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.     

Egg size and the evolution of phenotypic plasticity in larvae of the echinoid genus Strongylocentrotus

Planktotrophic larvae grow by utilizing energy obtained from food gathered in the plankton. Morphological plasticity of feeding structures has been demonstrated in multiple phyla, in which food-limited larvae increase feeding structure size to increase feeding rates. However, before larvae can feed exogenously they depend largely on material contained within the egg to build larval structures and to fuel larval metabolism. Thus, the capacity for plasticity of feeding structures early in development may depend on egg size. Using the congeneric sea urchins Strongylocentrotus franciscanus and S. purpuratus, which differ in egg volume by 5-fold, I tested whether the degree of expression of feeding structure (larval arm length) plasticity is correlated with differences in the size of the egg. I experimentally manipulated egg size of S. franciscanus (the larger-egged species) by separating blastomeres at the 2-cell stage to produce half-sized larvae. I reared half-size and normal-size larvae under high and low food treatments for 20 days. I measured arm and body lengths at multiple ages during development and calculated the degree of plasticity expressed by larvae from all treatments. Control and unmanipulated S. franciscanus larvae (from ∼ 1.0 nl eggs) had significantly longer arms relative to body size and a significantly greater degree of plasticity than half-sized S. franciscanus larvae (from < 0.18 nl eggs), which in turn expressed a significantly greater degree of plasticity than S. purpuratus larvae (from ∼ 0.3 nl eggs). These results indicate that egg size affects larval arm length plasticity in the genus Strongylocentrotus; larger eggs produce more-plastic larvae both in an experimental and a comparative context. However, changes in egg size alone are not sufficient to account for evolved differences in the pattern of plasticity expressed by each species over time and may not be sufficient for the evolutionary transition from feeding to non-feeding.     

DEVELOPMENTAL CONSEQUENCES OF AN EVOLUTIONARY CHANGE IN EGG SIZE: AN EXPERIMENTAL TEST

Larvae of two species of sea urchins (Strongylocentrotus droebachiensis and S. purpuratus) differ in initial form and in the rate of development. To determine whether these differences are attributable to the large interspecific difference in egg size, we experimentally reduced egg size by isolating blastomeres from embryos. The rate of development of feeding larvae derived from isolated blastomeres was quantified using a novel morphometric method. If the differences early in the life histories of these two species are due strictly to differences in egg size, then experimental reduction of the size of S. droebachiensis eggs should yield an initial larval form and rate of development similar to that of S. purpuratus. Our experimental manipulations of egg size produced three clear results: 1) smaller eggs yielded larvae that were smaller and had simpler body forms, 2) smaller eggs resulted in slower development through the early feeding larval stages, and 3) effects of egg size were restricted to early larval stages. Larvae from experimentally reduced eggs of the larger species had rates of development similar to those of the smaller species. Thus, cytoplasmic volumes of the eggs, not genetic differences expressed during development, account for differences in larval form and the rate of form change. This is the first definitive demonstration of the causal relationship between egg size (parental investment per offspring) and life-history characteristics in marine benthic invertebrates. Because larval form influences feeding capability, the epigenetic effects of egg size on larval form are likely to have important functional consequences. Adaptive evolution of egg size may be constrained by the developmental relationships between egg size and larval form: evolutionary changes in egg size alone can result in concerted changes in larval form and function; likewise evolutionary changes in larval form and function can be achieved through changes in egg size. These findings may have broader implications for other taxa in which larval morphology and, consequently, performance may be influenced by changes in egg size.     

The larval stages of the sea urchin, Strongylocentrotus purpuratus

The adult body plan of Strongylocentrotus purpuratus is established within the imaginal rudiment during the larval stages. To facilitate the study of these stages, we have defined a larval staging scheme, which consists of seven stages: Stage I, four-arm stage; Stage II, eight-arm stage; Stage III, vestibular invagination stage; Stage IV, rudiment initiation stage; Stage V, pentagonal disc stage; Stage VI, advanced rudiment stage; and Stage VI, tube-foot protrusion stage. Each stage is characterized by significant morphological features observed for the first time at that stage. This scheme is intended as a guide for determining the degree of larval development, and for identifying larval and adult structures. Larval anatomy was visualized using light and confocal microscopy as required on living material, whole mount fixed specimens, and serial sections. Antibody staining to localize specific gene products was also used. Detailed analysis of these data has furthered our understanding of the morphogenesis of the rudiment, and has suggested provocative questions regarding the molecular basis for these events. We intend this work to be of use to investigators studying gene expression and morphogenesis in postembryonic larvae.     

Potential for paired vestibules in plutei (Echinodermata, Echinoidea)

Abstract. Echinoids are generally thought to develop only a single ectodermal invagination (V1), which expands into a vestibule and contributes to the formation of the juvenile rudiment on the left side of the larval body. However, in 2 echinoid species, Strongylocentrotus purpuratus and Dendraster excentricus, a second invagination (V2) was observed to form on the right side of the larval body exactly opposite V1. Formation of the invaginations was followed and characterized using light microscopy and antibodies directed against the S2amide echinoderm neuropeptide as a morphological tag. V2 consistently formed later than V1, and appeared capable of contributing to the formation of a second rudiment when it received the appropriate mesodermal influence from contact with a hydrocoel. Otherwise, in both species, V2 formed a skeletal element that has not been previously described in this location. Because of the location, shape, and timing of its formation, V2, like V1, appears to have the innate ability of the larval ectoderm to contribute to juvenile rudiment formation.     

A comparison of fertilization envelope development in three species of Strongylocentrotus (S. franciscanus, S. droebachiensis, and S. purpuratus)

The ultrastructure of fertilization envelope (FE) development and the polypeptide spectra of Strongylocentrotus franciscanus and S. droebachiensis envelopes were compared to S. purpuratus. In S. franciscanus, the FE reached its maximum thickness of 67 nm by 3 minutes postinsemination (PI), and final structuralization was observed by 40 minutes PI. The fully formed FE did not have microvillar impressions (casts) and was symmetrical, with outer double laminar elements surrounding an amorphous central region. Isolated S. franciscanus FEs were soluble in reducing and denaturing solvents and the same set of 33 polypeptides ranging from 18.5 to 260 kD was detected in FEs isolated from 10 to 180 minutes PI. The S. droebachiensis FE retained microvillar casts, assumed its definitive form by 3 minutes PI, and was 70 nm thick between microvillar impressions. Isolated S. droebachiensis FEs were partially soluble in reducing and denaturing solvents, and the polypeptide spectra of FEs isolated between 10 and 60 minutes PI were identical and showed 14 polypeptides from 18.5 to 265 kD. Antisera against extracted FEs and the FE extract from S. purpuratus were immunologically cross-reactive (using an enzyme-linked immunosorbent assay) with S. franciscanus and S. droebachiensis FE preparations; immunoblots identified 13 and 5 cross-reactive polypeptides, respectively. Most of the cross-reactive polypeptides were of slightly different molecular weight. Based on comparative ultrastructural, solubility, and electrophoretic data, we suggest that S. droebachiensis FE development is most like that observed in S. purpuratus.     

Conserved pattern of embryonic actin gene expression in several sea urchins and a sand dollar

An examination of the size and relative abundance of actin-coding RNA in embryos of four sea urchins (Strongylocentrotus purpuratus, Strongylocentrotus droebachiensis, Arbacia punctulata, Lytechinus variegatus) and one sand dollar (Echinarachnius parma) reveals a generally conserved program of expression. In each species the relative abundance of these sequences is low in early embryos and begins to rise during late cleavage or blastula stages. In the four sea urchins, actin-coding RNAs increase between approximately 9- and 35-fold by pluteus or an earlier stage, and in the sand dollar about 5.5-fold by blastula. A major actin-coding RNA class of 2.0-2.2 kilobases (kb) is found in each species. A smaller actin-coding RNA class, which accumulates during embryogenesis, is also present in S. purpuratus (1.8 kb), S. droebachiensis (1.9 kb), and A. punctulata (1.6 kb), but apparently absent in L. variegatus and E. parma. In S. droebachiensis, actin-coding RNA is relatively abundant in unfertilized eggs and drops sharply by the 16-cell stage. This is in contrast to the other sea urchins where the actin message content is relatively low in eggs and does not change substantially in the embryos throughout early cleavage. The observations in this study suggest that the pattern of embryonic expression of at least some members of this gene family is ancient and conserved.     

Larval budding, metamorphosis, and the evolution of life-history patterns in echinoderms

Abstract. The oral surface and mouth of juvenile asteroids and echinoids with indirect development forms on the lower left side of the larval body, thus establishing a new axis of body symmetry. In contrast, the juvenile mouth of ophiuroids and holothuroids develops from the larval one, and the larval and adult body axes roughly coincide. Explaining how two such disparate modes of development arose in evolution has been a perennial problem for echinoderm biologists, but recent observations on larval budding in asteroids may provide an answer. The juvenile mouth of asteroids forms near the base of the left posterolateral lobe. The posterolateral lobes are also the principal site of bud formation in asteroid larvae that propagate asexually, and buds form mouths. By accelerating the development of oral and ectodermal structures belonging to the bud, and combining these with internal organs derived from the parent larva, a composite individual could be constructed with the same orientation and positioning as the juvenile rudiment in asteroids. Whether this also explains the position of the juvenile rudiment in echinoids is a more complex question, depending in part on whether asexual propagation is derived, and restricted to asteroids and ophiuroids, or is more primitive and hence widespread among stem echinoderms.     

Evolutionary dissociation between cleavage, cell lineage and embryonic axes in sea urchin embryos.

Using vital dye staining and the microinjection of fluorescent cell lineage-autonomous tracers, the relationship between the first cleavage plane and the prospective larval dorsoventral axis was examined in several sea urchin species, including: Strongylocentrotus purpuratus, S. droebachiensis, Lytechinus pictus, Clypeaster rosaceus, Heliocidaris tuberculata and H. erythrogramma. The results indicate that there is no single relationship between the early cleavage pattern and the dorsoventral axis for all sea urchins; however, specific relationships exist for individual species. In S. purpuratus the first cleavage plane occurs at an angle 45 degrees clockwise with respect to the prospective dorsoventral axis in most cases, as viewed from the animal pole. On the other hand, in S. droebachiensis, L. pictus and H. tuberculata, the first cleavage plane generally corresponds with the plane of bilateral symmetry. There does not appear to be a predominant relationship between the first cleavage plane and the dorsoventral axis in C. rosaceus. In the direct-developing sea urchin H. erythrogramma the first cleavage plane bisects the dorsoventral axis through the frontal plane. Clearly, evolutionary differences have arisen in the relationship between cleavage pattern and developmental axes. Therefore, the mechanism of cell determination is not necessarily tied to any particular pattern of cell cleavage, but to an underlying framework of axial systems resident within sea urchin eggs and embryos.     

Molecular phylogenies and divergence times of sea urchin species of Strongylocentrotidae,Echinoida

Sea urchins of the family Strongylocentrotidae have been important model systems in many fields of basic biology, yet knowledge of their evolutionary identities such as the phylogenetic relationships and divergence times remains limited. Here, I inferred molecular phylogenies of seven Strongylocentrotid species (Strongylocentrotus franciscanus, S. nudus, S. purpuratus, S. intermedius, S. droebachiensis, S. pallidus, and Hemicentrotus pulcherrimus) from the analyses of mitochondrial DNA sequences of 12SrDNA (349 nt), 12SrDNA-tRNA(gln) region (862 nt), and a combined sequence of cytochrome oxidase subunit I (COI, 1080 nt) and NADH dehydrogenase subunit I (NDI, 742 nt). The rate of sequence evolution and divergence times for each species were then estimated from the trees with reference to the time of separation between Strongylocentrotidae and Parechinidae, 35 to 50 MYA. The three trees agree well with each other, and the phylogeny is summarized by ((S. franciscanus, S. nudus), (H. pulcherrimus (S. purpuratus, S. intermedius (S. droebachiensis, S. pallidus)))). It is notable that the genus Strongylocentrotus consists of two distinct clades and that H. pulcherrimus branches off within Strongylocentrotus, implying assignment of a separate, monospecific genus to this species inappropriate. The rate of sequence evolution is calibrated to be 0.24%-0.34%/Myr in 12SrDNA, 0.25%-0.36%/Myr in 12SrDNA-tRNA(gln), and 0.65%-0.93%/Myr in COI-NDI combined sequences. S. purpuratus, in particular, shows the significantly higher rate of evolution in the 12SrDNA and 12SrDNA-tRNA(gln) regions compared to other species, suggesting careful use of its sequences in comparative studies. The two clades of Strongylocentrotidae seem to have split 13-19 MYA, and H. pulcherrimus branched off 7.2-14 MYA. In the former clade, S. franciscanus and S. nudus separated 5.7-8.1 MYA. In the latter clade, S. purpuratus, S. intermedius, and the clade of S. droebachiensis and S. pallidus diverged approximately 4.6-12 MYA, and the last two closest species separated 2.1-3.1 MYA.     

How do changes in parental investment influence development in echinoid echinoderms?

SUMMARY Understanding the relationship between egg size, development time, and juvenile size is critical to explaining patterns of life-history evolution in marine invertebrates. Currently there is conflicting information about the effects of changes in egg size on the life histories of echinoid echinoderms. We sought to resolve this conflict by manipulating egg size and food level during the development of two planktotrophic echinoid echinoderms: the green sea urchin, Strongylocentrotus droebachiensis and the sand dollar, Echinarachnius parma . Based on comparative datasets, we predicted that decreasing food availability and egg size would increase development time and reduce juvenile size. To test our prediction, blastomere separations were performed in both species at the two-cell stage to reduce egg volume by 50%, producing whole- and half-size larvae that were reared to metamorphosis under high or low food levels. Upon settlement, age at metamorphosis, juvenile size, spine number, and spine length were measured. As predicted, reducing egg size and food availability significantly increased age at metamorphosis and reduced juvenile quality. Along with previous egg size manipulations in other echinoids, this study suggests that the relationship between egg size, development time, and juvenile size is strongly dependent upon the initial size of the egg.     

Can salinity-induced mortality explain larval vertical distribution with respect to a halocline?

For the larvae of two echinoderm species that coexist in Atlantic Canada (bipinnaria of the sea star Asterias rubens and 4- and 6-arm echinoplutei of the sea urchin Strongylocentrotus droebachiensis), we examined the effect of short- and long-term exposure to salinity (ranging from 18 to 35) on the probability of larval survival in laboratory experiments. We also related larval vertical distributions in response to sharp haloclines generated in the laboratory to survival probability in the salinity of different layers in the water column. For both species and developmental stages, survival probability decreased with decreasing salinity, and a salinity range of 24-27 emerged as the critical threshold for larval tolerance. The relationship between the proportion of larvae that crossed a halocline into the top water layer and the survival probability of larvae in the salinity of that layer was significant for both species. Interestingly, the shape of this response was species-specific but not stage-specific for S. droebachiensis. Our findings suggest that larval avoidance of low-salinity water layers may be an adaptive behavior that increases survival and indirectly influences larval distribution.     

Evolutionary changes in sites and timing of actin gene expression in embryos of the direct- and indirect-developing sea urchins, Heliocidaris erythrogramma and H. tuberculata

 We describe an evolutionary comparison of expression of the actin gene families of two congeneric sea urchins. Heliocidaris tuberculata develops indirectly via a planktonic feeding pluteus that forms a juvenile rudiment after a long period of larval development. H. erythrogramma is a direct developer that initiates formation of a juvenile rudiment immediately following gastrulation. The developmental expression of each actin isoform of both species was determined by in situ hybridization. The observed expression patterns are compared with known expression patterns in a related indirect-developing sea urchin, Strongylocentrotus purpuratus. Comparisons reveal unexpected patterns of conserved and divergent expression. Cytoplasmic actin, CyIII, is expressed in the aboral ectoderm cells of the indirect developers, but is an unexpressed pseudogene in H. erythrogramma, which lacks aboral ectoderm. This change is correlated with developmental mode. Two CyII actins are expressed in S. purpuratus, and one in H. erythrogramma, but no CyII is expressed in H. tuberculata despite its great developmental similarity to S. purpuratus. CyI expression differs slightly between Heliocidaris and Strongylocentrotus with more ectodermal expression in Heliocidaris. Evolutionary changes in actin gene expression reflect both evolution of developmental mode as well as a surprising flexibility in gene expression within a developmental mode. Received: 27 July 1997 / Accepted: 30 December 1997     

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 .     

Localization and developmental fate of ovoperoxidase and proteoliaisin, two proteins involved in fertilization envelope assembly

Fertilization of the sea urchin egg leads to the assembly of an extracellular matrix, the fertilization envelope. Ovoperoxidase, the enzyme implicated in hardening the fertilization envelope, is inserted into the assembling structure via a Ca2+-dependent interaction with the protein proteoliasin (P. Weidman and B. M. Shapiro, 1987, J. Cell Biol. 105, 561-567). In the present report, polyclonal antisera were raised to ovoperoxidase and proteoliasin (purified from eggs of Strongylocentrotus purpuratus) and characterized by Western blot analysis and an enzyme-linked immunoabsorbent assay (ELISA). By indirect immunofluorescence microscopy all cortical granules of unfertilized eggs, as well as the fertilization envelope, contained both proteoliasin and ovoperoxidase. At the ultrastructural level both proteins are localized to the electron-dense spiral lamellae of the cortical granules. Western blot analysis revealed that ovoperoxidase and proteoliasin persist in early embryos until hatching, but are absent from later developmental stages. Homogenates of eggs of several other echinoderm species (Strongylocentrotus droebachiensis, Strongylocentrotus franciscanus, Pisaster ochraceus, Dendraster excentricus, and Lytechinus pictus) also contain proteins antigenically similar to ovoperoxidase and proteoliaisin, indicating that many echinoderms utilize a similar strategy for assembly of the fertilization envelope. The results underline the need for postsecretory controls in the extracellular matrix modifications that accompany the cortical reaction.     

Manipulation of Developing Juvenile Structures in Purple Sea Urchins (Strongylocentrotus purpuratus) by Morpholino Injection into Late Stage Larvae

Sea urchins have been used as experimental organisms for developmental biology for over a century. Yet, as is the case for many other marine invertebrates, understanding the development of the juveniles and adults has lagged far behind that of their embryos and larvae. The reasons for this are, in large part, due to the difficulty of experimentally manipulating juvenile development. Here we develop and validate a technique for injecting compounds into juvenile rudiments of the purple sea urchin, Strongylocentrotus purpuratus. We first document the distribution of rhodaminated dextran injected into different compartments of the juvenile rudiment of sea urchin larvae. Then, to test the potential of this technique to manipulate development, we injected Vivo-Morpholinos (vMOs) designed to knock down p58b and p16, two proteins involved in the elongation of S. purpuratus larval skeleton. Rudiments injected with these vMOs showed a delay in the growth of some juvenile skeletal elements relative to controls. These data provide the first evidence that vMOs, which are designed to cross cell membranes, can be used to transiently manipulate gene function in later developmental stages in sea urchins. We therefore propose that injection of vMOs into juvenile rudiments, as shown here, is a viable approach to testing hypotheses about gene function during development, including metamorphosis.     

HETEROCHRONIC DEVELOPMENTAL PLASTICITY IN LARVAL SEA URCHINS AND ITS IMPLICATIONS FOR EVOLUTION OF NONFEEDING LARVAE

Preexisting developmental plasticity in feeding larvae may contribute to the evolutionary transition from development with a feeding larva to nonfeeding larval development. Differences in timing of development of larval and juvenile structures (heterochronic shifts) and differences in the size of the larval body (shifts in allocation) were produced in sea urchin larvae exposed to different amounts of food in the laboratory and in the field. The changes in larval form in response to food appear to be adaptive, with increased allocation of growth to the larval apparatus for catching food when food is scarce and earlier allocation to juvenile structures when food is abundant. This phenotypic plasticity among full siblings is similar in direction to the heterochronic evolutionary changes in species that have greater nutrient reserves within the ova and do not depend on particulate planktonic food. This similarity suggests that developmental plasticity that is adaptive for feeding larvae also contributes to correlated and adaptive evolutionary changes in the transition to nonfeeding larval development. If endogenous food supplies have the same effect on morphogenesis as exogenous food supplies, then changes in genes that act during oogenesis to affect nutrient stores may be sufficient to produce correlated adaptive changes in larval development.     

Heterochronic developmental shift caused by thyroid hormone in larval sand dollars and its implications for phenotypic plasticity and the evolution of nonfeeding development

Recent work on a diverse array of echinoderm species has demonstrated, as is true in amphibians, that thyroid hormone (TH) accelerates development to metamorphosis. Interestingly, the feeding larvae of several species of sea urchins seem to obtain TH through their diet of planktonic algae (exogenous source), whereas nonfeeding larvae of the sand dollar Peronella japonica produce TH themselves (endogenous source). Here we examine the effects of TH (thyroxine) and a TH synthesis inhibitor (thiourea) on the development of Dendraster excentricus, a sand dollar with a feeding larva. We report reduced larval skeleton lengths and more rapid development of the juvenile rudiment in the exogenous TH treatments when compared to controls. Also, larvae treated with exogenous TH reached metamorphic competence faster at a significantly reduced juvenile size, representing the greatest reduction in juvenile size ever reported for an echinoid species with feeding larvae. These effects of TH on D. excentricus larval development are strikingly similar to the phenotypically plastic response of D. excentricus larvae reared under high food conditions. We hypothesize that exogenous (algae-derived) TH is the plasticity cue in echinoid larvae, and that the larvae use ingested TH levels as an indicator for larval nutrition, ultimately signaling the attainment of metamorphic competence. Furthermore, our experiments with the TH synthesis inhibitor thiourea indicate that D. excentricus larvae can produce some TH endogenously. Endogenous TH production might, therefore, be a shared feature among sand dollars, facilitating the evolution of nonfeeding larval development in that group. Mounting evidence on the effects of thyroid hormones in echinoderm development suggests life-history models need to incorporate metamorphic hormone effects and the evolution of metamorphic hormone production.