Genome resequencing clarifies phylogeny and reveals patterns of selection in the toxicogenomics model Pimephales promelas.

Background: The fathead minnow (Pimephales promelas) is a model species for toxicological research. A high-quality genome reference sequence is available, and genomic methods are increasingly used in toxicological studies of the species. However, phylogenetic relationships within the genus remain incompletely known and little population-genomic data are available for fathead minnow despite the potential effects of genetic background on toxicological responses. On the other hand, a wealth of extant samples is stored in museum collections that in principle allow fine-scale analysis of contemporary and historical genetic variation. Methods: Here we use short-read shotgun resequencing to investigate sequence variation among and within Pimephales species. At the genus level, our objectives were to resolve phylogenetic relationships and identify genes with signatures of positive diversifying selection. At the species level, our objective was to evaluate the utility of archived-sample resequencing for detecting selective sweeps within fathead minnow, applied to a population introduced to the San Juan River of the southwestern United States sometime prior to 1950. Results: We recovered well-supported but discordant phylogenetic topologies for nuclear and mitochondrial sequences that we hypothesize arose from mitochondrial transfer among species. The nuclear tree supported bluntnose minnow (P. notatus) as sister to fathead minnow, with the slim minnow (P. tenellus) and bullhead minnow (P. vigilax) more closely related to each other. Using multiple methods, we identified 11 genes that have diversified under positive selection within the genus. Within the San Juan River population, we identified selective-sweep regions overlapping several sets of related genes, including both genes that encode the giant sarcomere protein titin and the two genes encoding the MTORC1 complex, a key metabolic regulator. We also observed elevated polymorphism and reduced differentation among populations (FST) in genomic regions containing certain immune-gene clusters, similar to what has been reported in other taxa. Collectively, our data clarify evolutionary relationships and selective pressures within the genus and establish museum archives as a fruitful resource for characterizing genomic variation. We anticipate that large-scale resequencing will enable the detection of genetic variants associated with environmental toxicants such as heavy metals, high salinity, estrogens, and agrichemicals, which could be exploited as efficient biomarkers of exposure in natural populations.

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications.

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Keeping up with the neighbor: a novel mechanism of call synchrony in Neoconocephalus ensiger katydids.

During solo calling, pulse periods gradually changed by up to 15% over several minutes. Pairs of calling males synchronized their pulses. The pulse rate (10-14 Hz) was considerably faster than the rate of synchronized signal units in other insects (0.5-3 Hz). Within each pulse cycle, males made only small adjustments to their pulse period, leading to regular switches of leader and follower roles. Large-scale timing adjustments only occurred in response to large delays. Stimulation with single pulses had no predictable effect on the timing of the male's next pulse, resulting in a flat phase response curve. When entrained to a stimulus with a faster pulse period, males briefly interrupted calling; they resumed calling largely synchronized with the stimulus. Throughout the stimulus, males made gradual changes to their pulse period, similar to those during pair calling. After the stimulus ended, pulse periods increased over several minutes, but did not return to their pre-stimulus values. Thus social context and intrinsic state of the males influenced pulse period in Neoconocephalus ensiger. These results indicate that N. ensiger males synchronize calls by adjusting their intrinsic pulse period, instead of adjusting the timing of individual pulses, as described in other synchronizing insects.