卧龙自然保护区不同海拔的中国沙棘(Hippophae rhamnoides subsp. sinensis)天然群体的遗传多样性分析

中国沙棘是一种雌雄异株、风媒传粉的灌木或乔木，在中国西南的卧龙自然保护区有广泛的分布。本研究以采集于四川卧龙自然保护区5 个海拔(1800 m、2200 m、2600 m、3000 m、3400 m)梯度的中国沙棘天然群体为材料，以ISSR 和AFLP 标记技术研究其遗传多样性水平及其遗传结构，旨在了解卧龙地区中国沙棘天然群体的遗传多样性水平以及遗传多样性在群体间、群体内以及雌雄亚群体间的分布和特征，为中国沙棘树种的遗传改良及种质资源保存提供遗传研究背景与实验依据。同时探讨ISSR、AFLP 和RAPD三种标记对中国沙棘天然群体的遗传变异水平和群体间遗传结构的评估能力和各自的优缺点。研究得出以下主要结论： 1. ISSR和AFLP分析都表明卧龙自然保护区的中国沙棘群体拥有较高的遗传变异水平(h = 0.249，HT = 0.305)。出现这种结果的主要原因可能与卧龙自然保护区多变的气候条件和生境的异质度大有关。 2. ISSR 和AFLP 都揭示出卧龙自然保护区中国沙棘群体的遗传多样性随着海拔的增加发生显著的变化，表现为中海拔群体(2200 m 和2600 m)比高海拔群体(3000 m 和3400 m)和低海拔群体(1800 m)有更高的遗传多样性的趋势。出现这种趋势的可能解释是低海拔群体处在相对高温和相对干旱的环境，高海拔群体受到低温和紫外线胁迫，而中海拔群体存在中国沙棘生长的适宜环境。 3. ISSR 和AFLP 分析都表明：卧龙自然保护区中国沙棘的遗传结构遵循分布范围广、交配系统以异交为主的木本植物的通常模式，即大多数的遗传变异存在于群体内，只有少部分的遗传变异存在于群体间。 4. 经Mantel 检测表明，卧龙自然保护区中国沙棘群体间的海拔距离和对应遗传距离之间存在显著的正相关关系，即随着垂直海拔距离的增加，群体间的遗传距离也随之增加。Mantel 检测结果以及聚类分析将卧龙自然保护区5 个不同海拔的中国沙棘群体分为低、中、高海拔群体三组的研究结果都表明，海拔很可能是限制群体间基因交流的主要因素。 5. ISSR 分析发现同一海拔的雌雄亚群体首先聚类的研究结果表明，同一海拔的雌雄亚群体在遗传上最相似。方差分析结果表明只有3.8%的总遗传变异存在于雌雄亚群体间，这可能与雌雄植株间的交配和遗传物质的混合有关。 6. ISSR、AFLP 和RAPD 分析都表明卧龙自然保护区不同海拔的中国沙棘天然群体的遗传多样性水平较高。它们的分析结果估算得到的Nei's 平均基因多样度(h)分别为0.249、0.214 和0.170。从该结果可以看出ISSR 和AFLP 比RAPD 检测到更多的遗传多态性,这很可能是不同标记检测的基因组的位点不同所致。 7. 依据对不同标记系统的比较分析，认为ISSR、AFLP 和RAPD 三种分子标记系统都能成功地用于调查卧龙自然保护区不同海拔的中国沙棘群体的遗传变异水平及遗传变异结构，提供关于中国沙棘天然群体多态性水平和遗传变异分布的有用信息。在三者中，AFLP 具有最高效能指数和标记指数，在确定种间分类关系或鉴别个体方面是一种比较理想的标记。 Hippophae rhamnoides subsp. sinensis, a dioecious and deciduous shrub species,occupies a wide range of habitats in the Wolong Nature Reserve, Southwest China. Ourpresent study investigated the pattern of genetic variation and differentiation among fivenatural populations of H. rhamnoides subsp. sinensis, occurring along an altitudinal gradientthat varied from 1,800 to 3,400 m above sea level in the Wolong Natural Reserve, by usingISSR and AFLP markers to guide its genetic improvement and germplasm conservation. And,comparative study of ISSR, AFLP and RAPD was performed to detect their capacity toestimating the level and pattern of genetic variation occurring among the five elevationpopulations of H. rhamnoides subsp. sinensis, and to discuss their application to the study onplant genetics. The results were list following: 1. The ISSR and AFLP analysis conducted for the H. rhamnoides subsp. sinensispopulations located in the Wolong Natural Reserve of China revealed the presence of highlevels of genetic variation (h = 0.249, HT = 0.305). Besides such features as relatively widedistribution, dominantly outcrossing mating system, and effective seed dispersal by small animals and birds, it is sometimes argued that hard climatic conditions and heterogeneous habitats may also contribute to high levels of diversity. 2. Genetic diversity of H. rhamnoides subsp. sinensis populations was found to varysignificantly with changing elevation, showing a trend that mid-elevation populations (2,200m and 2,600 m) were genetically more diverse than both low-elevation (1,800 m) andhigh-elevation populations (3,000 m and 3,400 m). H. rhamnoides subsp. sinensis is thoughtto be stressed by drought and high temperature at low elevations, and by low temperature athigh elevations. The high genetic variability present in the mid-elevation populations of H.rhamnoides subsp. sinensis is assumed to be related to a greater plant density in the middlealtitudinal zone, where favorable ecological conditions permit its continuous distributioncovering the zone from 2,200 m to 2,600 m above sea level. 3. The genetic structure of H. rhamnoides subsp. sinensis revealed by ISSRs andAFLPs followed the general pattern detected in woody species with widespread distributionsand outcrossing mating systems. Such plants possess more genetic diversity withinpopulations and less variation among populations than species with other combinations oftraits. 4. In the present study, Mantel tests showed positive correlations between altitudinaldistances and genetic distances among populations or subpopulations. The observedrelationship between altitude and genetic distances, and the result of the cluster analysisincluding populations or male subpopulations and classifying the groups into three altitudeclusters suggest that altitude is a major factor that restricts gene flow between populationsand subpopulations. 5. The analysis of molecular variance showed that only 3.8% of the variability residedbetween female and male subpopulations. Such a very restricted proportion of the totalmolecular variance between female and male subpopulations is due to common sexuality andmixing of genetic material between females and males. 6. The analysis based on ISSRs, AFLPs and RAPDs all revealed relatively high levelsof genetic variation among different altitudinal populations of H. rhamnoides subsp. sinensisin Wolong Natural Reserve of China. Their estimates of mean Nei’s gene diversity is equal to0.249, 0.214 and 0.170 respectively, suggesting the higher capacity of detecting geneticvariation of ISSR and AFLP than RAPD. It might be ascribed to their distinct sensitivity todifferent type of genetic variation. 7. Based on the coparative study on ISSR, AFLP and RAPD, we drew a conclusion thatthey all successfully reveal some useful information concerning the level and pattern ofgenetic vatiation occurring among different elevation populations of H. rhamnoides subsp.sinensis. AFLP is a ideal tool to taxonomic study and individual identification for theirhighest efficiency index and marker index among the three marker systems.

中国沙棘(Hippophae rhamnoides subsp. sinensis)天然群体的遗传变异分析

近十年，植物群体遗传学的研究飞速发展，然而与海拔相关的植物群体遗传结构和遗传变异研究却相对较少。到目前为止，还不清楚遗传变异与海拔之间是否有一个通用的格局。在山区，各种生态因子，如温度、降水、降雪、紫外线辐射强度以及土壤成分都随海拔梯度急剧变化，造成了即使在一个小的空间区域，植被类型变化显著，这种高山环境的异质性和复杂性为我们研究植物群体遗传结构和分化提供了方便。沙棘(Hippophea)属于胡颓子科(Elaeagnaceae)为多年生落叶灌木或乔木，雌雄异株，天然种群分布极为广泛。中国沙棘(H. rhamnoides subsp. sinensis)是沙棘属植物中分布较广的一个亚种，种内形态变异非常丰富，加之其具有独特的繁育系统和广泛的生态地理分布，是研究沙棘属植物遗传变异和系统分化的理想材料。本文从1,800 m 到3,400 m 分5 个海拔梯度进行取样，用RAPD 和cpSSR 分子标记研究了卧龙自然保护区中国沙棘天然群体的遗传结构和遗传变异。5 个取样群体依次标记为A、B、C、D 和E，它们分别代表分布在海拔1,800，2,200，2,600，3,000 和3,400 m 的5 个天然群体。RAPD实验用11 条寡核苷酸引物，扩增得到151 个重复性好的位点，其中143 个多态位点，多态率达94.7%。在5 个沙棘群体中，总遗传多样性值(HT)为0.289，B群体内的遗传多样性值为0.315，这完全符合沙棘这种多年生、远交的木本植物具有高遗传变异的特性。5 个群体内遗传多样性随海拔升高呈低－高－低变异趋势，在2,200 m海拔处的B群体遗传多样性达最大值0.315，3,400 m海拔处的E群体则表现最小仅0.098。5 个群体间的遗传分化值GST＝0.406，也即是说有40.6%的遗传变异存在于群体间，1,800 m海拔处的A群体与其它群体的明显分离是造成群体间遗传分化大的原因。UPGMA聚类图和PCoA散点图进一步确证了5 个群体间的关系和所有个体间的关系。最后，经过Mantel检测，遗传距离与海拔表现了明显的相关性(r = 0.646, P = 0.011)。cpSSR 实验中，经过对24 对cpSSR 通用引物筛选，11 对引物能扩增出特异性条带，只有2 对引物(ccmp2 和ARCP4)呈现多态性。4 个等位基因共组合出4 种单倍型，单倍型Ⅰ出现在A 群体的所有个体和B 群体的8 个个体中，C、D、E 三个群体均不含有，而单倍型Ⅱ出现在C、D、E 三个群体的所有个体及B 群体的18 个个体中，A 群体不含有。另外两种单倍型Ⅲ和Ⅳ为稀有类型，仅B 群体中的4 个个体拥有。这种单倍型分布模式和TFPGA 群体聚类图揭示了，C、D、E 群体可能来源于同一祖先种，而A 群体却是由另一祖先种发展起来的，B 群体则兼具了这两种起源种的信息，这可能是因为在历史上的某一时期，在中国沙棘群体高山分化的过程中，B 群体处某个或者某些个体发生了基因突变，具备了适应高海拔环境的能力，产生了高海拔沙棘群体的祖先种。 In recent ten years, studies about population genetics of plants developed rapidly,whereas their genetic structure and genetic variation along altitudinal gradients have beenstudied relatively little. So far, it is uncleared whether there is a common pattern betweengenetic variation and altitudinal gradients. In the mountain environments, importantecological factors, e.g., temperature, rainfall, snowfall, ultraviolet radiation and soil substratesetc., change rapidly with altitudes, which cause the vegetation distribution varying typically,even on a small spatial scale. The mountain environments, which are heterogeneous andcomplex, facilitate and offer a good opportunity to characterize population genetic structureand population differentiation.The species of the genus Hippophae L. (Elaeagnaceae) are perennial deciduous shrubs ortrees, which are dioecious, wind-pollinated pioneer plants. The natural genus has a widedistribution extending from Northern Europe through Central Europe and Central Asia toChina. According to the latest taxonomy, the genus Hippophae is divided into six species and12 subspecies. The subspecies H. rhamnoides ssp. sinensis shows significant morphologicalvariations, large geographic range and dominantly outcrossing mating system. Thesecharacteristics of the subspecies are favourable to elucidate genetic variation and systemevolution. To estimate genetic variation and genetic structure of H. rhamnoides ssp. sinensisat different altitudes, we surveyed five natural populations in the Wolong Natural Reserve at altitudes ranging from 1,800 to 3,400 m above sea level (a.s.l.) using random amplifiedpolymorphic DNA markers (RAPDs) and cpSSR molecular methods. The five populations A,B, C, D, and E correspond to the altitudes 1,800, 2,200, 2,600, 3,000 and 3,400 m,respectively.Based on 11 decamer primers, a total of 151 reproducible DNA loci were yielded, ofwhich 143 were polymorphic and the percentage of polymorphic loci equaled 94.7%. Amongthe five populations investigated, the total gene diversity (HT) and gene diversity within population B equaled 0.289 and 0.315, respectively, which are modest for a subspecies of H.rhamnoides, which is an outcrossing, long-lived, woody plant. The amount of geneticvariation within populations varied from 0.098 within population E (3,400 m a.s.l.) to 0.315within population B (2,200 m a.s.l.). The coefficient of gene differentiation (GST) amongpopulations equaled 0.406 and revealed that 40.6% of the genetic variance existed amongpopulations and 59.4% within populations. The population A (1,800 m a.s.l.) differed greatlyfrom the other four populations, which contributes to high genetic differentiation. A UPGMAcluster analysis and principal coordinate analyses based on Nei's genetic distances furthercorroborated the relationships among the five populations and all the sampling individuals,respectively. Mantel tests detected a significant correlation between genetic distances andaltitudinal gradients (r = 0.646, P = 0.011).Eleven of the original 24 cpSSR primer pairs tested produced good PCR products, onlytwo (ccmp2 and ARCP4) of which were polymorphic. Four total length variants (alleles) werecombined resulting in 4 haplotypes. The haplotype was present in all individuals of Ⅰpopulation A and 8 individuals of populations B, the other three populations (C, D and Epopulations) did not share. The haplotype was present in all individuals of populations C, D Ⅱand E and 18 individuals of populations B, population A did not share. The other twohaplotypes and were rare haplotypes, which were only shared in 4 individuals of Ⅲ Ⅳpopulation B. The distribution of haplotypes and TFPGA population clustering map showedthat the populations C, D and E might be origined from one ancestor seed and population Amight be from another, whereas population B owned information of the two ancestor seeds. Itwas because that gene mutation within some individual or seed in the location of population Bwas likely to happen in the history of H. rhamnoides, which was the original ancestor of thehigh-altitude populations.

镇海棘螈繁殖群体生态和遗传结构研究

genetics, such as: population size, reproduction, mating system, growth, development,genetic structure and systematics status; The main results are presented below: The seasonal variation of the operational sexual ratio of this animal was found in the field and the ration always bias the female in the breeding season. Aiming at this character and considering the distance of time and space of both sexual habitat in breeding season, we census female population first by toe-clipping mark-recapture method, then estimated the population size with the definitive sexual ratio. Up to now, this species was found only at the Beilun district of the Ningbo City. The population size of the Ruiyan Temple Forest Park approximates to 369. The status of this population is extremely endangered, so besides protecting this population at the original locality, we also suggested to breed the salamander in fenced locality and to hatch embryos artificially, and send metamorphosed juveniles back to nature. We can transfer some individuals to other similar habitats or breed them under artificial conditions for saving this species from extinction. The early developmental stage of the Chinhai salamander is the same as its relative species, E. andersoni. Their balanceres are poorly developed and disappear very early. Temperature and moisture significantly influence the embryonic development of the Chinhai salamander. The embryonic stage is approx. 29 days under room temperature. The hatchling grows in a logarithmic curve. The larvae stage in water is approx. 58- 88 days. Many factors influence the nomal development, including two aspects of internal and external. Due to these factors, the effective protected measures were presented in detail. The breeding migration of E. chinhaiensis takes place at late March～late April every year. This salamander's hatching rate is high, but the rate of hatchling migrating into water is low. The average effectiveness of all the nest sites is 36.7%. The maternal self-conservation was contrary to the reproductive success of the egg-laying strategy. In the strategy of egg-laying behavior, the first factor selected by the female was its self-conservation, the second is embryonic survival rate, and the last is rate of hatchling survival rate. The oviposition selection is significant for the survival of the larvae. Based on the analysis of the evolutionary process of reproductive behaviors nad egg-laying site selections of all genera of the family Salamandridae, we deduced that perhaps Echinotriton is a transitional type in the evolutionary process from water to land. Due to its location in the adaptive stage in the terrestrial evolution, Echinotriton chinhaiensis's terrestrial nest may be one of important reason that causes this species to be endangered. The genetic deversity analysis shows that although the population size of the Chinhai salamander is quite small compared to other Chinese salamandrid species, the genetic diversity of this population is not reduce remarkably. We explain this phenomena with the polygamy mating system of this species. The result of 4 families' parenthood determinations shows that the parenhood determination can be taken without any paternal information. The "children" of every female include rich genetic information from at least two "fathers". It implies that female Chinhai salamander mates more than once with different males in a breeding season. The molecular evidence, the behavioral observation evidences and the sperm evidence in the female cloaca proved that this species has a polygamy mating system. The kin recognition in the mating of adult salamander was first discussed. The taxonomic status and phylogenetic relationships of 12 species representing 6 genera in the family Salamandridae were studied using DNA fingerprinting. The results showed that the DNA fingerprinting. The results showed that the DNA fingerprinting patterns demonstrated rich genetic diversity and species diversity, and also revealed the taxonomic status and phylogenetic relationshipes of higher taxa to a certain extent. The results are highly consistent with those obtained from the studies based on the morphology, ecology, cytology and molecular biology. The compreshensive analysis indicate that Tylototrition hainanensis and T. wenxianensis should be valid species; Echinotriton should be a valid genus;Tylotortriton is a natural cluster; Tylotortriton asperrimus should be put in Tylototrition rather than in Echinotriton, Hypselotriton and Allomestriton are synonyms of Cynops and Paramesotriton, respectively. There are three main groups in Chinese salamandride: Cynops, Paramesotriton and Pachytrition from the first group, the species of the Tylototriton from the second, and E. chinhaiensis composes the third.

Photo-sensitized oxidation in natural water via .OH radicals

A method is presented for determining production and consumption rates of .OH radicals produced photochemically in natural surface waters. It is based on the determination of the kinetics by which the concentration of a specified trace compound decreases during irradiation. In samples from Lake Greifensee (Switzerland) low production rates for .OH limit its possible effects. In addition, fast consumptions by the natural dissolved organic solutes and by the bicarbonate protect organic micropollutants from oxidation by .OH. Neither direct nor indirect H2O2 photolysis was a significant source of .OH in the lakewater studied lacking iron, whereas nitrate photolysis could have been a source. Comparison with reaction kinetic formulations allows generalizations for other types of waters.

Natural 13C abundance as a tracer for studies of soil organic matter dynamics

A method for measuring the long- and medium-term turnover of soil organic matter is described. Its principle is based on the variations of 13C natural isotope abundance induced by the repeated cultivations of a plant with a high 13C/12C ratio (C4 photosynthetic pathway) on a soil which has never carried any such plant. The 13C/12C ratio in soil organic matter being about equal to the 13C/12C ratio of plant materials from which it is derived, changing the 13C content of the organic inputs to the soil (by altering vegetation from C3 type into C4 type) is equivalent to a true labelling in situ of the organic matter. Two cases of continuous corn cultivation (Zea mays: δ13C = −12%.) on soils whose initial organic matter average δ13C is −26%. were studied. The quantity of organic carbon originating from corn (that is the quantity which had turned-over since the beginning of continuous cultivation) was estimated using the 13C natural abundance data. After 13 yr, 22% of total organic carbon had turned-over, in the system studied. Particle size fractions coarser than 50μm on the one hand, and finer than 2μm on the other. contained the youngest organic matters. The turnover rate of silt-sized fractions was slower

The Development of a Marine Natural Product-based Antifouling Paint

Problems with tin and copper antifouling compounds have highlighted the need to develop new environmentally friendly antifouling coatings. Bacteria isolated from living surfaces in the marine environment are a promising source of natural antifouling compounds. Four isolates were used to produce extracts that were formulated into ten waterbased paints. All but one of the paints showed activity against a test panel of fouling bacteria. Five of the paints were further tested for their ability to inhibit the settlement of barnacle larvae, Balanus amphitrite, and algal spores of Ulva lactuca, and for their ability to inhibit the growth of U. lactuca. Two paints caused a significant decrease in the number of settled barnacles. One paint containing extract of Pseudomonas sp. strain NUDMB50-11, showed excellent activity in all assays. The antifouling chemicals responsible for the activity of the extract were isolated, using bioassay guided fractionation, and their chemical structures determined.