Mitochondrial cox1 sequence data reliably uncover patterns of insect diversity but suffer from high lineage-idiosyncratic error rates

Background

The demand for scientific biodiversity data is increasing, but taxonomic expertise is often limited or not available. DNA sequencing is a potential remedy to overcome this taxonomic impediment. Mitochondrial DNA is most commonly used, e.g., for species identification (“DNA barcoding”). Here, we present the first study in arthropods based on a near-complete species sampling of a family-level taxon from the entire Australian region. We aimed to assess how reliably mtDNA data can capture species diversity when many sister species pairs are included. Then, we contrasted phylogenetic subsampling with the hitherto more commonly applied geographical subsampling, where sister species are not necessarily captured.

Methodology/Principal Findings

We sequenced 800 bp cox1 for 1,439 individuals including 260 Australian species (78% species coverage). We used clustering with thresholds of 1 to 10% and general mixed Yule Coalescent (GMYC) analysis for the estimation of species richness. The performance metrics used were taxonomic accuracy and agreement between the morphological and molecular species richness estimation. Clustering (at the 3% level) and GMYC reliably estimated species diversity for single or multiple geographic regions, with an error for larger clades of lower than 10%, thus outperforming parataxonomy. However, the rates of error were higher for some individual genera, with values of up to 45% when very recent species formed nonmonophyletic clusters. Taxonomic accuracy was always lower, with error rates above 20% and a larger variation at the genus level (0 to 70%). Sørensen similarity indices calculated for morphospecies, 3% clusters and GMYC entities for different pairs of localities was consistent among methods and showed expected decrease over distance.

Conclusion/Significance

Cox1 sequence data are a powerful tool for large-scale species richness estimation, with a great potential for use in ecology and β-diversity studies and for setting conservation priorities. However, error rates can be high in individual lineages.     

Comparative genomics of the social amoebae <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> and <Emphasis Type="Italic">Dictyostelium purpureum</Emphasis>

Background

The social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum.

Results

We have produced a draft genome sequence of another group dictyostelid, Dictyostelium purpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict.

Conclusions

The findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia.

     

Sheen Screen, a Miniaturized Most-Probable-Number Method for Enumeration of Oil-Degrading Microorganisms

Sheen Screen is a miniaturized method for enumerating oil-degrading microorganisms. The technique relies on the ability of oil-degrading microorganisms to emulsify oil when provided as a sole carbon source in 24-well tissue culture plates. Sediments that actively respire hydrocarbons have high numbers of Sheen Screen-positive microorganisms.     

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK a

Several small ribozymes employ general acid–base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK_a values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK_a values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid–base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid–base catalysis indicate that k₁, the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pK_a. The inference that k₁ is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK_a is that of the general acid and the higher pK_a is that of the general base.     

Synthesis, crystal structure and magnetic characterization of a series of four phenoxo-bridged binuclear manganese(III) Schiff base complexes

A family of four new phenoxo-bridged binuclear manganese(III) complexes of the general formula, [Mn(L)(X)]₂ where L = [N,N′-bis(salicylidene)]propane-1,2-diamine and X = salicylaldehyde anion (sal⁻) (1); NCS⁻ (2); NCO⁻ (3) and [Mn(L′)(N₃)]₂·2C₂H₅OH (4) where L′ = [N,N′-bis(2-hydroxyacetophenylidene)]propane-1,2-diamine has been prepared. The syntheses have been achieved by reacting manganese perchlorate with 1,2-diaminopropane and salicylaldehyde (or 2-hydroxyacetophenone for 4) or along with the respective pseudohalides so that the tetradentate Schiff base H₂L or H₂L′ is obtained in situ to bind the Mn(III) ion. The complexes have been characterized by IR spectroscopy, elemental analysis, crystal structure analysis and variable-temperature magnetic susceptibility measurements. The single crystal X-ray diffraction studies show that the compounds are isostructural containing dimeric Mn(III) units with bridging phenolate oxygen atoms. Low temperature magnetic studies indicate that the complexes 1-3 exhibit intradimer ferromagnetic exchange as well as single-molecule magnet (SMM) behavior while complex 4 is found to undergo an intradimer antiferromagnetic coupling.     

Distribution and Outcomes of a Phenotype-Based Approach to Guide COPD Management: Results from the CHAIN Cohort

RationaleThe Spanish guideline for COPD (GesEPOC) recommends COPD treatment according to four clinical phenotypes: non-exacerbator phenotype with either chronic bronchitis or emphysema (NE), asthma-COPD overlap syndrome (ACOS), frequent exacerbator phenotype with emphysema (FEE) or frequent exacerbator phenotype with chronic bronchitis (FECB). However, little is known on the distribution and outcomes of the four suggested phenotypes.ObjectiveWe aimed to determine the distribution of these COPD phenotypes, and their relation with one-year clinical outcomes.MethodsWe followed a cohort of well-characterized patients with COPD up to one-year. Baseline characteristics, health status (CAT), BODE index, rate of exacerbations and mortality up to one year of follow-up were compared between the four phenotypes.ResultsOverall, 831 stable COPD patients were evaluated. They were distributed as NE, 550 (66.2%); ACOS, 125 (15.0%); FEE, 38 (4.6%); and FECB, 99 (11.9%); additionally 19 (2.3%) COPD patients with frequent exacerbations did not fulfill the criteria for neither FEE nor FECB. At baseline, there were significant differences in symptoms, FEV₁ and BODE index (all p<0.05). The FECB phenotype had the highest CAT score (17.1±8.2, p<0.05 compared to the other phenotypes). Frequent exacerbator groups (FEE and FECB) were receiving more pharmacological treatment at baseline, and also experienced more exacerbations the year after (all p<0.05) with no differences in one-year mortality. Most of NE (93%) and half of exacerbators were stable after one year.ConclusionsThere is an uneven distribution of COPD phenotypes in stable COPD patients, with significant differences in demographics, patient-centered outcomes and health care resources use.     

Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells

Structural studies of asparagine-linked glycoproteins are complicated by the oligosaccharide heterogeneity inherent to individual glycosylation sites. Herein, we report the cloning of a novel isoform of avian Thy-1 and the subsequent expression, purification, and characterization of a soluble form of Thy-1 from Lec1 mammalian and Tn5 insect cells. The novel isoform of Thy-1 differs from the previously reported chicken isoform by eight amino acid residues, but these changes do not alter the secondary structure content, the disulfide bond pattern, or the sites of glycosylation. The disulfide linkage pattern and glycoform distribution on each N-glycosylation site of recombinant chicken Thy-1 from both cell lines were determined by a combination of amino-terminal sequencing and mass spectrometry. The mass spectral data showed that the amino-terminal glutamine was modified to pyroglutamate. Recombinant Thy-1 from Lec1 cells contained (GlcNAc)(2)(Man)(5) on asparagine 60, whereas the oligosaccharides on asparagine 23 and 100 contained approximately 80% (GlcNAc)(2)(Man)(4) and approximately 20% (GlcNAc)(2)(Man)(5). The glycoforms on Thy-1 expressed in Tn5 cells were more heterogeneous, with the oligosaccharides ranging over (GlcNAc)(2)(Fuc)(0-2)(Man)(2-3) on each site. The ability to generate recombinant glycoproteins with restricted carbohydrate heterogeneity is the first step toward the systematic study of structure-function relationships in intact glycoproteins.     

Developmental changes of FABP concentration,expression, and intracellular distribution in locust flight muscle

M-FABP from flight muscle of the locust,Schistocerca gregaria, is similar to mammalian H-FABP in its physical characteristics and amino acid sequence. We have studied developmental changes using ELISA, Northern Blotting, and EM/immuno-gold techniques. M-FABP is found in cytoplasm and nuclei, but not in mitochondria. It is the most abundant soluble muscle protein in fully developed adult locusts, comprising 18% of the total cytosolic protein. However, no FABP is detectable at the beginning of the adult stage. Its concentration rises dramatically during the next 10 days, after which it reaches its maximal value. Expression apparently is turned on after adult ecdysis and continues for 10 days; thereafter, FABP mRNA diminishes and reaches a constant, but low level, probably needed to maintain the current FABP level. From a series of experiments employing metamorphosis-controlling hormones and antihormones it is evident that the induction of FABP expression is directly linked to metamorphosis.Abbreviations ELISA Enzyme Linked Immuno Sorbent Assay - FABP Fatty Acid-Binding Protein - H-FABP mammalian Heart Fatty Acid-Binding Protein - M-FABP locust flight Muscle Fatty Acid-Binding Protein