Cloning and Sequence Analysis of 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo

[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.

16S rRNA Gene-Based Analysis of Ileal Bacterial Community and Phylogeny in Nursing and Weaned Piglets

Weaning of piglets is generally considered as a stressor which changes intestinal ecosystem and leads to clinical implications. Microbiota inhabiting in small intestine （especially ileum） are assumed to promote health, but their functional properties are yet poody dascdbed. As indicated by the 16S rRNA gene sequences of ileal micrebiota in nursing piglets （at the age of 21 and 28 d） and 28-day-old weaned piglets （weaned at 21 d of age）, the microbiota were mainly comprised of gram-positive bacteria. There were 40 operational taxonomic units （OTUs） （from 171 clones） in the ileum of nursing piglets aged 21 d, 61 OTUs （from 194 clones） in the ileum of nursing piglets aged 28 d, and 56 OTUs （from 171 clones） in the ileum of weaned piglets aged 28 d. The flea of nursing piglets aged 21 d were dominantly occupied by Lactobacilli （87.7%） as well as Streptococ cus （ 3.5 % ）. Lactobacillus amy/ovorus （41.5 % ）, Lactobaci/lus sp. （ 19.3 % ）, Lactobaci/lus reuteri （ 12.3 % ）, Lactobacillus salivarius （ 9.4 % ） and L. mucosae （4.7%） were the predominant species among Lactobacil/L Similar results were obtained in the nursing piglets at 28 d of age ex- cept that Lactobaci/li decreased to 71.1% and Streptococcus increased to 21.1% significantly. Lactobacillus （52.0%） and Streptococcus （26.3%） were the two major groups in the ileum of weaned piglets aged 28 d. Lactobacillus amylovorus （31.6%） and Lactobaci/lus reuteri （ 16.4% ） was the two most important species in Lactobacillus. Therefore, Lactobacilli were predominant in the ileum of nursing and weaned piglets, and they had the highest diversity, followed by Streptococcus. The diversity of ileal microbiota was not different remarkably between the nursing piglets and the weaned piglets, but the composition changed significantly. These findings are helpful to understand ileal bacterial ecophysiology and further develop nutritional regimes to prevent or counteract complications during the weaning transition.

Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of com-mon Chinese cuttlefish Sepiella maindroni: three from the South China Sea, one from East China Sea and one from Japan. The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals, and 13 positions are examined to be variable in all the sequences, which range from 494 to 509 base pairs. All of the individuals are grouped into 7 haplotypes (h1-h7). No marked genetic difference is observed among those populations. All of the individuals from Nagasaki belong to hl and the h3 haplotype is found only in the coastal waters of China. A(?)G transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.

16S rRNA Gene Sequence Analysis of Snow Leopard, Gray Wolf, Horse and Bactrian Camel in Mongolia

In this study, mitochondrial 16S rRNA sequences of snow leopard, gray wolf, domestic horse and Bactrian camel inhabited or domesticated in Mongolian territory were obtained by using polymerase chain reaction （PCR） based on universal primers for 16S rRNA （F-5＇-AACGAGCCTGGTGATA-3＇ and R-5＇-CTCCGGTCTGAACTCAGATCACGTA-3＇）. The 16S rRNA sequence was 1,048 bp to 1,086 bp in length, and each sequence was compared to other related species （Felidae, Camelidae, Equidae and Canidae） by using NCBI Basic Local Alignment Search Tool （BLAST）. Results showed that sequences were highly similar to sequences in GenBank database （93%-99%）. Then phylogenetic analysis was performed based on about 1,100 bp sequence of 16S rRNA for Panthera uncia, Canis lupus, Equus caballus, Camelus bactrianus and other related species. The result revealed that P. uncia and P. leo were sister species, C. bactrianus and C. ferus were more closely related species, and wolf and dog were the almost similar species. This finding could be important for designing species specific primers for PCR based analysis of animal species identification and forensic veterinary medicine.

16S rRNA Gene-Based Metagenomic Analysis of Soil Bacterial Diversity in Brazzaville, Republic of the Congo

Soil contains a great diversity of microorganisms, among which are bacteria. This study aimed to explore bacterial diversity in soil samples in Brazzaville in the Republic of the Congo. Environmental DNA was extracted. The illumina MiSeq sequencing was held and the diversity indices have been computed. Illumina MiSeq sequencing revealed 21 Phyla, four of which were abundant: Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes. Soil microbial communities in the studied samples were phylogenetically diverse but with a stable community structure. 17 classes are represented with relative abundances of Rihzobiales, Bacillales, Actinomycetales and Acidobacteriales. 40 families, the Alphaproteobacteria, the Bacilli and the 12 Actinobacteria. 83 orders among which the Rhizobiales are the most abundant followed by Bacillales and the least abundant followed by the Flavobacteriaceae. Of the 28 genera listed, the Bradyrhizobium is the most dominant in Mw3 and Mw4. 25 listed species, Bradyrhizobium, Bacillus, Actinoplanes, and Candidatu coribacter Acidobacterium are the most abundant species. The Shannon indices of Mw3 and Mw4 are equal, the H’max of Mw4 is greater than the H’max of Mw3. The Simpson index of Mw4 is equal to the Simpson index of Mw3, and the Pielou index (J) of Mw4 is less than the R of Mw3, but very close. This study opens interesting perspectives on the knowledge and exploitation of telluric bacteria in several areas of life.

16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea,Echinodermata)

Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease,pathogens of which are supposed to be bacteria by most researchers,is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao,China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies,and touchdown PCR was performed to amplify the target sequences. The results suggest that γ-proteobacteria(Alteromonadales and Vibrionales) of CFB group,many strains of which have been also determined as pathogens in other marine species,are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.

Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on ge- netic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the rela- tionship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K. bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

Isolation of Pectinase Producing Bacteria from the Rhizosphere of <i>Andrographis paniculata</i>Nees and 16S rRNA Gene Sequence Comparison of Some Potential Strains

Pectinases, the enzymes which break down pectic substances, have a wide range of applications in food, agriculture and environmental sectors. In the present study, attempts were made to isolate highly efficient pectinase producer from the rhizosphere of a medicinal plant, Andrographis paniculata Nees, known as the “King of bitters”. The total heterotrophic bacterial count of the rhizosphere soil of A. paniculata Nees ranged from 1.53 × 109 to 2.52 × 109 cfu/g. A total of 65 bacterial colonies were randomly selected from the nutrient agar plates, purified and assessed for pectinase activity. Out of the 65 isolates, 62 (95.38%) showed varying degree of pectinase activity in plate assay using pectin as a sole source of carbon. Among the pectinase producing strains, JBST36 showed best pectinase activity which is followed by the JBST22 and JBST27. Morphological characterization, biochemical tests and 16S rRNA gene sequencing were performed to identify the three most potential strains. Based on the morphological, biochemical and molecular data, JBST22 was identified as Bacillus flexus and the other two were identified as Bacillus subtilis. Furthermore, nucleotide sequences of the 16S rRNA gene of these 3 strains were compared and a phylogenetic tree was constructed. The study reveals that there are at least 66 base differences in the 16S rRNA gene sequences of B. flexus JBST22 and the B. subtilis JBST36.

Diversity of Microflora in Colonic Mucus from Severe Ulcerative Colitis Patients Analyzed by Terminal Restriction Fragment Length Polymorphism and Clone Libraries of Bacterial 16S rRNA Gene Sequences

Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microflora distribution;however, here we analyzed the bacterial diversity in colonic mucus from UC patients receiving colectomy surgery and control patients. The diversity of microflora was investigated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of the 16S rRNA gene sequences. In the T-RFLP analysis, the number of terminal restriction fragments (T-RFs) decreased significantly in UC patients when compared to control samples. Also in the clone library analysis, the number of operational taxonomic units (OTU) and the Shannon diversity index were reduced significantly in UC patients. These molecular analyses reveal an overall dysbiosis in UC patients. No specific pathogen was found, and a strong negative correlation in relative abundance of bacterial populations was observed between the phyla Bacteroidetes and Firmicutes in the UC patients. This is the first report showing a significant correlation between these two phyla, which may be important characteristics in the pathogenesis of UC.

Isolation of Swine Corynebacteria and Analysis of 16S rDNA Gene

A strain of gram-positive bacillus was isolated from suppurative lung organs of nursery pigs in a pig farm, which was further characterized by morphological observation, cultivation test, biochemical test, drug sensitivity test, pathogenicity test and 16S rDNA gene cloning and sequence analysis. The results showed that the isolate grew well in rabbit blood agar plate and horse serum tryptone soybean agar （TSA） plate under aerobic condition, which could lead to mortality of mice and were susceptible to cephalosporin antibiotics and fluoroquinolone antibiotics. Phylogenetic analysis showed that the isolate had close genetic evolutionary relationships with Corynebacterium bacteria, and the sequence of 16S rRNA gene shared the homology of 91.7% -98.3% with the representative strain of coryne- bacteria, indicating the isolated strain was eorynebactefium.

Phylogenetic Relationships among 12 Species of Tetrigidae (Orthoptera:Tetrigoidea) Based on Partial Sequences of 12S and 16S Ribosomal RNA

Mitochondrial 12S and 16S ribosomal RNA genes sequences were sequenced using dye-labeled terminator on an ABI 377 automated sequencer in 11 individuals and 1 species' sequences were gained from GenBank,representing 6 genera of family Tetrigidae.The collated sequences were aligned using Clustal X version 1.81 and then,the sequence variability and heredity distances based on Kimura 2-parameter model were calculated using Mega 2.1.In obtained sequences (736 bp),the average A+T content is 73.9%,ranging from 71.2% to 77.5%;the overall G+C content is 26.1%,ranging from 22.5% to 28.8%.Based on alignment of the combined sequences,185 parsimony-informative sites were revealed in 755 available base pairs.Phylogenetic trees were reconstructed using NJ,MP and ML methods with Cylindraustralia kochii as outgroup.The results indicated that the monophyletic nature of Tetrix is questioned and suggest that T.tubercarina may be given tribal rank.Our results also show that Coptltettix huanjiangensis and C.gongshanensis are the same species,i.e.Coptltettix gongshanensis Zheng,and C.huanjiangensis is the synonyms of C.gongshanensis.

High-throughput sequencing of 16S r RNA amplicons characterizes gut microbiota shift of juvenile sea cucumber Apostichopus japonicus feeding with three antibiotics

Sea cucumber Apostichopus japonicus is an important marine economic species in Asian countries due to its profound nutritional and medicinal value. So far, with the rapid development of intensifi ed artifi cial aquaculture of sea cucumbers, the use of antibiotics is still an inexpensive and dispensable way to treat pathogenic infections, especially during the nursery phase. However, there is little information on the eff ects of antibiotics on the intestinal microbiota of sea cucumber. Therefore an Illumina based sequencing method was used to examine the intestinal bacterial composition of juvenile A . japonicas following diets with three typical antibiotics (tetracycline, erythromycin, and norfl oxacin) under 15, 30, and 45 d. The fi ndings reveal that diff erent antibiotics have distinct eff ects on the growth performance of juvenile sea cucumbers. However, the richness and diversity of microbiota were barely aff ected by antibiotics but the community composition alterations indicated that the three antibiotics exhibited their respective patterns of reshaping the intestinal bacteria of juvenile sea cucumbers. In common, the abundance of some sensitive genera with helpful functions, such as Thalassotalea , Shewanella , Sulfi tobacter , and Halomonas decreased signifi cantly with exposure to antibiotics and the abundance of multiple potential pathogenic- and suspected antibiotic-resistant microorganisms like Arcobacter , Leucothrix , and Clostridium_sensu_stricto_1 was found increased signifi cantly in the antibiotic groups. These results suggest that low doses of antibiotics could aff ect the composition of the intestinal microbiota of sea cucumbers and might increase the risk of infection of the hosts. This study could help us to explore how antibacterial compounds modify the gut microbiota of sea cucumbers and provide theoretical guidance in hatchery management by scientifi c antibiotic use in sea cucumber mariculture.

Species identification and phylogenetic analysis of genus Nassarius(Nassariidae)based on mitochondrial genes

Genus Nassarius contains many subgenera, such as Zeuxis, Telasco, Niotha, Varicinassa, Plicarcularia, Nassarius s. str. and Reticunassa. On the basis of morphological characteristics of the shell and radula and sequences of mitochondrial cytochrome oxidase subunit I (COI) and 16S rRNA genes, Nassarius specimens collected from the South China Sea were identified and phylogenetically analyzed. Although Nassarius sp. and Nassarius (Varicinassa) variciferus were morphologically different in their shells, few variations were found among their radular teeth and sequences of mtCOI and mt16S RNA genes. Therefore, Nassarius sp. should be classified as N. (Varicinassa) variciferus. Nassarius (Zeuxis) sp. has only a subtle difference from Nassarius (Zeuxis) algidus on the shell, but it shows obvious differences in radular teeth and DNA sequence, indicating that they are two distinct species. Sequence divergence of mtCOI and mt16S RNA genes within Nassarius species was much lower than that between species, suggesting that these two genes are suitable for Nassarius species identification. Phylogenetic analysis (neighbor-joining and maximum parsimony) based on mtCOI and mt16S rRNA genes revealed the presence of two groups in genus Nassarius and a closest relationship between subgenera Zeuxis and Telasco. Species of subgenus Plicarcularia did not form a single clade. The molecular phylogeny was not congruent with the previous morphological phylogeny. The subgeneric divisions of genus Nassarius appear to be uncertain and unreliable.

16S rRNA Cloning and Identification of a Strain Isolated from Intestinal Tract of Rex Rabbit

This study was conducted to identify bacterial strain HeNan-001 isolated from intestinal tract of healthy rex rabbit. The strain was identified by gram staining, and OD600 values at different culture time were measured to develop a bacterial growth curve. The metabolic pathways of the bacterium using sugar in fermentation was identified with biochemical tubes. Total RNA of the strain was extracted, and total eDNA was obtained by Oligo （dT） method. Primers were designed using Primer 5.0 hip-software according to the 16S rRNA sequence published in GenBank, through cloning, ligation to vector PMD18T, construction of PMD18T/16S rRNA vector and transformation into DHSα, plasmid was extracted and sequenced, and the sequencing result was compared with sequences in NCBI followed by drawing of phylogenetic tree. The strain was verified to be double-stranded gram-positive cocci, which could ferment glucose, mannose, maltose and sorbitol, producing acids production, but failed to ferment sucrose, serum inulin and β-galactosidase. The phylogenetie tree analysis showed that the isolated bacterium shared the highest homology with an India isolate, and belongs to diplostreptococcus. This study provides significance experimental data and theoretical guidance for further development and utilization of the bacterial strain.

Phylogenetic Analysis of Epibacterial Communities on the Surfaces of Four Red Macroalgae

Macroalgal surfaces are prone to being attached by bacteria. Epibacterial community structures on marine macroalgae are host-specific but temporally and spatially variable. In this study, we investigated the structure of epibacterial communities on the surfaces of four red macroalgae, Gracilaria lemaneiformis, Gloiopeltisfurcata, Mazzaella sp. and Porphyra yezoensis, by analyzing the sequences of 16S rRNA gene libraries. Healthy individuals of all macroalgae species were collected in winter from a farm at Dalian, China. The results showed that the epibacterial communities were mainly dominated by ct-Proteobacteria, 7-Proteobacteria and Bacteroidetes. Deinococcus-Thermus, Spirochaetes and e-Proteobacteria were also found. The majority of cloned sequences shared the greatest similarity to those of culturable organisms. A large portion of sequences from the ct-Proteobacteria homed in Roseobacter clade, i.e., genera Ahrensia, Roseovarius, Litoreibacter, Octadecabacter, Thaiassobacter and Sulfitobacter, while members of Bacteroidetes mainly belonged to family Flavobacteriaceae. The cloned sequences could be separated into 66 OTUs at 0.01 distance value, and rare common OTUs were found among libraries. At genus level, Pseudoa#eromonas dominated Gr. lemaneiformis and GI. furcata libraries, accounting for 72.2% and 47.3%, respectively. Sulfitobacter dominated P. yezoensis library, accounting for 35.4%. A previously undefined cluster within Deinococcus-Thermus dominated Mazzaella sp. library, accounting for 24.6% of the all. These results indicated that a broad range of bacteria inhabited the surfaces of these macroalgae.

基于16S rRNA基因测序探究参地消银颗粒对血热湿滞毒蕴型银屑病患者肠道菌群的影响

目的:基于16S rRNA基因测序技术研究参地消银颗粒对血热湿滞毒蕴型银屑病患者肠道菌群的影响,进一步阐明血热湿滞毒蕴型银屑病患者肠道菌群与健康人群之间的差异。方法:选取符合纳入标准的血热湿滞毒蕴型银屑病患者24例,随机分为患病组、参地消银颗粒治疗组、复方青黛胶囊对照组,同时纳入12名健康志愿者为正常对照组。收集患者入院前及入院8周后的粪便样本以及健康志愿者体检当日的粪便样本,进行16S rRNA V3V4区段的基因测序分析不同菌属的相对丰度;利用QIIME2软件进行操作扩增子序列变异(ASV)的划分,对物种注释及丰度分析;通过α多样性(alpha diversity)和β多样性分析(beta diversity),揭示样本中物种组成和样本间群落结构的差异,通过功能预测分析在KEGG Pathway层面了解群落样本行使的生物学功能;对数据进行独立样本t检验和Mann-Whitney U检验以进行统计分析。结果:与正常对照组比较,患病组的肠道菌群在门水平上拟杆菌门丰度下降,而厚壁菌门丰度则富集;特别是考拉杆菌属的丰度在患病组中降低最明显。给予参地消银颗粒治疗后,显著改善患者的肠道菌群结构,特别是在厚壁菌门和拟杆菌门的比例上显示出较好的调节效果;此外,通过alpha和beta多样性分析,进一步揭示了治疗前后肠道菌群的多样性和群落结构的差异。功能预测分析显示,患病组的肠道菌群功能在核糖体、嘌呤代谢、嘧啶代谢以及氨基糖和核苷酸糖的代谢等生物学通路上与正常对照组存在一定程度差异。结论:参地消银颗粒通过调节肠道菌群的组成,尤其是在提高考拉杆菌属的丰度方面显示出显著效果,有助于恢复血热湿滞毒蕴型银屑病患者的肠道微生态平衡,从而可能影响疾病的进程和治疗效果。

Development of a 16S r RNA gene-based microarray for the detection of marine bacterioplankton community

A better understanding of bacterioplankton community shifts following change in marine environments is critical to predict the marine ecosystem function. In order to get a snapshot of the microbial taxonomy profiling of a wide range marine area, a quick, convenient and low cost method would be favorable. In this study, we developed a 16S rRNA gene-based microarray using ARB software, which contained 447 probes targeting 160 families of marine bacteria. The specificity, sensitivity and quantitative capability of this microarray were assessed by single cloned16S rRNA genes. The reliability of this microarray was tested by eight environmental samples. The results showed that the microarray was specific, only 1.16% false results were detected in five single-clone hybridization tests. The microarray could detect DNA samples as few as 1 ng/μL and the signal intensity could reflect the relative abundance of the bacteria in the range of 1 ng/μL to 100 ng/μL of DNA concentration. Hybridization with environmental samples showed that it can discriminate bacterioplankton communities by sites and time. High throughput sequencing results from the eight samples confirmed the hybridization results. It indicated that this developed microarray could be used as a convenient tool to monitor the bacterioplankton community in marine environment.

猪附红细胞体16S rRNA基因的序列测定和系统进化分析

从确诊为猪附红细胞体感染的猪场,无菌采集血样,抽提猪附红细胞体基因组DNA,采用真细菌的通用引物进行16S rRNA基因扩增,对扩增产物进行克隆和测序。从3 个地理位置不同的猪场均成功地扩增出长度为1 469 bp的核苷酸序列。系统进化分析表明,3个猪场样品所测序列一致性达99 52%以上,具有相同的基因型,但与国外报道的猪附红细胞体Illinois株同源性为95%,属于同一基因群,但基因型不同;所有种类的附红细胞体和血巴尔通氏体组成同一进化分支,这类血营养菌与支原体科,支原体属的病原最靠近(75%),而与立克次氏体目的病原较远(70%)。上述研究证实,广东所流行的猪附红细胞体是一种新基因型的猪附红细胞体,建议命名为猪附红细胞体广东株型;为反映进化关系,猪附红细胞体和其它血营养菌应划归于支原体科的支原体属。

奶牛子宫内膜炎化脓隐秘杆菌16SrRNA基因的鉴定与分析

对内蒙古呼和浩特地区奶牛子宫内膜炎化脓隐秘杆菌进行了分离鉴定。经生化实验鉴定的化脓隐秘杆菌利用PCR技术扩增其16S rRNA基因,得到1.5 kb目的片段;将目的片段与T载体连接,测定目的片段的基因序列,与GenBank公布的化脓隐秘杆菌16S rRNA基因序列进行比较分析,其同源率为93%～100%。本试验共分离到化脓隐秘杆菌32株。

基于线粒体12S和16S rRNA基因部分序列的角蟾亚科部分属种的系统发育关系

测定了角蟾亚科 2属 8种 (亚种 )和外群 3种的线粒体 12S和 16SrRNA基因部分DNA序列 ,比对后序列长共 94 9bp ,其中变异位点数 32 0 ,简约位点数 2 0 6。邻接法和最大简约法分析的系统关系树一致表明内群为一单系群 ,其中腺角蟾首先与其他物种分开 ;沙坪角蟾与宽头短腿蟾聚为一支 ;余下的 5种 (亚种 )角蟾组成一支 ,其中小角蟾短肢亚种的广西种群和香港种群聚为一亚支 ,另一亚支包括峨眉角蟾、小角蟾指名亚种、尾凸角蟾和重庆武隆的角蟾种 ,后两种角蟾进化关系最近。本结果支持短肢角蟾为有效种 ,同时提示腺角蟾、沙坪角蟾与宽头短腿蟾可能隶属 3个不同的亚属或属。