Morphology and molecular phylogeny of the marine diatom genus Nagumoea (Bacillariophyceae) from Japan

The canal-bearing diatom genus Nagumoea, described based on only morphological evidence, was tentatively assigned to the order Bacillariales, although its phylogenetic position remained unclear. Because three isolates of Nagumoea (SK002, SK024 and SK053) were successfully established from Japanese coasts, we performed their morphological observations and molecular phylogenetic analyses to discuss the phylogeny and taxonomic position of this genus. Strains SK002 and SK024 were identified as Nagumoea africana, whereas SK053 conformed with Nagumoea serrata. There was high interspecific divergence between N. africana and N. serrata in the rbcL sequences (8.03–8.17%), indicating their distinctness. Furthermore, intraspecific variations were detected within N. africana (2.35%) in the rbcL, implying its cryptic diversity. The maximum likelihood and Bayesian phylogenetic trees inferred from the plastid rbcL, psbC and nuclear 18S rDNA genes recovered Nagumoea as monophyletic with strong statistical support and embedded within an unresolved, poorly supported lineage containing Achnanthes, Craspedostauros, Staurotropis and Undatella in the canal-bearing order Bacillariales (= the family Bacillariaceae). Although the constrained tree based on the monophyly of Nagumoea and the other canal-bearing clade (Surirellales and Rhopalodiales) was statistically rejected by the topology tests, the phylogenetic position of Nagumoea with other Bacillarialean members remains equivocal. The possession of two plastids positioned fore and aft, observed in the present study, and lack of keel, typical of the Bacillariales, indicate the possibility of Nagumoea being part of the ingroup of the Bacillariales or its closely related outgroup.     

Analysis of ribosomal DNA restriction patterns in the genusKluyveromyces

Riboprinting was used to determine the relationships among strains belonging to 15 species of the genusKluyveromyces. The small subunit ribosomal RNA gene (SSU rDNA) was amplified using the Polymerase Chain Reaction (PCR) and subjected to a battery of nine restriction enzymes. Similarity coefficients between strains were calculated based on shared and unique restriction fragments. Cluster analysis revealed three major groups that generally correlated with previously reported relationships based on other molecular data. Variations in SSU rDNA restriction fragments may be used for differentiation of theKluyveromyces strains included in this study.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

基于核糖体基因的鳞钙皮菌种内分子关系分析

为探讨不同地域的鳞钙皮菌Didymium squamulosum种内分子亲缘关系,通过PCR扩增鳞钙皮菌子实体及原质团DNA,得到SSU、ITS1-5.8S-ITS2 rRNA基因区域,并以SSU、5.8S rRNA基因片段构建NJ亲缘关系树。     

Host identity and functional traits determine the community composition of the arbuscular mycorrhizal fungi in facultative epiphytic plant species

The epiphytic vascular mycobiota is scarce and facultative in semi-arid Mediterranean ecosystems. However, unlike in soil conditions, little is known about the factors driving mycorrhizal communities in epiphytic environments. Here, we investigated the arbuscular mycorrhizal fungi (AMF) harboured by 31 plant species occurring on the trunks of Phoenix dactylifera. We wanted to ascertain if host identity and plant functional traits shape mycorrhizal communities. Specifically, we tested the plant life-cycle (perennial versus annual), the plant life-form (herbaceous versus woody), the plant origin (exotic versus native) and the plant species. The plant affiliation to species strongly influenced the AMF community composition. Plant life-form and plant life-cycle also shaped indicator taxa. The AMF structure differed between annual and perennial species and higher AMF richness was detected in perennial plants. The epiphytic plants associated with AMF irrespective of whether they were native or not, probably because here no functional differences derive from plant origin.     

Morphology and molecular phylogeny of two new species of Spirostrombidium (Ciliophora,Oligotrichia), with a key to the species of Spirostrombidium

Microscopic methods were used to investigate the morphological characterization of two novel oligotrich ciliates, Spirostrombidium paraurceolare sp. nov. and Spirostrombidium faurefremieti sp. nov., isolated from a mangrove wetland in Zhanjiang and an intertidal sandy beach in Qingdao, respectively. Spirostrombidium paraurceolare sp. nov. is characterized by three thigmotactic and 8–10 buccal membranelles, the girdle kinety spiralling around cell with one and a half whorls, and located at right anterior third of dorsal side anteriorly. Spirostrombidium faurefremieti sp. nov. can be recognized by a prominently deep and broad buccal cavity, two thigmotactic and 15–19 buccal membranelles, and the girdle kinety spiralling around cell with two whorls. The small subunit ribosomal RNA genes of these two species were sequenced and compared with those of their congeners to reveal nucleotide differences. Phylogenetic analyses revealed that the genus Spirostrombidium is non-monophyletic. Spirostrombidium faurefremieti sp. nov. falls into a clade comprising most congeners, but Spirostrombidium paraurceolare sp. nov. branches off and groups with Varistrombidium kielum with moderate support. A key to the identification of Spirostrombidium species is also provided.www.zoobank.org/urn:lsid:zoobank.org:pub:AB96BEE6-BE3A-4B95-B75A-3469B1C53ABB2     

2型猪链球菌srtBCD菌毛岛SSU2100基因的克隆表达与免疫学特性

【目的】研究2型猪链球菌(Streptococcus suis serotype 2,S.suis 2)野毒株05ZYH33的srtBCD菌毛岛菌毛亚蛋白SSU2100的免疫保护性作用。【方法】通过PCR扩增出SSU2100基因片段,将目的基因克隆到表达载体pET28a上,转化入E.coli BL21感受态中表达,亲和层析法纯化目的蛋白;Western blot检测SSU2100蛋白的免疫原性,重组蛋白免疫BALB/c小鼠,ELISA法检测多抗血清的效价及IgG亚型,研究重组蛋白的免疫保护作用。【结果】在原核系统成功表达出了SSU2100蛋白;ELISA结果显示重组蛋白能够刺激小鼠产生高效价的免疫抗体;动物实验表明该蛋白具有良好的免疫保护作用。【结论】菌毛亚蛋白SSU2100可以作为S.suis 2亚单位疫苗的候选分子,为系统地阐释srtBCD菌毛岛在S.suis 2致病机制中的作用奠定基础。     

裳卷蛾变形孢虫SSU rRNA核心序列的克隆与系统发育分析

[目的]利用分子生物学手段探索裳卷蛾变形孢虫的遗传发育地位.[方法]微孢子虫的SSUrRNA序列是构建系统发育进化树的重要工具.试验通过T-A克隆法对裳卷蛾变形孢虫(Vairimorphaceraces)SSU rRNA核心序列进行了克隆,并采用近邻法构建了系统发育进化树.[结果]克隆得到了长为1228bp的核苷酸序列(GenBank EU267796).系统发育分析结果表明:裳卷蛾变形孢虫与分离于小菜蛾(Plutella xylostella)的Vairimorpha sp.Germany(GenBank AF124331)和Vairimorphaimperyecta(GenBank AJ131645)相似性最高,它们在系统发育进化树中与寄主为鳞翅目昆虫的Nosema属聚为一类,与纳卡变形孢虫(Vairimorpha necatrix)为代表的Vairimorpha属为相邻集.[结论]结合其生物学特征,裳卷蛾变形孢虫确实为Vairimorph a属的成员,但根据系统发育分析归入Nosematidae科可能更为合适.     

Combining Molecular Data with Classical Morphology for Uncultured Phagotrophic Euglenids (Excavata): A Single‐Cell Approach

Phagotrophic euglenids are one of the most diverse and important forms of heterotrophic flagellates in sediment systems, and are key to understanding the evolution of photosynthetic euglenids and ‘primary osmotrophs’, yet relatively little is known about their biodiversity and phylogenetic relationships. A wealth of light microscopy‐based information is available, but little progress has been made in associating this with molecular sequence data. We established a protocol to obtain light microscopy data and molecular data from single euglenid cells isolated from environmental samples. Individual cells from freshwater and marine benthic samples were isolated and rinsed by micropipetting, documented using high‐resolution photomicroscopy, then subjected to single‐cell nested PCR using taxon‐specific primers in combination with universal eukaryotic primers, generating > 75% or full‐length SSU rDNA sequences. As a proof‐of‐principle eight individuals were characterised and subjected to phylogenetic analyses. Many of these cells were identified as Anisonema or Dinema, and grouped with existing sequences assigned to these taxa, and with a ‘Peranema sp.’ sequence that we could now clearly demonstrate was misidentified or misannotated. Another cell is Heteronema c.f. exaratum, the first ‘skidding heteronemid’ for which sequence data are available. This is not closely related to Heteronema scaphurum, and intriguingly, branches as the sister group to primary osmotrophs. A cell similar to Ploeotia vitrea (the type of this genus), shows no particular phylogenetic affinity to Ploeotia costata, the best studied Ploeotia species. Our experimental protocol provides a useful starting point for future analyses on euglenid biodiversity (including environmental sequence surveys), and their evolution and systematics.     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.