Active eukaryotes in microbialites from Highborne Cay,Bahamas, and Hamelin Pool (Shark Bay), Australia

Microbialites are organosedimentary structures that are formed through the interaction of benthic microbial communities and sediments and include mineral precipitation. These lithifying microbial mat structures include stromatolites and thrombolites. Exuma Sound in the Bahamas, and Hamelin Pool in Shark Bay, Western Australia, are two locations where significant stands of modern microbialites exist. Although prokaryotic diversity in these structures is reasonably well documented, little is known about the eukaryotic component of these communities and their potential to influence sedimentary fabrics through grazing, binding and burrowing activities. Accordingly, comparisons of eukaryotic communities in modern stromatolitic and thrombolitic mats can potentially provide insight into the coexistence of both laminated and clotted mat structures in close proximity to one another. Here we examine this possibility by comparing eukaryotic diversity based on Sanger and high-throughput pyrosequencing of small subunit ribosomal RNA (18S rRNA) genes. Analyses were based on total RNA extracts as template to minimize input from inactive or deceased organisms. Results identified diverse eukaryotic communities particularly stramenopiles, Alveolata, Metazoa, Amoebozoa and Rhizaria within different mat types at both locations, as well as abundant and diverse signatures of eukaryotes with <80% sequence similarity to sequences in GenBank. This suggests the presence of significant novel eukaryotic diversity, particularly in hypersaline Hamelin Pool. There was evidence of vertical structuring of protist populations and foraminiferal diversity was highest in bioturbated/clotted thrombolite mats of Highborne Cay.     

Molecular phylogeny of Collembola inferred from ribosomal RNA genes

The classification of taxa within Collembola (Springtails, Hexapoda) has been controversial. In this study, we combined complete 18S rRNA gene with partial 28S rRNA gene (D7-D10) sequences to investigate the phylogeny of Collembola. About 2500 aligned sites of thirty species representing 29 genera from14 families of Collembola were analyzed, including one species of Neelipleona from which no sequence has been reported previously.The phylogenetic trees were obtained by different methods (maximum parsimony, maximum likelihood, and Bayesian analysis). Our results supported the monophyly of two of the four taxonomic groups of Collembola summarized by Deharveng [Deharveng, L., 2004. Recent advances in Collembola systematics. Pedobiologia 48, 415–433.], namely of Poduromorpha and of Symphypleona. Within Poduromorpha, Neanuridae was monophyletic with high support, but Hypogastruridae was not. Entomobryomorpha was paraphyletic, as the Tomoceroidea (Tomoceridae and Oncopoduridae) was found to be apart from the other entomobryomorphs. In the latter Isotomoidea and Entomobryoidea joined into a group with moderate support. Within Symphypleona, the phylogenetic relationship [(Sminthuridae + Bourletiellidae) + Sminthurididae] was consistent with traditional morphological studies. Neelipleona grouped with Symphypleona in all trees, with moderate support in the ML and Bayesian analyses.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

The diversity of fungi in aerobic sewage granules assessed by 18S rRNA gene and ITS sequence analyses

Aerobic sewage granules are dense, spherical biofilms, regarded as a useful and promising tool in wastewater treatment processes. Recent studies revealed that fungi can be implemented in biofilm formation. This study attempts to uncover the fungal diversity in aerobic granules by sequence analysis of the 18S and 5.8S rRNA genes and the internal transcribed spacer regions. For this purpose, appropriate PCR and sequencing primer sets were selected and an improved DNA isolation protocol was used. The sequences of 41 isolates were assigned to the taxonomic groups Pleosporaceae, Xylariales, Theleobolaceae, Claviceps, Aureobasidium, Candida boleticola , and Tremellomycetes within the fungi. It turned out that the fungal community composition in granules depended on the wastewater type and the phase of granule development.     

Archiascomycetes: detection of a major new lineage within the Ascomycota

For phylogenetic analysis of the higher fungi, we sequenced the nuclear small subunit rRNA (18S rRNA) gene fromTaphrina populina, the type species of the genusTaphrina, andProtomyces lactucae-debilis. The molecular phylogeny inferred from these 2 sequences and 75 sequences from the DNA data bank divided the Ascomycota into three major lineages: the hemiascomycetes, the euascomycetes, and the archiascomycetes, newly described herein. The former two lineages are monophyletic, whereas the archiascomycetes, which originated first and are comprised ofTaphrina, Protomyces, Saitoella, Schizosaccharomyces, andPneumocystis, may not be monophyletic. Among the archiascomycetes, theTaphrina/Protomyces branch is monophyletic. Confirmation of the archiascomycetes as a monophyletic taxonomic class will require comparison of additional genetically defined characters.This work was supported in part by grants 05454030 from the Ministry of Education, Science, and Culture of Japan (to J. S.) and 4369 from the Japan Society for the Promotion of Science Fellowship Programs (to H. N.).     

基于18S rRNA的中国大陆沿海石磺科贝类分类的初步分析

对采自上海崇明、福建宁德、海南海口等沿海地区9个群体的石磺科贝类进行外部形态特征差异分析和内部结构比较,在初步分类基础上利用核糖体小亚基18S rRNA基因部分序列对9个群体进行系统发育分析,以菊花螺为外群,结合GenBank上石磺科4个18S rRNA基因序列构建系统发生树来探讨我国大陆沿海石磺科属种间的亲缘关系.结果显示:我国石磺科贝类南方沿海种类多于北方沿海;除报道的瘤背石磺(Onchidium struma)和石磺(O.verruculatum)外,可能还有新记录5种:Onchidium属1种、Platevindex属2种、Peronia属1种和Paraoncidium属1种.分子系统发生树显示,我囝大陆沿海石磺科9个群体可分为4个亚群,分别为Onchidium、Platevindex、Paraoncidium、Peronia,其中Peronia亚群的置信度较高;Onchidium verruculatum应更名为Peronia verruculata.     

鸡球虫18S rRNA基因序列的测定与分析

为了利用18S rRNA基因进行鸡球虫系统进化分析,对巨型艾美耳球虫(Eimeria maxima)、柔嫩艾美耳球虫(E.tenella)、堆形艾美耳球虫(E.acervulina)3种共8个不同来源的虫株,分别提取总DNA进行18S rRNA基因的扩增和测序;将得到的序列登录GenBank进行同源性和趋异性分析,并结合GenBank中其它原虫的18S rRNA基因序列构建进化树.结果显示扩增获得8株鸡球虫18S rRNA基因长度为1746～1756 bp,序列比对显示同种不同株间的同源性大于不同种间的同源性,其中3株E.maxima株间同源性在98.7%～99.3%之间,4株E.tenella株间同源性在99.7%～99.9%之间;不同种间同源性为96.5%～98.1%,其中E.maxima与E.tenclla的遗传距离最大,为0.038;E.maxima与E.acervulina的遗传距离最小,为0.021.顶复器门9个不同属所构建的进化树结果显示,E.imeria和等孢属(Isospora)聚为一支,说明亲缘关系比较近.与GertBank中其它5株不同鸡球虫的18S rRNA基因共同构建的进化树显示,3株E.maxima聚为一支,与E.brunetti、E.mitis、E.mivati、E.praecox和E.acervulina聚为一大分支;4株E.tenella与1株E.necatrix共同形成一个分支,说明E.tenella与E.necattix的亲缘关系最近.本研究证实了在鸡球虫系统进化研究中,18S rRNA基因不仅可以区分不同种,而且有可能成为区分同种不同株的理想靶基因.     

产β-甘露聚糖酶Aspergillus sp. LQ21的分离、鉴定及发酵条件的研究

β-甘露聚糖酶是一类能够水解甘露聚糖、葡甘露聚糖、半乳甘露聚糖的半纤维素酶类,广泛存在于动植物和微生物中,此酶在食品、医药、饲料、造纸、石油等方面已得到广泛应用;近年来,其作为食品和饲料添加剂方面倍受关注。对采自土壤和树皮的样品通过富集培养、平板初筛和摇瓶复筛,得到1株具产β-甘露聚糖酶能力的曲霉属菌株LQ21,结合形态特征、培养特征及18S rRNA基因序列分析,将该菌株鉴定为Aspergillussp.真菌。考察了培养时间、起始pH、培养温度、碳源和氮源对该菌株产酶的影响。初步确定了其最适产酶培养基组成:魔芋粉0.5%,蛋白胨1%,NaNO30.2%,K2HPO40.1%,KCl 0.05%,MgSO4.7H2O 0.05%,FeSO4.7H2O 0.001%;最适培养条件:初始pH4.5,温度35℃,转速200 r/min,培养60 h发酵液上清中酶活达到最高。     

Relative diversity and community structure analysis of rumen protozoa according to T-RFLP and microscopic methods

Protozoa are common inhabitants of the rumen where they play roles in host nutrition and methanogenesis. Knowledge of how changes in the composition of protozoa communities affect these processes is limited in part due to a lack of efficient methods for protozoa community analysis. In this study, a terminal-restriction fragment length polymorphism (T-RFLP) assay targeting the 18S rRNA gene was developed for comparative analysis of rumen protozoa communities. Comparison of diversity and structure of protozoa communities from hay-fed versus silage/grain-fed cattle via T-RFLP analysis yielded similar overall results to microscopy analysis. According to both methods, Entodinium spp. were more abundant in the silage/grain-fed cattle and protozoa diversity (as calculated using the Shannon index) was higher for the hay-fed cattle due to greater species evenness. Type B protozoa were more prevalent in the hay-fed cattle, whereas Type A protozoa were more prevalent in the silage/grain-fed cattle. Analysis of similarity (ANOSIM) indicated that the protozoa communities from hay-fed and silage/grain-fed cattle were different, and multivariate analysis indicated that pen mates (i.e., cattle fed the same diet and housed together) tended to have similar protozoa communities types. In summary, we present a T-RFLP method for analyzing rumen protozoa communities which complements traditional microscopy approaches but has the advantage of being amenable to high-throughput.     

基于18S rRNA基因序列的直翅目主要类群系统发育关系研究

基于78种直翅目昆虫的18S rRNA基因全序列构建了直翅目各主要类群间的系统发育关系。本研究的结果支持直翅目的单系性,但不支持蝗亚目和螽亚目各自的单系性;直翅目下除蜢总科和蝗总科外各总科的划分多数与Otte系统相一致;蜢总科的单系性得不到支持;蝗总科的剑角蝗科、斑腿蝗科、斑翅蝗科、网翅蝗科和槌角蝗科5科均不是单系群,各物种间的遗传距离差异不大,应合并为一科,即蝗科;本研究支持将Otte系统中蚱总科和螽蟖总科下各亚科级阶元提升为科级阶元;18S rRNA基因全序列可以作为划分科级阶元的工具,当位于同一分支上互成姐妹群的类群间的遗传距离超过1%时,这几个类群属于不同的科;但由于其在进化上的保守性,18S rRNA基因只能用于纲目等高级阶元间关系的研究,而由其获得的总科以下阶元间的关系并不可靠。     

利用小亚基核糖体RNA 技术分析温室黄瓜近根土壤古菌和真菌多样性

土壤古菌和真菌在温室生态系统是仅次于细菌的微生物,具有类似于细菌的重要生态功能。通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库,分析温室黄瓜近根土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOTUR软件将古菌和真菌序列按照相似性97％的标准分成若干个可操作分类单元 (OTUs)。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs;真菌克隆文库包括真菌门的大多数亚门真菌,共24个OTUs,未发现担子菌亚门真菌。古菌多样性比较丰富,且发现少量的广域古菌 (甲烷菌),这一情况可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境有关;土壤真菌的优势种群为子囊菌,占到土壤真菌的80％以上,这可能与绝大多数植物真菌性病害属于土传病害,通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。     

Lateral transfer of eukaryotic ribosomal RNA genes: an emerging concern for molecular ecology of microbial eukaryotes

Ribosomal RNA (rRNA) genes are widely utilized in depicting organismal diversity and distribution in a wide range of environments. Although a few cases of lateral transfer of rRNA genes between closely related prokaryotes have been reported, it remains to be reported from eukaryotes. Here, we report the first case of lateral transfer of eukaryotic rRNA genes. Two distinct sequences of the 18S rRNA gene were detected from a clonal culture of the stramenopile, Ciliophrys infusionum. One was clearly derived from Ciliophrys, but the other gene originated from a perkinsid alveolate. Genome-walking analyses revealed that this alveolate-type rRNA gene is immediately adjacent to two protein-coding genes (ubc12 and usp39), and the origin of both genes was shown to be a stramenopile (that is, Ciliophrys) in our phylogenetic analyses. These findings indicate that the alveolate-type rRNA gene is encoded on the Ciliophrys genome and that eukaryotic rRNA genes can be transferred laterally.     

Biogeographic patterns in below-ground diversity in New York City's Central Park are similar to those observed globally

Soil biota play key roles in the functioning of terrestrial ecosystems, however, compared to our knowledge of above-ground plant and animal diversity, the biodiversity found in soils remains largely uncharacterized. Here, we present an assessment of soil biodiversity and biogeographic patterns across Central Park in New York City that spanned all three domains of life, demonstrating that even an urban, managed system harbours large amounts of undescribed soil biodiversity. Despite high variability across the Park, below-ground diversity patterns were predictable based on soil characteristics, with prokaryotic and eukaryotic communities exhibiting overlapping biogeographic patterns. Further, Central Park soils harboured nearly as many distinct soil microbial phylotypes and types of soil communities as we found in biomes across the globe (including arctic, tropical and desert soils). This integrated cross-domain investigation highlights that the amount and patterning of novel and uncharacterized diversity at a single urban location matches that observed across natural ecosystems spanning multiple biomes and continents.     

Protistan diversity estimates based on 18S rDNA from seawater incubations in the Western North Atlantic

Cloning/sequencing and fragment analysis of ribosomal RNA genes (rDNA) are becoming increasingly common methods for the identification of microbial taxa. Sequences of these genes provide many additional taxonomic characters for species that otherwise have few distinctive morphological features, or that require involved microscopy or laboratory culture and testing. These same approaches are now being applied with great success in ecological studies of natural communities of microorganisms. Extensive information on the composition of natural microbial assemblages is being amassed at a rapid pace through genetic analyses of environmental samples and comparison of the resulting genetic information with well-established (and rapidly growing) public databases. We examined microbial eukaryote diversity in a natural seawater sample from the coastal western North Atlantic Ocean using two molecular biological approaches: the cloning and sequencing of rRNA genes and by fragment analysis of these genes using the terminal restriction fragment length polymorphism (T-RFLP) method. A simple experiment was carried out to examine changes in the overall eukaryote (largely protistan) diversity and species composition (phylotype diversity) of a natural microbial assemblage when a seawater sample is placed in a container and incubated at ambient light and temperature for 72 h. Containment of the natural seawater sample resulted in relatively minor changes in the overall eukaryote diversity (species richness) obtained by either molecular method at three time points (time-zero, time-24 h, time-72 h). However, substantial changes in the dominance of particular eukaryote phylotypes took place between the three sampling times. Only 18% of the total number of phylotypes observed in the study were observed at all three time points, while 65% (108 of 165) phylotypes were observed only at a single time point (54 unique phylotypes initially, 37 more unique phylotypes at 24 h, and 17 more at 72 h). The results of this study indicate that a high diversity of protistan taxa existed in the original seawater sample at very low abundance, and thus were not observed in the initial characterization of community structure. Containment resulted in significant shifts in the dominance of these taxa, enabling the presence of previously unobserved phylotypes to be documented after 24 or 72 h of incubation.