Ugan古河道胡杨可培养内生细菌的多样性

摘要：【目的】为了了解塔河废弃古河道胡杨可培养内生细菌的多样性。【方法】从2棵胡杨树干部抽出其内存液,采用三种不同的培养基对样品的内生细菌进行了分离纯化;对它们进行16S rDNA测定和系统进化分析。【结果】分离纯化不同表型的细菌62株,对它们的16S rDNA序列分析表明,62株菌分别属于四个大类群;厚壁菌门（Firmicutes）、放线菌门(Actinobacteria)、α-变形菌纲(Alpha Proteobacteria) 、γ-变形菌纲(Gamma Proteobacteria),18个属,32个种;芽孢杆菌属和假单胞菌属是胡杨可培养内生细菌的优势细菌种群,它们分别占已测种群的40.32%、16.13%。其中菌株KTH-63为葡萄球菌科的潜在的新属新种,它与最近源菌株的16S rDNA序列相似率为92.491%;9株菌KLH-21、KLH-1、KTH-8、KTH-14、KNA-26、KLH-18、KTH-20、KNA-3、KLH-25是潜在的新种（16S rDNA相似率为96.089 %-97.769 %）,胡杨树干内存液中潜在新种的发现率高达总分离检测菌株的16.13 % 。本研究获得的胡杨可培养内生细菌的群落结构数据给植物内生细菌新增了10个属,18个种。【结论】胡杨具有多样性极其丰富的可培养内生细菌菌种资源,土著新种的发现频率超出了预期,胡杨可培养内生细菌的群落结构极大地刷新了植物内生细菌的种群记录,极具进一步发掘的潜力。     

天目山野生春兰根中可培养内生细菌多样性研究

为了解春兰植物内生细菌的多样性,采用稀释涂布法对表面灭菌的天目山野生春兰根内生细菌进行分离培养。通过R2A和TSA两种培养基共分离获得63株内生细菌。对16S rDNA序列测定结果进行系统发育分析可知,63株细菌分属于β-变形菌纲(31.74%)、γ-变形菌纲(7.94%)及厚壁菌门(60.32%)。其中厚壁菌门为最优势类群,芽孢杆菌属为最优势菌属,占分离总菌数的50.79%。天目山野生春兰根内生细菌多样性指数为1.56。结果表明,初春季节天目山野生春兰根内生细菌多样性较低。     

野生及温室盆栽蕙兰可培养根内生细菌的遗传多样性

惠兰(Cymbidium faberi)是中国兰属代表种之一,具有很高的观赏价值和经济价值,对其内生细菌进行研究不仅可以丰富植物内生细菌资源,还可以为探讨兰花与微生物之间的相互作用关系提供基础数据。本研究采用分离培养方法及16S r RNA基因序列测定对天目山野生蕙兰、在温室培养1年后的蕙兰根内生细菌遗传多样性进行了研究。结果表明:从野生蕙兰根内分离得到的97株细菌分属于变形菌门的α-变形菌纲、β-变形菌纲、γ-变形菌纲及厚壁菌门的13个属,其最优势类群为γ-变形菌纲(86.60%),Lelliottia(26.80%)为最优势菌属。从温室盆栽蕙兰根内分离得到的52株细菌分属于变形菌门的α-变形菌纲、β-变形菌纲、γ-变形菌纲及放线菌门的9个属,优势类群为β-变形菌纲(48.08%),优势菌属为草螺菌属(Herbaspirillum)(34.62%),其中菌株eh R17为潜在的新种。这些结果表明天目山野生蕙兰可培养根内生细菌多样性较其在温室培养1年后更为丰富,同时也说明植物内生细菌的群落结构与生长环境密切相关。     

新疆醉马草内生菌群落结构

【目的】揭示醉马草不同组织内生细菌和内生真菌种群组成和分布。【方法】采用液氮研磨法分别提取醉马草种子、叶、茎、根组织总DNA,采用通用引物扩增内生细菌16S rDNA和真菌ITS,通过限制性内切酶HhaⅠ和RsaⅠ对16S rDNA PCR产物酶切,HhaeⅢ和HinfⅠ对真菌rDNA ITS PCR产物酶切得到不同的TRFs片段。TRFs经T-RFLP分析程序结合基因文库比对后,分析醉马草不同组织中内生细菌和内生真菌的群落组成及内生菌群落相似性。【结果】研究表明醉马草根部内生细菌多样性最高,而种子内生真菌多样性最为丰富。醉马草各组织内生细菌优势菌属均为Bacillus(29%以上),种子、叶、茎、根内生真菌优势菌属分别为Mycosphaerella(6.5%),Teratosphaeria(4.5%),Fragum(1.1%),Sebacina(11.3%)。聚类分析表明茎和叶内生细菌群落结构相似,种子和其他组织内生细菌群落结构相似性较远,而茎和种子内生真菌群落结构相似,叶和其他组织内生真菌群落结构相似性较远。醉马草内生菌多样性丰富且存在尚未认识的新类群。     

春兰根中可分泌吲哚乙酸的内生细菌多样性

植物内生细菌可通过分泌吲哚乙酸(Indole-3-aceticacid,IAA)等方式促进植物生长。本研究以温室盆栽春兰(Cymbidium goeringii)为材料,采用分离培养方法对春兰根中可分泌IAA的内生细菌多样性进行了研究。从春兰根组织中共分离纯化得到了256株内生细菌,其中57株具有分泌IAA的能力,占总菌数的22.3%。根据ARDRA(amplified ribosomal DNA restriction analysis)及16SrDNA系统发育分析结果,将57株内生细菌划分为25个组,分属于6大类群,分别为变形菌门的α-变形菌纲(35.1%)、γ-变形菌纲(14.0%)和β-变形菌纲(8.8%)、厚壁菌门(33.3%)、放线菌门(7.0%)及拟杆菌门(1.8%)。其中变形菌门的α-变形菌纲和厚壁菌门为优势类群,类芽孢杆菌属(Paenibacillus)为优势菌属,且为高产IAA的主体菌属。另外,测序结果显示有4个菌株的序列与已知细菌的最高序列相似性低于97.0%,可能为潜在的新种或新属。研究结果表明春兰根中分泌IAA的内生细菌具有丰富的多样性。这一结果可为研究和开发植物促生细菌提供基础资料...     

南明河城区河段细菌多样性与环境因子的关系

摘要:【目的】了解南明河城区河段细菌群落结构及多样性变化,探讨城区河段环境因子对细菌群落结构的影响。【方法】应用对细菌16S rDNA V4区的高通量测序技术,分析和比较了南明河流经城区的5个样点的细菌群落多样性;然后采用冗余分析(RDA)探讨了水体环境因子与细菌多样性的关系。【结果】南明河贵阳区段细菌多样性指数( Shannon-Wiener 指数)分析结果显示,多样性指数平均达7.5,乌当桥采样点的细菌多样性＞水口寺采样点＞五眼桥采样＞花溪大桥采样点＞冠洲桥采样点。序列比对结果显示,南明河内细菌除了部分分类地位不明确的菌群和稀有菌群外,其余分布于11个门包含327个属的细菌,优势菌门为变形菌门(Proteobacterice,66.1%±3.30%),其中γ-变形菌纲(Gammaproteobacteria,54.76%±4.86%)为优势亚群,假单胞菌属(Pseudomonas,16.92%±0.02%)为优势菌属;RDA结果表明不同菌群受不同环境因子的影响 不同,菌属群落Ⅳ和环境中总氮、总磷显著正相关。【结论】高通量测序分析能获得更为全面的细菌群落多样性信息。河流经过城区后环境因子发生较大变化,影响河流细菌群落结构改变。这为研究河流城市区段的细菌结构多样性及与环境因子的关系提供了新的科学数据。     

两种不同生境中国沙棘种子内生细菌的多样性

植物内生菌广泛分布于植物的各种组织及器官中,对植物的生长表现出各种作用,而植物种子中的内生菌对植物的作用也越来越受到人们的关注。以榆中县和秦安县两种不同生境的中国沙棘种子为材料,分析中国沙棘种子内生细菌多样性,以期探究生境对种子内生菌多样性的影响,并为进一步研究种子内生菌与沙棘的相互作用提供依据。研究利用纯培养方法和高通量测序方法分别进行中国沙棘种子内生细菌多样性分析。对纯培养分离得到的内生细菌,利用16S rRNA基因序列分析法结合形态学特征进行内生细菌的鉴定;对高通量测序得到的数据进行基于OTUs(Operational Taxonomic units,可操作分类单元)的物种注释分析。通过纯培养方法从榆中县中国沙棘种子中分离得到4株内生细菌,分属于芽孢杆菌属(Bacillus)、葡萄球菌属(Staphylococcus)和假单胞菌属(Pseudomonas);秦安县中国沙棘种子中分离得到5株内生细菌,分属于芽孢杆菌属(Bacillus)、葡萄球菌属(Staphylococcus)和不动杆菌属(Acinetobacter)。采用高通量测序方法检测到榆中县中国沙棘种子内生细菌分属于7个门、68个属,秦安县中国沙棘种子内生细菌分属于5个门、30个属。在门分类水平,榆中县中国沙棘种子内生细菌的主要优势门类是蓝藻门(Cyanobacteria)和变形菌门(Proteobacteria),相对丰度分别为95.62%和2.03%;秦安县中国沙棘种子的主要优势门类是变形菌门(Proteobacteria)和蓝藻门(Cyanobacteria),相对丰度分别为91.68%和8.06%。在属分类水平,榆中县中国沙棘种子内生细菌的优势菌属为蓝藻细菌属(Cyanobacteria),相对丰度为95.09%;秦安县中国沙棘种子内生细菌的优势菌属为寡养单胞菌属(Stenotrophomonas),相对丰度为85.60%。榆中县和秦安县两种不同生境中国沙棘种子内生细菌有着丰富的多样性,且内生细菌的多样性和丰富度存在明显差异,表现为榆中县中国沙棘种子内生细菌的多样性和丰富度均高于秦安县中国沙棘种子内生细菌。     

不同生境下五唇兰根部可培养内生细菌多样性研究

兰科植物内生细菌与菌根真菌的协作对宿主植物的生长、抗病、抗逆及植物修复环境能力等具有重要意义,揭示其内生细菌多样性及与生境之间的关系有助于阐明兰科植物的适应与进化机制。本研究基于16SrDNA序列分析探讨了不同生境下东南亚特有种五唇兰根部可培养内生细菌多样性及其空间异质性。结果表明:从不同生境下五唇兰根部共分离出内生细菌59株,其中从土生型五唇兰根部分离出内生细菌45株(76.27%),从石生型五唇兰根部分离出内生细菌14株(23.73%);基于内生细菌16SrDNA序列同源性分析及构建的系统发育树显示,五唇兰根部内生细菌分属于7属,即芽孢杆菌属(Bacillus)、伯克氏菌属(Burkholderia)、草酸菌属(Pandoraea)、土壤杆菌属(Agrobacterium)、类芽孢杆菌属(Paenibacillus)、泛菌属(Pantoea)、欧文氏菌属(Erwinia),其中优势属为芽孢杆菌属,次优势属为泛菌属和伯克氏菌属;多样性分析显示,土生型五唇兰根部内生细菌群落的Shannon多样性指数大于石生型五唇兰,不同生境下五唇兰根部内生细菌群落结构差异极显著(P0.01)。土生型五唇兰根部内生细菌群落优势属为芽孢杆菌属和泛菌属,石生型五唇兰根部内生细菌群落优势属为芽孢杆菌属和伯克氏菌属。     

超积累和非超积累生态型东南景天茎、叶内生细菌多样性分析

【目的】探究和比较超积累和非超积累生态型东南景天茎、叶微生物群落结构的异同。【方法】采用高通量测序技术研究野外两种生态型东南景天茎和叶片的内生细菌群落结构。【结果】4个样品总共得到366 783条有效序列和39 948个OTU(97%相似度)。从Shannon指数得知:两种生态型东南景天叶片内生菌的多样性均高于茎;超积累生态型东南景天叶片内生菌的多样性高于非超积累生态型,但非超积累生态型东南景天茎组织中内生菌多样性高于超积累生态型东南景天。超积累生态型东南景天的叶片和茎中的内生菌分别包括26和21个门,123和117个科;非超积累生态型东南景天叶片和茎中的内生菌分别包括43和22个门,175和83个科,4个样品的优势菌群均为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和蓝藻细菌门(Cyanobacteria)。在属分类水平上,超累积生态型东南景天叶片和茎第一优势菌属分别为Synechococcus和Plesiomonas;非超积累生态型东南景天叶片和茎组织第一优势菌群分别为Pseudomonas和Dechloromonas。【结论】两种生态型东南景天的叶片和茎中均具有丰富的内生细菌,但超积累生态型东南景天叶片内生菌多样性最大,且存在一些独有的功能菌群。     

柑橘黄龙病寄主长春花内生细菌的分离及功能鉴定

【目的】感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构及功能对柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】利用兼性厌氧可培养技术以及植物内生菌功能特性分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。29种内生细菌进行功能分析,其中6株内生细菌至少具有4种功能特性,分属于马胃葡萄球菌、苏云金芽孢杆菌、巨大芽孢杆菌、短小杆菌属、摩氏摩根菌(Morganella morganii)及溶杆菌属(Lysobacter sp.)5个属。【结论】感病与健康长春花植株中均含有丰富的内生细菌且差异较大,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构。通过分析菌群的差异,有望找到与柑橘黄龙病菌生长相关的菌种。     

Species structure, contemporary distribution and status of the Siberian surgeon Acipenser baerii

A detailed analysis of the historical and contemporary range of the Siberian sturgeon, Acipenser baerii, shows that the contemporary status of its populations and forms can be described as threatened or endangered. Recently, the abundance of the nominal subspecies, A. b. baerii, which inhabits mainly the Ob River basin, decreased sharply. Due to construction of hydroelectric dams, up to 40% of the spawning grounds became inaccessible for migrating sturgeon of this subspecies. The Lake Baikal subspecies, A. baerii baicalensis, is extremely rare and was included in the Russian Federation Red Data Book in 1983. The abundance of the east Siberian subspecies, A. baerii stenorrhynchus, inhabiting the basins of the east Siberia rivers, has also significantly decreased during the last few years. Its range in the Yenisey and Lena River basins is gradually being reduced. Gametogenesis is anomalous in a high number of females from all populations of this subspecies (in the Kolyma and Indigirka river stocks 80–100% of females were anomalous in 1987–1989). These anomalies seem to be caused by high levels of water pollution.     

赤水河鱼类资源的现状与保护

赤水河是长江上游右岸一级支流,为长江上游珍稀特有鱼类国家级自然保护区的重要组成部分。为了了解赤水河鱼类资源现状,于2007年4-10月在赤水河流域进行了鱼类资源调查。在干、支流共51个采样点采集了鱼类标本;对茅台镇、赤水市和合江县三个江段的渔获物进行了统计和分析。共采集到鱼类119种(亚种),隶属于5目16科75属。其中25种为该水域的新记录;34种为长江上游特有鱼类,尤其是宽唇华缨鱼(Sinocrossocheilus labiatus)仅分布于赤水河。在上、中、下游分别采集到鱼类36、61和100种。宽鳍鱲(Zacco platypus)、中华倒刺鲃(Spinibarbus sinensis)、光泽黄颡鱼(Pelteobagrus nitidus)、张氏(Hemiculter tchangi)和蛇鮈(Saurogobio dabryi)为赤水河中主要的经济鱼类,同时,各江段渔获物的组成也存在一定的差别。本研究表明,赤水河流域鱼类种类相对丰富,但过度捕捞和涉水活动导致鱼类资源出现了一定程度的衰减。     

Ionic regulation and Na+–K+-ATPase activity in gills and kidney of the freshwater stingray Paratrygon aiereba living in white and blackwaters in the Amazon Basin

During low-water period, freshwater stingray Paratrygon aiereba collected in the whitewater (WW) of the River Amazon showed higher urea content, osmolality, Na⁺ and Cl⁻ concentrations in plasma and perivisceral fluid than those caught in blackwater (BW) of the River Negro. Gills and kidney Na⁺–K⁺-ATPase activities were significantly lower in WW than in BW fish. The high level of kidney Na⁺–K⁺-ATPase activity in P. aiereba may minimize ion loss and generate diluted solute-free urine in ion-poor BW environment.     

Biodiversity of floodplain forests in Europe and eastern North America: A comparative study of the Rhine and Mississippi Valleys

In this study, we investigate the mechanisms driving biodiversity in floodplain forests with a comparison of the composition and dynamics in the warm-temperate floodplain forests of the lower Mississippi Valley and the cool-temperate floodplain forests of the lower Wisconsin and Rhine River Valleys. We employ data from original research, as well as from the literature. We compare species, genus, and family diversity across regions with respect to species richness, numbers of species per family and genus, and a similarity index. We examine these results within a historical context, as well as with respect to river-floodplain dynamics. We also compare productivity data and successional stages for each region. We find a lower species, genus, and family richness in the cool-temperate forests of the Rhine compared to the cool-temperate forests of the Wisconsin, a probable result of the lack of available refugia for Rhine species in times of glacial expansion. We find the highest richness in the lower Mississippi Valley, likely a result of climatic factors and the availability of refugia in this region. In each of the regions, floodplain forests are more diverse than their upland counterparts, demonstrating the role of river-floodplain dynamics in maintaining species diversity. Each region maintains a high and relatively similar level of productivity in the floodplain forests. They also experience similar stages of succession, although succession becomes more complex in the warm-temperate forests of the Lower Mississippi.     

Growth and reproductive cycle of the zebra mussel in the River Rhine as studied in a river bypass

In a Rhine River bypass built at a water pollution control station, the growth and reproductive cycle of the zebra mussel (Dreissena polymorpha) were studied over a period of nearly 3 years. The experimental setup offered the possibility to manipulate the temperature of the continuously passing river water and to calculate influences of temperature and food on the growth of individually marked mussels. Shell growth rates were a function of algal density (measured as chlorophyll a). This function followed saturation curves with temperature-dependent plateau levels, and saturation occurred at values above 40 μg/l chlorophyll a. The relationship between shell growth rate and temperature followed the courses of bell-shaped curves with algal-food-dependent heights. No clear correlations could be found between shell growth and other environmental factors such as dissolved organic matter, total organic content, seston, and turbidity. Two spawning periods per year characterized the reproductive cycle of the zebra mussels at the study site. The first one covered nearly 3 months (April to July) comprising several spawning events. The second shorter one occurred in August. Young mussels of the first spawning period settled at the end of May, and they were able to reproduce a few months later (end of August) when their shell length exceeded 9 mm. This first spawning period of the young-of-the-years coincided with the second spawning period of the older generations. Through the combined analysis of the results of both body weight determinations and shell growth experiments, it was possible to model mussel growth over a period of nearly 3 successive years, and to suggest the presence of an endogenous factor that might be involved in determining the start of the annual shell growth period. Received: 8 April 1997 / Accepted: 3 November 1997     

Distribution of CH4 in the north basin of Lake Biwa and tributary rivers

To determine the origin of CH₄, the vertical distribution of CH₄ around the thermocline in the north basin of Lake Biwa and the horizontal distribution in the Yasu and Ado rivers were measured. In 1995, CH₄ concentrations (ranging from 200 to 1000 nM) exceeding the saturation level were observed just above or in the thermocline in the pelagic region off the Yasu River. These values were higher than those just below the thermocline and in the hypolimnion. CH₄ concentrations in the mouths of the Yasu and Ado rivers were much higher than at other stations, around 2500 and 2000 nM, respectively. Due to the drought in 1994 when there was no water from the Yasu River flowing into the north basin, CH₄ concentration just above or in the thermocline in the pelagic region off the Yasu River ranged from 49 to 74 nM. It is thus concluded that the high level of CH₄ observed in the mouth of the Yasu River is one of the sources of the high CH₄ concentrations in the pelagic region off the Yasu River. On the other hand, the CH₄ concentration in the pelagic region off the Ado River was about 50 nM. It is thought that water flowing in from the Ado River diffused readily into the lake water because the depth of the lake in the region off the Ado River declines steeply. The maximum concentration of CH₄ in the river mouths indicates that these areas are important sites for clarifying the mechanism of the decrease in dissolved oxygen in Lake Biwa and the Yodo River watershed. Received: October 25, 2000 / Accepted: May 8, 2001     

赣江和抚河南方鳅鮀的遗传多样性及遗传结构

南方鳅鮀（Gobiobotia meridionalis）是我国特有的一种小型底栖鱼类。本研究利用线粒体Cyt b基因对赣江巴邱镇江段55尾、万安县江段7尾和抚河抚州市江段48尾共计110尾南方鳅鮀样本的遗传多样性和遗传结构进行分析。结果表明,2个水系110尾南方鳅鮀的Cyt b基因序列共检测出45个单倍型,单倍型多样性和核苷酸多样性分别为0.967 ± 0.006和0.007 1 ± 0.000 2,其中,抚州种群分别为0.911 ± 0.022和0.004 3 ± 0.000 2,巴邱种群分别为0.950 ± 0.015和0.003 7 ± 0.000 2,万安种群分别为0.810 ± 0.130和0.002 6 ± 0.000 5。群体遗传分化指数（Fst）表明,抚州种群与巴邱种群之间存在高度分化。分子方差分析（AMOVA）结果表明,赣江和抚河南方鳅鮀种群的遗传变异主要来自于种群内部（68.42%）。南方鳅鮀群体整体遗传多样性较高,且赣江巴邱种群和抚河抚州种群之间存在高度分化,建议以局部种群为管理单元,加强赣江和抚河流域南方鳅鮀的遗传多样性及资源保护。     

Application of multivariate statistics and toxicity indices to evaluate the water quality suitability for fish of three rivers in the Kruger National Park,South Africa

The water quality of the Olifants, Letaba and Luvuvhu rivers within the Kruger National Park was assessed for their suitability to sustain fish populations. Since water quality assessments result in huge datasets, principal component analysis and an aquatic toxicity index (ATI) were employed to reduce these to more manageable indices. Water quality changed over time, with metal concentrations decreasing, and high flows resulted in increased concentrations of suspended metals. The ATI indicated that metals had no major effect on lowering index values in any of the rivers, and that these values were affected by turbidity in both the Olifants and Letaba rivers, whilst nutrients such as ammonium and orthophosphates negatively affected index scores in the Luvuvhu River. Water quality has improved from the 1990s to 2011.     

图们江流域河流生态系统健康评价

河流是重要的自然生态系统,也是重要的生态廊道之一,图们江流域河流生态系统的健康状况,对维护跨国界流域的水环境的管理和可持续发展有着重要的意义。基于河流水文、河流形态、河岸带状况、水体理化参数以及河流生物5个层面选取22个指标构建了图们江流域河流生态系统健康评价指标体系,运用层次分析法和加权平均法对其进行了健康评价。评价结果表明,虽然该地区处于"健康"级别,但也有28%的地区处于"亚健康"状态。河流健康综合指数(RHI`)与河流生物指标、水体理化指标、河流形态指标、河岸带指标等4项呈显著相关(P0.05),相关系数依次为0.847、0,757、0.740、0.547。研究结果表明图们江流域水生生物的生存环境遭到严重破坏、水体污染严重,河岸带生态退化、城市化影响严重等一系列影响河流健康的问题。     

甘肃省水洛河、清水河流域2009年旧石器考古调查

2009年6～7月在甘肃陇西盆地东部水洛河、清水河流域的旧石器考古调查共发现新地点16处; 采集石制品200余件, 动物化石60余件, 其中大部分采自地层断面。石制品个体多较小, 原料以脉石英为主, 类型包括石核、石片、断块和石器等。剥片方法以硬锤锤击法为主, 偶见砸击法; 石器以小型为主, 类型有刮削器、尖状器等。石器采用硬锤锤击法加工, 以单向为主, 存在少量两面加工者;毛坯以片状为主, 显示了中国北方石片石器工业的特点。地层观察、石制品特征以及14 C测年结果表明, 这些新地点时代应属于晚更新世晚期。