Best available science still supports an ancient common origin of Devils Hole and Devils Hole pupfish.

The age of DHP and how pupfish colonized Devils Hole have always been a topic of interest. Recently, two different publications (Martin, Crawford, Turner, & Simons, & Saglam et al., ) tackled this issue using genomic data sets and demographic models but came to widely different conclusions. In their comment, Martin and Höhne () argue that our results (Saglam et al., ) were misleading because we used inappropriate calibration information and biased a priori assumptions. They then re-analysed our data using a "biologically informed" mutation rate prior and concluded that our data support a much younger age of DHP (12.6 kya) as opposed to 60 kya reported in our study. Below we will summarize why their arguments do not hold up and explore some of the inconsistencies between their claims and what was actually presented in our study. Furthermore, we will demonstrate their re-analyses provide no new information compared to what was presented in our original manuscript and reinforce our estimate of a 60 kya divergence of DHP as outweighing competing hypotheses.

Phylogenetics support an ancient common origin of two scientific icons: Devils Hole and Devils Hole pupfish.

The Devils Hole pupfish (Cyprinodon diabolis; DHP) is an icon of conservation biology. Isolated in a 50 m(2) pool (Devils Hole), DHP is one of the rarest vertebrate species known and an evolutionary anomaly, having survived in complete isolation for thousands of years. However, recent findings suggest DHP might be younger than commonly thought, potentially introduced to Devils Hole by humans in the past thousand years. As a result, the significance of DHP from an evolutionary and conservation perspective has been questioned. Here we present a high-resolution genomic analysis of DHP and two closely related species, with the goal of thoroughly examining the temporal divergence of DHP. To this end, we inferred the evolutionary history of DHP from multiple random genomic subsets and evaluated four historical scenarios using the multispecies coalescent. Our results provide substantial information regarding the evolutionary history of DHP. Genomic patterns of secondary contact present strong evidence that DHP were isolated in Devils Hole prior to 20-10 ka and the model best supported by geological history and known mutation rates predicts DHP diverged around 60 ka, approximately the same time Devils Hole opened to the surface. We make the novel prediction that DHP colonized and have survived in Devils Hole since the cavern opened, and the two events (colonization and collapse of the cavern's roof) were caused by a common geologic event. Our results emphasize the power of evolutionary theory as a predictive framework and reaffirm DHP as an important evolutionary novelty, worthy of continued conservation and exploration.

Multiplex preamplification PCR and microsatellite validation enables accurate single nucleotide polymorphism genotyping of historical fish scales.

Incorporating historical tissues into the study of ecological, conservation and management questions can broaden the scope of population genetic research by enhancing our understanding of evolutionary processes and anthropogenic influences on natural populations. Genotyping historical and low-quality samples has been plagued by challenges associated with low amounts of template DNA and the potential for pre-existing DNA contamination among samples. We describe a two-step process designed to (i) accurately genotype large numbers of historical low-quality scale samples in a high-throughput format and (ii) screen samples for pre-existing DNA contamination. First, we describe how an efficient multiplex preamplification PCR of 45 single nucleotide polymorphisms (SNPs) can generate highly accurate genotypes with low failure and error rates in subsequent SNP genotyping reactions of individual historical scales from sockeye salmon (Oncorhynchus nerka). Second, we demonstrate how the method can be modified for the amplification of microsatellite loci to detect pre-existing DNA contamination. A total of 760 individual historical scale and 182 contemporary fin clip samples were genotyped and screened for contamination. Genotyping failure and error rates were exceedingly low and similar for both historical and contemporary samples. Pre-existing contamination in 21% of the historical samples was successfully identified by screening the amplified microsatellite loci. The advantages of automation, low failure and error rates, and ability to multiplex both the preamplification and subsequent genotyping reactions combine to make the protocol ideally suited for efficiently genotyping large numbers of potentially contaminated low-quality sources of DNA.

Single nucleotide polymorphisms unravel hierarchical divergence and signatures of selection among Alaskan sockeye salmon (Oncorhynchus nerka) populations.

BACKGROUND: Disentangling the roles of geography and ecology driving population divergence and distinguishing adaptive from neutral evolution at the molecular level have been common goals among evolutionary and conservation biologists. Using single nucleotide polymorphism (SNP) multilocus genotypes for 31 sockeye salmon (Oncorhynchus nerka) populations from the Kvichak River, Alaska, we assessed the relative roles of geography (discrete boundaries or continuous distance) and ecology (spawning habitat and timing) driving genetic divergence in this species at varying spatial scales within the drainage. We also evaluated two outlier detection methods to characterize candidate SNPs responding to environmental selection, emphasizing which mechanism(s) may maintain the genetic variation of outlier loci. RESULTS: For the entire drainage, Mantel tests suggested a greater role of geographic distance on population divergence than differences in spawn timing when each variable was correlated with pairwise genetic distances. Clustering and hierarchical analyses of molecular variance indicated that the largest genetic differentiation occurred between populations from distinct lakes or subdrainages. Within one population-rich lake, however, Mantel tests suggested a greater role of spawn timing than geographic distance on population divergence when each variable was correlated with pairwise genetic distances. Variable spawn timing among populations was linked to specific spawning habitats as revealed by principal coordinate analyses. We additionally identified two outlier SNPs located in the major histocompatibility complex (MHC) class II that appeared robust to violations of demographic assumptions from an initial pool of eight candidates for selection. CONCLUSIONS: First, our results suggest that geography and ecology have influenced genetic divergence between Alaskan sockeye salmon populations in a hierarchical manner depending on the spatial scale. Second, we found consistent evidence for diversifying selection in two loci located in the MHC class II by means of outlier detection methods; yet, alternative scenarios for the evolution of these loci were also evaluated. Both conclusions argue that historical contingency and contemporary adaptation have likely driven differentiation between Kvichak River sockeye salmon populations, as revealed by a suite of SNPs. Our findings highlight the need for conservation of complex population structure, because it provides resilience in the face of environmental change, both natural and anthropogenic.

Haplotype of growth hormone and angiotensin I-converting enzyme genes, serum angiotensin I-converting enzyme and ventricular growth: pathway inference in pharmacogenetics.

OBJECTIVES: An insertion/deletion (I/D) polymorphism in the angiotensin I-converting enzyme (ACE) gene is associated with variations in circulating and tissue angiotensin I-converting enzyme activity, and differences in exercise-induced left ventricular hypertrophic response. A genetic marker (CSH1.01) in the syntenic GH-CSH gene cluster correlates with metabolic syndrome in adult life in males. Approximately 24% linkage disequilibrium between CSH1.01T and D alleles of ACE I/D has also been reported. The objective was to examine the hypothesis that effects ascribed to ACE genotype may reflect causality in the GH-CSH cluster. METHODS: The ACE I/D polymorphism and GH-CSH BglII-B single nucleotide polymorphism (in strong linkage disequilibrium with CSH1.01) were determined in 847 British Army recruits. Serum angiotensin I-converting enzyme activity and left ventricular mass were measured before and after a 12-week physical training program. Genotype and haplotype analyses of both markers were performed in relation to these phenotypes. RESULTS: The ACE I/D polymorphism was in linkage disequilibrium with BglII-B (D'=0.3). Strong association was seen between ACE I/D genotypes and serum angiotensin I-converting enzyme activity (P<0.0001), but not left ventricular mass change. BglII-B genotypes also associated significantly with serum angiotensin I-converting enzyme level (P<0.0001). Haplotype analysis, however, showed that most of this association resulted from linkage disequilibrium between BglII-B and ACE I/D. BglII-B did not associate with left ventricular mass change. CONCLUSIONS: GH-CSH BglII-B genotype associates significantly with angiotensin I-converting enzyme levels, but only through linkage disequilibrium with ACE I/D. Every phenotype with which ACE I/D has been associated merits investigation of potential causal effects originating in the GH-CSH cluster (and vice versa), otherwise the chain of causality could be misinterpreted.

Prevalence and functionality of paucimorphic and private MC4R mutations in a large, unselected European British population, scanned by meltMADGE.

Identification of unknown mutations has remained laborious, expensive, and only viable for studies of selected cases. Population-based "reference ranges" of rarer sequence diversity are not available. However, the research and diagnostic interpretation of sequence variants depends on such information. Additionally, this is the only way to determine prevalence of severe, moderate, and silent mutations and is also relevant to the development of screening programs. We previously described a system, meltMADGE, suitable for mutation scanning at the population level. Here we describe its application to a population-based study of MC4R (melanocortin 4 receptor) mutations, which are associated with obesity. We developed nine assays representing MC4R and examined a population sample of 1,100 subjects. Two "paucimorphisms" were identified (c.307G>A/p.Val103Ile in 27 subjects and c.-178A>C in 22 subjects). Neither exhibited any anthropometric effects, whereas there would have been >90% power to detect a body mass index (BMI) effect of 0.5 kg/m(2) at P=0.01. Two "private" variants were also identified. c.335C>T/p.Thr112Met has been previously described and appears to be silent. A novel variant, c.260C>A/p.Ala87Asp, was observed in a subject with a BMI of 31.5 kg/m(2) (i.e., clinically obese) but not on direct assay of a further 3,525 subjects. This mutation was predicted to be deleterious and analysis using a cyclic AMP (cAMP) responsive luciferase reporter assay showed substantial loss of function of the mutant receptor. This population-based mutation scan of MC4R suggests that there is no severe MC4R mutation with high prevalence in the United Kingdom, but that obesity-causing MC4R mutation at 1 in 1,100 might represent one of the commonest autosomal dominant disorders in man.

Mutation scanning by meltMADGE: validations using BRCA1 and LDLR, and demonstration of the potential to identify severe, moderate, silent, rare, and paucimorphic mutations in the general population.

We have developed a mutation-scanning approach suitable for whole population screening for unknown mutations. The method, meltMADGE, combines thermal ramp electrophoresis with MADGE to achieve suitable cost efficiency and throughput. The sensitivity was tested in blind trials using 54 amplicons representing the BRCA1 coding region and a panel of 94 unrelated family breast cancer risk consultands previously screened in a clinical diagnostic laboratory. All 10 common polymorphisms, 15/15 previously identified disease-causing mutations, and three previously untested single base changes were identified. Assays of LDLR exons 3 and 8 were validated in 460 familial hypercholesteremics and detected 8/9 known variants. We then applied the exon 3 assay in several DNA banks representing approximately 8000 subjects with known cholesterol values and applied both assays in one DNA bank (n = 3600). In exon 3 we identified one previously reported moderate mutation, P84S (n = 1), also associated with moderate hypercholesteremia in this subject; an unreported silent variant, N76N (n = 1); and known severe hypercholesteremia splice mutation 313+1G-->A (n = 2). Around exon 8 we identified a paucimorphism (n = 35) at the splice site 1061-8T-->C (known to be in complete linkage disequilibrium with T705I) and unreported sequence variants 1186+11G-->A (n = 1) and D335N G-->A (n = 1). The cholesterol value for D335N was on the 96.2 percentile and for T705I, 2/35 carriers were above the 99th percentile. Thus, variants with predicted severe, moderate, and no effect were identified at the population level. In contrast with case collections, CpG mutations predominated. MeltMADGE will enable definition of the full population spectrum of rare, paucimorphic, severe, moderate (forme fruste), and silent mutations and effects.