江西庐山自然保护区两栖动物资源调查与评价

于2005—2006年先后3次对庐山自然保护区的两栖动物资源进行了实地调查,得知庐山自然保护区现已记录24种两栖动物,隶属2目8科,占江西省已知39种两栖动物的61．54％。有21种属东洋界种类,3种属广布种,无古北界种类。分别有6、6、3、5和4种两栖类的生态类型属静水型、陆栖-静水型、流水型、陆栖-流水型和树栖型。除大鲵、虎纹蛙为国家二级保护动物外,其余22种两栖动物均属“国家保护的有益的或者有经济、科学研究价值的陆生野生动物”。此次调查采集的小角蟾、阔褶蛙、虎纹蛙、竹叶蛙和花姬蛙为庐山自然保护区两栖动物新记录,并与江西省其它自然保护区讲行了两栖类相似件系数比较,提出了保护建议。     

中学动物学中有关两栖纲和爬行纲的两个问题

一、两栖纲:两栖动物在脊椎动物演化史上占有极为重要的位置,它是脊椎动物由水中向陆上发展的过渡类型。不了解两栖动物的特征,学生就不可能掌握脊椎动物由水中向陆上、由低级向高级发展的完整概念。从这个意义说,掌握两栖类的基本知识,也是完全必要的。现存的两栖动物,全世界有近3000种,其中无足目约170种;有尾目200余种;无尾目约2600种。我国有230余种(亚种),其中无足目1种;有尾目32种;无尾目197种。青蛙是泛指黑斑蛙、金线蛙、虎纹蛙等,是两栖纲,无尾目,蛙科动物。初中动物学课本图87,应该是黑斑蛙(Rana nigromaculata),体长可达8厘米。…     

棘胸蛙、虎纹蛙、黑斑蛙血细胞的比较

应用Giemsa染色法对棘胸蛙、虎纹蛙和黑斑蛙的血细胞进行显微观察和各形态参数测定,并对各类细胞作了图示和描述。结果表明,红细胞和白细胞的形态和数量比例有一定属和种的特征,并与其生长环境和进化地位有一定联系。     

黑斑蛙重盘吸虫的线粒体基因组特征及其系统发育研究

重盘科Diplodiscidae Cohn, 1904复殖吸虫是两栖动物最为常见的寄生虫,形态多样,目前未见基于线粒体基因组的遗传多样性研究。本研究记述并分析了寄生于滇蛙Dianrana pleuraden肠道的重盘科重盘属Diplodiscus Diesing, 1836黑斑蛙重盘吸虫D.nigromaculati的线粒体全基因组序列,为后续开展此类吸虫的遗传结构及遗传多样性研究提供了新的数据。黑斑蛙重盘吸虫线粒体基因组全长14 697 bp(GenBank登录号：MW698822),由22个tRNA基因、12个蛋白编码基因、2个rRNA基因和1个非编码区组成;AT含量(60%)高于GC含量(40%),但AT含量为目前已报道同盘总科Paramphistomoidea Fischoeder, 1901中最低;trnG基因和trnE基因的互换成为同盘总科吸虫线粒体基因组遗传结构的重要特征。基于12个蛋白编码基因的系统发育树支持重盘属隶属于重盘科。本研究中,滇蛙为黑斑蛙重盘吸虫的宿主新记录。     

两种蛙Sox基因的PCR-SSCP分析

采用PCR技术,以参考人SRI,基因HMG-box的保守区序列而设计一对特异引物,扩增了黑斑蛙和金线蛙的Sox基因。结果两种蛙的雌雄个体均扩增出217bp的基因片段,与人对照相同。对扩增产物进行SSCP分析显示,两种蛙雌雄个体间的单链迁移率相同,两种蛙之间差异较小,而与人有较大差异。测序表明,两种蛙的Sox序列之间以及与各类物种的Sox基因都有非常高的相似性,充分显示出Sox基因在系统进化上的高度保守性。     

云南昆明地区三种蛙皮肤显微结构的比较

对昆明地区的多疣狭口蛙、昭觉林蛙及黑斑蛙的背腹皮肤切片的显微结构进行了观察和比较,尤其对它们皮肤腺体的类型和功能进行了比较分析.结果 表明:多疣狭口蛙皮肤比昭觉林蛙及黑斑蛙皮肤含有更多的腺体,尤其是颗粒腺比后两者含量更为丰富,并且在皮下存在许多腺体团,背腹皮肤较后者厚.昭觉林蛙皮肤较黑斑蛙的含有更多的颗粒腺和粘液腺.3种蛙的背部皮肤都含有色素细胞,而除了在多疣狭口蛙雌性腹部观察到少量色素细胞外,另两种蛙在腹部皮肤中均未见色素层.相比之下,昭觉林蛙和黑斑蛙皮肤结构较相似,两者同多疣狭口蛙在皮肤腺体分布及数量上差别较大,这也体现了三者不同的生态适应机制.     

从12S rRNA基因序列研究中国蛙科24种的进化关系

测定了中国蛙科动物24种和蟾蜍科的中华大蟾蜍线粒体12S rRNA基因长约400bp片段的序列。采用邻接法,对其数据的系统发生分析表明24种可分成3个支系：第一个支系包括棘腹蛙、合江棘蛙、棘胸蛙、大头蛙、虎纹蛙、倭蛙、泽陆蛙共７种,其中３种棘蛙组成一个单系;第二个支系由３种湍蛙,即戴云湍蛙、华南湍蛙和武夷湍蛙组成,应属于湍蛙亚科;第三支系包含余下的１４种蛙,其中５种林蛙和４种臭蛙分别组成一个单系;２种侧褶蛙先聚合后,再与弹琴水蛙聚合,２种粗上先聚合在一起,再与由林蛙组成的单系群聚合。第三支系首先与第二支系相聚,然后它们与第一支系形成姐妹群,即由蛙亚科的７个物种组成的第一支系,不与蛙亚科另１４个物种组成的第三支系直接聚合,表明第一支系的物种可能不属于蛙亚科。此外,分子证据提示把陆蛙属、大头蛙属、虎纹蛙属、棘棘属、臭蛙属、粗皮蛙属和侧褶蛙属从原来的蛙属分出有其合理性。、     

中国大头蛙属3个种线粒体ND1基因全序列分析与亲缘关系

测定了大头蛙和脆皮大头蛙线粒体ND1基因全序列长度分别为978 bp和958 bp,(对应编码325和319个氨基酸)。对所测基因序列组分进行了分析,并与福建大头蛙同源序列进行比较发现,978个核苷酸位点中,有664个保守位点和多变位点294个。同时发现福建大头蛙与大头蛙该基因序列的同源性最高(核苷酸序列同源性为78.77%,氨基酸序列为92.62%)。基于ND1基因全序列的氨基酸和核苷酸两种数据形式,选用M ega3.1软件中的NJ法对大头蛙属3个种、黑斑蛙、泽陆蛙及外群中国大鲵共6条基因序列进行系统树重建分析,结果表明:所得的2个NJ树均将大头蛙属3个种聚于一支,其中大头蛙与福建大头蛙为姐妹群关系(自检值均高度支持),从而证实了大头蛙与福建大头蛙亲缘关系较近的观点。     

倭蛙属(无尾目∶蛙科)肩带研究

对倭蛙属(Nanorana)已知3种:高山倭蛙(Nanorana parkeri)、倭蛙(N.pleskei)和腹斑倭蛙(N.ventripunctata)的肩带作了连续切片后进行Masson染色,发现其上喙骨(epicoracoid)的融合与重叠的状况与虎纹蛙(Rana rugulosa)较相似,而与无尾目中其它物种已知的肩带形态存在较大的差异。倭蛙属和虎纹蛙等与已知肩带类型的蛙亚科其它的物种在系统进化中可能来源于不同的最近共同祖先。     

鲫鱼、黑斑蛙和家兔视网膜结构的比较组织学研究

为探讨鲫鱼Carassius auratus、黑斑蛙Rana nigromaculata和家兔Oryclagus cuniculus domestica视网膜组织结构与其生活环境的适应关系,测量了3种动物视网膜各层厚度、3个核层的胞核层数及胞核直径,并对数据进行了统计分析.结果表明:鲫鱼、黑斑蛙和家兔视网膜均由4层细胞...     

An insight into molecular taxonomy of bufonids,microhylids, and dicroglossid frogs: First genetic records from Pakistan

The current study was focused on documentation of amphibian assemblage in North Punjab and Islamabad Capital Territory, Pakistan, by using mitochondrial gene sequences of 16S rRNA. Our study entailed 37% of the known amphibian species of the country. We provided a phylogenetic analysis based on 74 newly generated mitochondrial 16S rRNAs from nine species of genus Microlyla, Duttaphrynus, Allopaa, Nanorana, Sphaerotheca, Minervarya, Hoplobatrachus, and Euphlyctis. We employed the maximum‐likelihood inference and Bayesian analysis to assess the taxonomic status of the samples obtained from Pakistan, with respect to other congeneric species from surrounding regions. Our findings confirmed the taxonomic status of South Asian anuran species Duttaphrynus stomaticus, Duttaphrynus melanostictus, Microhyla nilphamariensis, Allopaa hazarensis, Nanorana vicina, Sphaerotheca maskeyi (synonym: S. pashchima), Minervarya pierrei, Hoplobatrachus tigerinus, and Euphlyctis kalasgramensis in Pakistan. We have reported new country records of genus Minervarya ( M. pierrei). Minervarya pierrei was previously misidentified as Fejervarya limnocharis, due to dearth of genetic information. We provided the first genetic records of our endemic species N. vicina. The results revealed the taxonomic placement of N. vicina with respect to its congeners and validated the taxonomic status of N. vicina from its type locality (Murree) for the first time. The findings of the present study also indicated the paraphyletic relationship of A.‐ hazarensis with Nanorana species. So, based on our phylogenetic inferences, morphological characters, and habitat preferences, validity of generic status of A. hazarensis is undecided. As our data were not enough to resolve this issue, we suggest sequencing of additional mitochondrial and nuclear genes in the future studies to get a better resolution. We recommend carrying out extensive surveys throughout the country for proper scientific documentation of amphibians of Pakistan. Many new species, some of them might be endemic to Pakistan, are expected to be discovered, and taxonomic status of other species would be resolved.     

Molecular phylogeny of the ranid frogs from Southwest India based on the mitochondrial ribosomal RNA gene sequences

The Western Ghats of Southwestern India are known as one of the world's "hotspots" of biodiversity. We collected frog specimens from the family Ranidae and investigated the phylogenetic relationships among ranid species, particularly among the Fejervarya, a genus whose morphological diagnostic characteristics and phylogenetic features remain little known. We analyzed partial sequences of the mitochondrial 12S (428 bp) and 16S rDNAs (549 bp). Results showed that the members of Fejervarya form a monophyletic group with the genera Hoplobatrachus and Euphlyctis among ranid genera. This confirms the recent allocation of Fejervarya, including it within the subfamily Dicroglossinae. The mitochondrial rDNA data in our study also appeared to be useful as a marker to distinguish Fejervarya species without morphological differences. The phylogenetic tree based on the 16S rDNA sequence showed a correlation between Fejervarya phylogenies and their geographic distributions. Lastly, our results suggested the recent occurrence of a radiation event of Fejervarya species in the Indian-Sri Lankan region.     

采用未培养技术对荷斯坦奶牛瘤胃细菌多样性进行初步分析

采用未培养(Culture independent)技术直接从荷斯坦奶牛瘤胃液中提取瘤胃细菌微生物混合DNA(也叫元基因组DNA),利用细菌16SrDNA通用引物27F与1492R,扩增瘤胃混合微生物的16SrDNA,根据16SrDNA序列对瘤胃细菌多样性进行初步分析。通过16SrDNA序列同源性分析,发现有多于一半以上的序列与可培养的菌株的同源性小于90%,属于不可培养的菌株。选用45条测得序列与已知序列构建系统发育树,分析结果表明,它们分属于两大类LGCGPB(the lowG CGram positivebac teria)和CFB(Cytophaga_Flexibacter $CBacteroides group),剩下的克隆尚难确定其分类地位,可能是代表新属和种的序列,这些序列已向GenBank提交并得到序列号(AY986777_AY986791)。     

从线粒体基因探讨中国大头蛙群的分类及其属内地位

Partial sequences of the mitochondrial 12S rRNA and 16S rRNA gene were determined for 8 populations of three species of Chinese Limnonectes, and aligned with the published sequences of Limnonectes from other parts of the world. When Nanorana parker, Paa boulengeri, Fejervarya limnocharis and Hoplobatrachus rugulosus was used as outgroup taxa (Accession Nos. AY158705, AY313685, AF206111, AF206491, AY322311). The sequences of the 12S rRNA and 16S rRNA genes totaled 950 nueleotide positions with gaps including 510 variable sites. We reconstructed phylogenetie trees using Clustal X 1.8, Mega 2.1 and PHYLIP 3.5e software, and using the maximum parsimony and maximum likelihood methods, respectively. Our analyses suggest that these fanged frogs from China are another monophyletie group in addition to the four monophyletie groups identified by previous studies. The Chinese Limnonectes were grouped into three elades (BCL 55% ). The first elade contains one species (BCL 100% ), from a population of Limnoneetes fragilis from Hainan Province. The second contains four individuals (BCL 100% ), i. e. two populations of Limnonectes kuhlii from Yunnan Province. The third contains one species (BCL 100% ), i. e. five populations of Limnonectes fujianensis from Fujian Province and 1 from Taiwan Province. The resulted phylogenetie trees indicate L. fragilis is basal to L. kuhlii L. fujianensis 。     

Molecular differentiation of the microgastrine species commonly found in paddy fields from Southeast Asia,with additional data on their phylogeny （Hymenoptera：Braconidae）

Partial DNA sequences of three genes, that is, mitochondrial large ribosomal subunit (16S), nuclear large ribosomal subunit (28S D2) and mitochondrial NADH1 dehydrogenase (NADH1) gene, were sequenced from different microgas trine species(Braconidae: Microgastrinae) collected fresh from paddy fields. The DNA sequences were used to determine the extent of sequence variation among species in order to evaluate the specific status of each species. Cladistic analysis was also used to infer a phylogenetic relationship among these species. The results showed that sequence divergence among species of the same genus Cotesia was much lower than those among different genera, such as Cotesia, Exoryza and Apanteles; the sequence similarity of 16S rDNA and NADH 1 genes between Cotesia sp. and C. chilonis was higher than that between C. sp. and C. ruficrus.Phylogenetic analyses suggested that four species of Cotesia were always grouped in the same clade regardless of using different analysis methods; Cotesia sp. and C. chilonis are more closely related to each other than to C. ruficrus, different from previous morphological results. Additionally, sequence analyses indicated that NADH1 gene has more parsimony informative characters than 28S rDNA D2 and 16S rDNA at the species-level analysis,indicating that NADH1 gene might be a useful marker for species-level analysis.     

Exploring Species Level Taxonomy and Species Delimitation Methods in the Facultatively Self-Fertilizing Land Snail Genus Rumina (Gastropoda: Pulmonata)

Delimiting species in facultatively selfing taxa is a challenging problem of which the terrestrial pulmonate snail genus Rumina is a good example. These snails have a mixed breeding system and show a high degree of shell and color variation. Three nominal species (R. decollata, R. saharica and R. paivae) and two color morphs within R. decollata (dark and light) are currently recognized. The present study aims at evaluating to what extent these entities reflect evolutionary diverging taxonomic units, rather than fixed polymorphisms due to sustained selfing. Therefore, a phylogenetic analysis of nuclear (ITS1, ITS2) and mitochondrial DNA (COI, CytB, 12S rDNA, 16S rDNA) sequences was performed. Putative species in Rumina, inferred from the mitochondrial DNA phylogeny, were compared with those proposed on the basis of the COI gene by (1) DNA barcoding gap analysis, (2) Automatic Barcode Gap Discovery, (3) the species delimitation plug-in of the Geneious software, (4) the Genealogical Sorting Index, and (5) the General Mixed Yule Coalescent model. It is shown that these methods produce a variety of different species hypotheses and as such one may wonder to what extent species delimitation methods are really useful. With respect to Rumina, the data suggest at least seven species, one corresponding to R. saharica and six that are currently grouped under the name R. decollata. The species-level status of R. paivae is rejected.     

MALDI-TOF MS与16S rDNA方法对 弧菌科微生物的鉴定与系统分类学分析

目的比较基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)与16S rDNA方法对弧菌科微生物的鉴定及系统分类学分析能力。方法对19株弧菌科微生物,采用MALDI-TOF MS进行蛋白质图谱采集,通过对特征峰的分析,实现对微生物的鉴定和系统分类学分析;同时对19株微生物进行16S rDNA测序,用邻接法对16S rDNA序列进行鉴定和系统分类学分析,比较两种方法在弧菌科微生物鉴定和系统分类学分析中的异同。结果两种方法对19株弧菌科微生物的种属鉴定结果一致。系统分类学分析中,多株同种属的弧菌的两种方法分析结果一致,但对拟态弧菌和霍乱弧菌在树状图中的位置和亲缘关系,两种方法差异较大。结论 MALDI-TOF MS与16S rDNA均能够快速准确地鉴定弧菌科微生物,但利用MALDI-TOF MS进行系统分类学分析还有待数据库的扩大及算法的优化。     

Mitochondrial 12S rDNA sequence relationships suggest that the enigmatic bovid "Linh Duong" Pseudonovibos spiralis is closely related to buffalo

The phylogenetic position and taxonomic status of the recently described Southeast Asian endemic bovid Pseudonovibos spiralis were deduced from nearly complete 12S mitochondrial rDNA sequences of this species and Bubalus bubalis alongside 26 sequences of Bovidae from GenBank using Cervus elaphus (Cervidae) as outgroup. Maximum-likelihood analyses performed by PUZZLE and fastDNAml nested P. spiralis at the base of the subtribe buffalo Bovini, suggesting the close relationship of this enigmatic species with buffalo and enabling its distinction into the separate     

Phylogenetic analysis of 23S rRNA gene sequences of. Agrobacterium, Rhizobium and Sinorhizobium strains

The phylogenetic relationship among twelve Agrobacterium, four Rhizobium, and two Sinorhizobium strains originating from various host plants and geographical regions was studied by analysis of the 23S rDNA sequences. The study included Agrobacterium strains belonging to biovars 1, 2 (with tumor- or hairy-root inducing and non-pathogenic strains), A. vitis, A. rubi; representative species of the Rhizobium genus: R. galegae, R. leguminosarum and R. tropici and Sinorhizobium meliloti strains. The phylogenetic analysis showed that within Agrobacterium, the biovar designation was reflected in the 23S rDNA similarity and that strains of Agrobacterium and Rhizobium are closely related to each other. The results suggest that the taxonomic definition of Agrobacterium and Rhizobium should be considered for revision and that the Agrobacterium-biovar identity is probably a reliable taxonomic trait.     

A unique DNA repair and recombination gene (recN) sequence for identification and intraspecific molecular typing of bacterial wilt pathogen Ralstonia solanacearum and its comparative analysis with ribosomal DNA sequences

Ribosomal gene sequences are a popular choice for identification of bacterial species and, often, for making phylogenetic interpretations. Although very popular, the sequences of 16S rDNA and 16-23S intergenic sequences often fail to differentiate closely related species of bacteria. The availability of complete genome sequences of bacteria, in the recent years, has accelerated the search for new genome targets for phylogenetic interpretations. The recently published full genome data of nine strains of R. solanacearum, which causes bacterial wilt of crop plants, has provided enormous genomic choices for phylogenetic analysis in this globally important plant pathogen. We have compared a gene candidate recN, which codes for DNA repair and recombination function, with 16S rDNA/16-23S intergenic ribosomal gene sequences for identification and intraspecific phylogenetic interpretations in R. solanacearum. recN gene sequence analysis of R. solanacearum revealed subgroups within phylotypes (or newly proposed species within plant pathogenic genus, Ralstonia), indicating its usefulness for intraspecific genotyping. The taxonomic discriminatory power of recN gene sequence was found to be superior to ribosomal DNA sequences. In all, the recN-sequence-based phylogenetic tree generated with the Bayesian model depicted 21 haplotypes against 15 and 13 haplotypes obtained with 16S rDNA and 16-23S rDNA intergenic sequences, respectively. Besides this, we have observed high percentage of polymorphic sites (S 23.04%), high rate of mutations (Eta 276) and high codon bias index (CBI 0.60), which makes the recN an ideal gene candidate for intraspecific molecular typing of this important plant pathogen.