香港地区湍蛙遗传多样性研究

依据线粒体上ND2和CO1两个变异较大的基因序列分析了香港地区香港湍蛙7种群、华南湍蛙1种群，以及大陆其他地区华南湍蛙7种群，戴云湍蛙1种群，武夷湍蛙1种群的系统发育关系，进而探讨香港湍蛙的遗传多样性、香港湍蛙特有性、如何确定香港湍蛙最佳保护单元以及这四种湍蛙的物种分类地位。 1． 香港湍蛙保护遗传学研究 香港湍蛙核苷酸传多样性较低，从其遗传多样性信息、单倍型网络分析、中性检验值以及岐点分布结果一致显示香港湍蛙很可能经历了瓶颈后的扩张，种群正在由一个较小的有效种群大小迅速增长, 有足够的时间通过变异用于积累单倍型的多态性, 而对于提高核苷酸多样化而言, 时间尚短（Nei M et al，1975，Avise J C，2000；李明等，2003）。 分子变异分析结果显示香港湍蛙种群间存在较多的基因交流，且系统发育树上各种群间交叉在一起，没有形成与地理单元相关的分支，而从其单倍型网络看，他们源于共同的祖先，是一个单系群，与地理单元间没有形成显著的遗传分化。因此应作为一个进化显著单元（ESU）。结合其与其他湍蛙发育关系及遗传距离以及野外采集信息认为香港湍蛙只在香港地区有分布，属于香港特有种。该物种内遗传多样性较低，又属于世界自然保护联盟红皮书中的近危种，同时也是《野生动物保护条例》中的受保护野生动物，且由于香港城市建设等使得其栖息环境受到威胁，因此在香港特别行政区应该受到重点保护。 从单倍型分布和核苷酸多样性可以看出大榄涌种群和城门种群具有较高的单倍型多样性和核苷酸多样性，应该作为保护的重点区域。 2. 华南湍蛙东、南沿海种群间系统关系 华南湍蛙分布广，各种群存在着丰富的遗传多样性信息且中部种群广西龙胜和湖南张家界种群核苷酸多样性明显高于其他边缘种群华南湍蛙。种群间几乎没有基因交流，且各种群间无共享单倍型，可见已形成了显著的遗传分化。各种群间遗传距离都较远，其中广东南昆山种群以及福建三港种群与其他种群距离最远，因此可以推测其他种群（广东深圳、香港大屿山、广西龙胜和防城以及湖南张家界种群）可能为独立进化的种群。但是否是一新种或一隐存种，还需要结合形态学进行更深入的研究。 本研究中无论从系统关系看还是从遗传距离看，大屿山种群与深圳种群最近，支持陈坚峰等将其定为华南湍蛙，即华南湍蛙新增一个分布点：香港大屿山。 系统树上广西防城种群（支B）与龙胜和湖南种群（支A）形成姐妹群。香港大屿山种群与深圳种群先形成姐妹群（支C），但却没有与其距离很近的广东南岭及南昆山种群（支D）形成姐妹群，可能粤北和粤中的环境及气候较复杂因此与粤南其他种群形成了明显的隔离。同时可以看出华南湍蛙种群遗传分化与地理距离没有显著的相关性。 3. 四种湍蛙间的系统关系 根据线粒体CO1基因建立四种湍蛙间的系统关系及其遗传距离，很清楚地看到，香港湍蛙与戴云湍蛙关系很近，而华南湍蛙则与武夷湍蛙较近。然而，戴云湍蛙同一个种群内部共有两个单倍型DY1和DY2，且两个单倍型间遗传距离大于DY1与香港湍蛙间遗传距离，更远远大于香港湍蛙种群内部的距离，即戴云湍蛙内部两个单倍型间遗传距离达到了种级水平，同样在系统发育树上这两个单倍型与香港湍蛙形成并系。但是，戴云湍蛙种内在形态上差异不显著。因此，其是否属于萌芽物种分化形成（budding speciation）或已经完全分化为两个不同的种值得进一步研究？ 与戴云湍蛙香港湍蛙关系类似，从系统树上看华南湍蛙不形成单系，而是分成两个大支，与武夷湍蛙形成并系，且福建和南昆山的华南湍蛙与武夷湍蛙遗传距离远大于武夷湍蛙种内福建种群与浙江种群的遗传距离，达到了种级分化水平。由此，可以推断武夷湍蛙是有效种。系统树上广东深圳、香港大屿山、广西防城和龙胜以及湖南张家界种群与华南湍蛙福建及南昆山各种群间遗传距离已超出了种内各种群间的遗传距离，但是至于这一支是否应为另外一个种，有必要扩大采样，并结合核基因及形态信息进行进一步研究。 MtDNA of ND2 and CO1 gene were used to investigate genetic diversity of Amolops in Hongkong .We collected seven populations of A. hongkongensis,，one population of A.ricketti from Hong Kong and other seven populations of A.ricketti from East and South of Chinese mainland. As well as one population of A. daiyunensis and one population of A.wuyiensis Phylogenetic relationship were analyzed of four species. Discussed whether A.hongkongensis is an endemic species and how can we make the conservation and management decisions. 1. Conservation Genetics of A. hongkongensis A. hongkongensis has a low nucleotide diversity, the results of genetic diversity, haplotype network, neutrality test and the mismatch distributions indicate that A. hongkongensis experienced a recent expansion after a bottle neck. They had enough time to accumulated haplotype diversity, but it’s too short to have a high nucleotide diversity（Nei M et al，1975，Avise J C，2000；Li et al，2003）. The result of AMOVA reveals that it has much gene exchange among the populations of A. hongkongensis. The clades of the phylogenetic tree were mixed together, no significant genetic differentiation among 8 populations and they share the same ancestor from the network analysis, these indicate that they are monophyly and should be protected as one ESU. Combined with the information of relationships of interspecies, genetic distance and distribution investigate, We conclude that A. hongkongensis is an endemic species of Hong Kong. Considering on the status of low genetic diversity in A.hongkongensis, and this species was listed in the IUCN red list as near threatened, as well as listed in the . Furthermore, it’s habitat loss and degradation more rapidly as the human activity got higher and higher. So it’s urgent to protect them in Hong Kong. Our results suggest that Tai Lam Wu and TAI MO Shan -Shing Mun populations have the higher priority to be protected because their higher genetic diversity. 2．Phylogenetic relationships among populations of Amolops ricketti from the Southern and eastern China A. ricketti has the considerable genetic diversity of mitochondrial haplotypes within and among populations, and Mitochondrial DNA diversity was higher in populations at the central area of the present distribution range of the frog，i. e. the Longsheng population and Zhangjiajie population, than at the edges of their distribution range. They have no share haplotype among populations, and have a significant genetic differentiation. Genetic distance is high among the populations, especially the distance of Nankun and Sangang group with others, which suggested that they evolved independently. May be there is a cryptic species or a new species, a further study is needed. The results of gene tree and the genetic distance clearly demonstrate that the population from LanTau island is A. ricketti, so we agree with Chen et al(2005) . That means A.ricketti have a new distribution site: LanTau island, HongKong. Phylogenetic relationships were analyzed through NJ and Mrbayes methods and got a consistent topological structure, the structure indicated that the ingroup were comprised four groups. Populations Longsheng and Zhangjiajie were first clustered as clade A; Populations Fangcheng was clustered together (clade B) as a sister group to clade A；Populations Shenzhen and Lantau island were sister groups and clustered as clade C；Then the clade D included populations Nankunshan and Nanling in Guangdong province and Sangang in Fujian province. 3. Phylogenetic Relationships among these four specises Phylogenetic relationships based on 1503bp CO1 gene and the genetic distance show that A. hongkongensis close to A. daiyunensis whereas A.ricketti near to A.wuyiensis. Nevertheless, there are two haplotypes in A.daiyunensis and the genetic distance between them higher than the distance between DY1 with A. hongkongensis. A. hongkongensis is nested in the paraphyletic ancestral species A. daiyunensis. Without significant difference in the morphological characters, So, we considered both A.daiyunensis and A.hongkongensis are valid species, may be this represents a case of ‘budding speciation’ like Batrachuperus pinchonii(Fu and Zeng,2008) in the population of A. daiyunensis. Just like two species above A. wuyiensis and A. ricketti are not monophyly, instead, A.wuyiensis is nested in the paraphyletic ancestral species A.ricketti. We need do more research to make sure whether they are new species.

大熊猫“盛林1号”放归监测

大熊猫(Ailuropoda melanoleuca)是我国特有的珍稀濒危物种，国家Ⅰ级重点保护野生动物，被称为“国宝”。目前，大熊猫被局限在我国中西部的岷山、邛崃、大相岭、小相岭、凉山和秦岭6大山系中。对大熊猫的保护和研究，我国政府、保护生物学科研人员、社会各界及国际保护组织都做了大量的工作。根据全国三次大熊猫调查结果显示，大熊猫栖息地片段化现象依然存在，形成多个隔离的大熊猫小种群。尤其在小相岭、大相岭、岷山B和岷山C种群，大熊猫数量较少，且栖息地破碎，面临较大威胁。有的山系大熊猫种群数量些已低于最小可存活大熊猫种群的数量，如果不采取人工措施，这些种群的大熊猫存在灭绝的危险。 将圈养大熊猫放归野外，以补充野外大熊猫种群数量，增加其遗传多样性，复壮和扩大野生大熊猫种群，是大熊猫人工繁育的最终目标。为降低放归的风险性，在放归人工繁育大熊猫前，将救护存活的野生大熊猫先有计划放归野外，并对其进行跟踪监测，对积累大熊猫放归经验，进一步研究大熊猫野外生物学习性，丰富放归地大熊猫种群遗传多样性，为人工繁育大熊猫放归野外夯实基础，具有十分重要的意义。2005年8月8日，国家林业局和四川省人民政府联合将救护野生大熊猫“盛林1号”放归于龙溪－虹口国家级自然保护区内岷山B大熊猫种群栖息地，并进行系统监测研究。成功的积累了一些放归经验和放归大熊猫的生物学资料，为人工繁育大熊猫的放归奠定了一定基础。 2005年8月至2007年6月期间，我们采用GPS无线电项圈、粪便DNA检测和红外线自动触发相机陷阱的方法，对大熊猫“盛林１号”进行了追踪监测，获得了以下成果： 1.通过分析“盛林1号”放归后了活动趋势和采用两种贝叶斯方法，利用目前五大山系的已有微卫星遗传数据，检测“盛林１号”与五大山系的遗传关系的远近，推测其来源于邛崃山系的可能性较大。 2.收集了大量“盛林1号”野外生境选择数据。我们认为“盛林1号”放归后经历了应急期、初步稳定期、长途迁徙期三个阶段（这可能是今后放归大熊猫都必经的三个时期），并与当地大熊猫种群已发生交流。目前“盛林１号”仍在寻找适合的巢域。 3.结合过去监测数据分析，在放归区域大熊猫和羚牛尽管同域分布，但由于食性不同，对微生境选择还是有着很大差异，因此保护管理对策要有针对性。 4.“盛林1号”的放归是成功的。救护大熊猫异地放归工作应继续开展，但要改进放归后的监测技术。要改进现有对人工饲养大熊猫野化培训方法和放归方式，才能真正将人工繁殖个体放归野外。 Giant Panda (Ailuropoda melanoleuca) is an endangered species endemic to China. It was listed as National Protected I Class Species and is crowned as “National treasure” of China. The populations of Giant Panda are limited in 6 mountain system in Center-West of China, i.e. Mingshan, Mt. Qionglai, Mt. Daxiangling，Mt. Xiaoxiangling, Mt. Liangshan and Mt. Qinling. The results of the Third National Survey on Giant Panda showed that the habitats of Giant Panda is still fracted and Giant Panda population is divided into several isolated small populations. Population B from Mt. Daxiangling, Mt. Xiaoxiangling and Mt. Mingshan and Population C from Mt. Mingshan are very small with very fracted habitat and are more endangered. Several populations in those mountain systems are smaller than Minimum Viable Population of Giant Panda. It is very possible that those populations will be extinct without artificial help. The ultimate Goal of Reintroduction caged Giant Panda to wild is to increase wild population size and genetics diversity and rebuild and expand wild Giant Panda population. It is of significant to return rescued wild Giant Panda to wild and monitor their behavior before reintroduction artificial reproduced Giant Panda. It will increase our knowledge on reintroduction of Giant Panda. Aug 8th, 2005, “Shenglin 1”, a rescued wild Giant Panda was returned to Longxi-Hongkou National Nature Reservoir, which is habitat of Giant Panda Population B of Mt. Mingshan. A systematic monitor was carried out on “Shenglin 1”, and the successful return enriched our biological knowledge on Giant Panda reintroduction. It will be very help for future conservation work on reintroduce artificial reproduced Giant Panda. “Shenglin 1” was tracked with GPS collar, DNA in feces and infrared-trigged camera from Aug 2005 to Jun 2007. 1. Locomotion behavior and microsatellites comparison with Giant Panda from the 5 mountain systems indicated that “Shenglin 1” is possibly from Mt. Qionglai. 2. Habitat usage of “Shenglin 1” was studied. It was suggested that there were 3 phases after return, i.e. emergency response, preliminary stable phase and long distance locomotion, which could be a general process for other returned Giant Panda. It was indicated that there was some interaction between “Shenglin 1” and local population. “Shenglin 1” is seeking for suitable home range now. 3. Monitor data also indicated that microhabitat preference of Giant Panda and takin (Budorcas taxicolor) are different because of different diet, though they are sympatric. It was suggested that conservation management for the two species should be plan in particular. 4. The reintroduction of “Shenglin 1” is a successful case. The program of return rescued Giant Panda to other habitats is of value and should be continued. However, more improvement is needed for the monitor technique. More improvement is need for feralization and returning before we return artificial reproduced Giant Panda to wild.