高山松种实性状与生殖适应性

高山松(Pinus densata)是云南松(P. yunnanensis)与油松(P. tabulaeformis)自然杂交产生的二倍体杂种,是植物同倍体杂交物种形成的典型案例。高山松分布于青藏高原东南缘,占据了两个亲本种都不能正常生长的高海拔地带。良好生殖适应性的产生是物种形成的关键,但对高山松在高海拔生境下生殖适应性的变异与杂交起源遗传背景关系的研究尚未见报道。该研究通过对来自该种分布区内6个代表居群样本、13个反映高山松生殖适应性的种实性状的分析,探讨了其生殖性状在居群水平的变异模式,揭示了具有不同遗传组成和起源历史的高山松居群的生殖特性。研究结果表明:6个研究居群在种实性状上存在显著差异;可育种鳞数、总种鳞数、总种子数、可育种鳞率和可育种鳞密度等与纬度呈显著负相关关系;球果结种率与经度和生态梯度值呈显著的正相关关系;胚珠败育率则与经度和生态梯度值呈显著的负相关关系;球果长度也呈现了与经度的显著负相关。高山松球果结种率很高,平均为74%;球果的结种数、种子长和种翅长与云南松、油松及其它松属双维管亚属多数种接近;球果长度、球果总种鳞数接近油松的报道。这些结果表明,高山松在亲本种不能正常生长、繁衍的高原环境下具备正常的生殖能力;其种实特征在居群间分化显著并呈现一定的地理梯度变化。居群间的显著差异反映了其杂种起源特性,与高山松居群的遗传背景有关,也反映了处在异质生态环境中的高山松居群具有各自的生殖生态进化趋向。     

高山松及其亲本种油松和云南松DHAR基因的功能分化

高山松(Pinus densata)是油松(P. tabulaeformis)与云南松(P. yunnanensis)自然杂交产生的同倍性杂种, 分布于青藏高原东南缘, 占据了油松和云南松两个亲本种都不能正常生长的高海拔地带。为了揭示高山松、油松和云南松脱氢抗坏血酸还原酶(DHAR)基因的组成和功能分化, 分别从高山松、油松和云南松中克隆到2类DHAR基因(DHAR1与DHAR2)。组织表达模式分析表明, 这6个基因在根、韧皮部、叶和芽中均有表达; 通过系统发育分析发现, 高山松在物种形成过程中保留了油松的DHAR1拷贝以及云南松的DHAR2拷贝; 酶学性质分析则表明, 高山松与油松DHAR1蛋白对底物具有相似的催化活性、催化效率、最适pH和热力学稳定性, 但其催化活性比云南松DHAR1蛋白高约300倍。高山松DHAR2蛋白对底物的催化活性和热力学稳定性均高于油松DHAR2蛋白。高山松DHAR基因在生化功能上显示出优于或类似亲本DHAR, 这种优势功能的选择与杂种独特的生态适应性可能有重要的相关性。     

低温胁迫下二倍体杂交种高山松光系统Ⅱ功能稳定性研究

以人工气候室内的高山松(Pinus densata)及其亲本油松(P.tabulaeformis)和云南松(P.yunnanensis)实生苗为材料,通过测定低温胁迫和恢复后其幼苗的快速叶绿素荧光诱导动力学(JIP-test)参数,分析高山松及其亲本的光系统Ⅱ(PSⅡ)功能稳定性以及对低温胁迫的响应差异,以探讨高山松适应高海拔低温环境的光合生理生态机制。结果表明:经过33 d的低温(12℃/5℃,昼/夜)处理后,高山松PSⅡ功能参数单位截面积吸收的能量(ABS/CSo)、捕获的能量(TRo/CSo)、电子传递的能量(ETo/CSo)、耗散的能量(DIo/CSo)、有活性反应中心的数量(RC/CSo),以及原初光化学反应的最大量子产额(φPo)、吸收光能用于QA-以后的电子传递的能量比例(φEo)、捕获光能用于QA-以后的电子传递的能量比例(Ψo)、光能利用能力参数(PIabs)、2 ms时相对可变荧光(VJ)、荧光上升的初始斜率(Mo)、荧光曲线与Fm所围面积(Area)、介于F0和Fm之间的相对面积(Sm)和QA氧化还原次数(N)均与处理前无显著差异;油松和云南松的φPo、φEo、Ψo、PIabs、Area与对照差异显著,云南松的VJ、Mo、Sm、N、ETo/CSo、DIo/CSo、ABS/CSo也与对照存在显著差异;温度恢复之后(25℃/18℃,昼/夜),这些荧光参数的恢复程度表现为高山松>油松>云南松。可见,低温胁迫显著影响了油松和云南松的光能传递和光能利用能力以及受体库大小,且云南松遭受伤害的程度高于油松,而高山松的PSⅡ功能及其稳定性在遭受低温胁迫时明显高于亲本油松和云南松,且胁迫解除后恢复程度高,从而使得高山松能够在高海拔低温环境中累积代谢所需的有机物,保证其正常的生长和发育。     

同倍杂交种高山松与亲本种云南松的地理隔离研究

为了探讨横断山区同倍杂交种高山松与其亲本种云南松的地理隔离机制,采用实际的路线踏查方法,调查了高山松和云南松群体的地理分布情况和开花物候特征,并基于7个气象因子进行了聚类分析。调查研究表明高山松和云南松群体分布在不同的地貌类型中,以云南中甸为临界区,沿着金沙江河谷向北分布高山松,向南分布云南松。云南松的开花物候日期明显早于高山松,聚类分析表明两个物种分别聚为一支。在地质历史过程中,高大山系的形成以及海拔的升高将高山松和云南松从地理上隔离开来,两物种的适生区的气象因子的差异造成花期不遇,从而阻碍了两个物种问的基因交流,进而隔离开来。     

松属针叶角质层微形态特征在分类学中的应用

角质层是表皮细胞壁表面的一层不透水的脂肪性物质。角质层与表皮细胞紧密结合,植物表皮细胞形态和排列方式、气孔器的形态结构等微形态特征均能在角质层上反映出来。利用光学显微镜和扫描电镜对松属（Pinus）12种植物针叶角质层微形态特征进行观察和比较,详细描述20个性状,其中12个性状来自角质层内表面,8个性状来自角质层外表面。结果表明,这些特征可为该属属下分类和相似种的鉴别提供有用信息,具有重要的分类学意义：①表皮细胞长度、表皮毛长度、角质层外表面起伏程度、表皮细胞轮廓、有无气孔塞和针絮状物质等角质层微形态特征具有自身特异性,在属下可作为松属组级水平上的分类依据。角质层微形态 特征不支持将五针松组（P. Section Cembra）和白皮松组（P. Section Parrya）合并为P. Section Quinquefolius的观点,亦不支持将油松组（P. Section Pinus）分成P. Section Pinus和P. Section Trifolius的看法。②白皮松（P. bungeana）针叶角质层微形态特征既与五针松组有相同之处,又与油松组有相似之处,还有部分特征显示出不同于松属其他种类的独特性,可为白皮松亚属（P. Subgenus Parrya）的建立提供新依据。③扫描电镜下表皮细胞垂周壁纹路,气孔塞有无和外表面气孔形状等特征可为形态相似种火炬松（P. taeda）和湿地松（P. elliottii）提供种间界定依据。     

中国西南四种常见松树火适应性状比较

野火对松属植物的进化和分布有重要的影响。在与野火长期抗争中,松属植物形成了一系列性状提高在易火生境中的适合度,维持种群生存与繁衍。西南地区是中国野火高发区,作为这一地区特有松树,云南松、思茅松和高山松具有一些典型的火适应性状,如厚树皮。以这3种松树和东部常见的马尾松为研究对象,比较4种松树的火适应对策。结果表明：4种松树的火适应性状存在一定的差异。与火适应相关的13项性状主成分分析显示,4个种在空间上总体是分离的,也表现出一些重叠。其中云南松火适应生活史对策是火耐受型和火依赖型的中间类型,适应会发生林冠火的生境。高山松、思茅松和马尾松都是火耐受型,通过快速高生长、厚树皮等性状适应生境不同频度的地表火。四种松树火适应对策与分布区火险基本相符。表明野火在这一区域广泛存在,并对植物进化和森林格局有重要影响。野火在西南地区松属分布和种群维持中的生态作用应被给予足够重视。     

细叶云南松种实性状变异与地理气象因子的关联

细叶云南松(Pinus yunnanensis var. tenuifolia)是分布于滇、黔、桂交界处的一个云南松变种, 其自然生境独特, 属典型的干热河谷气候, 具有重要的生态、经济价值。该研究以分布于南盘江-红水河流域沿线的8个细叶云南松天然种群为材料, 采用描述性统计及巢式方差分析比较种群间及种群内种实性状差异, 运用Pearson相关及典型相关分析探究种实性状与地理气象因子间的相关性, 最后对种群进行主成分聚类和Mantel检验, 揭示其种实性状地理变异模式。结果显示: 细叶云南松11个种实性状在种群间和种群内都存在极显著差异(p < 0.001), 变异丰富。种实性状以种群内变异为主(平均表型分化系数V_ST = 18.65%), 球果性状种群间分化(24.22%-39.88%)高于种子及种翅性状(4.14%-13.80%), 表明球果性状受到更强的环境选择。尽管部分相关系数未达显著水平, 但整体上种实性状与经纬度、年平均气温呈正相关关系, 与相对湿度、年降水量呈负相关关系, 表明细叶云南松种实性状受到地理隔离、湿度和温度的协同选择作用, 使其能较好适应干热环境。主成分聚类将参试种群划分为3类, 位于东部的罗甸伍家坟(WJ)、罗甸大亭(DT)种群聚为一类, 其种实形态较大, 位于西南部的兴义坝汪(BW)种群单独一类, 其种实形态较小, 其他种群聚为一类, 种实形态介于前两类之间。总体上, 种实性状值有自西向东递增的趋势。Mantel检验表明, 参试种群存在明显的空间结构, 主要体现为渐变群模式。     

古尔班通古特沙漠固定沙丘上白梭梭和梭梭的空间分布及种间关联性

为了探讨沙漠中固沙灌木种群共存和演替机制, 本文基于古尔班通古特沙漠东南缘固定沙丘上白梭梭(Haloxylon persicum)和梭梭(H. ammodendron)种群的地理位置和生长发育阶段信息(幼株、营养株、生殖株和死株), 采用点格局分析方法(g(r)函数)及Monte-Carlo随机模拟检验和零模型选取的方法, 分析了固沙灌木白梭梭和梭梭种群不同生长发育阶段在0-20 m尺度内的空间分布格局及种间关联性。结果表明: (1)两个种群在研究尺度范围内呈聚集分布, 随着尺度的增大, 其聚集强度逐渐减弱; (2)两个种群整体上呈负关联关系, 尺度越大负关联关系越显著; (3)白梭梭种群生长发育阶段相差越大, 个体间正关联关系越显著; 梭梭种群生长发育阶段越接近, 个体间正关联关系越显著; (4)两个种群中龄级较大的个体(如营养株、生殖株和死株)会对对方种群中龄级较小的幼株产生一定的抑制作用; 同时, 随着两个种群中个体的成长, 双方受到的抑制作用逐渐减弱, 主要表现为正关联和无关联。总体而言, 古尔班通古特沙漠固定沙丘白梭梭和梭梭种群的分布格局整体上为聚集分布, 随龄级增加聚集性减弱, 受生境异质性和扩散限制的影响明显。种间关系多为负相关, 种内不同生长发育阶段之间均为正关联关系。     

Association of cone and seed traits of Pinus yunnanensis var. tenuifolia with geo-meteorological factors

细叶云南松(Pinus yunnanensis var. tenuifolia)是分布于滇、黔、桂交界处的一个云南松变种, 其自然生境独特, 属典型的干热河谷气候, 具有重要的生态、经济价值。该研究以分布于南盘江-红水河流域沿线的8个细叶云南松天然种群为材料, 采用描述性统计及巢式方差分析比较种群间及种群内种实性状差异, 运用Pearson相关及典型相关分析探究种实性状与地理气象因子间的相关性, 最后对种群进行主成分聚类和Mantel检验, 揭示其种实性状地理变异模式。结果显示: 细叶云南松11个种实性状在种群间和种群内都存在极显著差异(p < 0.001), 变异丰富。种实性状以种群内变异为主(平均表型分化系数V_ST = 18.65%), 球果性状种群间分化(24.22%-39.88%)高于种子及种翅性状(4.14%-13.80%), 表明球果性状受到更强的环境选择。尽管部分相关系数未达显著水平, 但整体上种实性状与经纬度、年平均气温呈正相关关系, 与相对湿度、年降水量呈负相关关系, 表明细叶云南松种实性状受到地理隔离、湿度和温度的协同选择作用, 使其能较好适应干热环境。主成分聚类将参试种群划分为3类, 位于东部的罗甸伍家坟(WJ)、罗甸大亭(DT)种群聚为一类, 其种实形态较大, 位于西南部的兴义坝汪(BW)种群单独一类, 其种实形态较小, 其他种群聚为一类, 种实形态介于前两类之间。总体上, 种实性状值有自西向东递增的趋势。Mantel检验表明, 参试种群存在明显的空间结构, 主要体现为渐变群模式。     

青藏高原有蹄类动物多样性和特有性: 演化与保护

青藏高原是地球上一个独特的自然地理单元, 具有丰富的生境类型和生物种类, 是生物多样性与全球环境变化领域的热点研究区域。青藏高原发育着独特的动物区系, 尤其是有蹄类动物, 如藏羚(Pantholops hodgsonii)、野牦牛(Bos mutus)、藏原羚(Procapra picticaudata)、普氏原羚(P. przewalskii)、白唇鹿(Przewalskium albirostris)等青藏高原特有动物。在本文中, 我们探讨了如下问题: 青藏高原有多少种有蹄类动物?有多少种特有有蹄类动物?其分布格局如何?生存状况如何?保护现状如何?我们首先确定了青藏高原动物地理区的地理边界。发现青藏高原有28种有蹄类, 其中10种是青藏高原特有种。青藏高原有蹄类种数占中国有蹄类的42%, 单位面积上的有蹄类物种密度比中国现生有蹄类物种平均密度高62%。特有种比例高达36%。然而, 青藏高原有蹄类物种分布不均匀, 其丰富度呈东部高西部低格局, 而高原特有有蹄类则分布于高原腹地。青藏高原有蹄类动物生存受威胁比例高, 其中71%为受威胁物种, 54%被列入濒危野生动植物种国际贸易公约(CITES)附录I或附录II。野外调查发现大额牛(Bos frontalis)已经野外灭绝, 目前仅在高黎贡山有人工养殖的群体。青藏高原有蹄类物种红色名录指数从1998-2015年呈现持续下降趋势, 全球气候变化将加剧这一趋势。青藏高原有蹄类受威胁局面仍在继续, 物种生存状况持续恶化。虽然经过40年的自然保护区建设, 但一些青藏高原有蹄类的重要种群与栖息地并没有被自然保护区和新建的三江源国家公园所覆盖。因此, 在开发青藏高原、实现人类社会经济发展目标的同时, 应尽量保存高原野生动物种群与生境, 实现青藏高原草地生态系统的可持续利用与生物多样性保护的双赢目标。     

Influence of ammonium and nitrate supply on growth,nitrate reductase activity and N-use efficiency in a natural hybrid pine and its parents

Aims Selection of tree species with a high capacity to assimilate N and efficiently utilize N resources would facilitate the success of initial tree seedling establishment in infertile soils. The preference for N forms was tested using three pine species (Pinus densata, Pinus tabuliformis and Pinus yunnanensis). Pinus densata is a natural diploid hybrid between P. tabuliformis and P. yunnanensis .Methods Seedlings of three pine species were supplied with nitrate-N, ammonium-N (at two different pH regimes) or combined ammonium and nitrate as a nitrogen source in perlite culture in a controlled environment.Important findings Seedlings of P. densata had higher total biomass and net photosynthesis when supplied with nitrate-N and ammonium nitrate than with ammonium-N. In parental species, total biomass and net photosynthesis for P. yunnanensis seedlings was higher in ammonium-N than in nitrate-N, whereas the other parental species P. tabuliformis had the highest total biomass among species for all treatments except ammonium with CaCO 3. Most morphological traits in P. densata seedlings were intermediate between its two parental species. However, N-use efficiency and photosynthetic N-use efficiency of P. densata significantly exceeded both parents when supplied with nitrate-N and ammonium nitrate. The results suggested that the diploid hybrid tree species P. densata has a preference for nitrate and is not well adapted to ammonium-N as a sole nitrogen source regardless of the growth medium pH. Based on changes in environmental conditions, such as predicted future temperature increases in high altitude areas associated with climate change, P. densata is likely to be increasingly competitive and have wide adaptation in high altitude regions.     

Maternal lineages of Pinus densata,a diploid hybrid

Previous morphological, allozyme and chloroplast DNA data have suggested that Pinus densata originated through hybridization between P. tabuliformis and P. yunnanensis. In the present study, sequence and restriction site analyses of maternally inherited mitochondrial nad1 intron were used to detect variation patterns in 19 populations of P. tabuliformis, P. yunnanensis and P. densata. A total of three mitotypes (A, B, C) were detected. All but one of the populations of P. yunnanensis possessed mitotype B while all populations of P. tabuliformis had mitotype A. Pinus densata populations, on the other hand, harboured both mitotypes A and B, which are characteristic of P. tabuliformis and P. yunnanensis, respectively. This result gives strong additional evidence supporting the hybrid origin of this diploid pine. The distribution of mitotypes indicated very different mating compositions and evolutionary history among P. densata populations. It seems that local founder populations and backcrosses may have played important roles in the early establishment of P. densata populations. The uplift of the Tibetan Plateau had a significant impact on the distribution of maternal lineages of P. densata populations.     

Cytoplasmic composition in Pinus densata and population establishment of the diploid hybrid pine

Sequence and restriction site analyses of the paternally inherited chloroplast rbcL gene and maternally inherited mitochondrial nad1 fragments from the same set of populations and individuals were used to investigate cytoplasmic composition and population establishment of Pinus densata, a diploid pine that originated through hybridization between P. tabuliformis and P. yunnanensis. Two variable sites and three chlorotypes (TT, TC and GC) were detected on the rbcL gene of the three pines. P. densata harboured the three chlorotypes, two of which (TT, GC) were characteristic of the parental species, respectively. The third chlorotype (TC) was distributed extensively in seven of the 10 P. densata populations analysed, and might represent a mutation type or have been derived from an extinct parent. The distribution of chlorotypes, together with that of mitotypes, indicated that significant founder effect and backcross happened during the population establishment of the hybrid pine. P. tabuliformis and P. yunnanensis had acted as both mother and father donors, i.e. bi-directional gene flow existed between the two parental species in the past. Population differentiation of P. densata is high, as detected from the cytoplasmic genomes: GST = 0.533 for cpDNA and GST = 0.905 for mtDNA. The differences in cytoplasmic composition among the hybrid populations suggest that the local populations have undergone different evolutionary histories.     

Genetic structure and evolutionary history of a diploid hybrid pine Pinus densata inferred from the nucleotide variation at seven gene loci

Although homoploid hybridization is increasingly recognized as an important phenomenon in plant evolution, its evolutionary genetic mechanisms are poorly documented and understood. Pinus densata, a pine native to the Tibetan Plateau, represents a good example of a homoploid hybrid speciation facilitated by adaptation to extreme environment and ecological isolation from the parents. Its ecologically and reproductively stabilized nature offers excellent opportunity for studying genetic processes associated with hybrid speciation. In this study, we investigated the levels and patterns of nucleotide variation in P. densata and its putative parents. Haplotype composition, gene genealogies, and the levels and patterns of nucleotide variation gave further support to the hybrid nature of P. densata. Allelic history, as revealed by our data, suggests the ancient nature of the hybrid preceding elevation of the Tibetan Plateau. We detected more deviations from neutrality in P. densata than in the parental species. Thus, at least some of the evolutionary forces that have shaped the genetic variation in P. densata are likely to be different from those acting upon parental species. We speculate that when populations of P. densata invaded new territories, they had elevated rates of response to selection in order to develop traits that help them to survive and adapt in the new environments.     

云南松及其近缘种的遗传变异与亲缘关系

The authors investigated the genetic diversity of 29 natural populations representing Pinus yunnanensis Franch. and its two close relatives, P. densata Mast. and P. kesiya Royle ex Gordn. var. langbianensis (A Chey.) Gaussen. Horizontal starch gel electrophoresis was performed for macrogametophytes collected from populations in Yunnan, Sichuan and Guangxi. Allozyme data for 33 loci of 14 enzymes demonstrated high levels of genetic variation at both population and species levels in comparison with other conifers, with the mean values for populations being P=0.694, A=2.0 and He=0.145 for P. yunnanensis; P=0.714, A=2.0 and He=0.174 for P. densata; and P=0.758, A=2.1 and He=0.184 for P. kesiya var. langbianensis. Based on Wright's F-statistics, the fixation index of P. yunnanensis, P. densata and P. kesiya var.langbianensis were 0.101, 0.054 and 0.143, respectively, indicating that the populations were largely under random mating. Based on Nei's genetic distance, the genetic differentiation was not obvious among the three species (i.e. the genetic distance was less than 0.075). Because of the wider distribution of P. yunnanensis with greater variety of habitats, it was shown that the genetic differentiation among the P. yunnanensis populations was larger than that of the populations of the other two species. According to morphological, geographic and allozymic evidences, the authors suggested that the three species be better treated as varieties under a single species. In addition, the extensive gene flow among the three pine species resulted in great genetic diversity and evolutionary potential. Also, high level of genetic variation of P. yunnanensis provides important basis for its genetic improvement and breeding in future.     

Genetic composition and diploid hybrid speciation of a high mountain pine, Pinus densata, native to the Tibetan plateau

Pinus densata has been suggested to have originated from hybridization events involving P. tabulaeformis and P. yunnanensis. In this study, allozyme differentiation at 12 loci was studied in 14 populations of P. tabulaeformis, P. densata, and P. yunnanensis from China. The observed genetic composition of P. densata supported the hybrid hypothesis and showed varying degrees of contribution from P. yunnanensis and P. tabulaeformis among its populations. These data, together with previous chloroplast DNA results, indicated different evolutionary histories among P. densata populations. To examine the possibility of ongoing hybridization among the three species, we analyzed patterns of linkage disequilibria between allozyme loci in ovule, pollen, and zygote pools. None of these tests suggested that there is significant ongoing gene exchange, implying that populations of P. densata have a stabilized hybrid nature. The normal fertility and high fecundity of P. densata indicate that this hybrid is maintained through sexual reproduction. P. densata represents an example of diploid hybrid speciation in an extreme ecological habitat that is both spatially and ecologically separated from that of its parents.     

Chromosomal localization of 5S and 18S–5.8S–25S ribosomal DNA sites in five Asian pines using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was employed on mitotic metaphase chromosome preparations of five Asian Pinus species: Pinus tabuliformis, Pinus yunnanensis, Pinus densata, Pinus massoniana and Pinus merkusii, using simultaneously DNA probes of the 18S rRNA gene and the 5S rRNA gene including the non-transcribed spacer sequences. The number and location of 18S rDNA sites varied markedly (5-10 pairs of strong signals) among the five pines. A maximum of 20 major 18S rDNA sites was observed in the diploid genome (2n = 24) of P. massoniana. The 5S rDNA FISH pattern was less variable, with one major site and one minor site commonly observed in each species. The differentiation of rDNA sites on chromosomes among the five pines correlates well with their phylogenic positions in Pinus as reconstructed from other molecular data. P. densata, a species of hybrid origin, resembles its parents ( P. tabuliformis and P. yunnanensis), including some components characteristic of each parent in its pattern. However, the species is unique, showing new features resulting possibly from recombination and genome reorganization.     

Distinct niche divergence characterizes the homoploid hybrid speciation of Pinus densata on the Tibetan plateau

Ecological divergence and selection for novel adaptations to new habitats have been theoretically proposed to play important roles in promoting homoploid hybrid speciation (HHS). The successful establishment of Pinus densata on the Tibetan Plateau is one of the few known examples of HHS. In this study, we carried out extensive field expeditions to obtain representative coverage of occurrence sites of P. densata and its two putative parents. We then applied a series of geographic information system-based analyses to define the patterns of environmental variation within and among the three pine species, to remove potentially confounding effects of spatial autocorrelation in the environmental data due to allopatric ranges, and to build species distribution models. All results consistently indicated that the ecological preferences of P. densata and its parental species have diverged, and they identified candidate ecological factors associated with habitat-specific adaptation. Projections from niche modeling indicated that P. densata could extend across a vast range along the parallel valley systems of the southeastern Tibetan Plateau. Our findings provide evidence of a distinct niche shift in P. densata and support the hypothesis that local adaptation and geographic isolation help maintain and reinforce between-species differences and reproductive isolation in the species complex.