Molecular characterization and identification ofSaprolegnia by restriction analysis of genes coding for ribosomal RNA

Restriction fragment length polymorphisms (RFLPs) in two regions of the ribosomal DNA (rDNA) repeat unit were examined in 33 strains representing 18 species ofSaprolegnia. The Polymerase Chain Reaction (PCR) was used to separately amplify the 18S rDNA and the region spanning the two internal transcribed spacers (ITS) and the 5.8S ribosomal RNA gene. Amplified products were subjected to a battery of restriction endonucleases to generate various fingerprints. The internal transcribed spacer region exhibited more variability than the 18S rDNA and yielded distinctive profiles for most of the species examined. Most of the species showing 100% similarity for the 18S rDNA could be distinguished by 5.8S + ITS restriction polymorphisms except forS. hypogyna, S. delica, S. lapponica, andS. mixta. The rDNA data indicate thatS. lapponica andS. mixta are conspecific withS. ferax, whereas there is no support for the proposed synonymies ofS. diclina withS. delica and ofS. mixta withS. monoica. Results from cluster analysis of the two data sets were very consistent and tree topologies were the same, regardless of the clustering method used. A further examination of multiple strains in theS. diclina-S. parasitica complex showed that restriction profiles are conserved across different strains ofS. parasitica originating from the U.K. and Japan.HhaI andBsaI restriction polymorphisms were observed in isolates from the U.S. and India. The endonucleaseBstUI was diagnostic forS. parasitica, generating identical fingerprints for all strains regardless of host and geographic origin. Except for the atypical strain ATCC 36144, restriction patterns were also largely conserved inS. diclina. Correlation of the rDNA data with morphological and ultrastructural features showed thatS. diclina andS. parasitica are not conspecific. Restriction polymorphisms in PCR-amplified rDNA provide a molecular basis for the classification ofSaprolegnia and will be useful for the identification of strains that fail to produce antheridia and oogonia.     

小鲵科线粒体16S rRNA基因序列分析及其系统发育

To study the phylogeny of Hynobiidae, we amplified DNA fragments of 470 bp 16S ribosomal RNA (16S rRNA) gene on mitochondrial DNA from Ranodon sibiricus and Ranodon tsinpaensis. PCR products were cloned into PMD18 T vector after purification. These sequences were determined and deposited in the GenBank (accession numbers: AY373459 for Ranodon sibiricus, AY372534 for Ranodon tsinpaensis). By comparing the nucleotide differences of 16S ribosomal RNA sequences among Liua shihi, Pseudohynobius flavomaculatus and Batrachuperus genus from GenBank database, we analyzed the divergences and base substitution among these sequences with the MEGA software. The molecular results support that B. tibetanus, B. pinchonii and B. karlschmidti are classified into three valid species. Liua shihi has closer phylogenetic relationships to Ranodon tsinpaensis than to other species. More our results reveal that Pseudohynobius flavomaculatus is not a synonym of Ranodon tsinpaensis. [Acta Zoologica Sinica 50 (3) : 464 - 469,2004].     

Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes

Lobose amoebae are abundant free-living protists and important pathogenic agents, yet their evolutionary history and position in the universal tree of life are poorly known. Molecular data for lobose amoebae are limited to a few species, and all phylogenetic studies published so far lacked representatives of many of their taxonomic groups. Here we analyze actin and small-subunit ribosomal RNA (SSU rRNA) gene sequences of a broad taxon sampling of naked, lobose amoebae. Our results support the existence of a monophyletic Amoebozoa clade, which comprises all lobose amoebae examined so far, the amitochondriate pelobionts and entamoebids, and the slime molds. Both actin and SSU rRNA phylogenies distinguish two well-defined clades of amoebae, the "Gymnamoebia sensu stricto" and the Archamoebae (pelobionts + entamoebids), and one weakly supported and ill-resolved group comprising some naked, lobose amoebae and the Mycetozoa.     

Phylogenetic relationships amongHomo sapiens and related species based on restriction site variations in rDNA spacers

A rapid method, using 12 restriction enzymes, was employed to analyze variations in ribosomal DNA (rDNA) spacers in a study of phylogenetic relationships betweenHomo sapiens and related species. We mapped restriction sites in the external and internal spacer regions and compared the arrangements of sites. The estimated sequence divergence betweenHomo sapiens andPan troglodytes, Pan paniscus, Gorilla gorilla, Pongo pygmaeus, Hylobates lar, H. agilis, andMacaca fuscata was 2.7, 2.3, 3.8, 7.3, 6.8, 7.8, and 14.1%, respectively. The genetic relationships inferred from these distances generally correspond to those inferred from analyses of other molecular markers in the literature. The divergence betweenH. lar andH. agilis and betweenH. lar andH. syndactylus was 0.34 and 2.4%, respectively.This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and also by the Cooperative Research Program of the Primate Research Institute, Kyoto University.     

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.     

基于18S rRNA和线粒体16S rRNA基因序列的柳蚕进化分析

为探讨柳蚕Actias selene Hübner与鳞翅目昆虫的系统发育关系,本研究利用PCR扩增获得了柳蚕核糖体18S rRNA和线粒体16S rRNA基因的部分序列,长度分别为391bp和428bp。并采用邻近距离法(NJ)、最大简约法(MP)、类平均聚类法(UPGMA)构建系统进化树。结果表明,柳蚕线粒体16SrRNA基因序列与大蚕蛾科昆虫的16SrRNA基因序列均表现出偏好于碱基AT的倾向。柳蚕与所研究的其它蚕类的遗传距离介于0.016至0.140之间,其中与温带柞蚕Antheraea roylii的遗传距离最小,与野桑蚕Bombyx mandarina的遗传距离最大。而基于鳞翅目昆虫18S rRNA基因部分序列的进化分析显示,柳蚕与柞蚕Antheraea pernyi之间的遗传距离最小(0.010),与蓖麻蚕Samia ricini的遗传距离最大(0.017)。     

Specialized avian predators repeatedly attack novel color morphs of Heliconius butterflies

The persistence of Müllerian mimicry and geographically distinct wing patterns, as observed in many Heliconius species (Lepidoptera: Nymphalidae), is difficult to explain from a predator's perspective: predator selection against locally rare patterns must persist despite avoidance learning. Maintaining spatial color-pattern polymorphism requires local pattern avoidance, fine-scale discrimination among similar wing patterns, and repeated attacks on novel color patterns. I tested for these behaviors by presenting 80 adult rufous-tailed jacamars (Galbula ruficauda) with three morphs of Heliconius butterflies, and then presenting the same suite of butterflies to 46 of these jacamars between four and 429 days later. These trials offer the first direct evidence of the selective predator behavior required to maintain aposematic polymorphism: jacamars avoid local aposematic morphs while repeatedly attacking similar but novel morphs over time.     

Cloning and restriction analysis of ribosomal RNA genes from Mycobacterium smegmatis

A genomic library of Mycobacterium smegmatis DNA was constructed in phage EMBL3. A clone (gamma HB85) containing rRNA genes was isolated using as probes, fragments of E. coli rRNA cistron B. This cloned DNA fragment was mapped by restriction analysis and was shown to contain one complete set of rRNA genes (rRNA A). The physical mapping of the second set of rRNA genes of M. smegmatis (rRNA B) was done by restriction analysis of total chromosomal DNA. The two sets of rRNA genes showed highly conserved restriction sites within the respective sets but not in the flanking regions. The two rRNA sets of genes are organised like in the other eubacteria in the order 16S-23S-5S.     

Phylogenetic analysis of the genera Pseudonocardia and Actinobispora based on 16S ribosomal DNA sequences

The 16S rDNA sequences of 11 strains, nine type strains of validated Pseudonocardia species and Actinobispora yunnanensis, and two strains of unnamed Pseudonocardia species, were determined and compared with those of representatives of the family Pseudonocardiaceae. The phylogenetic analysis indicated that all of the validated species of the genera Pseudonocardia and Actinobispora consistently formed a monophyletic unit and separated well from the other genera of the family Pseudonocardiaceae. One unnamed Pseudonocardia strain was related to members of the genus Pseudonocardia, whereas the other unnamed Pseudonocardia strain formed a distinct clade within the radiation of the genus Amycolatopsis.     

Effect of restriction endonucleases on assessment of biodiversity of cultivable polar marine planktonic bacteria by amplified ribosomal DNA restriction analysis

To choose a suitable restriction endonuclease for quick assessment of bacterial diversity in polar environments by ARDRA, we investigated the effect of restriction enzymes on ARDRA patterns of cultivable marine planktonic bacteria isolated from polar region. Thirty-three isolates were analyzed by ARDRA using five enzymes (HinfI, HaeIII, AluI, and the mix AfaI/MspI), respectively, resulting in different groups, each group corresponding to a particular genotype. A comparison of the ARDRA patterns was carried out, and phylogenetic position of all thirty-three bacteria was obtained by 16S rDNA sequencing. Consistent with phylogenetic analysis, ARDRA pattern comparison revealed that AluI, being sensitive and reliable enough to generate species-specific patterns, was a suitable restriction enzyme used for evaluating bacterial diversity, suggesting a combination of ARDRA with AluI and 16S rDNA sequencing can provide a simple, fast and reliable means for bacterial identification and diversity assessment in polar environments.     

Chloroplast DNA restriction site study of Verbesina (Asteraceae: Heliantheae)

Chloroplast DNA variation was examined for 79 species of Verbesina and 24 outgroups. Two independent analyses of the data were performed. An intergeneric study to elucidate the phylogenetic relationships of Verbesina used 22 genera, most of which are regarded in the literature as closely related to Verbesina. Coreopsis and Hymenoxys served as outgroups for this analysis. The 16 6-bp (base pair) restriction endonucleases used in the intergeneric study revealed 263 phylogenetically informative sites. Wagner analyses of these characters resulted in four equally parsimonious trees with a length of 857 steps and a Consistency Index of 0.492. Results from this study indicate that Verbesina is monophyletic, a member of the tribe Heliantheae, and that its sister taxa are the mostly Mexican genera Podachaenium, Squamopappus, and Tetrachyron. The infrageneric study of Verbesina included species belonging to all of its infrageneric taxa, except the monotypic sect. Stenocarpha. The 17 6-bp restriction endonucleases used in the infrageneric study revealed 137 sites 77 of which were phylogenetically informative. Wagner analyses of these characters generated 180 equally parsimonious trees with a length of 158 steps and a Consistency Index of 0.786. The genera Podachaenium, Squamopappus, and Tetrachyron served as outgroups. Two major clades, which correspond to traditional divisions of the genus based on leaf arrangement, are supported by the study. No support was found for the monophyly of seven of the 11 sections examined; further sampling of sections Lipactinia, Ochractinia, Verbesina, and Ximenesia is needed. Results support a major reassessment of the traditional infrageneric classification of the genus and provide the basis for the reevaluation of the sectional taxonomy of Verbesina. These studies support a North American origin for the genus with several independent introductions into South America producing significant diversity, especially in the Andean region.     

Phylogenetic analysis of pines using ribosomal DNA restriction fragment length polymorphisms

Phylogenetic relationships among 30 species of the genusPinus were studied using restriction site polymorphism in the large subunit of nuclear rDNA. Of the 58 restriction sites scored, 48 were phylogenetically informative, and the 30 species reduced to ten taxa when species with identical restriction site patterns were combined. These ten taxa corresponded to the currently recognized subsections of the genus, with the sole exception ofP. leiophylla, which was identical in its pattern of restriction sites to all three species included from subsect.Oocarpae despite its being in a different section of subg.Pinus (Pinea instead ofPinus). A measure of the proportion of phylogenetic information contained within the data set (Homoplasy Excess Ratio, or HER) revealed that the character states were significantly non-randomly distributed among the ten taxa (HER = 0.71, p < 0.01). Branchand-bound searches using either Wagner or Dollo parsimony as the optimization criterion were carried out using PAUP in order to estimate phylogenetic relationships among the ten taxa. Three taxa (Picea pungens, Tsuga canadensis, andLarix decidua) were used independently as outgroups for purposes of rooting the trees. Despite the extreme differences in the assumptions underlying the Wagner and Dollo parsimony, the two gave surprisingly similar estimates of phylogeny, with both analyses supporting the monophyly of the two major subgeneraPinus andStrobus and differing in topology only in the placement of subsect.Ponderosae within subg.Pinus. The likelihood for the Wagner tree was only slightly higher than that computed for the Dollo tree.     

Structure and variation in ribosomal RNA genes of pea

Summary A complete ribosomal DNA (rDNA) repeat unit has been cloned from the genome of Pisum sativum (garden pea) and used to construct a map containing a total of 58 cleavage sites for 23 different restriction enzymes. Regions encoding 18s and 25s ribosomal RNA (rRNA) were identified by R-loop analysis. A 180 bp sequence element is repeated eight times in the intergenic nontranscribed spacer (NTS) region, as defined by eight evenly spaced RsaI cleavage sites. Sequence heterogeneity among these elements (subrepeats) is indicated by the presence of an NcoI site within the five RsaI subrepeats distal to the 25s rRNA gene but not in the three subrepeats proximal to this gene, and also by the presence of an additional RsaI cleavage site in one subrepeat.The approximately 4000 copies of the rDNA repeat in the pea nuclear genome show considerable heterogeneity with respect to the length of the NTS region, and differences are also frequently observed between different genotypes. In both cases the length variation appears to be due primarily to differences in the number of subrepeat elements.Comparison of rDNA restriction maps for two pea genotypes separated for hundreds or perhaps thousands of generations reveals that they contain many rDNA identical repeat units. This data is consistent with the view that new rDNA variants are fixed only infrequently in the evolution of a species.Differences also exist between the rDNA repeats of a single genotype with respect to the degree of base modification at certain restriction sites. A large number of sites known to exist in the pea rDNA clone are not cleaved at all in genomic rDNA, or are cleaved in only some copies of the rDNA repeat. We believe these examples of incomplete cleavage results mostly from methylation, although it is difficult to rule out the possibility of sequence variation in all cases. Most putative modifications are best interpreted in terms of cytosine methylation in CG and CXG sequences, but at least one example is more consistent with adenine methylation.We also have constructed a more detailed restriction map of the wheat rDNA clone pTA71 and present a comparison of this map to our map of pea, pumpkin, and wheat in order to assess the amount of useful evolutionary information that can be obtained by comparison of such maps.     

Differentiation of restriction sites in ribosomal DNA in the genusApodemus

Southern blot analysis of ribosomal DNA (rDNA) from seven species ofApodemus was carried out in order to examine the genetic relationships between the species. Analysis of heterogeneity in rDNA spacers inA. sylvaticus, A. flavicollis, A. semotus, A. agrarius, A. argenteus, A. speciosus, andA. peninsulae, using 13 different restriction enzymes and cloned mouse rDNA probes, revealed that the families of rDNA in these species can be characterized by restriction maps which show the major constituents of rDNA repeating units (repetypes). Based on differences in the arrangement of restriction sites, sequence divergence among the different major repetypes was estimated. Among the seven species ofApodemus examined, the major repetypes ofA. flavicollis andA. sylvaticus were the most closely related, having only 1.0% sequence divergence. These repetypes and those of the remaining five species differ substantially from one another, with 4.3–8.5% divergence.This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.