The Molecular Evolution of Sperm Bindin in Six Species of Sea Urchins (Echinoida: Strongylocentrotidae)

Christiane H. Biermann1 Department of Ecology and Evolution, State University of New York at Stony Brook

The acrosomal protein bindin attaches sperm to eggs during sea urchin fertilization. Complementary to ongoing functional biochemical studies, I take a comparative approach to explore the molecular evolution of bindin in a group of closely related free-spawning echinoid species. Two alleles of the mature bindin gene were sequenced for each of six species in the sea urchin family Strongylocentrotidae. The nucleotide sequences diverged by at least 1% per Myr at both silent and replacement sites. Two short sections ¯anking the conserved block show an excess of nonsynonymous substitutions. Each is homologous to a region that had been identi®ed as a target of selection in other sea urchin comparisons. A large proportion of the bindin-coding sequence consists of a highly variable repeat region. Bindin sequences, even including the large intron, could not resolve the branching order among ®ve of the species.

Introduction For several sympatric groups of free-spawning ma- polypeptide backbone (Foltz 1994; Stears and Lennarz rine animals, gamete recognition proteins have been 1997). Bindin, on the contrary, is 100% protein (Minor, found to be under positive selection for interspeci®c di- Gao, and Davidson 1989), and therefore potentially con- vergence (Vacquier and Lee 1993; Swanson and Vac- tains speci®city information in its primary structure. quier 1995; Metz and Palumbi 1996). The sea urchin With recombinant deletion mutants, Lopez, Miraglia, family Strongylocentrotidae contains at least nine exter- and Glabe (1993) showed that either of the two variable nally fertilizing species, all of which occur in the North domains of bindin, on either side of the conserved mid- Paci®c with partly overlapping ranges (Jensen 1974; Ba- dle region, is suf®cient to impart speci®c gamete agglu- zhin 1998). If their sperm bindin is involved in repro- tination between Strongylocentrotus franciscanus and ductive isolation, evolutionary analyses of the gene may Strongylocentrotus purpuratus. Minor, Britten, and Da- indicate natural selection or functional domains. This is vidson (1993) identi®ed a number of peptides, small particularly interesting, as the genus Strongylocentrotus bindin segments of these two congeners, that interfere has been the primary model system for biochemical with sperm±egg binding. On average, the joint effect of analyses of bindin (e.g., Glabe and Clark 1991; Minor three peptides was necessary to inhibit fertilization. One et al. 1991). 30-residue peptide, matching the S. franciscanus se- Bindin is involved in the species-speci®c recogni- quence, showed inhibitory effects signi®cantly different tion between sperm and eggs in echinoid echinoderms, between the two species. serving to bind sperm cells to the egg surface. The pro- Strongylocentrotus franciscanus and S. purpuratus tein is exocytosed from the vesicle at the tip of the have overlapping ranges and spawning seasons (Strath- sperm head when sperm undergo the acrosome reaction mann 1987). However, they are separated evolutionarily (Vacquier and Moy 1977; Gao et al. 1986). Although by at least 20 Myr (Smith 1988), during which accu- other phases during sperm±egg recognition can be spe- mulation of silent or multiple substitutions may have cies-speci®c (SeGall and Lennarz 1979; unpublished obscured molecular traces of initial divergent selection. data), the interaction of bindin with the egg surface is a It may be more informative to compare mating barriers key stage, one that has received much recent attention in more recently derived species (Palumbi 1994; Coyne (e.g., Foltz and Lennarz 1993; Hofmann and Glabe and Orr 1997). This study analyzes sperm bindin for the 1994; Vacquier, Swanson, and Hellberg 1995; Cameron species that bridge the phylogenetic gap between S. et al. 1996; Metz and Palumbi 1996). franciscanus and S. purpuratus. Two alleles of the entire Ideally, both sides of a recognition system should mature bindin were sequenced for each of six species, be examined jointly, but it is probably not a straightfor- and their molecular evolution is compared between ex- ward matter to pinpoint key recognition sites in the gene ons and introns and to a protein-coding region of mi- for the putative sperm receptor on the egg surface (Gla- tochondrial DNA. be and Vacquier 1978; Giusti, Hoang, and Foltz 1997; Mauk et al. 1997). This is because the interaction of Materials and Methods bindin with its receptor is a multistep process involving Samples and Sequencing both the receptor's carbohydrate components and the The six sea urchin species were sampled from 1 Present address and address for correspondence and reprints: Christiane H. Biermann, Department of Biological Sciences, Univer- around the Paci®c Ocean between 1994 and 1996. The sity of Hull, Hull HU6 7RX, United Kingdom. E-mail: species abbreviations are as follows: drob, Strongylo- [email protected]. centrotus droebachiensis; pall, Strongylocentrotus pal- Key words: sea urchin, Strongylocentrotus, sperm bindin, fertil- lidus; purp, S. purpuratus; poly, Strongylocentrotus po- ization, reproductive isolation, tandem repeats. lyacanthus; Allo, Allocentrotus fragilis; Hemi, Hemi- Mol. Biol. Evol. 15(12):1761±1771. 1998 centrotus pulcherrimus. PurpCA and Allo3 were from ᭧ 1998 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038 California, Allo7 was from the Eastern Bering Sea, and

1761 1762 Biermann the Hemicentrotus individual (both bindin alleles se- don usage, as well as base composition percentages of quenced) was from Japan. ``F'' stands for Friday Harbor, the nucleotide sequences, were calculated for one allele Wash.; ``K'' for Kamchatka, Siberia, and ``R'' for Res- per species in MEGA (Kumar, Tamura, and Nei 1993). olute, Northwest Territories. The species identity of each The number of nonsynonymous (amino acid re- animal was con®rmed with mitochondrial DNA. Part of placement) substitutions per nonsynonymous site and the ATPase subunit 6 gene was sequenced for those in- the number of synonymous (silent) substitutions per si- dividuals that were not included in a larger mitochon- lent site (Dn and Ds, respectively; Lee, Ota, and Vacquier drial phylogenetic study (Kessing 1991; unpublished 1995; Hughes 1997) were estimated by Nei and Gojo- data). Sequences from the mitochondrial project provid- bori's (1986) method. Dn and Ds and their standard er- ed an independent locus for all of the species in this rors were computed in MEGA (Kumar, Tamura, and Nei study, referred to as ``mtDNA'' below. The sequences 1993) for sliding windows along the sequence. Windows include two regions of mitochondrial DNA: one cover- were 30 codons in length and moved by 10 codons for ing 145 codons of the CO1 gene, corresponding to po- each step, making the plot directly comparable to ®gure sitions 6835±7270 in the complete S. purpuratus mito- 3 in Metz and Palumbi (1996). When either Dn or Ds chondrial sequence (Jacobs et al. 1988), and the other exceeded 0.4 for a particular window, the p distance stretching from the end of CO2 through ATPase 8 into (proportion of differences) was used instead of the Jukes- ATPase 6 (positions 8356±9005 in Jacobs et al. 1988), Cantor corrected distance for both Dn and Ds in that excluding the Lys-tRNA. window (Kumar, Tamura, and Nei 1993). PCR primers were designed for near the 3Ј end of prebindin upstream of the cleavage site between prepro- Repeat Network and mature bindin, and, after RT-PCR, for the untrans- Because orthology/paralogy relationships in the re- lated region 3Ј of the stop codon. Sequencing primers peat region are uncertain, the individual segments were were spaced no more than 300 bp apart on both strands. taken apart. A name (single letter) was assigned to each Primer locations and sequences are available from the of the variations of the motif to visualize the order of author. their occurrence in the alleles. There are too few infor- DNA was isolated from gonad tissue, PCR-ampli- mative sites in the 21-nt repeat to construct an overall ®ed, and initially sequenced directly (with Sequenase bifurcating tree of the 29 different motifs. Therefore, a from Amersham, following the method of Khorana, Ga- mutational network was constructed from a pairwise dis- gel, and Cote 1994). However, because of the frequency tance matrix of individual repeats (Templeton, Crandall, of heterozygous length mutations, most PCR products and Sing 1992; Crandall and Templeton 1993). This had to be cloned before sequencing (pGEM-T vector method was developed for population-level data with from Promega, transformation into XL Blue Escherichia small numbers of changes; it evaluates parsimonious coli). In spite of using DNA polymerases with proof- connections between alleles on a pairwise basis. Net- reading ability (ExTaq and LATaq from TaKaRa, Bio- works are useful for population data (including a ``pop- X-Act from BioExpress, Expand High Fidelity from ulation'' of repeats within a gene), because they include Boehringer Mannheim), a substantial number of point ancestral sequences as internal nodes and allow the de- mutations and recombinations between alleles were ev- piction of ambiguities (Crandall and Templeton 1996; ident in the cloned PCR products (e.g., Bradley and Hil- Fitch 1996). lis 1997). Hence, at least two clones were sequenced in their entirety for each allele; whenever there was a dis- Phylogenetic Sequence Analyses crepancy, an additional clone was sequenced until two Phylogenetic trees were reconstructed with the help identical sequences were obtained. Mitochondrial DNA of PAUP* (test versions 4.0.0d59±63). A minimum- was always sequenced directly. The mature bindin se- evolution (distance) tree delineates the overall relation- quences have been deposited in GenBank under acces- ships of 13 bindin sequences, excluding the repeat re- sion numbers AF077309±AF077321. gion (®g. 6). The total number of nucleotides from the (nonrepeat) coding region and the intron, aligned in- Sequence Analyses cluding the S. franciscanus coding sequence (Minor et GCG (Wisconsin Package, Version 9.1, Madison, al. 1991), was 1,558. I employed LogDet distances and Wis.) was used to explore the sequence structure and rooted the tree at the midpoint. Regions with alignment provided the initial alignment (PileUp, endweighted), gaps were excluded from pairwise comparisons only. which was then corrected manually. Because of ques- The tree topology, and particularly the full bootstrap tionable homology, both the repeat region and the poly- support joining the two sequences from each species, glycine stretch were excluded from phylogenetic se- was robust under various distance measures. quence analyses or treated separately. Bindin sequence phylogenies were further explored Nucleotide diversity between the two alleles from with only one sequence per species, with Hemicentrotus each species (␲; Nei 1987) and net interspeci®c diver- as outgroup. Due to the small number of parsimony- gence (Da, Nei 1987) were calculated with DnaSP (Ro- informative sites in bindin (table 2), little con®dence can zas and Rozas 1997) in sliding windows along the se- be placed in parsimony-based phylogenies. Maximum- quence. Windows were 100 nt in length and moved in likelihood and distance trees were also constructed sep- steps of 25 nt. Both nonsynonymous and silent substi- arately for the nonrepeat bindin-coding sequences and tutions were included. Amino acid frequencies and co- for the intron. A 1,005-nt region of the mitochondrial Sperm Bindin in Strongylocentrotid Sea Urchins 1763

FIG. 1.ÐBindin amino acid sequences (one per species). The ®rst black line denotes the intron position and the start of the conserved block; the next two lines bracket the glycine-rich region. The repeatsÐarbitrarily right-alignedÐare separated by light lines. francis ϭ published S. franciscanus sequence (Minor et al. 1991). genome (Kessing 1991; unpublished data) was used to species. The only exception is the 5Ј half of the intron independently examine the relationships between the in A. fragilis, which is extremely divergent from this species and genera. region in the sibling species. Because positional homol- ogy is not attainable when aligning that region, Allocen- Results trotus was omitted from the main graphs in ®gure 2 (but General Structure see the dotted line), and for phylogenetic analyses em- ploying the intron, this 364-bp region was replaced by As expected from intergeneric comparisons (Glabe ``unknown'' characters. It contains multiple long runs of and Clark 1991; Minor et al. 1991; Metz and Palumbi single bases in Allocentrotus sample 3, which caused 1996), the central coding region of mature bindin is stuttering of both the PCR and sequencing enzymes. highly conserved within and across species (®g. 1). Just Probably for the same reason, it was not possible to upstream, a large intron inserts in strongylocentrotid sea determine this part of the intron sequence for the other urchins in a conserved position (®g. 2; Metz and Pal- Allocentrotus individual (sample 7). umbi 1996; Metz, Gomez-Gutierrez, and Vacquier 1998). It is more than twice as long as the intron found Nucleotide and Amino Acid Composition in the genus Echinometra (Metz and Palumbi 1996), and Base composition in bindin differs signi®cantly be- generally conserved enough to permit alignment across tween the three codon positions, with small numbers of 1764 Biermann

FIG. 2.ÐSliding-window comparison of nucleotide diversity ␲ (Nei 1987; between two alleles within each of ®ve species) and of net interspeci®c divergence Da (number of net nucleotide substitutions per site; Nei 1987). The repeated amino acid motifs (``repeats'') were right- aligned.

Ts and large numbers of Gs, especially in the ®rst po- this family: S. purpuratus, S. polyacanthus, and Hemi- sition, and many purines in the second codon position centrotus typically have 7 of these repeats, Allocentrotus (not shown). There is a signi®cant difference in base has 5, S. droebachiensis has between 6 and 9, and S. composition between the bindin-coding region and the pallidus has around 12 (®gs. 1 and 3). This region ap- intron (P Ͻ 0.00001, G-test with Williams correction; parently diversi®ed within this group. The basal stron- Sokal and Rohlf 1995); i.e., the bias against T's and for gylocentrotid, S. franciscanus, has only two repeats, and Gs is not speci®c to this part of the chromosome in Lytechinus variegatus, in a different family, has this mo- general. Base composition in coding mitochondrial tif only once (Minor et al. 1991). DNA differs signi®cantly from that in the coding bindin Figure 3 lists the nucleotide and amino acid se- sequence, but not from that in the bindin intron (P Ͻ quences of the individual repeats and the order in which 0.00001 and P 0.211, respectively). ϭ they occur in representatives of the different species. The conserved region, which is involved in the Letters with an asterisk represent repeats that differ from binding process (Miraglia and Glabe 1993), has a rela- tively balanced amino acid composition. It contains a the nonasterisked equivalents by a silent substitution; large number of nonpolar residues, many aspartic and different letters stand for repeats that differ at the protein glutamic acids, and positively charged residues. A tract level. The repeats contain 18±21 nt, three of which are of glutamic acid residues near the N-terminus of the completely invariant; hence, there is too little informa- conserved block (®g. 1) represents the only area of the tion to reconstruct their evolution unequivocally. protein that is clearly alpha-helical, negatively charged, Figure 4 shows the relationships between nucleo- and hydrophilic. The regions ¯anking the conserved tide sequences of individual repeats as a cladistic net- central part contain a large proportion of turn residues work. Small zeroes indicate inferred haplotypes that like glycines and prolines; i.e., bindin appears to not were not present among the repeat samples. Thick black form much secondary structure, and so far, it has not bars show direct connections between haplotypes which been possible to crystallize it for X-ray crystallography. are parsimonious (A. R. Templeton's Mathematica pro- Almost all codons are being used, in fairly equal pro- gram ParsProb; Templeton, Crandall, and Sing 1992). portions (not shown). Connections with triple lines are marginally parsimoni- Repeats ous (94% probability), with 2 bases out of 21 substituted The number of repeats of a seven-amino-acid motif (one inferred intermediate). Changes between repeats near the C-terminus of the protein is highly variable in that differ by three substitutions are not parsimonious Sperm Bindin in Strongylocentrotid Sea Urchins 1765

FIG. 4.ÐRelationships between nucleotide sequences of individ- ual repeats from the 3Ј region of sperm bindin (®g. 3). A cladistic network was constructed based on the parsimony criteria by Temple- ton, Crandall, and Sing (1992). Only the heavy bars and the triple lines symbolize parsimonious connections (see text). Small zeroes indicate inferred intermediate haplotypes. Repeat variants that differ by more than three changes from any others were not connected to the main network.

dispose bindin to rapid evolutionary change but, at the same time, make it dif®cult to recognize whether natural selection has promoted this mutability (Hughes 1991). Upstream of the tandem duplications is a glycine-rich stretch that is also very variable in length. Especially in these regions of bindin, blocks of similar sequence ap- pear to have been retained between species since recom- bination events during the divergence of these lineages (®g. 1). Synonymous and Nonsynonymous Substitution Rates

Dn and Ds increase linearly with the phylogenetic distance of the alleles compared (plot not shown). The rate (per available site) of amino-acid-altering substitu- FIG. 3.ÐIndividual repeats from the 3Ј region of sperm bindin. tions was slightly lower than that of silent changes in Each variation of the motif is given a one-letter name, and its nucle- almost all pairwise comparisons. It is nevertheless very otide and amino acid sequences are shown. Asterisks denote silent high compared to those for most other proteins: D /D changes. The order of occurrence of these motifs in representatives of n s the different species is shown below. For S. droebachiensis and S. ratios for bindin are generally an order of magnitude pallidus, individual ``1'' is from the Paci®c Ocean, and individual ``2'' higher than those for mitochondrial genes (see also ®g. is from the Atlantic Ocean. 5). The many insertions and deletions between bindin sequences can be considered nonsynonymous changes as well (AguadeÂ, Miyashita, and Langley 1992), and are according to Templeton's criteria; i.e., if 3 out of 21 not included in the Dn/Ds ratios. Moreover, the substi- bases have mutated, we expect that invisible multiple tutions among repeats are almost exclusively nonsynon- hits have occurred. These connections are tentatively ymous, but the repeat region was excluded from pair- marked by thin lines through two inferred intermediates. wise comparisons due to alignment ambiguity. Motifs that differ by more than three changes remain The sliding-window comparison clearly shows the unconnected. absence of replacement substitutions in the conserved The network permits inferences about whether region (®g. 5, codons 100±150). Both Dn and Ds vary identical motifs are more likely to be convergent (pos- widely along the rest of the gene, with Dn signi®cantly sibly the ``J'' repeats in S. droebachiensis-2 and S. po- exceeding Ds in three locations when the ®ve most lyacanthus) or orthologous to each other (repeats ``Q-T- closely related species are compared (marked by aster- A/B-I'' in S. droebachiensis-1 and S. pallidus-1; ®gs. 3 isks in ®g. 5, including two regions composed of two and 4). Only about one in seven mutations among the adjacent signi®cant windows each). Because one of repeats in ®gure 4 is a synonymous change. The unusual these three windows is in the repeat region, it was not amino acid composition of the repeats alone is not re- analyzed further. For the other two signi®cant windows sponsible for the excess of replacements: random point (white boxes in ®g. 5), Dn and Ds were contrasted on a mutations imposed on the motifs resulted, on average, pairwise basis between individual species (table 1). in 1 change out of 4.6 being silent. In spite of the uncertain homology when the repeats Phylogenetic Trees are aligned in the order found in the gene, the magnitude Bindin and mitochondrial DNA sequences indicate of both polymorphism and divergence in this region is that both Allocentrotus and Hemicentrotus fall phylo- striking (®g. 2). The repeated segments appear to pre- genetically within the genus Strongylocentrotus (®g. 6). 1766 Biermann

FIG. 5.ÐNonsynonymous changes per nonsynonymous site (Dn) and silent changes per silent site (Ds) in sliding windows 30 amino acids in length along the coding sequence of bindin. Pairwise differences between one individual each of S. purpuratus, S. droebachiensis, S. pallidus, S. polyacanthus, and A. fragilis are shown. Asterisks signify windows in which Dn was signi®cantly greater than Ds (Kumar, Tamura, and Nei 1993). The repeat region is marked by the cross-hatched rectangle below. The open rectangle covers a region that was identi®ed by Metz and Palumbi (1996) as being under positive selection in the sea urchin genus Echinometra, and the open square corresponds to a peptide recognized as species-speci®c in Strongylocentrotus by Minor, Britten, and Davidson (1993). The bars on the right show Jukes-Cantor corrected Dn and Ds among these ®ve species for 335 codons of mitochondrial sequence.

Strongylocentrotus franciscanus branched off well be- Collapsing branches with less than 50% bootstrap fore these two monospeci®c genera diverged from the support resulted in polytomies among the most closely other strongylocentrotid species. The phylogeny of this related species for both bindin and the mitochondrial family has been impossible to ascertain on the basis of data. Strongylocentrotus purpuratus, S. droebachiensis, morphological characters (O. Ellers and R. Mooi, per- S. pallidus, S. polyacanthus, and Allocentrotus fragilis sonal communication); hence, these sequences represent appear to have diverged so rapidly from each other that our best estimate of their relationships. the internodes may be too short to permit reconstruction of the branching order. This is consistent with the low Table 1 number of parsimony-informative sites: for example, out Pairwise Signi®cance t-tests (Kumar, Tamura, and Nei of 132 variable sites in the bindin intron, only 8 are 1993) of an Excess of Nonsynonymous Substitutions per parsimony-informative (table 2). Only a maximum-like- Site (Dn) Over Silent Substitutions per Site (Ds) lihood bootstrap analysis for the bindin intron supports any groupings among these ®ve species (not shown), but these are not retained in distance trees. The alignable coding regions of bindin do not strongly support any bifurcations (data not shown), and the overall bindin tree

NOTE.ÐComparisons are for regions identi®ed by sliding windows (®g. 5). FIG. 6.ÐMinimum-evolution tree based on LogDet distances be- To the upper right of the diagonal are tests for the upstream window (codons tween bindin sequences of strongylocentrotid sea urchins (midpoint- 61±100; see also Metz and Palumbi 1996), and tests in the lower left are for the rooted). See Materials and Methods for species names and sampling downstream window (codons 161±190; see also Minor, Britten, and Davidson locations. The sequences include the nonrepeat coding region and the 1993). An asterisk denotes signi®cance at P Ͻ 0.05. purp ϭ Strongylocentrotus intron of mature bindin (for S. franciscanus, only the coding region is purpuratus, drob ϭ Strongylocentrotus droebachiensis, pall ϭ Strongylocentrotus available; Minor et al. 1991). Numbers on nodes indicate bootstrap pallidus, Allo ϭ Allocentrotus fragilis, poly ϭ Strongylocentrotus polyacanthus. support (500 replications). Sperm Bindin in Strongylocentrotid Sea Urchins 1767

Table 2 Numbers of Characters, as well as Parameters Estimated by Maximum Likelihood, for Different Sequence Subsets in Six Sea Urchin Species (Strongylocentrotus droebachiensis, Strongylocentrotus pallidus, Strongylocentrotus purpuratus, Strongylocentrotus polyacanthus, Allocentrotus fragilis, and Hemicentrotus pulcherrimus) Nonrepeat Coding Bindin Bindin Intron mtDNA (coding) Number of aligned nucleotides ...... 549 979 1,005 Number of variable sites ...... 64 132 179 Parsimony-informative sites ...... 6 8 64 Observed proportion of constant sites ...... 0.883 0.865 0.822 Estimated proportion of invariable sites ..... 0.395 0.0 0.295 Estimated gamma shape parameter ...... 0.845 ϱ 0.253 Estimated transition/transversion ratio ...... 1.49 0.99 10.32

NOTE.ÐThe nonalignable part of the intron in Allocentrotus was excluded.

(®g. 6 shows a minimum-evolution tree) is dominated nonymous substitutions, and the lack of a conclusive by the intron sites. phylogenetic signal. More than a kilobase of mitochondrial coding se- quence (table 2) could not resolve the branching order Repeats among the ®ve most closely related species either: each The evolutionary dynamics of repeated DNA se- optimality criterion or evolutionary model tried resulted quences has become a major ®eld of investigation in in a multifurcation similar to that shown in ®gure 6. recent years. Generally, the focus has been on noncoding DNA, especially microsatellites (Stephan 1989; Nielsen Discussion 1997a). The attention is now shifting, since more and more coding repeats are found to be important in human Numerous length mutations in the mature bindin of diseases (Lesch et al. 1996; Mandel 1997). Interestingly, these six sea urchin species reduce the number of or- tandem repeats are generally found in adhesive proteins thologous sites available for evolutionary analysis. Al- like glue (Martin, Mayeda, and Meyerowitz 1988), bys- most one half of the coding region consists of repeated sus (Coyne, Qin, and Waite 1997), and silk (Guerette et residues and motifs (®g. 1) whose evolutionary dynam- al. 1996). Repeats in the latter proteins also form gly- ics are more complex and much less well understood cine-rich amorphous structures. The only other proteins than those of nucleotide substitutions. Another third of that commonly contain repetitive arrays are recognition the protein is extremely conserved, which further limits the inference of historical events. Nevertheless, inter- molecules, e.g., in parasitic protozoa (Hughes 1993). esting patterns can be gleaned from the sequences. Both its adhesive and recognition functions could there- Amino acid composition differs notably between fore have favored the repetitive character of bindin. the conserved region and the rest of the gene. The fact While in the strongylocentrotids, the repeats are most that the central conserved block contains the larger num- evident in the 3Ј part of bindin, in Echinometra, repeat ber of hydrophobic residues agrees with the ®nding that structures appear in the other half of bindin, upstream this part associates with phospholipid bilayers and, of the conserved block (Metz and Palumbi 1996). It will hence, presumably with the egg membrane in vivo be interesting to ®nd out whether the repeats are im- (Kennedy, DeAngelis, and Glabe 1989). This leaves the portant for the general or speci®c function of bindin. two variable ends of bindin to interact with the glyco- Repeat regions are thought to be hot spots for un- proteins of the egg in a potentially species-speci®c man- equal crossing over (Smith 1976), but in addition to ner (Lopez, Miraglia, and Glabe 1993). These glycine- length mutations, the bindin repeat region is character- rich ends do not contain many charged residues or ob- ized by numerous, largely nonsilent, point substitutions. vious features predicting secondary (and higher) struc- Although the parsimony network (®g. 4) and the simu- ture, consistent with bindin's function as an amorphous lations (see Results) are based on conventional models glue protein. of molecular evolution (random and independent nucle- Bindin is seasonally expressed in large amounts in otide substitutions), it is unlikely that recombination per the testes (Cameron et al. 1990; Nishioka et al. 1990), se would result in an excess of nonsynonymous substi- and the need for ef®cient translation is thought to select tutions. Generally, multiple copies of a sequence are for biased codon usage (Akashi 1997). Bindin's codon constantly being homogenized by gene conversion (Do- bias appears to be low, however. Without comparison ver et al. 1993; SchloÈtterer et al. 1994; Odorico and with other nuclear genes, it is dif®cult to assess whether Miller 1997). The extreme diversity of the 3Ј bindin the selection pressure for preferred codons on bindin is repeats, coupled with a scarcity of silent substitutions, weak, or just ineffective due to effectively small popu- is highly suggestive of natural selection as the agent lations (Akashi 1997). responsible for the maintenance of their varied identi- Below, I discuss three additional aspects of bindin ties. It appears that mutations induced by chromatid evolution: the repeat structure, the high rate of nonsy- breaks during recombination are not eliminated by the 1768 Biermann homogenizing effect of concerted evolution, but are Sliding-window comparisons do not identify an ex- maintained by positive selection. cess of nonsynonymous substitutions when all seven species are compared (not shown), but they do point to Rate of Nonsynonymous Substitutions three regions with signi®cantly large Dn values in the Two abalone sperm proteins have been shown to comparison of the ®ve most closely related species (®g. be subject to strong directional selection, especially in 5). This con®rms that proportionally more silent substi- closely related species (Lee and Vacquier 1992; Swan- tutions accumulated on the long branches leading to H. son and Vacquier 1995). This stimulated the exciting pulcherrimus and S. franciscanus. Sliding-window anal- notion that gamete recognition proteins may play a cru- yses are generally used for data exploration only (e.g., cial role in speciation in marine invertebrates (Palumbi Kreitman and Hudson 1991), because they essentially 1992, 1994). Gamete surfaces could be avoiding micro- consist of multiple comparisons (a certain proportion of bial attack (Vacquier and Lee 1993) or diversifying by windows are expected to be signi®cantly different by intraspeci®c processes such as sexual selection (Metz chance), and spatial autocorrelation makes statistical and Palumbi 1996). Alternatively, divergence of the sur- testing complex. They are nonetheless very useful in face molecules could be driven by reinforcement of mat- identi®ng pattern changes along a sequence. ing barriers upon secondary contact of two incipient Because one of the windows in which Dn signi®- species. In either case, the signature of positive Darwin- cantly exceeds Ds falls in the repeat region (cross- ian selection could be a large number of amino acid hatched bar below the graph in ®g. 5), it is not analyzed replacements between species at these recognition loci. further because of uncertain homology. It is fascinating Among the sea urchin bindins examined here, the that the other two windows (open boxes below the graph number of nonsynonymous substitutions is larger than in ®g. 5) coincide precisely with parts of bindin that the number of silent mutations, but on a per-site basis, have previously been pinpointed as species-speci®cÐ it is on average slightly lower. However, the mean Dn/ each for a different reason. Window 61±100, just up- Ds ratio is much higher for bindin than for mitochondrial stream of the conserved block, is in the same position proteins (®g. 5). Because Dn and Ds do not differ sig- as the region identi®ed as being under diversifying se- ni®cantly in bindin overall, the elevated Dn could be due lection in the genus Echinometra by Metz and Palumbi to a relaxation of purifying selection and not necessarily (1996). Window 161±190, just downstream of the poly- to the action of directional selection. The extreme con- Glu helix at the end of the conserved region, corre- servation of the central block speaks against complete sponds precisely to the only peptide of 24 tested that neutrality of the substitutions in bindin. Furthermore, all species-speci®cally inhibited fertilization between S. methods tend to underestimate the D /D ratio (Tsaur n s franciscanus and S. purpuratus (Minor, Britten, and Da- and Wu 1997), and the multiple insertion and deletion vidson 1993). differences were not counted (see AguadeÂ, Miyashita, Pairwise comparisons for these two windows re- and Langley 1992). veal ®ve combinations of species that show an excess Two phenomena complicate the interpretation of the observed pattern. We do not know whether certain of Dn in the ®rst window and two (different) species residues of bindin are of particular importance (as we combinations for the second, shorter window (table 1). do, e.g., for MHC molecules; Hughes, Ota, and Nei Neither of the windows shows a signi®cantly high Dn/ 1990). If this number is limited, selection may be re- Ds in comparisons between S. franciscanus or Hemicen- stricted to certain sites, and the number of replacement trotus and the other species (not shown). Only the pairs substitutions would be underestimated (Clark 1993; S. polyacanthus/S. purpuratus and S. polyacanthus/Al- Nielsen 1997b). Second, Lewontin (1989) suggested that locentrotus have signi®cantly more nonsynonymous it is impossible to estimate the number of evolutionary substitutions in the downstream window (lower left part events if the degree of constraint varies through time. of table 1), and this has to be interpreted with caution The divergence of the strongylocentrotid sea urchins ex- because of multiple comparisons. However, the mean amined here occurred at least between 3 and 20 MYA, Dn/Ds ratio between the seven species for this window much earlier than that of the tropical Echinometra spe- is extremely high at 1.98 (and this excludes the half of cies (Palumbi and Metz 1991; Metz and Palumbi 1996). the comparisons that had a Ds value of zero). Even though I sampled this set of species exhaustively, The upstream window, although its mean Dn/Ds ra- former diversifying selection could have been obscured tio is ``only'' 1.39 (excluding 9 of 21 comparisons be- by subsequent mutations. cause of zero in the denominator), has ®ve individual The two alleles sampled within each species dif- species pairs that show an excess of replacement sub- fered from each other, even on the amino acid level, stitutions. Positive selection is indicated between S. pur- except for S. polyacanthus. Substantial intraspeci®c puratus and its three sympatric close relatives, and be- polymorphism is incompatible with strong directional tween S. polyacanthus and the only included species it selection and would point to frequency-dependent or co-occurs with, S. droebachiensis and S. pallidus (Ba- balancing selection as the agent responsible for main- zhin 1998). In combination with the signal in Echino- tenance of diversity (Prout and Clark 1996). Clearly, metra (Metz and Palumbi 1996), this region should larger population samples are necessary to distinguish clearly be the target for more population genetic and modes of selection. biochemical investigations. Sperm Bindin in Strongylocentrotid Sea Urchins 1769

Phylogenetic Information because mtDNA is one linked unit, assuming that it is The maximum-likelihood parameters in table 2 a neutral marker may not be justi®ed (Ballard and Kreit- show important differences among data sets: the pro- man 1994; Hey 1997). Other nuclear genes may, of portion of invariable sites estimated and the gamma course, show a slower substitution rate than bindin. The exact evolutionary rate has no bearing on the fact that shape parameter (␣) differ dramatically between the bin- few mutations occurred between speciation events rel- din intron and exons and mtDNA. The inverse of ␣ is ative to the many autapomorphies that accumulated the variance of the gamma distribution; i.e., all sites in since then in both genomes. We may have to accept the the bindin intron evolve at essentially the same rate, existence of effectively hard polytomies (Hoelzer and while the distribution is much more skewed in coding Melnick 1994), potentially caused by biogeographic bindin and mtDNA. Combined with its extreme transi- events which may not be resolvable millions of years tion over transversion bias, mtDNA appears to require later. a very different substitution model. When the data sets were combined anyway (into 2.8 kb of ``total-evidence'' nucleotide sequence), an unequivocal resolution of the Acknowledgments branching order between these species was still not pos- Financial support was provided by the Fulbright sible (data not shown). This suggests that the inability Commission, the German Academic Exchange Service, to systematize them on the basis of adult morphology NSF Dissertation Improvement Grant DEB-9423662, may be due not only to ecological convergence and plas- the Quadrille Ball Committee of the Germanistic Society ticity, but also to a lack of synapomorphies in the mor- of North America, the Sigma Xi Society, the Lerner phological data set. Gray Fund for Marine Research at the American Mu- The lack of cladistic information indicates that the seum of Natural History, Friday Harbor Laboratories, time intervals between speciation events were relatively the Marine Biological Laboratory at Woods Hole, and brief (see the short internodes in ®g. 6). Interestingly, the Society for Molecular Biology and Evolution. Al- these two loci were expected to be particularly well suit- exander Bazhin, Kathy Conlan, Ron McConnaughey, ed to untangling the branching order. Mitochondrial Eric Munk, Robin Stears, Richard Strathmann, and Vic DNA has a low effective population size (e.g., Avise Vacquier generously supplied sea urchin samples. Peo- 1994) and is therefore expected to coalesce into recip- ple at the Dykhuizen, Eanes, Lennarz, Meyer, and Wray rocal monophyly faster than nuclear loci (Moore 1997). labs at Stony Brook, especially Walter Eanes, Brian Ver- Furthermore, bindin sequences, if involved in specia- relli, and Ing-Nang Wang, helped with advice and ma- tion, are also expected to separate before an average terials, as did Ed Metz, Steve Palumbi, and Vic Vac- nuclear gene (Hey 1994). This is because assortative quier. Bailey Kessing shared unpublished mtDNA se- mating within incipient species should occur earlier with quences. Many thanks to Alan Templeton, Dale Taney- respect to a mate recognition gene than with respect to hill, Dennis Slice, and David Swofford for software, and any other gene. to Walt Eanes, Dale Taneyhill, and two reviewers for Lee and Vacquier (1995) successfully inferred the comments on the manuscript. This is contribution num- phylogenetic relationships for over 20 species of Hali- ber 1020 from the Program in Ecology and Evolution otis gastropods from sequences of sperm lysin. In that at SUNY Stony Brook. study, the sperm protein locus contained enough infor- mation, even though the strong directional selection on Haliotis lysin (Lee, Ota, and Vacquier 1995) would LITERATURE CITED seem to violate the assumptions of some tree-building AGUADEÂ , M., N. MIYASHITA, and C. H. LANGLEY. 1992. Poly- methods. Indeed, S. Palumbi (personal communication) morphism and divergence in the Mst26A male accessory demonstrated that sperm bindin resolves the relation- gland gene region in Drosophila. 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Accepted August 28, 1998