Genetic, spatial, and temporal components of precise spawning synchrony in reef building corals of the Montastraea annularis species complex

When organisms release gametes into the sea, synchrony must be precise to increase fertilization and decrease hybridization. We tagged and genotyped over 400 spawning corals from the three species in the Montastraea annularis species complex. We report on the influence of species, individuals, and genotypes on timing of spawning from 2002 through 2009. During their annual spawning event M. franksi spawns on average 2 h after sunset, whereas M. annularis and M. faveolata spawn 3.5 h after sunset. Only M. franksi and M. annularis have compatible gametes. Individual colonies of the same genotype spawn at approximately the same time after sunset within and across years (within minutes), but different genotypes have significantly different spawning times. Neighboring colonies, regardless of genotype, spawn more synchronously than individuals spaced further apart. At a given distance, clone-mates spawn more synchronously than nonclone-mates. A transplant experiment indicates a genetic and environmental influence on spawn time. There is strong, but not absolute, concordance between spawn time, morphology, and genetics. Tight precision in spawning is achieved via a combination of external cues, genetic precision, and perhaps conspecific signaling. These mechanisms are likely to influence reproductive success and reproductive isolation in a density-dependent manner.     

Geographic differences in species boundaries among members of the Montastraea annularis complex based on molecular and morphological markers

The three members of the Montastraea annularis complex (M. annularis, M. franksi, and M. faveolata) are dominant reef builders in the western Atlantic whose species status has been controversial for over a decade. Although differences in colony morphology and reproductive characteristics exist, interspecific fertilizations are possible in the laboratory and genetic differentiation is slight. Here we compare the three taxa genetically and morphologically in Panama and the Bahamas, widely separated locations spanning most of their geographic ranges. In Panama, analyses of three AFLP loci, a noncoding region of the mitochondrial genome, and ITS sequences reveal that M. faveolata is strongly differentiated genetically. Discriminant function analysis also indicates no overlap with the other two species in the fine structure of the corallites that comprise the colony. Genetic analyses of larvae from interspecific crosses between M. faveolata and the other two taxa confirmed the hybrid status of the larvae, but no examples of the most probable F1 genotype were observed in the field. Although M. annularis and M. franksi were more similar, they also exhibited strong frequency differences at two AFLP loci and in the mitochondrial noncoding region, as well as distinct corallite structure. In the Bahamas, in contrast, the three taxa exhibited overlapping morphologies. Montastraeafranksi and M. annularis were indistinguishable genetically, and M. faveolata was distinct at fewer genetic loci. Once again, however, the most probable F1 genotype involving M. faveolata was not observed. Geographic differences between Panama and the Bahamas explain why past studies have come to different conclusions concerning the status of the three species. In general, the genetic and morphological data suggest a north to south hybridization gradient, with evidence for introgression strongest in the north. However, reproductive data show no such trend, with intrinsic barriers to gene flow comparable or stronger in the north.     

Molecular determination of species boundaries in corals: genetic analysis of the Montastraea annularis complex using amplified fragment length polymorphisms and a microsatellite marker

Analyses of DNA have not been widely used to distinguish coral sibling species. The three members of the Montastraea annularis complex represent an important test case: they are widely studied and dominate Caribbean reefs, yet their taxonomic status remains unclear. Analysis of amplified fragment length polymorphisms (AFLPs) and a microsatellite locus, using DNA from sperm, showed that Montastraea faveolata is genetically distinct. One AFLP primer yielded a diagnostic product (880 bp in M. faveolata 920 bp in M. franksi and M. annularis) whose homology was established by DNA sequencing. A second primer revealed a 630 bp band that was fixed in M. faveolata, and rare in M. franksi and M. annularis; in this case homologies were confirmed by Southern hybridizations. A tetranucleotide microsatellite locus with several alleles exhibited strong frequency differences between M. faveolata and the other two taxa. We did not detect comparable differences between M. annularis and M. franksi with either AFLPs (12 primers screened) or the microsatellite locus. Comparisons of AFLP patterns obtained from DNA from sperm, somatic tissues, and zooxanthellae suggest that the technique routinely amplifies coral (animal) DNA. Thus analyses based on somatic tissues may be feasible, particularly after diagnostic differences have been established using sperm DNA.     

Asymmetric conspecific sperm precedence in relation to spawning times in the Montastraea annularis species complex (Cnidaria: Scleractinia)

In broadcast spawners, prezygotic reproductive isolation depends on differences in the spatial and temporal patterns of gamete release and gametic incompatibility. Typically, gametic incompatibility is measured in no‐choice crosses, but conspecific sperm precedence (CSP) can prevent hybridization in gametes that are compatible in the absence of sperm competition. Broadcast spawning corals in the Montastraea annularis species complex spawn annually on the same few evenings. Montastraea franksi spawns an average of 110 min before M. annularis, with a minimum gap of approximately 40 min. Gametes are compatible in no‐choice heterospecific assays, but it is unknown whether eggs exhibit choice when in competition. Hybridization depends on either M. franksi eggs remaining unfertilized and in proximity to M. annularis when the latter species spawns or M. franksi sperm remaining in sufficient viable concentrations when M. annularis spawns. We found that the eggs of the early spawning M. franksi demonstrate strong CSP, whereas CSP appears to be lacking for M. annularis eggs. This study provides evidence of diverging gamete affinities between these recently separated species and suggests for the first time that selection may favour CSP in earlier spawning species when conspecific sperm is diluted and aged and is otherwise at a numeric and viability disadvantage with heterospecific sperm.     

Weak prezygotic isolating mechanisms in threatened Caribbean Acropora corals

The Caribbean corals, Acropora palmata and A. cervicornis, recently have undergone drastic declines primarily as a result of disease. Previous molecular studies have demonstrated that these species form a hybrid (A. prolifera) that varies in abundance throughout the range of the parental distribution. There is variable unidirectional introgression across loci and sites of A. palmata genes flowing into A. cervicornis. Here we examine the efficacy of prezygotic reproductive isolating mechanisms within these corals including spawning times and choice and no-choice fertilization crosses. We show that these species have subtly different mean but overlapping spawning times, suggesting that temporal isolation is likely not an effective barrier to hybridization. We found species-specific differences in gametic incompatibilities. Acropora palmata eggs were relatively resistant to hybridization, especially when conspecific sperm are available to outcompete heterospecific sperm. Acropora cervicornis eggs demonstrated no evidence for gametic incompatibility and no evidence of reduced viability after aging four hours. This asymmetry in compatibility matches previous genetic data on unidirectional introgression.     

Barriers to cross-fertilization between populations of a widely dispersed polychaete species are unlikely to have arisen through gametic compatibility arms-races

Although there are theoretical reasons to suspect that gametic incompatibility may develop readily among populations of broadcast spawning marine invertebrates, there have been very few studies documenting geographic patterns of interpopulation incompatibility for any species. To address this we determined how successfully individuals of the intertidal serpulid polychaete, Galeolaria caespitosa, can cross-fertilize within and among populations from across temperate Australia. Fertilization assays revealed asymmetrical differences between very distantly located populations from different coasts, with near-complete incompatibility between eggs from Sydney with sperm from Adelaide, but the reverse cross (Adelaide eggs, Sydney sperm) was reasonably compatible. Although that pattern was congruent with a clear difference in Cytochrome B sequences between worms on the south and east coasts of Australia, we also detected some indication of interpopulation incompatibility within the genetic grouping on east coast, between two populations separated by only 220 km. We then assessed whether commonly proposed gametic compatibility arms-races could account for these patterns. Our results suggest reduced gametic compatibility may reduce a female's maximum fertilization potential, resulting in a cost to this potential mechanism for reducing polyspermy. Consequently, the apparently rapid development of reproductive barriers here seems unlikely to have been driven by arms-races involving sexual conflict over fertilization rate.     

Sex-specific spawning behavior and its consequences in an external fertilizer

Identifying the target of sexual selection in externally fertilizing taxa has been problematic because species in these taxa often lack sexual dimorphism. However, these species often show sex differences in spawning behavior; males spawn before females. I investigated the consequences of spawning order and time intervals between male and female spawning in two field experiments. The first involved releasing one female sea urchin's eggs and one or two males' sperm in discrete puffs from syringes; the second involved inducing males to spawn at different intervals in situ within a population of spawning females. In both, fertilization success was measured as the fraction of eggs fertilized and the paternity share of each male. The results indicate that spawning after females imposes a cost on males but only during sperm competition. Further, the optimal interval between the initiations of male and female spawning depends on degree of sperm competition, distance between males and females, and water velocity. The results show that sex differences in spawning timing of marine invertebrates can be explained on the basis of the differential costs and benefits of spawning out of synchrony with the other sex and that the result of sexual selection on external fertilizers may be behavioral rather than morphological differentiation of the sexes.     

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.     

CRYPTIC CHOICE OF CONSPECIFIC SPERM CONTROLLED BY THE IMPACT OF OVARIAN FLUID ON SPERM SWIMMING BEHAVIOR

Despite evidence that variation in male–female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species’ ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species’ identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein‐rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior.     

High fertilization success in a surface-brooding Caribbean gorgonian

Colonies of the Caribbean gorgonian Pseudopterogorgia elisabethae release eggs that are retained on the colony surface where they are fertilized and then develop. In December 2001, spawning on San Salvador Island, Bahamas, occurred over 6 d, with spawning by any one colony limited to 1-3 d. With the exception of the first and last days of the spawning period, fertilization success was high, often greater than 90%. Eggs collected in December 2001 had an overall fertilization success of more than 66%. At one site, the increase in fertilization after the first day of spawning correlated with male spawning, but male gonad index was a poor predictor of fertilization success. The number of male colonies close to a female was not correlated with fertilization success. Surface brooding is an efficient mechanism for "harvesting" sperm released upstream of female colonies. By maintaining their eggs at a single location, surface-brooding species can extend the period over which eggs are likely to encounter sperm. As a result, fertilization success is summed across the temporal variance in sperm availability, and the need for very high densities of sperm, with its concomitant risk of polyspermy, may be reduced.     

Efficient utilization of very dilute aquatic sperm: sperm competition may be more likely than sperm limitation when eggs are retained

Fertilization success may be severely limited in marine invertebrates that spawn both male and female gametes. In a diverse group of aquatic organisms only sperm are released, with sperm-egg fusion occurring at the mother. Here, we report fertilization kinetics data for two such 'brooding' or 'spermcast' species--representing each major clade of the animal kingdom. High levels of fertilization were achieved at sperm concentrations of two or three orders of magnitude lower than is common with broadcast spawning species. At a concentration of 100 sperm ml(-1), fertilization rates of a bryozoan and colonial ascidian were near maximum, whereas most broadcast spawners would have displayed near complete reproductive failure. A further experiment looked at the rate of uptake of sperm under natural conditions. Results suggested that sperm released at ca. 0.9 m from an acting female could be collected at a rate of 3-12 times greater than the minimum required simply to avoid sperm limitation. Thus, evolutionary pressures on gametic and other reproductive characteristics of many species that release sperm but retain eggs may be quite different from those of broadcast spawners and may confer on the former an enhanced scope for sperm competition and female choice.     

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.     

On the causes of density banding in skeletons of corals of the genus Montastraea

To obtain a reliable climate reconstruction from coral skeletons it is first necessary to understand the way these grow and incorporate information. Thickness of skeletal elements (exothecal and endothecal dissepiments, costae, septa, and theca-wall) as well as the spacing between exothecal and endothecal dissepiments of the four extant Atlantic species of Montastraea (M. annularis, M. faveolata, M. franksi and M. cavernosa) were measured through high and low density bands. Our results show that growth periodicity, controlled by the effect of temperature, is expressed in changes in thickness of costae and exothecal dissepiments in the four studied Montastraea species, with no changes in endothecal elements and theca-wall thickness which, in turn, has implications for research on inclusive records using these species. Spacing between both exothecal and endothecal dissepiments resulted without changes along the high and low density bands, and we found evidence that there is a rhythmical formation of these structures linked somehow to lunar cycles.     

Bacterial communities associated with the mucopolysaccharide layers of three coral species affected and unaffected with dark spots disease

Dark spots disease (DSD) is a relatively new coral disease that has become one of the most prevalent afflictions in the Caribbean Sea. To partially characterize bacterial communities associated with DSD, carbon utilization patterns of bacterial strains isolated from the surface mucopolysaccharide layers of healthy and DSD-affected Montastraea annularis, Montastraea faveolata, and Siderastrea siderea were compared with each other and with bacterial strains isolated from the water column by using cluster analysis. Differences between healthy and diseased corals were found and were greatest for M. annularis than for the other species. A metabolic group of bacteria similar to Vibrio carchariae was found to be unique to diseased samples. Inoculation experiments on healthy corals did not result in the development of disease signs. However, our results support the hypothesis that stress (in this case disease) alters the normal microbiota in the coral surface mucopolysaccaride layers. Studies are continuing to determine the pathogenic agent(s) responsible for DSD.     

Larval development and survivorship in the corals Favia favus and Platygyra lamellina

Two Red Sea faviid species, Favia favus and Platygyra lamellina spawn eggs and sperm once a year, during the summer. External fertilization occurs 0.5 h after spawning, and mobile gastrulae appear 20 h later. Four stages in the early ontogenesis of these corals are described. The slow development (2–3 months) to the polyp stage in broadcasting species is attributed to the lack of zooxanthellae in their planulae and their appearance in the primary polyp only at a later stage. Survivorship of one-month-old primary polyps is ca 0.21% and 0.25% in F. favus and P. lamellina respectively, from the populations of 2–9-day-old planulae. Despite these low rates of survival, both species form dense populations in the Gulf of Eilat.     

Quantitative analysis of sperm plane circular movement in the blue mussels Mytilus edulis, M. trossulus and their hybrids

Fertilization success of free spawning organisms such as Mytilus species depends on gamete interactions. Therefore, gamete traits such as sperm movement are important for determining fertilization success in free spawning organisms. Since little is known about sperm movement pattern in Mytilus species, the purpose of this study was to investigate sperm movement pattern of blue mussel M. edulis, M. trossulus and their hybrids using computer-assisted sperm movement video analysis. Sperm of all genotypes were found to conduct circular movement in a two-dimensional plane. Furthermore, new sperm movement parameters, real time radius (R), angle change rate (θ) and the center of circular track (O(t)) were developed to verify and quantitatively describe the plane circular movement pattern using software (Image-J) that may be widely applied to sperm movement study in other organisms. Angle change rate was positively correlated to fertilization success. However, no correlation between fertilization and real time radius was detected. Although no interspecific differences were found in the radius, the F1 (first generation) hybrid sperm had a lower angle change rate than M. edulis and M. trossulus. Published studies have shown that sperm circular movement is more prevalent in aquatic broadcast spawning species than in species with mating behavior or internal fertilization. Therefore, a two-dimensional circular movement pattern in sperm may represent a trait that increases fertilization success for broadcast spawning species by either increasing gamete interaction rate at a small scale and/or avoiding swimming further away from the eggs before sperm detects the chemoattractant gradient.     

White plague disease outbreak in a coral reef at Los Roques National Park, Venezuela

Coral diseases have been reported as a major problem affecting Caribbean coral reefs. During August 2000, a coral mortality event of White Plague Disease-II (WPD-II) was observed at Madrizqui Reef in Los Roques National Park, Venezuela. This disease was identified as the major cause of coral mortality, affecting 24% of all colonies surveyed (n = 1 439). Other diseases such as Black Band Disease (BBD), Yellow Blotch Disease (YBD), Dark Spots Disease (DSD) and White Band Disease (WBD) were also recorded, but showed a lower incidence (0.14-0.97%). Two depth intervals, D1 (5.5-6.5 m) and D2 (9-9.5 m) were surveyed with two sets of three band transects 50 x 2 m long, placed parallel to the long axis of the reef. All healthy and injured corals, along each band transect, were counted and identified to species level. Additionally, all diseases and recent mortality that were still identifiable on each colony also were recorded. The incidence of colonies affected by WPD-II ranged from 12.8 to 33% among transects, where thirteen species of scleractinian corals showed several degrees of mortality. The species most affected were Montastraea annularis (39.13%), M. faveolata (26.67%), M. franksi (9.86%), Stephanocoenia intersepta (7.25%), Colpophyllia natans (6.96%), Diploria labyrinthiformis (2.99%), Mycetophyllia aliciae (2.03%), M. cavernosa (1.74%), and D. strigosa (1.45%). WPD-II was more common in the deeper strata (9-9.5 m), where 63% of the surveyed colonies were affected, although the disease was present along the entire reef. Presently, it is imperative to determine how fast the disease is spreading across the reef, how the disease spreads across the affected colonies and what the long-term effects on the reef will be.     

Deepwater broadcast spawning by Montastraea cavernosa, Montastraea franksi, and Diploria strigosa at the Flower Garden Banks, Gulf of Mexico

Broadcast spawning by corals is a tightly synchronized process characterized by co-ordinated gamete release within 30–60 min time windows once per year. In shallow water corals, annual water temperature cycles set the month, lunar periodicity the day, and sunset time the hour of spawning. This tight temporal regulation is critical for achieving high fertilization rates in a pelagic environment. Given the differences in light and temperature that occur with depth and the importance of these parameters in regulating spawn timing, it has been unclear whether deeper coral can respond to the same environmental cues that regulate spawning behaviour in shallower coral. In this report, a remotely operated vehicle was used to monitor coral spawning activity at the Flower Garden Banks at depths from 33 to 45 m. Three species Montastraea cavernosa, Montastraea franksi, and Diploria strigosa were documented spawning within this depth range. All recorded spawning events were within the same temporal windows as shallower conspecifics. These data indicate that deep corals at this location either sense the same environmental parameters, despite local attenuation, or communicate with shallower colonies that can sense such spawning cues.     

Ocean acidification alters sperm responses to egg-derived chemicals in a broadcast spawning mussel

The continued emissions of anthropogenic carbon dioxide are causing progressive ocean acidification (OA). While deleterious effects of OA on biological systems are well documented in the growth of calcifying organisms, lesser studied impacts of OA include potential effects on gamete interactions that determine fertilization, which are likely to influence the many marine species that spawn gametes externally. Here, we explore the effects of OA on the signalling mechanisms that enable sperm to track egg-derived chemicals (sperm chemotaxis). We focus on the mussel Mytilus galloprovincialis, where sperm chemotaxis enables eggs to bias fertilization in favour of genetically compatible males. Using an experimental design based on the North Carolina II factorial breeding design, we test whether the experimental manipulation of seawater pH (comparing ambient conditions to predicted end-of-century scenarios) alters patterns of differential sperm chemotaxis. While we find no evidence that male–female gametic compatibility is impacted by OA, we do find that individual males exhibit consistent variation in how their sperm perform in lowered pH levels. This finding of individual variability in the capacity of ejaculates to respond to chemoattractants under acidified conditions suggests that climate change will exert considerable pressure on male genotypes that can withstand an increasingly hostile fertilization environment.     

Density‐dependent patterns of multivariate selection on sperm motility and morphology in a broadcast spawning mussel

Sperm cells exhibit extraordinary phenotypic variation, both among taxa and within individual species, yet our understanding of the adaptive value of sperm trait variation across multiple contexts is incomplete. For species without the opportunity to choose mating partners, such as sessile broadcast spawning invertebrates, fertilization depends on gamete interactions, which in turn can be strongly influenced by local environmental conditions that alter the concentration of sperm and eggs. However, the way in which such environmental factors impact phenotypic selection on functional gamete traits remains unclear in most systems. Here, we analyze patterns of linear and nonlinear multivariate selection under experimentally altered local sperm densities (densities within the capture zone of eggs) on a range of functionally important sperm traits in the broadcast spawning marine mussel, Mytilus galloprovincialis. Specifically, we assay components of sperm motility and morphology across two fertilization environments that simulate either sperm limitation (when there are too few sperm to fertilize all available eggs), or sperm saturation (when there are many more sperm than required for fertilization, and the risk of polyspermy and embryonic failure is heightened). Our findings reveal that the strength, form, and targets of selection on sperm depend on the prevailing fertilization environment. In particular, our analyses revealed multiple significant axes of nonlinear selection on sperm motility traits under sperm limitation, but only significant negative directional selection on flagellum length under sperm saturation. These findings highlight the importance of local sperm densities in driving the adaptation of sperm phenotypes, particularly those related to sperm motility, in broadcast spawning invertebrates.