Bayesian mixed models and divergence time estimation of Chinese cavefishes (Cyprinidae : Sinocyclocheilus)

The genus Sinocyclocheilus is distributed in Yun-Gui Plateau and its surrounding region only, within more than 10 cave species showing different degrees of degeneration of eyes and pigmentation with wonderful adaptations. To present, published morphological and molecular phylogenetic hypotheses of Sinocyclocheilus from prior works are very different and the relationships within the genus are still far from clear. We obtained the sequences of cytochrome b (cyt b) and NADH dehydrogenase subunit 4 (ND4) of 34 species within Sinocyclocheilus, which represent the most dense taxon sampling to date. We performed Bayesian mixed models analyses with this data set. Under this phylogenetic framework, we estimated the divergence times of recovered clades using different methods under relaxed molecular clock. Our phyloegentic results supported the monophyly of Sinocyclocheilus and showed that this genus could be subdivided into 6 major clades. In addition, an earlier finding demonstrating the polyphyletic of cave species and the most basal position of S. jii was corroborated. Relaxed divergence-time estimation suggested that Sinocyclocheilus originated at the late Miocene, about 11 million years ago (Ma), which is older than what have been assumed.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Population differentiation of Procypris rabaudi (Tchang) in the upper reaches of the Yangtze River revealed by mitochondrial DNA and RAPD markers

Procypris rabaudi (Tchang) is a cyprinid fish endermic to middle and upper reaches of the Yangtze River. Besides in main stream and large tributaries, there exists an early matured, small-sized ecological type in a small tributary, Tang River. In this study, mitochondrial DNA cytochrome h (cyt b) gene sequence analysis and randomly amplified polymorphic DNA (RAPD) analysis were performed to investigate the differentiation of the Tang River population from the Mudong reach population of the Yangtze River, with the purpose of conservation and exploitation of this fish. In the 1140 bps of cyt b gene sequence surveyed, 20 sites were found polymorphic, which defined 23 haplotypes. Among them, four haplotypes accounted for 54.4% of all individuals, while population-specific haplotypes occurred in low frequencies. Analysis of molecular variation on cyt b data revealed no significant partition existing between Tang River population and Mudong reach population. Analyses of 132 RAPD loci suggested that genetic variation between populations was significant, though values of different F-ST were not very high. The results revealed low genetic diversity and the beginning of population differentiation, suggesting that Tang River population should be designated as a separate Management Unit.

Phylogenetic relationships of Danio within the order cypriniformes: A framework for comparative and evolutionary studies of a model species

The evolutionary relationships of species of Danio and the monophyly and phylogenetic placement of the genus within the family Cyprinidae and subfamily Rasborinae provide fundamentally important phyloinformatics necessary for direct evaluations of an array of pertinent questions in modern comparative biology. Although the genus Danio is not one of the most diverse within the family, Danio rerio is one of the most important model species in biology. Many investigations have used this species or presumed close relatives to address specific questions that have lasting impact on the hypothesis and theory of development in vertebrates. Largely lacking from this approach has been a holistic picture of the exact phylogenetic or evolutionary relationships of this species and its close relatives. One thing that has been learned over the previous century is that many organismal attributes (e.g., developmental pathways, ecologies, behaviors, speciation) are historically constrained and their origins and functions are best explained via a phylogenetic approach. Herein, we provide a molecular evaluation of the phylogenetic placement of the model species Danio rerio within the genus Danio and among hypothesized closely related species and genera. Our analysis is derived from data using two nuclear genes (RAG1, rhodopsin) and five mitochondrial genes (ND4, ND4L, ND5, COI, cyt b) evaluated using parsimony, maximum likelihood, and Bayesian analyses. The family Cyprinidae is resolved as monophyletic but the subfamily Rasborinae (priority over Danioinae) is an unnatural assemblage. Danio is identified as a monophyletic group sister to a clade inclusive of the genera Chela, Microrasbora, Devario, and Inlecypris, not Devario nor Esomus as hypothesized in previous studies. Danio rerio is sister to D. kyathit among the species of Danio evaluated in this analysis. Microrasbora and Rasbora are non-monophyletic assemblages; however, Boraras is monophyletic.

Origin of Gymnocypris przewalskii and phylogenetic history of Gymnocypris eckloni (Teleostei : Cyprinidae)

The origins and phylogenetic patterns were assessed for G. przewalskii and G. eckloni by analyzing the complete mtDNA cytochrome b gene sequence (1140bp). Phylogenetic analyses further supported that there were three mtDNA lineages (A-C) identified in G. przewalskii and G. eckloni, demonstrating that outer rakers of the first gill have little significance in the phylogeny of the Gymnocypris fishes. The network established showed that G. eckloni of the Yellow River specific haplotype A1 was a founder and it radiated all haplotypes of G. przewalskii which suggested G. przewalskii might only originate from one of two maternals of G. eckloni from the Yellow River. Fs test and mismatch analysis showed at least two expansion events in the population of G. przewalskii about 0.2734 Ma and 0.0658 Ma, while G. eckloni from Qaidam Basin could have experienced severe bottleneck effect about 0.0693 Ma. The population expansion was detected in subclades A1 and A21 with the most recent common ancestor (TMRCA) about 0.2308 +/- 0.01 Ma and 0.1319 +/- 0.015 Ma, respectively, which were within the geological age range of "Gonghe Movement" event that caused the separation of Lake Qinghai from the upper Yellow River. These results suggested the effect of the fish diversification by rapid uplift of the Qinghai-Tibetan Plateau in the Late Pleistocene.

Non-monophyly of fish in the Sinipercidae (Perciformes) as inferred from cytochrome b gene

The sinipercids represent a group of 12 species of freshwater percoid fish, including nine in Siniperca and three species in Coreoperca. Despite several classification attempts and a preliminary molecular phylogeny, the phylogenetic relationships and systematic position of sinipercids remained still unsolved. The complete cytochrome b gene sequences from nine sinipercid species four non-sinipercid fish species were cloned, and a total of 12 cyt b sequences from 10 species of sinipercids and 11 cyt b sequences from 10 species of non-sinipercid fish also in Perciformes were included in the phylogenetic analysis. As expected, the two genera Siniperca and Coreoperca within sinipercids are recovered as monophyletic. However, nine species representing Moronidae, Serranidae, Centropomidae, Acropomatidae, Emmelichtyidae, Siganidae and Centrarchidae included in the present study are all nested between Coreoperca and Siniperca, which provides marked evidence for a non-monophyly of sinipercid fishes. Coreoperca appears to be closest to Centrachus representing the family Centrarchidae. Coreoperca whiteheadi and C. herzi are sibling species, which together are closely related to C. kawamebari. In the Siniperca, the node between S. roulei and the remaining species is the most ancestral, followed by that of S. fortis. S. chuatsi and S. kneri are sibling species, sister to S. obscura. However, the sinipercids do not seem to have a very clear phylogenetic history, for different methods of phylogenetic reconstruction result in different tree topologies, and the only conclusive result in favor of a paraphyletic origin of the two sinipercid genera is the parametric bootstrap test. The paraphyly of Sinipercidae may suggest that the "synapomorphs" such as cycloid scales, upon which this family is based, were independently derived at least twice within sinipercid fishes, and further study should be carried out to include the other two Siniperca species and to incorporate other genes.

Genetic structure and low-genetic diversity suggesting the necessity for conservation of the Chinese Longsnout catfish, Leiocassis longirostris (Pisces : Bagriidae)

Genetic variation and phylogenetic relationship of Leiocassis longirostris populations from the Yangtze River were investigated at mitochondrial DNA level. The samples were collected from the upstream and mid-downstream of the Yangtze River. Three mitochondrial DNA fragments, ND5/6, cytochrome b (Cyt b) and control region (D-loop), were amplified and then digested by 10 restriction endonucleases. Twenty-three D-loop fragments randomly selected were sequenced. Digestion patterns of ND5/6 by AluI and HaeIII, D-loop by HinfI and RsaI, and Cyt b by HaeIII were polymorphic. Ten and eighteen haplotypes were obtained from RFLP data and sequence data, respectively. The individuals from upstream and mid-downstream of the Yangtze River were apparently divided into two groups. The average genetic distance was 0.008 and 0.010 according to the two data. Low diversities and decreasing abundance indicated that Leiocassis longirostris may be in severe danger and reasonable measures of fishery management should be taken.

Comparison of evolutionary rates in the mitochondrial DNA cytochrome b gene and control region and their implications for phylogeny of the Cobitoidea (Teleostei : Cypriniformes)

It is widely accepted that mitochondrial DNA (mtDNA) control region evolves faster than protein encoding genes with few exceptions. In the present study, we sequenced the mitochondrial cytochrome b gene (cyt b) and control region (CR) and compared their rates in 93 specimens representing 67 species of loaches and some related taxa in the Cobitoidea (Order Cypriniformes). The results showed that sequence divergences of the CR were broadly higher than those of the cyt b (about 1.83 times). However, in considering only closely related species, CR sequence evolution was slower than that of cyt b gene (ratio of CR/cyt b is 0.78), a pattern that is found to be very common in Cypriniformes. Combined data of the cyt b and CR were used to estimate the phylogenetic relationship of the Cobitoidea by maximum parsimony, neighbor-joining, and Bayesian methods. With Cyprinus carpio and Danio rerio as outgroups, three analyses identified the same four lineages representing four subfamilies of loaches, with Botiinae on the basal-most clade. The phylogenctic relationship of the Cobitoidea was ((Catostomidae + Gyrinocheilidae) + (Botiinae + (Balitorinae + (Cobitinae + Nemacheilinae)))), which indicated that Sawada's Cobitidae (including Cobitinae and Botiinae) was not monophyletic. Our molecular phylogenetic analyses are in very close agreement with the phylogenetic results based on the morphological data proposed by Nalbant and Bianco, wherein these four subfamilies were elevated to the family level as Botiidae, Balitoridae, Cobitidae, and Nemacheilidae. (c) 2005 Elsevier Inc. All rights reserved.

Molecular phylogenetics of Gymnocypris (Teleostei : Cyprinidae) in Lake Qinghai and adjacent drainages

149 complete mitochondrial DNA (mtDNA) cytochrome b (Cyt b) genes (1140 bp) of Gymnocypris przewalskii, Gymnocypris eckloni and Gymnocyptis scolistomus from the Lake Qinghai, Yellow River and Qaidam Basin were sequenced and analyzed. Consistent dendrogram indicated that the samples collected from the same species do not constitute a separate monophyletic group and all the samples were grouped into three highly divergent lineages (A, B and C). Among them, Lineage A contained all samples of G. przewalskii from the Lake Qinghai and partial samples of the G. eckloni from the Yellow River. Lineage B contained the remaining samples of G. eckloni from the Yellow River. Lineage C was composed of a monophyletic group by G. eckloni from the Qaidam Basin. Analysis of molecular variance (AMOVA) indicated that most of genetic variations were detected within these three mtDNA lineages (93.12%), suggesting that there are three different lineages of Gymnocypris in this region. Our Cyt b sequence data showed that G. przewalskii was not a polytypic species, and G. scolistomus was neither an independent species nor a subspecies of G. eckloni. The divergent mtDNA lineages of G. eckloni from the Yellow River suggested that gene flow between the different populations was restricted to a certain extent by several gorges on the upper reach of the Yellow River. Lineage B of G. eckloni might be the genetic effect from the ancestor which was incorporated with the endemic schizothoracine fishes when the headward erosion of the Yellow River reached to its current headwaters of late. The G. eckloni from Basin Qaidam was a monophyletic group (lineage C) and F-st values within G. eckloni from the Yellow River were higher than 0.98, suggesting that the gene flow has been interrupted for a long time and the G. eckloni from Basin Qaidam might have been evolved into different species by ecology segregation. The correlation between the rakers number of Gymnocypris and population genetic variation was not significant. All Gymnocypris populations exhibited a low nucleotide diversity (pi = 0.00096-0.00485). Therefore the Gymnocyptis populations from Basin Qaidam could have experienced severe bottleneck effect in history. Our result suggested Gym-nocypris populations of Basin Qaidam should give a high priority in conservation programs.

酶生物燃料电池载体选择及其应用基础研究

酶生物燃料电池（EBFC）是利用酶作为催化剂将化学能转化为电能的装置。由于氧化还原蛋白质和酶通常具有复杂的空间结构，活性中心深埋在它们的肽链中，很难与基底电极进行直接电子传递，从而影响了电池的性能。但使用适当的载体对电极表面进行修饰，可以实现直接的、快速的电子传递。因此，开发稳定性好、成本低、能够有效促进氧化还原蛋白质或酶与基底电极进行直接电子传递的载体成为EBFC发展中的重要课题之一。 本论文主要集中于EBFC中蛋白质或酶载体的选择方面的研究。探讨不同性质的载体，包括半导体电物质、生物相容性物质和导电物质对氧化还原蛋白质或酶的直接电子传递的影响。同时以SiO2纳米粒子为例，探讨了载体促进氧化还原蛋白质直接电子传递的作用机理。通过对不同载体的考察，最终选择了一种合适的材料组装成葡萄糖/O2 EBFC，并考察了EBFC的性能。主要结果如下： 1．将SiO2纳米粒子固定在GC电极上，成功实现了细胞色素c（Cyt c）的准可逆的直接电化学反应，并在这基础上提出双功能机理模型，说明了半导体对氧化还原蛋白质和电极之间的直接电子迁移的影响。 2．发现Cyt c能够在SBA-15膜修饰的电极上实现准可逆的直接电化学反应，并能够对H2O2产生较好的电催化还原效果。 3．以生物相容性物质壳聚糖为载体，分别研究了Cyt c、微过氧化物酶（MP-11）和葡萄糖氧化酶（GOD）的准可逆的直接电化学反应。并发现固载在壳聚糖上的Cyt c和MP-11对H2O2和O2还原有很好的电催化活性，而固载在壳聚糖上的GOD对葡萄糖氧化有很好的电催化活性。 4．以碳纳米管（CNT）为载体，实现了GOD的准可逆的直接电化学反应。并在氧化还原媒介体的作用下实现了其对葡萄糖的电催化氧化。 5．将筛选出的最佳载体组装成葡萄糖/O2 EBFC，分别以葡萄糖氧化酶和漆酶作为阳极和阴极的催化剂，制得有隔膜和无隔膜的EBFC。

藏鸡和姬鼠属(Apodemus )系统进化中的比较基因组学研究

在过去的十几年时间里，利用不同的分子标记促进了分子生态学和群 体遗传学的较大发展，其中线粒体DNA 和微卫星DNA 分析在分子生态学 研究中得到了最为广泛的应用，多核基因分子标记也越来越受到人们的关 注。本文采用了比较基因组学方法来研究了藏鸡(Tibetan Chickens)和姬鼠 属(Apodemus)的系统进化，对藏鸡的研究主要是为了解决藏鸡的系统进化 地位及其线粒体基因组的进化特征；对姬鼠属的研究目的在于发展建立一 种崭新的研究野生动物系统进化和生物地理的比较基因组学手段。主要结 果描述如下： (1)藏鸡(Tibetan Chickens)线粒体全基因组序列的测序和分析 通过利用PCR 扩增，测序，拼接，获得藏鸡线粒体全基因组序列并进 行数据分析处理。藏鸡线粒体全基因组序列全长16783bp，共有13 个蛋白 质编码基因、2 个rRNA 基因、22 个tRNA 基因和1 个D-loop 区。模拟电 子酶切结果显示，藏鸡DraI 酶的酶切结果和其他家鸡及红原鸡的酶切结果 都相同。基于D-loop 区全序列和13 个蛋白质编码基因序列，采用N-J 算 法与原鸡属4 个种，3 个亚种和3 个家鸡品系构建系统进化树：初步确定 藏鸡起源于红原鸡，与家鸡中的来航鸡、白洛克鸡亲缘关系最近，但是藏 鸡的进化与来航鸡、白洛克鸡这两个家鸡品系又显得相对独立。推测可能 原因是藏鸡的祖先在进入高原以后处于相对封闭的环境，从而形成了独特 群体遗传特性。 (2)姬鼠属(Apodemus)系统进化中的比较基因组学研究 本文中我们利用比较基因组学的研究方法寻找Exon-Primer-Intron- Crossing(EPIC)座位，并在中国四川省姬鼠属3 个种18 个个体中进行检验。 其方法是：通过比较人和小家鼠基因组，选择其中的外显子高度保守的单 拷贝基因，然后在500-1500bp 长度内含子的两端利用外显子序列设计了引 物，再进行PCR 扩增和克隆分析。通过PCR 扩增，我们在102 对引物选 择了6 对引物在18 个姬鼠属个体中进行PCR 产物克隆和测序。通过和 Cyt-b 相比较，在6 个座位当中，有5 个座位构建的系统进化树和Cyt-b 构建的系统进化树的拓扑结构高度一致，其中4 个座位可以很好的区分地 理不同的种群。通过计算核酸多样性，6 个座位的得到的结果都很接近， 说明6 个座位的突变率没有太大的差别。由此可见，我们利用比较基因组学的方法寻找EPIC 座位用于系统发育和群体遗传学的研究是可行的，通 过利用模式物种的基因组信息来研究野生非模式物种的系统发育和群体 遗传学将会提供前所未有的数据量和分辨率。

哀牢髭蟾（Vibrissaphora ailaonica）和红瘰疣螈(Tylototriton verrucosus)的分子系统地理学研究

本研究用线粒体细胞色素b 基因全序列和ND4 基因序列探讨了哀牢髭蟾 （Vibrissaphora ailaonica）分子亲缘地理学和保护遗传学，利用线粒体细胞色素b、 控制区全序列和部分12SrRNA 基因序列研究了分布于中国的红瘰疣螈 （Tylototriton verrucosus）分子亲缘地理学和保护遗传学。 哀牢髭蟾为生活于高海拔生境（大约2000-2600m）的濒危蛙类。各种致危因 素引起了关于对小的、隔离种群长期存活的关注，然而迄今没有关于该物种遗传 多样性的报道。本论文首次研究了分布于中国云南省的哀牢髭蟾的亲缘地理学和 保护遗传学。对采自于9 个种群81 个个体，我们应用线粒体mtDNA cyt b 和 ND4 基因共计1990bp, 获得了51 个单倍型。系统发育分析显示三个分化较深且互为单 系的族群，大致相应于分别被元江和藤条江河谷分开的三个地理单元（元江以东、 金平和藤条江以西地理单元），暗示了长期的地理隔离分化。分子变异等级分析 （AMOVA）显示遗传变异主要为三个地理单元之间（81.68％）和种群内的个体之 间（11.26%）。三个地理单元显著的地理分布暗示在空间和时间上的隔离，这与中 国西南地区在晚中新世到上新世的哀牢山的造山运动和隆升所引起的重要的气候 和古地质变化时间相一致。我们推测低海拔干热河谷可能是三个地理单元之间基 因交流受限的主要生态障碍。根据上述研究结果，我们建议对这三个遗传分化显 著的地理单元，元江以东地区、金平地区、藤条江以西地区作为独立的管理单元 分别加以保护。 本研究通过测定中国横断山区的红瘰疣螈（Tylototriton verrucosus）17个采集 点的123个红瘰疣螈标本的线粒体mtDNA Cyt b、D-loop和12SrRNA三个基因片段 (2347 bp)序列，首次研究了红瘰疣螈在横断山区的群体遗传结构和分子系统地理格 局，并讨论了T. shanjing的物种有效性。基于123个样品定义的49个单倍型的贝叶 斯和NJ系统发育分析表明：(1) T. verrucosus和T. shanjing均未各自构成单系，而是 共同构成一个单系群;(2) 横断山区的红瘰疣螈分为3个线粒体DNA地理单元，即滇 东南、滇中-滇西和片马地理单元，并且地理单元间不存在共享单倍型，说明红瘰 疣螈具有明显的系统地理分布格局。AMOVA分析同样表明3个地理单元之间存在 显著差异，并且分子变异主要发生在3个地理单元间（62.4%）。T. shanjing与T.verrucosus的mtDNA cyt b 序列差异平均值仅为1.1％,明显小于它们与两外群(贵 州疣螈和大凉疣螈)及外群间的遗传距离(6.5-9.9%)。因此，根据T. verrucosus和 T.shanjing的遗传差异以及系统发育分析结果都不支持T. shanjing的物种地位,T. shanjing为T. verrucosus的同物异名，并建议恢复T. verrucosus的中文名红瘰疣螈。 基于上述结果，我们建议将分布于滇东南、滇中－滇西、片马地区的红瘰疣螈作 为三个独立的管理单元分别加以保护。

中国菊头蝠科的系统分类及形态功能学研究

本文首先在查看了两千余号标本及整理已有文献的基础上，对中国菊头蝠属 (Rhinolophus)各种及亚种的分类进行了系统评述，并对各分类单位的形态特征进行了详尽描述。在中国分布的菊头蝠有8 个种组，19 个种，28 个亚种；重点讨论了角菊头蝠(R. cornutus)、浅褐菊头蝠(R. subbadius)和三叶菊头蝠(R. trifoliatus) 等物种的分类问题。其次，通过测量17 个种47 个菊头蝠标本的翼部(12 个指标) 和头骨(9 个指标)形态学指标及测定22 个种26 个菊头蝠组织样品的cyt-b 全序列，从形态学和分子系统学角度分析、探讨了中国菊头蝠系统发育关系。结果表明：中国菊头蝠包括了Aquias、非洲-古北和亚洲三大支系的物种，除了Aquias 支系的R. luctus 和R. formosae 以及非洲-古北支系的R. xinanzhongguoensis 和R. ferrumequinum，其余15 个种均属于亚洲支系。第三，在分类与系统发育关系研究的基础上，对分布于我国的19 种菊头蝠进行生物地理分析。在分布上，这些菊头蝠分属地中海-古北型和东洋型，后者又包括中国南方广布亚型、中国西南亚型和台湾特有分布亚型。中国菊头蝠的物种多样性呈现出由北向西南显著增加的趋势，云南、贵州和广西一带是中国菊头蝠最为集中分布的区域。R. ferrumequinum 等6 种菊头蝠在中国东部与西南的种群间存在种下分化，R. luctus 等4 种则因岛屿效应在海南和东部种群间形成种下分化。已有的研究成果表明翼手类的翼部形态反映了相应的飞行性能和生境利用特点。本文测量了6 种蹄蝠65 个标本和13 种菊头蝠229 个标本翼部骨骼的12 个长度变量以及后足长度，并摘抄原始标签上的体重数据，然后结合已发表的相关数据，对19 种蹄蝠和30 种菊头蝠的形态数据进行异速生长分析，以探讨菊头蝠和蹄蝠翼的特点及其适应性。结果表明，两者在翼部骨骼（前臂、第三指掌骨和第二指节以及第四、五指指节）存在显著性差异。其中部分骨骼长度的差异决定了它们在翼特征参数上的显著性差异：蹄蝠的前臂和第三指掌骨更长，使得翼展和展旋比要大于菊头蝠；而菊头蝠的第三指第二指节更长，使得翼尖长度和翼尖长度比值要大于蹄蝠。这些特点说明菊头蝠较蹄蝠更适应于在茂密生境中捕食。

澜沧江姬鼠（Apodemus ilex）种群遗传结构及其生物地理分析

澜沧江姬鼠（Apodemus ilex）为小型啮齿类动物，长期以来常被认为是中华姬鼠（A. draco）或长尾姬鼠（A. orestes）的同物异名，但近年基于线粒体Cyt b 的研究证明，该类群与中华姬鼠的遗传距离达到了种间水平，并被认为是一个独立种。本文提取、扩增并测定了来自云南14个地区42个产地156个澜沧江姬鼠的Cyt b 全序列，并将GenBank中的3条澜沧江姬鼠的Cyt b 全序列纳入分析，选取福建武夷山与四川峨眉山的2个中华姬鼠样品的Cyt b 全序列，以及台湾姬鼠、大耳姬鼠、高山姬鼠Cyt b 全序列作为外群，利用贝叶斯法和最大似然法构建系统发育树，并使用Network分析，分子变异分析（AMOVA）分析、错配分析（Mismatch analysis）与Fu`Fs检验等方法，探讨了这些地区澜沧江姬鼠的种群遗传结构，种群动态及生物地理格局。结果表明：云南地区过去被鉴定为中华姬鼠的样品均属于澜沧江姬鼠，澜沧江姬鼠与中华姬鼠间的遗传距离（K2P-ds+v）达到0.091，显著超过姬鼠属内种间差距（0.080），支持澜沧江姬鼠独立种的地位；该种不同地理种群可大致以澜沧江为界分为两大支系，且每个支系又各分为两个亚支系；进一步的AMOVA分析表明遗传变异主要来源于两大支系之间，而错配分析与Fu`Fs检验表明澜沧江姬鼠各地理种群及其两个支系都经历过多次扩张事件，这可能是和冰期与间冰期的轮回交替有关；分子系统树和中介网络图表明地理环境的隔离对于澜沧江姬鼠种群分化存在明显影响，其中澜沧江的阻隔作用最为显著。基于松散分子钟（Relax molecular clock）分析，中华姬鼠和澜沧江姬鼠的分歧时间大约在2.98 Ma（95％HPD: 4.32-1.88），而澜沧江姬鼠则在1.13 Ma（95％HPD: 1.95-0.65）分成两个支系。按照平均分歧时间来看，澜沧江姬鼠两个支系的的分歧事件发生在鄱阳冰期（1.2-0.9 Ma），两个支系内部进一步分化为两支的时间非常接近，分别为：Clade 1中为0.67 Ma （95％HPD: 1.17-0.34）和Clade 2 中为0.64 Ma (95％HPD: 1.17-0.36)，这种隔离种群各自产生分化的时间却非常接近，这提示其分化事件可能是受到古气候如冰期或间冰期的影响所产生。

凤鹛属鸟类（Yuhina, Aves）的系统发育研究

本研究以凤鹛属（Yuhina）鸟类为研究对象，分别使用分子生物学性状和形 态学性状来重建凤鹛属种间的系统发育关系，为东南亚地区鹛类的起源与进化研 究提出新的见解。分子生物学研究的内群不仅包括了凤鹛属（11 种中的10 种）， 而且还包括了菲律宾特有穗鹛属（Stachyris）和绣眼鸟属（Zosterops）等近缘种。 通过测定线粒体上两个蛋白编码基因Cyt b、ND3 的全长序列和两个RNA 编码 基因 12S, 16S 的部分序列，得到长度为2379bp 的联合序列，使用最大简约法、 最大似然法和贝叶斯法进行系统发育分析的结果表明，菲律宾岛特有的穗鹛（金 冠穗鹛S. dennistouni、纹穗鹛S. striata、怀氏穗鹛S. whiteheadi）与绣眼鸟属（暗 绿绣眼Z. japonicus、红胁绣眼Z. erythropleurus、灰腹绣眼Z. palpebrosus）聚为 一支，位于凤鹛属支系内部。通过凤鹛、菲律宾穗鹛、绣眼鸟三个类群羽冠性状 演化趋势的分析，发现羽冠性状在菲律宾特有穗鹛和绣眼鸟支系中发生丢失。与 前人的研究结果相比，新发现纹喉凤鹛Y. gularis 与棕肛凤鹛Y. occipitalis 是姐妹 群，黄颈凤鹛Y. flavicollis 和白项凤鹛Y. bakeri 是姐妹群，这两个姐妹群又聚为 一大支的关系。 在分类问题上，暗绿绣眼、红胁绣眼和灰腹绣眼在本研究中与鹛类表现出近 缘关系，与前人使用绣眼鸟属其它物种进行的相关研究显示出高度一致性，说明 绣眼鸟属的系统学地位存在很大疑问，应该扩大取样进行深入研究。栗冠凤鹛 Y. everetti 与栗耳凤鹛Y. castaniceps 聚为姐妹群的关系，但是二者之间较小的遗 传距离和形态学差别显示二者之间应该为亚种关系，而不是种级关系。依据栗耳 凤鹛特有的尾羽形态提出的Staphiada 属没有得到本分子生物学研究结果的支持 （Harrison, 1986a, b）。在白腹凤鹛Y. zantholeuca 不属于凤鹛属支系这一结果的 提示下，发现白腹凤鹛独有的形态学特征，例如羽冠由冠羽形成、鼻孔半裸露、 飞羽外翈没有异色羽缘，这些独有的特征与其特殊的系统学地位相对应。因此， 我们支持前人提出的将白腹凤鹛从凤鹛属中分出去，单独成Erpornis 属的建议 （Cibois et al., 2002）。 在历史生物地理学方面，根据菲律宾特有穗鹛和绣眼鸟的近缘关系，认为菲 律宾特有穗鹛的祖先具有与现在绣眼鸟一样的跨海迁移的能力，在中新世末期（5.74Mya）由喜马拉雅地区或印度支那起源地经大巽他地区扩散到菲律宾岛屿 上，经过长期独立进化后，形成现在高度特有的穗鹛支系。更新世冰期时，大巽 他地区海平面下降，使栗冠凤鹛得以扩散至婆罗州（1.66Mya）。在上新世初期台 湾岛成陆后，由于台湾岛与中国大陆相连，褐头凤鹛Y. brunneiceps 的祖先扩散 至台湾岛屿上（5.05Mya）。高黎贡山为第四纪冰期时凤鹛属鸟类的避难场所，因 此形成了现在凤鹛属鸟类在高黎贡山地区密集分布的格局。喜马拉雅山和青藏高 原的隆起导致青藏高原上植被带的变化和喜马拉雅山南麓在第四纪冰期中避难 所的作用，是纹喉凤鹛、棕肛凤鹛、黄颈凤鹛、白项凤鹛等种类现今在喜马拉雅 山南麓密集分布的可能原因。 在凤鹛属分子系统学的研究基础上，从形态学角度探讨凤鹛属除白领凤鹛外 其它物种之间的系统发育关系。使用最先分化出来的白领凤鹛为外群以外群比较 法进行51 个形态学特征的性状极化，使用最大简约法分析后，结果不仅支持一 些分子系统学揭示的物种之间的亲缘关系，例如，栗耳凤鹛与栗冠凤鹛姐妹群的 关系，黑颏凤鹛和褐头凤鹛姐妹群的关系，三种绣眼鸟聚为一支，再与怀氏穗鹛 聚为一个大支；而且发现了新的系统关系，黄颈凤鹛与缅甸凤鹛Y. humilis 是姐 妹群，具有浅色下体的凤鹛属物种具有较近的系统关系。分子数据和形态数据对 凤鹛属系统发育树中部节点解决能力的差异，说明凤鹛属物种之间分化间隔时间 很短。 通过综述近年来核基因在鸟类分子系统发育研究中的应用情况，建议未来研 究中增加核基因的内含子序列，例如肌球素基因内含子2（ myoglobin intron Ⅱ） 或β纤维蛋白原基因内含子7（β-fibrinogen intron7, β-fibint 7）来解决本研究 中未确定的末端分枝分歧顺序。