Molecular and morphological analysis of endangered species Gymnodiptychus integrigymnatus (Teleostei: Cyprinidae)

Gymnodiptychus integrigymnatus is a critically endangered species endemic to the Gaoligongshan Mountains. It was thought to be only distributed in several headwater-streams of the Longchuanjiang River (west slope of the Gaoligongshan Mountains, belonging to the Irrawaddy River drainage). In recent years, dozens of G. integrigymnatus specimens have been collected in some streams on the east slope of the Gaoligongshan Mountains (the Salween drainage). We performed a morphological and genetic analyses (based on cytochrome b and D-loop) of the newly discovered populations of G. integrigymnatus to determine whether the degree of separation of these populations warrants species status. Our analysis from the cytochrome b gene revealed that nine individuals from the Irrawaddy drainage area and seven individuals from the Salween drainage area each have only one unique haplotype. The genetic distance between the two haplotypes is 1.97%. Our phylogenetic analysis revealed that G. integrigymnatus is closely related to highly specialized schizothoracine fishes. Analysis from the mitochondrial control region revealed that G. integrigymnatus has relatively high genetic diversity (pi was 0.00891 and h was 0.8714), and individuals from different river drainages do not share the same haplotypes. The AMOVA results indicated 87.27% genetic variability between the Salween and Irrawaddy populations. Phylogenetic trees show two major geographic groups corresponding to the river systems. We recommend that G. integrigymnatus should be considered as a high priority for protected species status in the Gaoligongshan Mountains National Nature Reserve, and that the area of the Gaoligongshan Mountains National Nature Reserve should be expanded to cover the entire distribution of G. integrigymnatus. Populations of G. integrigymnatus from different river systems should be treated as evolutionarily significant units.

Genetic diversity and conservation of endangered animal species

The loss of biodiversity resulting from extinctions is receiving increasing attention. Over several thousands of animal species have been evaluated and recognized as endangered species. Inbreeding depression has been demonstrated in many wild animal speci

Ex situ conservation status of an endangered Yangtze finless porpoise population (Neophocaena phocaenoides asiaeorientalis) as measured from microsatellites and mtDNA diversity

The Yangtze finless porpoise, Neophocaena phocaenoides asiaeorientalis, is all endangered small cetacean that occurs only in the middle and lower reaches of the Yangtze River of China. The establishment of a breeding population of the porpoise in Tian-e-Zhou Baiji National Natural Reserve represents the first attempt at ex situ conservation efforts for a cetacean species. With the goal of effective protection, management, and monitoring of this preserved population, we examined its genetic diversity using 930 bp of mtDNA control region sequences and 13 polymorphic microsatellite loci. A very low level of genetic variation (h = 0.6010 +/- 0.0029 s.d.; pi = 0.0007 +/- 0.0000002 s.d.) in the mtDNA control region sequences and a moderate genetic diversity (Ho = 0.5740 +/- 0.2575 s.d.) in the microsatellites were detected in the population. It is necessary to introduce more individuals with representative genetic variations into the reserve ill order to foml a larger and healthier group structure for long-term survival of the population. Successful establishment of the Yangtze finless porpoise population in the Reserve also provides a useful model for an ex situ conservation programme for other rare and endangered species. (c) 2005 International Council for the Exploration ofthe Sea. Published by Elsevier Ltd. All rights reserved.

Pollinator shift and reproductive performance of the qinghai-tibetan plateau endemic and endangered Swertia przewalskii (Gentianaceae)

Reproductive failure results in many plant species becoming endangered. However, little is known of how and to what extent pollinator shifts affect reproductive performance of endangered species as a result of the artificial introduction of alien insects. In this study we examined breeding systems, visitor species, visiting frequency and seed set coefficients of Swertia przewalskii in two years that had different dominant pollinator species (native vs. alien). Flowers of this species were protandrous and herkogamous and insects were needed for the production of seeds. The stigmatic receptivity of this species was shorter than for other gentians. No significant difference in seed set coefficient was found for hand-pollinated plants between the two years, indicating that pollinator shift only had a minor effect on this plant's breeding system. The commonest pollinators in 2002 were native bumblebees, alien honeybees and occasional solitary bees, however, only alien honeybees were observed in 2004. The flower visitation rate in both years was relatively high, although the total visit frequency decreased significantly in 2004. The control flowers without any treatment produced significantly fewer seed sets in 2004 than in 2002. In the past decade the seed production of this species may have partly decreased due to pollination by alien honeybees, however, we suggest that they might have acted as alternative pollinators ensuring seed production of S. przewalskii when native pollinators were unavailable. The main reason that this plant is endangered is probably the result of habitat destruction, but changes in land use, namely intensified agricultural practice and unfavorable animal husbandry have also contributed to its decline. We recommend that in-situ conservation, including the establishment of a protected area, is the best way to preserve this species effectively.

濒危植物秦岭冷杉的种实特性和幼苗适应性

　　秦岭冷杉(Abies chensiensis)是松科冷杉属常绿乔木，我国国家二级濒危植物，主要分布在我国的秦岭。迄今为止，国内外有关秦岭冷杉的研究鲜有报道。本文研究了核心分布区和边缘区秦岭冷杉的结实特性、球果和种子的形态变异、种子萌发特性及幼苗的最初生长和适应状况，并通过与同属非濒危物种巴山冷杉相关特性的比较研究，初步探讨了秦岭冷杉的繁殖生物生态学特性, 为全面揭示秦岭冷杉濒危机制奠定基础。 　　与冷杉属其他种相比，核心区秦岭冷杉种子较大，出种量也较高，但空粒率很高，达到88%以上;边缘分布区的空粒率更达到95%以上，反映出极高的败育率。秦岭地区秦岭冷杉结实量和结实率随海拔高度升高而减少;同时，两个海拔高度的对比表明，低海拔秦岭冷杉具有较大的球果和种子，饱满度高。表明在其垂直分布范围内，低海拔地段更适合秦岭冷杉的生长和发育。 　　核心区（即秦岭地区）的秦岭冷杉球果和种子的绝大部分指标均值显著大于边缘区（即神农架地区）。相同海拔条件下，秦岭冷杉绝大部分球果和种子形态指标在地区间、地区内种群间、种群内部均有显著差异，约85%的变异来自地区内个体间和个体内，说明秦岭冷杉的球果和种子形态特征变化受遗传控制更显著。人工林与天然林相比，大部分人工林球果和种子指标均值和变异幅度大于天然林，表明通过人为经营可以改善秦岭冷杉的一些生殖性状。 　　秦岭冷杉具有浅休眠，除经典的冷层积方法可以打破休眠外，适当的物理、化学方法处理，如流动水冲洗、适宜浓度硝酸钾、赤霉酸浸种也可以起到相似的作用。各种促进萌发的方法所得到的最大萌发率间没有显著差异。核心区种子萌发率显著高于边缘区;此外，核心区种子在萌发速度和进程上也要显著高于边缘区，这也表明核心区秦岭冷杉活力更高。同样，由于巴山冷杉较高的饱满度，同等条件下，巴山冷杉种子萌发率约三倍于秦岭冷杉种子。 　　野外幼苗的适应性研究主要通过播种后观测出苗率及幼苗在各种环境条件下的存活率、生长状况来进行。通过一年的观测发现，秦岭冷杉野外种子出苗率平均只有16.9%，一年后幼苗死亡率高于50%。 林窗中、土壤覆盖厚度2~3cm的苗床中种子出苗率最高;较深土壤厚度出苗率低说明秦岭冷杉土壤种子库对更新贡献较小。层积30天种子出苗率高于未处理的种子，但对生长率没有影响。林外苗床中幼苗生长率较高。矿质土壤基质上幼苗生长率明显高于林地中。

濒危物种保护、生境破碎化及其恢复

保护区是否真的能起保护作用？保护区究竟如何布局？这是自然保护区的建设一直争论不休的课题。就大熊猫保护而言，目前面临的最紧迫问题可能不是保护区数量多少的问题，而是这些多个保护区组成的保护区群的配置或格局是否合理。因此，本研究以岷山大熊猫保护区群为研究对象，分析现有大熊猫及栖息地及其保护现状，指出目前保护区存在的问题，并对岷山大熊猫自然保护区群合理布局提出建议性设计，以期为大熊猫保护及自然保护区建设的理论提供借鉴。 到目前，为了保护大熊猫，在岷山地区已经建立了1 7个自然保护区，这些保护区的建立对大熊猫的保护起到了非常重要的重要。但是，大熊猫栖息地仍然持续下降。就其原因有两方面，即岷山地区人为活动的干扰，如旅游业的盲目发展。但，同时大熊猫自然保护区本身的布局存在一定的不合理性。最后，从景观尺度上提出岷山大熊猫保护区群合理建设的布局，即在分析现有保护布局基础上，找到优先保护的敏感地带建立廊道，将所有保护区连成布局合理的保护区群。 大熊猫受威胁的关键因子并不是竹子，而是森林．竹子复合系统的破碎化或质量的退化，故保护大熊猫应从保护森林、恢复森林．竹子复合生态系统的质量入手。但是，具体实践中究竞采取什么模式来恢复退化的大熊猫栖息地？选取不同模式的科学依据是什么？如何评判不同恢复模式的成本和效益？这些方面的工作到目前缺少系统研究。本研究在分析了岷山地区自然、社会与经济条件的基础上，从荒山荒地造林、天然林保护与低效林地改造主要植被恢复措施着手，全面分析各类植被恢复措施所形成植被的生态效益（保持水土、涵养水源、保持肥力等）、经济收益（木材、果品和林副特产品）、社会效益（种植结构、劳动力再分配等），总结出植被恢复成本和效益的构成要素，计算主要植被恢复模式的成本和效益，为选择适宜的植被恢复措施、典型植被恢复模式和合理确定植被恢复技术提供科学依据，从而推动岷山植被快速健康地恢复，为大熊猫提供适宜的生存空间。 三峡大坝的建设使其固有水域的人为改造导致水位上升，使原本连续的山地生态系统被分割成大小不等的岛屿。水位变化导致的这种生境岛屿化为栖息地破碎化和岛屿生物地理学提供了十分难得的实证研究的机会。然而，同时，这种栖息地的片段化无疑对三峡库区生物多样性保护和生态系统的完整性造成威胁。到目前，为了监测库区建设对生态环境的影响，已经建立了22个定位观测站，但没有一个是生境破碎化方面的，为此建议尽快建立生境破碎化方面的定位研究站。三峡库区生物多样性丰富，但保护区面积仅仅占库区面积的6.9%，而同期我国已经建立了1 999个自然保护区，占国土面积的15%，因此，非常迫切建立更多的保护区（至少达到全国平均水平）。为了从宏观尺度上加强对库区的综合保护，建议从景观生态学的角度出发，将库区作为一个自然、社会和经济复合生态系绕对待。

长期驯化环境下丁香属植物的光合生理生态学研究

本研究是在丁香属植物半个世纪的丰富引种基础上，采用光合生理的实验手段探讨该属重要物种长期驯化中的环境响应机制。 在迁地条件下，与广布种华北紫丁香相比， 濒危种羽叶丁香有较低的光需求，在中等光强下净光合速率显著低于广布种;气孔限制值较大，对光合作用的调节较弱;蒸腾作用亦较弱，导致中午叶温较高造成叶片伤害。功能叶片的净光合速率在8:00光强及气温较低时已达峰值，叶片生长历时短暂并于初夏前结束，长此以往可能影响光合产物积累。可见羽叶丁香以“逃避”的方式适应迁入地生长季节环境胁迫。 为了探究羽叶丁香在引种地“逃避”策略的环境驱动因子，我们分别选取种植在园内全光照曝晒和60% 遮荫、生物量差异极为显著，而其它条件相同的羽叶丁香开展实验。尽管曝晒单株比遮荫单株有着显著低下的生物量，但最大量子效率及叶绿素a/b的结果都表明曝晒并未对其PSⅡ及LHCⅡ造成损伤。遮荫单株也已经由高叶绿素含量表现出对低光环境的适应。可见，其光适应机制已经存在，而引种地生长季节的高温可能是羽叶丁香生长中要回避的主要因子。 为了进一步证实这个推断，并希望了解羽叶丁香在原产地的环境响应状况和致濒的生理原因，我们在其原产地贺兰山对该种进行了调查和试验。调查发现在贺兰山羽叶丁香分布在海拔1700m、西北坡向、近谷底处。由于坡度较缓，晴朗天气光照时数达8h，叶片厚重，净光合速率极显著地高于引种地;最大量子效率表明中午的强光并未带来严重光抑制，且小幅度的光抑制在次日清晨可完全恢复，并且有较高的实际量子效率。故在原产地强光仍旧不是其生长的限制因子;其极高的光合速率及高效的实际量子效率是对冷凉强光条件的特殊适应，说明羽叶丁香具有喜冷凉不惧光的固有特点，而冷凉条件于高温炎热的引种地不能得到满足时，便放弃了对光的要求而在遮荫的生境中适应下来。因此引种地的高温应该是其要“逃避”的重要因子。同时表明较强的光合能力及强光的适应特征不会使羽叶丁香在原产地成为濒危物种。 为了搞清羽叶丁香的濒危原因，我们将原产地野外分布状况连同两地水分利用效率棕合分析发现：在水分保证适当的北京植物园栽培条件下，即使是曝晒的植株也有着较高的水分利用效率，而原产地的羽叶丁香低下的水分利用效率表明其生存和分布对水分条件极为敏感。因此，羽叶丁香是良好水分条件的“享受者”，而不是干旱条件的“耐受者”，而这对于生长在干旱半干旱地区的物种来说在一定程度上限制了其散布的可能性，以致数量极为有限。 “全能” 的华北紫丁香恰是丁香属系统演化中较为进化的种，而“低能” 的羽叶丁香又恰是居于顶生花序系（原始）与欧丁香系（进化）之间的重要过渡种。在对丁香属起源及散布进行推断的基础上，这些已经得到的结果促使我们从区系和演化的层面去了解演化系在长期迁地驯化的光合生理差异。结果发现演化过渡种羽叶丁香的光合指征也多处于中间位置;顶生花序系（原始）种类光合效能相对较低，较强的水分平衡的保持能力可能成为它们能在跨气候带引种中成活的解释。欧丁香系（进化）种类气孔导度对光梯度反应敏感，但较强的蒸腾降低了其水分利用效率，在引种地干旱高温的生长季会因水分不能及时供给而可能在年周期尺度上直接影响生物量。小乔木型的种类可能由于较高的水分利用效率而不畏干旱，形成了拟女真亚组从西北到东北继而向日本的广泛分布。 驯化的现实意义之一是应用。丁香的盐碱耐受性是区域栽培中面对的问题。我们模拟氯化物硫酸盐型盐碱土（NaCl - MgSO4）与光照梯度交互的试验结果表明紫丁香系品种曝晒栽植能耐受0.2% 的土壤盐度，如果在相同盐度生境中遮荫种植会有更高的盐分耐受表现。 对迁地物种环境适应性的研究论证着眼点从“种” 的水平归结到“系”的层次，从对特殊代表物种当今适应机制的量化比较联系到其起源和散布的历史发展，在一定程度上超越了常规引种驯化对现存实体“种” 的定性描述;将迁地驯化与就地适应相联系，以较为科学的思路贯穿驯化保护研究，并最终将研究结果回归于驯化保护实践。

濒危植物疏花水柏枝种群生物学和回归引种的研究

　本文对三峡库区濒危植物疏花水柏枝的种子形成与传播、种子萌发与幼苗定居、植株生长动态等种群生物学特性及其与环境的关系，特别是对水淹胁迫的生理学与生态学响应进行了研究，并以此为基础结合疏花水柏枝抢救工程的实施，对疏花水柏枝回归引种的相关生态学问题进行了探讨。 　　疏花水柏枝植株的生殖分配极高，每株所形成的花枝、花序和种子数量都比较多，其种子传播的方式有风传播和水传播，种子直接传播是疏花水柏枝种群进行扩增的主要途径，自然分布疏花水柏枝种群因此而常常成群分布。种子通过江水的二次传播是沿长江呈星状分布的疏花水柏枝各种群间进行基因交流的主要途径。限制疏花水柏枝种子萌发的环境因素主要是土壤含水量，这也是导致该物种在自然条件下仅分布于消落带的关键因素。疏花水柏枝一年生幼苗的成活率以及衡量植株生长状况的生物量、枝条数和根系长度都随着密度的增加而显著下降，表明密度对疏花水柏枝一年生幼苗的定居与生长有明显的制约作用。疏花水柏枝每年夏天都要经历长时间的水淹，在该时期无论是否被水淹，其幼苗地上部的生长都处于停滞（休眠），水淹后植株生物量都有一定程度的减少，但在各种水淹程度处理与对照之间除根深有显著差异外，幼苗的存活率、生物量、一级枝数、一级枝长度、根冠比的差异都不显著，表明水淹对幼苗的定居和生长没有显著影响，疏花水柏枝是通过快速的生物量丧失和快速恢复来适应水淹阶段的环境胁迫的。夏季适当的水淹不仅不会影响植株的生长，反而有利于该物种躲避这个阶段河滩上的高温天气，提高幼苗的成活率。 　　疏花水柏枝对水淹的生理生态适应机制较复杂。夏季水淹过后，植株进入生长恢复阶段，恢复初期植株的日均蒸腾强度与日均光合速率随着其在夏季被淹没时间的增加而增加，说明夏季水淹有利于植株尽快恢复生长。生长季节的水淹胁迫会使疏花水柏枝植株最初的光合作用和蒸腾作用的能力有所下降，但是长期水淹后，植株会形成适应，其光合与水分生理又会恢复到正常状态。疏花水柏枝植株也会在生理生化上对水淹胁迫作出响应。在夏天洪水季节无论是否被水淹，植株的可溶性糖总糖与蔗糖的都含量上升，根系活力、丙二醛（MDA）含量、过氧化物酶(POD)活性、多酚氧化酶(PPO)活性在各种水淹处理与对照之间以及淹水后与淹水前之间没有显著差异，从生理上表明疏花水柏枝的生长在夏季处于生长停滞（休眠）状态，其生理生化变化是植株对休眠的响应。夏天洪水季节结束后，各处理植株的多酚氧化酶活性、过氧化物酶活性、蔗糖与总糖含量在生长恢复阶段显著下降，丙二醛含量、根系活力则分别有所增加，表明植株在生理生化上也得到恢复，且植株在夏季是否受水淹胁迫对生长恢复阶段植株的生理生化变化没有显著影响。生长阶段的水淹胁迫会导致丙二醛含量、多酚氧化酶、过氧化物酶活性上升，其中前两者的变化显著，而根系活力、可溶性总糖含量则在水淹后表现为下降。但是经长期水淹后，各种生理生化指标会逐渐恢复到正常水平，表明该物种对水淹的适应能力较强，水淹并不会对植株形成长期的生理压力。 　　疏花水柏枝被回归引种于长江三峡的几个主要支流上，形成几个相对隔离的重建种群。种群遗传多样性、年龄结构、分布格局、繁殖与扩展等生物学特性是种群持续发展的基础，疏花水柏枝种群的重建中必须予以考虑。各移栽点应分别从不同的地点采集移栽材料，且以生长旺盛、繁殖力强、遗传性状稳定的3-10年植株为主。在疏花水柏枝种群恢复与重建中目前所面临的主要问题是如何增强被隔离的种群间的基因交流、促进种群的种子扩散与萌发、协调新建种群与当地物种的关系、营造有利于新建种群定居与生长的生态环境。

Genetic diversity and population history of golden monkeys (Rhinopithecus roxellana)

Golden monkey (Rhinopithecus roxellana), namely the snub-nosed monkey, is a well-known endangered primate, which distributes only in the central part of mainland China. As an effort to understand the current genetic status as well as population history of this species, we collected a sample of 32 individuals from four different regions, which cover the major habitat of this species. Forty-four allozyme loci were surveyed in our study by allozyme electrophoresis, none of which was found to be polymorphic. The void of polymorphism compared with that of other nonhuman primates is surprising particularly considering that the current population size is many times larger than that of some other endangered species. Since many independent loci are surveyed in this Study, the most plausible explanation for our observation is that the population has experienced a recent bottleneck. We used a coalescent approach to explore various scenarios of population bottleneck and concluded that the most recent bottleneck could have happened within the last 15,000 years. Moreover, the proposed simulation approach could be useful to researchers who need to analyze the non- or low-polymorphism data.

Microsatellite polymorphisms of Sichuan golden monkeys

Previous study using protein electrophoresis shows no polymorphism in 44 nuclear loci of Sichuan golden monkey (Rhinopithecus roxellana), which limits our understandings of its population genetic patterns in the nuclear genome. In order to obtain sufficient information, we scanned 14 microsatellite loci in a sample of 32 individuals from its three major habitats (Minshan, Qinling and Shennongiia). A considerable amount of polymorphisms were detected. The average heterozygosities in the local populations were all above 0.5. The differentiations among local populations were significant. There was evidence of geneflow among subpopullations, but geneflow between Qinling and Shennongjia local populations was the weakest. Minshan and Qinling populations might have gone through recent bottlenecks. The estimation of the ratio of the effective population sizes among local populations was close to that from census sizes. Comparisons to available mitochondria data suggested that R. roxellana's social structures played an important role in shaping its population genetic patterns. Our study showed that the polymorphism level of R. roxellana was no higher than other endangered species; therefore, measures should be taken to preserve genetic diversity of this species.

Changes of urinary steroid conjugates and gonadotropin excretion in the menstrual cycle and pregnancy in the Yunnan snub-nosed monkey (Rhinopithecus bieti)

The Yunnan snub-nosed monkey (Rhinopithecus bieti) is one of the most endangered species in the world, and it is endemic to China. According to our knowledge, there was no information on reproduction for this species. The present study was designed to und