Disruption of the Orion molecular core 1 by wind from the massive star θ1 Orionis C.

Massive stars inject mechanical and radiative energy into the surrounding environment, which stirs it up, heats the gas, produces cloud and intercloud phases in the interstellar medium, and disrupts molecular clouds (the birth sites of new stars1,2). Stellar winds, supernova explosions and ionization by ultraviolet photons control the lifetimes of molecular clouds3-7. Theoretical studies predict that momentum injection by radiation should dominate that by stellar winds8, but this has been difficult to assess observationally. Velocity-resolved large-scale images in the fine-structure line of ionized carbon ([C II]) provide an observational diagnostic for the radiative energy input and the dynamics of the interstellar medium around massive stars. Here we report observations of a one-square-degree region (about 7 parsecs in diameter) of Orion molecular core 1-the region nearest to Earth that exhibits massive-star formation-at a resolution of 16 arcseconds (0.03 parsecs) in the [C II] line at 1.9 terahertz (158 micrometres). The results reveal that the stellar wind originating from the massive star θ1 Orionis C has swept up the surrounding material to create a 'bubble' roughly four parsecs in diameter with a 2,600-solar-mass shell, which is expanding at 13 kilometres per second. This finding demonstrates that the mechanical energy from the stellar wind is converted very efficiently into kinetic energy of the shell and causes more disruption of the Orion molecular core 1 than do photo-ionization and evaporation or future supernova explosions.

The Importance of Contact Zones for Distinguishing Interspecific from Intraspecific Geographic Variation.

With limited sampling, geographic variation within a single species can be difficult to distinguish from interspecific variation, confounding our ability to draw accurate species boundaries. We argue that thorough sampling and analysis of contact zones between putative taxa can determine if assortative mating or selection against hybrids exists (supporting the presence of two distinct species), or alternatively if mating is random among genotypes and admixture among adjacent populations is gradual and continuous (supporting geographic variation within a single species). Here, we test two alternative hypotheses for two pairs of named taxa at contact zones within the American milksnake (Lampropeltis triangulum) complex. A prior morphological analysis found areas of gradual intergradation among named taxa, and concluded that the taxa represented geographical races of a single polytypic species. In contrast, a subsequent analysis of gene sequence data, but with limited sampling near the contact zones, hypothesized distinct boundaries between species at the contact zones. At the contact zone between proposed species L. triangulum and Lampropeltis gentilis, we examined a $\sim$700 km-wide transect across the states of Kansas and Missouri, with thorough sampling and reduced-representation genomic-level sequencing, to test the two opposing taxonomic hypotheses. Our transect analyses included examinations of population structure, fixed differences, cline-fitting, and an admixture index analysis. These analyses all supported a gradual and continuous geographic cline across a broad intergrade zone between two geographic forms of L. triangulum, thus providing strong support for a single species in this region (and no support for the recognition of L. gentilis as a distinct species). At a second contact zone between proposed species L. triangulum and Lampropeltis elapsoides (but variously treated as species or subspecies by different researchers) in Kentucky and Tennessee, we re-evaluated morphological data. In this case, the contact zone analysis indicated sympatry and reproductive isolation of the two taxa, and thus strongly supported L. triangulum and L. elapsoides as distinct species. We conclude that detailed studies of contact zones, based on either genetic or morphological data, are essential for distinguishing intraspecific from interspecific variation in the case of widely and continuously distributed taxa. [Contact zones; speciation; species concepts; species delimitation; taxonomy.].

Species Persistence with Hybridization in Toad-Headed Lizards Driven by Divergent Selection and Low Recombination.

Speciation plays a central role in evolutionary studies, and particularly how reproductive isolation (RI) evolves. The origins and persistence of RI are distinct processes that require separate evaluations. Treating them separately clarifies the drivers of speciation and then it is possible to link the processes to understand large-scale patterns of diversity. Recent genomic studies have focused predominantly on how species or RI originate. However, we know little about how species persist in face of gene flow. Here, we evaluate a contact zone of two closely related toad-headed lizards (Phrynocephalus) using a chromosome-level genome assembly and population genomics. To some extent, recent asymmetric introgression from Phrynocephalus putjatai to P. vlangalii reduces their genomic differences. However, their highly divergent regions (HDRs) have heterogeneous distributions across the genomes. Functional gene annotation indicates that many genes within HDRs are involved in reproduction and RI. Compared with allopatric populations, contact areas exhibit recent divergent selection on the HDRs and a lower population recombination rate. Taken together, this implies that divergent selection and low genetic recombination help maintain RI. This study provides insights into the genomic mechanisms that drive RI and two species persistence in the face of gene flow during the late stage of speciation.

Molecular markers of metastatic disease in KRAS-mutant lung adenocarcinoma.

BACKGROUND: Prior studies characterized the association of molecular alterations with treatment-specific outcomes in KRAS-mutant (KRASMUT) lung adenocarcinoma (LUAD). Less is known about the prognostic role of molecular alterations and their associations with metastatic disease. PATIENTS AND METHODS: We analyzed clinicogenomic data from 1817 patients with KRASMUT LUAD sequenced at the Dana-Farber Cancer Institute (DFCI) and Memorial Sloan Kettering Cancer Center (MSKCC). Patients with metastatic (M1) and nonmetastatic (M0) disease were compared. Transcriptomic data from The Cancer Genome Atlas (TCGA) were investigated to characterize the biology of differential associations with clinical outcomes. Organ-specific metastasis was associated with overall survival (OS). RESULTS: KEAP1 (DFCI: OR = 2.3, q = 0.04; MSKCC: OR = 2.2, q = 0.00027) and SMARCA4 mutations (DFCI: OR = 2.5, q = 0.06; MSKCC: OR = 2.6, q = 0.0021) were enriched in M1 versus M0 tumors. On integrative modeling, NRF2 activation was the genomic feature most associated with OS. KEAP1 mutations were enriched in M1 versus M0 tumors independent of STK11 status (KEAP1MUT/STK11WT: DFCI OR = 3.0, P = 0.0064; MSKCC OR = 2.0, P = 0.041; KEAP1MUT/STK11MUT: DFCI OR = 2.3, P = 0.0063; MSKCC OR = 2.5, P = 3.6 × 10-05); STK11 mutations without KEAP1 loss were not associated with stage (KEAP1WT/STK11MUT: DFCI OR = 0.97, P = 1.0; MSKCC OR = 1.2, P = 0.33) or outcome. KEAP1/KRAS-mutated tumors with and without STK11 mutations exhibited high functional STK11 loss. The negative effects of KEAP1 were compounded in the presence of bone (HR = 2.3, P = 4.4 × 10-14) and negated in the presence of lymph node metastasis (HR = 1.0, P = 0.91). CONCLUSIONS: Mutations in KEAP1 and SMARCA4, but not STK11, were associated with metastatic disease and poor OS. Functional STK11 loss, however, may contribute to poor outcomes in KEAP1MUT tumors. Integrating molecular data with clinical and metastatic-site annotations can more accurately risk stratify patients.

How mitonuclear discordance and geographic variation have confounded species boundaries in a widely studied snake.

As DNA sequencing technologies and methods for delimiting species with genomic data become more accessible and numerous, researchers have more tools than ever to investigate questions in systematics and phylogeography. However, easy access to sophisticated computational tools is not without its drawbacks. Choosing the right approach for one's question can be challenging when presented with multitudinous options, some of which fail to distinguish between species and intraspecific population structure. Here, we employ a methodology that emphasizes intensive geographic sampling, particularly at contact zones between populations, with a focus on differentiating intraspecific genetic clusters from species in the Pantherophis guttatus complex, a group of North American ratsnakes. Using a mitochondrial marker as well as ddRADseq data, we find evidence of mitonuclear discordance which has contributed to historical confusion about the relationships within this group. Additionally, we identify geographically and genetically structured populations within the species Pantherophis emoryi that are congruent with previously described morphological variation. Importantly, we find that these structured populations within P. emoryi are highly admixed throughout the range of the species and show no evidence of any reproductive isolation. Our data support a revision of the taxonomy of this group, and we recognize two species within the complex and three subspecies within P. emoryi. This study illustrates the importance of thorough sampling of contact zones and consideration of gene flow when delimiting species in widespread complexes containing parapatric lineages.

The Multispecies Coalescent Over-Splits Species in the Case of Geographically Widespread Taxa.

Many recent species delimitation studies rely exclusively on limited analyses of genetic data analyzed under the multispecies coalescent (MSC) model, and results from these studies often are regarded as conclusive support for taxonomic changes. However, most MSC-based species delimitation methods have well-known and often unmet assumptions. Uncritical application of these genetic-based approaches (without due consideration of sampling design, the effects of a priori group designations, isolation by distance, cytoplasmic-nuclear mismatch, and population structure) can lead to over-splitting of species. Here, we argue that in many common biological scenarios, researchers must be particularly cautious regarding these limitations, especially in cases of well-studied, geographically variable, and parapatrically distributed species complexes. We consider these points with respect to a historically controversial species group, the American milksnakes (Lampropeltis triangulum complex), using genetic data from a recent analysis (Ruane et al. 2014). We show that over-reliance on the program Bayesian Phylogenetics and Phylogeography, without adequate consideration of its assumptions and of sampling limitations, resulted in over-splitting of species in this study. Several of the hypothesized species of milksnakes instead appear to represent arbitrary slices of continuous geographic clines. We conclude that the best available evidence supports three, rather than seven, species within this complex. More generally, we recommend that coalescent-based species delimitation studies incorporate thorough analyses of geographic variation and carefully examine putative contact zones among delimited species before making taxonomic changes.

Addressing the pitfalls when designing intervention studies to discover and validate biomarkers of habitual dietary intake.

INTRODUCTION: Dietary exposure monitoring within populations is reliant on self-reported measures such as Food Frequency Questionnaires and diet diaries. These methods often contain inaccurate information due to participant misreporting, non-compliance and bias. Urinary metabolites derived from individual foods could provide additional objective indicators of dietary exposure. For biomarker approaches to have utility it is essential that they cover a wide-range of commonly consumed foods and the methodology works in a real-world environment. OBJECTIVES: To test that the methodology works in a real-world environment and to consider the impact of the major sources of likely variance; particularly complex meals, different food formulations, processing and cooking methods, as well as the dynamics of biomarker duration in the body. METHODS: We designed and tested a dietary exposure biomarker discovery and validation strategy based on a food intervention study involving free-living individuals preparing meals and collecting urine samples at home. Two experimental periods were built around three consecutive day menu plans where all foods and drinks were provided (n = 15 and n = 36). RESULTS: The experimental design was validated by confirming known consumption biomarkers in urinary samples after the first menu plan. We tested biomarker performance with different food formulations and processing methods involving meat, wholegrain, fruits and vegetables. CONCLUSION: It was demonstrated that spot urine samples, together with robust dietary biomarkers, despite major sources of variance, could be used successfully for dietary exposure monitoring in large epidemiological studies.

Dendritic cell phenotype in severe asthma reflects clinical responsiveness to glucocorticoids.

BACKGROUND: Subsets of patients with severe asthma remain symptomatic despite prolonged, high-dose glucocorticoid therapy. We hypothesized that the clinical glucocorticoid sensitivity of these asthmatics is reflected in differences in peripheral blood dendritic cell subsets. OBJECTIVE: To compare peripheral blood leucocyte populations using flow cytometry at baseline and after 2 weeks of systemic glucocorticoid (steroid) treatment to identify immunological differences between steroid-sensitive (SS) and steroid-resistant (SR) asthmatics. METHODS: Adult severe asthmatics (SS n = 12; SR n = 23) were assessed for their response to 2 weeks of therapy with oral prednisolone. Peripheral blood was obtained before and after therapy and stained for lymphocyte (CD3, CD19, CD4, CD8 and Foxp3) and dendritic cell markers (Lineage negative [CD3, CD14, CD16, CD19, CD20, CD56], HLA-DR+, CD304, CD11c, ILT3 and CD86). RESULTS: A higher median frequency of myeloid DCs (mDCs) but not plasmacytoid DCs (pDCs) was observed in the blood of SR as compared to SS asthmatics (P = .03). Glucocorticoid therapy significantly increased median B cell, but not T cell numbers in both cohorts, with a trend for increased numbers of Foxp3+ Tregs in SS (P = .07), but not SR subjects. Oral prednisolone therapy significantly reduced the median numbers and frequencies of total DCs and pDCs in both SS and SR asthmatics. Interestingly, the expression of HLA-DR and ILT3 was also reduced on pDCs in all patients. In contrast, therapy increased the median frequency of mDCs in SS, but reduced it in SR asthmatics. CONCLUSIONS: Myeloid DC frequency is elevated in SR compared with SS asthmatics, and mDC shows a differential response to oral prednisolone therapy.

Glucagon increases energy expenditure independently of brown adipose tissue activation in humans.

AIMS: To investigate, for a given energy expenditure (EE) rise, the differential effects of glucagon infusion and cold exposure on brown adipose tissue (BAT) activation in humans. METHODS: Indirect calorimetry and supraclavicular thermography was performed in 11 healthy male volunteers before and after: cold exposure; glucagon infusion (at 23 °C); and vehicle infusion (at 23 °C). All volunteers underwent (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET)/CT scanning with cold exposure. Subjects with cold-induced BAT activation on (18)F-FDG PET/CT (n = 8) underwent a randomly allocated second (18)F-FDG PET/CT scan (at 23 °C), either with glucagon infusion (n = 4) or vehicle infusion (n = 4). RESULTS: We observed that EE increased by 14% after cold exposure and by 15% after glucagon infusion (50 ng/kg/min; p < 0.05 vs control for both). Cold exposure produced an increase in neck temperature (+0.44 °C; p < 0.001 vs control), but glucagon infusion did not alter neck temperature. In subjects with a cold-induced increase in the metabolic activity of supraclavicular BAT on (18)F-FDG PET/CT, a significant rise in the metabolic activity of BAT after glucagon infusion was not detected. Cold exposure increased sympathetic activation, as measured by circulating norepinephrine levels, but glucagon infusion did not. CONCLUSIONS: Glucagon increases EE by a similar magnitude compared with cold activation, but independently of BAT thermogenesis. This finding is of importance for the development of safe treatments for obesity through upregulation of EE.

The impact of insecticide-treated cloth targets on the survival of Stegomyia polynesiensis (= Aedes polynesiensis) under laboratory and semi-field conditions in French Polynesia.

The impact of deltamethrin-impregnated cloth targets on Stegomyia polynesiensis (= Aedes polynesiensis) (Marks) (Diptera: Culicidae) was assessed under laboratory and semi-field settings in French Polynesia. Stegomyia polynesiensis females were released into small laboratory cages and large field cages containing either a deltamethrin-treated or an untreated navy blue cloth, and mosquito knock-down and mortality were assessed. The 24-h mortality rate in mosquitoes exposed to the insecticide-treated target in small cages was 98.0%. These mosquitoes also demonstrated significantly higher levels of knock-down than those exposed to the untreated target. Mortality in field cages was assessed at 24 and 48 h. The 24-h mortality rate in mosquitoes exposed to the control target was 31.2%, whereas that in those exposed to the deltamethrin-treated target was 54.3%. The 48-h mortality rate was also elevated in mosquitoes exposed to the deltamethrin-treated target, but this result did not differ significantly from that observed in mosquitoes exposed to the control target. The significant suppression of female S. polynesiensis by deltamethrin-treated resting targets in this study indicates that these targets could play a role in the control of an important disease vector in the South Pacific region.

Lipolytic Markers, Insulin and Resting Fat Oxidation are Associated with Maximal Fat Oxidation.

The maximal capacity to oxidize fat during exercise (MFO) is associated with 24-h fat balance and insulin sensitivity. Understanding factors that influence MFO could have implications for metabolic health. We investigated relationships between selected plasma metabolites, hormones and overnight-fasted resting fat oxidation rates (Resting), with MFO. Resting fat oxidation and MFO was measured in 57 men with blood collected at rest and during exercise. Plasma glycerol (R=0.39, P=0.033), non-esterified fatty acids (NEFA: R=0.27, P=0.030) and insulin (R=- 0.36, P=0.007) measured at MFO correlated with MFO; only glycerol remained correlated when controlled for resting concentrations (R=0.36, P=0.008). The change in glycerol from rest to MFO correlated with exercise-induced fat oxidation (R=0.32, P=0.012). V˙O 2max correlated with resting fat oxidation (R=0.44, P=0.001) and MFO (R=0.52, P<0.001). Resting fat oxidation correlated with MFO (R=0.55, P<0.001); this remained when controlled for V˙O 2max (R=0.41, P=0.001). This study reports weak-to-moderate, albeit significant, relationships between plasma lipolytic markers, insulin and resting overnight-fasted fat oxidation with MFO and shows the plasma glycerol response to uniquely reflect exercise-induced fat oxidation. V˙O 2max correlates with fat oxidation but the relationship can be dissociated. Interventions to increase fat oxidation for optimal metabolic health would benefit from, but are not reliant on, increases in V˙O 2max.

The role of short chain fatty acids in appetite regulation and energy homeostasis.

Over the last 20 years there has been an increasing interest in the influence of the gastrointestinal tract on appetite regulation. Much of the focus has been on the neuronal and hormonal relationship between the gastrointestinal tract and the brain. There is now mounting evidence that the colonic microbiota and their metabolic activity have a significant role in energy homeostasis. The supply of substrate to the colonic microbiota has a major impact on the microbial population and the metabolites they produce, particularly short chain fatty acids (SCFAs). SCFAs are produced when non-digestible carbohydrates, namely dietary fibres and resistant starch, undergo fermentation by the colonic microbiota. Both the consumption of fermentable carbohydrates and the administration of SCFAs have been reported to result in a wide range of health benefits including improvements in body composition, glucose homeostasis, blood lipid profiles and reduced body weight and colon cancer risk. However, published studies tend to report the effects that fermentable carbohydrates and SCFAs have on specific tissues and metabolic processes, and fail to explain how these local effects translate into systemic effects and the mitigation of disease risk. Moreover, studies tend to investigate SCFAs collectively and neglect to report the effects associated with individual SCFAs. Here, we bring together the recent evidence and suggest an overarching model for the effects of SCFAs on one of their beneficial aspects: appetite regulation and energy homeostasis.

Cross-cultural perception of six commercial olive oils: A study with Spanish and US consumers.

A cross-cultural study was conducted with Spanish and US consumers to gain an insight into the preferred characteristics of olive oils in both countries. Six commercial olive oils (four samples from Spain and two samples from the US) were analyzed by a highly trained panel (descriptive analysis) and also by two consumers' groups (100 consumers from Spain and 100 from the US). Demographic, acceptability, and Just-About-Right data were collected to study the preferences of both groups, and the relationships with descriptive data were explored to determine the drivers of like/dislike. The Spanish extra virgin olive oils and the imported US extra virgin olive oil were characterized by having bitter, pungent, and more green notes, and were preferred by the Spanish consumers. The US consumers liked the bland Spanish refined olive oil, and the Californian olive oil that was characterized by fruity, floral, and sweet notes. The results showed that the Spanish consumers were more aware about olive oil quality in general than their US counterparts, maybe because of a higher usage of the product in Spain. The present study provides essential data which might help producers in designing and promoting olive oils matching US consumers' requirements, an emerging market for this Mediterranean product.

An Initial lexicon of sensory properties for nail polish.

OBJECTIVE: The objective of this study was to develop an initial lexicon for sensory properties of nail polish and to validate this lexicon using a descriptive analysis study of selected samples. METHODS: Seventeen commercial products from four categories (regular, flake-containing, water-based and gel) were used in this study. Descriptive sensory analysis was conducted in this study to characterize and evaluate application and removal properties of these nail polishes. Data was then processed by ANOVA, Principal Component Analysis (PCA) and Pearson's Correlation Coefficient analysis to explore the differences among samples and attributes. RESULTS: A lexicon of 21 sensory attributes was developed to describe the application of nail polish. It included three initial texture attributes, thirteen initial appearance attributes and five aroma attributes. A lexicon of five attributes in five stages was developed to describe the removal of nail polish. The results from ANOVA and PCA showed that attributes in the lexicon separated the different product categories. CONCLUSION: The results of this study indicated that descriptive sensory analysis can be used to evaluate nail polish. The results of this study present scientists who are working on nail polish an additional tool to describe application and removal properties of nail polish.

74 MHz nonthermal emission from molecular clouds: evidence for a cosmic ray dominated region at the galactic center.

We present 74 MHz radio continuum observations of the Galactic center region. These measurements show nonthermal radio emission arising from molecular clouds that is unaffected by free­free absorption along the line of sight. We focus on one cloud, G0.13-0.13, representative of the population of molecular clouds that are spatially correlated with steep spectrum (α(327MHz)(74MHz) = 1.3 ± 0.3) nonthermal emission from the Galactic center region. This cloud lies adjacent to the nonthermal radio filaments of the Arc near l 0.2° and is a strong source of 74 MHz continuum, SiO (2-1), and Fe I Kα 6.4 keV line emission. This three-way correlation provides the most compelling evidence yet that relativistic electrons, here traced by 74 MHz emission, are physically associated with the G0.13-0.13 molecular cloud and that low-energy cosmic ray electrons are responsible for the Fe I Kα line emission. The high cosmic ray ionization rate 10(­1)3 s(­1) H(­1) is responsible for heating the molecular gas to high temperatures and allows the disturbed gas to maintain a high-velocity dispersion. Large velocity gradient (LVG) modeling of multitransition SiO observations of this cloud implies H2 densities 10(4­5) cm(­3) and high temperatures. The lower limit to the temperature of G0.13-0.13 is 100 K, whereas the upper limit is as high as 1000 K. Lastly, we used a time-dependent chemical model in which cosmic rays drive the chemistry of the gas to investigate for molecular line diagnostics of cosmic ray heating. When the cloud reaches chemical equilibrium, the abundance ratios of HCN/HNC and N2H+/HCO+ are consistent with measured values. In addition, significant abundance of SiO is predicted in the cosmic ray dominated region of the Galactic center. We discuss different possibilities to account for the origin of widespread SiO emission detected from Galactic center molecular clouds.

Landing response of Aedes (Stegomyia) polynesiensis mosquitoes to coloured targets.

Aedes polynesiensis Marks (Diptera: Culicidae) is the primary vector of lymphatic filariasis (LF) in the island countries and territories of the South Pacific. In the development of a novel control tool, the response of Ae. polynesiensis to six different colours (three solid fabrics, two patterned fabrics and a plastic tarp) was measured using a digital photographic system. Adult mosquitoes were placed into an environmental chamber and allowed to choose between a white target and one of six experimental targets. Mosquito landing frequency and landing duration were calculated. Adult female Ae. polynesiensis preferred all of the experimental targets to the white control target. Mosquito landing frequency was highest for the solid targets (black, navy blue and red) followed in turn by the two colour pattern targets and the polyethylene target. Mosquito landing duration was greater for experimental targets when compared with white control targets. Mosquito landing frequencies did not change over time during the course of the assay. The response of male Ae. polynesiensis was also measured when exposed to a 100% cotton black target. Male mosquitoes preferred the black target to the white control target, although at levels lower than that observed in female mosquitoes. The results suggest that future investigations evaluating the visual responses of Ae. polynesiensis mosquitoes are warranted, with a special emphasis on semi-field and field-based experiments.

Individual-based landscape genomics for conservation: An analysis pipeline.

Landscape genomics can harness environmental and genetic data to inform conservation decisions by providing essential insights into how landscapes shape biodiversity. The massive increase in genetic data afforded by the genomic era provides exceptional resolution for answering critical conservation genetics questions. The accessibility of genomic data for non-model systems has also enabled a shift away from population-based sampling to individual-based sampling, which now provides accurate and robust estimates of genetic variation that can be used to examine the spatial structure of genomic diversity, population connectivity and the nature of environmental adaptation. Nevertheless, the adoption of individual-based sampling in conservation genetics has been slowed due, in large part, to concerns over how to apply methods developed for population-based sampling to individual-based sampling schemes. Here, we discuss the benefits of individual-based sampling for conservation and describe how landscape genomic methods, paired with individual-based sampling, can answer fundamental conservation questions. We have curated key landscape genomic methods into a user-friendly, open-source workflow, which we provide as a new R package, A Landscape Genomics Analysis Toolkit in R (algatr). The algatr package includes novel added functionality for all of the included methods and extensive vignettes designed with the primary goal of making landscape genomic approaches more accessible and explicitly applicable to conservation biology.

2b or not 2b? 2bRAD is an effective alternative to ddRAD for phylogenomics.

Restriction-site-associated DNA sequencing (RADseq) has become an accessible way to obtain genome-wide data in the form of single-nucleotide polymorphisms (SNPs) for phylogenetic inference. Nonetheless, how differences in RADseq methods influence phylogenetic estimation is poorly understood because most comparisons have largely relied on conceptual predictions rather than empirical tests. We examine how differences in ddRAD and 2bRAD data influence phylogenetic estimation in two non-model frog groups. We compare the impact of method choice on phylogenetic information, missing data, and allelic dropout, considering different sequencing depths. Given that researchers must balance input (funding, time) with output (amount and quality of data), we also provide comparisons of laboratory effort, computational time, monetary costs, and the repeatability of library preparation and sequencing. Both 2bRAD and ddRAD methods estimated well-supported trees, even at low sequencing depths, and had comparable amounts of missing data, patterns of allelic dropout, and phylogenetic signal. Compared to ddRAD, 2bRAD produced more repeatable datasets, had simpler laboratory protocols, and had an overall faster bioinformatics assembly. However, many fewer parsimony-informative sites per SNP were obtained from 2bRAD data when using native pipelines, highlighting a need for further investigation into the effects of each pipeline on resulting datasets. Our study underscores the importance of comparing RADseq methods, such as expected results and theoretical performance using empirical datasets, before undertaking costly experiments.

DNA barcoding of Chinese snakes reveals hidden diversity and conservation needs.

DNA barcoding has greatly facilitated studies of taxonomy, biodiversity, biological conservation, and ecology. Here, we establish a reliable DNA barcoding library for Chinese snakes, unveiling hidden diversity with implications for taxonomy, and provide a standardized tool for conservation management. Our comprehensive study includes 1638 cytochrome c oxidase subunit I (COI) sequences from Chinese snakes that correspond to 17 families, 65 genera, 228 named species (80.6% of named species) and 36 candidate species. A barcode gap analysis reveals gaps, where all nearest neighbour distances exceed maximum intraspecific distances, in 217 named species and all candidate species. Three species-delimitation methods (ABGD, sGMYC, and sPTP) recover 320 operational taxonomic units (OTUs), of which 192 OTUs correspond to named and candidate species. Twenty-eight other named species share OTUs, such as Azemiops feae and A. kharini, Gloydius halys, G. shedaoensis, and G. intermedius, and Bungarus multicinctus and B. candidus, representing inconsistencies most probably caused by imperfect taxonomy, recent and rapid speciation, weak taxonomic signal, introgressive hybridization, and/or inadequate phylogenetic signal. In contrast, 43 species and candidate species assign to two or more OTUs due to having large intraspecific distances. If most OTUs detected in this study reflect valid species, including the 36 candidate species, then 30% more species would exist than are currently recognized. Several OTU divergences associate with known biogeographic barriers, such as the Taiwan Strait. In addition to facilitating future studies, this reliable and relatively comprehensive reference database will play an important role in the future monitoring, conservation, and management of Chinese snakes.

Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high-performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage.

BACKGROUND AND OBJECTIVE: All pregnant women undergoing blood grouping at Southmead Hospital are offered haemoglobinopathy screening by high-performance liquid chromatography (HPLC). RhD-negative women who deliver RhD-positive infants are tested for fetomaternal haemorrhage (FMH) by acid elution (AE). The effectiveness of these two assays for quantitation of FMH was compared with flow cytometry (FC). MATERIALS AND METHODS: The relationship between expression of haemoglobin F (HbF) in individual cells by AE and FC and quantitation of HbF in haemolysates by HPLC was investigated, using maternal samples with unusually high levels of HbF-positive maternal cells (F cells) or with large FMH (fetal cells). Standard anti-D FC was performed to quantitate fetal D-positive cells in D-negative women and compared with FMH estimated by AE and HbF FC. RESULTS: AE overestimated FMH when maternal F cells were increased. HbF FC distinguished F cells from fetal cells. Values of HbF determined by HPLC were less than the level of 5% used for investigation of raised fetal haemoglobin, even in the maternal samples with elevated F cells or massive FMH. CONCLUSIONS: To quantitate FMH, measurement of HbF using FC was more sensitive and accurate than AE or HPLC. HbF FC is the method of choice when results from routine investigation using AE or standard anti-D FC are discrepant or when there is maternal and fetal RhD compatibility.