Gamete compatibility and sperm competition affect paternity and hybridization between sympatric Asterias sea stars

Gamete interactions may strongly influence speciation and hybridization in sympatric broadcast-spawning marine invertebrates. We examined the role of gamete compatibility in species integrity using cross-fertilization studies between sympatric Asterias sea stars from a secondary contact zone in the northwest Atlantic. In crosses between single males and single females, gametes of both species were compatible and produced viable, fertile hybrid offspring, but with considerable variation in the receptivity of eggs to heterospecific sperm. Differential compatibility of heterospecific gametes was detected in sperm competition studies in which we used a nuclear DNA marker to assign paternity to larval offspring. Several families showed conspecific sperm precedence in A. forbesi eggs, and one family showed competitive superiority of A. forbesi sperm fertilizing A. rubens eggs. Gametic interactions are an important component of prezygotic reproductive isolation in sympatric Asterias. The interaction between gametes of these closely related sea stars is consistent with the function of gamete recognition systems that are known to mediate fertilization success and speciation in other marine invertebrates.     

Quantitative analysis of gametic incompatibility between closely related species of neotropical sea urchins

Species of the sea urchin genus Echinometra found on the two coasts of Panamá are recently diverged and only partially isolated by incomplete barriers to interspecific fertilization. This study confirms previous work that revealed incompatibility between the eggs of the Atlantic E. lucunter and the sperm of the other two neotropical species, whereas eggs of its sympatric congener E. viridis and allopatric E. vanbrunti are largely compatible with heterospecific sperm. Here we quantify fertilization using a range of sperm dilutions. We demonstrate a much stronger block to cross-species fertilization of E. lucunter eggs than was previously shown at fixed sperm concentrations, and mild incompatibility of the other two species' eggs where previous crosses between species were not distinguishable from within-species controls. Additionally, we present evidence for intraspecific variation in egg receptivity towards heterospecific sperm. Our findings here again discount the "reinforcement model" as a viable explanation for the pattern of prezygotic isolation. Gamete incompatibility in these Echinometra has appeared recently-within the last 1.5 million years-but is weaker in sympatry than in allopatry. Accidents of history may help explain why incompatibility of eggs emerged in one species and not in others. Compensatory sexual selection on sperm in this species could follow, and promote divergence of proteins mediating sperm-egg recognition.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

Multiple gene genealogies reveal asymmetrical hybridization and introgression among strongylocentrotid sea urchins

The evolution of incompatibilities between eggs and sperm is thought to play important roles in establishing and maintaining reproductive isolation among species of broadcast-spawning marine invertebrates. However, the effectiveness of gametic isolation in initiating the speciation process and/or in limiting the introgression of genes among species at later stages of divergence remains largely unknown. In the present study, we collected DNA sequence data from five loci in four species of Strongylocentrotus sea urchins ( S. droebachiensis , S. pallidus , S. purpuratus , and S. franciscanus ) to test whether the susceptibility of S. droebachiensis eggs to fertilization by heterospecific sperm results in gene flow between species. Despite the potential for introgression, a small but statistically significant signal of introgression was observed only between the youngest pair of sister taxa ( S. pallidus and S. droebachiensis ) that was strongly asymmetrical (from the former into the latter). No significant gene flow was observed for either S. purpuratus or S. franciscanus despite the ability of their sperm to readily fertilize the eggs of S. droebachiensis . Our results demonstrate that asymmetrical gamete compatibilities in strongylocentrotids can give rise to asymmetrical patterns of introgression but suggest that gamete traits alone cannot be responsible for maintaining species integrities. The genetic boundaries between strongylocentrotid urchin species in the northeast Pacific appear to be related to postzygotic isolating mechanisms that scale with divergence times and not intrinsic gametic incompatibilities per se .     

GAMETIC INCOMPATIBILITY BETWEEN SPECIES OF THE SEA URCHIN ECHINOMETRA ON THE TWO SIDES OF THE ISTHMUS OF PANAMA

The Pliocene rise of the Central American Isthmus has resulted in numerous “geminate pairs,” i.e., closely related species, one on each coast. Such species pairs can provide information on the evolution of isolating mechanisms in allopatry and on the relationship between genetic divergence and reproductive isolation in populations separated at a known time. The sea urchin genus Echinometra has one species, E. vanbrunti, in the eastern Pacific, and two, E. lucunter and E. viridis, in the Caribbean. E. viridis is morphologically distinct from the other two species, leading to the conclusion that E. lucunter and E. vanbrunti constitute a geminate pair. Allozyme data, on the other hand, place the speciation event of the two currently sympatric species after the rise of the Isthmus. We report fertilization experiments between the gametes of the three species performed to determine degree of reproductive isolation. Crosses between E. viridis and E. vanbrunti produce rates of fertilization almost equal to those manifested in homogamic crosses. Sperm of E. lucunter can fertilize eggs of the other two species, but few of its eggs permit fertilization by heterospecific sperm. Contrary to the predictions of the “speciation by reinforcement” hypothesis, degree of incompatibility between the allopatric E. lucunter and E. vanbrunti is higher than between the sympatric E. lucunter and E. viridis. Despite the incomplete and unidirectional nature of their gametic isolation, E. lucunter and E. viridis maintain their genetic integrities. Consideration of the likely phylogenetic relationships between the three species suggests that incompatibility of E. lucunter eggs with heterospecific sperm has evolved in the last 3.5 million years, after the rise of the Isthmus. There is no correlation between genetic divergence and strength of reproductive isolation, either within Echinometra, or among the geminate species pairs of echinoids that have been studied to date. Because recognition between echinoid gametes depends on the chemical compositions of a sperm protein and an egg glycoprotein, the appearance of gametic isolation would require only the fixation of a few mutations in each population on either side of a geographic barrier and could be independent of any other kind of genetic divergence. Thus, in animals with external fertilization, speciation need not be accompanied by major genomic reorganization.     

Single-copy DNA distance between two congeneric sea urchin species exhibiting radically different modes of development

We have investigated the differences between nuclear genomes of two purportedly congeneric species of sea urchin that differ radically in early development. Heliocidaris tuberculata develops by means of a typical pluteus larva, whereas H. erythrogramma develops directly from an egg that is 100-fold the volume of the H. tuberculata egg. Reassociation kinetic analysis shows that the kinetic components of the genomic DNA from the two species are essentially the same. No single repeat component explains the 30% difference between the H. erythrogramma and H. tuberculata genomes. Reciprocal hybridization of tracer-labeled single-copy DNA fractions between these species indicates that approximately 50% of the single-copy DNA is sufficiently similar to form hybrids at standard hybridization criterion. Thermal denaturation profiles of the hybridized single-copy DNA sequence yields median (T50H) values of 13.8 degrees-16.5 degrees C. This result suggests a divergence time of 10-13 Mya, which is comparable to divergence times between congeneric sea urchin species in other genera that do not differ significantly in development. Radical differences in early developmental processes can evolve rapidly between closely related forms.     

Microtubule assembly is required for the formation of the pronuclei,nuclear lamin acquisition,and DNA synthesis during mouse,but not sea urchin,fertillization

Microtubule assembly is required for the formation of the male and female pronuclei during mouse, but not sea urchin, fertilization. In mouse oocytes, 50 μM colcemid prevents the decondensation of the maternal meiotic chromosomes and of the incorporated sperm nucleus during in vitro fertilization. Nuclear lamins do not associate with either of the parental chromatin sets although peripherin, the PI nuclear peripheral antigen, appears on both. DN A synthesis docs not occur in these fertilized, colcemid-arrested oocytes. This effect is limited to the first hours after ovulation, since colcemid added 4–6 hours later no longer prevents pronuclear development, lamin acquisition, or DNA synthesis. Neither microtubule stabilization with 10 μM taxol nor microfilament inhibition with 10 μM cytochalasin D or 2.2 μg/ml lalrunculin A prevent these pronuclear events; these drugs will inhibit the apposition of the pronuclei at the egg center. In sea urchin eggs, colcemid or griseofulvin treatment doe? not result in the same effect and the male pronucleus forms with the attendant accumulation of the nuclear lamins. The differences in the requirement for microtubule assembly during pronucleus formation may be related to the cell cycle: In mice the sperm enters a meiotic cytoplasm, whereas in sea urchin eggs it enters an interphase cytoplasm. Refertilization of mitotic sea urchin eggs was performed to test the possibility that this phenomenon is related to whether the sperm enters a meiotic/mitotic cytoplasm or one at interphase; during refertilization at first mitosis, the incorporated sperm nucleus is unable to decondense and acquire lamins. These results indicate a requirement for microtubule assembly for the progression from meiosis to first interphase during mouse fertilization and suggest that the cytoskeleton is required for changes in nuclear architecture necessary during fertilization and the cell cycle.     

Sperm polymorphism within the sea urchin Strongylocentrotus droebachiensis: divergence between Pacific and Atlantic oceans

The rapid evolution of traits related to fertilization such as sperm morphology may be pivotal in the evolution of reproductive barriers and speciation. The sea urchin Strongylocentrotus droebachiensis has a circumarctic distribution and shows substantial genetic subdivision between northeastern Atlantic populations and northwestern Atlantic and Pacific populations. Using transmission electron microscopy, we show here that sperm shape, size, and ultrastructure differ markedly among populations of S. droebachiensis from different oceans and reflect patterns of genetic divergence. Sperm nuclei from northwestern Atlantic and Pacific populations were longer and narrower than those from the northeastern Atlantic. We additionally demonstrate population-level differences in the amount and location of filamentous actin (F-actin) prior to the occurrence of the acrosome reaction. Sperm from Pacific and northwest Atlantic populations differed from that of all other echinoids examined in that intact sperm contains a partly preformed acrosomal process, a structure more closely resembling the acrosomal rod seen in some molluscs. Immunofluorescent studies using anti-bindin antibodies and the F-actin-specific stain phalloidin confirmed these findings. Divergence of reproductive traits such as sperm morphology may be related to divergence in gamete compatibility and genetic divergence, and could represent the first stages of speciation in free-spawning marine invertebrates.     

The structure of sulfated polysaccharides ensures a carbohydrate-based mechanism for species recognition during sea urchin fertilization

The evolution of barriers to inter-specific hybridization is a crucial step in the fertilization of free spawning marine invertebrates. In sea urchins, molecular recognition between sperm and egg ensures species recognition. Here we review the sulfated polysaccharide-based mechanism of sperm-egg recognition in this model organism. The jelly surrounding sea urchin eggs is not a simple accessory structure; it is molecularly complex and intimately involved in gamete recognition. It contains sulfated polysaccharides, sialoglycans and peptides. The sulfated polysaccharides have unique structures, composed of repetitive units of alpha-L-fucose or alpha-L-galactose, which differ among species in the sulfation pattern and/or the position of the glycosidic linkage. The egg jelly sulfated polysaccharides show species-specificity in inducing the sperm acrosome reaction, which is regulated by the structure of the saccharide chain and its sulfation pattern. Other components of the egg jelly do not possess acrosome reaction inducing activity, but sialoglycans act in synergy with the sulfated polysaccharide, potentiating its activity. The system we describe establishes a new view of cell-cell interaction in the sea urchin model system. Here, structural changes in egg jelly polysaccharides modulate cell-cell recognition and species-specificity leading to exocytosis of the acrosome. Therefore, sulfated polysaccharides, in addition to their known functions as growth factors, coagulation factors and selectin binding partners, also function in fertilization. The differentiation of these molecules may play a role in sea urchin speciation.     

Relationship between nuclear DNA synthesis and centrosome reproduction in sea urchin eggs

The importance of nuclear DNA synthesis for the doubling, or reproduction, of centrosomes in cells that are not growth-limited, such as sea urchin eggs, has not been clearly defined. Studies of enucleated, fertilized eggs show that nuclear activities are not required at each cell cycle for the normal reproduction of the complete centrosome. However, other studies report that the inhibition of nuclear DNA synthesis in intact eggs by the drug aphidicolin prevents centrosome reproduction and entry into mitosis as seen by nuclear envelope breakdown. To resolve this paradox, we systematically characterized the effect of aphidicolin on cell division in eggs from three species of sea urchins. Eggs were continuously treated with 5 or 10 micrograms/ml aphidicolin starting 5 min after fertilization. This blocked total incorporation of 3H-thymidine into DNA by at least 90%, as previously reported. We found that the sperm aster always doubles prior to first mitosis. Over a period of several hours, the centrosomes reproduce in the normal 2-4-8-16 fashion, with a period that is longer and more variable than normal. In every culture, a variable percentage of the eggs undergoes nuclear envelope breakdown. Once broken down, the nuclear envelope never visibly reforms even though centrosomes continue to double. Fluorescent labeling of DNA revealed that the chromatin does not condense into discrete chromosomes. Whether or not the nuclear envelope breaks down, the chromatin appears as an amorphous mass of fibers stretched between first two and then four asters. Later, the nuclear envelope/chromatin loses its association with some or all centrosomes. Our results were the same for all eggs at both drug concentrations. Thus, nuclear DNA synthesis is not required for centrosome reproduction in sea urchin eggs.     

Conspecific sperm precedence in two species of tropical sea urchins

Conspecific sperm precedence occurs when females are exposed to sperm from males of multiple species, but preferentially use sperm of a conspecific. Conspecific sperm precedence and its mechanisms have been documented widely in terrestrial species, in which complex female behaviors or reproductive tract morphologies can allow many opportunities for female choice and sperm competition, however, the opportunity for conspecific sperm precedence in free spawning marine invertebrates has been largely ignored. Two sea urchin species, Echinometra oblonga and E. sp. C, have high levels of interspecific fertilization in no-choice lab crosses, but no natural hybrids have been found. We performed competitive fertilization assays to test for conspecific sperm precedence and found that eggs of both species showed a marked preference for conspecific sperm when fertilized with heterospecific sperm mixtures. Strong rejection of heterospecific sperm would not have been predicted from no-choice assays and helps explain the lack of natural hybrids. We also found significant variation in hybridization success among crosses. Conspecific sperm precedence in free spawning invertebrates shows that the simple surfaces of eggs and sperm provide ample opportunity for egg choice and sperm competition even in the absence of intricate behavior or complex reproductive morphologies.     

No barriers to fertilization between sympatric colour morphs in the marine species flock Hypoplectrus (Serranidae)

Assortative mate choice is, so far, the only demonstrated isolating barrier between colour morphs in fish species flocks, such as freshwater cichlids and marine hamlets, suggesting an important role for sexual selection in speciation. However, there has been little consideration of post-copulatory mechanisms that are known to influence reproductive isolation in other taxa. Selective pressures through a cost to hybrid matings, or genetic drift, may lead to the evolution of fertilization barriers through gamete incompatability. In cases of incipient speciation, such as the hamlets, complete fertilization blocks may not have evolved. Instead, differential fertilization between species may reduce the success of interspecific fertilizations. We examined the fertilization dynamics of sympatric Hypoplectrus nigricans (black hamlet) and Hypoplectrus puella (barred hamlet). Experimental crosses were performed to score fertilization success in within-morph and between-morph crosses. Fertilization success did not differ between self-fertilized, within-morph or between-morph crosses. We therefore found no evidence of post-mating barriers through sperm:egg incompatibilities and potential for self-fertilization. Our results are consistent with a mechanism of colour morph divergence based on sexually selected morph mating preferences.     

Genome structure and divergence of nucleotide sequences in echinodermata

The arrangement of repetitive and single-copy DNA sequences has been studied in DNA of some species of Echinodermata — sea urchin, starfishes and sea-cucumber. Comparison of the reassociation kinetics of short and long DNA fragments indicates that the pattern of DNA sequence organization of all these species is similar to the so called Xenopus pattern characteristic of the genomes of most animals and plants. However, substantional variations have been found in the amount of repetitive nucleotide sequences in DNA of different species and in the length of DNA regions containing adjacent single-copy and repetitive sequences. Measurements of the size of S₁-nuclease resistant reassociated repetitive DNA sequences show a variability of ratios between long and short repetitive DNA sequences of different species. — The degree of divergence of short and long repetitive DNA sequences and single-copy DNA was studied by molecular hybridization of the sea urchin Strongylocentrotus intermedius ³H-DNA with the DNA of other species and by determination of the thermostability of the hybridized molecules so obtained. All three fractions of S. intermedius DNA contain sequences homologous to DNA of the other echinoderm species studied. The results obtained suggest that short repetitive DNA sequences are those which have been most highly conserved throughout the evolution of Echinodermata. A new hypothesis is proposed to explain the nature of the evolutionary changes in DNA sequence interspersion patterns.     

The relationship between conspecific fertilization success and reproductive isolation among three congeneric sea urchins

Few data are available on the effectiveness of reproductive isolating mechanisms in externally fertilizing taxa. I investigated patterns of conspecific and heterospecific fertilization among three coexisting sea urchin species, Strongylocentrotus droebachiensis, S.franciscanus, and S. purpuratus. In the laboratory, both among and within species, eggs from individual females whose eggs are more easily fertilized by conspecific sperm are also most susceptible to heterospecific fertilization. At one extreme, S. droebachiensis requires an order of magnitude fewer conspecific sperm to fertilize eggs than do the other two species and shows very little distinction between conspecific and heterospecific sperm in no choice experiments. Strongylocentrotus franciscanus has an intermediate susceptibility to fertilization by heterospecific sperm. At the other extreme, S. purpuratus rarely cross-fertilizes. Field observations indicate that S. droebachiensis is often surrounded by heterospecific sea urchins. Genetic analysis of larvae produced during heterospecific spawning events indicate that hybrids are generally produced if male conspecifics are more than 1 m from a spawning female S. droebachiensis. Laboratory cultures indicate that these hybrids suffer high mortality relative to conspecific larvae. Comparisons of reproductive success of S. droebachiensis during single-species and multispecies spawning events indicate that the benefits of producing easily fertilized eggs under conditions of sperm limitation may outweigh the costs of losing some offspring to hybrid fertilization. Patterns of variability in heterospecific fertilization are considered in light of three hypotheses: phylogenetic relatedness, reinforcement selection, and sexual selection.     

Dispersal barriers in tropical oceans and speciation in Atlantic and eastern Pacific sea urchins of the genus Echinometra

Echinometra is a pantropical sea urchin made famous through studies of phylogeny, speciation, and genetic structure of the Indo-West Pacific (IWP) species. We sequenced 630 bp of the cytochrome oxidase I (COI) mitochondrial gene to provide comparable information on the eastern Pacific and Atlantic species, using divergence between those separated by closure of the Isthmus of Panama 3.1 million years ago (Ma) to estimate dates for cladogenic events. Most recently (1.27-1. 62 Ma), the Atlantic species E. lucunter and E. viridis diverged from each other, at a time in the Pleistocene that sea levels fell and Caribbean coral speciation and extinction rates were high. An earlier split, assumed to have been coincident with the completion of the Isthmus of Panama, separated the eastern Pacific E. vanbrunti from the Atlantic common ancestor. Transisthmian COI divergence similar to that in the sea urchin genus Eucidaris supports this assumption. The most ancient split in Echinometra occurred between the IWP and the neotropical clades, due to cessation of larval exchange around South Africa or across the Eastern Pacific Barrier. Gene flow within species is generally high; however, there are restrictions to genetic exchange between E. lucunter populations from the Caribbean and those from the rest of the Atlantic. Correlation between cladogenic and vicariant events supports E. Mayr's contention that marine species, despite their high dispersal potential, form by means of geographical separation. That sympatric, nonhybridizing E. lucunter and E. viridis were split so recently suggests, however, that perfection of reproductive barriers between marine species with large populations can occur in less than 1.6 million years (Myr).     

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.     

Motility and centrosomal organization during sea urchin and mouse fertilization

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.     

Membrane conductance patterns during fertilization are sperm dependent in two sea urchin species

The influence of the egg and sperm on the conductance changes at fertilization in the sea urchin were investigated through cross-fertilization of two Hawaiian species, Tripneustes gratilla and Pseudoboletia indiana. The current-voltage (I-V) relation, measured in voltage-clamped eggs at intervals over the period 2-16 min following the rise to a positive membrane potential that signals sperm attachment, differs significantly in the two species. The magnitude of the conductance change depends on the species of the fertilizing sperm in both homologous and heterologous crosses. This supports the hypothesis that currents during this period arise from sperm membrane channels incorporated into the egg at sperm-egg fusion. Measurements of conductance during the first 90 sec, which includes the period of the major inward current correlated with cortical granule breakdown and elevation of the fertilization envelope, showed that the magnitude and timing of the maximum current also differed in the two species. This conductance change presumably involves an activation of egg membrane channels initiated by the sperm and would be expected to be characteristic of the egg species. However, in cross-fertilized eggs the magnitude and timing of the conductance change over this period also depends on the species of the sperm with little identifiable egg contribution, indicating that the fertilizing sperm can modulate the egg response to influence these events.