Molecular evidence for an Asian origin and a unique westward migration of species in the genus Castanea via Europe to North America

The genus Castanea (Fagaceae) is widely distributed in the deciduous forests of the Northern Hemisphere. The striking similarity between the floras of eastern Asia and those of eastern North America and the difference in chestnut blight resistance among species has been of interest to botanists for a century. To infer the biogeographical history of the genus, the phylogeny of Castanea was estimated using DNA sequence data from different regions of the chloroplast genome. Sequencing results support the genus Castanea as a monophyletic group with Castanea crenata as basal. The three Chinese species form a strongly supported sister clade to the North American and European clade. A unique westward expansion of extant Castanea species is hypothesized with Castanea originating in eastern Asia, an initial diversification within Asia during the Eocene followed by intercontinental dispersion and divergence between the Chinese and the European/North American species during the middle Eocene and a split between the European and the North American species in the late Eocene. The differentiation within North America and China might have occurred in early or late Miocene. The North America species are supported as a clade with C. pumila var. ozarkensis, the Ozark chinkapin, as the basal lineage, sister to the group comprising C. pumila var. pumila, the Allegheny chinkapin, and Castanea dentata, the American chestnut. Morphological evolution of one nut per bur in the genus may have occurred independently on two continents.     

中国板栗EST-SSR信息分析及其通用性

从已公布的壳斗科(Fagaceae)基因组数据库中获得48501条中国板栗(Castanea mollissima)EST序列,其中8479条EST含有12116个长度大于10 bp的SSR,EST-SSR的出现频率为24.98%。二核苷酸重复和三核苷酸重复是中国板栗EST-SSR最主要的重复类型,分别占总数的38.05%和42.20%。对中国板栗EST-SSR标记在茅栗(C.seguinii)和锥栗(C.henryi)上的通用性检测表明,677对中国板栗EST-SSR引物皆能扩增,证实这些引物在栗属中国特有种间具有很高的通用性。115个位点的多态性分析表明,中国板栗、茅栗和锥栗分别有106、108和101个位点表现出多态性。在575个等位基因中,有260个(45.22%)是3物种的共有等位基因,各物种都有各自特有的等位基因。这项研究为中国栗属植物EST-SSR标记的建立及应用奠定了基础。     

Comparative chloroplast and nuclear DNA analysis of Castanea species in the southern region of the USA

Boundaries between American Castanea species (Castanea dentata, the American chestnut and C. pumila var. pumila, the Allegheny chinkapin, and var. ozarkensis, the Ozark chinkapin) have been difficult to establish because of intraspecific variation, interspecific similarities and the incidence of chestnut blight, which has prevented trees from maturing. In this study, informative chloroplast (cp) DNA and nuclear sequences from Castanea taxa were analyzed to gain a better understanding of their phylogeography in North America. Our emphasis has been on the most southern Castanea population in the Appalachian region, known for its morphological diversity. This Ruffner Mountain (Alabama) population shows a high number of unique haplotypes, which can be divided into two main groups. One group shares homology with the widespread and evolutionarily recent C. dentata haplotype. The other group shares homology with American chestnuts and Allegheny chinkapin taxa from southern states. This group has been the result of recent and more ancient cp capture and hybridization, indicative of hybrid zone clustering and glacial refugial origin. The range of C. pumila must have been more extensive along the Coastal Plains region, since only a few mutations separate the Ozark chinkapin from the main Allegheny chinkapin haplotype. The geographic origin of the American Castanea species complex appears to be in the Gulf Coast region.     

栗属中国特有种居群的遗传多样性及地域差异

采用超薄平板聚丙烯酰胺等电聚焦技术对栗属（Ｃａｓｔａｎｅａ（Ｔｏｕｒｎ．）Ｌ．）３个中国特有种即中国板栗（Ｃ．ｍｏｌｉｓｉｍａＢＬ．）、茅栗（Ｃ．ｓｅｇｕｉｎｉＤｏｄｅ．）和锥栗（Ｃ．ｈｅｎｒｙｉ（Ｓｋａｎ）Ｒｅｈｄ．ｅｔＷｉｌｓ．）的３０个居群,１２个酶系统的２０个位点进行了遗传多样性与遗传结构分析。结果表明：中国板栗在种和居群水平都具有较茅栗、锥栗高的遗传多样性（Ｐ分别为９０．０％和８４．７％,Ｈｅ分别为０．３１１和０．２９５）,尤以长江流域居群表现显著,揭示了长江流域的神农架及周边地区为中国板栗的遗传多样性中心。Ｎｅｉ的遗传一致度测量结果显示中国板栗和茅栗的亲缘关系最近,并且地理距离和遗传距离有一定的相关性。中国板栗、茅栗和锥栗的遗传分化程度逐渐增大,Ｇｓｔ分别为７．５％、１０．９％和２２．１％;基因流（Ｎｍ）分别为３．２０、２．０５和０．８８。研究结果为探明栗属起源、系统进化及制定保育策略提供了科学依据。     

栗属中国特有种居群的遗传多样性及地域差异

Genetic variability in 30 populations of three endemic Castanea (Tourn.) L. species (C. moUissima BI., C. seguinii Dode. and C. henryi (Skan) Rehd. et Wils. ) in China was investigated using isoelectric focusing in thin-layer polyacrylamide slab gels at 20 loci coding for 12 enzyme systems. C. mollissima was found to possess a significant higher value of genetic variability than that of the other two species. The percentage of polymorphic loci (P) and the expected heterozygosity (He) were 90.0% and 0. 311 at the species level, respectively; while P = 84.7 %, He = 0.295 at the population level. Comparison of the population genetic variability of C. mollissima in three regions of China, the populations from the Yangtze River valley showed a markedly higher mean expected gene heterozygosity. The results provided additional evidence that the Yangtze River valley, particularly in Shennongjia area, was the center of the genetic diversity of C. moUissima. Genetic relationships among populations and species were assessed by Nei genetic identity (I) and standard genetic distance (D), suggesting that C. mollissima and C. seguinii have a closer relationship, and genetic distances were correlated with geographical distances among populations. Genetic differentiation between populations in C. mollissima, C. seguinii and C. henryi was 7.5%, 10.9% and 22.1%, respectively, and the gene flow rate (Nm) was 3.20, 2.05 and 0.88 respectively. The study provides useful imfonnation for understanding of the origin and evolutionary events in genus Castanea and planning an effective conservation strategy.     

Non-target effects of transgenic blight-resistant American chestnut (Fagales: Fagaceae) on insect herbivores

American chestnut [Castanea dentata (Marshall) Borkhausen], a canopy dominant species across wide swaths of eastern North America, was reduced to an understory shrub after introduction of the blight fungus [Cryphonectria parasitica (Murrill) Barr] in the early 1900s. Restoration of American chestnut by using biotechnology is promising, but the imprecise nature of transgenesis may inadvertently alter tree phenotype, thus potentially impacting ecologically dependent organisms. We quantified effects of genetic engineering and fungal inoculation of trees on insect herbivores by using transgenic American chestnuts expressing an oxalate oxidase gene and wild-type American and Chinese (C. mollissima Blume) chestnuts. Of three generalist folivores bioassayed, only gypsy moth [Lymantria dispar (L.)] was affected by genetic modification, exhibiting faster growth on transgenic than on wild-type chestnuts, whereas growth of polyphemus moth [Antheraea polyphemus (Cramer)] differed between wild-type species, and fall webworm [Hyphantria cunea (Drury)] performed equally on all trees. Inoculation of chestnuts with blight fungus had no effect on the growth of two herbivores assayed (polyphemus moth and fall webworm). Enhanced fitness of gypsy moth on genetically modified trees may hinder restoration efforts if this invasive herbivore's growth is improved because of transgene expression.     

栗的 PGI遗传和多样性

采用超薄聚丙烯酰４胺平板凝胶等电聚焦电泳和单株后代法,分析了栗属种的ＰＧＩ同工酶的遗传,研究发现Ｐｇｉ位点（Ｐｇｉ－ｉ）主要有３个等位基因并呈共显性遗传。在栗属的自然居群中还检测了出现频率较少的另外２个等位基因。     

栗与美国板栗化感作用的比较

以种子发芽率和发芽势为种子萌发参数, 以胚根、胚芽长度和鲜重变化为幼苗生长参数, 研究了栗(Castanea mollissima)叶水提取物对双子叶植物莴苣(Lactuca sativa)、萝卜(Raphanus sativus)、黄瓜(Cucumis sativus), 和单子叶植物洋葱(Allium cepa)、水稻(Oryza sativa)、小麦(Triticum aestivum)的化感作用; 比较了美国板栗(Castanea dentata)与栗叶水提取物及经X-5大孔树脂分离获得的11个洗脱组分间的化感作用强弱; 利用液相色谱-质谱联用技术结合标准物质的反证实验, 解析了美国板栗与栗化感作用最强分离组分的物质结构。研究结果表明, 栗叶化感效应强于美国板栗; 美国板栗与栗化感作用最强分离组分中(5:5洗脱组分)存在绿原酸、对羟基苯甲酸、原儿茶酸和没食子酸等物质。在利用栗基因恢复美国板栗种群时, 有必要考虑美国板栗与栗植物化学生态特性的差异。     

Nutrient data on chestnuts consumed in the United States

American chestnuts (Castanea dentata) and Chinese chestnuts (C. mollissima) grown in the United States and imported European (Italian) chestnuts (C. sativa) were analyzed for proximate, mineral, and amino acid composition. Unlike most other tree nuts, chestnuts are low in protein and fat but high in carbohydrate. American and Chinese chestnuts were higher than European chestnuts for protein, ash, crude fiber, iron, magnesium, phosphorus, and copper but lower in fat. Essential amino acid patterns of chestnuts, as measured by chemical scoring, were about the same for American, Chinese, and European chestnuts.     

Heterozygote advantage in the American chestnut, Castanea dentata (Fagaceae)

The American chestnut (Castanea dentata; Fagaceae) was a dominant canopy tree in the Appalachian Mountains of North America. Since the introduction of the chestnut blight fungus (Cryphonectria parasitica; Valsaceae) in America, the American chestnut has been reduced to a predominantly clonal, understory species. Our objective was to determine whether the ecological changes and absence of new recruits have influenced the population genetics of American chestnut. Leaf samples were collected from four populations in southwestern Virginia. Electrophoretic data from five polymorphic loci were used to determine the genetic diversity and population structure of the populations and subpopulations. Growth data and infection status were recorded for one of the populations to determine their relationship with heterozygosity. F statistics revealed a significant amount of differentiation among subpopulations and an excess of heterozygotes within subpopulations. Heterozygous individuals also had higher rates of vegetative growth. The superior performance and excess of heterozygotes suggests that selection favors heterozygous individuals. The prolonged absence of sexual reproduction in C. dentata has allowed subtle fitness differences to accumulate to the extent that they have had significant effects on the genetics of chestnut populations.     

Allozyme variation in endangered Castanea pumila var. pumila

Allozyme genetic variation in 12 populations of the endangered Castanea pumila var. pumila (Allegheny chinkapin), sampled across the natural range of the species in the United States, was evaluated using 11 loci from seven enzyme systems. At the species level, the percentage of polymorphic loci (Ps) was 72.7 %, the mean number of alleles per locus (As) was 1.9, the mean number of alleles per polymorphic locus (APs) was 2.3, the effective number of alleles per locus (Aes) was 1.5 and the genetic diversity (Hes) was 0.296. At the population level, Pp = 49.2 %, Ap = 1.5, Aep = 1.4, APp = 2.1 and Hep = 0.21. Most of the allozyme variation (70 %) in C. pumila var. pumila occurred within populations. Wright's gene flow rate [Nm(W)] was as low as 0.57. Population differentiation along the species range was not detected. Populations of C. pumila var. pumila in Florida had the most variable levels of genetic diversity, but populations in Virginia and Mississippi also showed high levels. Based on the results of this study, conservation management strategies are discussed.     

我国栗属物种(Castanea millissima)地理分布及其空间特征分析

在广泛收集我国栗属3个物种板栗(C.mollissimaBl.)、茅栗(C.seguinii)和锥栗(C.Henryi(skan)Rehd.etWils.)地理分布资料的基础上,利用地统计学方法对它们的上限和下限的分布幅度进行了分析,给出了分布高度的插值结果,并对其估值结果进行了分析。结果表明,板栗和茅栗的分布上限和下限呈现出由东向西逐渐升高的趋势。而且越向西其变化趋势越为明显;锥栗则表现出由东南向西北逐渐升高的趋势,其变化幅度也相对较小。对3种植物分布高度插值结果进行的误差分析表明茅栗和锥栗的估测值和实际值非常吻合,茅栗误差较小,大都在4%以下锥栗误差在6%以下;而板栗由于多为人工栽培,受人为因素影响大,因此,其估测值的误差相对较大,在0～20%之间波动,但其大部分误差也都小于10%。这些结果说明,地统计学方法能够良好地拟合自然条件下物种的地理分布规律,而人为因素影响强烈下的物种由于其分布的规律性受到破坏而影响了预测效果。     

湖北的主要板栗品种资源及其野生近缘种

在实地调查及引种观察的基础上 ,对湖北 1 2个主要地方板栗品种的经济性状进行比较 ,并对其中的 6个主栽优良大果形品种作较详细的描述。此外 ,对湖北境内的野生板栗 ( Castanea mollissima Bl.) ,茅栗 ( C. se-quinii Dode) ,锥栗 ( C.henryi Rehd.& Wils)的特性及利用价值进行评价 ,首次报道了一种适合于嫁接栽培板栗的茅栗居群。     

Molecular divergence in the eastern Asia– eastern North America disjunct section Rytidospermum of Magnolia (Magnoliaceae)

Molecular divergence in the eastern Asia—eastern North American disjunct section Rytidospermum of Magnolia was investigated by allozyme electrophoresis, chloroplast DNA (cpDNA) restriction site analysis, and gene sequencing. We calculated Nei's genetic identities between two Asian species, M. officinalis var. biloba and M. hypoleuca, and three American species, M. tripetala, M. fraseri var. fraseri, and M. macrophylla var. macrophylla, by using gene frequency data from 17 nuclear-encoded allozyme loci in 67 populations. We then estimated cpDNA sequence divergence between the five species by examining restriction site variation for ten endonucleases over the entire genome. Finally, nucleotide sequences of the chloroplast gene rbcL were compared between M. hypoleuca, M. tripetala, and M. macrophylla var. macrophylla. All three methods consistently yielded low divergence values between the American species M. tripetala and its Asian sister taxa, M. officinalis var. biloba and M. hypoleuca (Nei's I = 0.712 and 0.809, respectively; D-cpDNA = 0.083% for both pairs; D-rbcL = 0.000% between M. tripetala and M. hypoleuca). The other two American species, M. fraseri var. fraseri and M. macrophylla var. macrophylla, neither of which is sister to the Asian taxa, exhibited much higher divergence from the Asian taxa. We interpreted the low divergence between M. tripetala and its Asian sister taxa as a result of recent separation (the late Miocene to early Pliocene), based on time estimates from molecular data as well as geological and paleoclimatic evidence. A comparison of our results with those of the earlier studies revealed a diverse array of levels of divergence between several eastern Asian and eastern North American species pairs. Though different extinction patterns and variation in molecular evolutionary rates may be partly responsible, this heterogeneous pattern of divergence is best explained by different times of disjunction in different taxa, which in turn suggests that the floristic similarity between the two continents was most likely attained by multiple migrations via both Bering and North Atlantic land bridges, or possibly even with involvement of dispersal.     

栗种间杂交系的同工酶基因与形态标记的遗传连锁关系研究

探讨了美州栗（ＣａｓｔａｎｅａｄｅｎｔａｔａＢｏｒｋｈ）与中国栗（Ｃ．ｍｏｌｌｉｓｓｉｍａＢｌｕｍｅ）种间杂交的１个Ｆ２代和２个回交一代家系中８个同工酶基因,Ａｃｐ３、Ｅｓｔ５、Ｐｒｘ１、Ｐｒｘ２、Ｐｒｘ３,Ｍｅ和Ａｄｈ与２个形态标记：Ｉｎｈ和Ｔｗｈ的连锁遗传关系,研究结果表明,在所有家系中,Ｉｎｈ与Ｐｒｘ１和Ｅｓｔ５具有一致的连锁关系,其基因的连锁顺序为Ｉｎｙ－Ｐｒｘ１－Ｅｓｔ５。还发现有参试的     

基于mtDNA COI基因的系统分析表明栗属植物上隐藏瘿蜂种的存在

本研究利用mtDNACOI基因的部分序列对11种群不同寄主的栗瘿蜂Dryocosm us kuriphilus Yasumatsu遗传多态性进行了分析。在获得的660bp的序列中,变异位点(V)80个,包括简约信息位点(Pi)15个和自裔位点(Si)65个。T、A、C、G平均含量分别为30.6%、43.3%、13.7%和12.4%,A+T平均值为73.9%,远高于G+C的26.1%,表现出明显的A+T偏向性。为害锥栗的六步溪、大围山种群与茅栗、板栗的不同地理种群,以及为害欧洲栗的意大利Piedmont种群之间,基因序列完全一致或者遗传距离在0.3%以内,无论是NJ系统树还是UPGMA系统树均聚合在同一分支内,亲缘关系较近。而为害锥栗的采自福建省建瓯市水源镇的水源种群、福建省周宁县纯池镇的纯池种群,与其它各栗瘿蜂种群的基因序列差异较大,遗传距离均大于2%,在NJ和UPGMA系统树中水源种群和纯池种群均聚合为一单独的分支,与其它种群形成的分支互为姊妹分支。据此我们认为水源种群和纯池种群很有可能属于不同的物种,有进一步研究的必要。     

栗属植物基因组DNA的提取及RAPD、SSR分析

以中国板栗、茅栗和锥栗的叶片为材料,提取栗属的DNA。针对栗属植物富含多酚类等次生物质的特点对栗属DNA的提取方法进行了改良,采用CTAB-蛋白酶K法加重复抽提,去除栗属植物中的相关次生物质,获得了高质量的DNA。所提取的DNA完全满足PCR、RAPD、SSR等一些分子生物学实验要求。     

板栗和锥栗天然同域居群的叶表型变异研究

对分布于湖北省秭归县境内的一个由169株板栗（Castanea mollissima）和281株锥栗（C.henryi）组成的天然同域居群的20个叶表型性状的变异进行了研究。根据叶片背面和叶柄的被毛情况及叶缘形状3个定性性状,在总共450个个体中,共得到18个（4%）中间过渡类型个体。利用17个数量性状进行逐步判别分析（SDA）表明：根据定性性状得到的中间型个体中分别有7个和11个被归入板栗和锥栗;板栗和锥栗在叶形上的差异较大,有8个数量性状被选入判别方程,判别方程对板栗和锥栗的判别准确率都达93%以上,判别结果较为理想;综合20个表型性状进行主成分分析（PCA）,构建了3个主成分,主成分1和2的贡献率分别为53.5%和18.7%,累计贡献率达72.2%。前2个主成分的散布图中,有6个（1/3）中间型个体位于板栗和锥栗的重叠分布区域,可能是二者之间存在杂交的表型证据。     

Modelling chestnut biogeography for American chestnut restoration

Aim Chestnuts (Castanea spp.) are ecologically and economically important species. We studied the general biology, distribution and climatic limits of seven chestnut species from around the world. We provided climatic matching of Asiatic species to North America to assist the range‐wide restoration of American chestnut [C. dentata (Marsh.) Borkh.] by incorporating blight‐resistant genes from Asiatic species. Location North America, Europe and East Asia. Methods General chestnut biology was reviewed on the basis of published literature and field observations. Chestnut distributions were established using published range maps and literature. Climatic constraints were analysed for the northern and southern distribution limits and the entire range for each species using principal component analysis (PCA) of fourteen bioclimatic variables. Climatic envelope matching was performed for three Chinese species using Maxent modelling to predict corresponding suitable climate zones for those species in North America. Results Chestnuts are primarily distributed in the warm‐temperate and subtropical zones in the northern hemisphere. PCA results revealed that thermal gradient was the primary control of chestnut distribution. Climatic spaces of different species overlap with one another to different degrees, but strong similarities are shown especially between Chinese species and American species. Climatic envelope matching suggested that large areas in eastern North America have a favourable climate for Chinese species. Main conclusions The general biological traits and climatic limits of the seven chestnut species are very similar. The predictions of Chinese species climatic range corresponded with most of the historical American chestnut range. Thus, a regionally adapted, blight‐resistant, introgressed hybrid American chestnut appears feasible if a sufficiently diverse array of Chinese chestnut germplasm is used as a source of blight resistance. Our study provided a between‐continent climate matching approach to facilitate the range‐wide species restoration, which can be readily applied in planning the restoration of other threatened or endangered species.