Molecular insight into systematics,host associations,life cycles and geographic distribution of the nematode family Rhabdiasidae

Rhabdiasidae Railliet, 1915 is a globally distributed group of up to 100 known species of nematodes parasitic in amphibians and reptiles. This work presents the results of a molecular phylogenetic analysis of 36 species of Rhabdiasidae from reptiles and amphibians from six continents. New DNA sequences encompassing partial 18S rDNA, ITS1, 5.8S rDNA, ITS2 and partial 28S rDNA regions of nuclear ribosomal DNA were obtained from 27 species and pre-existing sequences for nine species were incorporated. The broad taxonomic, host and geographical coverage of the specimens allowed us to address long-standing questions in rhabdiasid systematics, evolution, geographic distribution, and patterns of host association. Our analysis demonstrated that rhabdiasids parasitic in snakes are an independent genus sister to the rest of the Rhabdiasidae, a status supported by life cycle data. Based on the combined evidence of molecular phylogeny, morphology and life cycle characteristics, a new genus Serpentirhabdias gen. nov. with the type species Serpentirhabdias elaphe (Sharpilo, 1976) comb. nov. is established. The phylogeny supports the monophyly of Entomelas Travassos, 1930, Pneumonema Johnston, 1916 and the largest genus of the family, Rhabdias Stiles and Hassall, 1905. DNA sequence comparisons demonstrate the presence of more than one species in the previously monotypic Pneumonema from Australian scincid lizards. The distribution of some morphological characters in the genus Rhabdias shows little consistency within the phylogenetic tree topology, in particular the apical structures widely used in rhabdiasid systematics. Our data suggest that some of the characters, while valuable for species differentiation, are not appropriate for differentiation among higher taxa and are of limited phylogenetic utility. Rhabdias is the only genus with a cosmopolitan distribution, but some of the lineages within Rhabdias are distributed on a single continent or a group of adjacent zoogeographical regions. Serpentirhabdias, Entomelas and Pneumonema show rather strict specificity to their host groups. The evolution of the Rhabdiasidae clearly included multiple host switching events among different orders and families of amphibians as well as switching between amphibians and squamatan reptiles. Only a few smaller lineages of Rhabdias demonstrate relatively strict associations with a certain group of hosts.     

Image analysis of Agaricus basidiospores for use in systematics

Basidiospore characters were measured using computer-based image analysis with greater speed (10 x) and objectivity than possible manually. This technology allowed measurement of additional variables for studies of spores, including area and circularity, as well as length, breadth and elongation. Principal component analysis of basidiospore characters aided definition of species and species groups.     

Molecular systematics of the neotropical shovelnose catfish genus Pseudoplatystoma Bleeker 1862 based on nuclear and mtDNA markers

Pseudoplatystoma is a commercially important genus of Neotropical migratory catfishes widely distributed in all major river basins of South America. Historically, only three species were recognized, but a recent revision proposed eight putative morphospecies for the genus. A molecular study based on mitochondria DNA (mtDNA) provided support for recognition of only some of the species and raised questions about species boundaries in this group. We present a more encompassing analysis based on mtDNA (cytochrome b, 818bp) and nuclear DNA-based phylogenies (Rag1 intron 1, 664bp and S7 intron 1, 635bp) for a more extensive sampling (279 individuals from 42 localities) of all putative species in all major river basins. Patterns generated by individual gene genealogies and a multispecies coalescent analysis provided evidence to suggest recognition of only four distinct species in this genus: Pseudoplatystoma magdaleniatum, Pseudoplatystoma corruscans, Pseudoplatystoma tigrimun (sensu lato) and Pseudoplatystoma fasciatum (sensu lato). The species phylogeny places P. magdaleniatum as the sister group to all the other species in the genus, but the relationships among P. fasciatum s.l, P. tigrimum s.l., and P. corruscans could not be resolved with confidence.     

Extreme Variation of mtDNA Neutral Substitution Rate across Mammalian Species - the Longevity Hypothesis

Mol. Biol. Evol. 25:120–130. 2008. doi:10.1093/molbev/msm248 Numerical values in     

Uncorrected nucleotide bias in mtDNA can mimic the effects of positive Darwinian selection

The relative rates of nucleotide substitution at synonymous and nonsynonymous sites within protein-coding regions have been widely used to infer the action of natural selection from comparative sequence data. It is known, however, that mutational and repair biases can affect rates of evolution at both synonymous and nonsynonymous sites. More importantly, it is also known that synonymous sites are particularly prone to the effects of nucleotide bias. This means that nucleotide biases may affect the calculated ratio of substitution rates at synonymous and nonsynonymous sites. Using a large data set of animal mitochondrial sequences, we demonstrate that this is, in fact, the case. Highly biased nucleotide sequences are characterized by significantly elevated dN/dS ratios, but only when the nucleotide frequencies are not taken into account. When the analysis is repeated taking the nucleotide frequencies at each codon position into account, such elevated ratios disappear. These results suggest that the recently reported differences in dN/dS ratios between vertebrate and invertebrate mitochondrial sequences could be explained by variations in mitochondrial nucleotide frequencies rather than the effects of positive Darwinian selection.     

The use of karyotyping in the systematics of yeasts

The use of electrophoretic karyotyping in systematics of yeasts is discussed. New data are provided on the karyotypes of the medically important fungiHortaea werneckii, Filobasidiella (=Cryptococcus)neoformans, andMalassezia species.Hortaea werneckii has twelve to eighteen bands of chromosomal DNA, ranging in size between 500 and 2300 kb. The karyotypes ofFilobasidiella neoformans consist of seven to fourteen bands of chromosomal DNA. The varietiesneoformans andbacillispora cannot be separated by their karyotypes, and no obvious correlation was found with serotypes, geography or habitat. All strains ofMalassezia pachydermatis studied have similar karyotypes consisting of five bands, whereas inM. furfur, four different karyotypes are prevalent. However, each of these karyotypes is stable.     

线粒体DNA高变区hv1快速测序方法及应用

本研究目的是使线粒体DNA hv1区的测序更加简单、快速和准确,以利于法医学检案。将线粒体DNA hv1高变区分别用三对重叠的引物进行全序列扩增,对这三对引物的扩增产物进行测序分析。用经过大量实验反复摸索得到的稳定的实验条件．对已确定为同一母亲所生的兄弟二人的样品进行分析,结果完全一样;对待确定姐弟关系的样品进行分析,结果是认定的;操作时间较原方法缩短一半。结果表明该方法是可替代原法的快速测序法。     

Phylogeography of the flat-tailed horned lizard (Phrynosoma mcallii) and systematics of the P. mcallii-platyrhinos mtDNA complex

Two species of horned lizards are sympatric along the periphery of the Salton Trough. Phrynosoma mcallii, endemic to the trough, is of conservation concern because its limited habitat has been fragmented by human activities. A more common and widespread species, Phrynosoma platyrhinos, occurs around the periphery of the trough and much further to the North. The two species are syntopic at a few localities, where morphological intermediates have also been found. Here, we used nested clade phylogeographical analysis (NCPA) and analysis of molecular variance (amova), to examine 781 bp of mitochondrial DNA (nad4 and two tRNAs) from 82 individuals of P. mcallii. We tested whether populations of this species were recently connected, or if they were historically isolated prior to human modification of the region. Our NCPA results indicated significant population structure associated with the Colorado River, suggesting limited gene flow and potential isolation across this barrier. Populations west of the Colorado River, currently isolated from one another by human development, show less genetic differentiation. We also collected homologous sequence data from 34 individuals of P. platyrhinos and seven specimens morphologically intermediate between P. mcallii and P. platyrhinos, as a preliminary investigation of hybridization between these two species. From phylogenetic results of these data, we identified a species (Phrynosoma goodei) previously recognized as a subspecies of P. platyrhinos. Six of the morphologically intermediate specimens shared mtDNA haplotypes with P. goodei, while one was nested among P. mcallii haplotypes.     

A dispenser for the rapid delivery of standard inocula of the cyst nematode Globodera rostochiensis

A simple machined dispenser is described, capable of providing rapid repetitive delivery of standard volumes of potato cyst nematode cysts. Standard volumes of cysts of mixed sizes, drawn from unsieved populations of G. rostochiensis, contain titres of infective eggs and larvae which are largely independent of cyst numbers; the method reduces inoculum variability compared with other cyst inoculation techniques. Standard volumes of cysts of mixed sizes may be used directly as the inoculum for plant resistance testing programmes without the operator error associated with hand-held scoops or the need for the subjective and time consuming selection of groups of individual cysts.     

Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida

Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.     

A modified procedure for quantitative analysis of mtDNA, detecting mtDNA depletion

Quantitative analysis of mitochondrial DNA (mtDNA) is crucial for proper diagnosis of diseases that are caused by or associated with mtDNA depletion. However, such a quantitative characterization of mtDNA is not a simple procedure and requires several laboratory steps at which potential errors can accumulate. Here, we describe a modified procedure for quantitative human mtDNA analysis. The procedure is based on using two PCR-amplified, fluorescein-labeled DNA probes, complementary to mtDNA (detection probe) and chromosomal 18S rDNA (reference probe), both of similar length. Thus, equal amounts of these probes can be used and, contrary to previously published procedures, no mtDNA purification (apart from total DNA isolation) or 18S rDNA cloning is necessary for probe preparation. Two separate hybridizations (each with one probe) are suggested instead of one hybridization with both probes; this decreases background signals and enables adjustment of the strength of specific signals from both probes, which is useful in the subsequent densitometric analysis after superimposing of both pictures. Using different DNA amounts for reactions, we have proved that the procedure is quantitative in a broad range of sample DNA concentrations. Moreover, we were able to detect mtDNA depletion unambiguously in tissue samples from patients suffering from diseases caused by dysfunction of mtDNA.     

A rapid,efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library

With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5′ end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing.     

The use of allozyme electrophoresis in invertebrate systematics

The role of enzyme electrophoresis is discussed as it applies to taxonomy and systematics. particularly of invertebrates. Details are given of methods for distinguishing and identifying cryptic or sibling species and the different approaches to sympatric and allopatric populations are reviewed. The calculation and uses of genetic distance measures are outlined. as are the empirical relationship of such measures to different levels of taxonomic separation. Defficulties. drawbacks and limitations of the technique are explained together with the advantages. Evidence for molecular clocks is outlined briefly and their role in systematic studies is discussed, as are methods of analysing genetic divergence data for systematic purposes. References to studies covering a wide range of invertebrate taxa are tabulated.     

Molecular systematics analysis of Lymantria dispar based on 18S rRNA and cox1 mtDNA sequence data

The complete sequence of the 18 S ribosomal RNA gene(18S rRNA) from Lymantria dispar was cloned and analysed here. 18 S rRNA and mitochondrial cytochrome c oxidasel(cox1) gene sequences of Lymantria dispar were compared with homologous sequences of other nine insects from different orders. Analytic results showed that 18 S rRNA of these insects had two conserved domains and the second domain was an even more conserved region. The phylogenetic trees based on the full-length sequence and the second domain fragment of 18 S rRNA as well as sequence of cox1 from different orders indicated that Lepidoptera and Trichoptera, which belongs to Amphiesmenoptera, had a closer phylogenetic relationship and fewer differences were observed comparing with traditional taxonomic results.     

Oxidants and not alkylating agents induce rapid mtDNA loss and mitochondrial dysfunction

Mitochondrial DNA (mtDNA) is essential for proper mitochondrial function and encodes 22 tRNAs, 2 rRNAs and 13 polypeptides that make up subunits of complex I, III, IV, in the electron transport chain and complex V, the ATP synthase. Although mitochondrial dysfunction has been implicated in processes such as premature aging, neurodegeneration, and cancer, it has not been shown whether persistent mtDNA damage causes a loss of oxidative phosphorylation. We addressed this question by treating mouse embryonic fibroblasts with either hydrogen peroxide (H(2)O(2)) or the alkylating agent methyl methanesulfonate (MMS) and measuring several endpoints, including mtDNA damage and repair rates using QPCR, levels of mitochondrial- and nuclear-encoded proteins using antibody analysis, and a pharmacologic profile of mitochondria using the Seahorse Extracellular Flux Analyzer. We show that a 60min treatment with H(2)O(2) causes persistent mtDNA lesions, mtDNA loss, decreased levels of a nuclear-encoded mitochondrial subunit, a loss of ATP-linked oxidative phosphorylation and a loss of total reserve capacity. Conversely, a 60min treatment with 2mM MMS causes persistent mtDNA lesions but no mtDNA loss, no decrease in levels of a nuclear-encoded mitochondrial subunit, and no mitochondrial dysfunction. These results suggest that persistent mtDNA damage is not sufficient to cause mitochondrial dysfunction.     

Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA

We present here the use of fluorescent methodologies for structural and functional studies of RNA in place of radioactivity. The methods are highly sensitive and quantitative with the use of an infrared fluorescence imaging system. IRD-700 and IRD-800 labels are used for fluorescence detection. Chemical probing methods are largely used for mapping RNA secondary structure and to monitor ligand interactions and conformational changes involving individual bases of RNA. The new fluorescent primer extension methodology allows simple and fast chemical probing of RNA with high sensitivity. IRD-700 and IRD-800 labeled primers can also be used to monitor protein-RNA interactions by fluorescent mobility shift assays. The speed and ease of these approaches are advantages over prior methods that used hazardous radioisotopes. Structural and biochemical investigations of RNA should benefit from the use of these fluorescent methodologies.