Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences.

Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis and feminization. While it is known that the bacterial strains responsible for these different host phenotypes form a single clade within the alpha-Proteobacteria, until now it has not been possible to resolve the evolutionary relationships between different Wolbachia strains. To address this issue we have cloned and sequenced a gene encoding a surface protein of Wolbachia (wsp) from a representative sample of 28 Wolbachia strains. The sequences from this gene were highly variable and could be used to resolve the phylogenetic relationships of different Wolbachia strains. Based on the sequence of the wsp gene from different Wolbachia isolates we propose that the Wolbachia pipientis clade be initially divided into 12 groups. As more sequence information becomes available we expect the number of such groups to increase. In addition, we present a method of Wolbachia classification based on the use of group-specific wsp polymerase chain reaction (PGR) primers which will allow Wolbachia isolates to be typed without the need to clone and sequence individual Wolbachia genes. This system should facilitate future studies investigating the distribution and biology of Wolbachia strains from large samples of different host species.     

Molecular phylogenetic study on the origin and evolution of Mustelidae

The family Mustelidae, which consists of Mustelinae, Lutrinae, Melinae, and Taxidiinae, is the largest family among Carnivora and is a highly diverse group. Recent molecular phylogenetic studies have clarified the phylogenetic relations among Mustelidae, but there remain several unresolved problems, particularly concerning the deep branchings. Whereas many studies support the monophyly of Mustelidae+Procyonidae among Musteloidea, the relations between Mustelidae+Procyonidae, Ailuridae, and Miphitidae are still unclear. To address these problems, we inferred a tree on the basis of the sequences of mitochondrial genomes and of multiple nuclear genes using the maximum likelihood method. Our results strongly support the hypothesis that the Taxidiinae branched at first, followed by the branching of the Melinae. After that, Mustelinae diversified, and Lutrinae evolved within Mustelinae. With respect to the deep branchings in Musteloidea, the Ailuridae/Mephitidae monophyly tree and the Mephitidae-basal tree are indistinguishable in log-likelihood score, and this problem remains unresolved.     

四种瘿蜂体内Wolbachia感染的PCR检测及感染株系的wsp基因序列分析

Wolbachia为节肢动物等的细胞质共生细菌,能对宿主的繁殖模式进行调控,包括诱导胞质不亲和、孤雌生殖、雌性化及雄性致死。本文采集了分布于美国的4种瘿蜂,利用Wolbachia的wsp基因特异性引物,对其Wolbachia的感染进行了PCR检测,证实了栎结瘤瘿蜂Callirhytis punctata Bassett和摇鼓栎瘿蜂Dryocosmus palustris Osten Sacken体内具Wolbachia共生,感染率分别为60%和36%。栎结瘤瘿蜂和摇鼓栎瘿蜂Wolbachia的wsp基因序列长度分别为564 bp和561 bp。栎结瘤瘿蜂与摇鼓栎瘿蜂Wolbachia的wsp基因序列的一致性为94%。栎结瘤瘿蜂与同为栎瘿蜂族的Andricus solitarius(strain 1)和Neuroterus macropterus,及客瘿蜂族的Synergus crassicorni的Wolbachia的wsp基因序列完全一致,与其他瘿蜂Wolbachia的wsp基因的序列一致性介于79%⁹9%之间。在NJ系统发育树中,栎结瘤瘿蜂与栎瘿蜂族的A.solitaries(strain 1),N.macropterus和B.pallida,以及客瘿蜂族的S.crassicornis的Wolbachia同属一分支,而摇鼓栎瘿蜂与栎瘿蜂族的麦氏安瘿蜂的Wolbachia聚集在同一分支。除客瘿蜂族的Ceroptres cerri感染的Wolbachia属于B群之外,其他瘿蜂感染的Wolbachia均属于A群。此外,本文采集的栎结瘤瘿蜂和摇鼓栎瘿蜂营有性生殖,说明Wolbachia的共生并不诱导其营产雌孤雌生殖。     

Non-concerted ITS evolution, early origin and phylogenetic utility of ITS pseudogenes in Pyrus

Molecular studies of 19 species of the genus Pyrus L. revealed different degrees of intra-individual polymorphism of the internal transcribed spacer (ITS 1, 5.8S rDNA and ITS 2) region due to the existence of putative non-functional copies (pseudogenes), putative recombinants and non-concerted evolution among functional copies. Different types of ITS pseudogenes displaying lower GC content and unstable secondary structure were preferentially amplified under normal PCR conditions. Functional ITS copies were successfully obtained in all investigated accessions under the modified PCR conditions. All pseudogenes were highly divergent from their corresponding functional copies and formed a monophyletic group in the phylogenetic tree based on all paralogs, indicating they were of relatively early origin. Functional ITS copies led to confused and poorly resolved phylogeny as a result of low sequence divergence, existence of unidentified ancient recombinants and a high degree of intra-individual functional ITS polymorphism, while certain types of pseudogenes and some relict pseudogenes offered more credible clues for the evolutionary history of Pyrus species.     

Molecular phylogenetics of soricid shrews (Mammalia) based on mitochondrial cytochrome b gene sequences: with special reference to the Soricinae

Molecular phylogeny of soricid shrews (Soricidae, Eulipotyphla, Mammalia) based on 1140 bp mitochondrial cytochrome b gene (cyt b ) sequences was inferred by the maximum likelihood (ML) method. All 13 genera of extant Soricinae and two genera of Crocidurinae were included in the analyses. Anourosorex was phylogenetically distant from the main groupings within Soricinae and Crocidurinae in the ML tree. Thus, it could not be determined to which subfamily Anourosorex should be assigned: Soricinae, Crocidurinae or a new subfamily. Soricinae (excluding Anourosorex ) should be divided into four tribes: Neomyini, Notiosoricini, Soricini and Blarinini. However, monophyly of Blarinini was not robust in the present data set. Also, branching orders among tribes of Soricinae and those among genera of Neomyini could not be determined because of insufficient phylogenetic information of the cyt b sequences. For water shrews of Neomyini ( Chimarrogale , Nectogale and Neomys ), monophyly of Neomys and the Chimarrogale – Nectogale group could not be verified, which implies the possibility of multiple origins for the semi-aquatic mode of living among taxa within Neomyini. Episoriculus may contain several separate genera. Blarinella was included in Blarinini not Soricini, based on the cyt b sequences, but the confidence level was rather low; hence more phylogenetic information is needed to determine its phylogenetic position. Furthermore, some specific problems of taxonomy of soricid shrews were clarified, for example phylogeny of local populations of Notiosorex crawfordi , Chimarrogale himalayica and Crocidura attenuata .     

基于wsp基因的叶螨体内Wolbachia株系的多样性与重组分析

胞内共生菌Wolbachia能对多种叶螨产生生殖调控作用.为更好地筛选有潜在应用价值的Wolbachia株系,本研究应用PCR技术对自然种群的截形叶螨Tetranychus truncatus、二斑叶螨T.urticae、神泽叶螨T.kanzawai 和山楂叶螨Amphitetranychus viennensis体内的Wolbachia感染情况进行检测,并对Wolbachia的wsp基因进行序列分析和基因重组检测.结果表明,叶螨中的Wolbachia株系具有较高的遗传多样性,其中截形叶螨感染两种分化较大的株系.不同叶螨感染特有的Wolbachia株系说明Wolbachia与其宿主存在一定的协同进化关系.二斑叶螨和截形叶螨感染同一株系的Wolbachia,可能由于水平传播造成.同时,不同株系Wolbachia的wsp基因间普遍存在着基因重组现象.     

Assessing the phylogenetic utility of sequence heterochrony: evolution of avian ossification sequences as a case study

The evolution of developmental sequences, or sequence heterochrony, is an emerging field of study that addresses the temporal interplay between evolution and development. Some phylogenetic signal has been found in developmental sequence data, but sampling has generally been limited to small numbers of taxa and few developmental events. Here we present the largest ossification sequence dataset to date. The sequences are composed of ossification events throughout the avian skeleton, and are used to address the evolutionary signal of ossification sequence data within this clade. The results indicate that ossification sequences are conserved in birds, and show a stronger phylogenetic signal than previous studies, perhaps due to the volume of data. Phylogenetic signal is not strong enough, however, to consider ossification sequence data to be any better at resolving phylogenetic hypotheses than other morphological data and just as prone to evolutionary convergence. There is no one-to-one correlation between ossification sequence and developmental stage. We discuss some methodological implications of our findings, as well as commonalities in avian ossification sequences such as early ossification of the long bones relative to the dermatocranium, and of the hindlimb over the forelimb.     

Mitochondrial cytochrome b gene sequences of old world monkeys: With special reference on evolution of Asian colobines

The classification and phylogenetic relationships of the Old World monkeys are still controversial. For Asian colobines, from three to nine genera were recognized by different primatologists. In the present study, we have sequenced a 424 bp mitochondrial tRNA^Thr gene and cytochrome b gene fragment fromMacaca mulatta, Mandrillus sphinx, Mandrillus leucophaeus, Semnopithecus entellus, Trachypithecus vetulus, T. johnii, T. phayrei, T. francoisi, Pygathrix nemaeus, Rhinopithecus roxellanae, R. bieti, R. avunculus, Nasalis larvatus, andColobus polykomos in order to gain independent information on the classification and phylogenetic relationships of those species. Phylogenetic trees were constructed with parsimony analysis by weighting transversions 5 or 10 fold greater than transitions. Our results support the following conclusions: (1) the Old World monkeys are divided into two subfamilies; (2) that among the colobines,Colobus, the African group, diverged first, andNasalis andRhinopithecus form a sister clade toPygathrix; (3) that there are two clades within leaf monkeys, i.e. 1)S. entellus, T. johnii, andT. vetulus, and 2)T. phayrei andT. francoisi; (4) thatRhinopithecus avunculus, R. roxellanae, andR. bieti are closely related to each other, and they should be placed into the same subgenus; (5) thatRhinopithecus is a distinct genus; and (6) that the ancestors of Asian colobines migrated from Africa to Asia during the late Pliocene or early Pleistocene.     

Molecular phylogeny and evolution of subsection Magnicellulatae (Erysiphaceae: Podosphaera) with special reference to host plants

The subsection Magnicellulatae of the genus Podosphaera section Sphaerotheca belongs to the tribe Cystotheceae of the Erysiphaceae, which has the characteristic of producing catenate conidia with distinct fibrosin bodies. In this study, we newly determined the nucleotide sequences of the D1/D2 domains of the 28S rDNA region and the sequences of the rDNA internal transcribed spacer (ITS) region to investigate the relationships between the phylogeny of this fungal group and their host plants. The results indicated that the 28S rDNA region is too conservative for phylogenetic analysis of this fungal group. The phylogenetic analysis using 95 ITS sequences demonstrated that two or more Magnicellulatae taxa often infect the same plant genus or species. Although there is a close relationship between Magnicellulatae and asteraceous hosts, this association seems to be not as strict as that between Golovinomyces and the Asteraceae. The difference between the two fungal groups may be explained by their different evolutionary timing.     

Kinetoplastid phylogenetics, with special reference to the evolution of parasitic trypanosomes

To fully understand the evolutionary history of parasitic kinetoplastids and to understand the context within which the evolution of each parasite group has developed, an understanding not just of the parasites, but of all kinetoplastids is required. Accordingly, this paper provides an overview of kinetoplastid evolution and systematics, including coverage of the proposal by Moreira et al. (2004) to divide kinetoplasts into Prokinetoplastina (Ichthyobodo and Perkinsiella) and Metakinetoplastina (other bodonids and trypanosomatids). The implications of such a revision, with regard to correctly identifying outgroup taxa for studies of evolution within taxa of medical importance, are addressed, together with a more detailed review of the evolution and origins of the trypanosomes in the light of new phylogenies, new approaches and revisions in kinetoplastid systematics.     

Molecular phylogeny of the genus Pieris (Ericaceae) with special reference to phylogenetic relationships of insular plants on the Ryukyu Islands

Phylogenetic relationships within the genus Pieris (Ericaceae) were investigated based on the rbcL and matK genes along with five spacer sequences of chloroplast DNA to address questions regarding the phylogeography of the genus in association with insular plants on the Ryukyu Islands. The most parsimonious trees indicated that P. floribunda from eastern North America is a sister taxon to the remaining taxa examined, and suggested that the East Asian taxa examined are monophyletic. A morphologically cohesive group, section Pieris, was revealed to be paraphyletic. Within the East Asian clade, insular endemics from the Ryukyu Islands, Taiwan, and mainland Japan formed a sister group to P. formosa from the Himalayas and southern China. Our data suggest that the insular endemics of the Ryukyu Islands and Taiwan arose via allopatric divergence as a result of a paleogeographical land configuration of a landbridge during the early–middle Pleistocene in the Quaternary Period.     

The evolution of proteinase substrates with special reference to dipeptidylpeptidase IV

Summary The design and development of specific substrates for proteolytic enzymes is reviewed. Particular attention is given to substrates containing the leaving groups 4-methoxy-2-naphthylamide (MNA) and 7-amino-4-trifluoromethylcoumarin (AFC). The MNA substrates are used for histochemical and cytochemical purposes, and they yield a coloured final reaction product when azo-coupled with a diazonium salt, an osmiophilic product for electron microscopy when coupled with hexazotized Pararosaniline, or a fluorescent final reaction product when coupled with 5-nitrosalicylaldehyde. AFC substrates are considerably more sensitive, and they yield the fluorescent product AFC after enzymatic cleavage of the substrate. AFC is not sufficiently water-insoluble to allow (intra)cellular localization, but AFC substrates are successfully used for incubations in microwells (Immu-Probe technique) and for the demonstration of banding patterns after gel electrophoresis (enzyme-directed overlay membrane technique). The methods are discussed with the example of the elucidation of the role of dipeptidylpeptidase IV in autoimmune diseases.As presented by R.E.S. for the Pearse Prize Lecture at the Annual Histochemistry Meeting of the Royal Microscopical Society on 8 January 1991 in London, UK. The oral presentation appeared in written form inProc. Roy. Microsc. Soc.26, 135–43 (1991).     

Molecular evolution and phylogenetic utility of the petD group II intron: a case study in basal angiosperms

Sequences of spacers and group I introns in plant chloroplast genomes have recently been shown to be very effective in phylogenetic reconstruction at higher taxonomic levels and not only for inferring relationships among species. Group II introns, being more frequent in those genomes than group I introns, may be further promising markers. Because group II introns are structurally constrained, we assumed that sequences of a group II intron should be alignable across seed plants. We designed universal amplification primers for the petD intron and sequenced this intron in a representative selection of 47 angiosperms and three gymnosperms. Our sampling of taxa is the most representative of major seed plant lineages to date for group II introns. Through differential analysis of structural partitions, we studied patterns of molecular evolution and their contribution to phylogenetic signal. Nonpairing stretches (loops, bulges, and interhelical nucleotides) were considerably more variable in both substitutions and indels than in helical elements. Differences among the domains are basically a function of their structural composition. After the exclusion of four mutational hotspots accounting for less than 18% of sequence length, which are located in loops of domains I and IV, all sequences could be aligned unambiguously across seed plants. Microstructural changes predominantly occurred in loop regions and are mostly simple sequence repeats. An indel matrix comprising 241 characters revealed microstructural changes to be of lower homoplasy than are substitutions. In showing Amborella first branching and providing support for a magnoliid clade through a synapomorphic indel, the petD data set proved effective in testing between alternative hypotheses on the basal nodes of the angiosperm tree. Within angiosperms, group II introns offer phylogenetic signal that is intermediate in information content between that of spacers and group I introns on the one hand and coding sequences on the other.