A search for phylogenetically informative wood characters within Lecythidaceae s.l

The wood structure of 71 species representing 24 genera of the pantropical Lecythidaceae s.l., including the edible Brazil nuts (Bertholletia excelsa) and the spectacular cannon-ball tree (Couroupita guianensis), was investigated using light and scanning electron microscopy. This study focused on finding phylogenetically informative characters to help elucidate any obscure evolutionary patterns within the family. The earliest diverging subfamily Napoleonaeoideae has mixed simple/scalariform vessel perforations, scalariform vessel-ray pitting, and high multiseriate rays, all features that are also present in Scytopetaloideae. The wood structure of Napoleonaea is distinct, but its supposed close relative Crateranthus strongly resembles Scytopetaloideae. The isolated position of Foetidia (Foetidioideae) can be supported by a unique type of vessel-ray pitting that is similar in shape and size to intervessel pitting (distinctly bordered, <5 μm). The more derived Planchonioideae and Lecythidoideae share exclusively simple perforations and two types of vessel-ray pitting, but they can easily be distinguished from each other by the size of intervessel pitting, shape of body ray cells in multiseriate rays, and the type of crystalliferous axial parenchyma cells. The anatomical diversity observed is clearly correlated with differences in plant size (shrubs vs. tall trees): the percentage of scalariform perforations, as well as vessel density, and the length of vessel elements, fibers, and multiseriate rays are negatively correlated with increasing plant size, while the reverse is true for vessel diameter.     

Automated scanning for phylogenetically informative transposed elements in rodents

Transposed elements constitute an attractive, useful source of phylogenetic markers to elucidate the evolutionary history of their hosts. Frequent and successive amplifications over evolutionary time are important requirements for utilizing their presence or absence as landmarks of evolution. Although transposed elements are well distributed in rodent taxa, the generally high degree of genomic sequence divergence among species complicates our access to presence/absence data. With this in mind we developed a novel, high-throughput computational strategy, called CPAL (Conserved Presence/Absence Locus-finder), to identify genome-wide distributed, phylogenetically informative transposed elements flanked by highly conserved regions. From a total of 232 extracted chromosomal mouse loci we randomly selected 14 of these plus 2 others from previous test screens and attempted to amplify them via PCR in representative rodent species. All loci were amplifiable and ultimately contributed 31 phylogenetically informative markers distributed throughout the major groups of Rodentia.     

A composite genome approach to identify phylogenetically informative data from next-generation sequencing

Background

Improvements in sequencing technology now allow easy acquisition of large datasets; however, analyzing these data for phylogenetics can be challenging. We have developed a novel method to rapidly obtain homologous genomic data for phylogenetics directly from next-generation sequencing reads without the use of a reference genome. This software, called SISRS, avoids the time consuming steps of de novo whole genome assembly, multiple genome alignment, and annotation.

Results

For simulations SISRS is able to identify large numbers of loci containing variable sites with phylogenetic signal. For genomic data from apes, SISRS identified thousands of variable sites, from which we produced an accurate phylogeny. Finally, we used SISRS to identify phylogenetic markers that we used to estimate the phylogeny of placental mammals. We recovered eight phylogenies that resolved the basal relationships among mammals using datasets with different levels of missing data. The three alternate resolutions of the basal relationships are consistent with the major hypotheses for the relationships among mammals, all of which have been supported previously by different molecular datasets.

Conclusions

SISRS has the potential to transform phylogenetic research. This method eliminates the need for expensive marker development in many studies by using whole genome shotgun sequence data directly. SISRS is open source and freely available at https://github.com/rachelss/SISRS/releases.

     

DNA sequence polymorphism within hominoid species exceeds the number of phylogenetically informative characters for a HOX2 locus.

Within- and between-species variability was examined in a noncoding 238-bp segment of the HOX2 cluster. DNA of 4-26 individuals of four species (Pongo pygmaeus, Pan troglodytes, Gorilla gorilla, and Homo sapiens) was PCR amplified and electrophoresed in a denaturing gradient gel to screen for variability. Coupled amplification and sequencing was used to determine the complete sequence for each of the different alleles identified, one each in humans and orangutans, two in chimpanzees, and four in gorillas. Maximum-parsimony methods were used to construct a gene tree for these sequences. Alleles in all four species cluster into groups consisting of only one species (i.e., alleles within a species are monophyletic). The number of base-pair differences observed among alleles within P. troglodytes and within G. gorilla is larger than the number of base-pair substitutions that phylogenetically link Pan with Homo. Given these and other published data, it is premature to accept any particular phylogenetic tree that relates these three genera through two separate speciation events.     

Trichome morphology provides phylogenetically informative characters for Tremandra, Platytheca and Tetratheca (former Tremandraceae)

Summary Trichomes of Tremandra R.Br. ex DC., Platytheca Steetz and Tetratheca Sm. (Elaeocarpaceae, former Tremandraceae), together with two outgroup species of Elaeocarpus L., are illustrated using scanning electron microscopy, and their distribution on various plant organs is documented. Various trichomes types were identified that relate taxa: simple hairs, stellate hairs, short glandular trichomes, long glandular trichomes, and three forms of tubercules. Both outgroup and ingroup taxa have simple hairs. Stellate hairs are confirmed as unique to Tremandra. Prominent and sculptured multicelled tubercules, some bearing a stout hair, are characteristic of Platytheca. Smaller multicelled tubercules occur in both Platytheca and Tetratheca, except for the Western Australian taxon Te. filiformis Benth. (possibly plesiomorphic). Unicellular tubercules (papilla) characterise two species of Tetratheca. Short glandular trichomes, usually found on the ovary, also occur in both of these genera but not in all species (possibly secondary losses), while long glandular trichomes, usually on stems and leaves, occur only in some groups of Tetratheca. Within Tetratheca, Western Australian taxa that have five-merous flowers fall into three ‘groups’: seven species (together with one from South Australia) that have short glandular trichomes but no long glandular trichomes; six species that have long glandular trichomes but no short glandular trichomes; and four species that have both trichome types. All other species of Tetratheca have four-merous flowers and form two ‘groups’: 12 eastern species (including one from South Australia) that have both short glandular trichomes and long glandular trichomes; 4 western species and six eastern species that lack short glandular trichomes. On the basis of these characters, a phylogenetic hypothesis for the three genera is presented.     

A multiplex bead-based suspension array assay for interrogation of phylogenetically informative single nucleotide polymorphisms for Bacillus anthracis

Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and represent informative DNA markers extensively used to analyze phylogenetic relationships between strains. Medium to high throughput, open methodologies able to test many SNPs in a minimum time are therefore in great need. By using the versatile Luminex® xTAG technology, we developed an efficient multiplexed SNP genotyping assay to score 13 phylogenetically informative SNPs within the genome of Bacillus anthracis. The Multiplex Oligonucleotide Ligation-PCR procedure (MOL-PCR) described by Deshpande et al., 2010 has been modified and adapted for simultaneous interrogation of 13 biallelic canonical SNPs in a 13-plex assay. Changes made to the originally published method include the design of allele-specific dual-priming-oligonucleotides (DPOs) as competing detection probes (MOLigo probes) and use of asymmetric PCR reaction for signal amplification and labeling of ligation products carrying SNP targets. These innovations significantly reduce cross-reactivity observed when initial MOLigo probes were used and enhance hybridization efficiency onto the microsphere array, respectively. When evaluated on 73 representative samples, the 13-plex assay yielded unambiguous SNP calls and lineage affiliation. Assay limit of detection was determined to be 2 ng of genomic DNA. The reproducibility, robustness and easy-of-use of the present method were validated by a small-scale proficiency testing performed between four European laboratories. While cost-effective compared to other singleplex methods, the present MOL-PCR method offers a high degree of flexibility and scalability. It can easily accommodate newly identified SNPs to increase resolving power to the canSNP typing of B. anthracis.     

Cophenetic correlation analysis as a strategy to select phylogenetically informative proteins: an example from the fungal kingdom

Background  

The construction of robust and well resolved phylogenetic trees is important for our understanding of many, if not all biological processes, including speciation and origin of higher taxa, genome evolution, metabolic diversification, multicellularity, origin of life styles, pathogenicity and so on. Many older phylogenies were not well supported due to insufficient phylogenetic signal present in the single or few genes used in phylogenetic reconstructions. Importantly, single gene phylogenies were not always found to be congruent. The phylogenetic signal may, therefore, be increased by enlarging the number of genes included in phylogenetic studies. Unfortunately, concatenation of many genes does not take into consideration the evolutionary history of each individual gene. Here, we describe an approach to select informative phylogenetic proteins to be used in the Tree of Life (TOL) and barcoding projects by comparing the cophenetic correlation coefficients (CCC) among individual protein distance matrices of proteins, using the fungi as an example. The method demonstrated that the quality and number of concatenated proteins is important for a reliable estimation of TOL. Approximately 40–45 concatenated proteins seem needed to resolve fungal TOL.     

Quality control of bacterial enumeration.

Standard bacterial suspensions can be used to assess test method performance, via control charts, and inhibition of recovery when analyzing water samples. Variability in standard suspensions prepared from different strains and species and the use of frozen environmental samples for quality control for spore and bacteriophage analyses are also discussed.     

Quantitative procedure for enumeration of bifidobacteria.

A membrane filter technique has been developed for the enumeration of bifidobacteria in natural aquatic environments. The technique is quantitative, selective, and differential. The medium (YN-6) contains: yeast extract, 2.0 g; agar, 1.5 g; polypeptone peptone, 1.0 g; vitamin-free Casamino Acids, 0.8 g; sodium chloride, 0.32 g; and L-cysteine hydrochloride, 0.003 g; in 100 ml of deionized water. The medium is adjusted to pH 7.0 before autoclaving. Nalidixic acid (80 micrograms/ml), neomycin sulfate (2.5 micrograms/ml), and bromcresol green (300 micrograms/ml) are included as selective and differential agents. After incubation for 48 h at 37 degrees C in an anaerobic environment, Gram-stained smears from green, glistening, smooth entire colonies are examined microscopically for typical bifidobacterial morphology. No significant difference in recoveries was observed when YN-6 was compared with reinforced clostridial agar, using bifidobacteria freshly isolated from feces and raw sewage. Using this technique with aquatic and fecal samples, less than 9% false-positive and 8% false-negative isolates were observed. These results indicated that the medium was able to satisfactorily recover organisms from a variety of situations.     

Structural analysis of phylogenetically conserved J domain protein gene.

Novel cDNAs encoding evolutionarily conserved J Domain Proteins (JDPs) were investigated from Drosophila and mouse. Each of the full coding sequences potentially encodes a conserved J domain, but lacks additional characteristic structures present in DnaJ family proteins. The expression was restricted to head in Drosophila. However, ubiquitous expression was observed in mice with the highest level in kidney.     

An Acanthamoeba sp. containing two phylogenetically different bacterial endosymbionts

Acanthamoebae are ubiquitous free-living amoebae and important predators of microbial communities. They frequently contain obligate intracellular bacterial symbionts, which show a worldwide distribution. All Acanthamoeba spp. described so far harboured no or only a single specific endosymbiont phylotype, and in some cases evidence for coevolution between the symbiotic bacteria and the amoeba host has been reported. In this study we have isolated and characterized an Acanthamoeba sp. (strain OEW1) showing a stable symbiotic relationship with two morphologically different endosymbionts. 16S rRNA sequence analysis assigned these symbionts to the candidate genus Procabacter (Betaproteobacteria) and the genus Parachlamydia (Chlamydiae) respectively. Fluorescence in situ hybridization and transmission electron microscopy confirmed the affiliation of the endosymbionts and showed their co-occurrence in the amoeba host cells and their intracellular location within separate compartments enclosed by host-derived membranes. Further analysis of this stable relationship should provide novel insights into the complex interactions of intracellular multiple-partner associations.     

Methodology for enumeration of coliphages in foods.

The effects of eluent composition, pH, and chaotropic agents on the recovery of T2, MS2, and indigenous coliphages from various foods were investigated. Additionally, methods of sample suspension and clarification were evaluated for coliphage recovery and application to various foods. Clarified sample suspensions were assayed for coliphages with a modified agar layer technique and appropriate Escherichia coli hosts. Centrifugation and polypropylene mesh filtration were more rapid and effective than glass wool filtration for clarification of sample suspensions and subsequent recovery of coliphages. Blending, stomaching, and shaking procedures were generally comparable for sample liquefaction and release of coliphages from foods. Complex basal eluents, EC medium and 1% casein, were generally more effective than a less complex eluent, phosphate buffer, for elution of coliphages from foods. For most foods, incorporation of sodium chloride or chaotropic agents, i.e., sodium trichloroacetate, urea, Tween 80, Triton X-100, and sodium nitrate, into basal eluents did not enhance recovery of coliphages. Indigenous coliphage recovery was not affected by sample suspension pH over a range of 6.0 to 9.0. With an optimal procedure, i.e., EC medium eluent, blending, and centrifugation, the recovery of T2 and MS2 ranged from 48 to 81% and from 58 to 100%, respectively, depending on the food type.     

Timed-release capsule method for coliform enumeration.

Wax-coated capsules containing selective ingredients (brilliant green and oxgall) were added at the time of inoculation of most-probable-number media (modified lactose broths). The inhibitory ingredients gradually diffused from the capsules into the nonselective media, imparting selectivity to the media. Concentrations of brilliant green did not reach inhibitory levels until 2 or more h had elapsed, which permitted repair of some injured cells. Resuscitation of heat-injured Escherichia coli B cells occurred in the capsule-containing media, but not in conventional brilliant green bile 2% broth or violet red bile agar. No statistically significant differences were noted between coliform counts obtained on two groups of water samples by using the capsule, most-probable-number, membrane filtration, and pour plate methods. The capsule method could be used, however, as a combined presumptive and confirmed test for the examination of water. Improvements are needed to adapt the capsule method to the analysis of some categories of food.     

Treatment of homozygous familial hypercholesterolaemia: an informative sibship.

In a family in which both parents had the heterozygous form of familial hypercholesterolaemia four of the children had the homozygous form. The three oldest homozygous children, two of whom did not receive any treatment and in one of whom treatment did not lower the plasma cholesterol concentration, developed xanthomas in early childhood and died aged 3, 9, and 10 years. The fourth homozygous child was treated with diet and drugs from the age of 1 and at the age of 15 had no xanthomas, no clinical evidence of heart disease, and a virtually normal coronary angiogram. His plasma cholesterol concentration was reduced by about 30% but remained considerably raised. It is concluded that treatment, if started before atherosclerosis develops, can delay the onset of atheroma and coronary heart disease even though normal plasma cholesterol concentrations are not achieved.     

Effects of betaine on enumeration of airborne bacteria.

The osmoprotectant betaine was incorporated into collection fluid and enumeration medium to determine its effects on the colony-forming abilities of airborne bacteria, which were collected from three separate locations: a wastewater treatment plant, the roof of a laboratory building, and an unobstructed farmland. At all locations, addition of 2 to 5 mM betaine caused a significant increase (from 21.6 to 61.3%) in colonial outgrowth, compared with the growth rate of controls without betaine. The presence of betaine in both the collection fluid and the enumeration medium had an additive effect on the colony-forming ability of airborne bacteria compared with the presence of betaine in either one alone. The effect of various betaine concentrations on the enumeration of aerosolized Pseudomonas syringae was determined. Betaine showed a threshold for maximum effect at a concentration of 2 to 5 mM. At higher concentrations (10 to 20 mM), the effects of betaine were negligible or possibly inhibitory. The significance of these results with respect to the development of protocols for monitoring airborne microorganisms, including genetically engineered microorganisms, is discussed.