摘要
夏蜡梅(Sinocalycanthus chinensis Cheng et S.Y.Chang)为蜡梅科夏蜡梅属落叶灌木,国家二级保护植物,第三纪孑遗种,夏蜡梅属唯一的代表,在蜡梅科中分类地位独特。夏蜡梅花大,花型奇特、观赏价值高,自然分布仅限于浙江临安、天台等非常狭窄的地域。由于生境破坏、种群衰退等原因,有限的夏蜡梅野生资源受到威胁,急待采取人工措施创造适合生境、扩大种群数量保护这一中国特有珍稀野生花卉。本研究对夏蜡梅分布地生境及分布现状进行了实地调查,运用群落学调查方法分析夏蜡梅种群生存及发展动态,从表型及分子标记遗传分析两方面探讨了我国目前分布夏蜡梅群体的遗传多样性,并以此为依据构建了遗传多样性种质采集保存策略,同时就夏蜡梅引种栽培中关键环境因子(光照、水分等)进行了不同处理比较,摸清了其适宜的栽培条件,为实施夏蜡梅迁地保护及园林开发利用奠定了基础。主要结果如下:
1.通过对夏蜡梅分布位置、海拔以及光照、气候、湿度、土壤等生境因子的综合调查与分析,总结出夏蜡梅资源分布及其生境特点,并首次在安徽绩溪龙须山附近发现夏蜡梅新分布,至此夏蜡梅不再是浙江特有种。
2.从种群生态学角度探究夏蜡梅的濒危机制,调查发现夏蜡梅种群结构存在波动性,幼年阶段个体较丰富,随年龄增长,种内种间对于光照、养分等生存资源及空间的竞争异常激烈,在6龄级前后种群死亡率陡增达到顶峰,其种群存活趋向于Deevey-Ⅱ型曲线,分布格局趋势为幼龄阶段为集群分布、中龄、老龄阶段向随机分布发展,由此提出缓解群内竞争适合的天然林抚育保护措施。
3.对夏蜡梅天然居群包括花色、花径、花梗长、内(外)轮花被片数目、长、宽在内的花部性状以及叶部(叶长、叶宽)、果部(果长、果宽)、种子(种长、种宽、种子重)等表型性状调查发现,夏蜡梅群体表型变异丰富,存在4种花色变异类型:白、浅粉、粉、粉红;20个表型数量性状在群体间与群体内差异均达到极显著水平,平均变异系数12.15%(6.4%～45.87%),平均表型分化系数为41.87%(1.14～85.07%),群体内变异大于群体间变异;花径、花梗长、外被片长度、形状(长宽比)以及种子重量、种长等性状变异与分布地经度、纬度相关性显著,而叶长、可育雄蕊数与土壤有机质含量、水解氮显著相关。
4.采用ISSR分子标记技术对夏蜡梅天然群体遗传多样性分析结果表明,夏蜡梅遗传多样性总体水平不低(多态性位点百分率P 63.86%、Shannon指数I 0.2687、Nei's基因多样性指数h 0.1713),但居群水平多样性偏低,居群间P值平均31.63%,I平均0.1805、h平均0.1234;居群间遗传分化系数G_(st)为0.2985,反映出夏蜡梅居群内遗传分化(70.15%)大于居群间遗传分化(29.85%),与表型分化结果相对应。
5.ISSR分子标记与表型数据UPGMA聚类分析均可将夏蜡梅8个天然群体划分2大类群,浙江天台大雷山夏蜡梅居群与近临安居群(包括安徽龙须山居群)距离最远,居群间无论是表型或是遗传分化均与地理距离显著相关,体现出连续性变异分布特征。
6.以夏蜡梅8个居群225个个体ISSR分子标记分析数据并结合表型多样性分析结果,通过计算机模拟随机取样建立基因捕获曲线,构建了夏蜡梅遗传多样性的种质保护取样策略:确定浙江临安大明山景区、顺溪横源、颊口前坑、清凉峰马啸、天台大雷山与安徽龙须山6个群体为遗传多样性优先保护的样本群体,迁地保护中按照30%比例采样即可代表夏蜡梅天然群体95%以上的遗传组成。
7.夏季高温下不同光照、土壤水分含量对夏蜡梅枝条生长及生理指标有显著影响,全光照与干旱(土壤含水量10%)诱导夏蜡梅体内膜脂过氧化加剧、丙二醛含量增加,POD、SOD活性升高;遮荫可部分减轻干旱胁迫对夏蜡梅的伤害,而补充水分又可适当补偿夏蜡梅对强光照的忍耐能力;通过对不同光照与土壤水分夏蜡梅生长及相关生理指标测试分析,筛选出夏季高温下夏蜡梅适合的光照及土壤含水量组合为:中度光照(遮荫50%)与45%的土壤含水量或低光照(遮荫75%)与25%的土壤含水量。
8.对夏蜡梅6个居群每居群20个单株实施种质采集迁地保护。种子播种发芽时间持续近16周,居群平均发芽率65.09%(47.35～84.18%);天台居群种子播种早期(第2周)即大量萌发,与临安周边居群夏蜡梅种子发芽模式显著不同。
通过以上研究,对我国特有珍稀野生花卉—夏蜡梅天然群体变异类型及分布特点有了更深入了解,完成从资源、生境调查、遗传多样性分析与采集策略制定及种子采集、播种直至栽培等系列过程并总结出一套迁地保护技术程序,初步建立了夏蜡梅迁地保护资源圃,为实现夏蜡梅遗传多样性保护及园林开发利用奠定基础。
Sinocalycanthus chinensis is a deciduous shrub of Calycanthaceae,Sinocalycanthus genus which has survived from the tertiary.As a second-grade protected wild plant in china and the only representative in the genus Sinocalycanthus,S.chinensis holds a special taxonomic status and high research value in system evolution field.New cultivars has been produced by crossing it with calycanthus floridus,the species belongs to calycanthus genus.S.chinensis inhabits narrowly in eastern or northwestern area of Zhejiang province.In the past forty years,the germplasm resources of S. chinensis were destructed greatly due to human factors and tourism development.Though most natural populations of S.chinensis have been conserved in Natural reserve,but the community of high canopy density do not favor it and the populations lead to a great regression.It has been detected that no genetic variation in the introduced population of S.chinensis in Tianmushan Natural reserve,shows that the ex-situation do not conserve its genetic diversity effectively and should not be considered as a success ex-situation of this endangered species.In this study,we conducted a series survey to habitat and resource of S.chinensis in its distribution area,analyzed the structure and dynamics of its natural population.On the basis of phenotypic and molecular marker data,sampling strategy of biodiversity as well as the suitable ex-situation conservation strategy was established.The main results were as follows:
1.By field survey in Zhejiang,Anhui Province of S.chinensis distribution area,we summarize its habitat as follows,lies in north or northwest slope of a mountain,over 400m altitude,with high humidity,moderate light,soil pH value range from 4.5 to 5.6.The environmental factors play an important role in its distribution and widespread.
2.The population structure and spatial distribution pattern analysis revealed that there were abundant seedlings but the population structure fluctured.The mortality rate significantly increased to the peak of 96.1%near ageⅥand the survival curve tend to be Deevey typeⅡ.With the age increased,the spatial distribution patterns presented the same trend from clusting to random among different populations.The interspecies and interspecific competition is one of the cause which lead S. chinensis to be an endangered species.
3.22 phenotypic traits were investigated by sample 8 populations.The results shows that significant variation existed among and within populations of S.chinensis.The outer petal color could be distinguished into four group,white,baby pink,pink and dark pink.The proportion among populations is different.There were a great correlation between longitude and latitude to flower diameter,floral shoot length,outer-perianth length,ratio of length to width,the weight and length of seed. The leaf length and number of fertile stamen varied with the content of organic matter and hydrolyzed nitrogen of soil.
4.By using inter-simple sequence repeat(ISSR) marker technique,the genetic diversity of 8 populations was studied.The results showed that at species level,the percentage of polymorphic loci(P) was 63.86%,Shannon's index(h) 0.2687,and Nei's gene diversity(I) was 0.1713 while at population level,they were 31.63%,0.1805,0.1234,respectively,indicating a low level of genetic diversity.The gene differentiation coefficient(G_(st) 0.2985) suggested low genetic differentiation among populations and high genetic diversity within populations.
5.According to the UPGMA cluster analysis on basis of phenotypic and molecular data,the 8 investigated populations could be divided into two groups.There was a great correlation between phenotypic and genetic differentiation to the geography distance.The distribution pattern of variation in S.chinensis natural population exhibited to be continuous.
6.The sample strategy of genetic biodiversity conservation was established based on data of ISSR detected by 255 individuals of 8 populations and phenotypic as well,which includes(1) 6 populations (2)30%sampling proportion.
7.The water content of soil and light density has a significant influence on growth of S.chinensis and the physical index differed correspondingly.Under high light density(100%full day light) or at drought soil(10%water content),the lipid peroxidation lead to malondialdehyde(MDA) accumulated which induced superoxide dismutase(SOD) and peroxidase(POD) activities significantly increasing. Shade could reduce the injury of lipid peroxidation while increasing water content(25%,45%) could enhancing the ability of tolerating high light density.The best combination of water content of soil and light density were of middle light density(50%full day light) and 45%water content,high-middle light density(25%full day light) and 25%water content.
8.Ex-situ conservation was conducted by sowing seeds collected from 120 individuals of 6 populations.Germination time lasting near to 16 weeks and the average germination rate was about 65.09%.The seeds from Tiantai population geminated earlier than other 5 population's near to Lin'an city.
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