First evidence for backcrossing of F1 hybrids in Acropora corals under sperm competition.

Acropora is a species-rich genus of reef-building corals with highly diverse morphologies. Hybridization among intercrossing species potentially influences species diversity within Acropora. However, the mechanisms that allow hybridization/backcrossing remain unknown. Although we tested a limited number of species, we hypothesized that Acropora gametes in the Indo-Pacific may preferentially fertilize conspecific gametes despite their compatibility with heterospecific gametes, leading to infrequent hybridization between potentially intercrossing species. In this study, F1 hybrids of Acropora florida and A. intermedia showed specific fertilization trends. For example, sperm had the ability to backcross with the parental species even in the presence of sperm from the parental species. Also, eggs of the hybrids produced from A. florida eggs and A. intermedia sperm ("FLOint") exhibited self-fertilization. Since a low ratio of hybridization between A. florida and A. intermedia is predicted, the population size of hybrids should be small. Therefore, self-fertilization would facilitate reproduction of the hybrid in nature, while remaining sperm could outcompete parental species sperm to backcross with eggs. Although we succeeded in breeding two colonies of hybrids, it is reasonable to speculate that hybrids show a high tendency to choose the most efficient sexual reproduction tactics.

Maternal inheritance of F1 hybrid morphology and colony shape in the coral genus Acropora.

BACKGROUND: The coral genus Acropora contains more than 150 species with very high morphological diversity. This high diversity may have been caused by repeated hybridization via mass spawning. However, we have little information whether hybrids are formed in these corals. Identifying morphological differences between hybrids and their parental species would provide an opportunity to find wild hybrids in the field and to understand how colony shapes of Acropora have become highly diversified throughout evolutionary history. In the two morphologically distinctive coral species Acropora florida and A. intermedia in the Indo-Pacific, their gametes show high rates of bi-directional intercrossing in vitro, and thus these two species are ideal species to investigate the morphological traits of the hybrids. METHODS: We examined morphological characters of F1 hybrids from A. florida to A. intermedia, which were produced from in vitro crossing experiments. To compare morphological differences, we grew juveniles and mature colonies of reciprocal F1 hybrids (FLOint: A. florida eggs × A. intermedia sperm, and INTflo: A. intermedia eggs × A. florida sperm) and of the parental species (purebreds of A. intermedia and A. florida). We analyzed skeletal morphology such as colony size, branch length, and branching number, and compared them with those of a putative F1 hybrid between A. florida and A. intermedia found in the field. We also confirmed the molecular phylogenetic position of F1 hybrids, parental species, and a putative F1 hybrid using the mitochondrial non-coding region. RESULTS: Our morphological analysis revealed that branching number of the F1 hybrids was intermediate relative to the parental species. Moreover, the FLOint hybrids were morphologically more closely related to the maternal species A. florida, and the INTflo hybrids were to A. intermedia. Molecular data showed that A. florida and A. intermedia were clearly divided into two clades, and that F1 hybrids grouped in the clade based on their maternal parent. A very similar pattern to the INTflo hybrids was obtained for the putative F1 hybrid in nature. DISCUSSION: Our results revealed that F1 hybrids between two Indo-Pacific species A. florida and A. intermedia had intermediate morphology relative to their parent species but reflected the maternal parent more. Similarity to maternal species in hybrids is opposite to the Caribbean Acropora species that had more paternal morphological characters in hybrids. These results further suggest that some genetic factor in eggs is likely to affect determination of colony shape in the Indo-Pacific. At present, we have considered colonies with intermediate morphs between different species to be intra-specific morphological variation, but they may be real F1 hybrids. Indeed, a putative F1 hybrid represented similar morphological and molecular features to the F1 hybrids, and thus it is plausible to be attributed as a "real" F1 hybrid in nature.

The reef-building coral Acropora conditionally hybridize under sperm limitation.

Multi-specific synchronous spawning risks both sperm limitation, which reduces fertilization success, and hybridization with other species. If available sperm of conspecifics are limited, hybridization with heterospecific sperm could be an alternative. Some species of the reef-building coral Acropora produce hybrid offspring in vitro, and therefore hybridization between such species does sometimes occur in nature. Here, we report that the interbreeding species Acropora florida and A. intermedia preferentially bred with conspecifics at optimal gamete concentrations (10(6) cells ml(-1)), but when sperm concentration was low (10(4) cells ml(-1)), A florida eggs displayed an increased incidence of fertilization by sperm of A intermedia However, A intermedia eggs never crossed with heterospecific sperm, regardless of gamete concentrations. It appears that A florida eggs conditionally hybridize with heterospecific sperm; in nature, this would allow A florida to cross with later-spawning species such as A intermedia These results indicate that hybridization between some Acropora species could occur in nature according to the number of available sperm, and the choice of heterospecific sperm for fertilization could be one of the fertilization strategies in the sperm-limited condition.

Possible natural hybridization of two morphologically distinct species of Acropora (Cnidaria, Scleractinia) in the Pacific: fertilization and larval survival rates.

Natural hybridization of corals in the Indo-Pacific has been considered rather rare. However, field studies have observed many corals with intermediate interspecific or unusual morphologies. Given that the existence of F1 hybrids with intermediate interspecific morphologies has been proven in the Caribbean, hybrids may also inhabit the Indo-Pacific and occur more frequently than expected. In this study, we focused on two morphologically different species, Acropora florida and A. intermedia, and performed crossing experiments at Akajima Island, Japan. Results showed that these species could hybridize in both directions via eggs and sperm, but that fertilization rates significantly differed according to which species provided eggs. These results are similar to those reported from the Caribbean. Although all embryos developed normally to the planular larval stage, the developmental processes of some hybrid embryos were delayed by approximately 1 h compared with conspecific embryos, suggesting that fertilization occurred 1 h later in interspecific crosses than in intraspecific crosses. More successful hybridization could occur under conditions with low numbers of conspecific colonies. Additionally, a comparison of survival rates between hybrid and intraspecific larvae revealed that intra- and interspecific larvae produced from eggs of A. florida survived for significantly longer than those produced from eggs of A. intermedia. Considering these data, under specific conditions, hybrids can be expected to be produced and survive in nature in the Pacific. Furthermore, we identified one colony with intermediate morphology between A. florida and A. intermedia in the field. This colony was fertilized only by eggs of A. florida, with high fertilization rates, suggesting that this colony would be a hybrid of these two species and might be backcrossed.

Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria).

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ss-tubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils.

High (210)Po atmospheric deposition flux in the subtropical coastal area of Japan.

Bulk atmospheric deposition fluxes of (210)Po and (210)Pb were measured at three coastal regions of Japan, the Pacific Ocean coastal area of the Japanese mainland (Odawa Bay), the Chinese continental side of Japanese coastal area (Tsuyazaki), and an isolated island near Okinawa (Akajima). Wet and dry fallout collectors were continuously deployed from September 1997 through August 1998 for periods of 3 to 31 days depending on the frequency of precipitation events. Annual (210)Pb deposition fluxes at Odawa Bay (35 degrees N 139 degrees E), Tsuyazaki (33 degrees N 130 degrees E) and Akajima (26 degrees N 127 degrees E) were 73.3+/-8.0, 197+/-35 and 78.5+/-8.0 Bqm(-2)y(-1), respectively. Higher (210)Pb deposition was observed at the Chinese continental side of Japanese coast than at the Pacific Ocean coastal site. The high (210)Pb atmospheric flux at the Chinese continental side coast was thought to be attributable to (222)Rn-rich air-mass transport from the Chinese continent during the winter monsoon. In contrast, the annual (210)Po deposition fluxes at the three study sites were 13.0+/-2.3 (Odawa Bay), 21.9+/-4.4 (Tsuyazaki) and 58.4+/-7.7 (Akajima)Bqm(-2)y(-1), respectively, indicating unusual high (210)Po deposition at Akajima during winter. Anomalous unsupported (210)Po input was observed during summer 1997, suggesting unknown source of (210)Po at this area.

Identification of symbiotically expressed coral mRNAs using a model infection system.

Hermatypic (or reef-building) corals live in obligatory mutualistic symbiosis with the symbiotic dinoflagellates Symbiodinium spp. (generally known as zooxanthellae). In an attempt to establish a model symbiosis system consisting of a coral host and a monoclonal population of zooxanthellae, infectivity of five cultured Symbiodinium cell lines was tested on naturally aposymbiotic juveniles of Acropora tenuis. A clade A3 strain (PL-TS-1) infected the juveniles at high density and promoted growth of the host. To identify host genes involved in the establishment or maintenance of symbiosis, mRNA expression patterns were compared between aposymbiotic and PL-TS-1-infected juvenile polyps using the suppression subtractive hybridization technique. Two mRNAs, the expression levels of which were augmented more than twofold by the presence of the symbionts, were thereby identified. One of the mRNAs, AtSym-02, encodes a novel protein of 322 amino acids which is predicted to be a glycosylated trans-membrane protein.

Conventional taxonomy obscures deep divergence between Pacific and Atlantic corals.

Only 17% of 111 reef-building coral genera and none of the 18 coral families with reef-builders are considered endemic to the Atlantic, whereas the corresponding percentages for the Indo-west Pacific are 76% and 39%. These figures depend on the assumption that genera and families spanning the two provinces belong to the same lineages (that is, they are monophyletic). Here we show that this assumption is incorrect on the basis of analyses of mitochondrial and nuclear genes. Pervasive morphological convergence at the family level has obscured the evolutionary distinctiveness of Atlantic corals. Some Atlantic genera conventionally assigned to different families are more closely related to each other than they are to their respective Pacific 'congeners'. Nine of the 27 genera of reef-building Atlantic corals belong to this previously unrecognized lineage, which probably diverged over 34 million years ago. Although Pacific reefs have larger numbers of more narrowly distributed species, and therefore rank higher in biodiversity hotspot analyses, the deep evolutionary distinctiveness of many Atlantic corals should also be considered when setting conservation priorities.

Isolation of actin-encoding cDNAs from symbiotic corals.

A cDNA (named LGfact) encoding actin was identified in planular larvae of the scleractinian coral Galaxea fascicularis, using the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. RNA from the adult coral that was inhabited by symbiotic dinophytes was subjected to a similar RT-PCR, and a cDNA fragment, named AGfact-p, was found to encode an actin form distinct from LGfact. In an expression study, LGfact transcripts were present at similar levels in asymbiotic larvae and symbiotic adults, indicating that LGfact was expressed by the host. On the other hand, the expression of AGfact-p was detected in adults but not in larvae. Partial cDNA sequences of orthologues of LGfact and AGfact-p were detected in another scleractinian coral, Favites chinensis. A sequence identical to a part of AFcact-p (an AGfact-p orthologue) was amplified from the genomic DNA extracted from asymbiotic larvae of F. chinensis, strongly suggesting that AFcact-p was a coral actin cDNA. Thus, we presume that A Gfact-p encodes an adult-specific form of actin in the host. A partial actin-encoding cDNA sequence (named Syact-p) obtained from Symbiodinium sp. did not exhibit high levels of similarity to the coral actin sequences.