小庙洪牡蛎礁巨蛎属牡蛎间生殖隔离研究

IOCAS-IR > 海洋环流与波动重点实验室

	小庙洪牡蛎礁巨蛎属牡蛎间生殖隔离研究
其他题名	Reproductive isolations between oysters of genus Crassostrea on the Xiaomiaohong oyster reef
	许飞
学位类型	博士
	2009-05-17
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	牡蛎分布牡蛎礁巨蛎属熊本牡蛎近江牡蛎杂交生殖隔离 Dna含量 C值 Coi Its1 遗传鉴定
摘要	中国牡蛎种类丰富，但是由于贝壳可塑性强，表型变化大，分类学及系统发生学研究进展缓慢，存在诸多争议。本文在全国部分海区采集牡蛎样品，并通过表型和分子生物学方法进行鉴定，重点调查潮下带牡蛎的分布情况。选取江苏南通小庙洪牡蛎礁作为典型海域，研究亲缘关系接近的巨蛎属牡蛎之间在繁殖上如何维持相互之间的关系。本研究的目的在于摸索牡蛎物种鉴定的实用方法，并阐明熊本牡蛎和近江牡蛎生殖隔离是如何维持的。研究结果对牡蛎等海洋贝类的分类及系统发生学研究具有一定的借鉴作用，为牡蛎礁的生态保护提供生物学参考依据。在辽宁营口、海洋岛，河北秦皇岛，山东西霞口、潍坊、青岛，江苏南通，浙江舟山、奉化，福建莆田，广东汕头等地区采集牡蛎样品，重点采集潮下带的牡蛎，通过地理分布、表型、DNA含量测定和COI种特异性探针鉴定对各地区的牡蛎物种组成进行分析。选取了南通小庙洪牡蛎礁作为研究牡蛎物种间相互关系的最佳地点。该地区牡蛎由主要分布在潮间带的熊本牡蛎和主要分布在潮下带的近江牡蛎组成。对南通小庙洪牡蛎礁上两种巨蛎属牡蛎在空间上的分布进行调查；定期取样，制作性腺切片，观察两种牡蛎的性腺发育情况；同时，定期在海区挂板，采用半人工采苗的方式采集海区牡蛎幼体，通过ITS1基因鉴定采集的稚贝所属物种。通过调查发现二者的分布存在重叠区域；两种牡蛎在2007年6月中旬至9月中旬存在两个明显的繁殖高峰期，在每个繁殖期内两种牡蛎同时发生大量繁殖行为的可能性极高。结果表明，两种牡蛎不存在明显的生态隔离和季节隔离等交配前生殖隔离机制。在室内对两种牡蛎进行2×2完全双列杂交，并通过ITS1基因的克隆测序验证杂种后代。双列杂交受精实验采用多亲本混合交配的方式（即每个物种的卵子或精子均来自多个个体），共进行7次重复。结果表明，熊本牡蛎的卵子能够被近江牡蛎的精子以较低的受精率（24.6%）受精，且形成的后代确实为两个物种杂交产生，而反方向不能受精。幼虫和稚贝生长存活的双列杂交实验共进行3次重复。估测受精率、受精卵的孵化率，第1至29日每两天测量一次D形幼虫的壳高和密度。结果表明杂交幼虫（熊本牡蛎♀与近江牡蛎♂的杂交，SA）的受精率（12.5%）显著低于（P < 0.05）熊本牡蛎（75.4%）和近江牡蛎（84.5%）纯种对照组；而孵化率与对照组无显著差异（P > 0.05）；自受精至29日龄，杂交幼虫壳高的生长速度明显低于纯种对照组（P < 0.05）；但是存活率与熊本牡蛎无显著差异（P > 0.05）。杂交幼虫附着变态期间死亡率高；93日龄，熊本牡蛎有10.08%的匍匐期面盘幼虫存活至稚贝，显著大于（P < 0.05）近江牡蛎（2.18%）和杂交幼虫（0.76%）；杂交稚贝壳高显著小于（P < 0.05）纯种对照组。稚贝在海区挂养3个月。与挂养前相比，杂交稚贝在潮下带的存活率为19.2%，125个稚贝仅存24个，壳高平均增加2.15 mm。熊本牡蛎在潮间带的存活率达到65%，壳高平均增加3.99 mm。近江牡蛎在潮下带的存活率达到85.7%，壳高平均增加5.22 mm，在潮间带的存活率仅10.9%，壳高平均增加3.28 mm。受精6个月后，所有杂交稚贝仅剩3枚，运回实验室暂养一段时间后全部死亡。设置20、25、30、35共4个盐度梯度，估算双列杂交组合在不同盐度下的受精率、孵化率、7日内的存活率和壳高生长，实验共进行3个重复。结果表明，杂交组合在4个盐度下的受精率（平均为11.2%）均显著低于（P < 0.05）熊本牡蛎（75.0%）和近江牡蛎（77.5%）纯种对照组；而孵化率与对照组无显著差异（P > 0.05）；杂交组合在盐度35时的孵化率（85.0%）显著大于20（49.6%）、25（51.3%）和30（55.4%）三个盐度梯度。7日龄杂交幼虫（10.5%）和近江牡蛎（11.4%）的存活率显著小于（P < 0.05）熊本牡蛎（30.3%）；杂交幼虫的壳高（75.7 μm）显著小于（P < 0.05）熊本牡蛎（81.5 μm）和近江牡蛎（85.6 μm）对照组。两因素方差分析表明，杂交组合方式对受精率、孵化率、7日龄存活率和7日龄壳高均有显著影响，盐度对7日龄壳高的影响显著。实验结果表明，江苏小庙洪牡蛎礁上的熊本牡蛎和近江牡蛎之间在分布空间上存在重叠区域，繁殖时间也存在重叠，并未产生明显的生态隔离和季节隔离。二者之间存在不对称性杂交，熊本牡蛎的卵子能够与近江牡蛎的精子以较低的受精率受精，而反方向完全不亲和。杂交幼虫在受精率上存在显著劣势；但是受精卵的孵化率与纯种受精卵无显著差异；杂交幼虫在生长上也存在显著劣势；而存活率与熊本牡蛎无显著差异；杂交幼虫附着变态期间大量死亡，存活下来的稚贝生长缓慢，死亡率高。表明熊本牡蛎和近江牡蛎之间即存在交配前的配子不亲和性隔离，又存在交配后隔离，主要表现为杂种不活。
其他摘要	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 (75.7 μm) was significantly less than Kumamoto oyster (81.5 μm) and Jinjiang oyster (85.6 μm). 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.
页数	140
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1519
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	许飞. 小庙洪牡蛎礁巨蛎属牡蛎间生殖隔离研究[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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