Attack of the PCR clones: Rates of clonality have little effect on RAD‐seq genotype calls

Interpretation of high‐throughput sequence data requires an understanding of how decisions made during bioinformatic data processing can influence results. One source of bias that is often cited is PCR clones (or PCR duplicates). PCR clones are common in restriction site‐associated sequencing (RAD‐seq) data sets, which are increasingly being used for molecular ecology. To determine the influence PCR clones and the bioinformatic handling of clones have on genotyping, we evaluate four RAD‐seq data sets. Data sets were compared before and after clones were removed to estimate the number of clones present in RAD‐seq data, quantify how often the presence of clones in a data set causes genotype calls to change compared to when clones were removed, investigate the mechanisms that lead to genotype call changes and test whether clones bias heterozygosity estimates. Our RAD‐seq data sets contained 30%–60% PCR clones, but 95% of RAD‐tags had five or fewer clones. Relatively few genotypes changed once clones were removed (5%–10%), and the vast majority of these changes (98%) were associated with genotypes switching from a called to no‐call state or vice versa. PCR clones had a larger influence on genotype calls in individuals with low read depth but appeared to influence genotype calls at all loci similarly. Removal of PCR clones reduced the number of called genotypes by 2% but had almost no influence on estimates of heterozygosity. As such, while steps should be taken to limit PCR clones during library preparation, PCR clones are likely not a substantial source of bias for most RAD‐seq studies.     

Leaf traits and performance vary with plant age and water availability in Artemisia californica

Background and AimsLeaf functional traits are strongly tied to growth strategies and ecological processes across species, but few efforts have linked intraspecific trait variation to performance across ontogenetic and environmental gradients. Plants are believed to shift towards more resource-conservative traits in stressful environments and as they age. However, uncertainty as to how intraspecific trait variation aligns with plant age and performance in the context of environmental variation may limit our ability to use traits to infer ecological processes at larger scales.MethodsWe measured leaf physiological and morphological traits, canopy volume and flowering effort for Artemisia californica (California sagebrush), a dominant shrub species in the coastal sage scrub community, under conditions of 50, 100 and 150 % ambient precipitation for 3 years.Key ResultsPlant age was a stronger driver of variation in traits and performance than water availability. Older plants demonstrated trait values consistent with a more conservative resource-use strategy, and trait values were less sensitive to drought. Several trait correlations were consistent across years and treatments; for example, plants with high photosynthetic rates tended to have high stomatal conductance, leaf nitrogen concentration and light-use efficiency. However, the trade-off between leaf construction and leaf nitrogen evident in older plants was absent for first-year plants. While few traits correlated with plant growth and flowering effort, we observed a positive correlation between leaf mass per area and performance in some groups of older plants.ConclusionsOverall, our results suggest that trait sensitivity to the environment is most visible during earlier stages of development, after which intraspecific trait variation and relationships may stabilize. While plant age plays a major role in intraspecific trait variation and sensitivity (and thus trait-based inferences), the direct influence of environment on growth and fecundity is just as critical to predicting plant performance in a changing environment.     

Thermal adaptations to extreme freeze–thaw cycles in the high tropical Andes

Temperature plays a key role in the biology of ectotherms, including anurans, which are found at higher elevations in the tropics than anywhere in the temperate zone. High elevation tropical environments are characterized by extreme daily thermal fluctuation including high daily maxima and nightly freezing. Our study investigated the contrasting operative temperatures of the anurans Telmatobius marmoratus and Pleurodema marmoratum in different environmental contexts at the same elevation and biome above 5,200 m. Telmatobius marmoratus avoids extremes of daily temperature fluctuation by utilizing thermally buffered aquatic habitat at all life stages, with minimal operative temperature variation (range: 4.6–8.0°C). Pleurodema marmoratum, in contrast, experienced operative temperatures from ?3.5 to 44°C and has one of the widest thermal breadths reported for any tropical frog, from >32°C (critical thermal maximum) to surviving freezing periods of 1 and 6 hr down to ?3.0°C. Our findings expand experimental evidence of frost tolerance in amphibians to the widespread Neotropical family Leptodactylidae, the first such evidence of frost tolerance in a tropical amphibian. Our study identifies three strategies (wide thermal tolerance breadth, use of buffered microhabitats, and behavioral thermoregulation), which allow these tropical frogs to withstand the current wide daily thermal fluctuation above 5,000 m.a.s.l. and which may help them adapt to future climatic changes. Abstract in Spanish is available with online material     

Mechanistic Phenotypes: An Aggregative Phenotyping Strategy to Identify Disease Mechanisms Using GWAS Data

A single mutation can alter cellular and global homeostatic mechanisms and give rise to multiple clinical diseases. We hypothesized that these disease mechanisms could be identified using low minor allele frequency (MAF<0.1) non-synonymous SNPs (nsSNPs) associated with “mechanistic phenotypes”, comprised of collections of related diagnoses. We studied two mechanistic phenotypes: (1) thrombosis, evaluated in a population of 1,655 African Americans; and (2) four groupings of cancer diagnoses, evaluated in 3,009 white European Americans. We tested associations between nsSNPs represented on GWAS platforms and mechanistic phenotypes ascertained from electronic medical records (EMRs), and sought enrichment in functional ontologies across the top-ranked associations. We used a two-step analytic approach whereby nsSNPs were first sorted by the strength of their association with a phenotype. We tested associations using two reverse genetic models and standard additive and recessive models. In the second step, we employed a hypothesis-free ontological enrichment analysis using the sorted nsSNPs to identify functional mechanisms underlying the diagnoses comprising the mechanistic phenotypes. The thrombosis phenotype was solely associated with ontologies related to blood coagulation (Fisher''s p = 0.0001, FDR p = 0.03), driven by the F5, P2RY12 and F2RL2 genes. For the cancer phenotypes, the reverse genetics models were enriched in DNA repair functions (p = 2×10−5, FDR p = 0.03) (POLG/FANCI, SLX4/FANCP, XRCC1, BRCA1, FANCA, CHD1L) while the additive model showed enrichment related to chromatid segregation (p = 4×10−6, FDR p = 0.005) (KIF25, PINX1). We were able to replicate nsSNP associations for POLG/FANCI, BRCA1, FANCA and CHD1L in independent data sets. Mechanism-oriented phenotyping using collections of EMR-derived diagnoses can elucidate fundamental disease mechanisms.     

Personal and Household Hygiene,Environmental Contamination,and Health in Undergraduate Residence Halls in New York City, 2011

Background

While several studies have documented the importance of hand washing in the university setting, the added role of environmental hygiene remains poorly understood. The purpose of this study was to characterize the personal and environmental hygiene habits of college students, define the determinants of hygiene in this population, and assess the relationship between reported hygiene behaviors, environmental contamination, and health status.

Methods

501 undergraduate students completed a previously validated survey assessing baseline demographics, hygiene habits, determinants of hygiene, and health status. Sixty survey respondents had microbiological samples taken from eight standardized surfaces in their dormitory environment. Bacterial contamination was assessed using standard quantitative bacterial culture techniques. Additional culturing for coagulase-positive Staphylococcus and coliforms was performed using selective agar.

Results

While the vast majority of study participants (n = 461, 92%) believed that hand washing was important for infection prevention, there was a large amount of variation in reported personal hygiene practices. More women than men reported consistent hand washing before preparing food (p = .002) and after using the toilet (p = .001). Environmental hygiene showed similar variability although 73.3% (n = 367) of subjects reported dormitory cleaning at least once per month. Contamination of certain surfaces was common, with at least one third of all bookshelves, desks, refrigerator handles, toilet handles, and bathroom door handles positive for >10 CFU of bacteria per 4 cm² area. Coagulase-positive Staphylococcus was detected in three participants'' rooms (5%) and coliforms were present in six students'' rooms (10%). Surface contamination with any bacteria did not vary by frequency of cleaning or frequency of illness (p>.05).

Conclusions

Our results suggest that surface contamination, while prevalent, is unrelated to reported hygiene or health in the university setting. Further research into environmental reservoirs of infectious diseases may delineate whether surface decontamination is an effective target of hygiene interventions in this population.     

Assessing Insecticide Hazard to Bumble Bees Foraging on Flowering Weeds in Treated Lawns

Maintaining bee-friendly habitats in cities and suburbs can help conserve the vital pollination services of declining bee populations. Despite label precautions not to apply them to blooming plants, neonicotinoids and other residual systemic insecticides may be applied for preventive control of lawn insect pests when spring-flowering weeds are present. Dietary exposure to neonicotinoids adversely affects bees, but the extent of hazard from field usage is controversial. We exposed colonies of the bumble bee Bombus impatiens to turf with blooming white clover that had been treated with clothianidin, a neonicotinoid, or with chlorantraniliprole, the first anthranilic diamide labeled for use on lawns. The sprays were applied at label rate and lightly irrigated. After residues had dried, colonies were confined to forage for six days, and then moved to a non-treated rural site to openly forage and develop. Colonies exposed to clothianidin-treated weedy turf had delayed weight gain and produced no new queens whereas those exposed to chlorantraniliprole-treated plots developed normally compared with controls. Neither bumble bees nor honey bees avoided foraging on treated white clover in open plots. Nectar from clover blooms directly contaminated by spray residues contained 171±44 ppb clothianidin. Notably, neither insecticide adversely impacted bee colonies confined on the treated turf after it had been mown to remove clover blooms present at the time of treatment, and new blooms had formed. Our results validate EPA label precautionary statements not to apply neonicotinoids to blooming nectar-producing plants if bees may visit the treatment area. Whatever systemic hazard through lawn weeds they may pose appears transitory, however, and direct hazard can be mitigated by adhering to label precautions, or if blooms inadvertently are contaminated, by mowing to remove them. Chlorantraniliprole usage on lawns appears non-hazardous to bumble bees.     

An NO Donor Approach to Neuroprotective and Procognitive Estrogen Therapy Overcomes Loss of NO Synthase Function and Potentially Thrombotic Risk

Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3^rd generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer''s transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events.     

Rivermouth Alteration of Agricultural Impacts on Consumer Tissue δ15N

Terrestrial agricultural activities strongly influence riverine nitrogen (N) dynamics, which is reflected in the δ¹⁵N of riverine consumer tissues. However, processes within aquatic ecosystems also influence consumer tissue δ¹⁵N. As aquatic processes become more important terrestrial inputs may become a weaker predictor of consumer tissue δ¹⁵N. In a previous study, this terrestrial-consumer tissue δ¹⁵N connection was very strong at river sites, but was disrupted by processes occurring in rivermouths (the ‘rivermouth effect’). This suggested that watershed indicators of N loading might be accurate in riverine settings, but could be inaccurate when considering N loading to the nearshore of large lakes and oceans. In this study, the rivermouth effect was examined on twenty-five sites spread across the Laurentian Great Lakes. Relationships between agriculture and consumer tissue δ¹⁵N occurred in both upstream rivers and at the outlets where rivermouths connect to the nearshore zone, but agriculture explained less variation and had a weaker effect at the outlet. These results suggest that rivermouths may sometimes be significant sources or sinks of N, which would cause N loading estimates to the nearshore zone that are typically made at discharge gages further upstream to be inaccurate. Identifying definitively the controls over the rivermouth effect on N loading (and other nutrients) will require integration of biogeochemical and hydrologic models.