Evolution of Bindin in the Pantropical Sea Urchin Tripneustes: Comparisons to Bindin of Other Genera
* Smithsonian Tropical Research Institute, Balboa, Panama@0/$'6, 百拇医药
Department of Biology, Duke University, Durham, North Carolina@0/$'6, 百拇医药
Abstract@0/$'6, 百拇医药
Bindin, a sea urchin sperm protein, mediates sperm-egg attachment and membrane fusion and is thus important in species recognition and speciation. Patterns of bindin variation differed among three genera that had been studied previously. In two genera of the superorder Camarodonta, Echinometra and Strongylocentrotus, both of which contain sympatric species, bindin is highly variable within and between species; a region of the molecule evolves at high rates under strong positive selection. In Arbacia, which belongs to the superorder Stirodonta and whose extant species are all allopatric, bindin variation is low, and there is no evidence of positive selection. We cloned and sequenced bindin from Tripneustes, a sea urchin that belongs to the Camarodonta but whose three species are found in different oceans. Worldwide sampling of bindin alleles shows that the bindin of Tripneustes (1) contains the highly conserved core characteristic of all other bindins characterized to date, (2) has an intron in the same position, and (3) has approximately the same length. Its structure is more like that of bindin from other camarodont sea urchins than to bindin from the stirodont Arbacia. The resemblances to other camarodonts include a glycine-rich repeat structure upstream of the core and lack of a hydrophobic domain 3' of the core, a characteristic of Arbacia bindin. Yet the mode of evolution of Tripneustes bindin is more like that of Arbacia. Differences between bindins of the Caribbean Tripneustes ventricosus and the eastern Pacific T. depressus, separated for 3 my by the Isthmus of Panama, are limited to four amino acid changes and a single indel. There are no fixed amino acid differences or indels between T. depressus from the eastern Pacific and T. gratilla from the Indo-Pacific. Bindin of Tripneustes, like that of Arbacia, also shows no evidence of diversifying selection that would manifest itself in a higher proportion of amino acid replacements than of silent nucleotide substitutions. When the rate of intrageneric bindin divergence is standardized by dividing it by cytochrome oxidase I (COI) divergence, Tripneustes and Arbacia show a lower ratio of bindin to COI substitutions between the species of each genus than exists between the species of either Echinometra or Strongylocentrotus. Thus, mode of bindin evolution is not correlated with phylogenetic affinities or molecular structure, but rather with whether the species in a genus are allopatric or sympatric. For a molecule involved in gametic recognition, this would suggest a pattern of evolution via reinforcement. However, in bindin the process that gave rise to this pattern is not likely to have been selection to avoid hybridization, because there is no excess of amino acid replacements between species versus within species in the bindins of Echinometra and Strongylocentrotus, as would have been expected if specific recognition were the driving force in their evolution. We suggest instead that the pattern of reinforcement is a secondary effect of the ability of species with rapidly evolving bindins to coexist in sympatry.
Key Words: sea urchins • bindin • speciation • gamete recognition • fertilization • Tripneustes.h+es, 百拇医药
Introductionh+es, 百拇医药
Explaining how reproductive isolation evolves between marine species is of crucial importance in understanding speciation in the sea. The availability of detailed information on phylogeographyg. on temporal isolation and gametic isolation , and on alpha taxonomy makes sea urchins good subjects for the study of marine speciation.h+es, 百拇医药
Most sea urchins are broadcast spawners, with external fertilization. Reproductive isolation between species could result from distinct spawning times or from species-specific gamete interactions. Congeneric sympatric sea urchins often have overlapping annual or monthly spawning periods. In such cases, reproductive isolation between closely related sea urchins is, at least in part, the product of gametic incompatibility ( (p. 522) ). Whereas gametic incompatibility could arise between a pair of sea urchin species at any step in the interactions between gametes (e.g., sperm activation, acrosomal reaction, sperm-egg attachment, sperm-egg fusion), it appears that in closely related species it generally emerges during sperm-egg attachment and membrane fusion (see for discussion). The sea urchin sperm protein bindin mediates both of these processes in sea urchins. Changes in the bindin locus can thus cause gametic incompatibility and convert populations into different species. Bindin is the major insoluble component of the acrosomal vesicle . It functions as glue between the acrosomal process and the glycoprotein bindin receptors of the vitelline layer of the egg. Bindin and bindin receptors often interact in a species-specific manner .
The evolution of bindin has been studied in three genera of sea urchins. studied bindin sequences of the central and western Pacific species of the cosmopolitan genus Echinometra. They found many sequence rearrangements and a higher number of nonsynonymous than synonymous substitutions, an indication of positive selection, in a region just 5' of the conserved bindin core. examined bindin in Strongylocentrotus and found evidence for positive selection in the same "hotspot" observed in Echinometra. studied the same molecule in Arbacia. In contrast to the previous studies, they found almost no sequence rearrangements and no evidence for positive selection. Changes in bindin are correlated with prezygotic isolation: species of Echinometra and Strongylocentrotus are often gametically incompatible ( [p. 522] ), whereas species of Arbacia are gametically compatible, at least in the single cross performed by . Patterns of bindin evolution within genera also correlate with the presence of sympatric species. Echinometra and Strongylocentrotus contain species with overlapping geographical distributions, whereas all species of Arbacia are distributed allopatrically.
examined three hypotheses that might explain the lack of variability of bindin in Arbacia: (1) Extensive gene flow over large distances within species of Arbacia may limit the potential for rapid bindin evolution. (2) Arbacia bindins may be functionally constrained. (3) Non-overlap in geographic distributions of the species of Arbacia may have obviated the necessity for the evolution of gamete recognition. A fourth hypothesis is that differences in the mode of evolution of bindin between Arbacia, on the one hand, and Echinometra and Strongylocentrotus, on the other, are due to phylogenetically inherited differences of their genomes. Arbacia belongs to the superorder Stirodonta, whereas Echinometra and Strongylocentrotus belong to the superorder Camarodonta. These superorders last shared a common ancestor approximately 160 MYA .+gm, http://www.100md.com
We examined bindin evolution in the genus Tripneustes, a member of the superorder Camarodonta . Tripneustes is pantropical and contains three allopatrically distributed species, T. ventricosus on both sides of the Atlantic, T. depressus in the eastern Pacific, and T. gratilla in the central and western Pacific as well as the Indian Ocean. The three species are morphologically very similar, to the degree that it has been suggested that they may constitute a single species . Indeed, there is no mitochondrial DNA differentiation between T. depressus and T. gratilla, which suggests that eastern and western Pacific populations of Tripneustes belong to the same species (Lessios, Kane, and Robertson, in preparation) Tripneustes mature bindin sequences were obtained from all species of the genus, and from all major regions of tropical oceans. We wanted to know whether bindin variation in this genus conformed to the pattern seen in other camarodonts, or whether it resembled that of Arbacia. Because Arbacia is more distantly related to Tripneustes but resembles this genus in containing no extant sympatric species, these comparisons offer insight into the relative roles of phylogeny (and resultant similarities in molecular structure) versus selection against hybridization in the evolution of this important gamete-recognition molecule.
Materials and Methods;hjt%, 百拇医药
Samples;hjt%, 百拇医药
Specimens of Tripneustes were collected from locations around the world . Our sampling was intended to cover all mitochondrial DNA (mtDNA) major clades (Lessios, Kane, and Robertson, in preparation), as well as all regions of the tropical oceans. Thus, we sampled two individuals from the eastern Atlantic (an mtDNA clade distinct from the western Atlantic), two from the Caribbean (including Florida), two from Brazil (a subclade of the western Atlantic clade in mtDNA), four from the eastern Pacific, two each from the central and western Pacific, and two from the Indian Ocean.;hjt%, 百拇医药
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FIG. 1. Localities from which bindin alleles were sampled. Tripneustes ventricosus (filled boxes) was sampled at Florida; Panama; Salvador, Brazil; and São Tomé. T. depressus (crosshatched boxes) was sampled at Isla del Coco and Clipperton Atoll. T. gratilla (open boxes) was sampled at Reunion, the Marquesas, Kiritimati, Guam, and Papua New Guinea
Identification of Bindin from cDNA|cyr, http://www.100md.com
To characterize the first bindin sequence of Tripneustes, we isolated poly-A mRNA from the testis of a ripe Tripneustes ventricosus from Buena Ventura, Panama, using a Micro Poly (A) Pure Kit (Ambion). Reverse transcription reactions were conducted using Superscript II reverse transcriptase (RT) (Life Technologies) according to the manufacturer's protocol, but with 1 µl of 100 µM MOBY (5'-AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGATTTTTTTTTTTTTTTTT-3') as the primer. Using primers MB1130+ (5'-TGCTSGGTGCSACSAAGATTGA-3') and core200- (5'-TCYTCYTCYTCYTGCATIGC-3'), we amplified a fragment of the core region of bindin from the RT reaction product. These primers correspond to amino acids VLGATKID and AMQEEEE of the core region of bindin . Based on the DNA sequence of the amplified fragment, exact Tripneustes bindin core primers were designed for use in 3' and 5' rapid amplification of cDNA ends (RACE) . Nested polymerase chain reactions (PCR) for 3' RACE were carried out on the MOBY-primed testis cDNA with a pair of exact match Tripneustes bindin core primers and the 3' RACE primers out2 (5'-GATCCGTCGACATCGATAATACG-3'), and in2 (5'-CGATAATACGACTCACTATAGG-3'). The 5' RACE was performed using the 5' RACE system (version 2.0, Life Technologies) according to the manufacturer's protocol.
Genomic Characterizations of Bindin2, http://www.100md.com
After the first Tripneustes bindin sequence was obtained from cDNA by RACE, we amplified other bindin alleles from genomic DNA. We extracted genomic DNA as described by from gonad tissue preserved in ethanol, NaCl-saturated 20% dimethyl-sulfoxide solution, or in liquid nitrogen. Using Tfl DNA polymerase (Epicentre Technologies), we amplified full-length mature bindin alleles from genomic DNA with primers TvF1 (5'-CTTTATCTCGGGGCATCGTC-3') and TvR1 (5'-CTGAACTTCCAATGGCTTCC-3'). Amplification conditions were as follows: 1 min at 96°C, then 39 cycles of 45 s at 94°C, 30 s at 50°C, 150 s at 72°C and finally 5 min at 72°C. We ran the amplification products in low-melting-point agarose gels. Bands were excised and treated with Gelase (Epicentre Technologies), then cloned using a pMOSBlue blunt-ended cloning kit (Amersham). We screened positive bacterial colonies according to the same PCR protocol stated above, with either the primer pair TvF1 and TvR1 or vector primers T7 and U19. The PCR product from a single positive colony per individual was gel purified and cycle sequenced as described in , with primers TvF1, TvR1, MB1130+, MB1136- (5'-ARGTCAATCTTSGTSGCCC-3'), TvF3 (5'-TGATGGACCTCAGCAGTGGTGT-3'), and TvR3 (5'-CACAAAATGATGGCTCACAGTT-3'). This combination of primers sequenced both strands of the full mature bindin and its intron. Sequencing was performed on an ABI 377 automated sequencer and edited using Sequencher 3.1 (Gene Codes Corp.). Sequences have been deposited in GenBank (accession numbers ).
A total of 12 mutations unique to a single allele (singletons) were observed among 16 mature bindin sequences with a combined length of 10,200 bp. Singleton mutations may represent true differences, or they may arise from cloning and polymerase error during amplification. Thus, the upper limit of sequencing error in the study was 0.12%.^/o$#}, http://www.100md.com
Phylogenetic Analysis^/o$#}, http://www.100md.com
Sequences were aligned by eye with the computer program Se-Al (version 1.0, written by A. Rambaut). A bindin sequence obtained from Lytechinus variegatus was used as an outgroup to root bindin phylogenetic trees. The L. variegatus bindin cDNA sequence reported by was not used because it differs in sequence at four amino acids of the core region, which are identical in 30 other Lytechinus bindin sequences (unpublished data) and all of the Tripneustes bindin alleles presented here. To calculate the best-fit model for constructing a tree, we entered the bindin coding sequences (full mature bindin plus 23 codons of preprobindin sequences) of Tripneustes in Modeltest version 3.06 . Comparison of the log-likelihood ratios of nested models performed by Modeltest indicated that the simplest model with a significantly better fit to the data than other models was that of . Allowing for site-specific rate categories according to codon position did not greatly improve the likelihood of the model. We used PAUP* 4.0b6 to conduct Neighbor-Joining phylogenetic analyses on the bindin coding sequences based on Tamura and Nei distances. Maximum parsimony and maximum likelihood (ML) trees were also constructed in PAUP. Five codons (62–66 in ) that could not be unambiguously aligned between Tripneustes and Lytechinus sequences were excluded from the phylogenetic analysis. Intron sequences were not used in the final analysis because they could not be aligned between Lytechinus and Tripneustes. Analyses that included the intron but left the Tripneustes tree unrooted produced the same intrageneric topology as analyses limited to the coding sequences.
fig.ommittedo^xf?{k, 百拇医药
FIG. 2. Amino acid sequence alignments of alleles in mature bindin of Tripneustes. Periods indicate identity to the first sequence, dashes indicate gaps. The three 10 amino acid repeats are overscored by open boxes, the hotspot region by a striped box, and the core by a black boxo^xf?{k, 百拇医药
Statistical Tests for Selectiono^xf?{k, 百拇医药
We divided the mature bindin sequences into three regions, based on previous observations of patterns of bindin variation in other genera: (1) a hotspot region 5' from the conserved core (amino acids 62–93 in ), corresponding to that observed in both Echinometra and Strongylocentrotus ); (2) the conserved core (amino acids 98–163); and (3) the rest of the molecule. We used MEGA version 2.1 to calculate the proportion of synonymous (dS) and nonsynonymous (dN) differences by the and (PBL) method in each of the three regions. Two separate analyses were carried out, one comparing all sequences to one another, the other comparing alleles from the Pacific species (T. depressus and T. gratilla) to those from the Atlantic (T. ventricosus). We tested for evidence of positive selection in each of the three regions of the molecule, using Fisher's exact tests on all pairwise comparisons , under model of evolution.
To test for the possibility that selection might be acting at sites scattered throughout the bindin molecule and not in specific regions, we implemented a series of models in PAML version 3.0 based on the neighbor-joining tree of the unique bindin alleles. We calculated the likelihood of this tree under two neutral models (M1 and M7) that do not allow for positively selected sites and under three alternate models (M2, M3, and M8) that do (see ). Then, we compared the log-likelihoods between the neutral and selection models. We also used PAML to test for evidence of changing dN/dS ratios along different lineages of the neighbor-joining tree by first calculating the likelihood for a model that kept the dN/dS ratio constant across the tree and then calculating the likelihood for a model that allowed each branch to have a separate dN/dS ratio. Finally, we tested for selection on bindin using the test to compare the dN/dS ratios within and between species.
Comparative Rates of Bindin Evolution6wv3&, http://www.100md.com
To ask whether bindin evolves faster in some genera than in others, we compared our data for Tripneustes bindin to all full-length mature bindin sequences of for Echinometra, of for Strongylocentrotus, and of for Arbacia. To calculate rates, we standardized intrageneric bindin differentiation by dividing dN and dS for the entire bindin molecule by the interspecific two-parameter (K2P) genetic distance of the mitochondrial cytochrome oxidase I (COI) gene. Average COI divergence between species of Tripneustes was calculated from all sequences included in Lessios, Kane, and Robertson (in preparation), of Echinometra from all sequences in , and of Strongylocentrotus from all sequences in . Because sequenced a different region of COI, we obtained our own sequences from 102 individuals of four species of Arbacia, covering the same 640 bp that were sequenced in Tripneustes. This region completely overlaps the 450 bp sequenced by in Echinometra and most of the 440 bp sequenced by in Strongylocentrotus.
Mature bindin sequences within the same genus were aligned by eye. Sequences from different genera were generally too divergent outside of the core region to be aligned with confidence. Thus, only intrageneric comparisons were feasible. In contrast to Tripneustes and Arbacia, in which alignments could be made for the whole mature bindin, the glycine-rich repeat regions in Echinometra and Strongylocentrotus bindins could not be aligned unambiguously. These regions (111 codons 3' of the core in Strongylocentrotus, 26 codons 5' of the core and 32 codons 3' of the core in Echinometra) were excluded from this analysis. Bindin intron sequences of Arbacia, Tripneustes, and Strongylocentrotus were aligned by eye for each genus. Intron sequences for Echinometra were not included, because they were unavailable in GenBank. Inversions in the bindin intron were aligned between sequences that shared them and were considered as gaps in sequences with the opposite inversion.l:3{if, 百拇医药
Strongylocentrotus polyacanthus was not included in the analysis, because COI data were not available for this species. S. franciscanus was also excluded, because its bindin sequences could not be unambiguously aligned with the sequences from the rest of the genus. T. depressus and T. gratilla sequences were treated as coming from the same species, because neither their bindin nor their COI sequences (Lessios, Kane, and Robertson, in preparation) are reciprocally monophyletic. Finally, we included Hemicentrotus pulcherrimus in the Strongylocentrotus analysis, because both COI and bindin data indicate that it is nested within this genus.
Codon Biasply?a, 百拇医药
We estimated codon bias for each full-length mature bindin allele from Arbacia, Tripneustes, Echinometra, and Strongylocentrotus with the program CODONS by calculating the effective number of codons (ENC) . These ENC values can range from 20 (same codon always used for an amino acid) to 61 (random use of each codon for every amino acid). A separate calculation was carried out for each allele, and then the values were averaged for each genus.ply?a, 百拇医药
Resultsply?a, 百拇医药
Structure of the Moleculeply?a, 百拇医药
We recovered 16 full-length mature bindin alleles from Tripneustes. Twelve of these alleles were unique sequences. Total aligned bindin sequences of Tripneustes include 69 bp of preprobindin, 633 or 663 bp of mature bindin, 835–855 bp of intron, and 31 bp of 3' untranslated region. Like the mature bindins from other genera, those from Tripneustes contain no cysteine or tryptophan residues . Their length (211 or 221 amino acids) is approximately the same as that of other known bindins .
Tripneustes bindins have the highly conserved core observed in bindins from all other studied genera. Of 66 amino acids defined as the core in this study (98–163 in ), 65 are identical to those of Lytechinus variegatus, and 64 are identical to the bindin sequences of S. purpuratus , S. franciscanus , and A. punctulata . All 18 amino acids (119–136 in ) implicated in membrane fusion are identical to those in all other known bindins.]?su, http://www.100md.com
At a point 5' of the core, Tripneustes bindins contain a glycine-rich repeat GG(Q/S/P) (V/G)GGG(G/S) (N/G) (S/M/G) reminiscent of glycine-rich motifs present in Echinometra and Strongylocentrotus but absent in Arbacia. This repeat is present in two copies in T. ventricosus, and in three copies in T. depressus and T. gratilla . This is the only insertion/deletion (indel) observed in Tripneustes mature bindin. Hydrophobicity plots of Tripneustes bindin are similar to those of other Camarodont sea urchins (Zigler and Lessios in preparation); they do not contain the 3' hydrophobic domain observed in Arbacia bindin .
The single intron is in the same location as in all other bindins studied to date (after amino acid 93 in ). It contains a region of approximately 75 bp just inside its 5' end that is inverted in half of the Tripneustes alleles with respect to other Tripneustes alleles. All T. ventricosus alleles, plus three alleles of T. gratilla ("Marquesas 80," "Guam 2," and "Papua New Guinea 1"), have one form of the inversion, whereas all T. depressus alleles and the rest of the T. gratilla alleles have the other form.-yalm, http://www.100md.com
Genealogy of Alleles-yalm, http://www.100md.com
depicts the genealogy of the sequenced bindin alleles, reconstructed by neighbor joining, using only coding sequences (mature bindin and preprobindin sequences). Neighbor joining, using distances, divides the genealogy into Atlantic and Pacific clades. Parsimony and ML methods place the "Guam 2," "Marquesas 80," and "Papua New Guinea 1" alleles with the Atlantic alleles. That different methods of phylogenetic reconstruction result in roots placed between different nodes of the Tripneustes bindin tree is probably due to the large number of changes between Tripneustes and Lytechinus, relative to the changes within Tripneustes. Mitochondrial DNA divides the Atlantic and Pacific haplotypes into reciprocally monophyletic clades (Lessios, Kane, and Robertson, in preparation)
fig.ommitted08*[}gk, 百拇医药
FIG. 3. Neighbor-Joining tree of sequences of preprobindin and mature bindin of Tripneustes, using genetic distances. Bootstrap support (1,000 replicates) of >50% is noted next to nodes. The tree is rooted on a bindin sequence of Lytechinus variegatus.08*[}gk, 百拇医药
There are four fixed amino acid substitutions and one indel that distinguish the Atlantic Tripneustes bindin alleles from all Pacific bindin alleles. Two of these substitutions (at amino acids 70 and 71 in ) occur in the "hotspot" region of Echinometra and Strongylocentrotus . Within the Atlantic clade, the two alleles from São Tomé off the coast of Africa form a well-supported monophyletic entity. Unlike the pattern seen in mtDNA, in which E. and W. Atlantic populations form reciprocally monophyletic sister clades (Lessios, Kane, and Robertson, in preparation), the São Tomé bindin alleles are nested within the ones from the W. Atlantic.08*[}gk, 百拇医药
In the Pacific, bindin alleles of T. depressus, off the coast of America, and of T. gratilla from the central and Indo-West Pacific do not sort according to nominal species or collection locality. One allele from Kiritimati, which, according to collecting location should belong to T. gratilla, actually groups with T. depressus. The T. depressus alleles (plus the Kiritimati allele) is a sister group to the T. gratilla sequences from Reunion, in the Indian Ocean. The other three T. gratilla sequences are basal in the Pacific clade. The basal position of these three alleles is consistent with the observed intron inversion: each of these alleles has the T. ventricosus form of the inversion, whereas the rest of the Pacific individuals have the other form. There are no fixed nonsynonymous changes between T. gratilla and T. depressus. The close affinity of bindin from the two Pacific species mirrors a similar lack of differentiation of mtDNA sequences (Lessios, Kane, and Robertson, in preparation). Apparently, gene flow between the eastern and central Pacific is either continuing or has been very recently interrupted.
Adaptive Evolution0:xdf, 百拇医药
Relative rates of change of the different regions of the Tripneustes bindin molecule are qualitatively similar to patterns of bindin evolution in other Camarodont sea urchins. Rates of nonsynonymous change are highest in the hotspot region, lowest in the core, and intermediate in the rest of the molecule . Only nonsynonymous changes are observed in the hotspot region. However, in contrast to the pattern in Echinometra and Strongylocentrotus, the excess of nonsynonymous changes in the hotspot region is not significant in any pairwise comparison. Tripneustes bindins also lack the large number of potentially important indels seen in the other Camarodont genera. In addition, using the models implemented in PAML, we found no evidence for positively selected sites dispersed throughout the molecule. The likelihood of models that allowed for positively selected sites was not significantly higher than that of models that did not . Nor did we find any evidence for significant variation in dN/dS ratios between lineages. Allowing a different dN/dS ratio for each branch in the phylogeny did not produce a significantly better model than a model with a single dN/dS ratio for the entire tree . Finally, a comparison of the ratios of replacement to silent differences within and between species was not significantly different from neutral expectation . Four replacement substitutions and zero silent substitutions are fixed between Pacific (T. gratilla and T. depressus) and Atlantic (T. ventricosus) individuals, and there are 12 replacement polymorphisms and 12 silent polymorphisms within the two groups (Fisher's exact test P = 0.11).
fig.ommittedxt, http://www.100md.com
Table 1 Replacement (dN) and Silent (dS) Substitutions per Site in Three Regions of Tripneustes Mature Bindin.xt, http://www.100md.com
fig.ommittedxt, http://www.100md.com
Table 2 Maximum Likelihood Testing for Variation in the Ratio of Replacement Substitutions to Silent Substitutions Among Sites and Lineages.xt, http://www.100md.com
Rate of Bindin Evolutionxt, http://www.100md.com
To compare the rate of relative change of amino acid replacement of bindin within each genus, we calculated interspecific K2P, dN and dS (PBL) for the full mature bindin and the intron, then divided these values by mitochondrial COI genetic distances (K2P) . If a universal calibration for a "COI clock" is assumed, then divergence in this molecule represents a proxy of the time that species have remained separate, and thus helps standardize the bindin divergence for the age of the species. The comparison of ratios of either bindin K2P or dN to COI genetic distance indicates that there are large differences between the genera in the overall rates of change in the bindin molecule. Because these are pairwise comparisons, each ratio is not independent of all others, so they cannot be compared statistically. Nevertheless, the trend is clear. All ratios of bindin K2P and especially dN values to COI distances in Arbacia and Tripneustes are smaller than all such ratios in Echinometra or Strongylocentrotus. Arbacia shows larger interspecific COI distances relative to those of the other genera, which could be due either to older ages of its species or to higher rates of COI divergence. The COI divergence between A. punctulata and A. stellata across the Isthmus of Panama is equivalent to that of six other genera thus separated , which would suggest that rate variation in COI is low, and thus that it correctly indicates most species of Arbacia to be older than those of the other genera. If as suggested, Atlantic and Pacific species of Arbacia were separated before the completion of the Panama isthmus, then rate of COI divergence in this genus would be slower than we assume, and the differences in rates of bindin evolution between the genera would be even more pronounced.
fig.ommitted.qz\n)l, 百拇医药
Table 3 Intrageneric Differences in Mature Bindin, Bindin Intron, and Mitochondrial Cytochrome Oxidase I in All Genera for Which Data Exist..qz\n)l, 百拇医药
Interestingly, the higher rate of bindin evolution in Strongylocentrotus and Echinometra relative to Arbacia or Tripneustes does not appear to be limited to adaptive changes. The comparison of bindin dS to COI divergence ratios follows the same pattern as that of comparisons of bindin dN to COI divergence. Every interspecific comparison in Arbacia or Tripneustes produces a lower ratio than in Strongylocentrotus or Echinometra . A positive correlation across molecules between rate of substitution in replacement and silent sites has been found in many organisms . Mutational bias, nonindependence of point mutations, and codon-level selection have been offered as explanations for the phenomenon. Almost all explanations involve some form of codon bias, but ENC values in bindin show that codon usage in this molecule is universally equitable (means per genus: Tripneustes, 59.2; Arbacia, 44.9; Strongylocentrotus, 52.2; Echinometra, 55.1).
The trend seen for the silent sites of the expressed region does not extend to the intron. Intron sequences are not available for Echinometra, but a comparison between Arbacia and Tripneustes, on the one hand, and Strongylocentrotus on the other, indicates that the ratio of intron to COI divergence is not different between the two groups, which is what would be expected if both the intron and COI evolve linearly with time.9ng@i, 百拇医药
Discussion9ng@i, 百拇医药
Tripneustes Bindin Structure and Divergence9ng@i, 百拇医药
The bindin of Tripneustes contains the highly conserved core characteristic of all other bindins studied to date, has an intron in the same position, and has approximately the same length. Its structure resembles that of the bindins of other Camarodont sea urchins (Echinometra, Strongylocentrotus, and Lytechinus) more than the bindin of the Stirodont Arbacia. The resemblances include the glycine-rich repeat structure 5' of the core and the lack of the 3' hydrophobic domain of Arbacia bindin. These similarities to the other Camarodonts are not surprising, given that Tripneustes last shared a common ancestor with Echinometra, Strongylocentrotus, and Lytechinus 25–60 MYA, and with Arbacia 120–180 MYA .
Despite the similarities in structure of Tripneustes bindin with that of other Camarodont sea urchins, its mode of evolution appears to be more like that of Arbacia. In contrast to Echinometra or Strongylocentrotus bindin, Tripneustes bindin has evolved slowly. For example, bindin of T. ventricosus from the Caribbean differs from bindin of the eastern Pacific T. depressus by only four fixed amino acid changes and a single indel. These two species were presumably separated from each other for more than 3 million years by the Isthmus of Panama There are no fixed amino acid differences or indels between T. depressus and T. gratilla from the Indo-Pacific, despite the tremendous geographical distance separating the eastern Pacific from the West Indian Ocean. This level of differentiation is almost as low as that seen in the bindin of Arbacia, in which only a single indel distinguishes species on either side of Central America . It contrasts with differentiation in Echinometra, in which species separated for less than 1.5 MY have 7 fixed amino acid differences and in Strongylocentrotus in which S. purpuratus and S. droebachiensis, separated for less than 3 MY, show 21 amino acid differences ). In addition, bindin of Tripneustes, like that of Arbacia, has only one indel, whereas indels are much more numerous in Echinometra and Strongylocentrotus. Such indels may be functionally important in gamete recognition . Bindin of Tripneustes, like that of Arbacia, shows no evidence of diversifying selection that would manifest itself in significantly more replacement substitutions than silent nucleotide substitutions. Finally, Tripneustes and Arbacia show a lower ratio of bindin to COI substitutions than Echinometra or Strongylocentrotus, despite the inevitable underestimation of bindin divergence in the latter two genera, arising from the exclusion from the analysis of the most variable regions, which could not be aligned. What could account for such similarities between distantly related taxa and differences between closely related ones?
One possible explanation might be the relative age of the species in different genera. By COI and intron divergence (but not by the number of silent substitutions in bindin) the extant species of Arbacia and Tripneustes diverged earlier from each other than did the species of Echinometra and Strongylocentrotus. If, as , have suggested for sex-related genes of Drosophila, the episodes of adaptive bindin evolution are concentrated to the time that new species are formed, and if positive selection on the molecule is subsequently relaxed, then older species may lose the signature of an excess of replacement substitutions relative to silent substitutions. This explanation cannot, however, be applied to bindin. A concentration of adaptive changes at the moment of speciation would decrease the ratio of replacement substitutions to silent substitutions in older species. Increased time since speciation would also decrease the ratio of bindin to COI divergence. However, longer time since speciation could not decrease the absolute number of amino acid changes fixed between species after they have been accumulated early in divergence. The low number of such replacements in Arbacia and Tripneustes must therefore indicate that they have never gone through a period of accelerated bindin divergence. This possibility is also supported by our analysis of the apportionment of replacement and silent substitutions along branches of the Tripneustes bindin tree, which failed to show a concentration of adaptive change in younger (or older) lineages.
considered three hypotheses as possible explanations for the decelerated bindin evolution in Arbacia: (1) high gene flow, (2) functional constraint, and (3) degree of overlap of species distributions. First, they suggested that high levels of gene flow and a lack of population subdivision within species of Arbacia may limit the rate of bindin evolution. Tripneustes, like Arbacia, shows low levels of biogeographic subdivision, at least in the Pacific (Lessios, Kane, and Robertson, in preparation). However, it is not clear that these two genera are exceptional among echinoids in that respect. High gene flow over thousands of kilometers is a standard feature of all sea urchin species with planktonic larvae . In particular, the observations of minimal population subdivision over a range of several thousand kilometers in Indo-West Pacific Echinometra and in eastern Pacific S. purpuratus or S. franciscanus indicate that the accelerated interspecific bindin divergence relative to Arbacia and Tripneustes cannot be attributable to this factor alone.
Second, proposed that slow bindin evolution in Arbacia might be due to functional constraints imposed by its molecular structure. They suggested that lack of repeat elements and indels, as well as the presence of a 3' hydrophobic domain, represent evolutionary constraints on Arbacia bindins that are not shared by bindins of Camarodonts. Tripneustes bindin, despite three repeats and a lack of 3' hydrophobic domain, evolves almost as slowly as Arbacia bindin. It appears, therefore, that these features of the molecule do not necessarily affect its evolutionary rate.hzn, 百拇医药
The third hypothesis of was that positive selection, arising from the presence of sympatric congeners, is operating on bindin of Echinometra and Strongylocentrotus, but is absent in Arbacia. Echinometra and Strongylocentrotus contain sympatric species, which are generally gametically incompatible . All extant species in Tripneustes and Arbacia are allopatric. There are no data about gametic compatibility between Tripneustes species, but A. punctulata and A. incisa are gametically compatible . Thus, it is possible that reinforcement of reproductive isolation among the sympatric species of Echinometra and Strongylocentrotus may explain the observed patterns of bindin evolution. This is a pattern of reinforcement on the genus level. However, as is also true for these patterns on the population level , we do not know the process that produced them. Did the presence of congeners create selective pressures for bindin divergence in Strongylocentrotus and Echinometra, or did divergence in bindin, caused by selection arising from another cause, permit species in these genera to invade the same area and coexist without fusing or becoming extinct? This question cannot be answered with certainty, but the patterns of intraspecific and interspecific divergence can provide a clue. If selection to avoid hybridization were responsible for the reinforcement pattern, we would have expected an excess of amino acid replacements between species (particularly between sympatric species) of Echinometra and Strongylocentrotus relative to within species. This however, is not the case in either genus. In the rapidly evolving 40 codon bindin region of Echinometra, the ratio of replacement substitutions to silent substitutions between alleles is larger than unity in both intraspecific and interspecific comparisons, and tests are not significant. In the same region of bindin, comparisons between S. franciscanus and either S. purpuratus or S. droebachiensis indicate that the ratio of replacement substitutions to silent substitutions is higher within than between species has shown experimentally that, in Echinometra, males carrying different bindin alleles have different rates of success in fertilizing females, depending on the female bindin genotype, and that intraspecific polymorphism is thus maintained by selection. Such selection could arise from male heterozygote advantage, nontransitive female preferences , or interlocus conflict evolution between the sexes . These processes can operate within species whether or not they are sympatric with a congener . Thus, it is possible that the selective force that accelerates bindin evolution in some genera is not avoidance of hybridization in sympatry. The pattern of reinforcement suggested by the comparison between genera with sympatric and allopatric species may be due to the ability of congeneric species with divergent bindin to coexist.
Because Echinometra was the first genus in which evolution of bindin was studied, and because its rapid evolution under positive selection fit patterns found in other genes related to sexual reproduction , it has been tacitly assumed that the patterns of positive selection and rapid allele divergence exemplify the mode of evolution of this locue.g.vidence from Strongylocentrotus reinforced this view. However, the information from Tripneustes now indicates that what found in Arbacia was not an isolated exception, and not a characteristic of stirodont versus camarodont sea urchins. Why bindin should evolve faster in certain genera of sea urchins and not others remains unclear, but the addition of data from more sea urchin taxa will help determine the factors that promote or retard evolution in this molecule.j.;vi&, 百拇医药
Acknowledgementsj.;vi&, 百拇医药
A. Calderón and L. Calderón helped in the laboratory. J. Kane and M. A. McCartney provided sampling and laboratory advice. M. A. McCartney also designed the RACE primers and provided instructions on the technique. T. Duda and B. Kessing helped with data analysis. D. R. Robertson collected specimens from Clipperton, the Marquesas, Reunion, and São Tomé. G. Paulay collected specimens from Guam and Papua New Guinea. R. Collin, A. Crawford, C. Cunningham, T. Duda, B. Kessing, C. Riginos, and three anonymous reviewers commented on the manuscript. This work was supported by a National Science Foundation predoctoral fellowship, a Duke University Zoology Department Dissertation Improvement Grant, and a Smithsonian Predoctoral fellowship to K.S.Z., and by the Smithsonian Molecular Evolution Program.
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Accepted for publication October 9, 2002.(K. S. Zigler and H. A. Lessios)
Department of Biology, Duke University, Durham, North Carolina@0/$'6, 百拇医药
Abstract@0/$'6, 百拇医药
Bindin, a sea urchin sperm protein, mediates sperm-egg attachment and membrane fusion and is thus important in species recognition and speciation. Patterns of bindin variation differed among three genera that had been studied previously. In two genera of the superorder Camarodonta, Echinometra and Strongylocentrotus, both of which contain sympatric species, bindin is highly variable within and between species; a region of the molecule evolves at high rates under strong positive selection. In Arbacia, which belongs to the superorder Stirodonta and whose extant species are all allopatric, bindin variation is low, and there is no evidence of positive selection. We cloned and sequenced bindin from Tripneustes, a sea urchin that belongs to the Camarodonta but whose three species are found in different oceans. Worldwide sampling of bindin alleles shows that the bindin of Tripneustes (1) contains the highly conserved core characteristic of all other bindins characterized to date, (2) has an intron in the same position, and (3) has approximately the same length. Its structure is more like that of bindin from other camarodont sea urchins than to bindin from the stirodont Arbacia. The resemblances to other camarodonts include a glycine-rich repeat structure upstream of the core and lack of a hydrophobic domain 3' of the core, a characteristic of Arbacia bindin. Yet the mode of evolution of Tripneustes bindin is more like that of Arbacia. Differences between bindins of the Caribbean Tripneustes ventricosus and the eastern Pacific T. depressus, separated for 3 my by the Isthmus of Panama, are limited to four amino acid changes and a single indel. There are no fixed amino acid differences or indels between T. depressus from the eastern Pacific and T. gratilla from the Indo-Pacific. Bindin of Tripneustes, like that of Arbacia, also shows no evidence of diversifying selection that would manifest itself in a higher proportion of amino acid replacements than of silent nucleotide substitutions. When the rate of intrageneric bindin divergence is standardized by dividing it by cytochrome oxidase I (COI) divergence, Tripneustes and Arbacia show a lower ratio of bindin to COI substitutions between the species of each genus than exists between the species of either Echinometra or Strongylocentrotus. Thus, mode of bindin evolution is not correlated with phylogenetic affinities or molecular structure, but rather with whether the species in a genus are allopatric or sympatric. For a molecule involved in gametic recognition, this would suggest a pattern of evolution via reinforcement. However, in bindin the process that gave rise to this pattern is not likely to have been selection to avoid hybridization, because there is no excess of amino acid replacements between species versus within species in the bindins of Echinometra and Strongylocentrotus, as would have been expected if specific recognition were the driving force in their evolution. We suggest instead that the pattern of reinforcement is a secondary effect of the ability of species with rapidly evolving bindins to coexist in sympatry.
Key Words: sea urchins • bindin • speciation • gamete recognition • fertilization • Tripneustes.h+es, 百拇医药
Introductionh+es, 百拇医药
Explaining how reproductive isolation evolves between marine species is of crucial importance in understanding speciation in the sea. The availability of detailed information on phylogeographyg. on temporal isolation and gametic isolation , and on alpha taxonomy makes sea urchins good subjects for the study of marine speciation.h+es, 百拇医药
Most sea urchins are broadcast spawners, with external fertilization. Reproductive isolation between species could result from distinct spawning times or from species-specific gamete interactions. Congeneric sympatric sea urchins often have overlapping annual or monthly spawning periods. In such cases, reproductive isolation between closely related sea urchins is, at least in part, the product of gametic incompatibility ( (p. 522) ). Whereas gametic incompatibility could arise between a pair of sea urchin species at any step in the interactions between gametes (e.g., sperm activation, acrosomal reaction, sperm-egg attachment, sperm-egg fusion), it appears that in closely related species it generally emerges during sperm-egg attachment and membrane fusion (see for discussion). The sea urchin sperm protein bindin mediates both of these processes in sea urchins. Changes in the bindin locus can thus cause gametic incompatibility and convert populations into different species. Bindin is the major insoluble component of the acrosomal vesicle . It functions as glue between the acrosomal process and the glycoprotein bindin receptors of the vitelline layer of the egg. Bindin and bindin receptors often interact in a species-specific manner .
The evolution of bindin has been studied in three genera of sea urchins. studied bindin sequences of the central and western Pacific species of the cosmopolitan genus Echinometra. They found many sequence rearrangements and a higher number of nonsynonymous than synonymous substitutions, an indication of positive selection, in a region just 5' of the conserved bindin core. examined bindin in Strongylocentrotus and found evidence for positive selection in the same "hotspot" observed in Echinometra. studied the same molecule in Arbacia. In contrast to the previous studies, they found almost no sequence rearrangements and no evidence for positive selection. Changes in bindin are correlated with prezygotic isolation: species of Echinometra and Strongylocentrotus are often gametically incompatible ( [p. 522] ), whereas species of Arbacia are gametically compatible, at least in the single cross performed by . Patterns of bindin evolution within genera also correlate with the presence of sympatric species. Echinometra and Strongylocentrotus contain species with overlapping geographical distributions, whereas all species of Arbacia are distributed allopatrically.
examined three hypotheses that might explain the lack of variability of bindin in Arbacia: (1) Extensive gene flow over large distances within species of Arbacia may limit the potential for rapid bindin evolution. (2) Arbacia bindins may be functionally constrained. (3) Non-overlap in geographic distributions of the species of Arbacia may have obviated the necessity for the evolution of gamete recognition. A fourth hypothesis is that differences in the mode of evolution of bindin between Arbacia, on the one hand, and Echinometra and Strongylocentrotus, on the other, are due to phylogenetically inherited differences of their genomes. Arbacia belongs to the superorder Stirodonta, whereas Echinometra and Strongylocentrotus belong to the superorder Camarodonta. These superorders last shared a common ancestor approximately 160 MYA .+gm, http://www.100md.com
We examined bindin evolution in the genus Tripneustes, a member of the superorder Camarodonta . Tripneustes is pantropical and contains three allopatrically distributed species, T. ventricosus on both sides of the Atlantic, T. depressus in the eastern Pacific, and T. gratilla in the central and western Pacific as well as the Indian Ocean. The three species are morphologically very similar, to the degree that it has been suggested that they may constitute a single species . Indeed, there is no mitochondrial DNA differentiation between T. depressus and T. gratilla, which suggests that eastern and western Pacific populations of Tripneustes belong to the same species (Lessios, Kane, and Robertson, in preparation) Tripneustes mature bindin sequences were obtained from all species of the genus, and from all major regions of tropical oceans. We wanted to know whether bindin variation in this genus conformed to the pattern seen in other camarodonts, or whether it resembled that of Arbacia. Because Arbacia is more distantly related to Tripneustes but resembles this genus in containing no extant sympatric species, these comparisons offer insight into the relative roles of phylogeny (and resultant similarities in molecular structure) versus selection against hybridization in the evolution of this important gamete-recognition molecule.
Materials and Methods;hjt%, 百拇医药
Samples;hjt%, 百拇医药
Specimens of Tripneustes were collected from locations around the world . Our sampling was intended to cover all mitochondrial DNA (mtDNA) major clades (Lessios, Kane, and Robertson, in preparation), as well as all regions of the tropical oceans. Thus, we sampled two individuals from the eastern Atlantic (an mtDNA clade distinct from the western Atlantic), two from the Caribbean (including Florida), two from Brazil (a subclade of the western Atlantic clade in mtDNA), four from the eastern Pacific, two each from the central and western Pacific, and two from the Indian Ocean.;hjt%, 百拇医药
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FIG. 1. Localities from which bindin alleles were sampled. Tripneustes ventricosus (filled boxes) was sampled at Florida; Panama; Salvador, Brazil; and São Tomé. T. depressus (crosshatched boxes) was sampled at Isla del Coco and Clipperton Atoll. T. gratilla (open boxes) was sampled at Reunion, the Marquesas, Kiritimati, Guam, and Papua New Guinea
Identification of Bindin from cDNA|cyr, http://www.100md.com
To characterize the first bindin sequence of Tripneustes, we isolated poly-A mRNA from the testis of a ripe Tripneustes ventricosus from Buena Ventura, Panama, using a Micro Poly (A) Pure Kit (Ambion). Reverse transcription reactions were conducted using Superscript II reverse transcriptase (RT) (Life Technologies) according to the manufacturer's protocol, but with 1 µl of 100 µM MOBY (5'-AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGATTTTTTTTTTTTTTTTT-3') as the primer. Using primers MB1130+ (5'-TGCTSGGTGCSACSAAGATTGA-3') and core200- (5'-TCYTCYTCYTCYTGCATIGC-3'), we amplified a fragment of the core region of bindin from the RT reaction product. These primers correspond to amino acids VLGATKID and AMQEEEE of the core region of bindin . Based on the DNA sequence of the amplified fragment, exact Tripneustes bindin core primers were designed for use in 3' and 5' rapid amplification of cDNA ends (RACE) . Nested polymerase chain reactions (PCR) for 3' RACE were carried out on the MOBY-primed testis cDNA with a pair of exact match Tripneustes bindin core primers and the 3' RACE primers out2 (5'-GATCCGTCGACATCGATAATACG-3'), and in2 (5'-CGATAATACGACTCACTATAGG-3'). The 5' RACE was performed using the 5' RACE system (version 2.0, Life Technologies) according to the manufacturer's protocol.
Genomic Characterizations of Bindin2, http://www.100md.com
After the first Tripneustes bindin sequence was obtained from cDNA by RACE, we amplified other bindin alleles from genomic DNA. We extracted genomic DNA as described by from gonad tissue preserved in ethanol, NaCl-saturated 20% dimethyl-sulfoxide solution, or in liquid nitrogen. Using Tfl DNA polymerase (Epicentre Technologies), we amplified full-length mature bindin alleles from genomic DNA with primers TvF1 (5'-CTTTATCTCGGGGCATCGTC-3') and TvR1 (5'-CTGAACTTCCAATGGCTTCC-3'). Amplification conditions were as follows: 1 min at 96°C, then 39 cycles of 45 s at 94°C, 30 s at 50°C, 150 s at 72°C and finally 5 min at 72°C. We ran the amplification products in low-melting-point agarose gels. Bands were excised and treated with Gelase (Epicentre Technologies), then cloned using a pMOSBlue blunt-ended cloning kit (Amersham). We screened positive bacterial colonies according to the same PCR protocol stated above, with either the primer pair TvF1 and TvR1 or vector primers T7 and U19. The PCR product from a single positive colony per individual was gel purified and cycle sequenced as described in , with primers TvF1, TvR1, MB1130+, MB1136- (5'-ARGTCAATCTTSGTSGCCC-3'), TvF3 (5'-TGATGGACCTCAGCAGTGGTGT-3'), and TvR3 (5'-CACAAAATGATGGCTCACAGTT-3'). This combination of primers sequenced both strands of the full mature bindin and its intron. Sequencing was performed on an ABI 377 automated sequencer and edited using Sequencher 3.1 (Gene Codes Corp.). Sequences have been deposited in GenBank (accession numbers ).
A total of 12 mutations unique to a single allele (singletons) were observed among 16 mature bindin sequences with a combined length of 10,200 bp. Singleton mutations may represent true differences, or they may arise from cloning and polymerase error during amplification. Thus, the upper limit of sequencing error in the study was 0.12%.^/o$#}, http://www.100md.com
Phylogenetic Analysis^/o$#}, http://www.100md.com
Sequences were aligned by eye with the computer program Se-Al (version 1.0, written by A. Rambaut). A bindin sequence obtained from Lytechinus variegatus was used as an outgroup to root bindin phylogenetic trees. The L. variegatus bindin cDNA sequence reported by was not used because it differs in sequence at four amino acids of the core region, which are identical in 30 other Lytechinus bindin sequences (unpublished data) and all of the Tripneustes bindin alleles presented here. To calculate the best-fit model for constructing a tree, we entered the bindin coding sequences (full mature bindin plus 23 codons of preprobindin sequences) of Tripneustes in Modeltest version 3.06 . Comparison of the log-likelihood ratios of nested models performed by Modeltest indicated that the simplest model with a significantly better fit to the data than other models was that of . Allowing for site-specific rate categories according to codon position did not greatly improve the likelihood of the model. We used PAUP* 4.0b6 to conduct Neighbor-Joining phylogenetic analyses on the bindin coding sequences based on Tamura and Nei distances. Maximum parsimony and maximum likelihood (ML) trees were also constructed in PAUP. Five codons (62–66 in ) that could not be unambiguously aligned between Tripneustes and Lytechinus sequences were excluded from the phylogenetic analysis. Intron sequences were not used in the final analysis because they could not be aligned between Lytechinus and Tripneustes. Analyses that included the intron but left the Tripneustes tree unrooted produced the same intrageneric topology as analyses limited to the coding sequences.
fig.ommittedo^xf?{k, 百拇医药
FIG. 2. Amino acid sequence alignments of alleles in mature bindin of Tripneustes. Periods indicate identity to the first sequence, dashes indicate gaps. The three 10 amino acid repeats are overscored by open boxes, the hotspot region by a striped box, and the core by a black boxo^xf?{k, 百拇医药
Statistical Tests for Selectiono^xf?{k, 百拇医药
We divided the mature bindin sequences into three regions, based on previous observations of patterns of bindin variation in other genera: (1) a hotspot region 5' from the conserved core (amino acids 62–93 in ), corresponding to that observed in both Echinometra and Strongylocentrotus ); (2) the conserved core (amino acids 98–163); and (3) the rest of the molecule. We used MEGA version 2.1 to calculate the proportion of synonymous (dS) and nonsynonymous (dN) differences by the and (PBL) method in each of the three regions. Two separate analyses were carried out, one comparing all sequences to one another, the other comparing alleles from the Pacific species (T. depressus and T. gratilla) to those from the Atlantic (T. ventricosus). We tested for evidence of positive selection in each of the three regions of the molecule, using Fisher's exact tests on all pairwise comparisons , under model of evolution.
To test for the possibility that selection might be acting at sites scattered throughout the bindin molecule and not in specific regions, we implemented a series of models in PAML version 3.0 based on the neighbor-joining tree of the unique bindin alleles. We calculated the likelihood of this tree under two neutral models (M1 and M7) that do not allow for positively selected sites and under three alternate models (M2, M3, and M8) that do (see ). Then, we compared the log-likelihoods between the neutral and selection models. We also used PAML to test for evidence of changing dN/dS ratios along different lineages of the neighbor-joining tree by first calculating the likelihood for a model that kept the dN/dS ratio constant across the tree and then calculating the likelihood for a model that allowed each branch to have a separate dN/dS ratio. Finally, we tested for selection on bindin using the test to compare the dN/dS ratios within and between species.
Comparative Rates of Bindin Evolution6wv3&, http://www.100md.com
To ask whether bindin evolves faster in some genera than in others, we compared our data for Tripneustes bindin to all full-length mature bindin sequences of for Echinometra, of for Strongylocentrotus, and of for Arbacia. To calculate rates, we standardized intrageneric bindin differentiation by dividing dN and dS for the entire bindin molecule by the interspecific two-parameter (K2P) genetic distance of the mitochondrial cytochrome oxidase I (COI) gene. Average COI divergence between species of Tripneustes was calculated from all sequences included in Lessios, Kane, and Robertson (in preparation), of Echinometra from all sequences in , and of Strongylocentrotus from all sequences in . Because sequenced a different region of COI, we obtained our own sequences from 102 individuals of four species of Arbacia, covering the same 640 bp that were sequenced in Tripneustes. This region completely overlaps the 450 bp sequenced by in Echinometra and most of the 440 bp sequenced by in Strongylocentrotus.
Mature bindin sequences within the same genus were aligned by eye. Sequences from different genera were generally too divergent outside of the core region to be aligned with confidence. Thus, only intrageneric comparisons were feasible. In contrast to Tripneustes and Arbacia, in which alignments could be made for the whole mature bindin, the glycine-rich repeat regions in Echinometra and Strongylocentrotus bindins could not be aligned unambiguously. These regions (111 codons 3' of the core in Strongylocentrotus, 26 codons 5' of the core and 32 codons 3' of the core in Echinometra) were excluded from this analysis. Bindin intron sequences of Arbacia, Tripneustes, and Strongylocentrotus were aligned by eye for each genus. Intron sequences for Echinometra were not included, because they were unavailable in GenBank. Inversions in the bindin intron were aligned between sequences that shared them and were considered as gaps in sequences with the opposite inversion.l:3{if, 百拇医药
Strongylocentrotus polyacanthus was not included in the analysis, because COI data were not available for this species. S. franciscanus was also excluded, because its bindin sequences could not be unambiguously aligned with the sequences from the rest of the genus. T. depressus and T. gratilla sequences were treated as coming from the same species, because neither their bindin nor their COI sequences (Lessios, Kane, and Robertson, in preparation) are reciprocally monophyletic. Finally, we included Hemicentrotus pulcherrimus in the Strongylocentrotus analysis, because both COI and bindin data indicate that it is nested within this genus.
Codon Biasply?a, 百拇医药
We estimated codon bias for each full-length mature bindin allele from Arbacia, Tripneustes, Echinometra, and Strongylocentrotus with the program CODONS by calculating the effective number of codons (ENC) . These ENC values can range from 20 (same codon always used for an amino acid) to 61 (random use of each codon for every amino acid). A separate calculation was carried out for each allele, and then the values were averaged for each genus.ply?a, 百拇医药
Resultsply?a, 百拇医药
Structure of the Moleculeply?a, 百拇医药
We recovered 16 full-length mature bindin alleles from Tripneustes. Twelve of these alleles were unique sequences. Total aligned bindin sequences of Tripneustes include 69 bp of preprobindin, 633 or 663 bp of mature bindin, 835–855 bp of intron, and 31 bp of 3' untranslated region. Like the mature bindins from other genera, those from Tripneustes contain no cysteine or tryptophan residues . Their length (211 or 221 amino acids) is approximately the same as that of other known bindins .
Tripneustes bindins have the highly conserved core observed in bindins from all other studied genera. Of 66 amino acids defined as the core in this study (98–163 in ), 65 are identical to those of Lytechinus variegatus, and 64 are identical to the bindin sequences of S. purpuratus , S. franciscanus , and A. punctulata . All 18 amino acids (119–136 in ) implicated in membrane fusion are identical to those in all other known bindins.]?su, http://www.100md.com
At a point 5' of the core, Tripneustes bindins contain a glycine-rich repeat GG(Q/S/P) (V/G)GGG(G/S) (N/G) (S/M/G) reminiscent of glycine-rich motifs present in Echinometra and Strongylocentrotus but absent in Arbacia. This repeat is present in two copies in T. ventricosus, and in three copies in T. depressus and T. gratilla . This is the only insertion/deletion (indel) observed in Tripneustes mature bindin. Hydrophobicity plots of Tripneustes bindin are similar to those of other Camarodont sea urchins (Zigler and Lessios in preparation); they do not contain the 3' hydrophobic domain observed in Arbacia bindin .
The single intron is in the same location as in all other bindins studied to date (after amino acid 93 in ). It contains a region of approximately 75 bp just inside its 5' end that is inverted in half of the Tripneustes alleles with respect to other Tripneustes alleles. All T. ventricosus alleles, plus three alleles of T. gratilla ("Marquesas 80," "Guam 2," and "Papua New Guinea 1"), have one form of the inversion, whereas all T. depressus alleles and the rest of the T. gratilla alleles have the other form.-yalm, http://www.100md.com
Genealogy of Alleles-yalm, http://www.100md.com
depicts the genealogy of the sequenced bindin alleles, reconstructed by neighbor joining, using only coding sequences (mature bindin and preprobindin sequences). Neighbor joining, using distances, divides the genealogy into Atlantic and Pacific clades. Parsimony and ML methods place the "Guam 2," "Marquesas 80," and "Papua New Guinea 1" alleles with the Atlantic alleles. That different methods of phylogenetic reconstruction result in roots placed between different nodes of the Tripneustes bindin tree is probably due to the large number of changes between Tripneustes and Lytechinus, relative to the changes within Tripneustes. Mitochondrial DNA divides the Atlantic and Pacific haplotypes into reciprocally monophyletic clades (Lessios, Kane, and Robertson, in preparation)
fig.ommitted08*[}gk, 百拇医药
FIG. 3. Neighbor-Joining tree of sequences of preprobindin and mature bindin of Tripneustes, using genetic distances. Bootstrap support (1,000 replicates) of >50% is noted next to nodes. The tree is rooted on a bindin sequence of Lytechinus variegatus.08*[}gk, 百拇医药
There are four fixed amino acid substitutions and one indel that distinguish the Atlantic Tripneustes bindin alleles from all Pacific bindin alleles. Two of these substitutions (at amino acids 70 and 71 in ) occur in the "hotspot" region of Echinometra and Strongylocentrotus . Within the Atlantic clade, the two alleles from São Tomé off the coast of Africa form a well-supported monophyletic entity. Unlike the pattern seen in mtDNA, in which E. and W. Atlantic populations form reciprocally monophyletic sister clades (Lessios, Kane, and Robertson, in preparation), the São Tomé bindin alleles are nested within the ones from the W. Atlantic.08*[}gk, 百拇医药
In the Pacific, bindin alleles of T. depressus, off the coast of America, and of T. gratilla from the central and Indo-West Pacific do not sort according to nominal species or collection locality. One allele from Kiritimati, which, according to collecting location should belong to T. gratilla, actually groups with T. depressus. The T. depressus alleles (plus the Kiritimati allele) is a sister group to the T. gratilla sequences from Reunion, in the Indian Ocean. The other three T. gratilla sequences are basal in the Pacific clade. The basal position of these three alleles is consistent with the observed intron inversion: each of these alleles has the T. ventricosus form of the inversion, whereas the rest of the Pacific individuals have the other form. There are no fixed nonsynonymous changes between T. gratilla and T. depressus. The close affinity of bindin from the two Pacific species mirrors a similar lack of differentiation of mtDNA sequences (Lessios, Kane, and Robertson, in preparation). Apparently, gene flow between the eastern and central Pacific is either continuing or has been very recently interrupted.
Adaptive Evolution0:xdf, 百拇医药
Relative rates of change of the different regions of the Tripneustes bindin molecule are qualitatively similar to patterns of bindin evolution in other Camarodont sea urchins. Rates of nonsynonymous change are highest in the hotspot region, lowest in the core, and intermediate in the rest of the molecule . Only nonsynonymous changes are observed in the hotspot region. However, in contrast to the pattern in Echinometra and Strongylocentrotus, the excess of nonsynonymous changes in the hotspot region is not significant in any pairwise comparison. Tripneustes bindins also lack the large number of potentially important indels seen in the other Camarodont genera. In addition, using the models implemented in PAML, we found no evidence for positively selected sites dispersed throughout the molecule. The likelihood of models that allowed for positively selected sites was not significantly higher than that of models that did not . Nor did we find any evidence for significant variation in dN/dS ratios between lineages. Allowing a different dN/dS ratio for each branch in the phylogeny did not produce a significantly better model than a model with a single dN/dS ratio for the entire tree . Finally, a comparison of the ratios of replacement to silent differences within and between species was not significantly different from neutral expectation . Four replacement substitutions and zero silent substitutions are fixed between Pacific (T. gratilla and T. depressus) and Atlantic (T. ventricosus) individuals, and there are 12 replacement polymorphisms and 12 silent polymorphisms within the two groups (Fisher's exact test P = 0.11).
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Table 1 Replacement (dN) and Silent (dS) Substitutions per Site in Three Regions of Tripneustes Mature Bindin.xt, http://www.100md.com
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Table 2 Maximum Likelihood Testing for Variation in the Ratio of Replacement Substitutions to Silent Substitutions Among Sites and Lineages.xt, http://www.100md.com
Rate of Bindin Evolutionxt, http://www.100md.com
To compare the rate of relative change of amino acid replacement of bindin within each genus, we calculated interspecific K2P, dN and dS (PBL) for the full mature bindin and the intron, then divided these values by mitochondrial COI genetic distances (K2P) . If a universal calibration for a "COI clock" is assumed, then divergence in this molecule represents a proxy of the time that species have remained separate, and thus helps standardize the bindin divergence for the age of the species. The comparison of ratios of either bindin K2P or dN to COI genetic distance indicates that there are large differences between the genera in the overall rates of change in the bindin molecule. Because these are pairwise comparisons, each ratio is not independent of all others, so they cannot be compared statistically. Nevertheless, the trend is clear. All ratios of bindin K2P and especially dN values to COI distances in Arbacia and Tripneustes are smaller than all such ratios in Echinometra or Strongylocentrotus. Arbacia shows larger interspecific COI distances relative to those of the other genera, which could be due either to older ages of its species or to higher rates of COI divergence. The COI divergence between A. punctulata and A. stellata across the Isthmus of Panama is equivalent to that of six other genera thus separated , which would suggest that rate variation in COI is low, and thus that it correctly indicates most species of Arbacia to be older than those of the other genera. If as suggested, Atlantic and Pacific species of Arbacia were separated before the completion of the Panama isthmus, then rate of COI divergence in this genus would be slower than we assume, and the differences in rates of bindin evolution between the genera would be even more pronounced.
fig.ommitted.qz\n)l, 百拇医药
Table 3 Intrageneric Differences in Mature Bindin, Bindin Intron, and Mitochondrial Cytochrome Oxidase I in All Genera for Which Data Exist..qz\n)l, 百拇医药
Interestingly, the higher rate of bindin evolution in Strongylocentrotus and Echinometra relative to Arbacia or Tripneustes does not appear to be limited to adaptive changes. The comparison of bindin dS to COI divergence ratios follows the same pattern as that of comparisons of bindin dN to COI divergence. Every interspecific comparison in Arbacia or Tripneustes produces a lower ratio than in Strongylocentrotus or Echinometra . A positive correlation across molecules between rate of substitution in replacement and silent sites has been found in many organisms . Mutational bias, nonindependence of point mutations, and codon-level selection have been offered as explanations for the phenomenon. Almost all explanations involve some form of codon bias, but ENC values in bindin show that codon usage in this molecule is universally equitable (means per genus: Tripneustes, 59.2; Arbacia, 44.9; Strongylocentrotus, 52.2; Echinometra, 55.1).
The trend seen for the silent sites of the expressed region does not extend to the intron. Intron sequences are not available for Echinometra, but a comparison between Arbacia and Tripneustes, on the one hand, and Strongylocentrotus on the other, indicates that the ratio of intron to COI divergence is not different between the two groups, which is what would be expected if both the intron and COI evolve linearly with time.9ng@i, 百拇医药
Discussion9ng@i, 百拇医药
Tripneustes Bindin Structure and Divergence9ng@i, 百拇医药
The bindin of Tripneustes contains the highly conserved core characteristic of all other bindins studied to date, has an intron in the same position, and has approximately the same length. Its structure resembles that of the bindins of other Camarodont sea urchins (Echinometra, Strongylocentrotus, and Lytechinus) more than the bindin of the Stirodont Arbacia. The resemblances include the glycine-rich repeat structure 5' of the core and the lack of the 3' hydrophobic domain of Arbacia bindin. These similarities to the other Camarodonts are not surprising, given that Tripneustes last shared a common ancestor with Echinometra, Strongylocentrotus, and Lytechinus 25–60 MYA, and with Arbacia 120–180 MYA .
Despite the similarities in structure of Tripneustes bindin with that of other Camarodont sea urchins, its mode of evolution appears to be more like that of Arbacia. In contrast to Echinometra or Strongylocentrotus bindin, Tripneustes bindin has evolved slowly. For example, bindin of T. ventricosus from the Caribbean differs from bindin of the eastern Pacific T. depressus by only four fixed amino acid changes and a single indel. These two species were presumably separated from each other for more than 3 million years by the Isthmus of Panama There are no fixed amino acid differences or indels between T. depressus and T. gratilla from the Indo-Pacific, despite the tremendous geographical distance separating the eastern Pacific from the West Indian Ocean. This level of differentiation is almost as low as that seen in the bindin of Arbacia, in which only a single indel distinguishes species on either side of Central America . It contrasts with differentiation in Echinometra, in which species separated for less than 1.5 MY have 7 fixed amino acid differences and in Strongylocentrotus in which S. purpuratus and S. droebachiensis, separated for less than 3 MY, show 21 amino acid differences ). In addition, bindin of Tripneustes, like that of Arbacia, has only one indel, whereas indels are much more numerous in Echinometra and Strongylocentrotus. Such indels may be functionally important in gamete recognition . Bindin of Tripneustes, like that of Arbacia, shows no evidence of diversifying selection that would manifest itself in significantly more replacement substitutions than silent nucleotide substitutions. Finally, Tripneustes and Arbacia show a lower ratio of bindin to COI substitutions than Echinometra or Strongylocentrotus, despite the inevitable underestimation of bindin divergence in the latter two genera, arising from the exclusion from the analysis of the most variable regions, which could not be aligned. What could account for such similarities between distantly related taxa and differences between closely related ones?
One possible explanation might be the relative age of the species in different genera. By COI and intron divergence (but not by the number of silent substitutions in bindin) the extant species of Arbacia and Tripneustes diverged earlier from each other than did the species of Echinometra and Strongylocentrotus. If, as , have suggested for sex-related genes of Drosophila, the episodes of adaptive bindin evolution are concentrated to the time that new species are formed, and if positive selection on the molecule is subsequently relaxed, then older species may lose the signature of an excess of replacement substitutions relative to silent substitutions. This explanation cannot, however, be applied to bindin. A concentration of adaptive changes at the moment of speciation would decrease the ratio of replacement substitutions to silent substitutions in older species. Increased time since speciation would also decrease the ratio of bindin to COI divergence. However, longer time since speciation could not decrease the absolute number of amino acid changes fixed between species after they have been accumulated early in divergence. The low number of such replacements in Arbacia and Tripneustes must therefore indicate that they have never gone through a period of accelerated bindin divergence. This possibility is also supported by our analysis of the apportionment of replacement and silent substitutions along branches of the Tripneustes bindin tree, which failed to show a concentration of adaptive change in younger (or older) lineages.
considered three hypotheses as possible explanations for the decelerated bindin evolution in Arbacia: (1) high gene flow, (2) functional constraint, and (3) degree of overlap of species distributions. First, they suggested that high levels of gene flow and a lack of population subdivision within species of Arbacia may limit the rate of bindin evolution. Tripneustes, like Arbacia, shows low levels of biogeographic subdivision, at least in the Pacific (Lessios, Kane, and Robertson, in preparation). However, it is not clear that these two genera are exceptional among echinoids in that respect. High gene flow over thousands of kilometers is a standard feature of all sea urchin species with planktonic larvae . In particular, the observations of minimal population subdivision over a range of several thousand kilometers in Indo-West Pacific Echinometra and in eastern Pacific S. purpuratus or S. franciscanus indicate that the accelerated interspecific bindin divergence relative to Arbacia and Tripneustes cannot be attributable to this factor alone.
Second, proposed that slow bindin evolution in Arbacia might be due to functional constraints imposed by its molecular structure. They suggested that lack of repeat elements and indels, as well as the presence of a 3' hydrophobic domain, represent evolutionary constraints on Arbacia bindins that are not shared by bindins of Camarodonts. Tripneustes bindin, despite three repeats and a lack of 3' hydrophobic domain, evolves almost as slowly as Arbacia bindin. It appears, therefore, that these features of the molecule do not necessarily affect its evolutionary rate.hzn, 百拇医药
The third hypothesis of was that positive selection, arising from the presence of sympatric congeners, is operating on bindin of Echinometra and Strongylocentrotus, but is absent in Arbacia. Echinometra and Strongylocentrotus contain sympatric species, which are generally gametically incompatible . All extant species in Tripneustes and Arbacia are allopatric. There are no data about gametic compatibility between Tripneustes species, but A. punctulata and A. incisa are gametically compatible . Thus, it is possible that reinforcement of reproductive isolation among the sympatric species of Echinometra and Strongylocentrotus may explain the observed patterns of bindin evolution. This is a pattern of reinforcement on the genus level. However, as is also true for these patterns on the population level , we do not know the process that produced them. Did the presence of congeners create selective pressures for bindin divergence in Strongylocentrotus and Echinometra, or did divergence in bindin, caused by selection arising from another cause, permit species in these genera to invade the same area and coexist without fusing or becoming extinct? This question cannot be answered with certainty, but the patterns of intraspecific and interspecific divergence can provide a clue. If selection to avoid hybridization were responsible for the reinforcement pattern, we would have expected an excess of amino acid replacements between species (particularly between sympatric species) of Echinometra and Strongylocentrotus relative to within species. This however, is not the case in either genus. In the rapidly evolving 40 codon bindin region of Echinometra, the ratio of replacement substitutions to silent substitutions between alleles is larger than unity in both intraspecific and interspecific comparisons, and tests are not significant. In the same region of bindin, comparisons between S. franciscanus and either S. purpuratus or S. droebachiensis indicate that the ratio of replacement substitutions to silent substitutions is higher within than between species has shown experimentally that, in Echinometra, males carrying different bindin alleles have different rates of success in fertilizing females, depending on the female bindin genotype, and that intraspecific polymorphism is thus maintained by selection. Such selection could arise from male heterozygote advantage, nontransitive female preferences , or interlocus conflict evolution between the sexes . These processes can operate within species whether or not they are sympatric with a congener . Thus, it is possible that the selective force that accelerates bindin evolution in some genera is not avoidance of hybridization in sympatry. The pattern of reinforcement suggested by the comparison between genera with sympatric and allopatric species may be due to the ability of congeneric species with divergent bindin to coexist.
Because Echinometra was the first genus in which evolution of bindin was studied, and because its rapid evolution under positive selection fit patterns found in other genes related to sexual reproduction , it has been tacitly assumed that the patterns of positive selection and rapid allele divergence exemplify the mode of evolution of this locue.g.vidence from Strongylocentrotus reinforced this view. However, the information from Tripneustes now indicates that what found in Arbacia was not an isolated exception, and not a characteristic of stirodont versus camarodont sea urchins. Why bindin should evolve faster in certain genera of sea urchins and not others remains unclear, but the addition of data from more sea urchin taxa will help determine the factors that promote or retard evolution in this molecule.j.;vi&, 百拇医药
Acknowledgementsj.;vi&, 百拇医药
A. Calderón and L. Calderón helped in the laboratory. J. Kane and M. A. McCartney provided sampling and laboratory advice. M. A. McCartney also designed the RACE primers and provided instructions on the technique. T. Duda and B. Kessing helped with data analysis. D. R. Robertson collected specimens from Clipperton, the Marquesas, Reunion, and São Tomé. G. Paulay collected specimens from Guam and Papua New Guinea. R. Collin, A. Crawford, C. Cunningham, T. Duda, B. Kessing, C. Riginos, and three anonymous reviewers commented on the manuscript. This work was supported by a National Science Foundation predoctoral fellowship, a Duke University Zoology Department Dissertation Improvement Grant, and a Smithsonian Predoctoral fellowship to K.S.Z., and by the Smithsonian Molecular Evolution Program.
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Accepted for publication October 9, 2002.(K. S. Zigler and H. A. Lessios)