Large-scale DNA sequencing in taxonomy and conservation : a case study with the mayfly family Heptageniidae (Ephemeroptera) in the Alps and Madagascar

Les larves aquatiques d'éphémères (Ephemeroptera) colonisent toutes les eaux douces du monde et sont couramment utilisées comme bio-indicateurs de la qualité de l'eau. Le genre Rhithrogena (Heptageniidae) est le deuxième plus diversifié chez les éphémères, et plusieurs espèces européennes ont une distribution restreinte dans des environnements alpins sensibles. Les espèces de Rhithrogena ont été classées en "groupes d'espèces" faciles à identifier. Cependant, malgré leur importance écologique et en terme de conservation, beaucoup d'espèces présentent des différences morphologiques ambiguës, suggérant que lataxonomie actuelle ne refléterait pas correctement leur diversité évolutive. De plus, aucune information sur leurs relations, leur origine, le taux de spéciation ou les mécanismes ayant provoqué leur remarquable diversification dans les Alpes n'est disponible. Nous avons d'abord examiné le statut spécifique d'environ 50% des espèces européennes de Rhithrogena en utilisant un large échantillonnage de populations alpines incluant 22 localités typiques, ainsi qu'une analyse basée sur le modèle général mixte de Yule et de coalescence (GMYC) appliqué à un gène mitochondrial standard (coxl) et à un gène nucléaire développé spécifiquement pour cette étude. Nous avons observé un regroupement significatif des séquences coxl en 31 espèces potentielles, et nos résultats ont fortement suggéré la présence d'espèces cryptiques et de fractionnements taxonomiques excessifs chez les Rhithrogena. Nos analyses phylogénétiques ont démontré la monophylie de quatre des six groupes d'espèces reconnus présents dans notre échantillonnage. La taxonomie ADN développée dans cette étude pose les bases d'une future révision de ce genre important mais cryptique en Europe. Puis nous avons mené une étude phylogénétique multi-gènes entre les espèces européennes de Rhithrogena. Les données provenant de trois gènes nucléaires et de deux gènes mitochondriaux ont été largement concordantes, et les relations entre les espèces bien résolues au sein de la plupart des groupes d'espèces dans une analyse combinant tous les gènes. En l'absence de points de calibration extérieurs tels que des fossiles, nous avons appliqué à nos données mitochondriales une horloge moléculaire standard pour les insectes, suggérant une origine des Rhithrogena alpins à la limite Oligocène / Miocène. Nos résultats ont montré le rôle prépondérant qu'ont joué les glaciations du quaternaire dans leur diversification, favorisant la spéciation d'au moins la moitié des espèces actuelle dans les Alpes. La biodiversité et le taux d'endémisme à Madagascar, notamment au niveau de la faune des eaux douces, sont parmi les plus extraordinaires et les plus menacés au monde. On pense que beaucoup d'espèces d'éphémères sont restreintes à un seul bassin versant (microendémisme) dans les zones forestières, ce qui les rendrait particulièrement sensibles à la réduction et à la dégradation de leur habitat. Mis à part deux espèces décrites, Afronurus matitensis et Compsoneuria josettae, les Heptageniidae sont pratiquement inconnus à Madagascar. Les deux genres ont une distribution discontinue en Afrique, à Madagascar et en Asie du Sud-Est, et leur taxonomie complexe est régulièrement révisée. L'approche standard pour comprendre leur diversité, leur endémisme et leur origine requerrait un échantillonnage étendu sur plusieurs continents et des années de travaux taxonomiques. Pour accélérer le processus, nous avons utilisé des collections de musées ainsi que des individus fraîchement collectés, et appliqué une approche combinant taxonomie ADN et phylogénie. L'analyses GMYC du gène coxl a délimité 14 espèces potentielles à Madagascar, dont 70% vraisemblablement microendémiques. Une analyse phylogénique incluant des espèces africaines et asiatiques portant sur deux gènes mitochondriaux et quatre gènes nucléaires a montré que les Heptageniidae malgaches sont monophylétiques et groupe frère des Compsoneuria africains. L'existence de cette lignée unique, ainsi qu'un taux élevé de microendémisme, mettent en évidence leur importance en terme de conservation. Nos résultats soulignent également le rôle important que peuvent jouer les collections de musées dans les études moléculaires et en conservation. - Aquatic nymphs of mayflies (Ephemeroptera) colonize all types of freshwaters throughout the world and are extensively used as bio-indicators of water quality. Rhithrogena (Heptageniidae) is the second most species-rich genus of mayflies, and several European species have restricted distributions in sensitive Alpine environments and therefore are of conservation interest. The European Rhithrogena species are arranged into "species groups" that are easily identifiable. However, despite their ecological and conservation importance, ambiguous morphological differences among many species suggest that the current taxonomy may not accurately reflect their evolutionary diversity. Moreover, no information about their relationships, origin, timing of speciation and mechanisms promoting their successful diversification in the Alps is available. We first examined the species status of ca. 50% of European Rhithrogena diversity using a widespread sampling scheme of Alpine species that included 22 type localities, general mixed Yule- coalescent (GMYC) model analysis of one standard mitochondrial (coxl) and one newly developed nuclear marker. We observed significant clustering of coxl into 31 GMYC species, and our results strongly suggest the presence of both cryptic diversity and taxonomic oversplitting in Rhithrogena. Phylogenetic analyses recovered four of the six recognized species groups in our samples as monophyletic. The DNA taxonomy developed here lays the groundwork for a future revision of this important but cryptic genus in Europe. Then we conducted a species-level, multiple-gene phylogenetic study of European Rhithrogena. Data from three nuclear and two mitochondrial loci were broadly congruent, and species-level relationships were well resolved within most species groups in a combined analysis. In the absence of external calibration points like fossils, we applied a standard insect molecular clock hypothesis to our mitochondrial data, suggesting an origin of Alpine Rhithrogena in the Oligocene / Miocene boundary. Our results highlighted the preponderant role that quaternary glaciations played in their diversification, promoting speciation of at least half of the current diversity in the Alps. Madagascar's biodiversity and endemism are among the most extraordinary and endangered in the world. This includes the island's freshwater biodiversity, although detailed knowledge of the diversity, endemism, and biogeographic origin of freshwater invertebrates is lacking. Many mayfly species are thought to be restricted to single river basins (microendemic species) in forested areas, making them particularly sensitive to habitat reduction and degradation. The Heptageniidae are practically unknown in Madagascar except for two described species, Afronurus matitensis and Compsoneuria josettae. Both genera have a disjunct distribution in Africa, Madagascar and Southeast Asia, and a complex taxonomic status still in flux. The standard approach to understanding their diversity, endemism, and origin would require extensive field sampling on several continents and years of taxonomic work. Here we circumvent this using museum collections and freshly collected individuals in a combined approach of DNA taxonomy and phylogeny. The cox/-based GMYC analysis revealed 14 putative species on Madagascar, 70% of which potentially microendemics. A phylogenetic analysis that included African and Asian species and data from two mitochondrial and four nuclear loci indicated the Malagasy Heptageniidae are monophyletic and sister to African Compsoneuria. The observed monophyly and high microendemism highlight their conservation importance. Our results also underline the important role that museum collections can play in molecular studies, especially in critically endangered biodiversity hotspots like Madagascar.

Authentication of vegetable oils by bulk and molecular carbon isotope analyses with emphasis on olive oil and pumpkin seed oil

The authenticity of vegetable oils consumed in Slovenia and Croatia was investigated by carbon isotope analysis of the individual fatty acids by the use of gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS), and through carbon isotope analysis of the bulk oil. The fatty acids from samples of olive, pumpkin, sunflower, maize, rape, soybean, and sesame oils were separated by alkaline hydrolysis and derivatized to methyl esters for chemical characterization by capillary gas chromatography/mass spectrometry (GC/MS) prior to isotopic analysis. Enrichment in heavy carbon isotope (C-13) of th, bulk oil and of the individual fatty acids are related to (1) a thermally induced degradation during processing (deodorization, steam washing, or bleaching), (2) hydrolytic rancidity (lipolysis) and oxidative rancidity of the vegetable oils during storage, and (3) the potential blend with refined oil or other vegetable oils. The impurity or admixture of different oils may be assessed from the delta C-13(16:0) VS. delta C-13(18:1) covariations. The fatty acid compositions of Slovenian and Croatian olive oils are compared with those from the most important Mediterranean producer countries (Spain, Italy, Greece, and France).

Subgroup And Quality Of Life Analyses Of The Phase Iii Clinical Trial Of Novottf-100a Versus Best Standard Chemotherapy For Recurrent Glioblastoma

BACKGROUND: NovoTTF is a portable device delivering low-intensity, intermediate-frequency, electric fields using noninvasive, disposable scalp electrodes. These fields physically interfere with cell division. Preliminary studies in recurrent and newly diagnosed glioblastoma (GBM) have shown promising results. A phase III study in recurrent GBM has recently been concluded. METHODS: Adults (KPS ≥ 70%) with recurrent GBM (any recurrence) were randomized (stratified by surgery and center) to either NovoTTF administered continuously (20-24 hours/day, 7 days/week) or the best available chemotherapy (best physician choice [BPC]). Primary endpoint was overall survival (OS); 6-month progression-free survival (PFS6), 1-year survival, and QOL were secondary endpoints. RESULTS: Two hundred thirty-seven patients were randomized (28 centers in the United States and Europe) to either NovoTTF alone (120 patients) or BPC (117 patients). Patient characteristics were balanced, median age was 54 years (range, 23-80 years), median KPS was 80% (range, 50-100). One quarter had surgery for recurrence, and over half were at their second or more recurrence. A survival advantage for the device group was seen in patients treated according to protocol (median OS, 7.8 months vs. 6.1 months; n = 185; p = 0.01). Moreover, subgroup analysis in patients with better prognostic baseline characteristics (KPS ≥ 80%; age ≤ 60; 1st-3rd recurrence) demonstrated a robust survival benefit for NovoTTF patients compared to matched BPC patients (median OS, 8.8 months vs. 6.6 months; n = 110; p < 0.01). In this group, 1-year survival was 35% vs. 20% and PFS6 was 25.6% vs. 7.7%. Interestingly, in patients who failed bevacizumab prior to the trial, OS was also significantly extended by NovoTTF (4.4 months vs. 3.1 months; n = 23 vs. n = 21; p < 0.02). Quality of life was equivalent or superior in NovoTTF patients. CONCLUSIONS: NovoTTF, a noninvasive, novel cancer treatment modality shows significant therapeutic efficacy with improved quality of life. The impact of NovoTTF was more pronounced when patients with better baseline prognostic factors were treated. A large scale phase III clinical trial in newly diagnosed GBM is currently being conducted.

Comparison between a high-resolution single-stage Orbitrap and a triple quadrupole mass spectrometer for quantitative analyses of drugs.

The capabilities of a high-resolution (HR), accurate mass spectrometer (Exactive-MS) operating in full scan MS mode was investigated for the quantitative LC/MS analysis of drugs in patients' plasma samples. A mass resolution of 50,000 (FWHM) at m/z 200 and a mass extracted window of 5 ppm around the theoretical m/z of each analyte were used to construct chromatograms for quantitation. The quantitative performance of the Exactive-MS was compared with that of a triple quadrupole mass spectrometer (TQ-MS), TSQ Quantum Discovery or Quantum Ultra, operating in the conventional selected reaction monitoring (SRM) mode. The study consisted of 17 therapeutic drugs including 8 antifungal agents (anidulafungin, caspofungin, fluconazole, itraconazole, hydroxyitraconazole posaconazole, voriconazole and voriconazole-N-oxide), 4 immunosuppressants (ciclosporine, everolimus, sirolimus and tacrolimus) and 5 protein kinase inhibitors (dasatinib, imatinib, nilotinib, sorafenib and sunitinib). The quantitative results obtained with HR-MS acquisition show comparable detection specificity, assay precision, accuracy, linearity and sensitivity to SRM acquisition. Importantly, HR-MS offers several benefits over TQ-MS technology: absence of SRM optimization, time saving when changing the analysis from one MS to another, more complete information of what is in the samples and easier troubleshooting. Our work demonstrates that U/HPLC coupled to Exactive HR-MS delivers comparable results to TQ-MS in routine quantitative drug analyses. Considering the advantages of HR-MS, these results suggest that, in the near future, there should be a shift in how routine quantitative analyses of small molecules, particularly for therapeutic drugs, are performed.

Multivariate analyses of genotype x environment interaction of popcorn

The objectives of this work were to evaluate the genotype x environment (GxE) interaction for popcorn and to compare two multivariate analyses methods. Nine popcorn cultivars were sown on four dates one month apart during each of the agricultural years 1998/1999 and 1999/2000. The experiments were carried out using randomized block designs, with four replicates. The cv. Zélia contributed the least to the GxE interaction. The cv. Viçosa performed similarly to cv. Rosa-claro. Optimization of GxE was obtained for cv. CMS 42 for a favorable mega-environment, and for cv. CMS 43 for an unfavorable environment. Multivariate analysis supported the results from the method of Eberhart & Russell. The graphic analysis of the Additive Main effects and Multiplicative Interaction (AMMI) model was simple, allowing conclusions to be made about stability, genotypic performance, genetic divergence between cultivars, and the environments that optimize cultivar performance. The graphic analysis of the Genotype main effects and Genotype x Environment interaction (GGE) method added to AMMI information on environmental stratification, defining mega-environments and the cultivars that optimized performance in those mega-environments. Both methods are adequate to explain the genotype x environment interactions.

Longitudinal analyses of renal lesions due to acute pyelonephritis in children and their impact on renal growth.

PURPOSE: Acute pyelonephritis is a common condition in children, and can lead to renal scarring. The aim of this study was to analyze the progression of renal scarring with time and its impact on renal growth. MATERIALS AND METHODS: A total of 50 children who had renal scarring on dimercapto-succinic acid scan 6 months after acute pyelonephritis underwent a repeat scan 3 years later. Lesion changes were evaluated by 3 blinded observers, and were classified as no change, partial resolution or complete disappearance. Renal size at time of acute pyelonephritis and after 3 years was obtained by ultrasound, and renal growth was assessed comparing z-score for age between the 2 measures. Robust linear regression was used to identify determinants of renal growth. RESULTS: At 6 months after acute pyelonephritis 88 scars were observed in 100 renal units. No change was observed in 27%, partial resolution in 63% and complete disappearance in 9% of lesions. Overall, 72% of lesions improved. Increased number of scars was associated with high grade vesicoureteral reflux (p = 0.02). Multivariate analysis showed that the number of scars was the most important parameter leading to decreased renal growth (CI -1.05 to -0.35, p <0.001), and with 3 or more scars this finding was highly significant on univariate analysis (-1.59, CI -2.10 to -1.09, p <0.0001). CONCLUSIONS: Even 6 months after acute pyelonephritis 72% of dimercapto-succinic acid defects improved, demonstrating that some of the lesions may be not definitive. The number of scars was significantly associated with loss of renal growth at 3 years.

2015 Iowa Fish Tissue Monitoring Program Summary of Analyses

To supplement other environmental monitoring programs and to protect the health of people consuming fish from waters within this state, the state of Iowa conducts fish tissue monitoring. Since 1980, the Iowa Department of Natural Resources (IDNR), the United States Environmental Protection Agency Region VII (U.S. EPA), and the State Hygienic Laboratory (SHL) have cooperatively conducted annual statewide collections and analyses of fish for toxic contaminants. From 1983 to 2014, this monitoring effort was known as the Regional Ambient Fish Tissue Monitoring Program (RAFT). Beginning in 2015, the only statewide fish contaminant-monitoring program in Iowa was changed to the Iowa Fish Tissue Monitoring Program (IFTMP). The IFTMP is administered by IDNR and the tissue analyses are completed at the SHL. Historically, the data generated from the IFTMP have enabled IDNR to document temporal changes in contaminant levels and to identify Iowa lakes and rivers where high levels of contaminants in fish potentially threaten the health of fish-consuming Iowans (see IDNR 2006). The IFTMP incorporates five different types of monitoring sites: 1) status, 2) follow-up, 3) trend, 4) turtle, and 5) random.

2014 Iowa Fish Tissue Monitoring Program Summary of Analyses

To supplement other environmental monitoring programs and to protect the health of people consuming fish from waters within this state, the state of Iowa conducts fish tissue monitoring. Since 1980, the Iowa Department of Natural Resources (IDNR), the United States Environmental Protection Agency Region VII (U.S. EPA), and the State Hygienic Laboratory (SHL) have cooperatively conducted annual statewide collections and analyses of fish for toxic contaminants. From 1983 to 2014, this monitoring effort was known as the Regional Ambient Fish Tissue Monitoring Program (RAFT). Beginning in 2015, the only statewide fish contaminant-monitoring program in Iowa was changed to the Iowa Fish Tissue Monitoring Program (IFTMP). The IFTMP is administered by IDNR and the analyses are completed at the SHL. Historically, the data generated from the IFTMP have enabled IDNR to document temporal changes in contaminant levels and to identify Iowa lakes and rivers where high levels of contaminants in fish potentially threaten the health of fish-consuming Iowans (see IDNR 2006). The IFTMP incorporates five different types of monitoring sites: 1) status, 2) follow-up, 3) trend, 4) turtle, and 5) random.

2013 Iowa Fish Tissue Monitoring Program Summary of Analyses

To supplement other environmental monitoring programs and to protect the health of people consuming fish from waters within this state, the state of Iowa conducts fish tissue monitoring. Since 1980, the Iowa Department of Natural Resources (IDNR), the United States Environmental Protection Agency Region VII (U.S. EPA), and the State Hygienic Laboratory (SHL) have cooperatively conducted annual statewide collections and analyses of fish for toxic contaminants. Beginning in 1983, this monitoring effort became known as the Regional Ambient Fish Tissue Monitoring Program (RAFT). Currently, the RAFT program is the only statewide fish contaminant-monitoring program in Iowa. Historically, the data generated from the RAFT program have enabled IDNR to document temporal changes in contaminant levels and to identify Iowa lakes and rivers where high levels of contaminants in fish potentially threaten the health of fish-consuming Iowans (see IDNR 2006). The Iowa RAFT monitoring program incorporates five different types of monitoring sites: 1) status, 2) follow-up, 3) trend, 4) turtle, and 5) random.

2012 Regional Ambient Fish Tissue Monitoring Program Summary of Analyses

To supplement other environmental monitoring programs and to protect the health of people consuming fish from waters within this state, the state of Iowa conducts fish tissue monitoring. Since 1980, the Iowa Department of Natural Resources (IDNR), the United States Environmental Protection Agency Region VII (U.S. EPA), and the State Hygienic Laboratory (SHL) have cooperatively conducted annual statewide collections and analyses of fish for toxic contaminants. Beginning in 1983, this monitoring effort became known as the Regional Ambient Fish Tissue Monitoring Program (RAFT). Currently, the RAFT program is the only statewide fish contaminant-monitoring program in Iowa. Historically, the data generated from the RAFT program have enabled IDNR to document temporal changes in contaminant levels and to identify Iowa lakes and rivers where high levels of contaminants in fish potentially threaten the health of fish-consuming Iowans (see IDNR 2006). The Iowa RAFT monitoring program incorporates five different types of monitoring sites: 1) status, 2) follow-up, 3) trend, 4) turtle, and 5) random.

2011 Regional Ambient Fish Tissue Monitoring Program Summary of Analyses

To supplement other environmental monitoring programs and to protect the health of people consuming fish from waters within this state, the state of Iowa conducts fish tissue monitoring. Since 1980, the Iowa Department of Natural Resources (IDNR), the United States Environmental Protection Agency Region VII (U.S. EPA), and the State Hygienic Laboratory (SHL) have cooperatively conducted annual statewide collections and analyses of fish for toxic contaminants. Beginning in 1983, this monitoring effort became known as the Regional Ambient Fish Tissue Monitoring Program (RAFT). Currently, the RAFT program is the only statewide fish contaminant-monitoring program in Iowa. Historically, the data generated from the RAFT program have enabled IDNR to document temporal changes in contaminant levels and to identify Iowa lakes and rivers where high levels of contaminants in fish potentially threaten the health of fish-consuming Iowans (see IDNR 2006). The Iowa RAFT monitoring program incorporates five different types of monitoring sites: 1) status, 2) trend, 3) follow-up, 4) turtle, and 5) random.

2010 Regional Ambient Fish Tissue Monitoring Program Summary of Analyses

To supplement other environmental monitoring programs and to protect the health of people consuming fish from waters within this state, the state of Iowa conducts fish tissue monitoring. Since 1980, the Iowa Department of Natural Resources (IDNR), the United States Environmental Protection Agency Region VII (U.S. EPA), and the State Hygienic Laboratory (SHL) have cooperatively conducted annual statewide collections and analyses of fish for toxic contaminants. Beginning in 1983, this monitoring effort became known as the Regional Ambient Fish Tissue Monitoring Program (RAFT). Currently, the RAFT program is the only statewide fish contaminant-monitoring program in Iowa. Historically, the data generated from the RAFT program have enabled IDNR to document temporal changes in contaminant levels and to identify Iowa lakes and rivers where high levels of contaminants in fish potentially threaten the health of fish-consuming Iowans (see IDNR 2006). The Iowa RAFT monitoring program incorporates five different types of monitoring sites: 1) status, 2) trend, 3) random, 4) follow-up and 5) turtle.

2009 Regional Ambient Fish Tissue Monitoring Program Summary of Analyses

To supplement other environmental monitoring programs and to protect the health of people consuming fish from waters within this state, the state of Iowa conducts fish tissue monitoring. Since 1980, the Iowa Department of Natural Resources (IDNR), the United States Environmental Protection Agency Region VII (U.S. EPA), and the University of Iowa Hygienic Laboratory (UHL) have cooperatively conducted annual statewide collections and analyses of fish for toxic contaminants. Beginning in 1983, this monitoring effort became known as the Regional Ambient Fish Tissue Monitoring Program (RAFT). Currently, the RAFT program is the only statewide fish contaminant-monitoring program in Iowa. Historically, the data generated from the RAFT program have enabled IDNR to document temporal changes in contaminant levels and to identify Iowa lakes and rivers where high levels of contaminants in fish potentially threaten the health of fish-consuming Iowans (see IDNR 2006). The Iowa RAFT monitoring program incorporates four different types of monitoring sites: 1) status, 2) trend, 3) random and 4) follow-up. New for 2009 was the one-time inclusion of snapping turtle tissue as part of the Iowa RAFT sampling program.

2008 Regional Ambient Fish Tissue Monitoring Program Summary of Analyses

To supplement other environmental monitoring programs and to protect the health of people consuming fish from waters within this state, the state of Iowa conducts fish tissue monitoring. Since 1980, the Iowa Department of Natural Resources (IDNR), the United States Environmental Protection Agency Region VII (U.S. EPA), and the University of Iowa Hygienic Laboratory (UHL) have cooperatively conducted annual statewide collections and analyses of fish for toxic contaminants. Beginning in 1983, this monitoring effort became known as the Regional Ambient Fish Tissue Monitoring Program (RAFT). Currently, the RAFT program is the only statewide fish contaminant-monitoring program in Iowa. Historically, the data generated from the RAFT program have enabled IDNR to document temporal changes in contaminant levels and to identify Iowa lakes and rivers where high levels of contaminants in fish potentially threaten the health of fish-consuming Iowans (see IDNR 2006). The Iowa RAFT monitoring program incorporates four different types of monitoring sites: 1) status, 2) trend, 3) random and 4) follow-up.