Cloning and sequencing of the growth hormone gene of large yellow croaker and its phylogenetic significance

Using conserved primers and the PCR reaction, the growth hormone (GH) gene and the 3'-UTR of the large yellow croaker (Pseudosciaena crocea) were amplified and sequenced. The gene structure was analyzed and compared to the GH genes of 5 other percoid fish downloaded from Genbank. Also the GH gene of the large yellow croaker and the genes from 14 Percoidei and 2 Labroidei species were aligned using Clustal X. A matrix of 564 bp was used to construct the phylogenetic tree using maximum parsimony and neighbor-joining methods. Phylogenetic trees by the two methods are identical in most of the clades with high bootstrap support. The results are also identical to those from morphological data. In general, this analysis does not support the monophyly of the families Centropomidae and Carangidae. But our GH gene tree indicates that the representative species of the families Sparidae and Sciaenidae are a monophyletic group.

东亚现代人群起源与史前迁移的群体遗传学和古DNA研究

现代人起源和史前迁移问题的遗传学研究是一个受到广泛关注的研究领 域。首先，它可以解答人类所关心的关于自身的起源以及我们祖先的史前活动问 题；其次，现代人祖先分开并扩散到世界各地，由于受到不同的外界环境和不同 的历史事件的影响，遗传上发生相应的变化，形成现有人群之间不仅在外表上存 在差异，同时在对疾病的易感性方面也存在差异。近年来的研究还发现，不同人 群之间在对药物的反应上也存在差异。 因此，人群起源与史前迁移研究对生物 医学研究也至关重要。目前，世界各地人群的起源和史前迁移问题的研究已经取 得很多进展，东亚之外的其它各个地区现有人群的起源和迁移已经相当清楚，东 亚现代人的起源对完整回答现代人的起源问题至关重要。 东亚现代人史前早期迁移的问题还存在很大争议，对现有研究结果的解释 甚至存在冲突。通过对东亚地区现有人群展开系统采样，并对东亚特有的Y 单 倍型遗传标记进行扫描，以东亚现代人特有的单倍型组O3-M122 下的各个特征 位点为遗传标记，我们对来自东亚地区的2332 份男性样本进行了研究。结果发 现，O3-M122 单倍型组为东亚现代人群的主导单倍型，其平均频率是44.3％。 以SNPs 突变特征为背景，展开SNPs-STRs 相结合的研究，通过大量的分析和统 计检验，从STRs 数据上可以看出东亚现代人南部人群的O3-M122 单倍型组的 多样性比北方人群高。这一证据揭示出东亚现代人特有单倍型O3-M122 突变为 东亚南部起源，然后随着东亚现代人早期的由南向北迁移活动而扩散到北方。利 用SNPs-STRs 相结合的遗传学分析手段，推导出东亚特有单倍型组O3-M122 伴 随人群由南向北的早期迁移事件发生于距今约25000～30000 年以前，这一结论 与东亚现代人的化石证据推断的结论相一致。O3-M122 单倍型组的史前早期迁 移事件有助于从遗传学角度透视东亚现代人早期迁移的路线和起源问题。 东亚地区现代人史前迁移活动遗留下神秘的悬棺文化一直令人费解，考古学的推论存在很大争议。我们利用遗传学手段对现存悬棺文化的保留人群展开研 究，采用父系和母系遗传标记相结合的方法探讨悬棺文化的传播人群的族源。从 遗传上看，悬棺葬俗的拥有人群不论从母系还是父系遗传学证据都为典型的东亚 南方人群。 为最终解决东亚现代人起源和史前迁移问题，我们对考古发掘出来的东亚 地区现代人化石展开古DNA（ancient DNA, aDNA）研究。目前，aDNA 分析的 技术和手段已经建立并逐步成熟，并且积累了一定量的东亚现代人的aDNA 数 据，这一研究还在继续。aDNA 最终会为解答东亚现代人起源和史前迁移问题提 供强有力的遗传学证据。

川金丝猴与滇金丝猴的群体遗传学研究

川金丝猴（RhinoPithecusroxellana）是我国著名的珍稀濒危保护动物，但是，现有的群体遗传学研究成果并不能满足科学合理的制定其遗传保护策略的要求。这主要是因为前人研究的遗传标记均是相对比较保守的功能基因，因此无法检测到足够的变异，进而也无法进行精细的分析。有鉴于此，我们选取了进化速率较快的线粒体控制区作为遗传标记，并对来自于三个主要栖息地的川金丝猴样本进行了群体遗传学研究。我们在线粒体控制区中观察到了大量的变异，这和前人的工作结果形成了强烈的反差。通过基于溯祖理论的最大似然法估算，我们得到现存川金丝猴群体的总的有效群体大小在700一2，300只以内，同时其最近共同祖先的年代在19，000-62，700年前左右。线粒体控制区的结果表明现存川金丝猴群体可能起源于眠山群体，我们称之为单起源假说，但是由于样本限制，多起源的可能性仍然存在。整个川金丝猴群体表现出显著的内部分化的信号，甚至氓山群体内部还存在一个序列分歧非常大的分枝，这可能是由其特殊的社群结构以及较弱的基因交流造成的，并且由于群体结构和分化信息的拮抗作用，我们并没有直接检测到瓶颈效应。通过和相关濒危与非濒危物种的比较，我们认为川金丝猴群体的遗传多样性水平并不低，提示其濒危的状态主要是由随机因素造成的而非遗传因素的结果。虽然我们在线粒体控制区中发现了大量的变异，但是前人在蛋白质电泳的研究中并未检出多态，同时，川金丝猴特殊的社群结构是否会影响其在核基因水平上的群体遗传结构也是十分有趣的问题，因此，为了从核基因组的角度进一步研究川金丝猴的遗传多样性，并实现对核基因和核外基因的比较，我们对样本的14个微卫星座位做了检测。我们发现这些微卫星座位均存在多态，所有地方群体的平均杂合度均大于0.5；地方群体间存在着显著的分化；地方群体间有效群体大小比率的估计值和群体大小比率的野外观测值非常接近。通过对比线粒体数据，我们认为川金丝猴的社群结构对其群体的遗传结构有着重要的影响，首先表现在核基因有更大的基因流水平，其次还表现在我们可以检测到眠山群体和秦岭群体在近期均经历了瓶颈效应，说明在核基因水平上群体结构信号的影响要弱于线粒体基因。但是，通过和其它物种比较，微卫星的数据却表明川金丝猴的遗传多样性水平并不高于其它的濒危动物，这和线粒体控制区的结果相反。这主要是当以微卫星为标记时，群体的历史动态对于当前群体的多态程度的影响力有限所致。微卫星的结果表明了保护川金丝猴群体的紧迫性。我们还通过细胞色素b基因证明了线粒体控制区中的巨大分歧不是由核假基因的造成的。巧合的是前人也在川金丝猴群体中对该基因进行过研究，但是结果却相反，即我们发现的变异水平远远高于他们的结果。为此，我们比较了两项工作的数据，我们推测在前人的工作中可能存在着潜在的测序错误。不过，如果没有出现污染的话，这两项工作的综合结果可以直接支持我们前面提到的单起源假说。此外，通过细胞色素b基因的分析，我们还估算出川金丝猴和滇金丝猴（R.bieti）的分歧时间大致在0.7-2.3百万年之间。镇金丝猴（R．biti）也是我国著名的濒危保护动物，它是川金丝猴的近缘种，其濒危等级甚至还要高于川金丝猴。迄今为止，关于滇金丝猴并基于DNA序列的的群体遗传学研究还没有见诸于报道。我们测定了来自于云南省维西县镇金丝猴群体样本的线粒体控制区全序列以及部分个体的细胞色素b全序列。在排除了核线粒体假基因存在的可能性之后，我们确认滇金丝猴维西群体内部也存在着两个序列分歧较大的分枝，但是，这可能是邻近群体迁移而来的产物。因此，如果考虑到群体结构和迁移的影响，我们认为维西群体的遗传多样性水平可能并不高。在以上的研究中，我们使用了大量的群体遗传学方法，但遗憾的是，目前还没有一套令人满意的侧重于群体遗传学分析的程序工具库。为了简化今后工作的复杂度和难度，我使用OCAAML语言编制了一套程序工具集（Pan'sToolKit，PTK）。OCAML是一门优秀的计算机语言，它既支持快速开发，又支持高速运行，是理想的算法实现语言。基于OCAML的PTK库集成了许多实用工具以及群体遗传学算法，尤其是溯祖理论的算法。PTK库不仅可以用于处理常规的实验数据，而且还可以用于辅助理论和算法的研究。

中国卧龙自然保护区不同海拔川滇高山栎(Quercus aquifolioides)群体的遗传变异

海拔梯度造成的环境异质性，如崎岖的地形、复杂的植被结构以及花期延迟等可能会极大地影响到物种的形态和遗传变异格局。理解物种形态和遗传变异的海拔格局对于物种多样性的管理和保护是非常重要的。尽管植物群体遗传学是一个飞速发展的研究领域，然而与海拔相关的形态变异、遗传变异及群体间遗传差异的研究却很少。到目前为止，还不清楚遗传变异与海拔之间是否必然的相关性。 川滇高山栎是一种重要的生态和经济型树种，广泛分布于中国西南的四川、西藏、贵州和云南省的高海拔地区，在保持水土、调节气候方面起着十分重要的作用。尽管主要受阳光限制而仅分布于阳坡，但其海拔梯度范围较大，表明川滇高山栎对不同的环境具有很强的适应性。本文通过叶型及生理响应、微卫星分子标记和扩增性片段长度多态性方法，试图探索川滇高山栎叶沿海拔梯度的形态和生理响应及其沿海拔梯度的遗传变异格局，为川滇高山栎的保护和利用提供进一步的遗传学理论依据和技术指导。 对叶形、含氮量及碳同位素的试验结果表明，平均比叶面积、气孔密度、气孔长度和气孔指数等气孔参数随海拔的升高呈非线性变化。在海拔大于2800 m时，川滇高山栎的比叶面积、气孔长度和气孔指数都随海拔升高而降低，但是在海拔小于2800 m时，这些指标都随海拔的升高而增大。相对而言，单位叶面积的含氮量和碳同位素则表现出相反的变化模式。另外，比叶面积是决定碳同位素沿海拔梯度变化的最重要参数。本研究结果表明，海拔2800 m附近是川滇高山栎生长和发育的最适地带，在这里生长的植物叶片厚度更薄、气孔更大、叶碳同位素值更小。 利用六对微卫星引物对五个不同海拔川滇高山栎群体遗传多样性进行研究，结果表明，群体内表现出较高的遗传多样性，平均每位点等位基因数11.33个，平均期望杂合度达0.820。群体间差异较小，分化仅为6.6％。聚类分析也并没有显示出明显的海拔格局。然而低频率等位基因却与海拔呈显著性正相关(R2=0.97, P < 0.01)，表明在高海拔处，川滇高山栎以更多的稀有基因来适应恶劣的环境条件。本试验结果表明由海拔梯度形成的选择性压力对川滇高山栎群体的遗传变异影响并不明显。 为了进一步探讨川滇高山栎群体遗传变异与海拔之间的相互关系，我们还对其进行了扩增性片段长度多态性分析。结果表明：(1)随海拔的升高(从群体WL2到群体WL5)，群体内遗传变异降低，而群体间遗传差异增加；(2)低海拔群体WL1表现出最低的遗传变异性(HE = 0.181)，同时与其余四个群体间呈现出最大的遗传差异性(平均FST = 0.0596)；(3)在除去低海拔群体WL1后，Mantel检测表明群体间遗传距离与海拔距离之间表现出正相关性。另外，研究结果还表明，遗传变异受生境条件(过度的湿热环境)及人为干扰(火烧、砍伐和放牧)的影响，这一点至少在低海拔群体WL1上发生了作用。 通过叶形态、生理及DNA分子水平的研究，结果表明叶形态特征和碳同位素与海拔紧密相关，与海拔之间呈非线性变化，海拔2,800 m附近是川滇高山栎生长和发育的最适地带。海拔梯度在一定程度上会影响到川滇高山栎群体的遗传变异结构，但在这样一个狭窄的地理分布区域里，这种影响并不足以导致群体间较大的遗传分化。同时生境条件及人为干扰也是影响遗传变异的限制性因子，不容忽视。 Altitudinal gradients impose heterogeneous environmental conditions, such as rugged topography, a complex pattern of vegetation and flowering delay, and they likely furthermore markedly affect the morphological and genetic variation pattern of a species. Understanding altitudinal pattern of morphological and genetic variation at a species is important for the management and conservation of species diversity. Although plant population genetics is a fast growing field of research, there are only few recent investigations, which analyzed the genetic differentiation and changes of intra-population variation along altitudinal gradients. At present, it is still unclear whether there are some common patterns of morphological and genetic variation with altitude. Quercus aquifolioides Rehder & E.H. Wilson, which is an important ecological and economical endemic woody plant species, is widely distributed in the Yunnan and Sichuan provinces, Southwest China. Its large range of habitat across different altitudes implies strong adaptation to different environments, although it is mainly restricted to sunny, south facing slopes. It plays a very important role in preventing soil erosion, soil water loss and regulating climate, as well as in retaining ecological stability. In this paper, we tried to understand the altitudinal pattern of morphological and genetic variation along altitudinal gradients through the experiments of leaf morphological and physiological responses, microsatellite analysis and AFLP markers. In leaf morphological and physiological responses experiment, we measured leaf morphology, nitrogen content and carbon isotope composition (as an indicator of water use efficiency) of Q. aquifolioides along an altitudinal gradient. We found that these leaf morphological and physiological responses to altitudinal gradients were non-linear with increasing altitude. Specific leaf area, stomatal length and index increased with increasing altitude below 2,800 m, but decreased with increasing altitude above 2,800 m. In contrast, leaf nitrogen content per unit area and carbon isotope composition showed opposite change patterns. Specific leaf area seemed to be the most important parameter that determined the carbon isotope composition along the altitudinal gradient. Our results suggest that near 2,800 m in altitude could be the optimum zone for growth and development of Q. aquifolioides, and highlight the importance of the influence of altitude in research on plant physiological ecology. Genetic variation and differentiation were investigated among five natural populations of Q. aquifolioides occurring along an altitudinal gradient that varied from 2,000 to 3,600 m above sea level in the Wolong Natural Reserve of China, by analyzing variation at six microsatellite loci. The results showed that the populations were characterized by relatively high intra-population variation with the average number of alleles equaling 11.33 per locus and the average expected heterozygosity (HE) being 0.779. The amount of genetic variation varied only little among populations, which suggests that the influence of altitude factors on microsatellite variation is limited. However, there is a significantly positive correlation between altitude and the number of low-frequency alleles (R2=0.97, P < 0.01), which indicates that Q. aquifolioides from high altitudes has more unique variation, possibly enabling adaptation to severe conditions. F statistics showed the presence of a slight deficiency of heterozygosity (FIS=0.136) and a low level of differentiation among populations (FST=0.066). The result of the cluster analysis demonstrates that the grouping of populations does not correspond to the altitude of the populations. Based on the available data, it is likely that the selective forces related to altitude are not strong enough to significantly differentiate the populations of Q. aquifolioides in terms of microsatellite variation. To further elucidate genetic variation pattern of Q. aquifolioides populations under sub-alpine environments, genetic variation and differentiation were investigated along altitudinal gradients using AFLP markers. The altitudinal populations with an average altitude interval of 400 m, i.e. WL1, WL2, WL3, WL4 and WL5, correspond to the altitudes 2,000, 2,400, 2,800, 3,200 and 3,600 m, respectively. Our results were as follows: (i) decreasing genetic variation (ranging from 0.253 to 0.210) and increasing genetic differentiation with altitude were obtained from the WL2 to the WL5 population; (ii) the WL1 population showed the lowest genetic variation (HE = 0.181) and the highest genetic differentiation (average FST = 0.0596) with the other four populations; (iii) the positive correlation was obtained using Mantel tests between genetic and altitude distances except for the WL1 population. Our results suggest that altitudinal gradients may have influenced the genetic variation pattern of Q. aquifolioides populations to some extent. In addition, habitat environments (unfavorable wet and hot conditions) and human disturbances (burning, grazing and felling) were possible influencing factors, especially to the low-altitude WL1 population. The present study shows that there were close correlations between morphological features and carbon isotope composition in our data. This indicates that a coordinated plant response modified these parameters simultaneously across different altitudes. Around 2,800 m altitude there seems to be an optimum zone for growth and development of Q. aquifolioides, as indicated by thinner leaves, larger stomata and more negative d13C values. All available evidence indicates altitudinal gradients may have influenced the genetic variation pattern of Q. aquifolioides to some extent. Decreasing genetic variation and increasing genetic differentiation with altitude was obtained except for the WL1 population. And the environment of habitats and human disturbances were also contributing factors, which impact genetic variation pattern, especially to the low-altitude WL1 population.

The complete mitochondrial genome of the mantid shrimp Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda): Novel non-coding regions features and phylogenetic implications of the Stomatopoda

The complete mitochondrial (mt) genome sequence of Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda) was determined; a circular molecule of 15,783 bp in length. The gene content and arrangement are consistent with the pancrustacean ground pattern. The mt control region of O. oratoria is characterized by no GA-block near the 3' end and different position of [TA(A)]n-blocks compared with other reported Stomatopoda species. The sequence of the second hairpin structure is relative conserved which suggests this region may be a synapomorphic character for the Stomatopoda. In addition, a relative large intergenic spacer (101 bp) with higher A + T content than that in control region was identified between the tRNA(Glu) and tRNA(Phe) genes. Phylogenetic analyses based on the current dataset of complete mt genomes strongly support the Stomatopoda is closely related to Euphausiacea. They in turn cluster with Penaeoidea and Caridea clades while other decapods form a separate group, which rejects the monophyly of Decapoda. This challenges the suitability of Stomatopoda as an outgroup of Decapoda in phylogenetic analyses. The basal position of Stomatopoda within Eumalacostraca according to the morphological characters is also questioned. (C) 2010 Elsevier Inc. All rights reserved.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.

Mining expressed sequences for single nucleotide polymorphisms in Pacific abalone Haliotis discus hannai

Although single nucleotide polymorphisms (SNPs) are important resources for population genetics, pedigree analysis and genomic mapping, such loci have not been reported in Pacific abalone so far. In this study, a bioinformatics strategy was adopted to discover SNPs within the expressed sequences (ESTs) of Pacific abalone, Haliotis discus hannai, and furthermore, polymerase chain reaction direct sequencing (PCR-DS) and allele-specific PCR (AS-PCR) were used for SNPs detection and genotype scoring respectively. A total of 5893 ESTs were assembled and 302 putative SNPs were identified. The average density of SNPs in ESTs was 1%. Fifty-two sets of sequencing primers were designed from SNPs flanking ESTs to amplify the genomic DNA, and 13 could generate products of expected size. Polymerase chain reaction direct sequencing of the amplification products from pooled DNA samples revealed 40 polymorphic SNP loci. Using a modified tetra-primer AS-PCR, seven mitochondrial and six nuclear SNPs were typed and characterized among 37 wild abalones. In conclusion, it is feasible to discover SNPs from number limited ESTs and the AS-PCR as a simple, robust and reliable assay could be a primary method for small- and medium-scale SNPs detection in abalones as well as other non-model organisms.

Yellow perch strain evaluation I: Genetic variance of six broodstock populations

As a prelude to strain selection for domestication and future marker assisted selection, genetic variation revealed by microsatellite DNA was evaluated in yellow perch, Perca flavescens, from four wild North American populations collected in 2003-2004 (Maine, New York, North Carolina, and Pennsylvania,), and two captive populations (Michigan and Ohio). For the loci examined, levels of heterozygosity ranged from H-e=0.04 to 0.88, genetic differentiation was highly significant among all population pairs, and effective migration ranged from low (N(e)m=0.3) to high (N(e)m=4.5). Deviation from Hardy-Weinberg equilibrium was regularly observed indicating significant departures from random mating. Instantaneous measures of inbreeding within these populations ranged from near zero to moderate (median F=0.16) and overall inbreeding levels averaged F-IS=0.18. Estimates of genetic diversity, Phi(ST), and genetic distance were highest between Michigan and all other broodstock groups and lowest between New York and Ohio. Genetic differentiation among groups did not correlate with geographic distance. Overall, the patterns of variation exhibited by the captive (Michigan and Ohio) populations were similar to patterns exhibited by the other wild populations, indicating that spawning and management practices to date have not significantly reduced levels of genetic variation. (C) 2007 Elsevier B.V. All rights reserved.

Nine microsatellite DNA primers for Hippophae rhamnoides ssp sinensis (Elaeagnaceae)

Hippophae rhamnoides ssp. sinensis occurs mainly in the and regions of northwest China. The wood stands of this subspecies play an important role in maintaining the local ecosystems in these regions. In addition, the genetic characteristics are essential to understand the historical range changes of this subspecies and its morphological differentiation with other subspecies. In this study, we developed nine microsatellite loci for this subspecies for the first time. We used the combining biotin capture method to enrich AG/CT/AC/GT/CG/GTG/CCA microsatellites. Twenty-six microsatellites were isolated from the enriching library and nine of them were found to be polymorphic through screening 12 distantly distributed individuals. The number of alleles per locus ranged from three to twelve and expected heterozygosity from 0.2659 to 0.4767, respectively. We further performed cross-priming tests in another subspecies and two congeneric species. These firstly isolated loci will provide a useful tool to investigate the genetic structure of this subspecies and its morphological differentiation from the other subspecies.

Alignment determination of symmetric top molecule using rotationally resolved LIF

This payer presents a concrete theoretical treatment which can be used for transforming the laser-induced fluorescence (LIF) intensity into the population and alignment parameters of a symmetric top molecule, The molecular population and alignment are described by molecular state multipoles. The results are presented in a general excitation-detection geometry and then specialized in some special geometries. The problem how to extract the initial molecular state multipoles from the rotationally resolved LIF intensity is discussed in detail. (C) 1999 Elsevier Science B.V. All rights reserved.

Theory for determining alignment parameters of symmetric top molecule using (n+1) LIF

Expressions used for extracting the population and alignment parameters of a symmetric top molecule from (n + 1) laser-induced fluorescence (LIF) are derived by employing the tensor density matrix method. The molecular population and alignment are described by molecular state multipoles. The LIF intensity is a complex function of the initial molecular state multipoles, the dynamic factors, and the excitation-detection geometrical factors. The problem of how to extract the initial molecular state multipoles from (2 + 1) LIF, as an example, is discussed in detail. (C) 2000 American Institute of Physics. [S0021-9606(00)30744-9].

Molecular phylogeography and population structure of a mid-elevation montane frog Leptobrachium ailaonicum in a fragmented habitat of southwest China

Leptobrachium ailaonicum is a vulnerable anuran restricted to a patchy distribution associated with small mountain streams surrounded by forested slopes at mid-elevations (approximately 2000-2600 m) in the subtropical Mount Wuliang and Mount Ailao ranges in southwest China (Yunnan Province) and northern Vietnam. Given high habitat specificity and lack of suitable habitat in lower elevations between these ranges, we hypothesized limited gene flow between populations throughout its range. We used two mitochondrial genes to construct a phylogeographic pattern within this species in order to test our hypothesis. We also examined whether this phylogeographic pattern is a response to past geological events and/or climatic oscillations. A total of 1989 base pairs were obtained from 81 individuals of nine populations yielding 51 unique haplotypes. Both Bayesian and maximum parsimony phylogenetic analyses revealed four deeply divergent and reciprocally monophyletic mtDNA lineages that approximately correspond to four geographical regions separated by deep river valleys. These results suggest a long history of allopatric separation by vicariance. The distinct geographic distributions of four major clades and the estimated divergence time suggest spatial and temporal separations that coincide with climatic and paleogeographic changes following the orogeny and uplift of Mount Ailao during the late Miocene to mid Pliocene in southwest China. At the southern distribution, the presence of two sympatric yet differentiated clades in two areas are interpreted as a result of secondary contact between previously allopatric populations during cooler Pleistocene glacial cycles. Analysis of molecular variance indicates that most of the observed genetic variation occurs among the four regions implying long-term interruption of maternal gene flow, suggesting that L ailaonicum may represent more than one distinct species and should at least be separated into four management units corresponding to these four geographic lineages for conservation. (C) 2009 Elsevier Inc. All rights reserved.