DNA barcoding in autotrophic euglenids: evaluation of COI and 18s rDNA

Autotrophic euglenids (Euglenophyceae) are a common and abundant group of microbial eukaryotes in freshwater habitats. They have a limited number of features, which can be observed using light microscopy, thus species identification is often problematic. Establishing a barcode for this group is therefore an important step toward the molecular identification of autotrophic euglenids. Based on the literature, we selected verified species and used a plethora of available methods to validate two molecular markers: COI and 18S rDNA (the whole sequence and three fragments separately) as potential DNA barcodes. Analyses of the COI gene were performed based on the data set of 43 sequences (42 obtained in this study) representing 24 species and the COI gene was discarded as a DNA barcode mainly due to a lack of universal primer sites. For 18S rDNA analyses we used a data set containing 263 sequences belonging to 86 taxonomically verified species. We demonstrated that the whole 18S rDNA is too long to be a useful marker, but from the three shorter analyzed variable regions we recommend variable regions V2V3 and V4 of 18S rDNA as autotrophic euglenid barcodes due to their high efficiency (above 95% and 90%, respectively).     

There are high levels of functional and genetic diversity in Oxyrrhis marina

Oxyrrhis marina, a widely distributed marine protist, is used to model heterotrophic flagellate responses in microbial food webs. Although clonal variability occurs in protists, assessments of intraspecific diversity are rare; such assessments are critical, particularly where species are used as models in ecological studies. To address the extent of intraspecific variation within O. marina, we assessed diversity among 11 strains using 5.8S rDNA and ITS sequences. The 5.8S rDNA and ITS regions revealed high divergence between strains: 63.1% between the most diverse. To compare O. marina diversity relative to other alveolates, 18S rDNA sequences for five strains were analysed with sequences from representatives of the major alveolate groups. 18S rDNA also revealed high divergence in O. marina. Additionally, consistent with phylogenies based on protein coding genes, maximum likelihood analysis indicated that O. marina was monophyletic and ancestral to the dinoflagellates. To assess ecophysiological differences, growth rates of seven O. marina strains were measured at 10 salinities (10-55 per thousand). Two salinity responses occurred: one group achieved highest growth rates at high salinities; the other grew best at low salinities. There was no clear correlation between molecular, ecophysiological, or geographical differences. However, salinity tolerance was associated with habitat type: intertidal strains grew best at high salinities; open-water strains grew best at low salinities. These data indicate the need to examine many strains of a species in both phylogenetic and ecological studies, especially where key-species are used to model ecological processes.     

Experimental fragmentation patterns of modern Nautilus shells and the implications for fossil cephalopod taphonomy

Shells of modern Nautilus pompilius from the Philippines were experimentally fragmented designed to mimic: (1) transport with sediment; (2) sediment loading; and (3) collision during floating. The breaking patterns by other mechanisms (predation and implosion by hydrostatic pressure) documented in the literature were also considered. The breaking patterns produced by various mechanisms are distinct and can therefore be differentiated. The results allow identification of the distinct mechanisms responsible for specific fragmentation not only in modern Nautilus but also in fossil cephalopods. This is a new approach for the more complete recognition of post-mortem transport of fossil cephalopods and their early taphonomic history, and contributes to our understanding of their palaeobiogeography and palaeoecology.     

A species complex within the isopod genus Haploniscus (Crustacea: Malacostraca: Peracarida) from the Southern Ocean deep sea: a morphological and molecular approach

Seven new species of the genus Haploniscus from the deep Scotia and Weddell Seas are presented, combining morphological and molecular data (mitochondrial 16S rDNA and nuclear 18S rDNA). Haploniscus cassilatus sp. nov. , H. cucullus sp. nov. , H. weddellensis sp. nov. , H. procerus sp. nov. and H. kyrbasia sp. nov. are characterized by a prominent rostral process, the size and shape of which vary among species. The rostrum of H. microkorys sp. nov. is distinctly smaller than that of the former species, while H. nudifrons sp. nov. does not possess a rostrum. The status of the latter as separate species is obvious, owing to the stronger morphological differences. DNA was sequenced from three of the other five species. Genetic distances together with the more subtle morphological variation justify the erection of separate species. Overall morphological variations between these species are small yet noticeable and include, among others, the rostrum, the shape of the pleotelson and setation of pereopods. Our molecular data sets reveal detailed phylogenetic insights within the Haploniscus cucullus complex, supporting the monophyly of all species. We found p -distances of at least 0.0732 (16S rDNA) and 0.0140 (complete 18S rDNA) between pairs of species and show that both genes can be used as a marker for DNA taxonomy.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society , 2008, 152 , 655–706.     

Cytogenetic features of rRNA genes across land plants: analysis of the Plant rDNA database

The online resource http://www.plantrdnadatabase.com/ stores information on the number, chromosomal locations and structure of the 5S and 18S‐5.8S‐26S (35S) ribosomal DNAs (rDNA) in plants. This resource was exploited to study relationships between rDNA locus number, distribution, the occurrence of linked (L‐type) and separated (S‐type) 5S and 35S rDNA units, chromosome number, genome size and ploidy level. The analyses presented summarise current knowledge on rDNA locus numbers and distribution in plants. We analysed 2949 karyotypes, from 1791 species and 86 plant families, and performed ancestral character state reconstructions. The ancestral karyotype (2n = 16) has two terminal 35S sites and two interstitial 5S sites, while the median (2n = 24) presents four terminal 35S sites and three interstitial 5S sites. Whilst 86.57% of karyotypes show S‐type organisation (ancestral condition), the L‐type arrangement has arisen independently several times during plant evolution. A non‐terminal position of 35S rDNA was found in about 25% of single‐locus karyotypes, suggesting that terminal locations are not essential for functionality and expression. Single‐locus karyotypes are very common, even in polyploids. In this regard, polyploidy is followed by subsequent locus loss. This results in a decrease in locus number per monoploid genome, forming part of the diploidisation process returning polyploids to a diploid‐like state over time.     

从核rDNA序列探讨隙蛛亚科Coelotinae的分类地位

隙蛛亚科Coelotinae主要分布于东亚地区,其中我国的已有种类占到全世界种数的一半以上,因此对于我国隙蛛类蜘蛛的研究已经成为世界暗蛛科研究的重点之一。隙蛛亚科属于无筛器类群,于1893年,由Cambridge以隙蛛属为模式属而建立,归属于无筛器的漏斗蛛科。之后,虽然经历了数次修订     

基于18S rDNA序列的蝽次目(半翅目：异翅亚目)

利用18SrDNA分子约1 912 bp的序列对蝽次目21个科53个种进行系统发育分析。运用MP法、ML法和NJ法分析后的结果表明:蝽次目的单系性得到很高的支持;扁蝽总科成为毛点类的姐妹群;毛点类基本确定为两大分支:一支包含蝽总科和红蝽总科;另一支主要由长蝽总科、缘蝽总科和南蝽总科组成;长蝽总科和缘蝽总科都是多系;长蝽总科中,跷蝽科和皮蝽科的关系最近,构成姐妹群,位于整个毛点类的基部;与长蝽总科中另外两个科长蝽科和地长蝽科的关系很远。说明利用18SrDNA分子对研究蝽次目的系统发育关系是适合的,能够重建蝽次目;扁蝽总科和蝽总科单系性的结果与形态学的研究以及Li et al (2005)的研究一致;但较Li et al(2005)的研究更进一步把红蝽总科从广义的缘蝽总科中分出来;并建议皮蝽科作为一个独立的总科更合适。     

New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. nov.

Up to few years ago, the phylogenies of tardigrade taxa have been investigated using morphological data, but relationships within and between many taxa are still unresolved. Our aim has been to verify those relationships adding molecular analysis to morphological analysis, using nearly complete 18S ribosomal DNA gene sequences (five new) of 19 species, as well as cytochrome oxidase subunit 1 (COI) mitochondrial DNA gene sequences (15 new) from 20 species, from a total of seven families. The 18S rDNA tree was calculated by minimum evolution, maximum parsimony (MP) and maximum likelihood (ML) analyses. DNA sequences coding for COI were translated to amino acid sequences and a tree was also calculated by neighbour-joining, MP and ML analyses. For both trees (18S rDNA and COI) posterior probabilities were calculated by MrBayes. Prominent findings are as follows: the molecular data on Echiniscidae (Heterotardigrada) are in line with the phylogenetic relationships identifiable by morphological analysis. Among Eutardigrada, orders Apochela and Parachela are confirmed as sister groups. Ramazzottius (Hypsibiidae) results more related to Macrobiotidae than to the genera here considered of Hypsibiidae. Macrobiotidae and Macrobiotus result not monophyletic and confirm morphological data on the presence of at least two large groups within Macrobiotus. Using 18S rDNA and COI mtDNA genes, a new phylogenetic line has been identified within Macrobiotus , corresponding to the ' richtersi-areolatus group'. Moreover, cryptic species have been identified within the Macrobiotus ' richtersi group' and within Richtersius . Some evolutionary lines of tardigrades are confirmed, but others suggest taxonomic revision. In particular, the new genus Paramacrobiotus gen. n. has been identified, corresponding to the phylogenetic line represented by the ' richtersi-areolatus group'.     

Variations of 18S rDNA Loci Among Six Populations of Paeonia obovata Maxim. （Paeoniaceae） Revealed by Fluorescence In Situ Hybridization

The localization of 18S ribosomal RNA genes （rDNA） by fluorescence in situ hybridization （FISH） had been performed for some species of Paeonla. However, the pattern of 18S rDNA loci among populations Is Indistinct. In the present study, we localized 18S rDNA loci on meiotic or mitotic chromosomes of six populations of Paeonla obovata Maxim. （Paeonlaceae）. Different numbers of rDNA loci were found with different diploid （2n=10） populations, namely eight （Lushl and Mt. JIuhua populations）, 10 （Mt. Talbal population）, and seven （Mt. Guandl population）, whereas tetraplold （2n=20） populations were all found with 16 loci. Aii rDNA loci were mapped near teiomeres of mitotic chromosomes and there was no chromosome with two loci. The present results show that molecular cytological polymorphlsm exists among P. obovata diploid populations, Indicating that structural variations occurred frequently during the evolutionary history of this species, accompanied with differentiation among populations.     

JENUFA GEN. NOV.: A NEW GENUS OF COCCOID GREEN ALGAE (CHLOROPHYCEAE,INCERTAE SEDIS) PREVIOUSLY RECORDED BY ENVIRONMENTAL SEQUENCING1

The diversity of eukaryotic microorganisms is far from fully described, as indicated by the vast number of unassigned genotypes retrieved by environmental sequencing or metagenomics. We isolated several strains of unicellular green algae from algal biofilms growing on tree bark in a Southeast Asian tropical rainforest and determined them to be relatives of an unidentified lineage of environmental 18S rDNA sequences, thus uncovering its cellular identity. Light, confocal, and electron microscope observations and sequencing the 18S rRNA gene revealed that the strains represent two different species within an apparently new genus, described here as Jenufa gen. nov. Both species formed minute coccoid cells with an irregular globular outline, a smooth cell wall, and a single parietal chloroplast without a pyrenoid. The two species, described herein as J. perforata and J. minuta, differed in chloroplast morphology and cell wall structure. Phylogenetic analyses of 18S rRNA gene sequences showed a firm relationship between the two species and placed the Jenufa lineage in an unresolved position within the CS clade (Chlamydomonadales + Sphaeropleales) of the class Chlorophyceae, although possible affinities to the genus Golenkinia were suggested both by maximum‐likelihood (ML) and Bayesian methods. Furthermore, two almost identical environmental 18S rDNA sequences from an endolithic microbial community occurring in dolomite rock in the central Alps turned out to be specifically related to, yet apparently distinct from, the sequence of J. minuta, indicating the existence of an undescribed Jenufa species occurring in the temperate zone.     

Chromosomal localization of 5S and 18S rDNA in five species of subgenus Strobus and their implications for genome evolution of Pinus

BACKGROUND AND AIMS: Studying the genome structure of pines has been hindered by their large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes in different groups of pines is extremely limited. However, techniques are now available that can surmount these difficulties. The purpose of this study was to exploit some of these techniques to characterize the genome differentiation between the two subgenera of Pinus: Pinus and Strobus. METHODS: Double-probe fluorescence in-situ hybridization (FISH) was used to localize the 5S and 18S rDNA loci on chromosomes of five species from the subgenus Strobus: P. bungeana, P. koraiensis, P. armandii, P. wallichiana and P. strobus. * KEY RESULTS: The rDNA FISH pattern varied considerably among the five species, with P. bungeana being the most distinct. By comparing the results obtained with those of previous rDNA FISH studies of members of the subgenus Pinus, several general features of rDNA loci distribution in the genus Pinus can be discerned: (a) species of subgenus Strobus generally have more rDNA loci than species of subgenus Pinus, correlating with their larger genomes in the subgenus Strobus; (b) there is a clear differentiation in 5S and 18S rDNA loci linkage patterns between the two subgenera; (c) variations in the rDNA FISH pattern correlate with phylogenetic relationships among species within the subgenus; (d) P. bungeana has fewer 18S rDNA sites than other pines investigated to date, but they give intense signals, and may reflect the primary distribution of the 18S-25S rDNA loci in the genus. CONCLUSIONS: The stable differentiation in rDNA FISH pattern between the subgenera suggests that chromosomal rearrangements played a role in the splitting of the two subgenera, and transpositional events rather than major structural changes are likely responsible for the variable rDNA distribution patterns among species of the same subgenus with conserved karyotypes.     

基于18S rDNA与ITS-5.8S rDNA对 重庆地区网状车轮虫的种内分化研究

本研究基于形态和分子数据对采自重庆地区5个地理株系的网状车轮虫(Trichodina reticulata)进行了比较研究及重描述。研究结果表明,网状车轮虫不同株系表现出不同的表型分化,含形态略有不同的齿体及有或无中央颗粒,因而具有明显的种内形态多样性。不同地理株系网状车轮虫的18S rDNA序列相似度在99.0%~100%之间,遗传距离为0.000~0.008,并在三大变异区(V4、V5与V7)均具一致的二级结构,表明不同株系的18Sr DNA相似度与遗传距离均属种内水平。综合18SrDNA和ITS-5.8S rDNA的变异位点和系统发育对种内分歧的研究分析显示,来自不同地理分布和宿主的网状车轮虫株系皆因相同的变异位点而聚为一枝,以此推断网状车轮虫的种内分化主要受其基因的影响,地理分布与宿主差异等环境影响在目前的种群分化阶段暂未突显。此外,本研究进一步验证了中央颗粒不能作为网状车轮虫的主要鉴别性特征的观点。     

Phylogeny of the Platyhelminthes and the evolution of parasitism

Robust phylogenies provide the basis for interpreting biological variation in the light of evolution. Homologous features provide phylogenetically informative characters whereas homoplasious characters provide phylogenetic noise. Both provide evolutionary signal. We have constructed molecular and morphologically based phylogenies of the phylum Platyhelminthes using a recently revised morphological character matrix and complete 18S and two partial 28S rRNA gene sequences in order to evaluate the emergence and subsequent divergence of parasitic forms. In total we examine 65 morphological characters, 97 18S rDNA, 41 Dl domain 28S rDNA, and 49 D3-D6 domain 28S rDNA sequences. For the molecular data there were 748, 132 and 249 phylogenetically informative sites for the 18S, Dl and D3-D6 28S rDNA data sets respectively. Morphological and molecular phylogenetic solutions are incongruent but not incompatible, and using the principles of conditional combination (18S rDNA + morphology passing Templeton's test) they demonstrate: a single and relatively early origin for the parasitic Neodermata (including the cestodes, trematodes and monogeneans); sister-group status between the cestodes and monogeneans, and between these taxa and the trematodes (digeneans and aspidogastreans). The sister-group to the Neodermata is likely to be a large clade of neoophoran turbellarians, based on combined evidence, or a clade consisting of the Fecampiid + Urastomid turbellarians, based on morphological evidence alone. The combined evidence solution for the phylogeny of fiatworms based on 18S rDNA and morphology is used to interpret morphological and life-history data and to support a model for the evolution and radiation of neodermatan parasites in the group.     

Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan

Endosymbiotic green algae of Japanese Paramecium bursaria were phylogenetically analyzed based on DNA sequences from the ribosomal DNA operon (18S rDNA, ITS1, 5.8S rDNA, and ITS2). Phylogenetic trees constructed using 18S rDNA sequences showed that the symbionts belong to the Chlorella sensu stricto (Trebouxiophyceae) group. They are genetically closer to the C. vulgaris Beijerinck group than to C. kessleri Fott et Nováková as proposed previously. Branching order in C. vulgaris group was unresolved in 18S rDNA trees. Compared heterogeneities of 18S rDNA, ITS1, 5.8S r, and ITS2 among symbionts and two Chlorella species, indicated that the ITS2 region (and probably also ITS1) is better able to resolve phylogenetic problems in such closely related taxa. All six symbiotic sequences obtained here (approximately 4000-bp sequences of 18S rDNA, ITS1, 5.8S rDNA, and ITS2) were completely identical in each, strongly suggesting a common origin.     

PHYLOGENETIC POSITION OF CHAETOSPHAERIDIUM (CHLOROPHYTA), A BASAL LINEAGE IN THE CHAROPHYCEAE INFERRED FROM 18S rDNA SEQUENCES

We sequenced the nuclear-encoded small-subunit ribosomal RNA gene (18S rDNA) of Chaetosphaeridium globosum (Nordst.) Klebahn, a microscopic freshwater epiphytic chlorophyte, to assess its phylogenetic affinities in the Chlorophyta. A phylogenetic analysis of a broad sampling of green algal taxa and Chaetosphaeridium confirmed that this alga is a member of the Charophyceae (Streptophyta) as earlier microscopical studies had suggested. However, more detailed phylogenetic analyses of the streptophyte lineage showed that contrary to expectations based on the ultrastructure of the zoospores, the presence of a unique type of setae, the oogamous mode of reproduction, and the occurrence of oscillatory rotations of the cytoplasm, Chaetosphaeridium and Coleochaete are not closely related and do not form a monophyletic clade. Instead, Chaetosphaeridium represents an early branch in the streptophyte lineage that had a near-simultaneous origin as the Charalean clade and a clade formed by all remaining streptophytes examined ( Klebsormidium, Coleochaete, Chlorokybus, Zygnematales, and bryophytes). All phylogenetic inference methods used (neighbor-joining analysis of Kimura distances, maximum likelihood, and maximum parsimony) resulted in essentially the same tree topology. No Group I introns were found in the 18S rDNA coding region of Chaetosphaeridium. Our molecular phylogenetic analysis of Chaetosphaeridium supports a recent cladistic classification of the Streptobionta by Kenrick and Crane in which Chaetosphaeridium is placed in a monotypic division and class, Chaetosphaeridiophyta and Chaetosphaeridiophyceae, respectively.     

Multiple origins of anural development in ascidians inferred from rDNA sequences

Ascidians exhibit two different modes of development. A tadpole larva is formed during urodele development, whereas the larval phase is modified or absent during anural development. Anural development is restricted to a small number of species in one or possibly two ascidian families and is probably derived from ancestors with urodele development. Anural and urodele ascidians constitute a model system in which to study the evolution of development, but the phylogeny of anural development has not been resolved. Classification based on larval characters suggests that anural species are monophyletic, whereas classification according to adult morphology suggests they are polyphyletic. In the present study, we have inferred the origin of anural development using rDNA sequences. The central region of 18S rDNA and the hypervariable D2 loop of 28S rDNA were amplified from the genomic DNA of anural and urodele ascidian species by the polymerase chain reaction and sequenced. Phylogenetic trees inferred from 18S rDNA sequences of 21 species placed anural developers into two discrete groups corresponding to the Styelidae and Molgulidae, suggesting that anural development evolved independently in these families. Furthermore, the 18S rDNA trees inferred at least four independent origins of anural development in the family Molgulidae. Phylogenetic trees inferred from the D2 loop sequences of 13 molgulid species confirmed the 18S rDNA phylogeny. Anural development appears to have evolved rapidly because some anural species are placed as closely related sister groups to urodele species. The phylogeny inferred from rDNA sequences is consistent with molgulid systematics according to adult morphology and supports the polyphyletic origin of anural development in ascidians. Correspondence to: W.R. Jeffery     

Phylogenetic analyses of various euglenoid taxa (euglenozoa) based on 18s rdna sequence data

18S rRNA genes (SSU rDNA) of five newly sequenced species were used as molecular markers to infer phylogenetic relationships within the euglenoids. Two members of the order Euglenales ( Lepocinclis ovata Playfair , Phacus similis Christen), two of the order Eutreptiales ( Distigma proteus Ehrenberg, , D. curvata Pringsheim) and Gyropaigne lefévrei Bourelly et Georges of the order Rhabdomonadales were used in parsimony, maximum likelihood, and distance analyses. All trees derived from SSU rRNA data strongly supported the monophyletic origin of the Euglenozoa, with kinetoplastids as sister clade to the euglenoids and Petalomonas cantuscygni Cann et Pennick diverging at the base of the monophyletic euglenoid lineage. The data also supported the theory that phagotrophic euglenoids arose prior to osmotrophs and phototrophs. A lineage of Peranema trichophorum Ehrenberg and all sequenced Euglenales formed a sister clade to the osmotrophs. This suggests that the evolution of phototrophy within the euglenoids radiated from a single event.     

Genetic diversity and phylogeny of the family Osteoglossidae by the nuclear 18S ribosomal RNA and implications for its conservation

The genetic structure of the family Osteoglossidae from different geographical regions was examined using the nuclear 18S ribosomal RNA (18S rDNA) sequence. The results showed that the partial length of 18S rDNA was 1277 bp, and they were relatively conserved in the species of the family Osteoglossidae. A total of 62 (4.83%) polymorphic sites were observed, and 28 haplotypes were defined, which were characterized by different haplotype diversity (0.00105–0.900) and nucleotide diversity (0.00321–3.000). Analysis of molecular variance (AMOVA) showed genetic divergence of these species (79.76%). Phylogenetic analysis revealed that 18S rDNA was a suitable genetic marker and was able to distinguish phylogenetic relationships among different Osteoglossidae species, and it also supported current taxonomy. In addition, low genetic diversity of several species provides genetic assessment information and should be taken into consideration to implement a conservation management planning for some species, such as Scleropages formosus green arowana.     

堆肥发酵菌种的分离、鉴定及其堆肥发酵效果研究

为获得适应性良好的菌种用于猪粪的堆肥处理,从新鲜猪粪中分离出一株酵母样菌株783,并采用PCR技术扩增其18S rDNA基因,并测定其基因序列.基于18S rDNA序列同源性比对以及系统发育树的分析,发现783是解脂耶罗威亚酵母.菌株783的18S rDNA序列已经被GenBank数据库收录,其序列号为EU434621.对猪粪堆肥发酵效果表明其在提高堆肥质量方面有着较好的效果.