【作者】 项小燕；

【导师】 张小平；

【作者基本信息】 安徽师范大学 ， 植物学， 2015， 博士

【摘要】 大别山五针松（Pinus dabeshanensis C.Y.Cheng&Y.W.Law）隶属于松科（Pinaceae）松属（Pinus）,是我国特有的珍稀濒危植物,仅局限分布在大别山地区。本文利用种群生态学和遗传学的方法,从群落物种多样性、区系特点、种群数量动态、种内和种间竞争、种群生殖力调查、转录组测序及微卫星引物开发、种群遗传多样性等方面对其进行了研究,以期为该濒危植物的保护和利用提供依据。主要研究结果如下:1 大别山五针松群落物种多性及区系特点大别山五针松分布区的群落内,共有维管植物72科141属218种。其中,蕨类植物4科4属6种,裸子植物3科3属4种,被子植物65科134属208种。蔷薇科、樟科、菊科、杜鹃花科、豆科和禾本科等科为优势科。区系分析显示,该群落区系起源古老,地理成分复杂,68个科中,世界分布、热带分布和温带分布的科分别有21、32和15个,显示由热带向温带过渡的特性。137个属中,热带成分共40属,占总属数（不包括15个世界广布属）的32.79%;温带成分共有82属,占总属数的67.21%,并以北温带为主。群落垂直结构分为乔木层、灌木层和草本层3层,其中,Margalef指数、Simpson指数和Shannon-Wiener指数均以灌木层最大,其次是乔木层,草本层最低。灌木层与草本层的Pielou指数相近,且均大于乔木层。2 大别山五针松种群数量动态用空间序列代替时间的方法分析种群结构,编制种群特定时间生命表,绘制死亡率曲线和消失率曲线,并用4个生存函数进行种群的生存分析;同时结合谱分析方法,分析了大别山五针松种群数量的动态变化。结果表明,大别山五针松种群数量少,结构存在波动性。幼苗阶段个体较丰富,幼树阶段个体较少,种群趋于衰退。生命表和存活曲线表明,大别山五针松存活曲线介于Deevey Ⅱ和Deevey Ⅲ型之间,种群死亡率和消失率曲线变化趋势基本一致,在第5龄级出现峰值。4个生存函数曲线表明,大别山五针松具有前期快速减少、中期稳定和后期衰退的特点。谱分析显示,大别山五针松种群动态除受基波影响外,还具有明显的小周期波动。3 大别山五针松种内、种间竞争关系用Hegyi单木竞争模型分析了大别山五针松的种内和种间竞争强度。结果表明,竞争主要来自种间。使大别山五针松受压的植物主要有短柄袍、黄山松、满山红和茅栗等。大别山五针松在胸径小于25 cm之前,受到的竞争压力较大;当胸径大于25 cm后,竞争强度变化不明显,竞争强度与胸径符合幂函数关系（CI=AD-B）。4 大别山五针松种群生殖力大小生殖力调查表明,自然种群种子的败育率为61.08%,饱满种子的活力为100%;栽培种群种子的败育率高达89.88%,饱满种子的活力为89.76%。低温冷藏更有利于种子活力的保存。种子可在较宽的温度范围内萌发,且室温储藏的种子萌发率比低温冷藏的要高;破壳处理有利于种子吸胀但对种子萌发没有明显的促进作用;自然种群种子萌发率显著高于栽培种群。5 大别山五针松的转录组测序及微卫星引物开发通过Illumina HiSeq^TM 2000测序平台,对大别山五针松针叶进行转录组测序。从所获得的unigene中共检测到SSR 1966个,平均每23.08 kb含一个SSR位点。SSR序列中最丰富的重复基元为三核苷酸,其次是二核苷酸、单核苷酸、六核苷酸、五核苷酸和四核苷酸,所占的比例分别为41.60%、23.25%、21.41%、7.83%、4.43%和1.53%。三核苷酸和二核苷酸中最丰富的基元类型分别是AGC/CTG和AT/AT。基于2-6核苷酸重复序列,共成功设计出引物431对。随机挑选82对扩增,共获得19对多态性引物,这些引物在24个个体中共检测到57个等位基因,平均每个位点的等位基因数NA=3.0个。引物的预期杂合度和观测杂合度范围分别为0-0.958和0.082-0.759,PIC值变化范围为0.077-0.707,平均值为0.389。6 大别山五针松的遗传多样性和遗传结构13对EST-SSR引物在233个个体中共检测到50个等位基因数,平均每个位点3.85个,自然种群和栽培种群的预期杂合度H_E分别为0.458和0.423,观察杂合度HO分别为0.481和0.510,表明大别山五针松种群的遗传多样性属于中等水平。当代基因流和历史基因流均显示种群间存在较大基因流,种群间遗传分化极小(F_ST=0.028)。MSVAR分析表明,有效种群从古代的大约10⁴-10⁵株下降至现在的102-103株,种群大小发生变化的时间大约在10000年前。基于BOTTLENECK软件和TPM及SMM两种模型检测结果显示,在TPM模型下,2个自然种群可能经历了近期的瓶颈效应。STRUCTURE聚类分析显示,自然种群和栽培种群被分为两组。其中,栽培种群主要含1个基因池,而自然种群包含2个。3个自然种群均含有稀有等位基因,而栽培种群没有,表示栽培种群不足以代表自然种群所包含的遗传信息。更新世气候的回升可能是导致种群大小发生变化的主要原因。7大别山五针松的濒危机制与保护策略鉴于上述研究,大别山五针松的濒危主要由以下几个方面导致:1)种群结实率低、种子败育率高,导致种群生殖力较低;2)幼苗死亡率高,难以向幼树转化;3)种群竞争力弱,在竞争中处于劣势;4)小种群效应导致近交衰退现象严重,难以抵御各类风险。针对以上研究结果,本文提出如下保护策略:1)就地保护,适当间伐杂木,为幼苗、幼树的生长提供光、营养等资源,促使幼苗形成幼树;同时减少生存竞争压力;2)人工控制授粉,提高种子品质;3)保护大王沟的3个种群,尤其注意保护含稀有等位基因的个体;4)从自然种群采集种子,并多采集大王沟1和大王沟3种群及含有稀有等位基因个体的种子,人工育苗扩大种群。更多还原

【Abstract】 Pinus dabeshanensis C.Y.Cheng&Y.W.Law is a rare endangered species that is endemic to the Dabieshan Mountains of China.In this study,we focused on the species diversity and floristic analysis of community,population dynamics,intra-and inter-specific competition,transcriptome sequence and development of EST-SSR markers,genetic diversity of populations by the methods of plant population ecology and population genetics.These results provided some evidence for conservation and management of the endangered species.The main results were as follows:1 Features of species diversity and floristic analysis of P.dabeshanensis communityIn P.dabeshanensis community,there were 218 vascular plant species belonging to 141 genera of 72 families,among which there are 4 families,4 genera,6 species in Pteridophyte;3 families,3 genera,4 species in Gymnosperm and 65 families,134 genera,208 species in Angiosperm.Rosaceae,Lauraceae,Compositae,Ericaceae,Leguminosae and Gramineae were the dominant families in P.dabeshanensis community.The floristic analysis revealed that the flora was ancient and the geographical components were complex.Among 68 families,21,32 and 15 families belonged to cosmopolitan,tropical and temperate distribution,respectively.40 and 82 genera belonged to tropical with 32.79%and temperate distribution with 67.21%in the total,respectively.The measurement of Margalef（R）,Simpson（D）,Shannon-Wiener（H）and Pielou（E）indices on arbor,shrub and herb layers was carried out.In addition to the diversity index E,the other diversity indices of shrub layer were the highest,followed by arbor layer,and those of herb layer were the lowest.The value of E of shrub layer had the similarity with that of herb layer,both of which were higher than that of arbor layer.2 Population dynamics of P.dabeshanensisWe used spatial series substituting for time sery to analyze the population structure.And the time-specific life table,mortality rate,vanish rate as well as the four survival functional curve of the population were calculated,respectively.Additionally,spectral analysis was used to analyze the population dynamics.The results showed that P.dabeshanensis population was extremely small and population structure fluctuated.Although the seedlings were more abundant,young trees were very deficient,suggesting that the population was decreasing.The survival curve of the population followed a pattern between Deevey Ⅱ and Deevey Ⅲ,the curves of mortality rate and vanish rate exhibited a similar variation tendency:peak exsisted in the 5th age class period.Four survival functional curves showed that the population decreased rapidly at the early ages,grew stably at the intermediate ages and declined in the late lifespan.Spectral analysis of the population showed that the population dynamics had a clear periodic fluctuation in the growth except for the influence of fundamental wave.3 Intraspecific and interspecific competition of P.dabeshanensisThe intraspecific and interspecific competition was studied using Hegyi individual competitive indices（CI）model.The results showed that intraspecific and interspecific competition intensity of P.dabeshanesis accounted for 17.11%and 82.89%in the total competition intensity,respectively,indicating a remarkable effect of the interspecific competition.The intraspecific competition mainly came from the roles of Quercus serrata var.brevipetiolata,Pinus taiwanensis,Rhododendron mariesii and Castanea seguinii.Notably,the competition intensity of P.dabeshanesis decreased inversely to diameter of the breast height（DBH）,which showed a stronger competition at DBH of<25 cm and appeared weaker at DBH over 25 cm.The relationship between the whole forest competition intensity and DBH of the target tree followed the power function（CI=AD-B）.4 Population fecundity of P.dabeshanensisThrough our investigations,we found that the percentages of abortive seeds were 61.08%and 89.88%,the viability percentages of full seeds were 100%and 89.76%from the natural population and cultivated population,respectively.Lower temperature was appropriate for the preservation of seed viability.Seeds could germinate in a wide range of temperatures and the final germination percentage of seeds storaged under room temperature was higher than that at 4℃.Moreover,the germination percentage of seeds from natural population was higher than that from the cultivated one.Scarifying seed coat promoted the water absorption of seeds while had non-significant promotion in rate of germination.5 Transcriptome sequence and development of EST-SSR markersUsing Illumina HiSeq^TM 2000 sequencing platform and de novo assembly,a total of 1,966 expressed sequence tag-simple sequence repeats（EST-SSRs）derived from unigenes obtained,with an average distribution density of one EST-SSR locus per 23.08 kilobases（kb）.The most abundant repeat motif was trinucleotide（41.6%）,followed by dinucleotide（23.25%）,mononucleotide（21.41%）,hexanucleotide（7.83%）,pentanucleotide（4.43%）and tetranucleotide repeats（1.53%）.The top tri-and di-nucleotide motif was AGC/CTG and AT/AT,respectively.After randomly selecting 82 from 431 for further validation,19 could reliably amplify polymorphic bands from 24 individuals of P.dabeshanensis.A total of 57 alleles were identified,with an average of 3.0 alleles per locus.The observed and expected heterozygosity per locus ranged from 0 to 0.958 and 0.082 to 0.759,respectively.PIC value ranged from 0.077 to 0.707,with an average of 0.380.6 Genetic diversity and structure of P.dabeshanensisA total of 50 alleles were identified across 233 individuals,with an average of 3.85 alleles per locus.Our results revealed that intermediate level of genetic diversity（H_E=0.458 vs.H_E=0.423）among natural and conserved populations and low genetic differentiation(F_ST=0.028)between populations.Both contemporary and historical migration rates among populations were high.Bayesian coalescent-based analyses suggested that 3 populations underwent reductions in population size ca.10,000 yr ago.Using BOTTLENECK software based on two models of TPM and SMM,two natural populations might have experienced recent genetic bottleneck effect under the TPM model.Bayesian clustering revealed that individuals from the natural populations and the conserved one were divided into two groups（K=2）,the conserved population was gathered into one gene pool while the natural ones were assigned to two.Additionally,our results identified private alleles in the natural populations but not in the ex situ population,suggesting that the ex situ conserved population insufficiently represents the genetic diversity present in the species.Past decline in population size is likely to be due to Holocene climate change.7 Endangered mechanism and conservation strategiesAccording to our aforementioned researches,the major endangered mechanisms for P.dabeshanensis may be as follows:1)The low percentage of seed setting and high percentage of abortive seeds resulted in lower fertility;2)High seedlings death rate and deficient young trees accelerated the population degeneration;3)The weak interspecific competition compelled population standing at a disadvantage;4)Small population effect resulted in severe inbreeding depression which attenuated the risk-resisting capabilities.As a result,we propose here the following conservation strategies:1)Adopting appropriate artificial disturbance including logging woods for the seedlings and young tree’s growth,and to reduce the competition;2)Utilizing artificial pollination in order to improve the seed quality;3)Conserving the three DWG populations,especially those individuals harboring private alleles so as to preserve the genetic diversity;4)Collecting seeds,especially from DWG1,DWG3 populations and those individuals with private alleles,for seedlings recruitment that might be an effective way to increase the population size.更多还原