Reproductive Isolations Between Oysters Of Genus Crassostrea On The Xiaomiaohong Oyster Reef

China owns abundant oyster resources. But taxonomy and phylogeny studies developed slowly and existed great controversy, as a result of the plasticity of oyster's shell and the large variety of their phenotypes. This study sampled oysters from several sites along China seas coast to investigate the distribution of various oyster species, especially oysters live at subtidal zones. All the samples were identified using either phenotypes or molecular markers. Basing the investigation result, we chose Xiaomiaohong oyster reef from Nantong, Jiangsu Province as a typical sea area to study the reproductive isolation between two oyster species belonging to the genus Crassostrea. The purpose is to explore applicable methods for identifying oysters, and clarify the reproductive isolation phenomenon between Kumamoto oyster C. sikamea and Jinjiang oyster C. ariakensis. Results of this result are useful for the taxonomy and phylogeny research of marine mollusca, such as oysters, as well as for the protection of the oyster reef.Oysters from several sites along China Seas were sampled. Through identifying the oysters according to their geographical distributions, phenotypes, DNA contents, as well as COI species-specific markers, clarify the oysters'species composition of every sampling site. Referring to the result, we chose Xiaomiaohong oyster reef of Nantong, Jiangsu Province as a typical research field to study interspecific relation of oysters. There are two oyster species including the Kumamoto oyster which mainly inhabit at intertidal zones and Jinjiang oyster which is mainly found at subtidal zones. We investigated the distribution on the reef of the two oyster species belonging to genus Crassostrea, the result shows that they were sympatric species on the reef;Oysters were sampled and gonad slices were made regularly to survey the gonad development; At the same time, adhesive substrates were located at the sea area to collect oyster spats, and the attached spats were identified using ITS1 gene to determine whether the two species spawned at the same time. The result indicated that there were two reproductive period. The two species were most likely to reproduce at the same time. It could be concluded that no ecological and seasonal isolation existed between the two species.Reciprocal diallel cross was conducted to confirm the hybrid possibility of the two oysters. Genetic confirmation was provided using clone sequencing of ITS1 gene attracted from hybrid spats. Reciprocal cross was designed to be a mass mating mode, that is, eggs or sperms from each species were mixed product from several individuals. There were a total of 7 replicates in this experiment. The result showed that eggs from Kumamoto oyster could be fertilized by Jinjiang oyster's sperm at a low fertilization (24.6%). Genetic confirmation showed that the larvae were indeed hybrids. But the gametes were incompatible at the negative direction.We designed another experiment to compare and monitor the growth and survival of larvae and spats both from hybrid and parental combination. There were a total of 3 replicates. The results indicated that the fertilization of hybrid combination (Kumamoto oyster♀and Jinjiang♂oyster, SA, 12.5%) was significantly (P<0.05) lower than the intraspecies cross of Kumamoto oyster (75.4%) and Jinjiang oyster (84.5%). But the hatching success was insignificant (P>0.05) with parent cross. Shell height of hybrids grew significantly (P<0.05) slower than the control groups, in 30 days after insemination (29 days age), but survival was insignificant (P>0.05) with Kumamoto oysters. Hybrids died numerously at the metamorphosis period. At 93 days age, 10.08% Kumamoto oyster pediveligers survived to juvenile period, which is significantly (P<0.05) larger than Jinjiang oyster (2.18%) and hybrids (0.76%).Hybrids shell height was significantly (P<0.05) less than the control groups. Juveniles were cultured at different sea areas for 3 months. Hybrids'survival was only 19.2%, with 24 juveniles in 125 survived at subtidal area. Their average shell height increased 2.15 mm in this period. Survival of Kumamoto oyster at intertidal area was 65%, and their shell height increased 3.99 mm. Survivals of Jinjiang oyster were 85.7% at subtidal area and 10.9% at intertidal area. Shell height of individuals lived at the former environment increased 5.22 mm, compared with 3.28 mm at the latter environment. Only 3 six-month age hybrids survived of all the 3 replicates. They were carried back and cultured at aquarium, but died at last.Four salinity grads was set at 20, 25, 30 and 35, to check the influence of salinity on fertilization, hatching, growth and survival of larvae produced by reciprocal cross. A total of 3 replicates were conducted. The results indicated that fertilization of hybrid combination (11.2%) was significantly (P<0.05) less than parent combination of both Kumamoto oyster (75.0%) and Jinjiang oyster (77.5%). But hatching rate did not show any significant (P>0.05). Hatching of hybrid at the salinity of 35 (85.0%) was higher than that at 20 (49.6%), 25 (51.3%), and 30 (55.4%). The survival of seven-day age larvae of hybrid (10.5%) and Jinjiang oyster (11.4%) was significantly (P<0.05) less than Kumamoto oyster (30.3%). Shell height of hybrids at day 7 (75.7μm) was significantly less than Kumamoto oyster (81.5μm) and Jinjiang oyster (85.6μm). A two-way ANOVA result showed that cross combination significantly influenced fertilization, hatching, seven-day age survival and seven-day age shell height. Effect of salinity on shell height at day 7 was significant.The results indicated that, Kumamoto oyster Crassostrea sikamea and Jinjiang oyster C. ariakensis coexist and reproducing at the same season. No significant ecological and seasonal isolation affect the two species. Kumamoto oyster's egg could be fertilized by Jinjiang oyster's sperm at low rate, but Jinjiang oyster's egg and Kumamoto oyster's sperm was absolutely incompatible. Cross of the two species showed hybrid weakness in both fertilization and growth, while zygotes'hatching success was similar with parental combinations, as well as the larvae survival which exist no significant with Kumamoto oyster larvae. Hybrids died numerously when metamorphosing and setting on the cultch. The survival hybrid spats grow poorly with high mortality. It can be concluded that gene flow between Kumamoto oyster and Jinjiang oyster was blocked either by premating isolation which was achieved mainly by the incompatible of gametes or by postmating isolation which was demonstrated by inviability of hybrids.