【作者】 顾钱洪；

【导师】 熊邦喜； 罗智；

【作者基本信息】 华中农业大学 ， 水产养殖， 2013， 博士

【摘要】 环棱螺属(Bellamya)隶属于软体动物门(Mollusca),腹足纲(Gastropoda),中腹足目(Mesogastropoda),田螺科(Viviparidae)。是淡水生态系统中大型底栖动物之一,广泛分布于亚洲,非洲和北美湖泊,在水生态系统中具有重要的生态功能。由于湖泊环境的恶化,加之人类对环棱螺资源的过度开发利用,在一定程度上对环棱螺的种质资源造成了破坏,种类不断减少。中国环棱螺属的分类主要依据螺壳形态特征,由于螺壳形态的变异性,容易导致同种异名和分类混淆。本研究从分子水平研究环棱螺遗传多样性和分类阶元,结合形态学研究探讨中国环棱螺的分类系统。通过研究环棱螺不同地理种群的遗传结构,为其种质资源保护提供分子遗传学基础。主要获得的结果如下：1.利用线粒体基因(COI、16S rRNA)和核基因(H3、28SrRNA)序列比较分析了环棱螺属系统进化关系。以Viviparus ater和V. contectus2个种类为外群,以采集中国15种环棱螺和GenBank下载的环棱螺的相关基因为内群,基于贝叶斯法构建系统发育树。系统发育分析结果表明,世界分布的环棱螺属聚为3大支(中国环棱螺种聚为一支,印度种聚为一支,非洲种聚为一支)。三大分支中中国环棱螺种位于进化树的基部,印度种和非洲种亲缘关系较近。非洲环棱螺种主要按湖泊形成3-4个分支。而在中国种的大分支里,既没有形成同种环棱螺聚类,也没有形成同一湖泊种聚类,种间和地理种群间均没有明显分化,种内和种间的遗传距离没有明显差异。2.采用FIASCO法成功构建了铜锈环棱螺基因组微卫星富集文库,获得单一微卫星序列2566条(Genbank登录号：JN555759-JN556037,JX018213-JX020499)。采用Primer5.0软件共设计了391对特异性微卫星引物。选择其中100对引物合成后进行PCR扩增,筛选获得了33个多态性微卫星标记,多态性分析结果表明,多态信息含量(PIC)值在0.244-0.889之间变化,平均值0.617；每个位点有5-13个等位基因,平均值8.636；期望杂合度(HE)和观测杂合度(Ho)分别在0.347-0.950和0.071-0.913之间变化,平均值分别是0.780和0.543。3.采用7个微卫星标记研究5种环棱螺(铜锈环棱螺,梨形环棱螺,方形环棱螺,双旋环棱螺,角形环棱螺)的种群遗传结构。遗传多样性分析：5种环棱螺均具有较高观测杂合度和期望杂合度值,表现出较丰富的遗传多样性,杂合子缺失导致了各个种群显著偏离哈迪温伯格平衡(HWE),平均观测杂合度均小于期望杂合度,种群内近交现象比较严重,导致了杂合体缺失。遗传分化分析：5种环棱螺各个种群间没有表现出明显的遗传分化,但少部分种群间的遗传距离较大。遗传变异分析(AMOVA)表明各个种类环棱螺群体的遗传变异主要发生在种群内,地理种群间的遗传分化程度很低,种群间存在明显的基因流。遗传结构分析：将不同地理种群按3大地理区域(长江以南,长江以北,长江流域)结构划分时,(?)MOVA结果显示,地理区域间的遗传变异所占百分比很小,种群间没有形成明显的系统地理结构。主成分判别分析结果显示,铜锈环棱螺12个种群被分成5个族群,梨形环棱螺8个种群被分成4个族群,方形环棱螺7个种群形成了3个族群,但各族群中优势种群的聚类并没有形成明显的地理格局分布。对种间的遗传变异分析(AMOVA),结果显示种间的遗传分化程度很低。5种环棱螺共34个种群形成了13个族群,各个种群在13个族群中的分布没有按种类形成明显聚类。4.利用线粒体COI基因研究3种环棱螺(铜锈环棱螺,梨形环棱螺,方形环棱螺)的种群遗传结构。遗传多样性分析：铜锈环棱螺116条序列中共发现81个单倍型和167个核苷酸多态性位点,种群间单倍型和核苷酸多样性分别在0.8670-0.9780和0.0130-0.0449之间；梨形环棱螺77条序列中有66个单倍型和155个核苷酸多态性位点,种群间单倍型和核苷酸多样性分别在0.8890-1.0000和0.0067-0.0416之间；方形环棱螺63条序列中有51个单倍型和158个核苷酸多态性位点,种群间的单倍型和核苷酸多样性分别在0.8210-1.000和0.0313-0.0474之间。三种环棱螺种群间的单倍型多样性和核苷酸多样性均较高,表现出较丰富的遗传多样性。群体历史动态分析：将所有种群当成一个整体时,3种环棱螺中性检验的Fu’s Fs值均为显著性负值,铜锈环棱螺Fs=-23.8828(P<0.001),梨形环棱螺Fs=-24.0457(P<0.001),方形环棱螺Fs=-12.9900(P=0.0110)。3种环棱螺的线粒体COI基因的核苷酸错配分布(mismatch distribution)均为单峰,表明这3种环棱螺均经历过明显的种群扩张。按照无脊椎动物分子变异率(μ=1.22%/MY)和环棱螺以1年为代时,计算的铜锈环棱螺种群扩张时间大约在2.197百万年(Ma),梨形环棱螺0.256Ma,方形环棱螺2.217Ma,三种环棱螺的扩张时间都发生在更新世。遗传结构分析：遗传变异分析(AMOVA)结果显示3种环棱螺各个种群的遗传分化不明显,主要的遗传变异来自种群内。三大地理区域间的遗传变异所占百分比小于0,说明种群间没有形成明显的地理格局分布,从单倍型网络图也可以看出,不同地理种群的单倍型相连,没有按着采样点形成谱系分支。种间的AMOVA结果显示,种间的遗传变异只占1.05%,并且种间的遗传距离值很小(0.004-0.030),说明3种环棱螺种间的遗传分化程度也很低。5.根据螺壳的6个形态性状对中国5种环棱螺32个种群的形态变异进行主成分分析和聚类分析,结果显示：环棱螺同种不同地理种群间形态具有一定的差异,但并没有形成明显的形态分化。判别分析结果表明,各个种群的判别函数的判别准确率均较低,说明种群间的形态变异不大。5种环棱螺32个种群的主成分分析和聚类分析,结果显示32个种群形成了3个形态类群,铜锈环棱螺所有种群单独为一个类群,角形环棱螺和梨形环棱螺种群聚成一个类群,方形环棱螺和双旋环棱螺种群聚成一个类群。6.对采自同一湖泊的5种环棱螺齿舌结构的电镜扫描观测比较发现,5种环棱螺。具有相同的齿式：2·1·1·1·2,由4个部分组成,中央齿,侧齿,内缘齿和外缘齿。齿舌各个部分的形态差异较小,但各部分的尖齿数和排列上存在较明显的差异,因此,齿舌可以作为环棱螺种类鉴定的良好材料。综合本研究结果,中国环棱螺种间和地理种群间没有形成明显的遗传分化,系统发育和种群遗传结构的研究结果均不支持中国环棱螺种的分类阶元。螺壳形态在种间具有一定的形态分化,并且齿舌结构在种间形成了明显的差异。因此,螺壳的形态特征和齿舌显微结构是重要的分类依据,但分子水平研究环棱螺的分类阶元有待进一步深入。更多还原

【Abstract】 Bellamya is a member of Viviparidae, Mesogastropoda, Gastropoda, Mollusca. It is one of macrobenthos in freshwater ecosystem, and wildly distributed in various lakes of Asia, Africa and North America. As an important genus of freshwater gastropods, Bellamya plays a significant role in the ecosystem. Due to environmental deterioration in lakes and overexploitation of resource of Bellamya by human in the late years, germplasm resources of Bellamya was damaged to some extent and the number of species was in decline. The classification of Bellamya was mostly based on the shell characteristics. Because of the variability of shell morphology, synonyms and taxon confusion were often found in reports. In this study, the genetic diversity and taxonomy research were investigated on the basis of molecular genetics, combined with morphological study. It was essential to investigate the population genetic structure, which could provide molecular genetic basis for genetic resource conservation. The main results were as follows:1. The partial sequence of the mitochondrial (COI and16S rRNA) and nuclear (H3and28S rRNA) of Bellamya genus were compared to determine their phylogenetic relationships, including Bellamy species from India, Africa and China. The phylogenetic trees were reconstructed based on Bayesian Inference methods. Viviparus ater and V. contectus were used as outgroup, and the sampled8species(Bellamya aeruginosa, B. angularis, B. quadrata, B. dispiralis, B. purificata, B. turritus, B. lapillorum, B. lapided) and7species(Bellamya sp.) in China, and Bellamy species from India and Africa download from GeneBank were used as ingroup. The phylogenetic analysis revealed that the worldwide Bellamya species form into three major clades, Indian species-clade, African species-clade and Chinese species-clade. Chinese Bellamya was the basal among the three clades. The African species was close to Indian species. Most of the African Bellamya species formed into three or four major lake-clades. However, in China Bellamya species-clade, neither the same species formed a cluster, nor the species in the same lake formed a cluster, which indicated that there was no significant genetic differentiation among populations and species. There was no significant difference between intra and interspecific genetic distance, further illustrating that the interspecific genetic differentiation was very low.2. A microsatellite-enriched genomic library of B. aeruginosa was successfully constructed by using the method of FIASCO (Fast Isolation by AFLP Sequences Containing Repeat), and2566microsatellite sequences were captured and deposited in GeneBank (Genbank assession No:JN555759-JN556037, JX018213-JX020499).391pairs of SSR-primers were designed by Primer v5.0, and100pairs of them were synthesized.33pairs of SSR-primers were successful amplified and had high polymorphic information content value (PIC), which ranged from0.244to0.889(average0.617). Allele number per locus ranged from5to13(average8.863). The expected heterozygosity (HE) and observed heterozygosity (Ho) varied from0.347to0.950and0.071to0.913with an average of0.780and0.543, respectively.3. The genetic structure of5Bellamy a species (B. aeruginosa, B. purificata, B. quadrata, B. angularis, B. dispiralis) were analyzed using7microsatellite locus.The genetic diversity analysis showed that all the5species had high observed heterozygosities and expected heterozygosities, indicating high genetic diversity among populations. Heterozygote deficiency was indicated with a significant deviation from the from Hardy-Weinberg equilibrium observed over all populations, while average HO value was consistently lower than HE value. Heterozygote deficiency had close relation with the high degree of inbreeding in population.There was no significant genetic differentiation among populations for each of the5Bellamya species, though high genetic distance was found between a very few populations. Analysis of molecular variance (AMOVA) indicated that the genetic variance mainly presented in individuals within populations, there were no significant genetic differences among populations. Based on the molecular analysis, the investigated populations showed obvious gene flow among populations.AMOVA from microsatellite data indicated that no significant genetic structure of Bellamya existed at various hierarchical levels (among regions, among populations within regions, and within population). The proportions of genetic variance among regions were very low, indicating that populations had not form a pattern of obvious geographical distribution. The discriminant analysis of principal components (DAPC) method revealed no clear separation of regional clusters,12populations of B. aeruginosa were divided into five clusters,8populations of B. purificata were divided into four clusters, and7population of B. quadrata were divided into three clusters. However, the dominant populations in each cluster also couldn’t form obvious geographical distribution pattern. AMOVA revealed very low amount of genetic differentiation among the five species. The 34populations of the5species were divided into13clusters, and no species clade was found in these clusters.4. The genetic structure of B. aeruginosa, B. quadrat a and B. purificata were studied using mtDNA COI gene.The3Bellamya species showed high genetic diversity among populations.12populations of B. aeruginosa had116sequences, in which81haplotypes and167polymorphic sites were detected, and the haplotype and nucleotide diversity ranged from0.8670to0.9780and0.0130to0.0449, respectively.8populations of B. purificata had77sequences, in which66haplotypes and155polymorphic sites were detected, and the haplotype and nucleotide diversity ranged from0.8890to1.0000and0.0067to0.0416, respectively.7population of B. quadrata had63sequences, in which51haplotypes and158polymorphic sites were detected, and the haplotype and nucleotide diversity ranged from0.8210to1.0000and0.0313to0.0474, respectively.When all populations pooled in one group for each of the three species, All the Fu’s Fs value of the three species were significant negative, for B. aeruginosa Fs=-23.8828(P<0.001), for B. purificata Fs=-24.0457(P<0.001), for B. quadrata Fs=-12.9900(P=0.011). Besides, Mismatch distribution of pairwise nucleotide differences of mtDNA COI showed unimodal pattern for each of the three species. The results supported that for B. aeruginosa, B. purificata and B. quadrata all had ever experienced demographic expansion. Based on1.22%±0.27%per million years (Myr), the demographic expansion time for B. aeruginosa, B. purificata and B. quadrata was about2.197Ma,0.256Ma and2.217Ma respectively, which were in Pleistocene.AMOVA analysis indicated that there was no obvious genetic structure of the three species existed at various hierarchical levels (among regions, among populations within regions, and within population). The proportion of genetic variance among regions was negative, indicating that populations had not form a pattern of obvious geographical distribution. The network generated from haplotype data set showed that the haplotype of different populations linked together, and displayed no apparent lineage clades association with sampling locations. AMOVA also revealed very low amount of genetic differentiation among the three species,1.05%of the total variation was distributed among the three species, and genetic distance between the three species ranged from0.004to0.030.5. According to six morphological characters, principal component analysis and cluster analysis were used to investigate morphological variations of32populations of5Bellamya species (B. aeruginosa, B. purificata, B. quadrata, B. angularis, B. dispiralis). The result showed that there were different degrees of morphological variation among populations, but no obvious morphological differentiation was found in the same Bellamya species. Discriminant analysis also indicated that there were no significant differences among populations, with low disciminant accuracy in most populations. The disciminant accuracy was33.33%-66.67%for B. aeruginosa;45%-80%for B. purificata;33.33%-86.67%for B. quadrata;53.33%-93.33%for B. angularis,46.15%-76.19%for B. dispiralis. The results of principal component analysis and cluster analysis for32populations showed that the5species could be classified into three morphologically different groups. The first group just included B. aeruginosa, the second group included B. purificata and B. angularis, and the third group included B. quadrata and B. dispiralis.6. The radula of five Bellamya species (B. aeruginosa, B. purificata, B. angularis, B. quadrata and B. dispiralis) samping from the same lake was compared on the basis of scanning electron microscopic observation. Results showed that all of them have the same dentition formula2·1·1·1·2, consists of central teeth, lateral teeth, inner marginal teeth and outer marginal teeth. There was no remarkable interspecific morphological difference in the four parts of radula, but significant differences in the amount of the denticles on the central teeth, lateral teeth and inner marginal teeth among the five species.In conclusion, there was no obvious interpopulation and interspecific genetic differentiation for Bellamya in China. The phylogenetic inference and analysis of genetic structure could not support the taxonomic category of Bellamya in China. Morphological analysis indicated that there were differences among species, and the difference of radula structure was obvious. Consequently, the shell morphology characteristic and radula structure is still the most important basis for Bellamya classification, it is necessary to find better molecular marker to do research on the taxonomic category of Bellamya.更多还原