From the Field to the Lab: Best Practices for Field Preservation of Bat Specimens for Molecular Analyses

Studies in molecular ecology depend on field-collected samples for genetic information, and the tissue sampled and preservation conditions strongly affect the quality of the DNA obtained. DNA yields from different tissue types have seldom been compared, and the relative performance of storage media has never been directly tested, even though these media may influence DNA degradation under field conditions. We analyzed DNA yield from buccal swabs and wing punches harvested from live bats using nucleic acid quantification as well as quantitative PCR for a single-copy nuclear locus. We also compared DNA yields from wing tissue preserved in three media: ethanol, NaCl-saturated dimethyl sulfoxide (DMSO), and silica desiccant. Wing punches yielded more total DNA than did buccal swabs, and wing tissues preserved in silica beads yielded significantly more total and nuclear DNA than those preserved in DMSO or ethanol. These results show that tissue type and preservation media strongly influence the quantity of DNA obtained from non-lethal genetic samples, and based on these effects we provide recommendations for field collection of tissues for genetic analyses

Evolutionary implications of Liebig's law of the minimum : Selection under low concentrations of two nonsubstitutable nutrients

Interactions between different axes of an organism's niche determine the evolutionary trajectory of a population. An extreme case of these interactions is predicted from ecological theory in Liebig's law of the minimum. This law states that in environments where multiple nutrients are in relatively low concentrations, only one nutrient will affect the growth of the organism. This implies that the evolutionary response of the population would be dictated by the most growth-limiting nutrient. Alternatively, it is possible that an initial adaptation to the most limiting nutrient results in other nutrients present in low concentration affecting the evolutionary dynamics of the population. To test these hypotheses, we conducted twelve evolution experiments in chemostats using Escherichia coli populations: four under nitrogen limitation, four under magnesium limitation, and four in which both nitrogen and magnesium are in low concentrations. In the last environment, only magnesium seems to limit growth (Low Nitrogen Magnesium Limited environment, LNML). We observe a decrease in nitrogen concentration in the LNML environment over the course of our evolution experiment indicating that nitrogen might become limiting in these environments. Genetic reconstruction results show that clones adapted to magnesium limitation have genes involved in nitrogen starvation, that is, glnG (nitrogen starvation transcriptional regulator) and amtB (transport protein) to be upregulated only in the LNML environment as compared to magnesium-limiting environments. Together, our results highlights that in low-nutrient environments, adaptation to the growth-limiting nutrient results in other nutrients at low concentrations to play a role in the evolutionary dynamics of the population

Pervasive Selection for Clinically Relevant Resistance and Media Adaptive Mutations at Very Low Antibiotic Concentrations

Experimental evolution studies have shown that weak antibiotic selective pressures (i.e., when the antibiotic concentrations are far below the minimum inhibitory concentration, MIC) can select resistant mutants, raising several unanswered questions. First, what are the lowest antibiotic concentrations at which selection for de novo resistance mutations can occur? Second, with weak antibiotic selections, which other types of adaptive mutations unrelated to the antibiotic selective pressure are concurrently enriched? Third, are the mutations selected under laboratory settings at subMIC also observed in clinical isolates? We addressed these questions using Escherichia coli populations evolving at subMICs in the presence of either of four clinically used antibiotics: fosfomycin, nitrofurantoin, tetracycline, and ciprofloxacin. Antibiotic resistance evolution was investigated at concentrations ranging from 1/4th to 1/2000th of the MIC of the susceptible strain (MICsusceptible). Our results show that evolution was rapid across all the antibiotics tested, and selection for fosfomycin- and nitrofurantoin-resistant mutants was observed at a concentration as low as 1/2000th of MICsusceptible. Several of the evolved resistant mutants showed increased growth yield and exponential growth rates, and outcompeted the susceptible ancestral strain in the absence of antibiotics as well, suggesting that adaptation to the growth environment occurred in parallel with the selection for resistance. Genomic analysis of the resistant mutants showed that several of the mutations selected under these conditions are also found in clinical isolates, demonstrating that experimental evolution at very low antibiotic levels can help in identifying novel mutations that contribute to bacterial adaptation during subMIC exposure in real-life settings

Supplementary Figures

Figures S1–S9. Descriptions for the figures are contained in the file "Rojas_et_al_supplementary_material.docx

timing_speciation

R script to quantify the number and timing of speciation events between extant sister species for the Quaternary and pre-Quaternary (Neogene + Paleogene) periods on a posterior sample of time-calibrated phylogenies. The code can be modified to accommodate older periods if necessary. The file "my_trees.nex" is a generic example that refers to any file including a posterior distribution of trees

Rojas_et_al_supplementary_material

Descriptions of figures S1–S9. Descriptions of Tables S1, S2 and S5. Tables S3, S4, S6 and S7

Different adaptive strategies in E-coli populations evolving under macronutrient limitation and metal ion limitation

Background: Adaptive responses to nutrient limitation involve mutations that increase the efficiency of usage or uptake of the limiting nutrient. However, starvation of different nutrients has contrasting effects on physiology, resulting in different evolutionary responses. Most studies performed to understand these evolutionary responses have focused only on macronutrient limitation. Hence our understanding of adaptation under limitation of other forms of nutrients is limited. In this study, we compared the evolutionary response in populations evolving under growth-limiting conditions for a macronutrient and a major cation. Results: We evolved eight populations of E. coli in nutrient-limited chemostats for 400 generations to identify the genetic basis of the mechanisms involved in efficient usage of two nutrients: nitrogen and magnesium. Our population genomic sequencing work, based on this study and previous work, allowed us to identify targets of selection under these nutrient limiting conditions. Global transcriptional regulators glnGL were targets of selection under nitrogen starvation, while proteins involved in outer-membrane biogenesis (genes from the lpt operon) were targets of selection under magnesium starvation. The protein involved in cell-cycle arrest (yhaV) was a target of selection in both environments. We re-constructed specific mutants to analyze the effect of individual mutations on fitness in nutrient limiting conditions in chemostats and in batch cultures. We further demonstrated that adaptation to nitrogen starvation proceeds via a nutrient specific mechanism, while that to magnesium starvation involves a more general mechanism. Conclusions: Our results show two different forms of adaptive strategies under limitation of nutrients that effect cellular physiology in different ways. Adaptation to nitrogen starvation proceeds by upregulation of transcriptional regulator glnG and subsequently of transporter protein amtB, both of which results in increased nitrogen scavenging ability of the cell. On the other hand, adaptation to magnesium starvation proceeds via the restructuring of the cell outer-membrane, allowing magnesium to be redistributed to other biological processes. Also, adaptation to the chemostat environment involves selection for loss of function mutations in genes that under nutrient-limiting conditions interfere with continuous growth

Rojas_et_al_Table_S5

Distribution of New World Noctilionoidea in Central and North America, South America, and the Antilles. Taxa were assigned to one or more of the three biogeographic regions based on their current distribution determined using the mammal distributions compiled by the IUCN (data downloaded on October 2013), and the literature since 2008. 1 = present, 0 = absent