Slow molecular evolution in an ancient asexual ostracod

Genetic variability of the non-marine ostracod species Darwinula stevensoni was estimated by sequencing part of the nuclear and the mitochondrial genome. As Darwinulidae are believed to be ancient asexuals, accumulation of mutations should have occurred, both between alleles within lineages and between lineages, during the millions of years of parthenogenetic reproduction. However, our sequence data show the opposite: no variability in the nuclear ITS1 region was observed within or among individuals of D. stevensoni, despite sampling a geographical range from Finland to South Africa. Lack of allelic divergence might be explained by concerted evolution of rDNA repeats. Homogeneity among individuals may be caused either by slow molecular evolution in ITS1 or by a recent selective sweep. Variability of mitochondrial cytochrome oxidase (COI) was similar to intraspecific levels in other invertebrates, thus weakening the latter hypothesis. Calibrating interspecific, genetic divergences among D. stevensoni and other Darwinulidae using their fossil record enabled us to estimate rates of molecular evolution. Both COI and ITS1 evolve half as fast, at most, in darwinulids as in other invertebrates, and molecular evolution has significantly slowed down in ITS1 of D. stevensoni relative to other darwinulids. A reduced ITS1 mutation rate might explain this inconsistency between nuclear and mitochondrial evolution in D. stevensoni.     

Genomic clues to an ancient asexual scandal

Despite abandoning meiosis, the bdelloid rotifers have persisted for millions of years and given rise to hundreds of species. Several mechanisms - allelic variants with different functions, high effective population size, and resistance to radiation - may contribute to their success.     

PROSOFT: A general purpose software in protein chemistry

Applesoft and 6502 Assembler software was designed to quicklyperform operations commonly encountered in protein chemistry.It was not designed for a specific application but can be convenientlyused to speed up the determination of protein primary structure. Received on October 6, 1987; accepted on November 10, 1987     

OntoVisT: A general purpose Ontological Visualization Tool

Ontologies have emerged as a fast growing research topic in the area of semantic web during last decade. Currently there are 204 ontologies that are available through OBO Foundry and BioPortal. Several excellent tools for navigating the ontological structure are available, however most of them are dedicated to a specific annotation data or integrated with specific analysis applications, and do not offer flexibility in terms of general-purpose usage for ontology exploration. We developed OntoVisT, a web based ontological visualization tool. This application is designed for interactive visualization of any ontological hierarchy for a specific node of interest, up to the chosen level of children and/or ancestor. It takes any ontology file in OBO format as input and generates output as DAG hierarchical graph for the chosen query. To enhance the navigation capabilities of complex networks, we have embedded several features such as search criteria, zoom in/out, center focus, nearest neighbor highlights and mouse hover events. The application has been tested on all 72 data sets available in OBO format through OBO foundry. The results for few of them can be accessed through OntoVisT-Gallery. AVAILABILITY: The database is available for free at http://ccbb.jnu.ac.in/OntoVisT.html.     

The use of an inexpensive,general purpose microcomputer in quantitative cytochemistry

Summary The histochemical localization of alkaline phosphatase in sheep parathyroid gland was investigated using light and electron microscopy. The reaction products of enzyme activity were observed by light microscopy in pericytes. By electron microscopy they were limited to the intercellular spaces between the gland cells, being exclusively confined to the external surface of plasma membrane.     

ArrayD: A general purpose software for Microarray design

Background  

Microarray is a high-throughput technology to study expression of thousands of genes in parallel. A critical aspect of microarray production is the design aimed at space optimization while maximizing the number of gene probes and their replicates to be spotted.     

A general purpose computer analysis system for chromatographic data

A manual integration system for the analysis of chromatographicdata is described. The analog output produced by an HPLC absorbancemonitor is passed to a non-inverting signal amplifier. Thisamplified signal is sent to an IBM PC where an analog to digitalconverter is used to digitize the data. A set of six computerprograms which collect, store and analyze these data are presented.This system was used to analyze the nucleotide content of theanaerobic organism Clostridium aceto-butylicum by strong anion-exchangeHPLC. Received on May 26, 1987; accepted on November 15, 1987     

A general purpose method for extracting RNA from Dictyostelium cells

Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods; however, it has been adjusted because of the large quantities of carbohydrate and nucleases found in Dictyostelium cells. After cell lysis and phenol:chloroform extraction, the resulting high-quality RNA isolated with the described protocol allows the molecular genetic analysis of wild-type and genetically modified cells. The purified RNA can be used for analyses such as northern blotting, RT-PCR and microarrays. This procedure requires approximately 2 h to complete.     

Recombination in Glomus intraradices, a supposed ancient asexual arbuscular mycorrhizal fungus

Background  

Arbuscular mycorrhizal fungi (AMF) are important symbionts of most plant species, promoting plant diversity and productivity. This symbiosis is thought to have contributed to the early colonisation of land by plants. Morphological stasis over 400 million years and the lack of an observed sexual stage in any member of the phylum Glomeromycota led to the controversial suggestion of AMF being ancients asexuals. Evidence for recombination in AMF is contradictory.     

Cryptic species in putative ancient asexual darwinulids (Crustacea, Ostracoda)

Background

Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied.

Methodology/Principal Findings

We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies.

Conclusions/Significance

Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.     

Clonal diversity in the ancient asexual ostracod Darwinula stevensoni assessed by RAPD-PCR

As Darwinulidae (Ostracoda) are considered to be ancient asexuals with a wide geographical and ecological distribution, they are expected to have accumulated mutations during a long timeframe. However, previous studies on genetic variability suggested a low genetic divergence within the darwinulid species Darwinula stevensoni. Here, overall genotopic diversity of D. stevensoni is estimated with the Random Amplified Polymorphic DNA (RAPD) technique. Using six primers revealing 47 consistently scorable polymorphic loci, substantial clonal diversity within this species is detected. Five of the seven surveyed populations are multiclonal. Moreover, the seven populations have a different clonal composition with almost all of the observed clonal genotypes being restricted to single populations, indicating the absence of a single widespread 'clone'. The observed clonal diversity seems to refute the existence of a widespread general purpose genotype for D. stevensoni. However, in light of previously detected uniformity at functional loci, we reconsider the definition of a GPG. We suggest that it need not imply a genome-wide fixed genotype, but rather consists of a set of ecologically relevant genes.     

Transposon proliferation in an asexual parasitoid

The widespread occurrence of sex is one of the most elusive problems in evolutionary biology. Theory predicts that asexual lineages can be driven to extinction by uncontrolled proliferation of vertically transmitted transposable elements (TEs), which accumulate because of the inefficiency of purifying selection in the absence of sex and recombination. To test this prediction, we compared genome-wide TE load between a sexual lineage of the parasitoid wasp Leptopilina clavipes and a lineage of the same species that is rendered asexual by Wolbachia-induced parthenogenesis. We obtained draft genome sequences at 15-20× coverage of both the sexual and the asexual lineages using next-generation sequencing. We identified transposons of most major classes in both lineages. Quantification of TE abundance using coverage depth showed that copy numbers in the asexual lineage exceeded those in the sexual lineage for DNA transposons, but not LTR and LINE-like elements. However, one or a small number of gypsy-like LTR elements exhibited a fourfold higher coverage in the asexual lineage. Quantitative PCR showed that high loads of this gypsy-like TE were characteristic for 11 genetically distinct asexual wasp lineages when compared to sexual lineages. We found no evidence for an overall increase in copy number for all TE types in asexuals as predicted by theory. Instead, we suggest that the expansions of specific TEs are best explained as side effects of (epi)genetic manipulations of the host genome by Wolbachia. Asexuality is achieved in a myriad of ways in nature, many of which could similarly result in TE proliferation.     

A general purpose parallel block structured open source incompressible flow solver

A general purpose incompressible flow solver, called caffa3d.MBRi, is presented which features a block structured framework to accommodate both a flexible approach to geometry representation and a straightforward implementation of parallel capabilities through the MPI library. Representation of complex geometries can be handled semi automatically through a combination of body fitted blocks of grids and the immersed boundary condition strategy over both Cartesian and body fitted grid blocks. The parallelization strategy is based on the same block structured framework, by means of encapsulated MPI calls performing a set of conceptually defined high level communication tasks. A set of real world applications ranging from bioengineering to microclimate scenarios is presented to demonstrate the capabilities of the solver, which is open source and freely available through the web page.     

Chloroformates in gas chromatography as general purpose derivatizing agents

Chloroformates with simplest alkyls, i.e. methyl, ethyl or isobutyl, already known as favourable reagents for treating amino groups in gas chromatography for years, were revealed randomly as exceptionally rapid esterification agents. Unlike the rather poor results achieved with chloroformate-mediated ester formation in organic chemistry, the pyridine-catalyzed esterification of carboxylic acids appeared to proceed at the analytical microscale smoothly. Along with the catalyzer, an alcohol should also be present in the medium, accompanied by acetonitrile or water, according to the character of the compounds treated. Reaction conditions were optimized for various classes of carboxylic acids and a uniquely rapid derivatization of amino acids in aqueous ethanol was shown to be possible. Most of the analytes, e.g. acidic metabolites in physiological fluids, could be treated directly in the aqueous matrix. A simultaneous analysis of, e.g., amino and fatty acids or amines and their acidic catabolytes was proven to be possible. Along with the low-molecular-mass reagents, still some others, i.e. the hexyl, menthyl or pentafluorobenzyl ones, found their application fields. Results of optimized reaction conditions and a wide range of applications of chloroformate-mediated derivatization in various disciplines have been summarized in this review.     

Ecological strategies in the ancient asexual animal group Darwinulidae (Crustacea, Ostracoda)

• 1 We investigated the relationship between geographical distribution and ecological tolerance within the ancient asexual family Darwinulidae. Distribution maps were compiled based on data from the literature, the Non‐marine Ostracod Distribution in Europe database and personal collections. Ecological tolerance was assessed experimentally by exposing individual ostracods to a combination of eight different salinities (range from 0 to 30 g L^?1) and three different temperatures (10, 20 and 30 °C).
• 2 The type species of the family, Darwinula stevensoni, is ubiquitous and cosmopolitan; the two species Penthesilenula brasiliensis and Microdarwinula zimmeri also have an intercontinental distribution. Two other darwinulid species tested here (Vestalenula molopoensis and P. aotearoa) are known only from their type localities. The latter is also true for most extant darwinulids.
• 3 Darwinula stevensoni and P. brasiliensis had a broad salinity tolerance, tolerating distilled water and also salinity up to 25–30 g L^?1, whereas the maximum salinity tolerance of V. molopoensis was 12 g L^?1 and of P. aotearoa, 20 g L^?1.
• 4 The results indicate that both ecological specialists and generalists, as well as intermediate forms, exist in the Darwinulidae and that taxa with the broadest ecological tolerance also have the widest distribution.

     

A reliable general purpose method for extracting genomic DNA from Dictyostelium cells

In this protocol, we present a standard method for extracting DNA from cells of the social amoeba Dictyostelium discoideum. While this procedure is similar to other phenol:chloroform-based purification methods, it is modified to account for the high level of carbohydrate and nucleases found in Dictyostelium cells. Genomic DNA can be isolated from wild-type and genetically modified cells using the described protocol, allowing molecular genetic analyses to be performed. Following cell lysis, nucleic acid extraction, and precipitation, the isolated DNA is suitable for digestion by restriction enzymes, amplification by PCR and Southern blotting. This procedure takes approximately 3 h to complete.     

A general purpose non-linear curve fitting program for the British Broadcasting Corporation Microcomputer

Software for non–linear curve fitting has been writtenin BASIC to execute on the British Broadcasting CorporationMicrocomputer. The program uses the direct search algorithmPattern–search, a robust algorithm that has the additionaladvantage of needing specification of the Junction without inclusionof the partial derivatives. Although less efficient than gradientmethods, the program can be readily configured to solve low–dimensionaloptimization problems that are normally encountered in lifesciences. In writing the software, emphasis has been placedupon the ‘user interface’ and making the most efficientuse of the facilities provided by the minimal configurationof this system. Received on March 4, 1985; accepted on March 14, 1985     

A general purpose computerized display and analysis system for image processing in nuclear medicine.

In this work a general purpose image processor is described, which takes into account the special features and the various requirements for analysing images in nuclear medicine. A new approach for system structure involving encoding and representing medical images is given. This encoder, which is part of the image processor, is efficient especially for long-term storage and for certain pattern recognition in medical imaging. In this processor a statistical package is incorporated for collecting medical statistics using the acquired patient data. This information is used together with the encoder for an automated diagnostic system.