Characterization of acid-sensing ion channels in dorsal horn neurons of rat spinal cord

Acid-sensing ion channels (ASICs) are ligand-gated cation channels activated by extracellular protons. In periphery, they contribute to sensory transmission, including that of nociception and pain. Here we characterized ASIC-like currents in dorsal horn neurons of the rat spinal cord and their functional modulation in pathological conditions. Reverse transcriptase-nested PCR and Western blotting showed that three ASIC isoforms, ASIC1a, ASIC2a, and ASIC2b, are expressed at a high level in dorsal horn neurons. Electrophysiological and pharmacological properties of the proton-gated currents suggest that homomeric ASIC1a and/or heteromeric ASIC1a + 2b channels are responsible for the proton-induced currents in the majority of dorsal horn neurons. Acidification-induced action potentials in these neurons were compatible in a pH-dependent manner with the pH dependence of ASIC-like current. Furthermore, peripheral complete Freund's adjuvant-induced inflammation resulted in increased expression of both ASIC1a and ASIC2a in dorsal horn. These results support the idea that the ASICs of dorsal horn neurons participate in central sensory transmission/modulation under physiological conditions and may play important roles in inflammation-related persistent pain.

FEEDING AND VOCAL BEHAVIORS OF BLACK GIBBONS (HYLOBATES-CONCOLOR) IN YUNNAN - A PRELIMINARY-STUDY

Feeding and vocal behaviours of wild black gibbons (Hylobates concolor) were observed from 1987 to 1989 in south-western Yunnan, notably H. concolor jingdongensis at Mt. Wuliang (24-degrees 18-42'N, 100-degrees 30-50'E) in the early spring of 1989. 12 plant species were observed to have been eaten by the gibbons; these included tree species, lianas and epiphytes. Approximately 21 % of feeding time was devoted to eating fruits, 61 % to leaf buds and shoots, 7 % to flowers and 11 % to leaves. The gibbons preferred fruits to leaves even though they commonly ate leaves. In this study, the morning songs (duet and solo), reponsive (territorial) songs, alarm calls and communication calls were recorded. The gibbons sang their morning songs mainly in the early morning, with a single bout lasting more than 10 min on average. The singing of a group would trigger other groups, and all groups in an area tended to sing sequentially. The morning duet song bout was dominated by an adult male. The male emitted booms, aa notes, early multimodulated figures, intermediate multimodulated figures and codas, the latter occurring only in duets following the female's great call. The female uttered great calls and abortive great calls. The subadults or juveniles also took part in the morning songs. Lone males were heard to utter solos which lasted longer than the duets of the pairs.

Prenatal exposures to persistent organic pollutants as measured in cord blood and meconium from three localities of Zhejiang, China

Prenatal exposures to persistent organic pollutants were assessed using the levels of PCBs and organochlorine pesticides (OCPs) measured in cord blood and meconium samples from Luqiao and two other localities of the Zhejiang province in China. Luqiao is a town with the largest site for disassembly of PCB-containing obsolete transformers and electrical waste in China. The other two localities Pingqiao (100 km NW of Luqiao) and Lin'an (500 km NW of Luqiao) are towns without known electronic or electrical waste sites. A total of 23 PCB congeners (including 12 dioxin-like) and 6 OCPs were measured using the traditional GC-mu ECD technique. Micro-EROD bioassay was additionally used to measure TCDD-based TEQ levels of the 12 dioxin-like PCBs. Significant correlations were found between the TEQs measured by the two methods, supporting the application of micro-EROD as a practical toot for complementing the chemical analysis. The data showed that beta-HCH, p,p'-DDE, and 6 PCB congeners (101, 138 153, 180, 183, and 187) were the predominant pollutants, with PCB 138 being the best indicator (predictor) for total PCB levels. Cord blood and meconium from Luqiao have higher levels of PCBs than those from the other two localities, suggesting that a disassembly site for electronic and electric waste would provide an environment for greater exposure to these chemicals. The cord blood or meconium levels of beta-HCH, though likewise considerably high, were comparable in the three localities. Similar findings were observed for p,p'-DDE. Pollution by these OCPs might have come from past use of agricultural pesticides in the three localities. (c) 2007 Published by Elsevier B.V.

Differential expression of the Brunol/CELF family genes during Xenopus laevis early development

The BRUNOL/CELF family of RNA-binding proteins plays important roles in post-transcriptional regulation and has been implicated in several developmental processes. In this study, we describe the cloning and expression patterns of five Brunol genes in Xenopus laevis. Among them, only Brunol2 is maternally expressed and the zygotic expression of the other four Brunol genes starts at different developmental stages. During Xenopus development, Brunol1, 4-5 are exclusively expressed in the nervous system including domains in the brain, spinal cord, optic and otic vesicles. Brunol2 and 3 are expressed in both the somatic mesoderm and the nervous system. Brunol2 is also extensively expressed in the lens. In transfected Hela cells, BRUNOL1, 2 and 3 proteins are localized in both the cytoplasm and the nucleus, while BRUNOL4 and 5 are only present in the cytoplasm, indicating their different functions.

Lentivirus介导神经营养因子- 3促进脊髓损伤大鼠功能恢复

　目的　探讨Lentivirus介导分泌神经营养因子- 3 (NT - 3)直接体内转基因治疗大鼠脊髓损伤作用和机制。　方法　将28只Wistar大鼠在T10水平制成半横断损伤模型,随机分为体内转基因治疗组和损伤对照组,每组14只;用携带NT - 3和绿色荧光蛋白(GFP)的Lentivirus对大鼠行直接体内转基因治疗;荧光显微镜观察体内转基因表达,后肢运动功能评分法(BBB评分) 检测大鼠后肢功能恢复情况。　结果　用荧光显微镜检测到损伤脊髓内有较多的持续表达转基因的细胞; BBB评分结果显示,从伤后第4周开始,实验组大鼠后肢功能较对照组明显恢复( P < 0. 01) ,至第10周时,实验组和对照组BBB分差增大,达3. 3分。　结论　Lentivirus是一种有效的转基因载体,由Lentivirus介导分泌NT - 3的直接体内转基因治疗能够明显促进损伤脊髓的功能恢复。

人胚神经干细胞移植治疗大鼠脊髓损伤

　目的　探讨人胚神经干细胞( hNSC) 移植治疗脊髓损伤(SCI) 的可行性。方法　分 离、培养和鉴定hNSC;用5 溴22 脱氧尿苷嘧啶(BrdU) 标记hNSC ,并将其移植到14 只T10 半横断 的Wistar 大鼠损伤脊髓内(另外14 只T10 半横断损伤的大鼠作为对照组,仅损伤脊髓内注射 DMEM/ F12 培养液) ,用BrdU 的FITC 免疫荧光染色检测移植细胞的存活和迁徙,用NF2200 、 GFAP 免疫组织化学鉴定移植细胞的分化,BBB 评分评定大鼠功能恢复情况。结果　(1) 获得了大 量的hNSC; (2) 用免疫组织化学可以检测到移植的hNSC 能在体内长时间存活(达2 个月) 并向远 处迁徙,并分化为神经元和胶质细胞; (3) 检测到实验组大鼠BBB 得分明显高于对照组大鼠( P < 0. 01) ,在SCI 后第10 周时实验组和对照组BBB 得分最大差距达到2. 1分。结论　hNSC 移植能促 进SCI 大鼠后肢功能恢复,它是SCI 移植治疗较有价值的细胞资源。

Mechanisms of H+ modulation of glycinergic response in rat sacral dorsal commissural neurons

Many ionotropic receptors are modulated by extracellular H+. So far, few studies have directly addressed the role of such modulation at synapses. In the present study, we investigated the effects of changes in extracellular pH on glycinergic miniature inhibitory postsynaptic currents (mIPSCs) as well as glycine-evoked currents (I-Gly) in mechanically dissociated spinal neurons with native synaptic boutons preserved. H+ modulated both the mIPSCs and I-Gly, biphasically, although it activated an amiloride-sensitive inward current by itself. Decreasing extracellular pH reversibly inhibited the amplitude of the mIPSCs and I-Gly, while increasing external pH reversibly potentiated these parameters. Blockade of acid-sensing ion channels (ASICs) with amiloride, the selective antagonist of ASICs, or decreasing intracellular pH did not alter the modulatory effect of H+ on either mIPSCs or I-Gly, H+ shifted the EC50 of the glycine concentration-response curve from 49.3 +/- 5.7 muM at external pH 7.4 to 131.5 +/- 8.1 muM at pH 5.5, without altering the Cl- selectivity of the glycine receptor (GlyR), the Hill coefficient and the maximal I-Gly, suggesting a competitive inhibition of I-Gly by H+. Both Zn2+ and H+ inhibited I-Gly. However, H+ induced no further inhibition of I-Gly in the presence of a saturating concentration of Zn2+. In addition, H+ significantly affected the kinetics of glycinergic mIPSCs and I-Gly. It is proposed that H+ and/or Zn2+ compete with glycine binding and inhibit the amplitude of glycinergic mIPSCs and I-Gly. Moreover, binding of H+ induces a global conformational change in GlyR, which closes the GlyR Cl- channel and results in the acceleration of the seeming desensitization of IGly as well as speeding up the decay time constant of glycinergic mIPSCs. However, the deprotonation rate is faster than the unbinding rate of glycine from the GlyR, leading to reactivation of the undesensitized GlyR after washout of agonist and the appearance of a rebound I-Gly. H+ also modulated the glycine cotransmitter, GABA-activated current (I-GABA). Taken together, the results support a 'conformational coupling' model for H+ modulation of the GlyR and suggest that W may act as a novel modulator for inhibitory neurotransmission in the mammalian spinal cord.

Amolops bellulus: A new species of stream-breeding frog from western Yunnan, China (Anura : Ranidae)

We describe a new species of stream-breeding frog of the genus Amolops, Amolops bellulus, from the mountainous region of Lushui County, Yunnan Province, China, near the boundary between Yunnan and Myanmar. This species differs from all its congeners in a combination of morphological characteristics including the absence of a circummarginal groove at the tip of first finger, lack of a supratympanic fold, a white band along the upper jaw extending to shoulder, a black band starting from tip of snout along upper loreal region and extending to anterior flank of body, absence of a vocal sac, tympanum distinct and feebly concave, and the presence of vomerine teeth.

Murine MyaK, a member of a family of yeast YAK1-related genes, is highly expressed in hormonally modulated epithelia in the reproductive system and in the embryonic central nervous system

We have cloned a mouse homologue (designated Myak) of the yeast protein kinase YAK1. The 1210 aa open reading frame contains a putative protein kinase domain, nuclear localization sequences and PEST sequences. Myak appears to be a member of a growing family of YAK1-related genes that include Drosophila and human Minibrain as well as a recently identified rat gene ANPK that encode a steroid hormone receptor interacting protein. RNA blot analysis revealed that Myak is expressed at low levels ubiquitously but at high levels in reproductive tissues, including testis, epididymis, ovary, uterus, and mammary gland, as well as in brain and kidney. In situ hybridization analysis on selected tissues revealed that Myak is particularly abundant in the hormonally modulated epithelia of the epididymis, mammary gland, and uterus, in round spermatids in the testis, and in the corpora lutea in the ovary, Myak is also highly expressed in the aqueduct of the adult brain and in the brain and spinal cord of day 12.5 embryos, Mol. Reprod. Dev. 55:372-378, 2000. (C) 2000 Wiley-Liss, Inc.

Why do Chinese alligators (Alligator sinensis) form bellowing choruses: A playback approach

Crocodilians are quite vocal relative to other reptile groups, and the alligators are among the most vocal of the crocodilians. The Chinese alligator, Alligator sinensis, is usually solitary but engages in bellowing choruses in certain waters during the mating season. This paper reports the organization of Chinese alligator's bellowing choruses based upon field observations and playback experiments. Alligators of both genders engaged in the choruses, remaining immobile throughout and inclining toward bellowing synchronously (i.e., starting and finishing at about the same time). The choruses lasted about 10 min with abrupt onset and offset. Moreover, playback experiments revealed that both male and female alligators responded equally to bellowing stimuli from the same and opposite sexes and that none of the tested alligators approached the loudspeaker in spite of playback of male or female stimuli. These suggest that Chinese alligators. may not bellow to compete for or attract mates during the choruses. Instead, when their ecological behaviors, namely, dispersed inhabitation, multi-copulation, restricted mating season, etc., are considered, we hypothesize that they may synchronize bellows to enhance group detectability for assembling individuals into certain waters for subsequent copulations. (C) 2009 Acoustical Society of America. [DOI: 10.1121/1.3203667]

Developmental expression of CagMdkb during gibel carp embryogenesis

Midkine (Mdk) genes have been revealed to have different expression patterns in vertebrates and therefore, additional studies on Mdk expression patterns are required in more species. In this study, CagMdkb has been cloned and characterized from a SMART cDNA library of 10-somite stage embryos of Carassius auratus gibelio. Its full length cDNA is 1091 bp and encodes a sequence of 147 amino acids, which shows 97.3% identity to zebrafish Mdkb on the amino acid level. RT-PCR analysis reveals that CagMdkb is first transcribed in gastrula embryos and maintains a relatively stable expression level during subsequent embryogenesis. Western blot analysis reveals a 19 kDa maternal CagMdkb protein band and the zygotic CagMdkb protein is expressed from gastrula stage. At around 10 somite stage, the 19 kDa CagMdkb is processed to another protein band of about 17 kDa, which might be the secreted form with the 21-residue signal peptide removed. With immunofluorescence analysis, maternal CagMdkb protein was found to be localized in each blastamere cell of early embryos. The zygotic CagMdkb positive fluorescence signal was detected from a pair of large neurons at 18-somite stage. At the later stages, CagMdkb protein was also extended to numerous small neurons in the forebrain, midbrain and hindbrain, as well as to nerve fibers in the spinal cord. Co-localization with 3A10 antibody revealed CagMdkb immunoreactivity on developing Mauthner neurons, a member of reticulospinal neurons. In addition, ectopic expression of CagMdkb in early embryos of gibel carp and zebrafish suppressed head formation and CagMdkb function was found to depend on secretory activity. All these findings indicate that CagMdkb plays an important role in neural development during gibel carp embryogenesis and there is functional conservation of Mdkb in fish head formation.

Biotransfer of persistent organic pollutants from a large site in China used for the disassembly of electronic and electrical waste

Samples of groundwater, river water, river sediment, paddy soil, rice seeds, hen eggs, fish, umbilical cord blood, and newborn meconium were collected from October 2002 to October 2003 near a large site in China used for the disassembly of obsolete transformers and other electronic or electrical waste. Six indicator PCB congeners, three non-ortho dioxin-like PCB congeners, and six organochlorine pesticides were determined in the samples by GC with electron capture detector. The results demonstrated that the local environment and edible foods had been seriously polluted by toxic PCBs and organochlorine pesticides. The actual daily intakes (ADIs) of these pollutants were estimated for local residents living in the area. The intake data showed that the contents of PCBs in these local residents were substantial, as the ADI estimates greatly exceed the reference doses set by the World Health Organization and the United States Agency for Toxic Substances and Disease Registry. The presence of the indicator PCB congeners in the cord blood and the meconium samples, as well as significant correlations (r(2) > 0.80, p < 0.05) between these levels, suggests a potential biotransfer of these indicators from mothers to their newborns. This preliminary study showed that obsolete transformers and other electronic or electrical waste can be an important source for the emission of persistent organic pollutants into the local environment, such as through leakage, evaporation, runoff, and leaching. Contamination from this source appears to have reached the level considered to be a serious threat to environmental and human health around the disassembly site.

隆肛蛙属Feirana种群的系统学与动物地理学研究

对隆肛蛙属的物种构成进行了订正，建立新属肛刺蛙属Yerana gen. nov.；订正后的隆肛蛙属现仅隶2种， 即隆肛蛙F. quadrana和太行隆肛蛙F. taihangnicus。运用形态学分析探讨了隆肛蛙属物种及种群的形态差异和分类关系，通过分子系统学研究探讨了隆肛蛙属物种及种群的分类和系统发育关系，运用动物地理学方法结合系统发育关系探讨了隆肛蛙属种群的地理分布格局成因与历史过程。主要结果和推论如下： 1．隆肛蛙属物种构成的订正及一新属建立 建立新属肛刺蛙属，将隆肛蛙属中的原叶氏隆肛蛙F. yei归隶新属肛刺蛙属并更名为叶氏肛刺蛙Y. yei，，新属建立的主要依据为：（1）雄性肛部隆起，肛孔下方有两个布满黑刺的大的白色球形隆起，具单咽下内声囊， 第一指具婚刺；（2）形态量度分析表明叶氏肛刺蛙与隆肛蛙和太行隆肛蛙的形态差异远大于后两者之间的差异；（3）叶氏肛刺蛙的分布区与隆肛蛙和太行隆肛蛙的分布区距离较远且呈隔离状态；（4）分子系统学研究资料（Jiang et al.,2005）证明叶氏肛刺蛙与隆肛蛙和太行隆肛蛙非单系发生；叶氏肛刺蛙在第二支中位于基部。因此，隆肛蛙属现仅隶2种，即隆肛蛙和太行隆肛蛙。 2．隆肛蛙属种群形态学研究 对隆肛蛙属中隆肛蛙和太行隆肛蛙的15个地理种群565只标本的28项形态性状进行了测量，运用典型判别分析法对其分析的结果表明：（1）太行隆肛蛙与隆肛蛙形态差异明显，支持其为不同的物种；（2）原隆肛蛙河南伏牛山种群和山西中条山种群应为太行隆肛蛙的地理种群；（3）隆肛蛙不同地理种群之间形态差异明显，其中四川安县种群、陕西周至种群和湖北利川种群与模式产地重庆巫山种群的差异可能达到了亚种或亚种以上分化水平。对隆肛蛙属量度分析的15个种群进行定性形态分析表明其分为三种形态型，对应隆肛蛙、过渡型和太行隆肛蛙，其变异特征主要为内跗褶、雄性肛部隆起及疣粒分布、第五趾外侧缘膜等，这与量度分析结果相似。 3．隆肛蛙属种群分子系统学研究 测定隆肛蛙属Feirana的2种19种群的线粒体12S rRNA和16S rRNA基因片段、ND2基因的DNA序列，比对后共计1953bps。（1）遗传多样性与距离分析：结果表明，隆肛蛙属种群具很高的遗传多样性，19个种群样品表现出19种单倍型（遗传多样性指数Hd＝1.0）； ND2基因的进化信息含量远高于12SrRNA和16SrRNA。隆肛蛙属2种群组内的种群间的遗传距离远小于两种群组间的距离，种群在不同基因上的遗传距离表现的关系与对应的系统树一致。（2）系统发育关系分析：结果表明，不同基因片断基于不同方法构建的隆肛蛙属种群系统发育树结构基本一致，基本表明隆肛蛙属种群为单系发生；它们在系统树中分为两大支，分别对应于隆肛蛙和太行隆肛蛙；支持中条山种群（沁水、历山和济源种群）和伏牛山种群（栾川和内乡种群）为太行隆肛蛙的地理种群，而原隆肛蛙秦岭中东段的部分种群（柞水、宁陕、长安大坝沟种群）也应为太行隆肛蛙的地理种群。（3）亚种分化分析：根据遗传距离分析和系统发育关系分析结果，并考虑形态上的差异情况以及地理分布信息，隆肛蛙所隶种群组可分为2亚种，即隆肛蛙指名亚种F. quadrana quadrana包括四川盆地东缘大巴山东段-巫山-武陵山北麓种群和秦岭中段（周至板房子和长安广货街）种群，他们在系统关系树上聚为一支；安县亚种F. quadrana anxianensis包括四川盆地西缘岷山东麓-龙门山-大巴山和秦岭西段的种群（安县、青川、文县、南江和凤县种群），他们在系统关系树上聚为一支。太行隆肛蛙所隶种群组也可分为2亚种，即太行隆肛蛙指名亚种F. taihangnicus taihangnicus包括中条山的种群（沁水、历山和济源种群）和中东秦岭的部分种群（柞水、长安大坝沟和宁陕种群），他们在系统关系树上聚为一支；太行隆肛蛙伏牛亚种F. taihangnicus funiuensis，为伏牛山地区的种群（栾川和内乡种群），他们在系统关系树上聚为一支。 4．隆肛蛙属种群动物地理学研究 隆肛蛙属19种群的分歧年代分析： 以长江巫山段和黄河三门峡段的形成历史时期为参考点，根据已测隆肛蛙属19种群及其外群包括N. pleski、P. yunnanesis、P. robertingeri、F. limnocharis的1953bps DNA序列构建分子钟，获得各支系的分歧年代。结果表明：①棘蛙族在70Ma左右开始其独立演化历程，这与Roelants et al.（2004）的分析结果～60±15Ma左右开始分化基本一致，后者印证了本文的分子钟。②隆肛蛙属的起始分化年代较早，隆肛蛙和太行隆肛蛙两种群组的最近祖先种群大概在46Ma～50Ma左右；隆肛蛙和太行隆肛蛙种群组内的种群分化年代相对两种群组间晚得多， 隆肛蛙种群组内两亚种分化起始年代约为10Ma左右，而太行隆肛蛙种群组内两亚种分化起始年代约为6Ma。 隆肛蛙属种群分布格局形成过程分析： ①隆肛蛙属的系统关系与地理分布格局密切相关，大部分系统分支分级与地理距离成正比；②隆肛蛙属最近祖先种群的分化中心可能位于秦岭中部地区， 隆肛蛙属的种群分布格局的形成表现为隔离分化与扩散相结合的机制，由隔离分化产生的隆肛蛙祖先种群主要从秦岭中部向西南方向扩散，后隔离分化为两亚种；太行隆肛蛙祖先种群向东北方向扩散也分化为两亚种。 隆肛蛙属种群分布区域地质历史的探讨：本文所建分子钟和种群分化方式印证了该区域的几次主要地质事件，包括岷山-龙门山-西秦岭等地区的快速差异隆起、第四纪冰期等。 The specific composition of the genus Feirana should be revised. A new genus Yerana gen. nov.（Ranidae：Dicroglossinae）was established based on morphological data-set and molecular phylogeny, as a result, only two species F. quadrana and F. taihangnicus are classified into Feirana now. Morphological differences and taxonomy of populations of Feirana were investigated based on morphological and morphometric data; phylogenetic relationships and taxonomy of populations of Feirana were elucidated using molecular data, and then the proceeding of the distribution pattern of populations of Feirana were discussed. The main results and conclusions and proposals were presented as following: 1. Revising of the specific composition of the genus Feirana and establishment of a new genus The new genus Yerana, only containing the type species Y. yei, was established based on the following evidences: (1) In adult male, distinct up-heaved circular vesicle presents around the anal, and under anal there are two white balls on which black spines exist, black horny spines scatter on the upper side of first finger, and internal single subgular vocal sac presents; (2) there is obvious morphometric differences between Yerana and Feirana; (3) Yerana is distributed far from Feirana; (4) evidences of molecular phylogeny（Jiang et al.,2005）suggested that Yerana take a special phylogenetic clade which is different from other genus included in the tribe Paini. As a result, there are only two species in Feirana, i.e., F. quadrana and F. taihangnicus. 2. Morphological research of populations of Feirana Twenty-eight characters of 565 individuals of 15 populations of the genus Feirana were measured, the results of Canonical Discriminant analysis of the morphometric data-set indicated that: (1) there are very prominent differences between the two species F. quadrana and F. taihangnicus. The validity of species F. taihangnicus was approved here; (2) Mt. Funiu population and Mt. Zhongtiao population should belong to the species F. taihangnicus; (3) Obvious differences exist among 12 populations of F. quadrana, the differentiation among Zhouzhi population, Anxian population, Lichuan population, and Wushan population together with the others probably reach sub-specific or specific level. Result of morphological comparison between 15 different populations show that 3 morphological types are recogenized in according with F. quadrana, F. taihangnicus and intergradation, this result conform to the result of morphometric analysis. 3. Molecular phylogenetic study on populaions of Feirana Fragment of 12SrRNA and 16SrRNA genes, and ND2 gene of 19 populations of two species of Feirana were sequenced and aligned, from which 1953 bps were received. (1) analyses of genetic distance and hereditary diversity indicated that: genetic distance between populations in each group were less than distance between two groups of Feirana, 19 haplotypes were recognized from 19 samples of 19 populations, so the hereditary diversity of populations of Feirana was very high (Hd=1.0), phylogenetic information in ND2 gene is more than fragment sequence of 12SrRNA and 16SrRNA genes. (2) Result of molecular phylogeny indicate that the phylogenetic trees constructed using different methods based on different sequence data sets showed the revised genus Feirana is monophyletic since the 19 populations of Feirana were firstly clustered together as one large clade, which was further clustered into two major clades, corresponding to F. quadrana(GroupⅠ) and F. taihangnicus(GroupⅡ), respectively. So populations of Qinshui and Lishan in Mt. Zhongtiao, populations of Luanchuan and Neixiang in Mt. Funiu, and populations of Zhashui, Dabagou of Chang’an and Ningshan in eastern Mt. Qinling should belong to the species F. taihangnicus; (3) Subspecific differentiation. on the basis of genetic distance, phylogenetic trees and geographical distribution, F. quadrana should have two subspecies, i.e., F. quadrana qudadrana, consisting of the populations Guanghuojie of Chang’an and Zhouzhi in Mid-Mt. Qinling, populations in Wushan area and northern Mt. Wuling (Lichuan), and F. qudadrana anxianensis, consisting of the populations in eastern Mt. Ming shan-Mt. Longmen-western Mt. Daba-western Mt. Qinling (Anxian, Qingchuan, Wenxian, Nanjiang and Fengxian); F. taihangnicus should also has two subspecies, i.e., F. taihangnicus taihangnicus, consisting of the populations in Mt. Zhongtiao and eastern Mt. Qinling, and F. taihangnicus funiuensis, consisting of the populations in Mt. Funiu. 4. Zoogeography of populaions of Feirana Analysis for divergent time of 19 populations of Feirana: Using the dates of run-through of Wushan segment of Changjiang River as the time when the population of Lichuan started differentiated from the populations of Wushan and Shennongjia, and the dates of Sanmenxia segment of Yellow River as the time when the populations in Mt. Zhongtiao started differentiated from the population of Dabagou in Chang’an, molecular clock was established using sequences with 1953 bps of 19 populations of Feirana and outgroup including N. pleski, P. yunnanesis, P. robertingeri, F. limnocharis in order to estimate divergent time of all clades. Result of that indicated that: ① the tribe Paini started to evolve independently at about 70Ma when is in consistent with that estimated by Roelants et al.（2004）with result of about ～60±15Ma, they were corroborated by each other, this confirms the validity of this molecular clock; ② divergent time for speciation of Feriana is early, ancestral populations of F. quadrana and F. taihangnicus were found about 46Ma～50Ma; differentiation of populations within species is greatly late to the divergence of the two species, divergent time for F. quadrana is 10Ma and divergent time for F. taihangnicus is 6Ma. Proceeding of distribution pattern of Feirana. Phylogenetic relationships of populations of Feirana matched quite with distribution pattern of them, the relationships among clades showed in phylogenetic trees is direct ratio to geographical distance of them; the estimated date of speciation between two species of Feirana was as early as speciation of Paa yunnanesis and Nanara pleski; middle part of Mt. Qinling is the center of speciation of Feirana, combination of mult-events of dispersal and vicariance are probably the mechanism of speciation of Feirana, F. quadrana colonized the mid-Mt. Qinling and then differentiated into two subspecies in southwest direction, ancestral population of F. taihangnicus colonized the mid-Mt. Qinling and then differentiated into two subspecies in northeast direction. On geological history of the distribution of Feirana. According to molecular clock and speciation model of populations of Feirana, some geological events are confirmed, including special rise of Mt. Minshan- Mt. Longmen-western Mt. Qinling, glacial age.