Acute toxicity of cadmium to six intertidal invertebrates

Following a static bioassay techniques the acute toxicity of cadmium to six species of intertidal invertebrates was determined. The sensitivity of the animals to cadmium was of the following order: Emerita sp. (burrowing crustacean) Donax spiculum (burrowing bivalve) Perna viridis (sedentary bivalve) Sabellaria clandestinus (tube-dwelling polychaete) Modiolus carvalhoi and Modiolus sp. (sedentary bivalves). The above observation was based on the median lethal concentrations recorded for the different species, Emerita sp. 1.35 p.p.m., Donax spiculum 1.8 p.p.m., Perna viridis 2.5 p.p.m., Sabellaria clandestinus 2.8 p.p.m., Modiolus carvalhoi 5.6 p.p.m. and Modiolus sp. 9.6 p.p.m. The findings throw insight into the toxicity of cadmium to the common intertidal animals which are either suspension or detritus feeders.

Phylogenetic relationships of the Chinese brown frogs (Genus rana) inferred from partial mitochondrial 12S and 16S rRNA gene sequences

Based on partial sequences of the 12S and 16S ribosomal RNA genes, we estimated phylogenetic relationships among brown frogs of the Rana temporaria group from China. From the phylogenetic trees obtained, we propose to include Rana zhengi in the brown frog

A New Odorrana (Amphibia: Ranidae) from Vietnam and China

Eastern and western populations of the ranid frog Odorrano chapaensis from Vietnam and China are readily differentiated by morphology and mtDNA, and weakly differentiated by morphometrics. The western population contains the type localities of O. chopoens

Diversity, abundance distribution and secondary production of intertidal and subtidal benthic macrofauna of Bushehr Province with emphasis on annelida groups

The present investigation was undertaken to establish a reference situation for future use, to identify temporal and spatial composition of macrofauna and estimate some ecological indices in the sub tidal waters along the Bushehr coastal waters in Persian Gulf. Six transects were selected including Genaveh, Farakeh, Shif, Bandargah, Rostami and Asalouyeh, at each transect 3 station were sampled in depths of zero, 5 and 10 metres. Sampling was seasonally carried out by a VAN VEEN grab 0.0225 m2, during summer 2008 until spring 2009. Samples were wet sieved immediately using 0.5 mm mesh size sieves and sediment retained in the sieve was preserved in 4% buffered formalin solution. Macrofauna specimen were separated from the sediments using decantation and elutriation methods, enumerated and identified up to the Genus level. Environmental factors such as temperature. pH, and salinity were recorded in field using sensitive probs and refractometer (for salinity) and also sediment samples were taken for TOM and grain size analysis in all the stations. 5611 specimens belonging to 66 genera were collected during the present study. Polychaetes were dominant both in terms of genus number (31) and relative abundance (74 % of total macrofaunal abundance). The other dominant groups were Artheropoda, (16.1%), Molusca (2.8%), Echinodermata (1.29%) and others including Nematoda, Nemertina, Echiura and Turbellaria (5.8%). Thirty one Genera belong of 27 families of polychaeta, one genus and family of Subphylum Chlicerata,19 genera belong to 14 families of Crustacea, 8 genera belong to 6 families of Molusca, were indentified in the studied region. 1 family (Polygordidae) and 3 genera (Flabeligera, Pilargis and Polygordius) of Polychaeta, 1 family (Nymphonidae) and genus (Nymphon) of Chelicerata, 1 Family (Nematoplanidae) and genus (Nematoplana) of Turbellaria, were identified for the first time in Persian Gulf area. The result indicated that macrofauna organism have strong relationship with the grain size characteristics of the sediments they inhabit. The most surface deposit feeder specimens such as Prionospio and Cossura were found in zero meters depth of Genaveh, Farakeh, Bandargah, Rostami and Asalouyeh stations with sandy substratum, however the most burrowing deposit feeder and scavenger specimens such as Capitella and Petaloproctus were collected in 5 and 10 meter depths of stations with silty–clay substratum. The annual mean abundance, Shanon- weiner diversity and evenness of macrofauna were estimated1152.73 N/ m² , 2.72 and 0.792 respectively .The annual average biomass and secondary production were computed 1.797 gDW m² and 3.594 gDW m² y-1 .The average of water temperature, salinity, pH and oxygen concentration were recorded between 16.37-36.05 °C, 38-42 g/l, 7.89-8.76 and 4.23-8.23 mg/l, respectively during this study in 6 studied region. Among of investigated stations Asalouyeh adjacent of effluent canal of Gas and petrochemical industry sewage and Farakeh regions adjacent the Helleh estuary had the lowets and the highest community indices. The average of diversity and density in 5 meters depth stations with moderate of sand, silt and clay were slightly more than 2 other depths stations, it seems that 5 meters stations are made a transition habitats between 2 sandy and clay habitats, that can be used by 2 groups of surface and borrowing deposit feeders. Based on the data provided in this survey, the temperature variation, sediment texture, TOM, type habitat and manmade factors of Gas and petrochemical industries have had the most effect on the macrofauna community structure in the studied region during sampling periods.

西藏湍流蛙类一新种

在西藏墨脱采集到1种与Odorrana graminea、O.chloronota和O.livida体背绿色和指端膨大具沟的湍流蛙类相近的物种,经鉴定为新种,命名为墨脱臭蛙Odorrana zhaoi.新种具有以下主要特征:上唇缘具有金黄色条纹;鼓膜圆形,显著,TD:ED为0.56;背侧褶弱,背部皮肤除后部有小疣粒外光滑,体侧有疣粒和小刺;4肢背面有横斑纹;无外蹠突;雄性具1对外声囊和咽胸部椭圆形小刺团,无肱腺.

Invertebrate communities of Lake Nabisojjo

Invertebrates are organisms without a backbone on the basis of body size. Aquatic invertebrates can be divided into two broad categories: micro-and macro-invertebrates. The former commonly known as zooplankton, ranging in size from < 100um 10 ca.1500um and are mainly planktonic (i.e living suspended in the water column). The latter, also known as benthos, (bottom dwelling) are associated with bottom sediments(ie, living on sediment surface or burrowing in sediments), are much bigger organism greater than 1500um in body size.

The nature and origin of deep ocean clay crust from the Gulf of Guinea

Deep ocean sediments off the west coast of Africa exhibit a peculiar undrained strength profile in the form of a crust, albeit of exceptionally high water content, overlying normally consolidated clay. Hot-oil pipelines are installed into these crustal sediments, so their origins and characteristics are of great interest to pipeline designers. This paper provides evidence for the presence of burrowing invertebrates in crust material, and for the way sediment properties are modified through their creation of burrows, and through the deposition of faecal pellets. A variety of imaging techniques are used to make these connections, including photography, scanning electron microscopy and X-ray computer tomography. However, the essential investigative technology is simply the wet-sieving of natural cores, which reveals that up to 60% by dry mass of the crustal material can consist of smooth, highly regular, sand-sized capsules that have been identified as the faecal pellets of invertebrates such as polychaetes. Mechanical tests reveal that these pellets are quite robust under effective stresses of the order of 10 kPa, acting like sand grains within a matrix of fines. Their abundance correlates closely with the measured strength of the crust. While this can easily be accepted in the context of a pellet fraction as high as 60%, the question arises how a smaller proportion of pellets, such as 20%, is apparently able to enhance significantly the strength of a sediment that otherwise appears to be normally consolidated. A hypothesis is suggested based on the composition of the matrix of fines around the pellets. These appear to consist of agglomerates of clay platelets, which may be the result of the breakdown of pellets by other organisms. Their continued degradation at depths in excess of 1 m is taken to explain the progressive loss of crustal strength thereafter.

Establishment, characterization, and virus susceptibility of a new marine cell line from red spotted grouper (Epinephelus akaara)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25 degrees C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with 2n = 8st + 40t. A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10(8.5) TCID50 ml(-1)), while the viral titer of frog Rana grylio virus 9807 (RGV(9807)) reached 10(3.5) TCID50 ml-1. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.

Characterization of an early gene encoding for dUTPase in Rana grylio virus

dUTPase (DUT) is a ubiquitous and important enzyme responsible for regulating levels of dUTP. Here, an iridovirus DUT was identified and characterized from Rana grylio virus (RGV) which is a pathogen agent in pig frog. The DUT encodes a protein of 164aa with a predicted molecular mass of 17.4 kDa, and its transcriptional initiation site was determined by 5'RACE to start from the nucleotide A at 15 nt upstream of the initiation codon ATG. Sequence comparisons and multiple alignments suggested that RGV DUT was quite similar to other identified DUTs that function as homotrimers. Phylogenetic analysis implied that DUT horizontal transfers might have occurred between the vertebrate hosts and iridoviruses. Furthermore, its temporal expression pattern during RGV infection course was characterized by RT-PCR and Western blot analysis. It begins to transcribe and translate as early as 4 h postinfection (p.i.), and remains detectable at 48 h p.i. DUT-EGFP fusion protein was observed in the cytoplasm of pEGFP-N3-Dut transfected EPC cells. Immunofluorescence also confirmed DUT cytoplasm localization in RGV-infected cells. Using drug inhibition analysis by a de novo protein synthesis inhibitor (cycloheximide) and a viral DNA replication inhibitor (cytosine arabinofuranoside), RGV DUT was classified as an early (E) viral gene during the in vitro infection. Moreover, RGV DUT overexpression was shown that there was no effect on RGV replication by viral replication kinetics assay. (c) 2006 Published by Elsevier B.V.

Environmental signals: Synthetic humic substances act as xeno-estrogen and affect the thyroid system of Xenopus laevis

According to outdated paradigms humic substances (HS) are considered to be refractory or inert that do not directly interact with aquatic organisms. However, they are taken up and induce biotransformation activities and may act as hormone-like substances. In the present study, we tested whether HS can interfere with endocrine regulation in the amphibian Xenopus laevis. In order to exclude contamination with phyto-hormones, which may occur in environmental isolates, the artificial HS 1500 was applied. The in vivo results showed that HS 1500 causes significant estrogenic effects on X. laevis during its larval development and results of semi-quantitative RT-PCR revealed a marked increase of the estrogenic biomarker estrogen receptor mRNA (ER-mRNA). Furthermore, preliminary RT-PCR results showed that the thyroid-stimulating hormone (TSH beta-mRNA) is enhanced after exposure to HS1500, indicating a weak adverse effect on T3/T4 availability. Hence, HS may have estrogenic and anti-thyroidal effects on aquatic animals, and therefore may influence the structure of aquatic communities and they may be considered environmental signaling chemicals. (c) 2005 Elsevier Ltd. All rights reserved.

Cyclin A2 is differentially expressed during oocyte maturation between gynogenetic silver crucian carp and gonochoristic color crucian carp

Silver crucian carp (Carassius auratus gibelio) is a unique gynogenetic fish. Because of its specific genetic background and reproduction mode, it is an intriguing model system for understanding regulatory mechanism of oocyte maturation division. It keeps its chromosomal integrity by inhibiting the first meiotic division (no extrusion of the first pole body). The spindle behavior during oocyte maturation is significantly different from that in gonochoristic fish. The chromosomes are first arranged in a tripolar spindle, and then they turn around and are reunited mutually to form a normal bipolar spindle. A new member of the fish A-type cyclin gene, cyclin A2, has been isolated by suppression of subtractive hybridization on the basis of its differential transcription in fully-grown oocytes between the gynogenetic silver crucian carp and gonochoristic color crucian carp. There are 18 differing amino acids in the total 428 residues of cyclin A2 between the two forms of crucian carps. In addition, cDNAs of cyclin A1 and cyclin B have also been cloned from them. Thus two members of A-type cyclins, cyclin A1 and cyclin A2, are demonstrated to exist in fish, just as in frog, humans, and mouse. Northern blotting reveals that cyclin A2 mRNA is more than 20-fold and cyclin A1 mRNA is about 2-fold in fully grown oocytes of gynogenetic silver crucian carp compared to gonochoristic color crucian carp. However, cyclin B does not show such a difference between them. Western blot analysis also shows that the cyclin A2 protein stockpiled in fully grown oocytes of gynogenetic crucian carp is much more abundant than in gonochoristic crucian carp. Moreover, two different cyclin A2 expression patterns during oocyte maturation have been revealed in the two closely related crucian carps. For color crucian carp, cyclin A2 protein is translated only after hormone stimulation. For silver crucian carp, cyclin A2 protein can be detected throughout the process of maturation division. The different expression of cyclin A2 may be a clue to understanding the special maturation division of gynogenetic silver crucian carp.

Studies on morphogenesis and cellular interactions of Rana grylio virus in an infected fish cell line

A pathogenic virus (RGV), isolated from diseased pig frog Rana grylio with lethal syndrome, was investigated with regard to morphogenesis and cellular interactions in EPC cells, a cell Line from fish. Different stages of virus amplification, maturation and assembly were observed at nucleus, cytoplasm and cellular membranes. The matured virus particles, were not only distributed diffusely in nucleus, cytoplasm and cellular surface, but also aggregated as pseudocrystalline arrays in the cytoplasm. Virions were released by budding from the plasma membranes, or following cell lysis. Various types of cell damage, such as small vacuoles, spherical inclusions, and swollen and empty mitochondria, were also found. Some typical characteristics of RGV, such as the symmetrical shape of the virions, replication process involving both nuclear and cytoplasmic phases, budding release from cellular membrane and intracellular membrane, viromatrix and paracrystalline aggregation in cytoplasm, and its acute pathogenic effects, were observed to be similar to that of other iridoviruses. Therefore, the RGV appears to be a member of the Iridoviridae based on these studies. (C) 1999 Elsevier Science B.V. All rights reserved.

雨蛙皮肤分泌液中两种活性蛋白结构和功能的研究

在本研究中我们首次从雨蛙皮肤分泌液中分离得到了一种神经毒素（命名为Anntoxin）和一种干细胞自我更新支持因子（命名为AnSF）。随后，我们通过构建雨蛙皮肤cDNA 文库，利用特异引物筛选到Anntoxin 和AnSF 的cDNA 编码序列，前者的Gene Bank 登录号为FJ598043，后者还在等待分配登录号。Anntoxin 具有60 个氨基酸，是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，构建Anntoxin 的3D-NMR 溶液结构，证实Anntoxin 不同于有三对二硫键（键组合模式：1-6，2-4，3-5）的Kunitz 类型丝氨酸蛋白酶抑制剂，它只有两对二硫键（组合模式：1-4，2-3）。AnSF 具有123 个氨基酸，在C 端具有和Calmadolin 同源的两个EF 手指结构，能够支持人类胚胎干细胞（hESC）和猴神经干细胞（rNSC）的自我更新。为了进行Anntoxin 的生物活性和结构分析，我们在体外成功表达了 Anntoxin，获得了大量的重组Anntoxin（rAnntoxin）。经过生物活性分析， rAnntoxin 和天然分离到的Anntoxin 生物活性相当，都具有很强的胰蛋白酶抑制剂活性。Anntoxin 是一种Kunitz 类型的丝氨酸蛋白酶抑制剂，和来源于芋螺（Cone Snail）的神经毒素Conkunitzin-S1，黑色眼镜蛇毒液（black cobra, Dendroaspis polylepis polylepis）的树突毒素δ-DaTX 或蛋白酶抑制剂K 分别具有32.8%和36.7%的相同序列，和鱼类（fish）来源的Stonustoxin 也有一定的同源性。利用膜片钳技术分别检测Anntoxin 对大鼠背根神经节（rat DRG）上Na+通道，K+通道，Ca2+通道的作用，结果证明Anntoxin 对河豚毒素敏感（TTX-S）的钠离子通道（Nav）有较强的抑制活性，对 K+通道，Ca2+通道作用不明显。随后我们在非洲爪蟾卵母细胞上表达几种典型和常用于测试对亚型K+通道作用的Kv1.1，Kv1.2，Kv1.3，Kv2.1 和 Kv4.2，Kv4.3，Anntoxin 对这些亚型K+通道上的K+电流都没有明显影响。我们成功构建了Anntoxin 的3D-NMR 溶液结构（NMR 号：PDB ID 2KCR， BMRB ID 16094），证实Anntoxin 具有典型的Kunitz 结构，由反向平行的 β–折叠片和α–螺旋及转角组成梨形结构。利用RT-PCR，WesternBlot 以及 ELISA 技术，发现在皮肤、脑、肝、胃和肠中都能检测Anntoxin mRNA 转录，但只在皮肤、脑、肝和胃中有蛋白表达，表达量分别为29.5、5.39、 4.80 和2.02 微克/克鲜重，可以看出Anntoxin 在皮肤中大量表达，是皮肤分泌液中非常重要的组成部分。因为皮肤是雨蛙接触外界的第一屏障，雨蛙的生存环境中存在很多潜在威胁，比如微生物、吸血昆虫、鸟类、爬行动物、哺乳动物等，所以Anntoxin 有可能是雨蛙适应环境的重要化学武器，于是我们测试了Anntoxin 对甜菜夜蛾幼虫（Laphygma exigua Hubner）、水蛇（Enhydris plumbea）、鹌鹑（Coturnix coturnix）、昆明小鼠（Kunming mice）的急性毒性，其LD50 分别为50，450，2500 和3000 微克/千克体重，说明在华西雨蛙皮肤中大量表达的Anntoxin 对几类潜在天敌确实有较强的杀灭作用。为了检测AnSF 的生物学活性，我们在体外成功表达了AnSF，获得了大量rAnSF。设计三个浓度梯度10、100 和500ng/ml，把AnSF 和hESC 共培养，发现在10~100 ng/ml 浓度时对hESC 的自我更新有支持作用；设计三个浓度梯度10、100 和500ng/ml，把AnSF 和rNSC 共培养，发现在 10ng/ml 时对rNSC 的自我更新有较强的支持作用。在超过500ng/ml 高浓度时，AnSF 对hESC 和rNSC 都有明显的细胞毒性作用，对rNSC 的毒性作用更明显。利用RT-PCR 技术，我们检测了雨蛙的皮肤、肌肉、肝脏、胰脏、胃、肠、心脏和脑，AnSF 只在皮肤中有少量表达。这表明AnSF 可能只参与雨蛙皮肤干细胞库的维持，保持皮肤内环境稳定，因为蛙类的皮肤细胞要负责产生大量活性物质参与先天免疫和抗氧化等重要的生理活动，需要经常更新，而AnSF 的存在可能保证雨蛙皮肤干细胞库容量稳定，不断分化出各种成熟的皮肤细胞来使皮肤能够得到足够和及时的更新，保证其功能的正常行使。所以AnSF 是维持华西雨蛙皮肤内环境稳定的重要物质。

若尔盖自然保护区高原林蛙(Rana kukunoris)扩散模型与景观连接分析

揭示水体中繁殖的两栖动物在异质性景观中的空间扩散特点，探讨景观面积丧失和破碎化对于两栖动物的影响，为两栖动物的保护提供理论依据。本文以四川西北部若尔盖湿地自然保护区的高原林蛙(Rana kukunoris)为研究对象，通过运用地理信息系统及建立景观模型等方法，在分析若尔盖湿地自然保护区范围内现有景观格局的基础上，建立了高原林蛙的景观扩散模型，并模拟了“沼泽→草甸”的湿地逆演化过程下高原林蛙的空间分布与景观连接的变化特点。主要结果是： 1．若尔盖湿地自然保护区呈典型的沼泽—草甸式斑块—基质景观格局。草甸面积占整个景观面积的79.42%，景观蔓延度指数（CONTAG）为79.00远离最小值0而更趋向于最大值100，面积和景观蔓延度指数表明草甸是整个景观中面积占绝对优势且景观连接好的类型，构成了景观的基质，对景观的动态格局演变起主导作用。沼泽面积仅占整个景观面积的18.08%，但却是整个景观中斑块数目最多的单元，占所有斑块数的82.9%。因此沼泽斑块与草甸基质之间的动态结构对高原林蛙的扩散起着决定性的作用。 2．空间扩散模型表明，其它类型的景观不但扩展了高原林蛙的活动范围，而且也为高原林蛙在不同沼泽斑块间的连接提供了通道。高原林蛙的空间扩散区域使得彼此间成斑块化隔离状分布的沼泽形成了潜在景观功能连接，促进了不同斑块间物种的交流。小型沼泽作为垫脚石（stepping-stone），使得整个景观中的相隔距离较远的大型斑块联结为一个功能整体，促进了高原林蛙在整个景观中的相互动态联系。 3．模拟“沼泽→草甸”的湿地逆演化过程表明，大量小型沼泽湿地的消失将会 对在沼泽中繁殖并扩散到其它景观类型中去的高原林蛙造成潜在影响。逆演化过程不仅使沼泽斑块的分布范围，沼泽源斑块的面积和空间扩散面积减少，而且对景观连接也有很大影响。小型沼泽的消失，将使得景观斑块的功能连接变小，使得依靠小型沼泽作为跳板的动物在沼泽斑块之间的移动将变得更加困难。 本文是对生境丧失与破碎化影响下两栖动物的行为反应的一种尝试。影响模型的因素很多，包括动物对各种类型景观的偏好程度，地理数据的精度，及模型的可靠程度都是制约模型准确度的因素。 The spatial diffusion of water—breeding amphibian through heterogeneous landscape and the effects of landscape losing and fragmentation to amphibian were the core theory of the landscape ecology of amphibian. Geographical information system (GIS) and landscape model were used to model the diffused area of Rana kukunoris in Zoige Wetland Natural Reserve. Model was also used to analysis the spatial distribution variation of R. kukunoris and the change of landscape connectivity when simulated the retrogressive succession of landscape. The main results are below: 1. There was peatland—meadow pattern which was typical patch—matrix landscape pattern in Zoige Natural Reserve. The meadow area occupied 79.42% of the entire landscape area, contagion index (CONTAG) was 79.00 which was far away the minimum value (0) but tend to the maximum value (100). Both of these showed that meadow was the largest part and the most continue units. It was shown that meadow was matrix of the landscape, which evolved the leading role to the landscape dynamic pattern. Though their area only occupies 18.08% of entire landscape area, peatlands were according to 82.9% of the total patches. Dynamic of the pattern between peatlands and meadows decided the spatial diffusion of R. kukunoris. 2．The model indicated that the other types of landscape not only expanded diffusion of R. kukunoris, but also have provided the potential channels for frog's connections among different peatlands. The spatial diffusion zone of R. kukunoris forced isolated patch peatlands to be potential landscape functional connectivity. The small peatlands, as stepping-stone, made the large peatlands connect as a functional one and promoted the integrated and dynamic connectivity of R. kukunoris in the whole landscape. 3. The simulation of “peatlands→meadows”retrogressive succession process indicated that the decrease of small peatlands will have potential effect to R. kukunoris because they must bred in peatlands and diffuse to other type of the landscape. Retrogressive succession process not only made the decrease of distribution of peatlands, patches number of peatlands and diffused area of R. kukunoris, but also reduced the connectivity among source patches. As stepping-stone, the disappearance of small peatlands will made the migration of R. kukunoris among patches more difficult. The model was an experiment of the amphibian behavior reaction to habitat losing and fragmentation. There were many factors that could influence the accuracy of model, such as the preference of animals to each type of landscape, the geographical data precision, reliable degree of model.

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.