Past and present: Phylogeography of the Bufo gargarizans species complex inferred from multi-loci allele sequence and frequency data

Using multi-locus DNA sequence and frequency data, we examined the phylogeographic patterns of the Asian toad Bufo gargarizans species complex. A total of 166 individuals from 56 sites were genotyped for one mitochondrial locus (516 base pairs, ND2 gene) and five nuclear intron loci (Sox9-2, Rho-3, CCNB2-3, UCH-2, DBI-2; 250-350 base pairs each). We conducted a phylogenetic analysis of the allele sequence data to construct gene trees for each individual locus using Bayesian inference, a multi-locus phylogeny based on all five nuclear loci using POFAD, and a phylogenetic network using the NeighborNet algorithm. Furthermore, we used population-genetic analysis of the allele frequency data to reveal ongoing processes, including an analysis of molecular variance (AMOVA), a Bayesian assignment analysis, and a non-equilibrium Bayesian method for estimating recent migration rate. Our phylogenetic analyses showed that the observed divergence in the B. gargarizans species complex likely dated back to 7-8 million years ago. Repeated range expansions during the inter-glacial periods of Pleistocene likely established the current distribution of genetic diversity, although historical vicariant events were still evidenced. Both the west and the southeast regions may have served as refugia during the glaciation, and the range expansion was in general from west to northeast. Additionally, we detected strong ongoing migration both from west to the other regions and from south to north, which likely represents a meta-population dynamic that has emerged over the past ～10,000 years. The morphologically identified species Bufo tibetanus is not genetically diagnosable, and therefore should be considered a junior synonym of B. gargarizans.     

中华蟾蜍中脑组织学研究

采用HE染色和Holmes银染法对蟾蜍中脑的显微结构进行了研究.中脑背侧,视叶可分为顶盖和被盖,顶盖从外侧到内侧依次分为:带状层、外灰质层、浅白质层、中灰质层、中白质层、深灰质层、深白质层和中央灰质.被盖前端分层与顶盖相同,后端分层不明显.中脑腹侧包括被盖和大脑脚,HE染色和Holmes银染法显示,大脑脚从外向内颜色由浅变深,存在大量纵向神经纤维束,两脚底分界处有横向交错的神经纤维.被盖外侧细胞不分层,聚集形成核团.被盖内侧,细胞和纤维以中脑水管为中心,呈同心圆环分8层.通过比较蟾蜍中脑背腹差异程度,了解背腹功能不同.同时对中华蟾蜍中脑同其他脊椎动物的进行了比较.     

镉在中华蟾蜍组织中的分布规律

利用原子吸收分光光度计测定了镉在中华蟾蜍不同组织中的含量,并对其在不同器官组织中的分布规律进行了分析。结果表明：镉在中华蟾蜍不同组织中具有选择性,主要积累在肠和肝脏中,其次是精巢、皮肤、卵巢和肌肉,而骨骼中没有积累。     

中华蟾蜍糖原含量的季节变化

于2005年11月至2006年10月,用硫酸-蒽酮比色法和比重法测定了中华蟾蜍各月份的肝糖原和肌糖原含量及肝比重。结果显示:冬眠期间(11月至次年的2月),糖原含量逐月下降;2月份时出现临时回升,然后继续下降;4月份时肝糖原含量最低;5月份起,肝糖原含量逐渐上升;5月份时肌糖原含量为最低;6月份起,肌糖原含量逐渐上升。虽然在7-8月间出现过下降,两种糖原的含量在10月份时达到一年中的最高值。这些结果表明,蟾蜍糖原含量在一年中呈现显著的季节性波动。越冬前所储备糖原的一部分可能用于越冬期间维持高水平的血糖,一部分用于基础代谢。2月份时糖原含量的临时上升,可能是血液中作为防冻保护剂的葡萄糖运回肝脏和肌肉中再合成糖原的结果。7-8月间糖原含量降低可能与蟾蜍夏蛰有关。雌性5月至10月期间的肝糖原总体水平显著低于雄性,可能与依赖可得到葡萄糖的卵母细胞中的糖原合成有关。糖原含量的季节变化与蟾蜍的生活状态(越冬、繁殖等)有关,并与血糖含量有联动关系。     

中华大蟾蜍卵母细胞成熟过程中膜电位变化的实验分析

The full-grown oocytes obtained from toad (bufo bufo gargarizans) submitted in hibernation state or reared at 25-30 degrees C for several months, named hibernation oocyte or high temperature oocyte, had a membrane potential of -41.51 +/- 0.77 mV and -43.83 +/- 1.39 mV in Ringer's solution respectively. The hibernation oocytes underwent GVBD (germinal vesicle breakdown) and membrane depolarization at 19 +/- 1 degree C after progesterone stimulation. The membrane potential was about -20 mV at the period of GVBD, and -10 mV or so at 20 hours after the hormone treatment. However, the high temperature oocytes did not undergo GVBD, their membrane potential decreased before the fourth hour after treatment with progesterone and then recovered. If the hibernation oocytes were preincubated at 37-38 degrees C for 13 hours prior to the culture in the medium containing progesterone (10(-6)M, 37-38 degrees C), no GVBD was observed and the membrane depolarized before the fourth hour after treatment with progesterone then recovered, but MPF was detectable in the cytoplasm (unpublished). Both GVBD and membrane depolarization appeared in the hibernation oocytes and high temperature oocytes after injection of MPF. The time required for the hibernation oocytes injected MPF to attain the membrane potential about -20 mV was 4 hours earlier than that of progesterone treatment. It was just the time required for the appearance of MPF in the cytoplasm of oocytes treated with the hormone. It was noticed in our precedent article that a factor which appeared in the cytoplasm of high temperature oocytes differed from MPF. The factor was called Hibernation Oocyte Mature Promoting Factor (HOMPF).(ABSTRACT TRUNCATED AT 250 WORDS)     

中华蟾蜍颈动脉腺的组织学结构

2005年10月～2006年5月,用组织学技术研究中华蟾蜍颈动脉腺结构。结果表明,颈动脉腺位于外颈动脉基部,圆球形,深红色至棕褐色。组织结构显示：颈动脉腺的外壁是动脉管壁的延续,包括外膜、中膜和内膜。整个外壁厚薄不均。颈动脉腺的最大特点是中膜和内膜并不像一般血管形成环圈状,而是从不同部位向管腔突出延伸、相互连接构成大小不一、形状各异、迂回曲折的网状血管。管腔大者,管壁厚,弹性纤维、平滑肌纤维多,内膜靠腔面内皮细胞多成立方状,细胞核端位近圆形;管腔小者,管壁薄,弹性纤维、平滑肌纤维少,内皮细胞扁平、排列稀疏,胞核长梭形;一些区域管径极小,管壁极薄,成为开放的血窦,只允许一个血细胞通过。网状管壁间有密集成团的大型类上皮细胞等细胞分布。据结构推测中华蟾蜍颈动脉腺有调节血压等功能。     

除草剂草甘膦对中华大蟾蜍的慢性毒性

为揭示除草剂对农林生态系统中脊椎动物天敌潜在的、长期的慢性毒性,测定了草甘膦对中华大蟾蜍（Bufo gargarizans Cantor）蝌蚪生长发育和运动频率的影响以及对中华大蟾蜍成体空胃率的影响。结果表明,亚致死剂量的草甘膦溶液对中华大蟾蜍蝌蚪的生长发育和运动频率具有明显的抑制作用,蝌蚪体长、体宽、尾长、尾宽和体质量的增长率均与草甘膦浓度呈极显著负相关;蝌蚪的运动频率亦与草甘膦浓度显著负相关。同时,草甘膦的喷施对中华大蟾蜍成体的捕食造成负面影响,其空胃率与草甘膦浓度呈极显著正相关,即使在草甘膦的推荐使用浓度范围之内．中华大蟾蜍（成体）处理组亦出现30％～60％的空胃率。     

中华蟾蜍血液生理生化指标的测定

本文对中华蟾蜍（Bufo gargarizans）血液生理和生化指标进行了测定,并对其测定结果进行了t检验。结果显示,中华蟾蜍血液生理生化指标中白蛋白（ALB）、总蛋白（TP）、甘油三酯（TG）、总胆固醇（CHO）、丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、乳酸脱氢酶（LDH）、葡萄糖（Glu）、血清钙（Ca）、血清氯（Cl）、血清磷（P）、血清镁（Mg）、尿素（Urea）、血清钠（Na）雌雄问均差异不显著（P〉0．05）;碱性磷酸酶（ALP）、血清钾（K）两项指标雌雄之间差异显著（P〈0．05）;肌酐（Cre）在雌雄之间差异极显著（P〈0．01）,从测定结果可看出中华蟾蜍雌雄间血液生理生化值差异不大。     

异丙甲草胺、醚菌酯和咪鲜胺锰盐对中华大蟾蜍蝌蚪的急性致死效应

采用静态换水方法研究了异丙甲草胺、醚菌酯和咪鲜胺锰盐3种农药对中华大蟾蜍(Bufo gargarizans)蝌蚪的急性毒性及其联合毒性.结果表明,在急性毒性和两两联合毒性实验中,不同浓度农药对蝌蚪的死亡率影响均显著.在24 h、48h及72 h急性毒性实验中,异丙甲草胺对中华大蟾蜍蝌蚪的半致死浓度(LC50)分别为29.81、28.81和25.83mg/L,醚菌酯为1.72、1.46和1.41 mg/L,咪鲜胺锰盐为7.43、3.75和3.22 mg/L.异丙甲草胺、醚菌酯和咪鲜胺锰盐3种农药的安全浓度(SC)分别为8.07、0.32和0.29 mg/L.异丙甲草胺为中等毒性农药,而醚菌酯和咪鲜胺锰盐均为剧毒性农药.在两两联合的毒性实验中,异丙甲草胺-醚菌酯和异丙甲草胺-咪鲜胺锰盐均表现为中等毒性,醚菌酯-咪鲜胺锰盐表现为剧毒性.在24 h、48 h及72h两两联合毒性实验中,异丙甲草胺-醚菌酯对中华大蟾蜍蝌蚪的半致死浓度(LC50)分别为18.41、15.69和13.38 mg/L,异丙甲草胺-咪鲜胺锰盐为15.56、10.45和8.11 mg/L,醚菌酯-咪鲜胺锰盐为4.17、2.84和2.00 mg/L,且其相对应的安全浓度分别为3.42、1.41和0.40 mg/L.在联合毒性评价中,异丙甲草胺-醚菌酯联合组在24 h和48 h表现为拮抗作用,醚菌酯-咪鲜胺锰盐联合组在48 h表现为加和作用,其余均表现为协同作用.同样,重复方差分析结果表明,农药浓度、染毒时间及两者相互作用对蝌蚪的存活率影响均显著.与前期发表数据进行比较,表明中华大蟾蜍蝌蚪对污染物具有很强的敏感性.这些结果将为无尾类动物的毒理学研究提供科学依据.     

高效氯氰菊酯对中华蟾蜍蝌蚪的毒性影响

以中华蟾蜍蝌蚪为实验对象,采用半静态毒性试验方法研究了高效氯氰菊酯对其的急性毒性和生理毒性。结果表明:高效氯氰菊酯对中华蟾蜍蝌蚪的96h半致死浓度(96h-LC50)为1.147mg/L,其毒性为中级;蝌蚪的总抗氧化能力(T-AOC)随染毒时间的延长而逐渐降低,当高效氯氰菊酯质量浓度达到1μg/L以上时,蝌蚪的T-AOC在出现兴奋效应后随即下降;蝌蚪体内的谷胱甘肽-S转移酶(GSH-ST)活性随着农药浓度的增大和染毒时间的延长呈现出不断增加的趋势,当高效氯氰菊酯质量浓度达到1μg/L以上时,蝌蚪的GSH-ST活性较对照组有极显著的提高。研究表明,高效氯氰菊酯对中华蟾蜍蝌蚪会造成一定的毒性影响。     

Phylogeographic study reveals the origin and evolutionary history of a Rhododendron species complex in Taiwan

This study infers a single origin and a once-widespread distribution of the Rhododendron pseudochrysanthum species complex in Taiwan based on chloroplast DNA (cpDNA) variation. In total, 124 individuals from five endemic Rhododendron species were used for amplifications of two chloroplast intergenic spacers: trnL-trnF and atpB-rbcL. The haplotype and nucleotide diversities were much lower for the R. pseudochrysanthum complex, comprised of the species R. pseudochrysanthum, R. morii, Rhododendron rubropunctatum, and Rhododendron hyperythrum, than for Rhododendron formosanum. Two measures of pairwise population differentiation, N(ST) and F(ST), consistently revealed mostly non-significant levels of genetic divergence between populations of the R. pseudochrysanthum complex. No genetic difference was found among the four species of the R. pseudochrysanthum complex by analysis of molecular variance (AMOVA), which is concordant with the parsimonious topology of cpDNA haplotypes for the complex. Nested clade analysis (NCA) of the cpDNA haplotypes indicated that restricted gene flow with isolation-by-distance characterized the recolonization pattern of the R. pseudochrysanthum complex. In contrast, the NCA analysis indicated a contiguous range expansion for cpDNA haplotypes of R. formosanum. This research suggests a once-widespread distribution of the R. pseudochrysanthum complex probably via north-to-south colonization of mid-elevations during low-temperature periods of the Pleistocene. Population fragmentation followed the warmer climate which began in the Holocene and resulted in the present-day range contraction into high elevations.     

三氯杀螨醇对中华蟾蜍卵巢发育的影响

选取健康活跃的中华蟾蜍Bufo gargarizans雌性个体分成5个试验组,每组7只,分别以0.00(对照组,注射任氏液)、0.25、0.50、1.00和2.00 mg/kg浓度的三氯杀螨醇(DCF)行体腔注射染毒,每3 d一次,连续30 d,于第31 d对各组蟾蜍称量体重,之后杀死动物取其卵巢称重计算卵巢系数,并对卵巢以Bouin's液固定,石蜡切片(厚7 μm),H.E染色,用Axioskop40显微镜统计单位面积内(0.02 cm2)卵泡数量,观察卵巢内卵泡发育进程和卵泡结构.用t检验得出各试验组卵巢系数和卵泡数量差异的显著性.     

Population Genetics and Phylogeography of Bufo gargarizans in China

The Chinese toad Bufo gargarizans has an extensive range, covering nearly all of China. This study addresses the population genetic structure of this toad and the relation to geographic location. Partial sequences of the mtDNA control region were obtained from 14 populations, 29 haplotypes were defined, and 73 variable sites were found, including 66 parsimony informative sites. All population genetic analyses indicated no clear geographic pattern in the distribution of haplotypes. The genetic divergence between the populations was significant. Phylogeographic analyses suggested that past fragmentation and/or long-distance colonization seemed to have shaped the present-day distribution of the haplotypes of B. gargarizans.     

Phylogeographic inference using Bayesian model comparison across a fragmented chorus frog species complex

Fragmented species complexes provide an interesting system for investigating biogeographic history and the present distribution of genetic variation. Recent advances in sequencing technology and statistical phylogeography enable the collection and rigorous analysis of large multilocus data sets, but designing studies that produce meaningful phylogeographic inferences remains challenging. We implemented a Bayesian model comparison approach to investigate previous biogeographic hypotheses while simultaneously inferring the presence of genetic structure in a chorus frog species complex. The Illinois chorus frog (Pseudacris illinoensis), originally described as a subspecies of the broadly distributed Strecker's chorus frog (Pseudacris streckeri), occurs in small, disjunct regions associated with scarce sand prairie habitats that have been impacted by human development. We used high‐throughput sequencing to develop and collect a multitiered genetic data set comprised of three different marker types (23 anonymous nuclear sequence loci, four mitochondrial genes and 14 microsatellite loci) designed to address questions across different evolutionary timescales. Phylogenetic analyses uncovered a deep divergence between populations in the Edwards Plateau of central Texas and all other P. streckeri/P. illinoensis populations, but suggest the disjunct distribution of P. illinoensis occurred more recently. Our best‐supported migration model is consistent with the hypothesis that central Texas represented a refugium from which populations expanded via multiple routes. This model also indicates that disjunct northern and southern regions of P. illinoensis should be considered genetically distinct management units. Our study provides an evolutionary context for future studies and conservation efforts in P. illinoensis and demonstrates the utility of model‐based approaches for phylogeographic inference.     

中华大蟾蜍TAGLN2 cDNA的克隆与其组织分布

为研究蟾蜍(Bufo)基原的中药材中的多肽类有效成分,开展从日本蟾蜍(B.japonicus formosus)皮肤cDNA质粒文库中筛选蛋白编码基因的工作.由于后续拓展蟾蜍药用价值的研究中需要大量的实验材料,开始以中华大蟾蜍(B.gargarizans)皮肤第一链cDNA为模板,克隆其相关基因的工作.通过DNA聚合酶链式反应获得编码转胶蛋白-2(transgelin2,TAGLN2)的转录子(GenBank登录号为KF432856).该cDNA全长1 207 bp,其5′、3′端非翻译区域和开放阅读框分别为16 bp、597 bp和594 bp,编码由197个氨基酸残基组成的蛋白质,与日本蟾蜍的TAGLN2(GenBank登录号为AGQ03775.1)相比,仅一个氨基酸发生替换,与人的同源性为82%,与其他动物的同源性介于70%⁷5%.RT-PCR组织分布检测表明TAGLN2在所检测的中华大蟾蜍各器官中均有表达,在肺、肝和肾中表达量较多.这些研究结果为后续中华大蟾蜍TAGLN2的生物学功能研究以及相关药物研发提供基础数据.     

乙草胺对蟾蜍心电活动的影响

采用生物机能实验系统,对乙草胺胁迫条件下蟾蜍Bufo gargarizans的心率和心电活动的相关指标进行了测定和分析.试验结果表明,随着乙草胺处理浓度的增大,蟾蜍的心率减慢,心电图中P波、R波和T波的电压峰值降低,而P-R间期、QRS期和Q-T间期的时值延长.乙草胺胁迫条件下,蟾蜍心率与心电图的各项指标有显著相关性.由此可见,乙草胺的喷施对蟾蜍心电活动周期和机械活动周期均造成了一定影响.     

TGA对蟾蜍血清生理指标的影响

本试验利用生化检测方法,以健康的中华蟾蜍为实验动物,研究注射巯基乙酸对血液生理指标的影响,并对其作用机理进行了初步探讨.试验结果表明,随染毒TGA剂量增加,蟾蜍血清总蛋白、葡萄糖含量下降,胆固醇、甘油三酯含量上升;随染毒TGA时间的延长,血清总蛋白、葡萄糖含量先下降后上升但均低于对照组,胆固醇、甘油三酯含量先上升后下降但均高于对照组.因此提示化工产品对生物的影响不容忽视.