Detecting parallel polygenic adaptation to novel evolutionary pressure in wild populations: a case study in Atlantic cod (Gadus morhua).

Populations can adapt to novel selection pressures through dramatic frequency changes in a few genes of large effect or subtle shifts in many genes of small effect. The latter (polygenic adaptation) is expected to be the primary mode of evolution for many life-history traits but tends to be more difficult to detect than changes in genes of large effect. Atlantic cod (Gadus morhua) were subjected to intense fishing pressure over the twentieth century, leading to abundance crashes and a phenotypic shift toward earlier maturation across many populations. Here, we use spatially replicated temporal genomic data to test for a shared polygenic adaptive response to fishing using methods previously applied to evolve-and-resequence experiments. Cod populations on either side of the Atlantic show covariance in allele frequency change across the genome that are characteristic of recent polygenic adaptation. Using simulations, we demonstrate that the degree of covariance in allele frequency change observed in cod is unlikely to be explained by neutral processes or background selection. As human pressures on wild populations continue to increase, understanding and attributing modes of adaptation using methods similar to those demonstrated here will be important in identifying the capacity for adaptive responses and evolutionary rescue. This article is part of the theme issue 'Detecting and attributing the causes of biodiversity change: needs, gaps and solutions'.

The practice and promise of temporal genomics for measuring evolutionary responses to global change.

Understanding the evolutionary consequences of anthropogenic change is imperative for estimating long-term species resilience. While contemporary genomic data can provide us with important insights into recent demographic histories, investigating past change using present genomic data alone has limitations. In comparison, temporal genomics studies, defined herein as those that incorporate time series genomic data, utilize museum collections and repeated field sampling to directly examine evolutionary change. As temporal genomics is applied to more systems, species and questions, best practices can be helpful guides to make the most efficient use of limited resources. Here, we conduct a systematic literature review to synthesize the effects of temporal genomics methodology on our ability to detect evolutionary changes. We focus on studies investigating recent change within the past 200 years, highlighting evolutionary processes that have occurred during the past two centuries of accelerated anthropogenic pressure. We first identify the most frequently studied taxa, systems, questions and drivers, before highlighting overlooked areas where further temporal genomic studies may be particularly enlightening. Then, we provide guidelines for future study and sample designs while identifying key considerations that may influence statistical and analytical power. Our aim is to provide recommendations to a broad array of researchers interested in using temporal genomics in their work.

Simulation-Based Evaluation of Methods, Data Types, and Temporal Sampling Schemes for Detecting Recent Population Declines.

Understanding recent population trends is critical to quantifying species vulnerability and implementing effective management strategies. To evaluate the accuracy of genomic methods for quantifying recent declines (beginning <120 generations ago), we simulated genomic data using forward-time methods (SLiM) coupled with coalescent simulations (msprime) under a number of demographic scenarios. We evaluated both site frequency spectrum (SFS)-based methods (momi2, Stairway Plot) and methods that employ linkage disequilibrium information (NeEstimator, GONE) with a range of sampling schemes (contemporary-only samples, sampling two time points, and serial sampling) and data types (RAD-like data and whole-genome sequencing). GONE and momi2 performed best overall, with >80% power to detect severe declines with large sample sizes. Two-sample and serial sampling schemes could accurately reconstruct changes in population size, and serial sampling was particularly valuable for making accurate inferences when genotyping errors or minor allele frequency cutoffs distort the SFS or under model mis-specification. However, sampling only contemporary individuals provided reliable inferences about contemporary size and size change using either site frequency or linkage-based methods, especially when large sample sizes or whole genomes from contemporary populations were available. These findings provide a guide for researchers designing genomics studies to evaluate recent demographic declines.

Complex genetic patterns and distribution limits mediated by native congeners of the worldwide invasive red-eared slider turtle.

Non-native (invasive) species offer a unique opportunity to study the geographical distribution and range limits of species, wherein the evolutionary change driven by interspecific interactions between native and non-native closely related species is a key component. The red-eared slider turtle, Trachemys scripta elegans (TSE), has been introduced and successfully established worldwide. It can coexist with its native congeners T. cataspila, T. venusta and T. taylori in Mexico. We performed comprehensive fieldwork, executed a battery of genetic analyses and applied a novel species distribution modelling approach to evaluate their historical lineage relationships and contemporary population genetic patterns. Our findings support the historical common ancestry between native TSE and non-native (TSEalien ), while also highlighting the genetic differentiation of the exotic lineage. Genetic patterns are associated with their range size/endemism gradient; the microendemic T. taylori showed significant reduced genetic diversity and high differentiation, whereas TSEalien showed the highest diversity and signals of population size expansion. Counter to our expectations, lower naturally occurring distribution overlap and little admixture patterns were found between TSE and its congeners, exhibiting reduced gene flow and clear genetic separation across neighbouring species despite having zones of contact. We demonstrate that these native Trachemys species have distinct climatic niche suitability, probably preventing establishment of and displacement by the TSEalien . Moreover, we found major niche overlap between TSEalien and native species worldwide, supporting our prediction that sites with closer ecological optima to the invasive species have higher establishment risk than those that are closer to the niche-centre of the native species.

Land use and life history constrain adaptive genetic variation and reduce the capacity for climate change adaptation in turtles.

BACKGROUND: Rapid anthropogenic climate change will require species to adapt to shifting environmental conditions, with successful adaptation dependent upon current patterns of genetic variation. While landscape genomic approaches allow for exploration of local adaptation in non-model systems, most landscape genomics studies of adaptive capacity are limited to exploratory identification of potentially important functional genes, often without a priori expectations as to the gene functions that may be most important for climate change responses. In this study, we integrated targeted sequencing of genes of known function and genotyping of single-nucleotide polymorphisms to examine spatial, environmental, and species-specific patterns of potential local adaptation in two co-occuring turtle species: the Blanding's turtle (Emydoidea blandingii) and the snapping turtle (Chelydra serpentina). RESULTS: We documented divergent patterns of spatial clustering between neutral and putatively adaptive genetic variation in both species. Environmental associations varied among gene regions and between species, with stronger environmental associations detected for genes involved in stress response and for the more specialized Blanding's turtle. Land cover appeared to be more important than climate in shaping spatial variation in functional genes, indicating that human landscape alterations may affect adaptive capacity important for climate change responses. CONCLUSIONS: Our study provides evidence that responses to climate change will be contingent on species-specific adaptive capacity and past history of exposure to human land cover change.

Genome-wide diversity and habitat underlie fine-scale phenotypic differentiation in the rainbow darter (Etheostoma caeruleum).

Adaptation to environmental change requires that populations harbor the necessary genetic variation to respond to selection. However, dispersal-limited species with fragmented populations and reduced genetic diversity may lack this variation and are at an increased risk of local extinction. In freshwater fish species, environmental change in the form of increased stream temperatures places many cold-water species at-risk. We present a study of rainbow darters (Etheostoma caeruleum) in which we evaluated the importance of genetic variation on adaptive potential and determined responses to extreme thermal stress. We compared fine-scale patterns of morphological and thermal tolerance differentiation across eight sites, including a unique lake habitat. We also inferred contemporary population structure using genomic data and characterized the relationship between individual genetic diversity and stress tolerance. We found site-specific variation in thermal tolerance that generally matched local conditions and morphological differences associated with lake-stream divergence. We detected patterns of population structure on a highly local spatial scale that could not be explained by isolation by distance or stream connectivity. Finally, we showed that individual thermal tolerance was positively correlated with genetic variation, suggesting that sites with increased genetic diversity may be better at tolerating novel stress. Our results highlight the importance of considering intraspecific variation in understanding population vulnerability and stress response.

Rapture-ready darters: Choice of reference genome and genotyping method (whole-genome or sequence capture) influence population genomic inference in Etheostoma.

Researchers studying nonmodel organisms have an increasing number of methods available for generating genomic data. However, the applicability of different methods across species, as well as the effect of reference genome choice on population genomic inference, remain difficult to predict in many cases. We evaluated the impact of data type (whole-genome vs. reduced representation) and reference genome choice on data quality and on population genomic and phylogenomic inference across several species of darters (subfamily Etheostomatinae), a highly diverse radiation of freshwater fish. We generated a high-quality reference genome and developed a hybrid RADseq/sequence capture (Rapture) protocol for the Arkansas darter (Etheostoma cragini). Rapture data from 1,900 individuals spanning four darter species showed recovery of most loci across darter species at high depth and consistent estimates of heterozygosity regardless of reference genome choice. Loci with baits spanning both sides of the restriction enzyme cut site performed especially well across species. For low-coverage whole-genome data, choice of reference genome affected read depth and inferred heterozygosity. For similar amounts of sequence data, Rapture performed better at identifying fine-scale genetic structure compared to whole-genome sequencing. Rapture loci also recovered an accurate phylogeny for the study species and demonstrated high phylogenetic informativeness across the evolutionary history of the genus Etheostoma. Low cost and high cross-species effectiveness regardless of reference genome suggest that Rapture and similar sequence capture methods may be worthwhile choices for studies of diverse species radiations.

Characterizing coral reef biodiversity: genetic species delimitation in brachyuran crabs of Palmyra Atoll, Central Pacific.

Coral reefs are highly threatened ecosystems, yet there are numerous challenges in conducting inventories of their vanishing biodiversity, partly because many taxa remain difficult to detect and describe. Genetic species delimitation methods provide a standardized means for taxonomic classification including of cryptic, rare, or elusive groups, but results can vary by analytical method and genetic marker. In this study, a combination of morphological and genetic identification methods was used to estimate species richness and identify taxonomic units in true crabs (Infraorder Brachyura; n = 200) from coral reefs of Palmyra Atoll, Central Pacific. Genetic identification was based on matches between mitochondrial 16S ribosomal RNA (16S rRNA) and/or cytochrome c oxidase subunit I (COI) sequences to GenBank data, while morphological work relied on the taxonomic literature. Broad agreement in the number of candidate species delimited by genetic distance thresholds and tree-based approaches was found, although the multi-rate Poisson tree process (mPTP) was less appropriate for this dataset. The COI sequence data identified 30-32 provisional species and the 16S data revealed 34-35. The occurrence of 10 families, 20 genera, and 19 species of brachyurans at Palmyra was corroborated by at least two methods. Diversity levels within Chlorodiella laevissima indicated possible undescribed or cryptic species in currently lumped taxa. These results illustrate the efficacy of DNA sequences in identifying organisms and detecting cryptic variation, and underscore the importance of using appropriate genetic markers and multiple species delimitation analyses, with applications for future species descriptions.

Does gene flow aggravate or alleviate maladaptation to environmental stress in small populations?

Environmental change can expose populations to unfamiliar stressors, and maladaptive responses to those stressors may result in population declines or extirpation. Although gene flow is classically viewed as a cause of maladaptation, small and isolated populations experiencing high levels of drift and little gene flow may be constrained in their evolutionary response to environmental change. We provide a case study using the model Trinidadian guppy system that illustrates the importance of considering gene flow and genetic drift when predicting (mal)adaptive response to acute stress. We compared population genomic patterns and acute stress responses of inbred guppy populations from headwater streams either with or without a recent history of gene flow from a more diverse mainstem population. Compared to "no-gene flow" analogues, we found that populations with recent gene flow showed higher genomic variation and increased stress tolerance-but only when exposed to a stress familiar to the mainstem population (heat shock). All headwater populations showed similar responses to a familiar stress in headwater environments (starvation) regardless of gene flow history, whereas exposure to an entirely unfamiliar stress (copper sulfate) showed population-level variation unrelated to environment or recent evolutionary history. Our results suggest that (mal)adaptive responses to acutely stressful environments are determined in part by recent evolutionary history and in part by previous exposure. In some cases, gene flow may provide the variation needed to persist, and eventually adapt, in the face of novel stress.

Geography best explains global patterns of genetic diversity and postglacial co-expansion in marine turtles.

For many species, climate oscillations drove cycles of population contraction during cool glacial periods followed by expansion during interglacials. Some groups, however, show evidence of uniform and synchronous expansion, while others display differences in the timing and extent of demographic change. We compared demographic histories inferred from genetic data across marine turtle species to identify responses to postglacial warming shared across taxa and to examine drivers of past demographic change at the global scale. Using coalescent simulations and approximate Bayesian computation (ABC), we estimated demographic parameters, including the likelihood of past population expansion, from a mitochondrial data set encompassing 23 previously identified lineages from all seven marine turtle species. For lineages with a high posterior probability of expansion, we conducted a hierarchical ABC analysis to estimate the proportion of lineages expanding synchronously and the timing of synchronous expansion. We used Bayesian model averaging to identify variables associated with expansion and genetic diversity. Approximately 60% of extant marine turtle lineages showed evidence of expansion, with the rest mainly exhibiting patterns of genetic diversity most consistent with population stability. For lineages showing expansion, there was a strong signal of synchronous expansion after the Last Glacial Maximum. Expansion and genetic diversity were best explained by ocean basin and the degree of endemism for a given lineage. Geographic differences in sensitivity to climate change have implications for prioritizing conservation actions in marine turtles as well as for identifying areas of past demographic stability and potential resilience to future climate change for broadly distributed taxa.

Genetic effects of landscape, habitat preference and demography on three co-occurring turtle species.

Expanding the scope of landscape genetics beyond the level of single species can help to reveal how species traits influence responses to environmental change. Multispecies studies are particularly valuable in highly threatened taxa, such as turtles, in which the impacts of anthropogenic change are strongly influenced by interspecific differences in life history strategies, habitat preferences and mobility. We sampled approximately 1500 individuals of three co-occurring turtle species across a gradient of habitat change (including varying loss of wetlands and agricultural conversion of upland habitats) in the Midwestern USA. We used genetic clustering and multiple regression methods to identify associations between genetic structure and permanent landscape features, past landscape composition and landscape change in each species. Two aquatic generalists (the painted turtle, Chrysemys picta, and the snapping turtle Chelydra serpentina) both exhibited population genetic structure consistent with isolation by distance, modulated by aquatic landscape features. Genetic divergence for the more terrestrial Blanding's turtle (Emydoidea blandingii), on the other hand, was not strongly associated with geographic distance or aquatic features, and Bayesian clustering analysis indicated that many Emydoidea populations were genetically isolated. Despite long generation times, all three species exhibited associations between genetic structure and postsettlement habitat change, indicating that long generation times may not be sufficient to delay genetic drift resulting from recent habitat fragmentation. The concordances in genetic structure observed between aquatic species, as well as isolation in the endangered, long-lived Emydoidea, reinforce the need to consider both landscape composition and demographic factors in assessing differential responses to habitat change in co-occurring species.

Linking Genetic Kinship and Demographic Analyses to Characterize Dispersal: Methods and Application to Blanding's Turtle.

Characterizing how frequently, and at what life stages and spatial scales, dispersal occurs can be difficult, especially for species with cryptic juvenile periods and long reproductive life spans. Using a combination of mark-recapture information, microsatellite genetic data, and demographic simulations, we characterize natal and breeding dispersal patterns in the long-lived, slow-maturing, and endangered Blanding's turtle (Emydoidea blandingii), focusing on nesting females. We captured and genotyped 310 individual Blanding's turtles (including 220 nesting females) in a central Wisconsin population from 2010 to 2013, with additional information on movements among 3 focal nesting areas within this population available from carapace-marking conducted from 2001 to 2009. Mark-recapture analyses indicated that dispersal among the 3 focal nesting areas was infrequent (<0.03 annual probability). Dyads of females with inferred first-order relationships were more likely to be found within the same nesting area than split between areas, and the proportion of related dyads declined with increasing distance among nesting areas. The observed distribution of related dyads for nesting females was consistent with a probability of natal dispersal at first breeding between nearby nesting areas of approximately 0.1 based on demographic simulations. Our simulation-based estimates of infrequent female dispersal were corroborated by significant spatial genetic autocorrelation among nesting females at scales of <500 m. Nevertheless, a lack of spatial genetic autocorrelation among non-nesting turtles (males and females) suggested extensive local connectivity, possibly mediated by male movements or long-distance movements made by females between terrestrial nesting areas and aquatic habitats. We show here that coupling genetic and demographic information with simulations of individual-based population models can be an effective approach for untangling the contributions of natal and breeding dispersal to spatial ecology.

Finding the right coverage: the impact of coverage and sequence quality on single nucleotide polymorphism genotyping error rates.

Restriction-enzyme-based sequencing methods enable the genotyping of thousands of single nucleotide polymorphism (SNP) loci in nonmodel organisms. However, in contrast to traditional genetic markers, genotyping error rates in SNPs derived from restriction-enzyme-based methods remain largely unknown. Here, we estimated genotyping error rates in SNPs genotyped with double digest RAD sequencing from Mendelian incompatibilities in known mother-offspring dyads of Hoffman's two-toed sloth (Choloepus hoffmanni) across a range of coverage and sequence quality criteria, for both reference-aligned and de novo-assembled data sets. Genotyping error rates were more sensitive to coverage than sequence quality and low coverage yielded high error rates, particularly in de novo-assembled data sets. For example, coverage ≥5 yielded median genotyping error rates of ≥0.03 and ≥0.11 in reference-aligned and de novo-assembled data sets, respectively. Genotyping error rates declined to ≤0.01 in reference-aligned data sets with a coverage ≥30, but remained ≥0.04 in the de novo-assembled data sets. We observed approximately 10- and 13-fold declines in the number of loci sampled in the reference-aligned and de novo-assembled data sets when coverage was increased from ≥5 to ≥30 at quality score ≥30, respectively. Finally, we assessed the effects of genotyping coverage on a common population genetic application, parentage assignments, and showed that the proportion of incorrectly assigned maternities was relatively high at low coverage. Overall, our results suggest that the trade-off between sample size and genotyping error rates be considered prior to building sequencing libraries, reporting genotyping error rates become standard practice, and that effects of genotyping errors on inference be evaluated in restriction-enzyme-based SNP studies.

More precisely biased: increasing the number of markers is not a silver bullet in genetic bottleneck testing.

In response to our review of the use of genetic bottleneck tests in the conservation literature (Peery et al. 2012,Molecular Ecology, 21, 3403­3418), Hoban et al. (2013, Molecular Ecology, in press) conducted population genetic simulations to show that the statistical power of genetic bottleneck tests can be increased substantially by sampling large numbers of microsatellite loci, as they suggest is now possible in the age of genomics. While we agree with Hoban and co-workers in principle, sampling large numbers of microsatellite loci can dramatically increase the probability of committing type 1 errors(i.e. detecting a bottleneck in a stable population) when the mutation model is incorrectly assumed. Using conservative values for mutation model parameters can reduce the probability of committing type 1 errors, but doing so can result in significant losses in statistical power. Moreover, we believe that practical limitations associated with developing large numbers of high-quality microsatellite loci continue to constrain sample sizes, a belief supported by a literature review of recent studies using next generation sequencing methods to develop microsatellite libraries. conclusion, we maintain that researchers employing genetic bottleneck tests should proceed with caution and carefully assess both statistical power and type 1 error rates associated with their study design.

Resolving the phylogenetic history of the short-necked turtles, genera Elseya and Myuchelys (Testudines: Chelidae) from Australia and New Guinea.

Phylogenetic relationships and taxonomy of the short-necked turtles of the genera Elseya, Myuchelys, and Emydura in Australia and New Guinea have long been debated as a result of conflicting hypotheses supported by different data sets and phylogenetic analyses. To resolve this contentious issue, we analyzed sequences from two mitochondrial genes (cytochrome b and ND4) and one nuclear intron gene (R35) from all species of the genera Elseya, Myuchelys, Emydura, and their relatives. Phylogenetic analyses using three methods (maximum parsimony, maximum likelihood, and Bayesian inference) produce a single, well resolved, and strongly corroborated hypothesis, which provides support for the three genera, with the exception that the genus Myuchelys is paraphyletic - Myuchelys purvisi is the sister taxon to the remaining Elseya, Myuchelys and Emydura. A new genus is proposed for the species Myuchelys purvisi to address this paraphyletic relationship. Time-calibration analysis suggests that diversification of the group in Australia coincides with periods of aridification in the late Eocene and between the mid-Miocene and early Pliocene. Other speciation events occurred during the faunal exchange between Australia and the island of New Guinea during the late Miocene and early Pliocene. Lineages distributed in New Guinea are likely influenced by the complex geologic history of the island, and include cryptic species diversity.

Reliability of genetic bottleneck tests for detecting recent population declines.

The identification of population bottlenecks is critical in conservation because populations that have experienced significant reductions in abundance are subject to a variety of genetic and demographic processes that can hasten extinction. Genetic bottleneck tests constitute an appealing and popular approach for determining if a population decline has occurred because they only require sampling at a single point in time, yet reflect demographic history over multiple generations. However, a review of the published literature indicates that, as typically applied, microsatellite-based bottleneck tests often do not detect bottlenecks in vertebrate populations known to have experienced declines. This observation was supported by simulations that revealed that bottleneck tests can have limited statistical power to detect bottlenecks largely as a result of limited sample sizes typically used in published studies. Moreover, commonly assumed values for mutation model parameters do not appear to encompass variation in microsatellite evolution observed in vertebrates and, on average, the proportion of multi-step mutations is underestimated by a factor of approximately two. As a result, bottleneck tests can have a higher probability of 'detecting' bottlenecks in stable populations than expected based on the nominal significance level. We provide recommendations that could add rigor to inferences drawn from future bottleneck tests and highlight new directions for the characterization of demographic history.

Decreased lipoprotein clearance is responsible for increased cholesterol in LDL receptor knockout mice with streptozotocin-induced diabetes.

OBJECTIVE: Patients with diabetes often have dyslipidemia and increased postprandial lipidmia. Induction of diabetes in LDL receptor (Ldlr(-/-)) knockout mice also leads to marked dyslipidemia. The reasons for this are unclear. RESEARCH DESIGN AND METHODS: We placed Ldlr(-/-) and heterozygous LDL receptor knockout (Ldlr(+/-)) mice on a high-cholesterol (0.15%) diet, induced diabetes with streptozotocin (STZ), and assessed reasons for differences in plasma cholesterol. RESULTS: STZ-induced diabetic Ldlr(-/-) mice had plasma cholesterol levels more than double those of nondiabetic controls. Fast-performance liquid chromatography and ultracentrifugation showed an increase in both VLDL and LDL. Plasma VLDL became more cholesterol enriched, and both VLDL and LDL had a greater content of apolipoprotein (apo)E. In LDL the ratio of apoB48 to apoB100 was increased. ApoB production, assessed using [(35)S]methionine labeling in Triton WR1339-treated mice, was not increased in fasting STZ-induced diabetic mice. Similarly, postprandial lipoprotein production was not increased. Reduction of cholesterol in the diet to normalize the amount of cholesterol intake by the control and STZ-induced diabetic animals reduced plasma cholesterol levels in STZ-induced diabetic mice, but plasma cholesterol was still markedly elevated compared with nondiabetic controls. LDL from STZ-induced diabetic mice was cleared from the plasma and trapped more rapidly by livers of control mice. STZ treatment reduced liver expression of the proteoglycan sulfation enzyme, heparan sulfate N-deacetylase/N-sulfotrasferase-1, an effect that was reproduced in cultured hepatocytyes by a high glucose-containing medium. CONCLUSIONS: STZ-induced diabetic, cholesterol-fed mice developed hyperlipidemia due to a non-LDL receptor defect in clearance of circulating apoB-containing lipoproteins.

Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis.

Hepatic steatosis is often associated with insulin resistance and obesity and can lead to steatohepatitis and cirrhosis. In this study, we have demonstrated that hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), two enzymes critical for lipolysis in adipose tissues, also contribute to lipolysis in the liver and can mobilize hepatic triglycerides in vivo and in vitro. Adenoviral overexpression of HSL and/or ATGL reduced liver triglycerides by 40-60% in both ob/ob mice and mice with high fat diet-induced obesity. However, these enzymes did not affect fasting plasma triglyceride and free fatty acid levels or triglyceride and apolipoprotein B secretion rates. Plasma 3-beta-hydroxybutyrate levels were increased 3-5 days after infection in both HSL- and ATGL-overexpressing male mice, suggesting an increase in beta-oxidation. Expression of genes involved in fatty acid transport and synthesis, lipid storage, and mitochondrial bioenergetics was unchanged. Mechanistic studies in oleate-supplemented McA-RH7777 cells with adenoviral overexpression of HSL or ATGL showed that reduced cellular triglycerides could be attributed to increases in beta-oxidation as well as direct release of free fatty acids into the medium. In summary, hepatic overexpression of HSL or ATGL can promote fatty acid oxidation, stimulate direct release of free fatty acid, and ameliorate hepatic steatosis. This study suggests a direct functional role for both HSL and ATGL in hepatic lipid homeostasis and identifies these enzymes as potential therapeutic targets for ameliorating hepatic steatosis associated with insulin resistance and obesity.