A Candidate Complex Approach to Study Functional Mitochondrial DNA Changes: Sequence Variation and Quaternary Structure Modeling of Drosophila simulans Cytochrome c Oxidase

A problem with studying evolutionary dynamics of mitochondrial (mt) DNA is that classical population genetic techniques cannot identify selected substitutions because of genetic hitchhiking. We circumvented this problem by employing a candidate complex approach to study sequence variation in cytochrome c oxidase (COX) genes within and among three distinct Drosophila simulans mtDNA haplogroups. First, we determined sequence variation in complete coding regions for all COX mtDNA and nuclear loci and their isoforms. Second, we constructed a quaternary structure model of D. simulans COX. Third, we predicted that six of nine amino acid changes in D. simulans mtDNA are likely to be functionally important. Of these seven, genetic crosses can experimentally determine the functional significance of three. Fourth, we identified two single amino acid changes and a deletion of two consecutive amino acids in nuclear encoded COX loci that are likely to influence cytochrome c oxidase activity. These data show that linking population genetics and quaternary structure modeling can lead to functional predictions of specific mtDNA amino acid mutations and validate the candidate complex approach. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A hierarchical Bayesian model for next-generation population genomics

The demography of populations and natural selection shape genetic variation across the genome and understanding the genomic consequences of these evolutionary processes is a fundamental aim of population genetics. We have developed a hierarchical Bayesian model to quantify genome-wide population structure and identify candidate genetic regions affected by selection. This model improves on existing methods by accounting for stochastic sampling of sequences inherent in next-generation sequencing (with pooled or indexed individual samples) and by incorporating genetic distances among haplotypes in measures of genetic differentiation. Using simulations we demonstrate that this model has a low false-positive rate for classifying neutral genetic regions as selected genes (i.e., Φ(ST) outliers), but can detect recent selective sweeps, particularly when genetic regions in multiple populations are affected by selection. Nonetheless, selection affecting just a single population was difficult to detect and resulted in a high false-negative rate under certain conditions. We applied the Bayesian model to two large sets of human population genetic data. We found evidence of widespread positive and balancing selection among worldwide human populations, including many genetic regions previously thought to be under selection. Additionally, we identified novel candidate genes for selection, several of which have been linked to human diseases. This model will facilitate the population genetic analysis of a wide range of organisms on the basis of next-generation sequence data.     

Invasion genetics of the Mediterranean fruit fly: variation in multiple nuclear introns

Biological invasions generally start from low initial population sizes, leading to reduced genetic variation in nuclear and especially mitochondrial DNA. Consequently, genetic approaches for the study of invasion history and population structure are difficult. An extreme example is the Mediterranean fruit fly, Ceratitis capitata (Medfly), for which successive invasions during this century have resulted in a loss of 60% of ancestral genetic variation in isozymes and 75% of variation in mitochondrial DNA. Using Medflies as an example, we present a new approach to invasion genetics that measures DNA sequence variation within introns from multiple nuclear loci. These loci are so variable that even relatively recently founded Medfly populations within California and Hawaii retain ample genetic diversity. Invading populations have only lost 35% of the ancestral genetic variation. Intron variation will allow high-resolution genetic characterization of invading populations in both natural and managed systems, although non-equilibrium methods of analysis may be necessary if the genetic diversity represents sorting ancestral polymorphism.     

Human populations show reduced DNA sequence variation at the factor IX locus

Levels and patterns of human DNA sequence variation vary widely among loci. However, some of this variation may be due to the different populations used in different studies. So far, few studies of diverse human populations have compared different genetic loci for the same samples of populations and individuals. Here, we present new polymorphism data from intron 4 of the Factor IX gene (FIX) sequenced in diverse Old World populations. An explicit comparison is made with another X-linked gene, PDHA1, for which the sampling of individuals was very similar. Despite having a similar amount of divergence from chimpanzees, as do other nuclear genes, FIX has comparatively much less DNA sequence variation among humans. Nucleotide diversity at FIX is the lowest among the existing non-Y chromosome nuclear gene datasets and is less than 10% of the diversity found at PDHA1. Estimates of effective population size based on FIX are 8,558, about half of the value obtained for PDHA1, and the time to the most recent common ancestry among human FIX gene copies (282,000 years) is one of the most recent estimates reported for human genes. Analyses presented here suggest a history for the FIX region that includes recent positive directional selection, or background, selection. The general conclusion emerging is that very large variations can exist between the histories of similar genomic regions, even when sampling differences are minimized.     

Urbanization as a facilitator of gene flow in a human health pest

Urban fragmentation can reduce gene flow that isolates populations, reduces genetic diversity and increases population differentiation, all of which have negative conservation implications. Alternatively, gene flow may actually be increased among urban areas consistent with an urban facilitation model. In fact, urban adapter pests are able to thrive in the urban environment and may be experiencing human‐mediated transport. Here, we used social network theory with a population genetic approach to investigate the impact of urbanization on genetic connectivity in the Western black widow spider, as an urban pest model of human health concern. We collected genomewide single nucleotide polymorphism variation from mitochondrial and nuclear double‐digest RAD (ddRAD) sequence data sets from 210 individuals sampled from 11 urban and 10 nonurban locales across its distribution of the Western United States. From urban and nonurban contrasts of population, phylogenetic, and network analyses, urban locales have higher within‐population genetic diversity, lower between‐population genetic differentiation and higher estimates of genetic connectivity. Social network analyses show that urban locales not only have more connections, but can act as hubs that drive connectivity among nonurban locales, which show signatures of historical isolation. These results are consistent with an urban facilitation model of gene flow and demonstrate the importance of sampling multiple cities and markers to identify the role that urbanization has had on larger spatial scales. As the urban landscape continues to grow, this approach will help determine what factors influence the spread and adaptation of pests, like the venomous black widow spider, in building policies for human and biodiversity health.     

Assessing cell-specific effects of genetic variations using tRNA microarrays

Background

Short-read resequencing of genomes produces abundant information of the genetic variation of individuals. Due to their numerous nature, these variants are rarely exhaustively validated. Furthermore, low levels of undetected variant miscalling will have a systematic and disproportionate impact on the interpretation of individual genome sequence information, especially should these also be carried through into in reference databases of genomic variation.

Results

We find that sequence variation from short-read sequence data is subject to recurrent-yet-intermittent miscalling that occurs in a sequence intrinsic manner and is very sensitive to sequence read length. The miscalls arise from difficulties aligning short reads to redundant genomic regions, where the rate of sequencing error approaches the sequence diversity between redundant regions. We find the resultant miscalled variants to be sensitive to small sequence variations between genomes, and thereby are often intrinsic to an individual, pedigree, strain or human ethnic group. In human exome sequences, we identify 2–300 recurrent false positive variants per individual, almost all of which are present in public databases of human genomic variation. From the exomes of non-reference strains of inbred mice, we identify 3–5000 recurrent false positive variants per mouse – the number of which increasing with greater distance between an individual mouse strain and the reference C57BL6 mouse genome. We show that recurrently miscalled variants may be reproduced for a given genome from repeated simulation rounds of read resampling, realignment and recalling. As such, it is possible to identify more than two-thirds of false positive variation from only ten rounds of simulation.

Conclusion

Identification and removal of recurrent false positive variants from specific individual variant sets will improve overall data quality. Variant miscalls arising are highly sequence intrinsic and are often specific to an individual, pedigree or ethnicity. Further, read length is a strong determinant of whether given false variants will be called for any given genome – which has profound significance for cohort studies that pool datasets collected and sequenced at different points in time.

     

Anchored pseudo-de novo assembly of human genomes identifies extensive sequence variation from unmapped sequence reads

The human genome reference (HGR) completion marked the genomics era beginning, yet despite its utility universal application is limited by the small number of individuals used in its development. This is highlighted by the presence of high-quality sequence reads failing to map within the HGR. Sequences failing to map generally represent 2–5 % of total reads, which may harbor regions that would enhance our understanding of population variation, evolution, and disease. Alternatively, complete de novo assemblies can be created, but these effectively ignore the groundwork of the HGR. In an effort to find a middle ground, we developed a bioinformatic pipeline that maps paired-end reads to the HGR as separate single reads, exports unmappable reads, de novo assembles these reads per individual and then combines assemblies into a secondary reference assembly used for comparative analysis. Using 45 diverse 1000 Genomes Project individuals, we identified 351,361 contigs covering 195.5 Mb of sequence unincorporated in GRCh38. 30,879 contigs are represented in multiple individuals with ~40 % showing high sequence complexity. Genomic coordinates were generated for 99.9 %, with 52.5 % exhibiting high-quality mapping scores. Comparative genomic analyses with archaic humans and primates revealed significant sequence alignments and comparisons with model organism RefSeq gene datasets identified novel human genes. If incorporated, these sequences will expand the HGR, but more importantly our data highlight that with this method low coverage (~10–20×) next-generation sequencing can still be used to identify novel unmapped sequences to explore biological functions contributing to human phenotypic variation, disease and functionality for personal genomic medicine.     

Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [gamma-(32)P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.     

Plant genome organisation and diversity: the year of the junk!

Having gained a thorough understanding of the structure and organization of model plant genomes, such as those of Arabidopsis thaliana and rice, we have now started to investigate the most interesting aspect of genome structure - its variations. Variation in DNA sequence is responsible for the genetic component of phenotypic variation (i.e. the component upon which both natural and artificial selection act). Recent studies have started to shed light on sequence variation outside of the genic regions, owing mainly to large insertion/deletion (indel) polymorphisms caused by the presence or absence of transposable elements of different classes. In addition to long terminal repeat retrotransposons, DNA transposons have been shown to be responsible for these polymorphisms. These comprise Helitrons, CACTA and Mu-like elements that are capable of acquiring and piecing together fragments of plant genes and are often expressed. Future analyses of the functional roles of intergenic sequence variation will tell us if we will need to pay more attention not only to genes, but also to the 'junk' DNA surrounding them.     

From famine to feast? Selecting nuclear DNA sequence loci for plant species-level phylogeny reconstruction

Phylogenetic analyses of DNA sequences have prompted spectacular progress in assembling the Tree of Life. However, progress in constructing phylogenies among closely related species, at least for plants, has been less encouraging. We show that for plants, the rapid accumulation of DNA characters at higher taxonomic levels has not been matched by conventional sequence loci at the species level, leaving a lack of well-resolved gene trees that is hindering investigations of many fundamental questions in plant evolutionary biology. The most popular approach to address this problem has been to use low-copy nuclear genes as a source of DNA sequence data. However, this has had limited success because levels of variation among nuclear intron sequences across groups of closely related species are extremely variable and generally lower than conventionally used loci, and because no universally useful low-copy nuclear DNA sequence loci have been developed. This suggests that solutions will, for the most part, be lineage-specific, prompting a move away from 'universal' gene thinking for species-level phylogenetics. The benefits and limitations of alternative approaches to locate more variable nuclear loci are discussed and the potential of anonymous nongenic nuclear loci is highlighted. Given the virtually unlimited number of loci that can be generated using these new approaches, it is clear that effective screening will be critical for efficient selection of the most informative loci. Strategies for screening are outlined.     

RNA association defines a functionally conserved domain in the nuclear pore protein Nup153

Traffic between the nucleus and cytoplasm takes place through a macromolecular structure termed the nuclear pore complex. To understand how the vital process of nucleocytoplasmic transport occurs, the contribution of individual pore proteins must be elucidated. One such protein, the nucleoporin Nup153, is localized to the nuclear basket of the pore complex and has been shown to be a central component of the nuclear transport machinery. Perturbation of Nup153 function was demonstrated previously to block the export of several classes of RNA cargo. Moreover, these studies also showed that Nup153 can stably associate with RNA in vitro. In this study, we have mapped a domain within Nup153, encompassing amino acids 250-400 in human Nup153, that is responsible for RNA association. After cloning this region of Xenopus Nup153, we performed a cross-species analysis. Despite variation in sequence conservation between Drosophila, Xenopus, and human, this domain of Nup153 displayed robust RNA binding activity in each case, indicating that this property is a hallmark feature of Nup153 and pointing toward a subset of amino acid residues that are key to conferring this ability. We have further determined that a recombinant fragment of Nup153 can bind directly to RNA and that this fragment can interact with endogenous RNA targets. Our findings identify a functionally conserved domain in Nup153 and suggest a role for RNA binding in Nup153 function at the nuclear pore.     

An Empirical Analysis of mt 16S rRNA Covarion-Like Evolution in Insects: Site-Specific Rate Variation Is Clustered and Frequently Detected

The structural and functional analysis of rRNA molecules has attracted considerable scientific interest. Empirical studies have demonstrated that sequence variation is not directly translated into modifications of rRNA secondary structure. Obviously, the maintenance of secondary structure and sequence variation are in part governed by different selection regimes. The nature of those selection regimes still remains quite elusive. The analysis of individual bacterial models cannot adequately explore this topic. Therefore, we used primary sequence data and secondary structures of a mitochondrial 16S rRNA fragment of 558 insect species from 15 monophyletic groups to study patterns of sequence variation, and variation of secondary structure. Using simulation studies to establish significance levels of change, we found that despite conservation of secondary structure, the location of sequence variation within the conserved rRNA structure changes significantly between groups of insects. Despite our conservative estimation procedure we found significant site-specific rate changes at 56 sites out of 184. Additionally, site-specific rate variation is somewhat clustered in certain helices. Both results confirm what has been predicted from an application of non-stationary maximum likelihood models to rRNA sequences. Clearly, constraints on sequence variation evolve and leave footprints in the form of evolutionary plasticity in rRNA sequences. Here, we show that a better understanding of the evolution of rRNA sequences can be obtained by integrating both phylogenetic and structural information.     

Detecting ancient admixture in humans using sequence polymorphism data

A debate of long-standing interest in human evolution centers around whether archaic human populations (such as the Neanderthals) have contributed to the modern gene pool. A model of ancient population structure with recent mixing is introduced, and it is determined how much information (i.e., sequence data from how many unlinked nuclear loci) would be necessary to distinguish between different demographic scenarios. It is found that approximately 50-100 loci are necessary if plausible parameter estimates are used. There are not enough data available at the present to support either the "single origin" or the "multiregional" model of modern human evolution. However, this information should be available in a few years.     

Multilocus Phylogeography and Species Delimitation in the Cumberland Plateau Salamander,Plethodon kentucki: Incongruence among Data Sets and Methods

Species are a fundamental unit of biodiversity, yet can be challenging to delimit objectively. This is particularly true of species complexes characterized by high levels of population genetic structure, hybridization between genetic groups, isolation by distance, and limited phenotypic variation. Previous work on the Cumberland Plateau Salamander, Plethodon kentucki, suggested that it might constitute a species complex despite occupying a relatively small geographic range. To examine this hypothesis, we sampled 135 individuals from 43 populations, and used four mitochondrial loci and five nuclear loci (5693 base pairs) to quantify phylogeographic structure and probe for cryptic species diversity. Rates of evolution for each locus were inferred using the multidistribute package, and time calibrated gene trees and species trees were inferred using BEAST 2 and *BEAST 2, respectively. Because the parameter space relevant for species delimitation is large and complex, and all methods make simplifying assumptions that may lead them to fail, we conducted an array of analyses. Our assumption was that strongly supported species would be congruent across methods. Putative species were first delimited using a Bayesian implementation of the GMYC model (bGMYC), Geneland, and Brownie. We then validated these species using the genealogical sorting index and BPP. We found substantial phylogeographic diversity using mtDNA, including four divergent clades and an inferred common ancestor at 14.9 myr (95% HPD: 10.8–19.7 myr). By contrast, this diversity was not corroborated by nuclear sequence data, which exhibited low levels of variation and weak phylogeographic structure. Species trees estimated a far younger root than did the mtDNA data, closer to 1.0 myr old. Mutually exclusive putative species were identified by the different approaches. Possible causes of data set discordance, and the problem of species delimitation in complexes with high levels of population structure and introgressive hybridization, are discussed.     

Preliminary assessment of population structure in the mackerel icefish (Champsocephalus gunnari)

The mackerel icefish (Champsocephalus gunnari Lönnberg E (1905) The Fishes of the Swedish South Polar Expedition. Wiss. Ergebnisse Schwedische Südpol- Exped. 1901–1903, vol 5, p 37 is widely distributed south of the Antarctic convergence and over shelf areas surrounding sub-Antarctic Islands. In order to evaluate global population structure in this species, we examined DNA sequence variation in four mitochondrial regions and four nuclear genes in icefish from four locations in the Atlantic Ocean sector and one location in the Indian Ocean. Despite small sample sizes, mitochondrial and nuclear gene data indicated the existence of at least three genetically distinct stocks: Heard Island, South Shetland Islands, and the remaining Atlantic populations (Shag Rocks, South Georgia, and Bouvet Island). The mitochondrial and nuclear SNP markers developed here will be useful for more extensive analyses of population structure in this species.