克隆植物对异质性环境的生态适应对策

时空异质性是生境的基本特征。几乎所有植物都是在一定尺度的时空异质性环境中完成其生活史的。在进化过程中，植物可能形成了各种有效利用环境异质性的生态适应对策。克隆生长使得克隆植物在理论上更容易适应于异质性环境。本文以匍匐茎和根状茎型草本为材料，应用实验生态学方法研究了游击型克隆植物对异质性环境的生态适应对策。 在沙丘生境（如毛乌素沙地）中，沙埋是植物常常遭遇的事件。由于沙埋在水平空间表现出非均匀性，克隆植物的基株或克隆片断常常经历局部沙埋。通过温室和野外实验，研究了克隆整合作用对鹅绒委陵菜和沙鞭沙埋分株忍受沙埋能力的影响。结果表明，克隆整合显著提高鹅绒委陵菜和沙鞭沙埋分株的存活。进一步的耗-益分析表明，克隆整合使鹅绒委陵菜沙埋分株显著受益，而对非沙埋分株却没有显著耗损，故整个克隆片断的生长得到显著提高。因此，克隆整合是沙丘生境中克隆植物对局部沙埋胁迫的生态适应对策之一。 通过2个温室实验，研究了金戴戴对光照、基质养分和盐分的克隆可塑性。结果表明：光照强度、基质养分和盐分对金戴戴克隆生长和克隆形态均有十分显著的影响。深度遮光、低养和高盐均显著削弱金戴戴的生长，其生物量、叶面积、分株数、匍匐茎长及叶柄长和根冠比对基质盐分的可塑性大小和格局显示出基株间的差异。在低养分条件下，金戴戴匍匐茎节间显著伸长，而分枝强度显著减弱。这些结果与克隆植物觅食模型相符合，表明当生长于异质性环境，金戴戴可凭借克隆可塑性实现的觅养行为来增加对养分资源的摄取。因此，克隆可塑性是克隆植物利用环境异质性的另一条途径。 在另一温室实验中，研究了三种匍匐茎克隆草本鹅绒委陵菜、金戴戴和绢毛匍匐委陵菜对光照和养分资源交互斑块性环境的反应。当置于高光低养下的分株与低光高养下的分株相连时，高光低养分株、低光高养分株以及克隆片断的生物量均得到显著提高。同时，低光高养下分株的根冠比相对增加，而高光低养下分株的根冠比相对下降。这表明，三种克隆植物发生了环境诱导的克隆内分工行为。这种环境诱导的克隆内分工行为有利于整个基株对资源交互斑块性环境的利用，是克隆植物对异质性环境的生态适应对策。

异质性环境中两种匍匐茎草本植物的克隆可塑性

　　克隆植物常常生长在资源异质性分布的环境中，克隆植物特有的生活史特征使其对环境和资源变化产生的反应规范（Reaction norm）具有特殊的表现和适应意义。本文以匍匐茎草本植物蛇莓(Duchesnea indica)和活血丹(Glechoma longituba)为材料，研究克隆植物在不同异质性环境中的反应规范，即可塑性（plasticity）和克隆分株间相互作用（即克隆整合（clonal integration））对表型的影响及其适应意义。 蛇莓和活血丹是具有不同分枝类型的两种匍匐茎草本植物。对于匍匐茎草本植物而言，生境中光资源分布在时间和空间上的变化极为丰富。本文主要研究在不同环境中，这两种克隆植物克隆分株间的相互作用是如何通过影响其形态特征，从而影响其光资源获取策略。 实验涉及了4种光照条件，分为5个部分，分别在大棚和野外进行。 1．同质光环境中蛇莓和活血丹克隆片段的可塑性 在大棚中，将整个克隆片段（clonal fragment）都放置在光照强度分别为自然光照PPFD（Photosynthetic Photon Flux Density）的100％、50％和10％三种不同的同质光环境中，目标是通过比较不同光照强度同质环境中不同基因型的形态特征，揭示匍匐茎草本克隆植物的反应规范及遗传差异。实验结果发现，当光照强度从100% PPFD 降到 50% PPFD时，活血丹增加对地上部分生物量投资，增加部分多投入到匍匐茎上，用于支持匍匐茎实现觅食行为，叶片和叶柄的变化不大。同样条件下，蛇莓的生物量投资却向根部转移，地上部分叶片和叶柄形态上的变化比匍匐茎的形态变化显著，出现增强对光资源吸收能力的变化。当光照强度从50%降到 10%时，活血丹继续增加对地上部分的生物量投资，但由于光照强度弱，几乎不足以维持新的匍匐茎的发生，所有增加的生物量部分都投入到叶片和叶柄的变化上。这种条件下，蛇莓的生物量投资依然向根部转移，叶片和叶柄为增加对光资源吸收能力而发生了相应变化，匍匐茎的新增数减少，形态变化也不大。实验证明，可能由于匍匐茎的发生方式不同，活血丹的匍匐茎节间长度，叶片和叶柄的关联程度比蛇莓相应性状的关联程度强。不同基因型的蛇莓和活血丹对光照梯度的反应有显著差异。 2．异质光环境中蛇莓克隆片段的可塑性 针对克隆植物分株间在相当长的时间里形体相连，而环境异质性可能存在于分株大小的空间尺度，设置异质环境，将蛇莓的不同相连分株放置在不同的光资源斑块中，研究其整合作用在适应异质性环境过程中的作用及其对表型的影响。首先设置同一种资源（光资源）的梯度差异斑块。 在光斑块中，整合作用发生与否、作用方式与资源梯度、分株自身所处斑块的资源条件和分株的年龄结构有关。在不同的条件下，整合作用可能造成对表型的不同影响，从而可能加剧或削弱分株对本地资源的反应（局部反应，local response）。整合作用对分株局部反应的影响强度和方向，在不同基因型间存在明显差异。这些基因型间的差异暗示，整合作用可能也是具有遗传基础的一种独立性状。 3．光块斑和养分斑块共存环境中活血丹克隆片段的可塑性 设置光和养分的资源互补性斑块。将相连分株种植在不同斑块中。当生长在高光低养斑块中的分株与其互补斑块（低光高养斑块）的分株相连时，其适合度相关性状的值增加，根冠比是可塑的，通过相应的形态变化，高光照斑块中的分株捕获光资源的能力增强，但两种斑块中植株的吸收养分的能力却没有大的变化。收益－损耗分析显示整合作用有益于异质环境中的植株，低光高养斑块中植株的生物量获益。实验结果证明了异质环境中相连分株间存在光合产物和养分传递。在这种环境种，分株形态的变化对本地斑块发生趋富反应，形态的改变有助于对本地丰富资源的吸收。这种反应有利于克隆片段对资源的吸收。 4．野外环境中蛇莓克隆片段的可塑性 基于大棚实验资料，对野外林下蛇莓种群进行监测。首先对单株在林下复杂的光环境中的表现进行了跟踪，结果发现，随着分株数目的增加，适合度相关性状值有所提高，而处于不同位置的相连分株在形态上的差异不显著。大棚实验中发现的显著形态变化在具有显著差异的自然环境条件下没有发生，这可能暗示着在资源条件变化频率较高的环境中整合作用的作用方式。分株不是对某一时刻资源条件进行形态特化，而是通过对所扩展的总体区域的总的环境条件调整表型。这样，从克隆片段的水平上看，资源的吸收可能达到较高水平。在密度增大，植株间互相荫蔽使得光照减弱的情况下，匍匐茎的变化没有表现出觅食行为，叶片和叶柄出现增大增长等增强吸收光能力的相应表型变化，证明叶片叶柄是克服光照不足的主要器官。这些结果与大棚实验结果一致。蛇莓基株对于相连分株内部可能具有密度调节功能，从而通过减弱叶片增大的趋势，增长匍匐茎，分散新生分株，使密度保持在一定程度，不对种群的发展造成阻碍作用。非相连分株间随着密度的增大，相互间的作用方式类似于非克隆植物：出现叶片增大，叶柄增长的现象，同时密度制约了适合度相关性状如分株数目等的增加。 5．野外环境中蛇莓种群格局动态 最后，在进化的单位，种群水平上对蛇莓种群在自然界的动态进行了调查。Spearman相关分析，没有发现光资源和测量指标在量上的相关。在自然界中整合作用使大量分散分布、相互连接的分株相互作用，以促进基株对资源的获取。Moran’s I指数分析显示，随时间的推进，各个指标在相关尺度上有所增加。这暗示了整合作用的存在，分株间联系的加强。大棚实验中观察到的关于各个器官的作用的结论在自然界中得到进一步的验证。蛇莓匍匐茎在遮荫环境中不是作为觅食器官。为了争取对更多光资源的吸收，蛇莓在叶片数和叶形态上发生改变。对分株数、叶片数、匍匐茎数目的分布格局的调查显示出没有一定的规律性，而且处于不断的变化中。这一结果可能暗示种群发展过程中处理异质性分布资源的对策的变化。 此外，本文还揭示了可塑性和整合作用在基因型间的差异，针对所发现的现象，从作用的遗传机制上对可塑性及其适应意义进行了讨论。结合分子生物学和地统计学等学科的研究成果，对进一步的实验提出了方法和路线。

克隆植物绢毛匍匐委陵菜的生态对策研究

本文以匍匐茎草本绢毛匍匐委陵菜(Potentilla reptans L var. sericophylla)为研究对象，基于实验生态学研究方法和野外调查研究了该植物对异质性环境的生态适应对策。 　　对落叶松和油松群林下的绢毛匍匐委陵菜的基株特征和种群特征研究表明：在落叶松林下，绢毛匍匐委陵菜克隆分株具有更大叶面积和更长叶柄;在油松林下，绢毛匍匐委陵菜具有更多的匍匐茎和更多的克隆分株。两个生境中的绢毛匍匐委陵菜形成与其生境相适应的克隆形态和克隆生长特征。在两个群落中的绢毛匍匐委陵菜分株种群的密度和地上生物量没有差异。 　　应用GS+统计软件分析了绢毛匍匐委陵菜分株种群特征及其与所处的土壤基质九个指标（土壤有机质（SOC）、土壤全氮（STN）、土壤全磷（STP）、有效钾（K+）、有效磷（PO43-）、NH4+、NO3-、土壤含水量（SWC）、pH）的空间分布格局的关系，结果显示，绢毛匍匐委陵菜克隆分株种群呈斑块状分布，其克隆分株数的空间自相关尺度为1米;其生长的土壤基质的9个土壤养分及土壤性状指标也都显示了强烈的空间自相关，其自相关尺度（变程）范围在0.24 m ～1.05 m之间，大小顺序为STP>K+>SOC>pH>STN> NO3-> PO43->SWC> NH4+。 其中除STP外，其他8个土壤指标的空间自相关尺度都在0.5 m 左右，NH4+仅为24.4 cm。绢毛匍匐委陵菜野外克隆分株的匍匐茎节间长平均在7 cm 左右。由此表明绢毛匍匐委陵菜克隆分株种群的空间分布尺度大于其所在土壤基质养分及其物理性状的分布尺度，相互联结的克隆分株对异质性土壤资源表现出较大的缓冲能力。其原因之一有可能是克隆生理整合作用使克隆分株局部反应减弱，克隆分株表现趋于一致，克隆分株间实现资源共享，从而高效利用异质性土壤资源。 　　绢毛对匍匐委陵菜克隆特征的局部特化实验将生长在北京东灵山油松（Pinus tabulaeformis）林林窗和林下的绢毛匍匐委陵菜，进行生境间的交互移植-重植野外生态实验以验证绢毛匍匐委陵菜林窗和林下种群间的行为差异是完全由表型可塑性引起，还是局部分化的结果。研究结果表明，实验植物的叶片长度、叶片宽度、叶柄长度和匍匐茎节间长度等克隆形态特征在两生境间无差异。两个来源的植株，其基株生物量、基株分株数和基株匍匐茎总长度等克隆生长特征在林下生境中都比在林窗生境中小，表现出显著的可塑性。所研究的克隆形态特征和克隆生长特征及其可塑性在不同生境来源的实验植物间没有差异。绢毛匍匐委陵菜克隆形态特征和克隆生长特征及其可塑性在林下和林窗生境间没有发生局部分化，林窗为其较适生境，克隆生长特征的可塑性对绢毛匍匐委陵菜利用生境异质性可能具有主要意义。 　　绢毛匍匐委陵菜对土壤养分的克隆可塑性共进行了两个实验。实验1 探讨绢毛匍匐委陵菜对8个土壤基质养分梯度(N, P, K)的克隆可塑性。其结果表明，在植株能够生长的土壤基质养分条件下，随着土壤养分浓度增加，基株生物量、匍匐茎数、分株数增加;生物量分配可塑性反应格局为：随着土壤基质养分浓度的增加，对根生物量投资降低，对叶片的生物量投资增加，而对匍匐茎的投资没有改变。匍匐茎节间长、比叶片重没有对土壤养分梯度发生可塑性反应。随着土壤基质养分的增加，植株根冠比和比根重下降，而根系总长和叶片面积增加。实验2 设置与实验1相同的8个土壤养分梯度，分三个时期收获以探讨绢毛匍匐委陵菜不同发育时期对土壤养分梯度的克隆可塑性，其结果表明：随着植株发育年龄的增加，绢毛匍匐委陵菜的克隆可塑性有增强的趋势。生物量分配格局对土壤养分梯度的可塑性反应早于基株总生物量的可塑性反应;叶片面积、根系总长度和比根重在植物生长后期对土壤养分梯度发生可塑性反应;比叶面积在植物生长早期对土壤养分梯度发生可塑性反应，而在后期则没有;匍匐茎比节间重在生长之初和生长后期均对养分梯度发生可塑性反应。 　　绢毛匍匐委陵菜对局部遮荫的克隆可塑性实验是将采自林窗和林内生境的绢毛匍匐委陵菜“分株对”（即由一匍匐茎节间相连着的两个分株，其一为“目标分株”另一为“相连分株”）在一户外实验中进行全不遮荫、全部遮荫和局部遮荫处理。其结果表明，绢毛匍匐委陵菜基株生物量、匍匐茎总长度、分株数、匍匐茎比节间重、叶柄长、比叶柄重在遮荫条件下较小。匍匐茎节间长度没有对遮荫处理发生反应。在局部遮荫处理，遮荫斑块的分株的叶柄长度由于连着未遮荫斑块中分株而变得更长。这种克隆整合对克隆形态可塑性的修饰作用只在林窗生境来源的实验植物中观察到。其它克隆生长和克隆形态特征的可塑性在不同生境来源的实验植物间没有差异。

克隆植物东方草莓对异质生境的适应对策

克隆植物被认为比非克隆植物更宜于利用异质性环境。在复杂的空间异质环境中，克隆植物可能形成了各种有效利用环境异质性的适应对策。对于克隆植物适应机制的研究，前人已做了大量的工作，特别是从形态和生物量分配等方面对简单异质生境下克隆植物的克隆整合和克隆分工进行了详细的研究。本研究以分布广泛的克隆植物东方草莓（Fragaria orientalis）作为研究对象，应用野外调查和实验生态学方法，采用多对比度单资源模型和不同向双资源模型，从形态和生理生态的角度，研究复杂异质生境下克隆植物的整合和分工及其耗益问题，分析不同类型的生境对克隆植物整合和分工的修饰作用，进而探讨克隆植物对异质生境的适应策略。克隆构型和分株种群特征是植物克隆生长及其生态适应对策研究的基本内容。本文通过野外调查，研究在不同光照条件下东方草莓克隆构型、分株种群特征以及点分布格局。结果表明：东方草莓的克隆构型随光照发生相应的变化，低光照下其匍匐茎节间长和分枝角度均增大而分枝强度减小；随光照减弱，东方草莓分株种群的生物量、根冠比和分株种群密度显著降低；不同光照下东方草莓分株均以随机分布为主但不同尺度下有所差异，其分布格局强度依次为旷地<林缘<林下。结合克隆植物对资源的利用对策，探讨了克隆构型和分株种群特征以及分布格局随环境条件变化的生态适应意义。不同生境斑块条件下克隆植物可能采取不同的适应对策。采用盆栽实验，研究不同水分对比度下克隆整合及其生理生态特征，并对单向和交互资源中东方草莓的克隆整合做了对比研究。结果显示：高的水分对比度能够促进东方草莓的克隆整合，并能刺激相连分株增加光合作用，东方草莓体内的氧化—抗氧化系统也II随对比度做出相应的反应。耗-益分析表明胁迫分株的受益是以供给分株的损耗为代价的，但从克隆片段总体来说是受益的。单向资源中东方草莓生长的绝对值高于交互资源，但耗-益分析表明生长于交互资源下东方草莓的克隆整合获益大于生长于单向资源下东方草莓的克隆整合获益。长期生长于特定生境的克隆植物，在进化过程中其克隆整合和克隆分工在对资源异质性的适应策略方面可能有所侧重。采用盆栽实验对来自不同海拔梯度的东方草莓的克隆整合和克隆分工对异质资源的适应对策进行了研究。实验结果表明，来自高海拔的东方草莓可塑性较差。来自两个海拔的东方草莓对切断匍匐茎的表现有所差异，总体上切断匍匐茎对来自高海拔的东方草莓影响更大些。另外，来自高海拔的东方草莓表现出更高的克隆分工。IIIClonal plants are known to be more suitable for the habitats of heterogeneousresources than nonclonal plants, perhaps due to their well developed adaptivestrategies to environmental heterogeneity. Many studies have been done on theadaptive mechanisms of clonal plants, especially on the clonal integration anddivision of labor with morphology and biomass allocation under simpleheterogeneous habitats. Based on field surveys, laboratory experiments, multi-contrastunidirectional resource model and reciprocal resource model, Fragaria orientalis, aRosaceae stoloniferous herb that widely distributes in China, was used to study thisplant’s morphological and physiological responses to complicated heterogeneoushabitats in terms of its clonal integration, division of labor and cost-benefit, as well astheir modifications by different habitats, so as to better understand the adaptivestrategies of clonal plants under heterogeneous environments.Clonal architecture and ramet population characteristics are of the major concernin the studies on growth and adaptive strategies of clonal plants. Clonal architecture,ramet population characteristics and spatial point pattern of F. orientalis underdifferent light intensity were studied with field observations. The results showed that,clonal architecture changed with light availability: Internode-lengths and branchangels of stolons were larger while branch intensities were smaller under lower lightintensity than those under higher light intensity; Biomass of ramet population,root-shoot ratio and density of ramet population decreased significantly with reduce oflight intensity; Under all light intensities, spatial pattern of ramets was mainlyrandomly distributed but it changed with different scales, with pattern intensity as:open space < forest edge < understory. Adaptation significance of the clonal architecture, the ramet population characteristics and the spatial pattern changing withdifferent environments was discussed according to these results.Clonal plants may take different adaptive strategies under different patches. Withpot culture, clonal integration and physiological parameters of F. orientalis underdifferent water contrasts were studied, and clonal integration under unilateralresources and reciprocal resources were also compared. The results suggested that,high water contrast improve the clonal integration of F. orientalis and increase thephotosynthesis of connected ramets. Oxidative and antioxidative system of F.orientalis also responded with changing water contrasts. According to cost-benefitanalysis, the drought-stressed ramets obtained benefits from the connectedwell-watered ramets, and as a whole, the clonal fragment could also get benefits.Growth of F. orientalis in homogeneous resources was better than that inheterogeneous resources, but the whole plant got more benefit through clonalintegration in heterogeneous resources than in homogeneous resources.Pot culture experiments were also used to study the adaptive strategies inutilizing heterogeneous resources by the plant populations from different altitudes.The results showed that, F. orientalis from alpine zones were shorter and lessexpanded with poorer clonal plasticity than those from middle mountains. F.orientalis from two different altitudes showed different responses to stolon severing,and as a whole, stolon severing had more influence on F. orientalis from alpine zones.In addition, F. orientalis from alpine zones exhibited higher division of labor, whichsuggested that clonal plants from different habitats develop their own adaptivemechanisms in their clonal integration and division of labor in response toenvironmental heterogeneity.

Invasive clonal plant species have a greater root-foraging plasticity than non-invasive ones

Clonality is frequently positively correlated with plant invasiveness, but which aspects of clonality make some clonal species more invasive than others is not known. Due to their spreading growth form, clonal plants are likely to experience spatial heterogeneity in nutrient availability. Plasticity in allocation of biomass to clonal growth organs and roots may allow these plants to forage for high-nutrient patches. We investigated whether this foraging response is stronger in species that have become invasive than in species that have not. We used six confamilial pairs of native European clonal plant species differing in invasion success in the USA. We grew all species in large pots under homogeneous or heterogeneous nutrient conditions in a greenhouse, and compared their nutrient-foraging response and performance. Neither invasive nor non-invasive species showed significant foraging responses to heterogeneity in clonal growth organ biomass or in aboveground biomass of clonal offspring. Invasive species had, however, a greater positive foraging response in terms of root and belowground biomass than non-invasive species. Invasive species also produced more total biomass. Our results suggest that the ability for strong root foraging is among the characteristics promoting invasiveness in clonal plants.

Breast cancer stem cells and epithelial mesenchymal plasticity : implications for chemoresistance

Tumour heterogeneity is a key characteristic of cancer and has significant implications relating to tumour response to chemotherapy as well as patient prognosis and potential relapse. It is being increasingly accepted that tumours are clonal in origin, suggestive of a tumour arising from a deregulated or mutated cell. Cancer stem cells (CSC) possess these capabilities, and with appropriate intracellular triggers and/or signalling from extracellular environments, can purportedly differentiate to initiate tumour formation. Additionally through epithelial mesenchymal plasticity (EMP), where cells gain and maintain characteristics of both epithelial and mesenchymal cell types, epithelial-derived tumour cells have been shown to de-differentiate to acquire cancer stem attributes, which also impart chemotherapy resistance. This new paradigm places EMP centrally in the process of tumour progression and metastasis, as well as modulating drug response to current forms of chemotherapy. Furthermore, EMP and CSCs have been identified in cancers arising from different tissue types making it a possible generic therapeutic target in cancer biology. Using breast cancer (BrCa) as an example, we summarise here the current understanding of CSCs, the role of EMP in cancer biology - especially in CSCs and different molecular subtypes, and the implications this has for current and future cancer treatment strategies.

Expression des allèles spécifiques chez l'hybride clonal Phoxinus eos-neogaeus (Pisces : Cyprinidae)

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Converse Bergmann cline in a eucalyptus herbivore, paropsis atomaria olivier (coleoptera: chrysomelidae) : phenotypic plasticity or local adaptation?

Aim To measure latitude-related body size variation in field-collected Paropsis atomaria Olivier (Coleoptera: Chrysomelidae) individuals and to conduct common-garden experiments to determine whether such variation is due to phenotypic plasticity or local adaptation. Location Four collection sites from the east coast of Australia were selected for our present field collections: Canberra (latitude 35°19' S), Bangalow (latitude 28°43' S), Beerburrum (latitude 26°58' S) and Lowmead (latitude 24°29' S). Museum specimens collected over the past 100 years and covering the same geographical area as the present field collections came from one state, one national and one private collection. Methods Body size (pronotum width) was measured for 118 field-collected beetles and 302 specimens from collections. We then reared larvae from the latitudinal extremes (Canberra and Lowmead) to determine whether the size cline was the result of phenotypic plasticity or evolved differences (= local adaptation) between sites. Results Beetles decreased in size with increasing latitude, representing a converse Bergmann cline. A decrease in developmental temperature produced larger adults for both Lowmead (low latitude) and Canberra (high latitude) individuals, and those from Lowmead were larger than those from Canberra when reared under identical conditions. Main conclusions The converse Bergmann cline in P. atomaria is likely to be the result of local adaptation to season length.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.