Chromosome-Level Genome Assembly of the Blacktail Brush Lizard, Urosaurus nigricaudus, Reveals Dosage Compensation in an Endemic Lizard.

Urosaurus nigricaudus is a phrynosomatid lizard endemic to the Baja California Peninsula in Mexico. This work presents a chromosome-level genome assembly and annotation from a male individual. We used PacBio long reads and HiRise scaffolding to generate a high-quality genomic assembly of 1.87 Gb distributed in 327 scaffolds, with an N50 of 279 Mb and an L50 of 3. Approximately 98.4% of the genome is contained in 14 scaffolds, with 6 large scaffolds (334-127 Mb) representing macrochromosomes and 8 small scaffolds (63-22 Mb) representing microchromosomes. Using standard gene modeling and transcriptomic data, we predicted 17,902 protein-coding genes on the genome. The repeat content is characterized by a large proportion of long interspersed nuclear elements that are relatively old. Synteny analysis revealed some microchromosomes with high repeat content are more prone to rearrangements but that both macro- and microchromosomes are well conserved across reptiles. We identified scaffold 14 as the X chromosome. This microchromosome presents perfect dosage compensation where the single X of males has the same expression levels as two X chromosomes in females. Finally, we estimated the effective population size for U. nigricaudus was extremely low, which may reflect a reduction in polymorphism related to it becoming a peninsular endemic.

Reference Genome of the Northwestern Pond Turtle, Actinemys marmorata.

The northwestern pond turtle, Actinemys marmorata, and its recently recognized sister species, the southwestern pond turtle, A. pallida, are the sole aquatic testudines occurring over most of western North America and the only living representatives of the genus Actinemys. Although it historically ranged from Washington state through central California, USA, populations of the northwestern pond turtle have been in decline for decades and the species is afforded state-level protection across its range; it is currently being considered for protection under the US Endangered Species Act. Here, we report a new, chromosome-level assembly of A. marmorata as part of the California Conservation Genomics Project (CCGP). Consistent with the reference genome strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technology to produce a de novo assembled genome. The assembly comprises 198 scaffolds spanning 2,319,339,408 base pairs, has a contig N50 of 75 Mb, a scaffold N50 of 146Mb, and BUSCO complete score of 96.7%, making it the most complete testudine assembly of the 24 species from 13 families that are currently available. In combination with the A. pallida reference genome that is currently under construction through the CCGP, the A. marmorata genome will be a powerful tool for documenting landscape genomic diversity, the basis of adaptations to salt tolerance and thermal capacity, and hybridization dynamics between these recently diverged species.

Response to Comment on "Individual heterozygosity predicts translocation success in threatened desert tortoises".

Hansson et al argue that our main finding could provide an overly simplistic metric for maximizing genetic rescue. They agree that translocating the most genetically diverse individuals led to a large increase in translocated tortoise survival, but recommend instead moving individuals that have low genetic load and the greatest representation of metapopulation diversity. Their recommendation is based on specific model assumptions and fitness effects that are often unknown and are not generalizable to many endangered species applications.

Response to Comment on "Individual heterozygosity predicts translocation success in threatened desert tortoises".

Hedrick brings up several potential concerns that he feels challenge or limit our main finding. Hedrick does not comment on our empirical results, but rather argues that several factors may confound or invalidate our conclusion. Many of these concerns focus on unknown ecological aspects of the translocated tortoises, but we believe there is no reason to conclude that they bias the results or interpretation as presented in our original paper.

Genome-wide Association Analysis of Proinflammatory Cytokines and Gene-lifestyle Interaction for Invasive Breast Cancer Risk: The WHI dbGaP Study.

Immune-related etiologic pathways to influence invasive breast cancer risk may interact with lifestyle factors, but the interrelated molecular genetic pathways are incompletely characterized. We used data from the Women's Health Initiative Database for Genotypes and Phenotypes Study including 16,088 postmenopausal women, a population highly susceptible to inflammation, obesity, and increased risk for breast cancer. With 21,784,812 common autosomal single-nucleotide polymorphisms (SNP), we conducted a genome-wide association (GWA) gene-environment interaction (G × E) analysis in six independent GWA Studies for proinflammatory cytokines [IL6 and C-reactive protein (CRP)] and their gene-lifestyle interactions. Subsequently, we tested for the association of the GWA SNPs with breast cancer risk. In women overall and stratified by obesity status (body mass index, waist circumference, and waist-to-hip ratio) and obesity-related lifestyle factors (exercise and high-fat diet), 88 GWA SNPs in 10 loci were associated with proinflammatory cytokines: 3 associated with IL6 (1 index SNP in MAPK1 and 1 independent SNP in DEC1); 85 with CRP (3 index SNPs in CRPP1, CRP, RP11-419N10.5, HNF1A-AS1, HNF1A, and C1q2orf43; and two independent SNPs in APOE and APOC1). Of those, 27 in HNF1A-AS1, HNF1A, and C1q2orf43 displayed significantly increased risk for breast cancer. We found a number of novel top markers for CRP and IL6, which interacted with obesity factors. A substantial proportion of those SNPs' susceptibility influenced breast cancer risk. Our findings may contribute to better understanding of genetic associations between pro-inflammation and cancer and suggest intervention strategies for women who carry the risk genotypes, reducing breast cancer risk. PREVENTION RELEVANCE: The top GWA-SNPs associated with pro-inflammatory biomarkers have implications for breast carcinogenesis by interacting with obesity factors. Our findings may suggest interventions for women who carry the inflammatory-risk genotypes to reduce breast cancer risk.

Individual heterozygosity predicts translocation success in threatened desert tortoises.

Anthropogenic environmental modification is placing as many as 1 million species at risk of extinction. One management action for reducing extinction risk is translocation of individuals to locations from which they have disappeared or to new locations where biologists hypothesize they have a good chance of surviving. To maximize this survival probability, the standard practice is to move animals from the closest possible populations that contain presumably related individuals. In an empirical test of this conventional wisdom, we analyzed a genomic dataset for 166 translocated desert tortoises (Gopherus agassizii) that either survived or died over a period of two decades. We used genomic data to infer the geographic origin of translocated tortoises and found that individual heterozygosity predicted tortoise survival, whereas translocation distance or geographic unit of origin did not. Our results suggest a relatively simple indicator of the likelihood of a translocated individual's survival: heterozygosity.

Historical museum collections and contemporary population studies implicate roads and introduced predatory bullfrogs in the decline of western pond turtles.

The western pond turtle (WPT), recently separated into two paripatrically distributed species (Emys pallida and Emys marmorata), is experiencing significant reductions in its range and population size. In addition to habitat loss, two potential causes of decline are female-biased road mortality and high juvenile mortality from non-native predatory bullfrogs (Rana catesbeiana). However, quantitative analyses of these threats have never been conducted for either species of WPT. We used a combination of historical museum samples and published and unpublished field studies shared with us through personal communications with WPT field researchers (B. Shaffer, P. Scott, R. Fisher, C. Brown, R. Dagit, L. Patterson, T. Engstrom, 2019, personal communications) to quantify the effect of roads and bullfrogs on WPT populations along the west coast of the United States. Both species of WPT shift toward increasingly male biased museum collections over the last century, a trend consistent with increasing, female-biased road mortality. Recent WPT population studies revealed that road density and proximity were significantly associated with increasingly male-biased sex ratios, further suggesting female-biased road mortality. The mean body size of museum collections of E. marmorata, but not E. pallida, has increased over the last 100 years, consistent with reduced recruitment and aging populations that could be driven by invasive predators. Contemporary WPT population sites that co-occur with bullfrogs had significantly greater average body sizes than population sites without bullfrogs, suggesting strong bullfrog predation on small WPT hatchlings and juveniles. Overall, our findings indicate that both species of WPT face demographic challenges which would have been difficult to document without the use of both historical data from natural history collections and contemporary demographic field data. Although correlational, our analyses suggest that female-biased road mortality and predation on small turtles by non-native bullfrogs are occurring, and that conservation strategies reducing both may be important for WPT recovery.

Does Body Shape in Fundulus Adapt to Variation in Habitat Salinity?

Understanding the ecological pressures that generate variation in body shape is important because body shape profoundly affects physiology and overall fitness. Using Fundulus, a genus of fish that exhibits considerable morphological and physiological variation with evidence of repeated transitions between freshwater and saltwater habitats, we tested whether habitat salinity has influenced the macroevolution of body shape at different stages in development. After accounting for phylogenetic inertia, we find that body shape deviates from the optimal streamlined shape in a manner consistent with different osmoregulatory pressures exerted by different salinity niches at every stage of ontogeny that we examined. We attribute variation in body shape to differential selection for osmoregulatory efficiency because: (1) saline intolerant species developed body shapes with relatively low surface areas more conducive to managing osmoregulatory demands and (2) inland species that exhibit high salinity tolerances have body shapes similar to saline tolerant species in marine environments.

Adapterama III: Quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD).

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

Formation of a recent hybrid zone offers insight into the geographic puzzle and maintenance of species boundaries in musk turtles.

Speciation is the result of an accumulation of reproductive barriers between populations, but pinpointing the factors involved is often difficult. However, hybrid zones can form when these barriers are not complete, especially when lineages come into contact in intermediate or modified habitats. We examine a hybrid zone between two closely related riverine turtle species, Sternotherus depressus and S. peltifer, and use dual-digest RAD sequencing to understand how this hybrid zone formed and elucidate genomic patterns of reproductive isolation. First, the geographical extent and timing of formation of the hybrid zone is established to provide context for understanding the role of extrinsic and intrinsic reproductive isolating mechanisms in this system. The strength of selection on taxon-specific contributions to maintenance of the hybrid zone is then inferred using a Bayesian genomic cline model. These analyses identify a role for selection inhibiting introgression in some genomic regions at one end of the hybrid zone and promoting introgression in many loci at the other. When selective pressures necessary to generate outliers to the genomic cline are considered with the geographical and temporal context of this hybrid zone, we conclude that habitat-specific selection probably limits introgression from S. depressus to S. peltifer in the direction of river flow. However, selection is mediating rapid, unidirectional introgression from S. peltifer to S. depressus, which is probably facilitated by anthropogenic habitat alteration. These findings indicate a potentially imminent threat of population-level genomic extinction for an already imperiled species due to ongoing human-caused habitat alteration.

Follow-up ecological studies for cryptic species discoveries: Decrypting the leopard frogs of the eastern U.S.

Cryptic species are a challenge for systematics, but their elucidation also may leave critical information gaps about the distribution, conservation status, and behavior of affected species. We use the leopard frogs of the eastern U.S. as a case study of this issue. We refined the known range of the recently described Rana kauffeldi, the Atlantic Coast Leopard Frog, relative to the region's two other leopard frog species, conducted assessments of conservation status, and improved methods for separating the three species using morphological field characters. We conducted over 2,000 call and visual surveys and took photographs of and tissue samples from hundreds of frogs. Genetic analysis supported a three-species taxonomy and provided determinations for 220 individual photographed frogs. Rana kauffeldi was confirmed in eight U.S. states, from North Carolina to southern Connecticut, hewing closely to the Atlantic Coastal Plain. It can be reliably differentiated in life from R. pipiens, and from R. sphenocephala 90% of the time, based on such characters as the femoral reticulum patterning, dorsal spot size and number, and presence of a snout spot. However, the only diagnostic character separating R. kauffeldi from R. sphenocephala remains the breeding call described in 2014. Based on our field study, museum specimens, and prior survey data, we suggest that R. kauffeldi has declined substantially in the northern part of its range, but is more secure in the core of its range. We also report, for the first time, apparent extirpations of R. pipiens from the southeastern portion of its range, previously overlooked because of confusion with R. kauffeldi. We conclude with a generalized ecological research agenda for cryptic species. For R. kauffeldi, needs include descriptions of earlier life stages, studies of niche partitioning with sympatric congeners and the potential for hybridization, and identification of conservation actions to prevent further declines.

Resolving taxonomic turbulence and uncovering cryptic diversity in the musk turtles (Sternotherus) using robust demographic modeling.

Accurate and consistent delimitation of species and their relationships provides a necessary framework for comparative studies, understanding evolutionary relationships, and informing conservation management. Despite the ever-increasing availability of genomic data, evolutionary dynamics can still render some relationships exceedingly difficult to resolve, including underlying speciation events that are rapid, recent, or confounded by post-speciation introgression. Here we present an empirical study of musk turtles (Sternotherus), which illustrates approaches to resolve difficult nodes in the Tree of Life that robust species-tree methods fail to resolve. We sequence 4430 RAD-loci from 205 individuals. Independent coalescent-based analyses, corroborated with morphology and geography, strongly support the recognition of cryptic species within Sternotherus, but with conflicting or weak support for some intraspecific relationships. To resolve species-tree conflict, we use a likelihood-based approach to test support for alternative demographic models behind alternative speciation scenarios and argue that demographic model testing has an important role for resolving systematic relationships in recent, rapid radiations. Species-tree and demographic modeling strongly support the elevation of two nominal subspecies in Sternotherus to species and the recognition of a previously cryptic species (S. intermedius sp. nov.) described within. The evolutionary and taxonomic history of Sternotherus is discussed in the context of these new species and novel and well-supported systematic hypotheses.

Epilepsy service provision in the National Health Service in England in 2012.

PURPOSE: The National Health Service in the United Kingdom compares favourably on many international measures of quality and cost-effectiveness. It has many centres of excellence for epilepsy care and in some areas is a world-leader. However, for over twenty years there have been concerns that the provision of good quality care is variable and in many areas major improvements are required. We report the results of the latest major survey into the quality of epilepsy care in the NHS. METHODS: Four target groups were sent questionnaires: acute hospital trusts, Clinical Commissioning Groups, local authorities and patients. Each questionnaire was specifically designed by Epilepsy Action after reviewing national guidance and quality standards. RESULTS: We present the key results of the survey and we discuss them in the context of the latest national guidelines, quality standards, the organisational structure of the NHS and the research literature. CONCLUSION: Although there are some examples of excellent services for people with epilepsy these results show that overall there has been little improvement in recent years and there continues to be significant geographical variability in quality with many areas offering sub-optimal care.

A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways.

Obesity and type 2 diabetes are characterized by decreased insulin sensitivity, elevated concentrations of free fatty acids (FFAs), and increased macrophage infiltration in adipose tissue (AT). Here, we show that FFAs can cause activation of RAW264.7 cells primarily via the JNK signaling cascade and that TLR2 and TLR4 are upstream of JNK and help transduce FFA proinflammatory signals. We also demonstrate that F4/80(+)CD11b(+)CD11c(+) bone marrow-derived dendritic cells (BMDCs) have heightened proinflammatory activity compared with F4/80(+)CD11b(+)CD11c(-) bone marrow-derived macrophages and that the proinflammatory activity and JNK phosphorylation of BMDCs, but not bone marrow-derived macrophages, was further increased by FFA treatment. F4/80(+)CD11b(+)CD11c(+) cells were found in AT, and the proportion and number of these cells in AT is increased in ob/ob mice and by feeding wild type mice a high fat diet for 1 and 12 weeks. AT F4/80(+)CD11b(+)CD11c(+) cells express increased inflammatory markers compared with F4/80(+)CD11b(+)CD11c(-) cells, and FFA treatment increased inflammatory responses in these cells. In addition, we found that CD11c expression is increased in skeletal muscle of high fat diet-fed mice and that conditioned medium from FFA-treated wild type BMDCs, but not TLR2/4 DKO BMDCs, can induce insulin resistance in L6 myotubes. Together our results show that FFAs can activate CD11c(+) myeloid proinflammatory cells via TLR2/4 and JNK signaling pathways, thereby promoting inflammation and subsequent cellular insulin resistance.