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.     

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。     

Inadequate Cold Tolerance as a Possible Limitation to American Chestnut Restoration in the Northeastern United States

The American chestnut (Castanea dentata (Marshall) Borkh.), once a major component of eastern forests from Maine to Georgia, was functionally removed from the forest ecosystem by chestnut blight (an exotic fungal disease caused by Cryphonectria parasitica (Murr.) Barr), first identified at the beginning of the twentieth century. Hybrid‐backcross breeding programs that incorporate the blight resistance of Chinese chestnut (Castenea mollissima Blume) and Japanese chestnut (Castenea crenata Sieb. & Zuc.) into American chestnut stock show promise for achieving the blight resistance needed for species restoration. However, it is uncertain if limitations in tissue cold tolerance within current breeding programs might restrict the restoration of the species at the northern limits of American chestnut's historic range. Shoots of American chestnut and hybrid‐backcross chestnut (i.e., backcross chestnut) saplings growing in two plantings in Vermont were tested during November 2006, February 2007, and April 2007 to assess their cold tolerance relative to ambient low temperatures. Shoots of two potential native competitors, northern red oak (Quercus rubra L.) and sugar maple (Acer saccharum L.), were also sampled for comparison. During the winter, American and backcross chestnuts were approximately 5°C less cold tolerant than red oak and sugar maple, with a tendency for American chestnut to be more cold tolerant than the backcross chestnut. Terminal shoots of American and backcross chestnut also showed significantly more freezing damage in the field than nearby red oak and sugar maple shoots, which showed no visible injury.     

Effects of transgenic American chestnut leaf litter on growth and survival of wood frog larvae

Biotechnology offers a new approach for the restoration of tree species affected by exotic pathogens; however, nontarget impacts of this novel strategy on other organisms have not been comprehensively assessed. We evaluated the effect of transgenic American chestnut (Castanea dentata) leaf litter on the growth and survival of larval wood frogs (Lithobates sylvaticus), a forest‐dwelling amphibian species widely sympatric with American chestnut, that forage almost entirely on periphyton and litter detritus that accumulate in temporary vernal pools in forests. We reared wood frog larvae on Castanea leaf litter (American chestnut genetically engineered for blight tolerance, nontransgenic American chestnut, Chinese chestnut [Castanea mollissima], and an American–Chinese chestnut hybrid) and litter from two non‐Castanea, nontransgenic “control” tree species, coupled with two levels of supplementary food. We observed no differences in growth or survival of wood frog larvae reared on transgenic versus nontransgenic American chestnut leaves. Without supplementary food, wood frog larvae provided leaves from American chestnut (both types) developed faster and grew larger than those exposed to other leaf litter treatments. Results of this study provide preliminary evidence that (1) American chestnut may have formerly been an important source of food for forest‐dwelling amphibians and (2) transgenic American chestnut litter generated as part of chestnut restoration efforts is unlikely to present direct novel risks to developing amphibian larvae in the forest environment.     

Interactions between a fungal pathogen, foliar properties, and generalist herbivores

American [Castanea dentata (Marsh) Borkh.] × Chinese [Castanea mollissima Blume] chestnut (Fagac, ae) hybrids are a novel system in which to study influences of phytopathogenic fungi and woody plant hybridization on herbivore susceptibility, as the hybrids are well characterized with regard to resistance to the chestnut blight fungus [Cryphonectria parasita (Murr) Barr (Endothia) Diaporthales: Valsaceae] and variability is present. We chose two groups of resistance‐rated backcross chestnut that shared an F1 parent and had different American parents. Foliage from both backcross groups and the parent trees was sampled on three dates for use in feeding assays with gypsy moth larvae [Lymantria dispar (L.) [Lepidoptera: Lymantriidae], adult Japanese beetles [Popillia japonica Newman (Coleoptera: Scarabaeidae)], and fall webworm larvae [Hyphantria cunea Drury (Lepidoptera: Arctiidae)], respectively. Foliar analyses were performed concurrently and included carbohydrate, tannin, and nitrogen content, toughness, and density. Blight resistance had almost no effect on herbivore performance or foliar chemistry. When the parent trees and backcross groups were compared, however, significant differences in gypsy moth performance and Japanese beetle consumption were evident. There were no differences in fall webworm consumption. Most foliar characteristics measured differed among chestnut genotypes at some point in the season, and all varied seasonally. No clear pattern emerges with respect to the relationship among blight resistance, herbivore susceptibility, foliar properties, and plant genotype, and more research is needed to separate these effects.     

Comparisons of Ectomycorrhizal Colonization of Transgenic American Chestnut with Those of the Wild Type,a Conventionally Bred Hybrid,and Related Fagaceae Species

American chestnut (Castanea dentata [Marsh.] Borkh.) dominated the eastern forests of North America, serving as a keystone species both ecologically and economically until the introduction of the chestnut blight, Cryphonectria parasitica, functionally eradicated the species. Restoration efforts include genetic transformation utilizing genes such as oxalate oxidase to produce potentially blight-resistant chestnut trees that could be released back into the native range. However, before such a release can be undertaken, it is necessary to assess nontarget impacts. Since oxalate oxidase is meant to combat a fungal pathogen, we are particularly interested in potential impacts of this transgene on beneficial fungi. This study compares ectomycorrhizal fungal colonization on a transgenic American chestnut clone expressing enhanced blight resistance to a wild-type American chestnut, a conventionally bred American-Chinese hybrid chestnut, and other Fagaceae species. A greenhouse bioassay used soil from two field sites with different soil types and land use histories. The number of colonized root tips was counted, and fungal species were identified using morphology, restriction fragment length polymorphism (RFLP), and DNA sequencing. Results showed that total ectomycorrhizal colonization varied more by soil type than by tree species. Individual fungal species varied in their colonization rates, but there were no significant differences between colonization on transgenic and wild-type chestnuts. This study shows that the oxalate oxidase gene can increase resistance against Cryphonectria parasitica without changing the colonization rate for ectomycorrhizal species. These findings will be crucial for a potential deregulation of blight-resistant American chestnuts containing the oxalate oxidase gene.     

Restoration of threatened species: a noble cause for transgenic trees

Some of the first applications of transgenic trees in North America may be for the conservation or restoration of threatened forest trees that have been devastated by fungal pathogens or insect pests. In some cases, where resistance has yet to be found in the natural population of a tree species, incorporating genes from other organisms may offer the only hope for restoration. In others, transgenics may play a role as part of an integrated approach, along with conventional breeding or biocontrol agents. American chestnut (Castanea dentata) was wiped out as a canopy species by a fungal disease accidentally introduced into the United States around 1900. Similarly, American elm (Ulmus americana) virtually disappeared as a favored street tree from Northeastern U.S. cities after the introduction of the Dutch elm disease fungus in the 1940s. In both cases, progress has been made toward restoration via conventional techniques such as selection and propagation of tolerant cultivars (American elm) or breeding with a related resistant species (American chestnut). Recently, progress has also been made with development of systems for engineering antifungal candidate genes into these “heritage trees.” An Agrobacterium-leaf disk system has been used to produce transgenic American elm trees engineered with an antimicrobial peptide gene that may enhance resistance to Dutch elm disease. Two gene transfer systems have been developed for American chestnut using Agrobacterium-mediated transformation of embryogenic cultures, setting the stage for the first tests of potential antifungal genes for their ability to confer resistance to the chestnut blight fungus. Despite the promise of transgenic approaches for restoration of these heritage trees, a number of technical, environmental, economic, and ethical questions remain to be addressed before such trees can be deployed, and the debate around these questions may be quite different from that associated with transgenic trees developed for other purposes.     

A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests

Cystatin CsC, a cysteine proteinase inhibitor from chestnut (Castanea sativa) seeds, has been purified and characterized. Its full-length cDNA clone was isolated from an immature chestnut cotyledon library. The inhibitor was expressed in Escherichia coli and purified from bacterial extracts. Identity of both seed and recombinant cystatin was confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis, two-dimensional electrophoresis and N-terminal sequencing. CsC has a molecular mass of 11275 Da and pI of 6.9. Its amino acid sequence includes all three motifs that are thought to be essential for inhibitory activity, and shows significant identity to other phytocystatins, especially that of cowpea (70%). Recombinant CsC inhibited papain (Ki 29 nM), ficin (Ki 65 nM), chymopapain (Ki 366 nM), and cathepsin B (Ki 473 nM). By contrast with most cystatins, it was also effective towards trypsin (Ki 3489 nM). CsC is active against digestive proteinases from the insect Tribolium castaneum and the mite Dermatophagoides farinae, two important agricultural pests. Its effects on the cysteine proteinase activity of two closely related mite species revealed the high specificity of the chestnut cystatin.     

中国板栗EST-SNP和抗栗疫病候选基因分析及同源比对

从壳斗科基因组数据库下载中国板栗的Unigene序列和基于这些Unigene序列开发获得的EST-SNP数据,分析发现,中国板栗EST-SNP的发生频率为4个/kb,在碱基置换类型上,C-T置换发生的频率最高,C-G置换发生频率最低,转换和颠换的比值为1.74∶1.对211个在中国板栗健康组织和感染栗疫病的染病组织中差异表达的基因进行基因注释和蛋白质结构域分析,结果表明,参与蛋白质代谢、对胁迫、生物和非生物刺激发生响应的基因所占的比例较多,并发现了大量的跨膜结构、信号肽、卷曲螺旋和蛋白激酶相关结构域.对这211个中国板栗抗栗疫病相关的候选基因和其Unigene序列进行同源比对,统计整理定位在具有同源性的Unigene序列上的EST-SNP,共有3023个EST-SNP标记.这批EST-SNP标记可为今后开展基于候选基因途径的中国板栗抗栗疫病居群基因组学研究和关联作图研究奠定重要的基础.     

Isolation of wound inducible genes from Castanea sativa stems and expression analysis in the bark tissue

In vitro shoot cultures of chestnut (Castanea sativa Mill.) were used to identify wound-responsive genes. cDNA fragments of genes induced 3 and 24 h after wounding were isolated from stem tissue by the differential mRNA display method. Corresponding partial and full-length clones were isolated from a cDNA library of wounded stems. Putative wound-responsive signalling genes (serine–threonine protein kinase, two calmodulin genes), a novel wound-responsive putative chaperon gene (Csp13.9), and a new family of proline-rich proteins were identified. Northern analysis of bark tissue from 14-month-old seedlings revealed strong induction of these genes upon wounding in a temporal manner. Therefore we conclude that these early wound-inducible genes are involved in the wound response of bark tissue.     

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.     

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.     

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.     

Selection,caching, and consumption of hardwood seeds by forest rodents: implications for restoration of American chestnut

Recent field trials on blight‐resistant hybrids (BC₃F₃) of American chestnut (Castanea dentata) and Chinese chestnut (C. mollissima) have intensified planning for widespread restoration of Castanea to eastern U.S. forests. Restoration will likely rely on natural seed dispersal from sites planted with chestnut; however, we do not know how dispersal agents such as granivorous rodents will respond to hybrid chestnuts. At one extreme, excessive seed consumption may impede restoration. Alternatively, scatter‐hoarding rodents might facilitate the spread of chestnut by dispersal of seeds from restoration plantings. We conducted trials with five rodent species to quantify foraging preferences and to evaluate the potential role of granivores in chestnut restoration. Specifically, we presented seeds from American and hybrid chestnuts (BC₃F₂) with other common mast species and recorded the proportion of seeds removed and the fates of tagged seeds. Mice, chipmunks, and flying squirrels harvested both chestnut types preferentially over larger, tougher black walnut, hickory, and red oak seeds, but fox squirrels and eastern gray squirrels preferred larger seeds to chestnuts. All rodents consumed a greater proportion of the chestnuts than other seed types. American and hybrid chestnut also differed in important ways: except for fox squirrels, rodents preferentially removed American chestnuts over hybrid chestnuts, but we estimated that fox squirrels carried a greater proportion of hybrid chestnuts beyond our tag search area, suggesting that hybrids may be dispersed farther and cached more often than American chestnut. These differences indicate that hybrid chestnut may not be functionally equivalent to American chestnut with regard to seed–granivore interactions.