892AF250909.
Sequence Analysis
Sequences were aligned using CLUSTAL W (http://www2.ebi.ac.uk/clustalw). Maximum-likelihood analyses were performed using the PAML package (Yang 1999
: http://abacus.gene.ucl.ac.uk/ziheng/paml.html). For site variation, three likelihood ratio tests were used. First, we compared a neutral model (M1) with two dN/dS ratios (0 for conserved sites and 1 for neutral sites) to a selection model (M2) with an additional class of sites with a dN/dS ratio estimated from the data (Nielsen and Yang 1998
). The second test compared the neutral model (M1) to a selection model (M3) with three dN/dS classes estimated from a discrete distribution (Yang et al. 2000
). The third test compared a neutral model (M7) assuming a beta distribution of dN/dS among sites (Yang et al. 2000
). This is a flexible distribution, but dN/dS is limited in the interval (0, 1), where 0 indicates complete constraint and 1 is the expectation under no selective constraint. The alternative selection model (M8) adds an extra class of sites with dN/dS estimated from the data, thus allowing for positively selected sites (Yang 1999
). Twice the log likelihood difference between the two models was compared with the chi-square distribution, with the degrees of freedom based on the difference between the number of parameters estimated from the models (Nielsen and Yang 1998
; Yang 1999
; Yang et al. 2000
). For the lineage variation, one VERL repeat from each species was randomly chosen for the analysis. Twice the difference between the log likelihood differences of the models was compared with the chi-square distribution with 10 df (Yang 1998
). The analysis was repeated several times with different VERL repeats from each species, and similar results were obtained. A neighbor-joining tree was constructed using MEGA (Kumar, Tamura, and Nei 1993
) with 1,000 bootstrap replicates and pairwise deletion of gaps. Tajima's (1989)
D statistic, Fu and Li's (1993)
D* statistic, Hudson, Kreitman, and Aguadé (1987
; HKA) tests, and the McDonald-Kreitman (MK) test (McDonald and Kreitman 1991
) were all computed using DnaSP 3.0 (Rozas and Rozas 1999
). Significance values for Tajima's D statistic were obtained by coalescence simulations. The HKA test compared the last VERL repeat region with the C-terminal nonrepeat region.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
The VERL polymorphism and divergence data were used to test for departures from neutrality using a variety of statistical tests; no departures from neutrality were detected. First, the nonrepeat C-terminal region of VERL was compared to the last VERL repeat with the HKA test (table 3 ). If evolution is neutral, the ratio of divergence to polymorphism should be the same for the two regions. In all comparisons using the HKA test there was no departure from neutrality. The pink abalone HKA comparison remained nonsignificant when the two subdivided VERL sequences were analyzed separately (table 3 ). Our HKA tests used linked regions of VERL; the comparison to an unlinked neutral locus will be important goal for future work. The ratios of silent and replacement changes between species was then compared with the MK test. If evolution is neutral, the silent/replacement ratios should be similar for both the polymorphic changes and fixed divergent changes. For VERL, the ratios were identical, indicating no departure from neutrality. Finally, we calculated dN/dS for all possible pairwise comparisons between the polymorphic VERL repeats. In no case was dN significantly greater than dS (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It had been hypothesized that the redundant and repetitive nature of VERL repeats could lead to relaxed selection on individual VERL repeats within a single VERL molecule (Swanson and Vacquier 1998
). Since there may be 28 repeats in each VERL molecule, mutations in one repeat may be only weakly selected against (if at all), since the remaining 27 VERL repeats would remain functional. As long as the mutation does not disrupt the construction of the VE, from the female's perspective it may be tolerated. This would be particularly likely for mutations that affect VERL-lysin interaction, since there is potentially an excess of sperm (as suggested by observed spawning [Stekoll and Shirley 1993
] and comparison of other taxa [Yund 2000
]), such that even eggs with a reduced VERL-lysin affinity would most likely be fertilized.
It was also hypothesized that the neutral drift of the egg receptor VERL leads to a continually changing target to which lysin must adapt in order to maintain optimal interaction of sperm and egg cognate proteins. Selection would favor lysin variants that bind optimally, since once lysin is released, if the sperm does not fertilize the egg encountered, its genetic material would not be passed to the next generation. Thus, there may be considerable competition among sperm to fertilize the egg. The redundant nature of the VERL molecule is a key part of this hypothesis, as is the homogenization of VERL repeats by concerted evolution for the maintenance of the redundant nature of VERL and thus a single target to which lysin can adapt.
The analyses presented here are consistent with previous results demonstrating neutral evolution of the abalone egg receptor, VERL (Swanson and Vacquier 1998
), despite strong positive Darwinian selection on the cognate sperm protein lysin (Lee, Ota, and Vacquier 1995
; Metz, Robles-Sikisaka, and Vacquier 1998
). It appears that lysin is evolving to match changes in a neutrally drifting VERL. Lysin exhibits two to three times the number of amino acid substitutions of VERL (Nei and Zhang 1998
). The simplest model of lysin-VERL coevolution may predict a one-to-one correlation for the divergence of lysin and VERL. However, there is no a priori reason to expect a one-to-one correlation between lysin and VERL divergence. Lysin may need to undergo multiple substitutions for each substitution in VERL. When the repeated nature of VERL is considered, it is possible to construct scenarios in which lysin may require fixation of multiple changes for every single change in VERL in order to maintain optimal interaction with VERL. For example, a mutation in one VERL repeat may not produce a selective force for lysin to change. However, if by chance this becomes more prevalent among the VERL repeats, there would be strong selection for a corresponding change in lysin. At the point where the mutation represents 50% of the repeats, selection may become strong, favoring lysins which interact with the two types of repeats, which may not be optimal for either type alone. When the mutation is present in the majority of repeats, selection may then favor the single lysin variant best adapted to a single VERL repeat type. Thus, one mutation in VERL could lead to multiple rounds of adaptation in lysin.
Lysin functions specifically to dissolve the egg VE and probably does not have other functions (Lewis, Talbot, and Vacquier 1982
). Likewise, VERL is a major component of the egg VE and most likely does not have other functions (Swanson and Vacquier 1997
). Therefore, the evolutionary forces driving their divergence are most likely related to their roles in fertilization. Although we cannot rule out other molecules being involved in the dissolution of the VE, extensive biochemical data suggest the dissolution of the VE occurs though the specific interaction of lysin and its egg receptor, VERL (Swanson and Vacquier 1997
). The binding kinetics of isolated VERL and lysin show high affinity and the same species specificity as lysin-mediated dissolution of intact VEs. Furthermore, the addition the remaining VE components does not alter the binding kinetics of lysin and VERL, indicating that lysin does not have high affinity for the other VE components (unpublished data).
The pink abalone population subdivision observed at the VERL locus suggests the possibility that assortative mating is being established. If so, this process may be in progress, because we do not observe similar subdivision at other pink abalone loci. However, we studied only the last repeat of the repeat array. This repeat is least likely to be subjected to the homogenizing effects of concerted evolution due to its being at the end of the repeat array (McAllister and Werren 1999
). It is unknown if other repeats in the repeat array will show similar subdivision. However, subdivision is also observed in the nonrepeat carboxyl-terminal region in silent sites (data not shown). Currently, we are not able to detect corresponding changes in pink abalone lysin. Elucidation of the entire pink lysin genomic structure (or working from cDNA for lysin) will permit these types of analyses. We prefer to pursue the use of genomic DNA, because sampling can be performed noninvasively, which is important given the recent dramatic declines in abalone populations. It is also of interest that the levels of polymorphism tend to correlate between abalone lysin and VERL. For example, low VERL polymorphism in red abalone is associated with a lack of red lysin polymorphism. In contrast, moderate pink lysin polymorphism is associated with relatively high levels of pink VERL polymorphism. Taken together, these observations suggest the coevolution of VERL and lysin. However, polymorphism levels at other loci will have to be studied in order to determine if this is a genomewide effect relating to effective population size or specific to these two loci.
From their analysis of the sea urchin sperm protein bindin, Metz and Palumbi (1996)
first suggested the hypothesis that assortative mating in marine invertebrates could evolve through the interaction of male gamete recognition protein with a "tolerant" female receptor. However, evolutionary analysis of the female receptor for bindin has not been possible. Our analyses of the abalone egg VERL is consistent with this hypothesis. The "tolerant" nature of the egg receptor in abalone may result from the egg receptor's being largely a highly repeated structure. Furthermore, relaxed selection on the female locus of a mate recognition system is consistent with a theoretical model for the evolution of assortative mating (Wu 1985
). Future studies on the evolution of gamete recognition proteins could provide insights into their role in speciation.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
1 Keywords: lysin
VERL
Darwinian selection
speciation
fertilization
abalone
gamete recognition
sperm-egg interaction
sperm competition
2 Address for correspondence and reprints: Willie J. Swanson, Department of Molecular Biology and Genetics, Cornell University, 403 Biotechnology Building, Ithaca, New York. 14853-2703. E-mail: wjs18{at}cornell.edu
![]() |
literature cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aguadé, M. 1998. Different forces drive the evolution of the Acp26Aa and Acp26Ab accessory gland genes in the Drosophila melanogaster species complex. Genetics 150:10791089.
Aquadro, C. F. 1997. Insights into the evolutionary process from patterns of DNA sequence variability. Curr. Opin. Genet. Dev. 7:835840.[ISI][Medline]
Elder, J. F., and B. J. Turner. 1995. Concerted evolution of repetitive DNA sequences in eukaryotes. Q. Rev. Biol. 70:297320.[ISI][Medline]
Ferris, P. J., C. Pavlovic, S. Fabry, and U. W. Goodenough. 1997. Rapid evolution of sex-related genes in Chlamydomonas. Proc. Natl. Acad. Sci. USA 94:86348639.
Fu, Y. X., and W. H. Li. 1993. Statistical tests of neutrality of mutations. Genetics 133:693709.
Herndon, L. A., and M. F. Wolfner. 1995. A Drosophila seminal fluid protein, Acp26Aa, stimulates egg laying in females for 1 day after mating. Proc. Natl. Acad. Sci. USA 92:1011410118.
Howard, D. 1999. Conspecific sperm and pollen precedence and speciation. Annu. Rev. Ecol. Syst. 30:10932.[ISI]
Hudson, R. R., M. Kreitman, and M. Aguadé. 1987. A test of neutral molecular evolution based on nucleotide data. Genetics 116:153159.
Kreitman, M., and H. Akashi. 1995. Molecular evidence for natural selection. Annu. Rev. Ecol. Syst. 26:403422.[ISI]
Kumar, S., K. Tamura, and M. Nei. 1993. MEGA: molecular evolutionary genetics analysis. Version 1.01. Pennsylvania State University, University Park.
Lee, Y.-H., and V. D. Vacquier. 1992. The divergence of species-specific abalone sperm lysin is promoted by positive Darwinian selection. Biol. Bull. 182:97104.
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. 12:231238.[Abstract]
Leighton, D. L., and C. A. Lewis. 1982. Experimental hybridization in abalones. Int. J. Invertebr. Reprod. 5:273282.[ISI]
Lewis, C. A., C. F. Talbot, and V. D. Vacquier. 1982. A protein from abalone sperm dissolves the egg vitelline layer by a nonenzymatic mechanism. Dev. Biol. 92:227239.[ISI][Medline]
Lillie, F. R. 1919. Problems of fertilization. University of Chicago Press, Chicago.
Litscher, E. S., H. Qi, and P. M. Wassarman. 1999. Mouse zona pellucida glycoproteins mZP2 and mZP3 undergo carboxy-terminal proteolytic processing in growing oocytes. Biochemistry 38:1228012287.
Loeb, J. 1916. The organism as a whole. Putnam, New York.
Lyon, J. D., and V. D. Vacquier. 1999. Interspecies chimeric sperm lysins identify regions mediating species-specific recognition of the abalone egg vitelline envelope. Dev. Biol. 214:151159.[ISI][Medline]
McAllister, B. F., and J. H. Werren. 1999. Evolution of tandemly repeated sequences: what happens at the end of an array? J. Mol. Evol. 48:469481.[ISI][Medline]
McDonald, J. H., and M. Kreitman. 1991. Adaptive protein evolution at the Adh locus in Drosophila. Nature 351:652654.
MacDonald, R. J., G. H. Swift, A. E. Przybyla, and J. M. Chirgwin. 1987. Isolation of RNA using guanidinium salts. Methods Enzymol. 152:219227.[ISI][Medline]
Messier, W., and C. B. Stewart. 1997. Episodic adaptive evolution of primate lysozymes. Nature 385:151154.
Metz, E. C., R. E. Kane, H. Yanagimachi, and S. R. Palumbi. 1994. Fertilization between closely related sea urchins is blocked by incompatibilities during sperm-egg attachment and early stages of fusion. Biol. Bull. 187:2334.
Metz, E. C., and S. R. Palumbi. 1996. Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin. Mol. Biol. Evol. 13:397406.[Abstract]
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 95:1067610681.
Nei, M., and J. Zhang. 1998. Molecular origin of species. Science 282:14281429.
Nielsen, R., and Z. Yang. 1998. Likelihood models for detecting positively selected amino acid sites and applications to the HIV-1 envelope gene. Genetics 148:929936.
Owen, B., J. H. McLean, and R. J. Meyer 1971. Hybridization in the eastern Pacific abalones (Haliotis). Los Angeles Co. Mus. Nat. Hist. Bull. 9.
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 96:1263212637.
Rozas, J., and R. Rozas. 1999. DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174175.
Schmid, K. J., and D. Tautz. 1997. A screen for fast evolving genes from Drosophila. Proc. Natl. Acad. Sci. USA 94:97469750.
Stekoll, M. S., and T. C. Shirley. 1993. In situ spawning of an Alaskan population of pinto abalone, Haliotis kamtschatkana Jones, 1845. Veliger 36:9597.
Swanson, W. J., E. C. Metz, C. D. Stout, and V. D. Vacquier. 1998. Rapid evolution of acrosomal proteins and species-specificity of fertilization in abalone. Pp. 139146 in C. Gagnon, ed. The male gamete: from basic science to clinical applications. Cache River Press, Ill.
Swanson, W. J., and V. D. Vacquier. 1995. Extraordinary divergence and positive Darwinian selection in a fusagenic protein coating the acrosomal process of abalone spermatozoa. Proc. Natl. Acad. Sci. USA 92:49574961.
. 1997. The abalone egg receptor for sperm lysin is a giant multivalent molecule. Proc. Natl. Acad. Sci. USA 94:67246729.
. 1998. Concerted evolution in an egg receptor for a rapidly evolving abalone sperm protein. Science 281:710712.
Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:585595.
Tian, J., H. Gong, and W. J. Lennarz. 1999. Xenopus laevis sperm receptor gp69/64 glycoprotein is a homolog of the mammalian sperm receptor ZP2. Proc. Natl. Acad. Sci. USA 96:829834.
Tsaur, S. C., and C.-I. Wu. 1997. Positive selection and the molecular evolution of a gene of male reproduction, Acp26Aa of Drosophila. Mol. Biol. Evol. 14:544549.[Abstract]
Vacquier, V. D. 1998. Evolution of gamete recognition proteins. Science 281:19951998.
Vacquier, V. D., and Y.-H. Lee. 1993. Abalone sperm lysin: Unusual mode of evolution of a gamete recognition protein. Zygote 1:181196.
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 87:57925796.
Wassarman, P. M., and S. Mortillo. 1991. Structure of the mouse egg extracellular coat, the zona pellucida. Int. Rev. Cytol. 130:85110.[Medline]
Wu, C.-I. 1985. A stochastic simulation study on speciation by sexual selection. Evolution 39:6682.
Wyckoff, G. J., W. Wang, and C.-I. Wu. 2000. Rapid evolution of male reproductive genes in the descent of man. Nature 403:304309.
Yanagimachi, R. 1988. Sperm-egg fusion. Curr. Top. Mem. Trans. 32:343.
Yang, Z. 1998. Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. Mol. Biol. Evol. 15:568573.[Abstract]
. 1999. Phylogenetic analysis by maximum likelihood (PAML). Version 2.0. University College London, London.
Yang, Z., R. Nielsen, N. Goldman, and A.-M. Krabbe Pedersen. 2000. Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics 155:431449.
Yund, P. O. 2000. How severe is sperm limitation in natural population of marine free-spawners? Trends Ecol. Evol. 15:1013.