方向四热带生物多样性比较基因组学研究

方向四：热带生物多样性比较基因组学研究中国科学院西双版纳热带植物园2012年6月29日实施方案1.申报情况申报方向四的有4个研究组：生态进化生物学组、植物地理学研究组、植物分子生物学组、植物系统发育与保护研究组。

2.总目标利用新一代测序技术等分子生物学手段，探讨热带植物基因组多样化及基因组进化；开展热带植物比较基因组学研究，结合植物的系统分析，探讨热带植物功能性状进化及基因组差异；对特定功能基因开展系统深入研究，揭示功能基因发挥作用的分子机制、调控网络及应用途经。

3.主要任务23456784.保障措施1)通过自愿申报、交流讨论，选择符合目标任务要求的人员梯队及其研究团队，确定责任人，签订任务书或责任书。

2)制定总目标与年度工作计划，分步骤完成既定任务。

3)建立动态考核机制，适时对各研究组的工作进展进行考查。

对于考查结果不佳的团队，给予及时约谈或警示谈话，限期整改，并加强监督检查的频率。

如果整改效果不好，将向全园通报警示。

4)资金分配以均衡为原则，每组支持35万元。

预留10万元机动费。

5)在人力资源分配、条件保障、政策导向等相关方面给予倾斜。

5.附件一：各研究组申报材料9附件一：各研究组申报材料方向四：热带生物多样性比较基因组学研究目录生态进化生物学研究组 (11)植物地理学研究组 (18)植物分子生物学研究组 (29)植物系统发育与保护研究组 (41)生态进化生物学研究组1. 题目Genomic adaptations to environmental gradients in tropical trees2. 研究背景（≦500字）All species have a finite geographic distribution. The limits to their geographic distribution are often related to barriers to their dispersal, like large bodies of water, but many species are limited by environmental factors, like freezing temperatures and long periods of drought. The range of such species is therefore sensitive to changes in environmental conditions, and in case of abrupt climate change as currently observed, species need either to adapt to their new conditions, or migrate into favorable habitats (Burrows et al. 2011, Early et Sax, 2011, Chen et al, 2011). Species that cannot adapt or migrate will become extinct. Hence, it is of crucial importance to understand the genomic consequences of adaptation to marginal habitat to be able to predict the effects of climate change on tropical biodiversity. Here, we propose to investigate these genomic adaptations, by comparing species with restricted vs. widespread distributions, and species distributed at the margin vs. at the center of their favorable habitat.The Fagaceae are ecologically prominent in the tropical forests of Asia. Two genera are ecologically dominant and taxonomically diverse in the Asian tropics (Lithocarpus and Castanopsis) and form two biodiversity hotspots. The current distribution of these trees suggests that they are sensitive to seasonal rainfall and temperature. They are often used in the pollen record to indicate cooler wet climates in the past (Anshari et al., 2001; Morley, 2000; Zheng & Li, 2000). For these reasons, the family is a good model group for the historical distribution of evergreen tropical forests in Southeast Asia (Cannon & Manos, 2003) and the future response of these trees in global climate change.3. 拟解决的科学问题（≦500字）1) is genetic diversity limited in marginal vs core populations?2) what are the genomic differences between species with restricted vs. widespread ranges?3) what are the loci involved in adaptation to marginal habitat, and are they shared among species?4. 研究目标（≦200字）By comparing stone oak species and communities along a strong environmental gradient, we will test several hypotheses about the dynamics and evolution of genomic and phenotypic diversity in response to environmental conditions that are clearly limiting.5. 科学假说(Hypotheses)（≦500字）We hypothesize that the capacity of a species to adapt to very different conditions and hence have a wide distribution range depends on it's capacity to acquire adaptive genes through hybridization with other related species. If this hypothesis is true, widespread species should share a larger part of their genome with other species than restricted species.Additionally, we hypothesize that adaptive genes will be under strong purifying selection at the environmental limits of a group's distribution allowing us to directly identify those important genomic regions.6. 研究内容（≦1000字）The stone oaks (Lithocarpus) comprise the second largest genus in the Fagaceae, a globally important family of trees. In the tropical and subtropical forests of Indochina, the stone oaks are frequently ecologically dominant, particularly in the transition zone from tropical to subtropical forests. The subtropics form a natural boundary to the distribution of several genera in the family, including the stone oaks. The genera, Lithocarpus, Castanopsis, and Trigonobalanus, are only found to the south of the subtropical boundary while the genera, Quercus and Castanea, are only found in temperate forests (Zhou et al. 1995). An additional group, sometimes placed in the genus Quercus and sometimes recognized as a separate genus, Cyclobalanopsis, is only found south of the subtropical boundary. These Asian oaks are the only windpollinated trees in the family that persist in the tropical zone. The tropical genera of the Fagaceae are unique to Asia, being absent from Africa and the Americas.Because the boundary between temperate and subtropical forests is primarily defined by the ability of trees to withstand freezing temperatures, local or global climate change should have a direct impact on the geographic location of this boundary. Warming conditions should drive the boundary north while cooling conditions should drive the boundary south. The Hengduan Mts., in the foothills of the Himalayas, provide an excellent natural laboratory for examining the adaptation of forest trees to environmental extremes. The gradual uplift of the Himalayas has created a steep environmental gradient, from the warm evergreen forests of Xishuangbanna to the montane forests in Tibet over a relatively small geographic scale of ~700 kilometers.7. 研究方案（≦1000字）Sampling strategy.We will sample forest communities at three locations: 1) in lowland evergreen forests of Xishuangbanna, where taxonomic diversity is greatest; 2) in submontane evergreen forests of Ailoshan, where taxonomic diversity is relatively low but ecological dominance is high; and 3) at the limits of their elevational distribution in the Hengduan Mts of Northwest Yunnan and Tibetan Plateau. We will record and sample Lithocarpus individuals in each location (3 vegetation zones X 2 locations) along three parallel transects. All trees > 5 cm DBH will be sampled until twenty trees are recorded along each transect. Finally, for the population level analyses, we will first select 2-5 widespread Lithocarpus species (distributed across many different habitat types), and then we will collect 10-15 population samples (>10 inds.) for these species for each of the three general forest types.Objective 1: Comparison of genomic diversity between core and marginal species. This study will take advantage of our large existing database of whole genomic DNA sequence from 13 species of Fagaceae (Table 1), sequenced on the Illumina next gen platform since 2007. The DNA sequence coverage ranges from 3-10x for each species. We will sequence the whole genome of ~3-5 more species on the Illumina platform, including one Quercus/Cyclobalanopsis species. We have developed novel bioinformatic techniques for comparing genomic diversity and composition among these many species without having to first completely assemble a reference genome (Cannon et al., 2010). We will use these bioinformatic techniques together with referenced comparative techniques to identify important elements of genomic variation and polymorphism, and to compare the genomic diversity among the samples. To improve genome assembly of a few selected species, we will sequence their genome at a low coverage (1x) using PacBio SMRT sequencing technology that generate much longer reads than the Illumina platform (currently modal read length ~ 2000bp, with some reads much longer). The Illumina reads will be used to correct the errors in the PacBio reads before assembly. The long reads from PacBio combined with the high-coverage and high accuracy of Illumina will allow the assembly of much longer contigs than with any of these technology separately.Objective 2: Comparison of genomic diversity between species with restricted and widespread ranges. We will use similar approaches as for objective 1, but by focusing on the comparison of genomes of species with restricted vs. widespread ranges to identify genomic regions that may be involved in the wider ecological tolerance of widespread species.Objective 3: Identification of loci involved in adaptation to marginal habitats. Two different approaches will be used to fulfill this objective:1) Population genomics of adaptation to marginal habitats (population-level analysis): Recently, powerful methods of genotyping by sequencing such as RAD-seq (Miller et al. 2007) have allowed the simultaneous identification and genotyping of thousands of SNPs for any species. Using this method, we will first genotype at least 10,000 polymorphic SNPs for all individuals of the population sample. Populations from distinct habitats are expected to be under strong divergent selection, while populations from similar habitats are expected to be under balancing selections forloci providing a selective advantage in a given habitat. The identification of these loci (or at least genomic regions linked to these loci) is possible using genome scan methods (identification of outlier loci). For instance, a similar approach allowed the identification of loci involved in the parallel adaptation to freshwater habitats in sticklebacks (Hohenlohe et al. 2010). RAD loci length is typically limited to read length of the Illumina platform (typically 100bp currently), but by using pair-end sequencing, it is possible to assemble much longer loci linked to the RAD loci (Etter et al. 2011) that can allow the annotation of loci of interests, and the design of PCR primers for further in-depth analyses.2) Direct identification of loci under purifying selection: We will use local de novo assembly, centered on the informative elements identified for objectives 1 & 2, and the large amount of set of contigs and scaffolds generated by whole genome de novo assembly of Illumina data. We will use standard techniques to discover open-reading frames and to examine whether significant signal for stabilizing selection can be found. The main evidence for stabilizing selection is the ratio of non-synonymous (dN) changes in the nucleotide sequence compared to synonymous (dS) changes. Those genomic regions under stabilizing selection will exhibit very low dN/dS ratios (Hurst, 2002), and we will design PCR primers for further in-depth analyzes.We will then design primers for ~ 50 regions identified using these two approaches, and sequence them in numerous other individuals to verify their sequence. Forhigh-throughput sequencing, we will use the Roche 454 platform that allows sequencing of fragments with a modal length of 700bp. Such long reads will allow to sequence entire haplotypes and keep the phase between multiple SNPs at a given locus. Important loci under selection can then be confirmed by identifying selective sweeps (using Tajima's D test), and shifts in dN/dS ratios across the phylogeny (using PAML). We will perform bioinformatic analysis to annotate these loci using other plant genome databases.In parallel, we will use potted seedlings in a common garden setup to conduct high-and low-temperature treatments using the growth chambers, and measure physiology (the effect on photosystem 1 and photosystem 2).If the adult plants of these target species are available in a common garden such as in XTBG or Kunming Institute of Botany, we can analyze their hydraulic traits and wood anatomy to elucidate their adaptation to cold and dry habitats in terms of water transportation efficiency and safety.8. 年度目标Year 1: Comparative genomics with existing data and new genomes. Start field collection. Write paper on genome structure and phylogenomics. Setup seedlings in common garden.Year 2: Start RAD-sequencing and finish field collection. Design markers for the genomic regions of interest and start screening them on different populations. Write comparative genomics paper. Do physiology measurements.Year 3: Finish RAD-sequencing and screening of population samples. Write papers on population genomics and synthesis paper.9. 创新点（≦200字）This study represents the first effort to combine novel comparative genomic techniques, population genomics and ecophysiology of non-model forest tree species. The results from this study will provide a deep understanding of how forest tree species adapt to extremes in climate, particularly the effects of freezing temperatures at the boundary between subtropical and temperate forests. The stone oaks (Lithocarpus) are excellent model organisms for the study of forest tree evolution and adaptation. The genetic and taxonomic databases established during this study will provide the basis for abundant subsequent research into the forest dynamics in response to both current and historical climate change.10. 前期研究基础（≦1000字）1) In the last 5 years, Chuck Cannon have obtained whole genomic sequence data from the Illumina platform for over >12 species of Fagaceae from different geographic locations, including equatorial rainforest in Borneo and upland seasonal forest in Ailoshan. He has extensive experience in comparative genomics and developped a pipeline for comarative genomics without assembly. He also has morphometric databases of fruit morphology and nutritional analysis of seed variation.2) Yann Surget-Groba has extensive experience in bioinformatics, and onpopulation genomics approaches. He previously did comparative genomics studies to identify genes under selection, and used next-generation sequencing for population genomics approaches.3) Gao Jie has good experience in population genetics, and nextgeneration sequencing for population genomics approaches.4) Lang Tiange has experience in comparative genomics and bioinformatics.5) Joeri S. Strijk is currently conducting a comparative genomics study of Fagaceae.6) Cao Kunfang has been working for several years on the ecophysiology of several plant groups in relation to environmental stresses and adaptation to different environments.11. 是否需要与其它研究组合作We will collaborate with Cao Kunfang to obtain physiological data that will be correlated with genomic data to find genes involved in adaptation to stress, drought & cold tolerance. Established field stations at XTBG and Ailaoshan will immensely facilitate field work.12. 预期结果（论文发表、人才培养、队伍建设等）We are planing on publishing ~2 publications per year, several of these into journals like Molecular Ecology or higher (PNAS, PLoS Genetics, PLoS Biology, Genome Research...).We also plan to train several students and interns in the field of genomics and bioinformatics. This project will develop long term collaboration between the Ecological Evolution Group and the Group for Plant Physiological Ecology.The genomic and bioinformatic resources that we will build during this project will help building collaborations with other groups involved in plant molecular genetics.13. 经费估算We request 1,400,000 RMB (220,000 for library preparation equipment, 200,000 for genome sequencing, 200,000 for RAD sequencing, 200,000 for physiology, 140,000 for workshop organization, 120,000 for travel and conferences, 100,000 for high memory workstation, 100,000 for fieldwork, 80,000 for small laboratory equipment, 40,000 for publication costs).Our group has held the Workshop in Tropical Biodiversity and Genomics for two years, supported by the Yunnan High-End Talent grant awarded to Dr. Cannon. Theworkshop has been quite successful and well-received by its participants. We feel that continuing this international workshop, on a unique and compelling topic, is worthwhile, so we are requesting funding for the next several years. Previously, we have received a small grant (40,000 RMB) from the Bureau of International Cooperation in support of this workshop and we will continue to apply for this money each year.14. 需要我园提供的其它支持No15. 核心成员及所属研究组Yann Surget-Groba, Ecological Evolution GroupChuck Cannon, Ecological Evolution GroupGao Jie, Ecological Evolution GroupLang Tiange, Ecological Evolution GroupJoeri S. Strijk, Ecological Evolution GroupCao Kunfang, Group for Plant Physiological Ecology植物地理学研究组1. 题目Adaptive and plastic responses across trophic levels by natural populations to environmental gradients.2. 研究背景（≦500字）Many plant and animal species are shifting their geographical range in response to anthropogenic global change and has enormous consequences for our reliance on biodiversity (Parmesan 2006; Moss et al. 2010). Insect-plant science incorporates understanding genetic structure, ecological, and evolutionary significance of phenotype variation among interacting trophic levels. These attributes are critical in the elucidation of crop yield improvement, pest and invasive species management, biological organisation, and ecosystem function (Colautti et al. 2012). Populations on the limits of a species geographic range are excellent candidates for identifying ecologically meaningful genetic variation, as these individuals often show characteristics at the extremes of phenotype expression that are suited to marginal habitat (Kawecki 2008; Hill et al. 2011). These traits include: drought tolerance (Villar et al. 2011), pollinator service decline (Moeller et al. 2012), herbivore defense (DiGuistini et al. 2011), flowering phenology (Huang et al. 2011), dispersal (Mueller et al. 2011), mating/breeding system (Cheptou & Donohue 2011), invasiveness (Colautti et al. 2010), etc. A long-standing hypothesis predicts maladaptation with gene flow from central to marginal populations or alternatively, increases effective population size and genetic variation increasing fitness at the range limit of a species (Haldane 1948; Alleaume-Benharira et al. 2006; Hansen et al. 2012). However, it is not known whether plants and insects that rely on each other for survival and reproduction display common attributes in this regard. These hypotheses have not been tested in natural systems at geographic range limits.Ficus are a keystone component of tropical forests and host to an obligate pollination mutualism (Janzen, 1979). Spatial variation in plant species distributions are predominantly explained in terms of climate and energy variables (Wright, 1983; Hawkins et al., 2003). Climate and environmental change in conjunction with physiological tolerances (Warren et al., 2010) and dispersal capabilities (Ahmed et al., 2009) of its obligate fig wasp pollinator (Agaonidae) are also expected to strongly structure the distribution of Ficus among these biomes. The evolution of climate tolerance and biological invasion into apotypic environmental conditions by the obligate mutualism has not been empirically tested. One way to do this is to test thehypothesis that populations are differentially structured amongst distinct environments, depending on intrinsic genetic architecture that either adapts to novel conditions or tolerates them.Identifying sources of ecological variation within clades across the environments they inhabit is a challenging task (Hutchinson, 1959; Rosenzweig, 1987; Hawkins et al., 2003; Willig et al., 2003). The difficulty of separating one causal influence on species diversity over another is clouded by direct and indirect interdependencies among climate, physical environment, and species life history at different spatial scales (Algar et al., 2009; Field et al., 2009). Processes believed to contribute to the broad-scale distribution of lineages include the non-exclusive influences of latitude (Pianka, 1966), habitat type and area relationships (Rosenzweig, 1995),energy/climate/productivity variables (Wright, 1983), environmental heterogeneity, biotic interactions and life history (Ricklefs, 2006), and historical factors in general (Webb et al., 2002). Connecting ecologically meaningful species traits with genetic variation represents are clear way forward in the untangling of causal effects (Webb et al., 2002; Westoby, 2006; Graham & Fine, 2008; Fontaine et al., 2011). Mutualisms are rich sources of species diversity where host (plant) ecology is crucial to community organization and represent excellent case studies (Janzen, 1979; Herre, 1989; Kissling et al., 2007; Ghara et al., 2011).3. 拟解决的科学问题（≦500字）1) Which Ficus species are distributed across the most extreme environmental gradients?2) Is the genetic structuring of neutral markers between the range-limit and centralrange populations of both Ficus and wasp populations equivalent?3) Dispersal to environmentally favourable sites is different between Ficus and wasps?4) Do selection pressures on range-limit populations of Ficus and fig wasp populations result in population divergence?4. 研究目标（≦200字）The goal of the proposed study is to develop a conceptual basis that can differentiate between responses to long-term environmental change and short-term landscape disturbance. Different organisms have varied dispersal and intrinsic genetic architecture able to cope with such changes. This property is especially cryptic among interacting species such as in trophic webs. The undeniable changes to biodiversity that are driven by climate change and habitat disturbance have importantconsequences for sustainable economics. Understanding genetic and phenotypic variation in natural populations is an important strategy used to face these ultimate challenges.5. 科学假说(Hypotheses)（≦500字）We hypothesize that species with contrasting dispersal abilities and generation times have different strategies in coping with environmental shifts. Population responses to environmental shifts will either occur by tolerance (phenotypic plasticity), or adaptation (natural selection) to increasingly hostile environments such as for populations at the limits of a species distribution. Unlike insects, trees are unable to easily disperse away from conditions outside their physiological tolerance thresholds. These differences should manifest in population structure differences between those at the edge of the range compared to central-range populations. These contrasting life history characteristics will have consequence for interactions between insects and plants on the limits of their ranges where environmental change is most pronounced. This hypothesis postulates that i) genetic diversity of range-limit Ficus population will be divergent compared to central-range populations, and ii) that fig wasp population structuring will be increasingly less-dependent on host genetic structuring for species with increasingly more relaxed host-ranges.6. 研究内容（≦1000字）Ficus is a pantropical genus comprising approximately 800 species and is especially diverse in South-East Asia (Corner 1965; Rønsted et al. 2008). China includes 100 known species of Ficus. Ficus occupy a broad range of habitats growing as trees, shrubs, hemi-epiphytes, lithophytes, and vines, and are of eitherdioecious or monoecious breeding systems (Harrison 2005). Differences in species diversity exist among tropical Ficus communities in Australasia, Indo-Malay, Neotropical, and Afrotropical regions (Berg & Wiebes, 1992) and are characteristic of the abiotic and biotic environments they have evolved with. Beyond tropical distributions, Ficus display evolutionary conservatism of other habitat categories across mesic to xeric regions, each characterised by different phenotypes and environmental variables (Berg, 1990; McLeish et al., 2011). Ficus (Moraceae) is synonymous with the canopies of most tropical rainforest habitat types (Janzen, 1979). Figs are also host to the iconic fig wasp (Chalcidoidea) pollination mutualism (Weiblen, 2002). In many respects, the fig syconium is central to the reproductive viology of the pollination mutualism. Ecological scale influences on local fig species diversity have been investigated and showed that figs generally have high diversity and low abundance in tropical communities (Herre, 1996; Harrison et al., 2003). For any region, species diversity is an emergent property of habitat differentiation and climatic tolerance(Whittaker, 1975). For example, it has been useful to evaluate traits pertaining to soil moisture, light, or pH differences between habitats: Whittaker’s (1975) niche. Similarly, environmental water variables should have a strong bearing on regionalscale dimensions because they relate to climate. This highlights the difficulties in reconciling contributions of abiotic factors to diversity at different geographical and temporal scales (Whittaker et al., 2001; Graham & Fine, 2008; Field et al., 2009). Previous studies have demonstrated a clear link between Ficus phenotypes and the environment that has important consequences for the ecology and evolution of the fig wasp pollination mutualism (Patiño et al., 1994; Patiño et al., 1995; Hao et al., 2010). This evidence provides strong a priori expectations that environmental gradients influence hydraulic architecture and are directly related to environmental water variables and habitat qualities.Fig wasp genera and species have been classified in six subfamilies of wasps (Agaoninae, Epichrysomallinae, Sycoecinae, Sycophaginae, Sycoryctinae, and Otitesellinae) that are associated exclusively with Ficus syconia. Non-pollinating fig wasps (Sycoecinae & Otitesellinae: Pteromalidae) and pollinating fig wasps (Agaonidae) depend on host fig trees (Ficus Moraceae) for oviposition and reproduction, shelter, and nutrition. Non-pollinating and pollinating fig wasps are believed to maintain a high degree of specificity with their hosts. The Sycoryctinae (Pteromalidae) is a poorly understood group that includes distinct behavioural suites such as inquilines, parasitoids, and possibly hyperparasitoids. The Sycoecinae and Otitesellinae are all gall-inducers like the pollinators (Agaonidae). The Sycoryctinae are all parasitoids. The density, phenology, and reproductive characterisitcs of Ficus (Herre, 1989) are critical to the maintenance and stability of the pollination mutualism (Dunn et al., 2008; Cook & Segar, 2010). Transitions among life history strategies along environmental gradients have been observed in Southern African Ficus species (Burrows & Burrows, 2003). The dispersal ability of pollinating fig wasps, crucial to Ficus reproduction, is considerable (Ahmed et al., 2009) and risky due to predation, desiccation, and host density variation. Life inside the fig syconium also comes with risks due to predation by parasitoids and physiological tolerance limitations. Fig wasps are especially sensitive to heat and do not survive temperatures inside the syconium that vary much from the immediate surroundings (Patiño et al., 1994). Large syconia must transpire to maintain tolerable temperatures suited to wasp survival within the syconium while small syconia do not require transpiration for cooling to maintain tolerable temperatures. Drought tolerance traits should bestow Ficus with the ability to withstand water stress in environments outside the tropical forest. Phenotypic plasticity associated with the demands of contrasting water-related stresses should develop into lineage divergence along environmental gradients (Hao etal., 2010).7. 研究方案（≦1000字）Sampling strategy.Pilot studies using environmental niche modelling (Zang Ming Gang, Plant Geography Group) are currently underway. This approach allows specific targeting of the most suitable specie’s for revealing differences in genomes betw een populations on range-limits compared to central-range populations. The pilot study will also identify regions where the highest sample sizes are available. Twenty Ficus species have been identified that are not influenced by sample bias that causes error in the prediction of species distributions (Table 1). Actual locality information (herbarium records that have been georeferenced) will also be used to determine the best regions for field location collections. It is anticipated that species that are found commonly (high densities) such as F. hispida, and that are distributed both in environments at and adjacent to XTBG as well as in regions close to the Tibetan plateau, will be the focal species. Ficus species found commonly provide the best opportunity to collect and capture both plant and insect genetic variation. The speices modelling will indicate which species are most likely present across environmental gradients that show distinct characteristics in abiotic variables. McLeish et al. (in review A) have uncovered 2 key environmental variables that are significantly correlated with the presence of Ficus phenotypes across contrasting habitats. McLeish et al. (in review B) have also demonstrated the dimensions of host Ficus species-specificity for several fig wasp behavioural groups. This information is critical in the identification of factors that influence gene flow within species at different tropic levels. Environmental niche modelling will be combined with phylogeny to identify Ficus lineages that have been most influenced by historical environmental shifts (McLeish et al. 2011). This step is important because it can quickly and effectively identify those lineages that are know to possess phenotypes, and thus genotypes, that have either adapted to or tolerated environmental shifts. These data are already in hand and the process will be completed rapidly, and generate preliminary publication(s). Table 1: Candidate Ficus species that show significant non-bias in sampling.F.abeliiF.altissimaF.auriculataF.benjaminaF.filicauda。