reproductive protein evolution

17 Oct 2002 7:58 AR AR173-ES33-07.tex AR173-ES33-07.SGM LaTeX2e(2002/01/18)P1: IBD10.1146/annurev.ecolsys.33.010802.150439

Annu. Rev. Ecol. Syst. 2002. 33:161–79doi: 10.1146/annurev.ecolsys.33.010802.150439

Copyright c© 2002 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on August 6, 2002

REPRODUCTIVE PROTEIN EVOLUTION

Willie J. Swanson1,2 and Victor D. Vacquier21Department of Biology, University of California, Riverside, California 92521;email: [email protected] of Genome Sciences, University of Washington, Box 357730, Seattle,Washington 98195-77303Center for Marine Biotechnology and Biomedicine, Scripps Institution ofOceanography, University of California, San Diego, La Jolla, California 92093;email: [email protected]

Key Words positive Darwinian selection, fertilization, speciation, sexual conflict,lysin

■ Abstract The evolution of proteins involved in reproduction is only now begin-ning to be studied. A reoccurring observation is the rapid evolution of the moleculesmediating reproductive events following the release of gametes. We review the exam-ples where rapid evolution of reproductive proteins has been documented, coveringtaxa ranging from diatoms to humans. The selective pressures causing this divergenceremain unknown, but several hypotheses are presented. The functional consequencesof this rapid divergence could be involved in speciation.

INTRODUCTION

An emerging trend in the study of reproductive proteins is the observation ofrapid, adaptive evolution in many genes that mediate post-copulatory gameteusage/storage, signal transduction and fertilization (Singh & Kulathinal 2000,Swanson & Vacquier 2002). For example, mammalian egg coat proteins are amongthe 10% most rapidly evolving proteins in comparison of orthologs between hu-mans and rodents (Makalowski & Boguski 1998). Likewise, the most rapidlyevolving proteins in the genomeDrosophilaare involved in reproduction (Schmid& Tautz 1997, Ting et al. 1998, Tsaur & Wu 1997). In this review, we demonstratethat this is a general phenomenon that occurs in diverse taxa. We present severalhypotheses about the selective forces that may be driving this rapid evolution, butstress that the selective pressure remains unknown and may differ between taxa.Finally, we point out potential functional consequences of this rapid evolution,including the possibility of speciation.

We define rapidly evolving genes as those that have a higher than average per-centage of amino acid substitutions—typically in the 10% most rapidly evolvingclass of genes within a genome. We recognize that this is a crude measure, because

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many rapidly evolving genes may show extreme divergence only in a portion oftheir sequence, such as a binding site. Additionally, defining rapid evolution solelyby percent divergence does not provide information about the potential causes ofrapid evolution. For example, rapid evolution might be due to a lack of functionalconstraint, e.g., a pseudogene accumulating mutations. Alternatively, rapid evo-lution might be due to positive Darwinian selection, which occurs when naturalselection promotes amino acid divergence resulting in protein adaptation. A clearsignal of positive Darwinian selection is an excess in the number of nonsynony-mous substitutions per nonsynonymous sites (dN; amino acid altering) comparedwith the number of synonymous substitutions per synonymous sites (dS; silentchanges). BecausedN anddS are normalized to the number of sites, in cases of noselection,dN/dS = 1 (for example in a pseudogene). For most proteins,dN/dS ismuch less than one [averagedN/dSratio of∼0.2 is found between humans and mice(Li 1997)] indicating purifying selection. Averaging thedN/dS ratio across allsites and lineages is a conservative test for positive Darwinian selection (Yang &Bielawski 2000). The classic example is the class one major histocompatibilitylocus, where the averagedN/dSratio across all sites is∼0.5, but those sites directlyinvolved in binding foreign antigens havedN/dS ratios>1 (Hughes & Nei 1988,Yang & Swanson 2002).

If sequences are available from multiple species, new methods are available touse maximum likelihood predictions to detect selection acting upon a subset ofcodons (Nielsen & Yang 1998, Yang et al. 2000a). These methods can find a subsetof sites showing extreme divergence, if there are sufficient data (Anisimova et al.2001). Importantly, these new methods do not require a priori knowledge of the sitesunder selection. They can also be used to predict functionally important sites of agene subject to positive Darwinian selection (Swanson et al. 2001c). There are twosteps in using these methods. First is the identification of the presence, or absence,of a subset of sites being subjected to positive selection. This is accomplished bya likelihood ratio test comparing the likelihood of a neutral model with that of aselection model. The neutral models range from those having two classes ofdN/dS

ratios set at 0 and 1 (Nielsen & Yang 1998), to modeling a beta distribution ofdN/dS

ratios between the values of 0 and 1 (Yang et al. 2000a). The selection models addan additional class of sites with adN/dS ratio that is estimated from the data andcan be greater or less than one. This appears to be a robust and powerful methodfor detecting positive selection, as indicated by analysis of simulated (Anisimovaet al. 2001) and empirical data (Yang & Swanson 2002). The second step occursif a subset of sites is predicted to be under positive selection. Then, an empiricalBayesian approach is used to assign the posterior probabilities of sites fallingwithin the selected class of codons (Nielsen & Yang 1998, Yang et al. 2000a). Asignal of positive Darwinian selection indicates that there is an adaptive advantageto changing the amino acid sequence, and this signal can be used to identifyfunctionally important gene regions, such as binding sites (Swanson et al. 2001c,Yang & Swanson 2002).


REPRODUCTIVE PROTEIN EVOLUTION 163

Rapidly Evolving Reproductive Proteins

Many eukaryotes have reproductive proteins, the sequences of which have exten-sively diverged between closely related species (Table 1). Many of these genes arerapidly evolving but do not show a clear signal of positive Darwinian selection.However, future analyses, perhaps with improved statistical methods or additional

TABLE 1 Rapidly evolving genes involved in reproduction

Gene (locus) Organism Evidence for+ selection Reference

Pheromones Euplotes None (Luporini et al. 1995)(ciliate protozoa)

mid1 Chlamydomonas None (Ferris et al. 1997)(green alga)

fus1 Chlamydomonas None (Ferris et al. 1996)(green alga)

Sig1 Thalassiosira None (Armbrust & Galindo 2001)(Diatoms)

Pheromones Basidiomycete None (Brown & Casselton 2001)(fungi)

SCR Brassicaceae None (Schopfer et al. 1999)

S-locus Solanaceae None (Richman & Kohn 2000)

Pollen coat Arabidopsis None (Mayfield et al. 2001)proteins

Lysin Tegula & Haliotis OveralldN/dS> 1 (Hellberg & Vacquier 1999,(turban snails Lee et al. 1995)and abalone)

sp18 Haliotis(abalone) OveralldN/dS> 1 (Swanson & Vacquier 1995a)

TMAP Tegula (turban OveralldN/dS> 1 (Hellberg et al. 2000)snails)

Bindin Sea urchins Region withdN/dS> 1 (Metz & Palumbi 1996)

Acp26Aa Drosophila Lineage withdN/dS> 1 (Tsaur & Wu 1997)

Acp36DE Drosophila Polymorphism survey (Begun et al. 2000)

ZP3 Mammals Class of sites withdN/dS> 1 (Swanson et al. 2001c)

ZP2 Mammals Class of sites withdN/dS> 1 (Swanson et al. 2001c)

OGP Mammals Class of sites withdN/dS> 1 (Swanson et al. 2001c)

Zonadhesin Mammals None (Gao & Garbers 1998)

TCTE1 Mammals None (Juneja et al. 1998)

Protamines Mammals dN/dS> 1 (Wyckoff et al. 2000)



data, may reveal the signal of positive Darwinian selection. Nevertheless, theyare of interest because their rapid evolution may result in functional differencesaffecting reproduction.

Marine ciliates of the genusEuplotessecrete protein pheromones of 40–43amino acids that mediate sexual conjugation and vegetative growth. An alignmentof these pheromone sequences from different mating types of one species showsthat the mature protein has diverged extensively (Luporini et al. 1995). How-ever, the signal sequences of these genes remain highly conserved. Additionally, aregion that is cleaved off to make the mature pheromone also shows high conser-vation, suggesting different selective pressures acting upon the different regionsof this molecule (Figure 1). These pheromones are involved in both cell mitoticproliferation and mating pair formation inE. raikovi. Interestingly, the receptor forthe pheromones has been identified as an alternatively spliced, membrane boundform of the pheromone, with the external region structurally identical to the sec-reted form. It has been hypothesized that binding of the same pheromone inducesmitotic division, while binding of an alternative pheromone induces mating pairformation (Ortenzi et al. 2000).

Two genes that control mating in the unicellular green alga,Chlamydomonasreinhardtii, show extensive divergence between species. The product of theChla-mydomonas midgene determines if a cell will be of mating type plus or minus,whereasfus1 encodes a protein needed for fusion of plus and minus cells. Nohomologues ofC. reinhardtii fus1, and only one homologue ofmid, were found in

Figure 1 Alignments of two reproductive proteins. TheEuplotesmating pheromones(Luporini et al. 1995) and the plant SCR proteins (Schopfer et al. 1999). In both cases,the functional secreted proteins are extensively divergent while the portions cleaved offto make the secreted proteins are well conserved. Signal sequences of both proteins areshown in bold. The pre-region that is cleaved off of theEuplotes functional secretedpheromone is underlined. Dots denote identity to the first sequence and dashes are inserted foralignment.



12 otherChlamydomonasspecies (Ferris et al. 1996, 1997). These genes arelocated in the mating type locus (MT), a∼1 Mb domain under recombinationalsuppression. This region is extremely dynamic, having large indels, translocatedgenes, gene duplications, and gene inactivation events. Although this region doeshave a generally high level of mutatgenic change, there are also several conservedhousekeeping genes in the region (Ferris et al. 2002).

An extracellular matrix protein encoded by theSig1gene of the diatomTha-lassiosirais upregulated during mating, and is thought to function in the matingprocess.Sig1 is highly divergent both within and between species and there arewell-documented differences that distinguish betweenSig1from the Atlantic andthe Pacific Oceans (Armbrust & Galindo 2001).Sig1 is present as a multicopygene, with some individuals displaying as many as 19 gene variants ofSig1, sug-gesting a minimum of 10 loci.Sig1shows extreme intraspecific, interspecific, andinter-individual variation. Although the exact function of the Sig1 protein remainsunknown, its extreme divergence suggests that it might be a barrier to reproductionbetween different diatom strains.

Mating compatibility in Basidiomycetefungi requires secretion of proteinpheromones that bind to cell surface receptors to mediate signal transduction to in-duce expression of mating genes (Brown & Casselton 2001). The pheromones andtheir receptors show extreme sequence variation (Casselton & Olesnicky 1998),which could underlie species-specific gamete interaction.

The reproductive proteins fromEuplotes, Chlamydomonas, Thalassiosira, andBasidiomyceteshow extreme divergence between species; however, there is noevidence that this divergence is promoted by positive Darwinian selection. Thereare currently not enough sequences available, or those that are available are toodivergent to reliably align (Figure 1), making it impossible to perform likelihoodratio tests to determine if any sites are subjected to positive selection.

Many species of flowering plants cannot self-fertilize because their pollen(male) is incompatible with stylar (female) tissue, and this self-incompatibilityprevents inbreeding depression. In sporophytic self-incompatibility in the genusBrassica, the pollen component is encoded by the highly variable S-locus cys-teine rich geneSCR(Schopfer et al. 1999). The stylar recognition S-locus receptorkinase,SRK, is also highly variable and encodes a membrane-spanning proteinkinase (Schopfer & Nasrallah 2000) that has been shown to bind SCR in an allele-specific manner (Kachroo et al. 2001). SCR is similar to theEuplotespheromonesin that, although the signal sequences of SCR proteins are relatively conserved,the mature SCR proteins have diverged extensively (Figure 1) (Nasrallah 2000).In gametophytic self-incompatibility in theSolanaceae, the pollen component hasyet to be identified, but the stylar product of the self-incompatibility gene encodesan extracellular RNase encoded by theSlocus. However, the single S-allele RNaseis sufficient to determine self-incompatibility (Richman & Kohn 2000), indicatingit may function as a receptor and ligand in a manner analogous to theEuplotesmating pheromone.Salleles can differ by 50% in amino acid identity within thesame species and can show a clear signature of positive Darwinian selection by



TABLE 2 Example reproductive proteins withoveralldN/dS ratios>1a

Gene dN dS dN/dS

ProtamineHsp-OWM 13.1 4.6 2.89

LysinHr-Hs 5.8 1.8 3.63

sp18Hr-Hs 8.1 1.8 4.50

TMAPTb-Tr 17.6 5.0 3.51

S-alleleswithin Pc 40.2 16.1 2.49

Acp26AaDm-Dy 47.7 30.2 1.57

aData from: protamine (Wyckoff et al. 2000); lysin and sp18(Vacquier et al. 1997); TMAP (Hellberg et al. 2000); S-alleles(Richman et al. 1996); and Acp26Aa (Tsaur & Wu 1997). Speciesabbreviations are:Homo sapiens(Hsp), Old world monkey(OWM), Haliotis sorenseni(Hs), H. rufescens(Hr), Tegulabrunnea(Tb),T. regina(Tr), Physalis crassifolia(Pc),Drosophilamelanogaster(Dm), andD. yakuba(Dy).

havingdN/dS ratios>1 (Table 2) (Richman & Kohn 2000). This indicates thatthere is an advantage for sequence diversity at this locus.

Components of the pollen coat fromArabidopsis thalianaalso show extensivevariability (Mayfield et al. 2001). A recent study identified all abundant pollen coatproteins greater than 10 kDa fromArabidopsisby protein sequencing and com-parison with the completed genome. A total of 10 proteins were isolated, severalof which were duplicated genes arranged in clusters. For example, five oleosingenes were found that clustered together in bothA. thaliana andBrassica oler-acea. Like the diatom geneSig1, there was extensive intraspecific, interspecific,and inter-individual sequence variation (Mayfield et al. 2001).

Immediately before fertilization, sperm of marine gastropods of the genusHaliotis (abalone) andTegula(turban snail) release a soluble protein called lysinonto the surface of the egg vitelline envelope (VE). In a species-specific, nonen-zymatic process, lysin dissolves a hole in the VE through which the sperm passesto reach the egg cell membrane. The amino acid sequences of lysins from differ-ent species are extremely divergent, which has resulted from adaptive evolution(Hellberg & Vacquier 1999, Metz et al. 1998b, Yang et al. 2000b). Pairwise com-parisons showdN/dS ratios significantly greater than one (Table 2). Additionally,site-specific analyses using maximum likelihood ratio tests (Yang et al. 2000a)show that a subset of sites on the surface of lysin have been the target of natural



selection (Yang & Swanson 2002, Yang et al. 2000b). Some of these sites, such asthe hypervariabe N-terminal, have been implicated in the species-specific functionof lysin-mediated VE dissolution by site directed mutagenesis (Lyon & Vacquier1999). Sites predicted to be under positive selection from five California abalonespecies are located primarily on the N- and C-terminal regions (Figure 2). Al-though the driving force behind this rapid evolution is not yet clear, it has beensuggested that the rapid diversification of sperm lysin is driven by the need forits adaptation to a constantly changing egg receptor that is evolving neutrally(Swanson et al. 2001a, Swanson & Vacquier 1998). The VE receptor for lysin,VERL, is known from several closely related species of the abalone. Having beenreleased from the acrosome, lysin binds to VERL molecules of the egg vitellineenvelope in a species-specific manner (Kresge et al. 2001a, Swanson & Vacquier1997). The fibrous VERL molecules lose their cohesion and splay apart, creatinga hole through which the sperm swims (Kresge et al. 2001a, Swanson & Vacquier1998). At ∼1 million Daltons, VERL is a large glycoprotein that contains22 tandem repeats of a∼153–amino acid sequence. In contrast to lysin, VERLshows no evidence of positive Darwinian selection; instead, it appears to be evolv-ing neutrally. VERL repeatdN/dS ratios are less than one, and there is no evidencefor a class of sites subjected to positive Darwinian selection using likelihood ratiotests (Swanson et al. 2001a). Additionally, several tests of neutrality based on poly-morphism surveys also failed to reject equilibrium neutral expectations (Swansonet al. 2001a). The tandem VERL repeats show high levels of DNA sequenceidentity (>95%) within a molecule, indicating the repeats are subject to concertedevolution (Swanson & Vacquier 1998). Concerted evolution is the process in whichunequal crossing over and gene conversion randomly homogenize a sequence oftandem repeats within the gene and within a population, a mechanism exemplifiedby ribosomal genes (Elder & Turner 1995, McAllister & Werren 1999). The endresult is that the repeats within a molecule from one species are more similar toeach other than they are to the homologous repeats from other species.

Abalone sperm also release a protein (sp18) that is thought to mediate the fusionof the sperm and egg (Swanson & Vacquier 1995b). In five Californian abalonespecies, sp18 proteins are up to 73% different at the amino acid sequence level(Swanson & Vacquier 1995a) and there is evidence that this protein might evolve upto 50 times faster than the fastest evolving mammalian proteins (Metz et al. 1998b).Like lysin, estimates ofdN/dSratios show evidence for positive Darwinian selectionwith dN/dS ratios as high as 4.7 averaged across the entire molecule (Table 2).Likelihood ratio tests also indicate a subset of sites subjected to positive selection,which when placed on the 3-D structure of sp18, fall near the top and bottomof the molecule (Figure 1). Comparison of sp18 to the database using BlastP orPsi-Blast reveals no similarity to other proteins. However, more detailed analysesdetected similarity to abalone lysin (Swanson & Vacquier 1995a), which was laterconfirmed by intron mapping (Metz et al. 1998a) and determination of the three-dimensional structure of both molecules (Kresge et al. 2001b). Figure 2 shows the3D structure of both molecules. The similarity of the alpha-helical arrangement



Figure 2 Three-dimensional structures of abalone sperm lysin and sp18. The lysin structureis from the red abalone (Kresge et al. 2000a), and the structure of sp18 is from the greenabalone (Kresge et al. 2000b). The black residues shown in spacefill (displaying the size ofthe residue side-chains) are the sites predicted to be subjected to positive Darwinian selectionusing maximum likelihood methods. Only sites with a posterior probability greater than 0.95are shown. The backbone alpha-helices of the two molecules are strikingly similar, indicatingthe two proteins arose by a gene duplication event. However, the angles of the helices arequite different, perhaps indicating selection for different functions.



is apparent. However, the angles of the helices are significantly different resultingin a root mean square deviation of 6.45 angstroms for the comparison of thealpha-carbon traces of the two molecules. The structural high similarity betweenthe two molecules indicates that the two abalone fertilization proteins arose bygene duplication. In addition to lysin and sp18,Tegulasperm also release a majoracrosomal protein of unknown function that is highly divergent and subject toadaptive evolution (Table 2) (Hellberg et al. 2000).

Rapid, extensive evolution of reproductive proteins has also been seen in seaurchins, the sperm of which use a protein called bindin to attach to the egg surfaceand possibly to fuse with the egg cell membrane (Vacquier et al. 1995).Echi-nometra mathaeiandE. oblongaare two sympatric sea urchin species that livein the Pacific, and based on the mitochondrial DNA sequence comparisons, theyare the most closely related sea urchin species from all known urchin species.Because adhesion of bindin to eggs has evolved to be species-specific (Palumbi &Metz 1991) few interspecies hybrids are formed. Bindin sequences show remark-able divergence both within and betweenEchinometraspecies (Metz & Palumbi1996), as well as between species of another sea urchin genus,Strongylocentrotus(Biermann 1998). In bothEchinometraandStrongylocentrotusbindin (Biermann1998), a region with elevateddN/dS ratio has been identified as a target of posi-tive selection. The exact function of this region remains unknown, but it might beinvolved in the species-specific adhesion of sperm to eggs.

Nonmarine invertebrates also show rapid adaptive evolution of reproductiveproteins, and the accessory gland proteins ofDrosophilaare the best characterizedexample (Partridge & Hurst 1998, Wolfner 1997). During copulation, an estimated83 protein products of theDrosophila male accessory glands (Swanson et al.2001b) are transferred along with sperm to the female reproductive tract (Wolfner1997). These proteins have a variety of effects on the physiology of the matedfemale (Wolfner 1997). It has been shown that the accessory gland proteins aretwice as diverse between species as are the nonreproductive proteins (Civetta &Singh 1995, Singh & Kulathinal 2000). Although DNA analysis confirms this two-fold increase in the rate of amino acid replacement between species (Swanson et al.2001b), the molecular evolution of only a few accessory gland proteins has beenstudied in detail. In particular, the accessory gland proteinAcp26Aais one of thefastest evolving genes in theDrosophilagenome, with adN/dSratio of 1.6 betweenD. melanogasterandD. yakuba(Table 2). This indicates that its evolution is drivenby positive Darwinian selection (Tsaur et al. 1998, Tsaur & Wu 1997). Otheraccessory gland proteins that show signs of positive selection includeAcp36DE(Begun et al. 2000) andAcp29AB(Aguade 1999). The divergence of accessorygland proteins has been shown to be partly responsible for species-specific usageof gametes in someDrosophilaspecies, where injections of conspecific accessorygland extracts can rescue an infertile hybrid cross (Fuyama 1983).

The rapid, adaptive evolution of reproductive proteins and species-specific fer-tilization is not limited to invertebrates, as similar phenomena have also beendescribed in mammals (Swanson et al. 2001c, Wyckoff et al. 2000). This wasfirst demonstrated in mammalian protamines (Rooney et al. 2000, Wyckoff et al.



2000). An excess of amino acid replacements was observed in primates, whichwas also accompanied by low amino acid polymorphism within species (Wyckoffet al. 2000). Comparison between humans and Old World monkeys showeddN/dS

ratios>1 (Table 2). Adaptive evolution has also been observed in mammalianzona pellucida egg coat proteins ZP2 and ZP3 by using maximum likelihood ratiotests to identify a subset of sites with adN/dS ratios greater than one (Swansonet al. 2001c). The zona pellucida is an elevated glycoproteinaceous envelope thatprotects the mammalian egg (Wassarman et al. 2001). The zona pellucida glyco-proteins bind sperm and induce the acrosome reaction, one of the first steps inmammalian fertilization. A region of ZP3 that was previously implicated in thespecies-specific induction of the acrosome reaction (Chen et al. 1998) was identi-fied to be the target of positive selection by predicting the sites subjected to positiveselection using an empirical Bayes approach (Swanson et al. 2001c).

NOT ALL REPRODUCTIVE PROTEINS EVOLVE RAPIDLY

We have stressed the generality of rapidly evolving reproductive proteins above.However, it is clear that not all reproductive proteins show rapid evolution. Addi-tionally, reproductive proteins may evolve rapidly in some lineages, but be con-served in other lineages. This was exemplified by a study of the sea urchin proteinbindin (Metz et al. 1998a). As detailed above, bindin was shown to be subjected topositive Darwinian selection from an analysis of species in the generaEchinometraandStrongylocentrotus. However, a study of bindin in the genusArbaciashowedno signs of positive Darwinian selection. In fact, bindin sequences are quite con-served among species ofArbacia. Consistent with this observation, two differentspecies, living in different oceans, are interfertile. Why is bindin so conserved inthe genusArbacia? One possibility is that the four species ofArbaciaexaminedare all allopatric, so there is no chance for hybridization. Species of the generaEchinometraandStrongylocentrotushave overlapping habitats with congenericspecies, which may increase the chance of hybridization. Alternatively, bindinfrom Arbacia species may be under increased functional constraint as indicatedby a lack of indels and acquisition of a hydrophobic domain not found in bindinfrom other genera (Metz et al. 1998a). Further comparisons of nonrapidly evolvingversus rapidly evolving reproductive proteins, may shed light on the forces affect-ing their evolution. For example, are there some characteristics (either ecologicalor molecular) that are associated with rapidly evolving reproductive proteins?

WHAT DRIVES THE EVOLUTION OFREPRODUCTIVE PROTEINS?

The selective pressure driving the evolution of reproductive proteins remains un-known. However, several hypotheses have been proposed. Below, we list someof these hypotheses and discuss how they may apply to the systems described



above. We stress that no single selective pressure may account for all the examplesof rapidly evolving reproductive proteins. Additionally, several of these selectivepressures may be acting simultaneously. Finally, we note that many of the hypothe-ses have overlapping predictions and could be considered a subset of each other.For example, sperm competition may be intimately coupled with sexual conflictin some situations (Rice & Holland 1997).

Relaxed Constraint

It is possible that the repetitive nature of some reproductive proteins leads to re-laxed constraint and neutral evolution. This would produce a continually changingsequence. For example, the abalone VERL protein may change as a result of amutation that occurs in one of the 22 VERL repeats. This change might result ina lower affinity of the mutant repeat for lysin, but the mutant repeat is toleratedand fertilization occurs because there are still 21 unchanged VERL repeats in eachVERL molecule. Thus, the redundant nature of VERL leads to relaxed selectionon each repeat unit, such that mutations do not have any fitness consequences,being neither beneficial, nor harmful. Such tolerance has been suggested for ga-mete recognition in sea urchins (Metz & Palumbi 1996). In successive generations,concerted evolution randomly spreads the mutant repeat within the VERL geneby unequal crossing over and gene conversion (Elder & Turner 1995, McAllister& Werren 1999, Swanson & Vacquier 1998). Sperm would be selected for thatinteract most efficiently with the new forms of VERL, because there would beenormous competition between sperm to be the first to fertilize the egg. Addi-tionally, because specificity of sperm-egg interaction is not absolute, the eggs willmost likely be fertilized if there is sperm around—which may further reduce theselective pressure on eggs. This creates a continuous selective pressure on lysinto adapt to the ever-changing VERL, and provides an explanation for the adaptiveevolution of lysin (Metz et al. 1998b, Yang et al. 2000b). In other organisms thereare also repeated, redundant, reproductive genes within the genome—such as inthe diatomSig1(Armbrust & Galindo 2001) gene and theArabidopsispollen coatproteins (Mayfield et al. 2001). It may be that positive Darwinian selection willbe found to enhance diversity at these loci, which would refute this hypothesis incertain cases.

Reinforcement

If two allopatric populations come back in contact, hybrids may be formed if thereproductive recognition system has not diverged. If the hybrid mating results inless fit offspring owing to genomic incompatibilities, there would be selection tofavor differentiation of the reproductive recognition system (Dobzhansky 1940,Howard 1993). This process has been referred to as reinforcement, and could leadto the rapid evolution of proteins mediating reproduction. It should be possible totest this hypothesis by comparisons of reproductive proteins from allopatric andsympatric population/species. The finding that bindin from sea urchins of the genus



Arbaciaare not rapidly evolving may be consistent with reinforcement, becausethis represents a clear case of allopatric speciation. However, other scenarios couldalso be consistent with this observation (Metz et al. 1998a).

Gene Duplication

One hypothesis for the rapid, adaptive evolution of these molecules could beselection for new function. This may have occurred following the gene duplicationevent that gave rise to abalone sp18 and lysin. Presumably, there was an ancestralmollusk that had one sperm acrosomal protein. This protein may have performedtwo tasks—dissolution of the egg VE and fusion between the sperm and egg.Following a gene duplication event, the two molecules could have specialized—sp18 for fusion, and lysin for VE dissolution. Currently, lysin dissolves the VE,but sp18 has no effect on the integrity of the VE. Both molecules are capable offusing artificial liposomes, but sp18 is a much more potent fusagen and localizesto the acrosomal process—the region of the abalone sperm that fuses with theegg (Swanson & Vacquier 1995b). However, we feel this would not lead to thecontinual evolution that has been observed. Rather it should lead to a burst of posi-tive selection followed by purifying selection to maintain function. It is of interestthat several of the reproductive proteins described above are members of duplicatedgene families. It will be of interest to determine if reproductive proteins arise fromgene duplication events more frequently than nonreproductive proteins.

Sperm Competition

Sperm competition (Clark et al. 1999) occurs because every sperm competes withall the other sperm to be the first to fuse with the egg. This competition can befierce; for example, in the male sea urchin there are 200 billion sperm cells perfive milliliters of semen (a typical amount of semen released in a spawning event).Sperm competition can exert a selective pressure at many steps in the fertilizationcascade. Individual sperm could be selected for being the best or the fastest atinitiating and maintaining swimming, responding to chemoattractants that diffusefrom the egg, binding to the egg’s surface, binding to the egg components thatinduce the acrosome reaction, penetrating the egg envelope, and fusing with theegg. Sperm competition can also occur in internal fertilizers when the female matesmultiple times. In many cases, the majority of offspring are sired by one of the maleswith whom the female mated. Sperm precedence is a similar but distinct mechanismthat occurs when females preferentially utilize sperm from homo-specific males(Howard 1999). One question regarding sperm competition and sperm precedenceis the involvement of the female. Genetic studies have shown that female genotypecan play an important role in sperm competition (Clark & Begun 1998, Clark et al.1999, Price 1997). What provides the selective force for the female reproductiveproteins to change? Possibilities include neutrally drifting targets as mentionedabove for abalone VERL, or perhaps sexual conflict as discussed below.



Sexual Conflict

Sexual conflict could come into play when sperm cells are too abundant (Gavrilets2000, Rice & Holland 1997). Sperm competition presents some problems for theegg. For example, fusion with multiple sperm (polyspermy) will result in arrestof the zygote’s development. How do eggs prevent polyspermy? Eggs can preventmultiple fusions by a variety of methods. For example, some eggs have a fastblock to polyspermy that is accomplished by reversing the electrical potentialof the egg membrane that prevents fusion with additional sperm (Gould-Somero& Jaffe 1984). Additionally, some eggs undergo a slow block to polyspermy byaltering the egg receptors to prevent further sperm-egg interaction. It is intriguingthat in organisms like mammals with only a slow block to polyspermy (Gould-Somero & Jaffe 1984, Jaffe et al. 1983), we see adaptive evolution in egg coatproteins. However, in organisms that have a fast block to polyspermy such asabalone (Gould-Somero & Jaffe 1984) we see relaxed selection on the egg coatproteins. It may be that adaptive evolution is observed in mammals to slow downfertilization, allowing the egg to more efficiently regulate sperm entry to preventpolyspermy. However, in organisms with fast blocks to polyspermy this selectivepressure is absent resulting in neutral evolution of egg coat proteins.

Sexual Selection

Sexual selection at the cellular level is known as cryptic female choice (Eberhard1996). Cryptic female choice might come into play when one egg preferentiallybinds with a sperm that carries a particular allele of a sperm surface protein, asopposed to the alleles carried by other sperm. One example of assortative femalechoice is the preference ofEchinometraeggs to be fertilized by sperm that carrythe same bindin allele as they do (Palumbi 1999). However, the question remainswhy females might change their preference. This question is being explored at theorganismal level, using models of Fisherian and/or good genes sexual selection(Iwasa & Pomiankowski 1995) and empirical evidence of preference change inresponse to environmental factors. Could some of these hypotheses be appropriateat the cellular level?

Microbial Attack

It has been proposed that eggs may be subjected to invasion by pathogens, partic-ularly in marine invertebrates where the eggs settle into microrganism-rich marinesediments to undergo development (Vacquier et al. 1997). In such cases, theremay be selective pressure to change egg coats to prevent invasion by pathogens.This would lead to a continual selective pressure on sperm to evolve to keep upwith the ever-changing egg surface. This could also occur in internal fertilizers,perhaps mediated by sexually transmitted microbial diseases. The potential forparasites to impact mating preferences has been long discussed in a variety ofsystems (Hamilton & Zuk 1989).



Self-/Non-Self-Recognition

Molecules involved in self-recognition often show extreme variability within aspecies. It is possible that molecules involved in reproduction could also havebeen involved in self-recognition. For example, theEuplotesmating pheromoneis involved in both self and non-self-recognition (Luporini et al. 1995), and thediffering conditions result in either induction of cell growth or sexual mating.In plants, the selective pressure to prevent self-fertilization is thought to be onemechanism by which great diversity arises at theSCR(Schopfer et al. 1999) andS-allele loci (Richman & Kohn 2000).

Within-Population Variation

The rapid evolution between species discussed above suggests that there may beinteresting evolutionary dynamics occurring at these loci within a species. Severalpopulation surveys have been performed on reproductive proteins, and the resultscan be quite different. For example, the abalone sperm proteins lysin and sp18show signs of selective sweeps with little or no variation within a species (Metzet al. 1998b). In contrast, the sea urchin sperm protein bindin shows extensive poly-morphism within species (Metz & Palumbi 1996). High divergence accompaniedby high polymorphism is also observed in theDrosophilaaccessory gland proteins(Begun et al. 2000). An intriguing question is whether the extensive polymorphismand divergence of some reproductive proteins could be involved in speciation.

Population surveys of abalone fertilization are starting to hint at the possibilityof assortative mating based upon reproductive loci. The last abalone egg VERLrepeat in the repeat array of 22 repeats was identified and sequenced from 11sympatric pink abalone (H. corrugata) and two different variants of the last VERLrepeat sequences were found (Figure 3). These types are different by several aminoacids as well as a large indel. Five individuals were homozygous for each of thetwo variants, and only one was heterozygous for both variants (Swanson et al.2001a). The small number of heterozygotes indicates that assortative mating maytake place in the same population of pink abalone, or that there is postmatingselection against heterozygotes. Theoretically, this molecular differentiation couldeventually lead to a sympatric speciation event—the splitting of the current pinkabalone population into two new species. Larger samples of abalone need to beanalyzed before we can say whether this prediction will be borne out.

A similar assortative mating phenomenon has also been found in theEchi-nometrasea urchins. IndividualE. mathaeihave two different alleles of bindin,and homozygotes for each variant can be distinguished by PCR and restrictionmapping. Eggs ofE. mathaeiare preferentially fertilized by sperm that carry thesame bindin allele (Palumbi 1999). This result indicates that the genes that en-code bindin and its egg surface receptor might be linked and inherited as one unit,as occurs in reproductive gene pairs in fungi (Casselton & Olesnicky 1998) andplants (Schopfer & Nasrallah 2000, Schopfer et al. 1999). Quantitative fertilizationspecificity has also been documented in the sea urchinStrongylocentrotus pallidus



Figure 3 Neighbor-joining tree of the two pink abalone haplotypes discovered bysequencing the last VERL repeat from a population of pink abalone. The haplotypesare rooted with the VERL repeat from the red abalone. The separation of the two pinkclades is supported by 100% bootstrap values. The scale bar shows total nucleotidedivergence.

(Biermann 2000) and otherEchinometraspecies (Rahman & Uehara 2000), in-dicating that the differentiation of the gamete recognition system might have acrucial role in reproductive isolation in many sea urchin genera.

CONCLUSIONS

In this review, we have demonstrated that the rapid evolution of reproductiveproteins occurs in several taxonomic groups. In some instances, the rapid evolutionis driven by positive Darwinian selection, but in other cases the patterns do notdepart from neutrality (Table 1). This variation in selective pressure occurs at alltaxonomic levels as well as varying temporally. The selective pressure driving theevolution of reproductive loci remains unknown. We have listed a few, nonexclusivehypotheses that several researchers are in the process of testing. Several of these



hypotheses have overlapping predictions, making it difficult to distinguish betweenthem. Undoubtedly, there are other selective pressures not discussed here that coulddrive the rapid evolution of reproductive proteins. Further studies on the evolutionand function of reproductive proteins could provide answers to the nature of theselective pressure driving the evolution of reproductive proteins. We may also learnwhat the functional consequence of the rapid evolution could mean.

ACKNOWLEDGMENTS

We thank the National Institutes of Health and National Science Foundation forgrants supporting this work: NIH Grant HD12896 to V.D.V. and NSF grant DEB-0111613 to W.J.S. Drs. C.F. Aquadro, M.F. Wolfner, J.D. Calkins, and J.P. Vacquierare thanked for their criticisms of the manuscript.

The Annual Review of Ecology and Systematicsis online athttp://ecolsys.annualreviews.org

LITERATURE CITED

Aguade M. 1999. Positive selection drives theevolution of the Acp29AB accessory glandprotein in Drosophila.Genetics152:543–51

Anisimova M, Bielawski JP, Yang Z. 2001. Ac-curacy and power of the likelihood ratio testin detecting adaptive molecular evolution.Mol. Biol. Evol.18:1585–92

Armbrust EV, Galindo HM. 2001. Rapid evo-lution of a sexual reproduction gene in cen-tric diatoms of the genusThalassiosira.Appl.Environ. Microbiol.67:3501–13

Begun DJ, Whitley P, Todd BL, Waldrip-DailHM, Clark AG. 2000. Molecular populationgenetics of male accessory gland proteins inDrosophila. Genetics156:1879–88

Biermann CH. 1998. The molecular evolu-tion of sperm bindin in six species of seaurchins (Echinodia: Strongylocentrotidae).Mol. Biol. Evol.15:1761–71

Biermann CH. 2000. Geographic divergence ofgamete recognition systems in two speciesin the sea urchin genusStrongylocentrotus.Zygote8:S86–87

Brown AJ, Casselton LA. 2001. Mating inmushrooms: increasing the chances but pro-longing the affair.Trends Genet.17:393–400

Casselton LA, Olesnicky NS. 1998. Molecu-lar genetics of mating recognition in basid-iomycete fungi.Microbiol. Mol. Biol. Rev.62:55–70

Chen J, Litscher ES, Wassarman PM. 1998.Inactivation of the mouse sperm receptor,mZP3, by site-directed mutagenesis of indi-vidual serine residues located at the combin-ing site for sperm.Proc. Natl. Acad. Sci. USA95:6193–97

Civetta A, Singh RS. 1995. High divergence ofreproductive tract proteins and their associa-tion with postzygotic reproductive isolationin Drosophila melanogaster and Drosophilavirilis group species.J. Mol. Evol.41:1085–95

Clark AG, Begun DJ. 1998. Female genotypesaffect sperm displacement in Drosophila.Genetics149:1487–93

Clark AG, Begun DJ, Prout T. 1999. Female×male interactions in Drosophila sperm com-petition.Science283:217–20

Dobzhansky T. 1940. Speciation as a stage inevolutionary divergence.Am. Nat.312–21

Eberhard WG. 1996.Female Control: SexualSelection by Cryptic Female Choice. Prince-ton, NJ: Princeton Univ. Press



Elder JF Jr, Turner BJ. 1995. Concerted evolu-tion of repetitive DNA sequences in eukary-otes.Q. Rev. Biol.70:297–320

Ferris PJ, Armbrust EV, Goodenough UW.2002. Genetic structure of the mating-typelocus of Chlamydomonas reinhardtii.Genet-ics160:181–200

Ferris PJ, Pavlovic C, Fabry S, GoodenoughUW. 1997. Rapid evolution of sex-relatedgenes in Chlamydomonas.Proc. Natl. Acad.Sci. USA94:8634–39

Ferris PJ, Woessner JP, Goodenough UW. 1996.A sex recognition glycoprotein is encoded bythe plus mating-type gene fus1 of Chlamy-domonas reinhardtii.Mol. Biol. Cell7:1235–48

Fuyama Y. 1983. Species-specificity of parag-onial substances as an isolating mechanismin Drosophila.Experientia39:190–92

Gao Z, Garbers DL. 1998. Species diversityin the structure of zonadhesin, a sperm-specific membrane protein containing mul-tiple cell adhesion molecule-like domains.J.Biol. Chem.273:3415–21

Gavrilets S. 2000. Rapid evolution of reproduc-tive barriers driven by sexual conflict.Nature403:886–89

Gould-Somero M, Jaffe LA. 1984. Control ofcell fusion at fertilization by membrane po-tential. InCell Fusion and Transformation,ed. RF Beers, EG Bassett, pp. 27–38. NewYork: Raven

Hamilton WD, Zuk M. 1989. Parasites and sex-ual selection.Nature341:289–90

Hellberg ME, Moy GW, Vacquier VD. 2000.Positive selection and propeptide repeatspromote rapid interspecific divergence of agastropod sperm protein.Mol. Biol. Evol.17:458–66

Hellberg ME, Vacquier VD. 1999. Rapid evo-lution of fertilization selectivity and lysincDNA sequences in teguline gastropods.Mol. Biol. Evol.16:839–48

Howard DJ. 1993.Reinforcement: Origin, Dy-namics, and Fate of an Evolutionary Hypoth-esis, pp. 46–69. Oxford, England: OxfordUniv. Press

Howard DJ. 1999. Conspecific sperm and

pollen precedence and speciation.Annu. Rev.Ecol. Syst.30:109–32

Hughes AL, Nei M. 1988. Pattern of nucleotidesubstitution at major histocompatibility com-plex class I loci reveals overdominant selec-tion. Nature335:167–70

Iwasa Y, Pomiankowski A. 1995. Continualchanges in mate preferences.Nature 377:420–22

Jaffe LA, Sharp AP, Wolf DP. 1983. Absence ofan electrical polyspermy block in the mouse.Dev. Biol.96:317–23

Juneja R, Agulnik SI, Silver LM. 1998. Se-quence divergence within the sperm-speci-fic polypeptide TCTE1 is correlated withspecies-specific differences in sperm bindingto zona-intact eggs.J. Androl.19:183–88

Kachroo A, Schopfer CR, Nasrallah ME, Nas-rallah JB. 2001. Allele-specific receptor-ligand interactions inBrassicaself-incompa-tibility. Science293:1824–26

Kresge N, Vacquier VD, Stout CD. 2000a. 1.35and 2.07 A resolution structures of the redabalone sperm lysin monomer and dimer re-veal features involved in receptor binding.Acta Crystallogr. D Biol. Crystallogr.56:34–41

Kresge N, Vacquier VD, Stout CD. 2000b.The high resolution crystal structure ofgreen abalone sperm lysin: implications forspecies-specific binding of the egg receptor.J. Mol. Biol.296:1225–34

Kresge N, Vacquier VD, Stout CD. 2001a.Abalone lysin: the dissolving and evolvingsperm protein.Bioessays23:95–103

Kresge N, Vacquier VD, Stout CD. 2001b. Thecrystal structure of a fusagenic sperm proteinreveals extreme surface properties.Biochem-istry 40:5407–13

Lee YH, Ota T, Vacquier VD. 1995. Positive se-lection is a general phenomenon in the evolu-tion of abalone sperm lysin.Mol. Biol. Evol.12:231–38

Li W-H. 1997. Molecular Evolution. Sunder-land, MA: Sinauer

Luporini P, Vallesi A, Miceli C, Bradshaw RA.1995. Chemical signaling in ciliates.J. Eu-karyot. Microbiol.42:208–12



Lyon JD, Vacquier VD. 1999. Interspecieschimeric sperm lysins identify regions me-diating species-specific recognition of theabalone egg vitelline envelope.Dev. Biol.214:151–59

Makalowski W, Boguski MS. 1998. Evolution-ary parameters of the transcribed mammaliangenome: an analysis of 2,820 orthologous ro-dent and human sequences.Proc. Natl. Acad.Sci. USA95:9407–12

Mayfield JA, Fiebig A, Johnstone SE, PreussD. 2001. Gene families from the Arabidop-sis thaliana pollen coat proteome.Science292:2482–85

McAllister BF, Werren JH. 1999. Evolution oftandemly repeated sequences: What happensat the end of an array?J. Mol. Evol.48:469–81

Metz EC, Gomez-Gutierrez G, Vacquier VD.1998a. Mitochondrial DNA and bindin genesequence evolution among allopatric speciesof the sea urchin genus Arbacia.Mol. Biol.Evol.15:185–95

Metz EC, Palumbi SR. 1996. Positive selectionand sequence rearrangements generate ex-tensive polymorphism in the gamete recogni-tion protein bindin.Mol. Biol. Evol.13:397–406

Metz EC, Robles-Sikisaka R, Vacquier VD.1998b. Nonsynonymous substitution in aba-lone sperm fertilization genes exceeds sub-stitution in introns and mitochondrial DNA.Proc. Natl. Acad. Sci. USA95:10676–81

Nasrallah JB. 2000. Cell-cell signaling in theself-incompatibility response.Curr. Opin.Plant Biol.3:368–73

Nielsen R, Yang Z. 1998. Likelihood modelsfor detecting positively selected amino acidsites and applications to the HIV-1 envelopegene.Genetics148:929–36

Ortenzi C, Alimenti C, Vallesi A, Di PB, LaTA, Luporini P. 2000. The autocrine mito-genic loop of the ciliate Euplotes raikovi:The pheromone membrane-bound forms arethe cell binding sites and potential signalingreceptors of soluble pheromones.Mol. Biol.Cell 11:1445–55

Palumbi SR. 1999. All males are not created

equal: Fertility differences depend on gameterecognition polymorphisms in sea urchins.Proc. Natl. Acad. Sci. USA96:12632–37

Palumbi SR, Metz EC. 1991. Strong reproduc-tive isolation between closely related tropicalsea urchins (GenusEchinometra). Mol. Biol.Evol.8:227–39

Partridge L, Hurst LD. 1998. Sex and conflict.Science281:2003–8

Price CS. 1997. Conspecific sperm precedencein Drosophila.Nature388:663–66

Rahman MA, Uehara T. 2000. Experimentalhybridization between two tropical speciesof sea urchins (genusEchinometra) in Oki-nawa.Zygote8:S90

Rice WR, Holland B. 1997. The enemieswithin: intergenomic conflict, interlocus con-test evolution (ICE), and the intraspecific RedQueen.Behav. Ecol. Sociobiol.41:1–10

Richman AD, Kohn JR. 2000. Evolutionarygenetics of self-incompatibility in the Sola-naceae.Plant Mol. Biol.42:169–79

Richman AD, Uyenoyama MK, Kohn JR. 1996.Allelic diversity and gene genealogy at theself-incompatibility locus in the Solanaceae.Science273:1212–16

Rooney AP, Zhang J, Nei M. 2000. An unusualform of purifying selection in a sperm pro-tein.Mol. Biol. Evol.17:278–83

Schmid KJ, Tautz D. 1997. A screen for fastevolving genes from Drosophila.Proc. Natl.Acad. Sci. USA94:9746–50

Schopfer CR, Nasrallah JB. 2000. Self-incompatibility. Prospects for a novel puta-tive peptide-signaling molecule.Plant Phys-iol. 124:935–40

Schopfer CR, Nasrallah ME, Nasrallah JB.1999. The male determinant of self-incompa-tibility in Brassica.Science286:1697–700

Singh RS, Kulathinal RJ. 2000. Sex gene poolevolution and speciation: a new paradigm.Genes Genet. Syst.75:119–30

Swanson WJ, Aquadro CF, Vacquier VD.2001a. Polymorphism in abalone fertiliza-tion proteins is consistent with the neutralevolution of the egg’s receptor for lysin(VERL) and positive Darwinian selection ofsperm lysin.Mol. Biol. Evol.18:376–83



Swanson WJ, Clark AG, Waldrip-Dail HM,Wolfner MF, Aquadro CF. 2001b. Evolution-ary EST analysis identifies rapidly evolv-ing male reproductive proteins in Drosophila.Proc. Natl. Acad. Sci. USA98:7375–79

Swanson WJ, Vacquier VD. 1995a. Extraordi-nary divergence and positive Darwinian se-lection in a fusagenic protein coating theacrosomal process of abalone spermatozoa.Proc. Natl. Acad. Sci. USA92:4957–61

Swanson WJ, Vacquier VD. 1995b. Liposomefusion induced by a M(r) 18,000 protein lo-calized to the acrosomal region of acrosome-reacted abalone spermatozoa.Biochemistry34:14202–8

Swanson WJ, Vacquier VD. 1997. The abaloneegg vitelline envelope receptor for spermlysin is a giant multivalent molecule.Proc.Natl. Acad. Sci. USA94:6724–29

Swanson WJ, Vacquier VD. 1998. Concertedevolution in an egg receptor for a rapidlyevolving abalone sperm protein.Science281:710–12

Swanson WJ, Vacquier VD. 2002. Rapid evo-lution of reproductive proteins.Nat. Rev. Ge-netics3:137–44

Swanson WJ, Yang Z, Wolfner MF, AquadroCF. 2001c. Positive Darwinian selectiondrives the evolution of several female repro-ductive proteins in mammals.Proc. Natl.Acad. Sci. USA98:2509–14

Ting C-T, Tsaur S-C, Wu M-L, Wu CI. 1998.A rapidly evolving homeobox at the site of ahybrid sterility gene.Science282:1501–4

Tsaur SC, Ting CT, Wu CI. 1998. Positive selec-tion driving the evolution of a gene of malereproduction, Acp26Aa, of Drosophila: II.Divergence versus polymorphism.Mol. Biol.Evol.15:1040–46

Tsaur SC, Wu CI. 1997. Positive selection andthe molecular evolution of a gene of malereproduction, Acp26Aa of Drosophila.Mol.Biol. Evol.14:544–49

Vacquier VD, Swanson WJ, Hellberg ME.1995. What have we learned about sea urchinsperm bindin?Dev. Growth Differ.37:1–10

Vacquier VD, Swanson WJ, Lee YH. 1997.Positive Darwinian selection on two homol-ogous fertilization proteins: What is the se-lective pressure driving their divergence?J.Mol. Evol.44:S15–22

Wassarman PM, Jovine L, Litscher ES. 2001. Aprofile of fertilization in mammals.Nat. CellBiol. 3:59–64

Wolfner MF. 1997. Tokens of love: functionsand regulation of Drosophila male accessorygland products.Insect Biochem. Mol. Biol.27:179–92

Wyckoff GJ, Wang W, Wu CI. 2000. Rapid evo-lution of male reproductive genes in the de-scent of man.Nature403:304–9

Yang Z, Bielawski JP. 2000. Statistical methodsfor detecting molecular adaptation.TrendsEcol. Evol.15:496–503

Yang Z, Nielsen R, Goldman N, Pedersen AM.2000a. Codon-substitution models for het-erogeneous selection pressure at amino acidsites.Genetics155:431–49

Yang Z, Swanson WJ. 2002. Codon-substi-tution models to detect adaptive evolutionthat account for heterogeneous selectivepressures among site classes.Mol. Biol. Evol.19:49–57

Yang Z, Swanson WJ, Vacquier VD. 2000b.Maximum-likelihood analysis of molecularadaptation in abalone sperm lysin revealsvariable selective pressures among lineagesand sites.Mol. Biol. Evol.17:1446–55

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