Expression of major piroplasm protein (p33) of Theileria sergenti (Korean isolate) and its immunogenicity in guinea pigs

To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein band of p33. The antigenicity of expressed polypeptide was further examined through the inoculation of a guinea pig. The sera of guinea pigs immunized with p33 expressed cell lysate showed similar fluorescent antibody patterns and reacted with the same molecular weight protein of T. sergenti in immunoblotting analysis, thus indicating that this protein can be a promising candidate for a subunit vaccine in the future.     

Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene

Theileria sergenti is a tick-borne parasite found in many parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a marker for epidemiological and phylogenetic studies of benign Theileria species. In this study, Chinese species of T. sergenti were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the MPSP gene. Using universal or allele-specific primer sets for PCR amplification of the MPSP gene, 98 of 288 cattle blood samples, collected from 6 provinces in China, were found to be positive. Among the positive samples, only 3 allelic MPSP gene types (Chitose [C]-, Ikeda [I]-, and buffeli [B]-type) were successfully amplified. Moreover, the results revealed that the majority of the parasites sampled in this study were C- and I-type (prevalence of 84 and 69%, respectively), whereas the B-type was less common (prevalence of 36%). Co-infections with C-, I-, and B-type T. sergenti also were found. An additional known allele, Thai-type, was not detected. Phylogenetic analysis based on the MPSP gene sequences, including 3 standard stocks generated in the laboratory ( T. sergenti Wenchuan, T. sergenti Ningxian, and T. sergenti Liaoyang), revealed that the isolates of Chinese sergenti were comprised of at least 4 allelic MPSP gene types, i.e., C-, I-, B1-, and B2-type, and these parasites with 6 MPSP types 1-5 and 7 were present in China.     

Phylogenetic relationships of 34 passerines based on mitochondrial Cytochrome b sequences

Phylogenetic relationships of 34 passerines were studied based on mitochondrial Cytochrome b (Cyt b) sequences. Phylogenetic trees were constructed using Neighbor-Joining, Maximum-Parsimony and Minimum evolution methods. The results show that the divergence between Fringillids and Emberizids reaches a family level and they should be grouped into family Fringillidae and Emberizidae, respectively; Accentors has a relatively close relationship with Fringillids and Emberizids; the divergence between robins and flycatchers does not reach a family level and they should be member to family Muscicapidae; long-tailed tits and sylviids should all be listed into families; barn swallow, crowtits and long-tailed tits have close relationships with Sylviidae; in the Fringillidae, brambling should be member to one subfamily Fringillinae, several other birds under the subfamily Carduelinae; in the Sylviidae, although lanceolated warblers and scaly-headed stubtails have a relatively far relationship, they should be member to one subfamily Acrocephalinae and warblers to Phylloscopinae. Muscicapidae, Fringillidae and Emberidae are all monophylies, but Sylviidae is not. The substitution rates of major clades are thought to be the same according to relative rate tests. Divergence time of major clades is estimated at the rate of 1.6% per million years, thus the estimated divergence time between Fringillidae and Emberizidae is 10.5 million years, robins and flycatchers 9.0 Myr, Acrocephalinae and Phylloscopinae 9.0 Myr, Carduelis flammea and Carpodacus erythrinus 7.5 Myr, Luscinia cyane and Tarsiger cyanurus 7.8 Myr, two outgroups 13.5 Myr.     

Phylogenetic relationships among the black fly species (Diptera: Simuliidae) of Thailand based on multiple gene sequences

Simulium is a very speciose genus of the black fly family Simuliidae that includes many important pests of humans and animals. Cytotaxonomic and morphological studies have made substantial progress in Simulium systematics. 16S rRNA and ITS-1 DNA sequence studies have assisted this progress. Intensive multi-gene molecular systematic investigations will, however, be required for a comprehensive understanding of the genus’ taxonomy and evolution. Our research was conducted to investigate the relationships of Thai Simulium at the subgeneric, species group and species levels. We also examined the possibility of using mitochondrial DNA sequences to facilitate Simulium species identification. Data were collected from three mitochondrial genes (COI, ND4 and 16S rRNA) and two segments of the nuclear 28S ribosomal RNA (the D1 to D2 and the D4 expansion regions). The subgenera Simulium and Gomphostilbia were monophyletic in most analyses. Nevermannia included Montisimulium but was otherwise monophyletic in multigene analyses. In most analyses, Simulium and Nevermannia were more closely related to each other than to Gomphostilbia which was usually basal. Species groups were generally monophyletic. Within Gomphostilbia, however, the batoense species group was always paraphyletic to the other two species groups found in Thailand. Three species groups in Simulium were not monophyletic. The tendency to gill filament number reduction for some species groups in the subgenus Simulium was associated with a derived position in multigene analyses. Most species were monophyletic with two exceptions that probably represent species complexes and will present difficulties for rapid mitochondrial DNA identification.     

Detecting and differentiating Theileria sergenti and Theileria sinensis in cattle and yaks by PCR based on major piroplasm surface protein (MPSP)

Theileria sergenti and Theileria sinensis are closely related members of benign Theileria species found in cattle and yaks in China. They are morphologically indistinguishable. A polymerase chain reaction (PCR) targeting major piroplasm surface protein of T. sergenti and T. sinensis was developed in this study. The newly developed oligonucleotide primer set was able to specifically amplify the DNA of T. sinensis and in conjunction with primers for T. sergenti and these two species could be detected and distinguished. Specificity testing also revealed that there was no cross-reaction with the other tick-borne diseases Theileria annulata, Babesia ovata, Anaplasma marginale as well as bovine white blood cells. Phylogenetic analysis based on the MPSP gene sequences confirmed the specificity of PCR assays. The sensitivity of the methods was 0.1 pg DNA for the T. sergenti PCR and 1 pg DNA for T. sinensis PCR. Two hundred and thirty-six field blood samples from of cattle and yaks were collected from five different geographical regions in China where benign Theileria species have been found. T. sergenti was found in all five provinces but was absent from one county in Gansu Province. T. sinensis was only found in Gansu Province. In both counties in Gansu where the parasites co-existed, mixed infections were detected. Our results indicate that the PCR methods developed in this study are suitable for the detection and differentiation of T. sergenti and T. sinensis.     

Phylogenetic relationships in Gleditsia (Leguminosae) based on ITS sequences

We used nucleotide sequences from the internal transcribed spacers and 5.8S gene of nuclear ribosomal DNA to test competing phylogenetic and biogeographic hypotheses in Gleditsia. Eleven of 13 Gleditsia species were sampled, along with two species of its sister genus, Gymnocladus. Analyses of ITS data and of a combined data set that included sequences of ITS and two chloroplast genes supported several conclusions that were interpreted in light of fossil data and current legume phylogeny. Gleditsia and Gymnocladus appear to have originated in eastern Asia during the Eocene. Eastern North American species of both genera most likely evolved from ancestors that migrated across the Bering land bridge, but the eastern Asian/eastern North American disjunction appears to be much older in Gymnocladus than in Gleditsia. Gleditsia amorphoides, from temperate South America, is sister to the rest of the genus, suggesting early long-distance dispersal from Asia. The remainder of Gleditsia is divided into three unresolved clades, possibly indicating a split early in the evolution of the genus. Two of those clades contain only Asian species, and one contains Asian and North American species. The North American species, Gleditsia triacanthos and Gleditsia aquatica, are polymorphic and paraphyletic with respect to their ITS and cpDNA sequences, which suggests recent diversification.     

基于Cytb基因序列探讨蝽亚科11种昆虫的系统发育关系

对蝽亚科6属11种昆虫线粒体DNA细胞色素b基因部分序列进行PCR扩增和序列测定。比较其同源性,统计密码子使用频率并应用生物学软件构建分子系统树。在获得的432bp序列中,碱基T,C,A和G的平均含量分别是38．1,18．2,31．9和11．8％,表现出强烈的AT偏向性;就每个氨基酸密码子来看,第3位点的A＋T含量较高,达到85．5％。该序列片段中共有162个核甘酸位点发生变异（约占37．5％）,种间序列差异范围为0．9％～19．4％,平均为16．6％,变异较大。以筛豆龟蝽Megaeopta cribraria为外群,通过多种方法构建的系统发育树拓扑结构一致,这6属11种昆虫大致形成4个分支：珀蝽属、碧蝽属、菜蝽属3属的关系最接近,形成一个分支;真蝽属的3种聚为1支,与第1支形成姊妹群;曼蝽属的2种形成1支;麻皮蝽位于系统树的基部,为分化较早的1支,是蝽亚科中较为原始的类群。     

Phylogenetic relationships of Betula species (Betulaceae) based on nuclear ADH and chloroplast matK sequences

The phylogenetic relationships within the genus Betula (Betulaceae) were investigated using a part of the nuclear ADH gene and DNA sequences of the chloroplast matK gene with parts of its flanking regions. Two well-supported phylogenetic groups could be identified in the chloroplast DNA sequence: one containing the three American species B. lenta, B. alleghaniensis, and B. papyrifera and the other including all the other species studied. The ADH gene displayed more variation, and three main groups could be identified. In disagreement with the classical division of the genus Betula, B. schmidtii and B. nana grouped with the species in subgenus Betula, and B. ermanii grouped with species in subgenus Chamaebetula, including B. humilis and B. fruticosa. The ADH phylogeny suggests that several independent polyploidizations within the genus Betula could have taken place. The ADH and chloroplast phylogenies were in part incongruent due to the placement of B. papyrifera. The most likely reason for this seems to be cytoplasmic introgression.     

Adjuvant effect of bovine heat shock protein 70 on piroplasm surface protein, p33, of Theileria sergenti

In this study, we investigated the immunological effects of bovine heat shock protein 70 (HSP70) on the major Theileria sergenti surface protein (p33).The gene encoding p33 was expressed as a fusion protein with bovine HSP70 from a plasmid vector. The adjuvant function of HSP70 on p33 was evaluated with regard to antibody response, cytokine production, and a challenge experiment in mice or cattle. HSP-p33 fusion protein provoked higher humoral and cellular immunity than either Escherichia coli-expressed p33 or piroplasm soluble protein. The HSP adjuvant activity toward p33 was also possible to detect in the inoculated cattle. The overall growth of parasites in cattle was significantly restrained in the HSP-p33-inoculated group, up to 50–52 days longer than in the controls.The present results indicate that HSP-p33 fusion protein is a promising candidate vaccine for clinical theileriosis in the field.     

Phylogenetic relationships of the sweetpotato in Ipomoea series Batatas (Convolvulaceae) based on nuclear beta-amylase gene sequences

Phylogenetic relationships of 13 accessions and a cultivar representing the sweetpotato, Ipomoea batatas (L.) Lam., and its wild progenitors, were investigated using the nucleotide sequence variation of a nuclear-encoded beta-amylase gene. A 1.1-1.3 kb fragment of the gene spanning two exons separated by a long intron was PCR-amplified, cloned, and sequenced. Exon sequences proved highly conservative, while intron sequences yielded large differences. Intron analyses grouped species in a phylogenetic context according to the presence of two genome types: A and B. These groups are consistent with results of previous analyses, save for the novel placement of I. tiliacea, among the A-genome species. Sequences specific to both A and B genome species have been identified. Exon sequences indicate that I. ramosissima and I. umbraticola are quite different from other A-genome species. Placement of I. littoralis is questionable; its intron is similar to other B-genome species, but its exons are quite different. Exon evolution indicates that the B-genome has evolved faster than the A-genome. Interspecific intron and exon variation indicates I. trifida, I. tabascana, and I. batatas form a monophyletic group.     

Phylogenetic relationships among Lactuca (Asteraceae) species and related genera based on ITS-1 DNA sequences

Internal transcribed spacer (ITS-1) sequences from 97 accessions representing 23 species of Lactuca and related genera were determined and used to evaluate species relationships of Lactuca sensu lato (s.l.). The ITS-1 phylogenies, calculated using PAUP and PHYLIP, correspond better to the classification of Feráková than to other classifications evaluated, although the inclusion of sect. Lactuca subsect. Cyanicae is not supported. Therefore, exclusion of subsect. Cyanicae from Lactuca sensu Feráková is proposed. The amended genus contains the entire gene pool (sensu Harlan and De Wet) of cultivated lettuce (Lactuca sativa). The position of the species in the amended classification corresponds to their position in the lettuce gene pool. In the ITS-1 phylogenies, a clade with L. sativa, L. serriola, L. dregeana, L. altaica, and L. aculeata represents the primary gene pool. L. virosa and L. saligna, branching off closest to this clade, encompass the secondary gene pool. L. virosa is possibly of hybrid origin. The primary and secondary gene pool species are classified in sect. Lactuca subsect. Lactuca. The species L. quercina, L. viminea, L. sibirica, and L. tatarica, branching off next, represent the tertiary gene pool. They are classified in Lactuca sect. Lactucopsis, sect. Phaenixopus, and sect. Mulgedium, respectively. L. perennis and L. tenerrima, classified in sect. Lactuca subsect. Cyanicae, form clades with species from related genera and are not part of the lettuce gene pool.     

Phylogenetic relationships of palaearctic formica species (hymenoptera, formicidae) based on mitochondrial cytochrome B sequences

Ants of genus Formica demonstrate variation in social organization and represent model species for ecological, behavioral, evolutionary studies and testing theoretical implications of the kin selection theory. Subgeneric division of the Formica ants based on morphology has been questioned and remained unclear after an allozyme study on genetic differentiation between 13 species representing all subgenera was conducted. In the present study, the phylogenetic relationships within the genus were examined using mitochondrial DNA sequences of the cytochrome b and a part of the NADH dehydrogenase subunit 6. All 23 Formica species sampled in the Palaearctic clustered according to the subgeneric affiliation except F. uralensis that formed a separate phylogenetic group. Unlike Coptoformica and Formica s. str., the subgenus Serviformica did not form a tight cluster but more likely consisted of a few small clades. The genetic distances between the subgenera were around 10%, implying approximate divergence time of 5 Myr if we used the conventional insect divergence rate of 2% per Myr. Within-subgenus divergence estimates were 6.69% in Serviformica, 3.61% in Coptoformica, 1.18% in Formica s. str., which supported our previous results on relatively rapid speciation in the latter subgenus. The phylogeny inferred from DNA sequences provides a necessary framework against which the evolution of social traits can be compared. We discuss implications of inferred phylogeny for the evolution of social traits.     

Phylogenetic relationships of catostomid fishes (Actinopterygii: Cypriniformes) based on mitochondrial ND4/ND5 gene sequences

Family Catostomidae is a diverse group of benthic freshwater fishes that are distributed across North America and in parts of East Asia. In this study, the phylogenetic relationships of Catostomidae is examined using 3436 nucleotides of mitochondrial ND4 and ND5 protein coding genes and intervening tRNAs. All 13 genera and 60 species of catostomids were sampled to represent diversity of the family. Catostomidae and its four subfamilies were found to be monophyletic; however, relationships of the subfamilies are not strongly supported with bootstrapping. The analysis provides strong support for recognizing four tribes in subfamily Catostominae.     

Phylogenetic relationships among Passiflora species based on the glutamine synthetase nuclear gene expressed in chloroplast (ncpGS)

This paper presents the first molecular phylogeny of the genus Passiflora encompassing almost all sections of this large genus. The nuclear-encoded chloroplast-expressed glutamine synthetase gene (ncpGS) was used to examine the relationships among Passiflora species (passionflowers), which was then compared with the new classification proposed by Feuillet and MacDougal. The resulting Bayesian, likelihood, and parsimony trees are congruent and well supported. The 90 Passiflora species examined apparently split into eight main subgenera: Plectostemma, Granadilla, Astrophea, Deidamioides, Polyanthea, Dysosmia, Tetrapathea, and Tryphostemmatoides. These results are in overall agreement with the Feuillet and MacDougal's classification but here we propose that three additional subgenera, Polyanthea, Dysosmia, and Tetrapathea, should be maintained. We observe a striking overall correlation between the phylogenetic position of the different species and their chromosome number. The first clade contains the arborescent species of the subgenus Astrophea, with n=12. The second clade, subgenus Plectostemma, includes species from four subgenera of Killip's classification with n=6 chromosomes. The last clade, subgenus Granadilla, includes species of seven old subgenera with n=9. Subgenus Dysosmia, with a variable chromosome number of n=9-11, is considered here as a separate subgenus closely related to the subgenus Granadilla.     

基于ITS序列探讨杜鹃花属马银花组的系统发育

以杜鹃花属叶状苞亚属Rhododendron　subg. Therorhodion (Maxim.) Gray 的叶状苞杜鹃R. redowskianum Maxim.为外类群,使用PAUP4.0b8软件对马银花亚属sub g. Azaleastrum Planch.的马银花组sect. Azaleastrum (Planch.) Maxim. 7种1 变种和长蕊杜鹃组sect. Choniastrum Planch. 2种及2个相关的单型亚属（异蕊杜鹃 亚属subg. Mumeazalea Max     

Phylogenetic relationships in Myrceugenia (Myrtaceae) based on plastid and nuclear DNA sequences

Myrceugenia is a genus endemic to South America with a disjunct distribution: 12 species occurring mainly in central Chile and approximately 25 in southeastern Brazil. Relationships are reconstructed within Myrceugenia from four plastid markers (partial trnK-matK, rpl32-trnL, trnQ-5'rps16 and rpl16) and two ribosomal nuclear regions (ETS and ITS) using maximum parsimony and Bayesian analyses. Relationships inferred previously from morphological data are not completely consistent with those from molecular data. All molecular analyses support the hypothesis that Myrceugenia is monophyletic, except for M. fernadeziana that falls outside the genus. Chilean species and Brazilian species form two separate lineages. Chilean species form three early diverging clades, whereas Brazilian species are a strongly supported monophyletic group in a terminal position. Least average evolutionary divergence, low resolution, short branches, and high species diversity found in the Brazilian clade suggest rapid radiation. Geographical distributions and phylogenetic reconstructions suggest that extant Myrceugenia species arose in northern Chile followed by colonization southward and finally to the Juan Fernández Islands and southeastern Brazil.     

Phylogenetic relationships among major species of japanese coleoid cephalopods (Mollusca: Cephalopoda) using three mitochondrial DNA sequences

Phylogenetic relationships among 36 species of major coleoid cephalopods from Japanese waters were studied using partial sequences of three mitochondrial genes, 16S rDNA, 12S rDNA, and cytochrome c oxidase subunit I gene. Octopoda and Decapoda were monophylic groups. Within Sepioidea, Sepiadariidae and Sepiolidae were not closely related to Sepiidae, but rather related to Teuthoidea. Sepiidae with a distinct calcareous shell formed a single cluster. Myopsida was closely related to Oegopsida. Within Octopoda, Opisthoteuthis depressa and Argonauta argo diverged earlier than Octopodiidae. The common octopuses in Japanese waters were separated into three clusters. The first cluster occupied a basal position, and includes large-sized octopuses, such as Enteroctopus dofleini and Octopus (Paroctopus) conispadiceus from the continental shelf and upper slope. The second cluster consisted of long-armed octopuses, such as O. ornatus, O. minor, and O. sasakii. The third cluster contained small- to medium-sized octopus, such as Amphioctopus fangsiao, A. areolatus, O. cyaneus, and O. vulgaris, in which several species possess ocelli on the web. The second cluster formed the sister group to the third cluster.