Validating sonication as a DNA extraction method for use with carrion flies.

Entomological evidence can be critical in establishing a postmortem interval estimate. DNA-based species identification can be an extremely valuable tool for forensic entomology. The problem of processing samples in a consistent, cost-effective manner that retains the morphological attributes of the specimen for vouchering has led us to investigate sonication as a primary means of non-destructive DNA extraction from carrion flies. We analyze the efficacy of this technique and compare it to an established DNA extraction technique - the Qiagen DNeasy tissue kit. Our results indicate that sonication produces a significant reduction in the sequence length and lower PHRED quality scores when compared to sequences using DNA obtained using the DNeasy kit, but species identification and phylogenetic inferences between sonication and DNeasy extractions are equivalent.

Mutations to Less-Preferred Synonymous Codons in a Highly Expressed Gene of Escherichia coli: Fitness and Epistatic Interactions.

Codon-tRNA coevolution to maximize protein production has been, until recently, the dominant hypothesis to explain codon-usage bias in highly expressed bacterial genes. Two predictions of this hypothesis are 1) selection is weak; and 2) similar silent replacements at different codons should have similar fitness consequence. We used an allele-replacement strategy to change five specific 3rd-codon-position (silent) sites in the highly expressed Escherichia coli ribosomal protein gene rplQ from the wild type to a less-preferred alternative. We introduced the five mutations within a 10-codon region. Four of the silent sites were chosen to test the second prediction, with a CTG to CTA mutation being introduced at two closely linked leucine codons and an AAA to AAG mutation being introduced at two closely linked lysine codons. We also introduced a fifth silent mutation, a GTG to GTA mutation at a valine codon in the same genic region. We measured the fitness effect of the individual mutations by competing each single-mutant strain against the parental wild-type strain, using a disrupted form of the araA gene as a selectively neutral phenotypic marker to distinguish between strains in direct competition experiments. Three of the silent mutations had a fitness effect of |s| > 0.02, which is contradictory to the prediction that selection will be weak. The two leucine mutations had significantly different fitness effects, as did the two lysine mutations, contradictory to the prediction that similar mutations at different codons should have similar fitness effects. We also constructed a strain carrying all five silent mutations in combination. Its fitness effect was greater than that predicted from the individual fitness values, suggesting that negative synergistic epistasis acts on the combination allele.

Sequencing human mitochondrial hypervariable region II as a molecular fingerprint for environmental waters.

To protect environmental water from human fecal contamination, authorities must be able to unambiguously identify the source of the contamination. Current identification methods focus on tracking fecal bacteria associated with the human gut, but many of these bacterial indicators also thrive in the environment and in other mammalian hosts. Mitochondrial DNA could solve this problem by serving as a human-specific marker for fecal contamination. Here we show that the human mitochondrial hypervariable region II can function as a molecular fingerprint for human contamination in an urban watershed impacted by combined sewer overflows. We present high-throughput sequencing analysis of hypervariable region II for spatial resolution of the contaminated sites and assessment of the population diversity of the impacting regions. We propose that human mitochondrial DNA from public waste streams may serve as a tool for identifying waste sources definitively, analyzing population diversity, and conducting other anthropological investigations.

DNA-based identification of forensically important Lucilia (Diptera: Calliphoridae) in the continental United States.

Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA-based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100-bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA-based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA-based identification is likely to be successful for the other seven species.

Microbial survey of a full-scale, biologically active filter for treatment of drinking water.

The microbial community of a full-scale, biologically active drinking water filter was surveyed using molecular techniques. Nitrosomonas, Nitrospira, Sphingomonadales, and Rhizobiales dominated the clone libraries. The results elucidate the microbial ecology of biological filters and demonstrate that biological treatment of drinking water should be considered a viable alternative to physicochemical methods.

mtDNA-based identification of Lucilia cuprina (Wiedemann) and Lucilia sericata (Meigen) (Diptera: Calliphoridae) in the continental United States.

Existing data suggest that the forensically important dipteran species Lucilia cuprina (Wiedemann) and Lucilia sericata (Meigen) may be particularly difficult to discriminate using DNA sequence data. L. cuprina is paraphyletic with respect to L. sericata in mtDNA phylogenies, with some L. cuprina having mtDNA haplotypes that are very similar to those of L. sericata. We examine this problem by providing the first DNA data for L. cuprina from North America, including portions of both the mitochondrial COI gene and the nuclear 28S rRNA gene. With the new data, L. cuprina remains monophyletic for 28S but paraphyletic with respect to L. sericata for COI. However, we find that all flies that are identified as L. cuprina by morphology and have L. sericata-like mtDNA form a distinctly monophyletic mtDNA clade. This clade may possibly have originated by hybridization between L. cuprina and L. sericata, but its wide geographic distribution strongly suggests a singular origin as opposed to repeated incidents of hybridization. The phylogenetic results strongly support the hypothesis that L. cuprina and L. sericata can be discriminated using mtDNA sequence data. We find that a fragment of COI spanning approximately 1200 base pairs is sufficient to discriminate between the two species with greater than 95% bootstrap support.

The nocturnal ovipositing behavior of carrion flies in Cincinnati, Ohio.

The behavioral patterns of nocturnal oviposition represent a window of time that potentially has a large impact on postmortem interval estimations. We investigated the behavioral patterns of carrion flies at night by exposing euthanized rats between sunset and sunrise to see if carrion flies oviposited upon the carrion over two consecutive summers. We investigated urban and rural locations, in both lit and unlit conditions with n = 125. We found that nocturnal ovipositing did not occur in the Cincinnati metropolitan area. We conclude that nocturnal oviposition is an unlikely event in the Cincinnati metropolitan area.

Rodent phylogeny revised: analysis of six nuclear genes from all major rodent clades.

BACKGROUND: Rodentia is the most diverse order of placental mammals, with extant rodent species representing about half of all placental diversity. In spite of many morphological and molecular studies, the family-level relationships among rodents and the location of the rodent root are still debated. Although various datasets have already been analyzed to solve rodent phylogeny at the family level, these are difficult to combine because they involve different taxa and genes. RESULTS: We present here the largest protein-coding dataset used to study rodent relationships. It comprises six nuclear genes, 41 rodent species, and eight outgroups. Our phylogenetic reconstructions strongly support the division of Rodentia into three clades: (1) a "squirrel-related clade", (2) a "mouse-related clade", and (3) Ctenohystrica. Almost all evolutionary relationships within these clades are also highly supported. The primary remaining uncertainty is the position of the root. The application of various models and techniques aimed to remove non-phylogenetic signal was unable to solve the basal rodent trifurcation. CONCLUSION: Sequencing and analyzing a large sequence dataset enabled us to resolve most of the evolutionary relationships among Rodentia. Our findings suggest that the uncertainty regarding the position of the rodent root reflects the rapid rodent radiation that occurred in the Paleocene rather than the presence of conflicting phylogenetic and non-phylogenetic signals in the dataset.

The systematic component of phylogenetic error as a function of taxonomic sampling under parsimony.

The effect of taxonomic sampling on phylogenetic accuracy under parsimony is examined by simulating nucleotide sequence evolution. Random error is minimized by using very large numbers of simulated characters. This allows estimation of the consistency behavior of parsimony, even for trees with up to 100 taxa. Data were simulated on 8 distinct 100-taxon model trees and analyzed as stratified subsets containing either 25 or 50 taxa, in addition to the full 100-taxon data set. Overall accuracy decreased in a majority of cases when taxa were added. However, the magnitude of change in the cases in which accuracy increased was larger than the magnitude of change in the cases in which accuracy decreased, so, on average, overall accuracy increased as more taxa were included. A stratified sampling scheme was used to assess accuracy for an initial subsample of 25 taxa. The 25-taxon analyses were compared to 50- and 100-taxon analyses that were pruned to include only the original 25 taxa. On average, accuracy for the 25 taxa was improved by taxon addition, but there was considerable variation in the degree of improvement among the model trees and across different rates of substitution.

Identifying conflicting signal in a multigene analysis reveals a highly resolved tree: the phylogeny of Rodentia (Mammalia).

Homoplasy among morphological characters has hindered inference of higher level rodent phylogeny for over 100 years. Initial molecular studies, based primarily on single genes, likewise produced little resolution of the deep relationships among rodent families. Two recent molecular studies (Huchon et al., 2002, Mol. Biol. Evol. 19:1053-1065; Adkins et al., 2003, Mol. Phylogenet. Evol. 26:409-420), using larger samples from the nuclear genome, have produced phylogenies that are generally concordant with each other, but many of the deep superfamilial nodes were still lacking substantial statistical support. Data are presented here for a total of approximately 3,600 base pairs from portions of three different nuclear protein-coding genes, CB1, IRBP, and RAG2, from 19 rodents and three outgroups. Separate analyses, with data partitioned according to both genes and codon position, produced conflicting results. Trees obtained from all partitions of CB1 and RAG2 and those obtained from the first- plus second-position sites of IRBP were generally concordant with each other and the trees from the two recent studies, whereas trees obtained from the third-position sites of IRBP were not. Although the IRBP third-position sites represent only 1/9 of the total data set, combined analyses using either parsimony or likelihood resulted in trees in agreement with the IRBP third-position sites and in disagreement with the remaining 8/9 of the sites from this data set and the two recent multigene studies. In contrast, maximum-likelihood analysis using a site-specific rates model did recover a tree that is highly congruent with the trees in the two recent studies. If the IRBP third-position sites are removed from the current data set, then combined likelihood analyses obtain a tree that is highly congruent with those of the two recent studies. This analysis also provides, for the first time in a study of rodent phylogeny, robust statistical support for every bipartition, with just one exception. This tree divides rodents into two major clades. The first contains Myodonta (Muroidea plus Dipodidae) and the only unresolved trichotomy, from which descend Geomyoidea, Pedetidae, and Castoridae. On the other side of the root is a clade containing Sciuroidea plus Gliridae, and Hystricognathi. Some uncertainty remains on the placement of the root. Trees on which the Hystricognathi are the basal sister group to Myodonta, Geomyoidea, Pedetidae, and Castoridae are also found within a Bayesian 95% credible set, as estimated by Metropolis-coupled Markov chain Monte Carlo sampling.

BIOGEOGRAPHY OF NEW WORLD TAIGA-DWELLING MICROTUS (MAMMALIA: ARVICOLIDAE): A HYPOTHESIS TEST THAT ACCOUNTS FOR PHYLOGENETIC UNCERTAINTY.

If we adopt a statistical approach to systematics and recognize that phylogenies are estimated with error, then we can begin to explore statistically justified methods for testing a variety of comparative hypotheses, including those concerning the evolution of life-history characters and biogeography. In this paper I examine two biogeographic hypotheses concerning the rodent genus Microtus. Like many comparative hypotheses, these can be phrased so that each predicts the existence of a particular monophyletic group. Neither of the predicted groups appear on the single best phylogeny as determined by both Dollo parsimony and maximum likelihood analysis of restriction site maps of mitochondrial DNA. Simulation studies, however, suggest that often the best phylogeny from a single data set has only a low probability of being exactly correct. We must also examine those trees that, while not the single best-supported tree, are not rejected by the data. If we find the best phylogeny for which a hypothesis is satisfied, then likelihood methods can be used to test whether that phylogeny is significantly worse then the best tree overall. If that tree can be rejected, then so can the hypothesis. Computational constraints limit the use of likelihood methods for searching among topologies, so parsimony is used as a data exploratory tool. One of the predicted groups cannot be rejected, even though the most parsimonious tree which includes that group requires 11 more steps than does the most parsimonious tree.