An age-of-allele test of neutrality for transposable element insertions not at equilibrium

An age-of-allele test of neutrality for transposable element insertions not at equilibrium

Justin P. Blumenstiel, Miaomiao He, Casey M. Bergman
(Submitted on 16 Sep 2012)

How natural selection acts to limit the proliferation of transposable elements (TEs) in genomes has been of interest to evolutionary biologists for many years. To describe TE dynamics in populations, many previous studies have relied on the assumption of equilibrium between transposition and selection. However, since TE invasions are known to happen in bursts through time, this assumption may not be reasonable. Here we derive a test of neutrality for TE insertions that does not rely on the assumption of transpositional equilibrium. We consider the case of TE insertions that have been ascertained from a single haploid reference genome sequence and have had their allele frequency estimated in a population sample. By conditioning on age information provided within the sequence of a TE insertion in the form of the number of substitutions that have occurred within the fragment since insertion into a reference genome, we derive the probability distribution for the TE allele frequency in a population sample under neutrality. Taking models of population fluctuation into account, we then test the fit of predictions of our model to allele frequency data from 190 retrotransposon insertion loci in North American and African populations of Drosophila melanogaster. Using this non-equilibrium model, we are able to explain about 80% of the variance in TE insertion allele frequencies. Controlling for nonequilibrium dynamics of transposition and host demography, we demonstrate how one may detect negative selection acting against most TEs as well as evidence for a small subset of TEs being driven to high frequency by positive selection. Our work establishes a new framework for the analysis of the evolutionary forces governing large insertion mutations like TEs or gene duplications.

Our paper: A faster-X effect for gene expression in Drosophila embryos

[This author post is by Alex Kalinka and Pavel Tomancak on their paper, An excess of gene expression divergence on the X chromosome in Drosophila embryos: implications for the faster-X hypothesis, posted to the arXiv here.]

We have been working towards publishing our study of gene expression evolution on the X chromosome in Drosophila embryos since the beginning of March this year. Recently, Casey Bergman suggested that we upload our manuscript to the arXiv, and after we did so, we were kindly invited by Graham Coop to write a guest post about our work for Haldane's Sieve.

It makes sense to post here since the roots of our study go back to Haldane in 1924 [1]; he recognised that the unusual inheritance pattern of the X chromosome, in which a single copy is present in the heterogametic sex versus two copies in the homogametic sex, could in turn lead to unusual evolutionary patterns on the X relative to the autosomes. If, for example, a beneficial mutation is recessive, then it would be more exposed to natural selection in the heterogametic sex where, relative to an equivalent autosomal allele, it would spend less time being masked by the dominant, less beneficial allele [1]. The prediction that adaptive evolution might proceed more quickly on the X than on the autosomes has been dubbed the faster-X hypothesis. However, the X chromosome might also be expected to evolve more rapidly for non-adaptive reasons. In each mating pair there will be 3 copies of the X chromosome versus 4 copies of each autosome, which might in turn lead to a lower chromosomal effective population size for the X thereby increasing the strength of random genetic drift.

While some studies have reported evidence for a faster-X effect for adaptive protein evolution in Drosophila, other studies have reported that there is no difference between the X and the autosomes, and to date the evidence is somewhat inconclusive. As we focused our study on gene expression, we had an opportunity to relax the implicit assumption that the majority of adaptive evolution occurs in coding regions. To help disentangle adaptive and non-adaptive evolutionary signatures in our data, we used both between-species measures of gene expression divergence and within-species measures of gene expression variation using inbred strains of D. melanogaster generated by the Drosophila Genetic Reference Panel (DGRP).

We found an excess of gene expression divergence on the X chromosome between six Drosophila species (a mean increase of ~20%). In contrast, we found that the X exhibits a significantly lower level of gene expression variation between inbred strains of D. melanogaster (a mean decrease of ~10%). Taken together, these results suggest that the divergence that we find between species is not driven by a relaxation of selective constraint on the X chromosome. To further explore whether such a signature could be driven by the hemizygosity of the X, we analysed gene expression in mutation accumulation lines of D. melanogaster. If the single copy of the X in males is driving the excess of expression divergence that we found on the X, then we would expect to find an excess of expression variation between lines that have independently accumulated mutations. In fact, we found the opposite was the case – the X chromosome displayed a significantly lower rate of mutation accumulation than the autosomes suggesting that the hemizygosity of the X alone is not sufficient to drive a higher rate of fixation of gene expression mutations.

Overall, we argue that the excess divergence we find on the X is best understood within the framework of the faster-X hypothesis. In support of this interpretation, we find that there is an excess of gene expression divergence on Muller's D element along the branch leading to the obscura sub-group; Muller's D element segregates as a neo-X chromosome in the obscura sub-group, and hence provides a powerful, independent test of faster evolution of the X chromosome.

Several questions remain, however, and we hope that our findings will help to stimulate further research into the details underpinning the differences we find on the X. In particular, work needs to be done to discover the genetics underlying divergence on the X, such as the relative importance of cis versus trans-acting factors, and, crucially, we need to develop a better understanding of how variation in gene expression impacts organismal fitness. Research into the latter question is essential if we are to bridge the conspicuous gaps between sequence variation, gene expression variation, and organismal fitness.

Dave Gerrard initially found elevated gene expression divergence on the X in the course of analysing data for our developmental hourglass paper, and spoke about his findings at the 43rd population genetics conference; that was more than two years ago. Since then we collected new data, and it took a while to put the paper together although it is still not certain that it will be published in a traditional journal. The arXiv is a great way to let the scientific community know about your results before the academic process runs its course. We only regret we didn't make use of this excellent outlet back in March.

[1] Haldane JBS (1924) A mathematical theory of natural and artificial selection. Part I. Trans Camb Phil Soc 23: 19-41.

Our paper: Population genomics of sub-Saharan Drosophila melanogaster: African diversity and non-African admixture

[This author post is by John Pool on his paper: Population genomics of sub-Saharan Drosophila melanogaster: African diversity and non-African admixture arXived here.]

We are in the process of publishing this analysis of >100 sequenced Drosophila melanogaster genomes (largely haploid genomes at >25X depth).  These genomes come from more than 20 geographic locations, largely within sub-Saharan Africa, where the species is thought to originate.  Truth be told, this sampling scheme was somewhat accidental – we wanted to identify a population representing a “center of genetic diversity” for the species, which for us involved sequencing small numbers of genomes from many different population samples (some from previous lab stocks, others from newly collected lines).  Ultimately we did find the sample we were looking for, and we are in the process of sequencing ~300 genomes from this Zambian population.  Still, it seemed more than worthwhile to analyze the “geographic scatter” of genomes we had obtained from across sub-Saharan Africa (as well as one small sample from Europe).

Our ambitions for this paper were largely descriptive – a preliminary analysis of genetic variation within and among the sampled populations.  We envisioned being able to compare diversity levels and genetic structure across Africa (much as I once did with a dramatically smaller data set), and to identify specific loci with signatures of selection.  And we were able to do that.  We found the highest levels of genetic diversity in and around Zambia, raising the prospect of a southern-central African origin for D. melanogaster.  We found low-to-moderate levels of genetic structure across most of sub-Saharan Africa, with only Ethiopian populations showing stronger genetic differentiation (along with some morphological differentiation, but that’s another story).  Analyses of allele frequencies within and between populations revealed a substantial number of loci with evidence of recent natural selection – many GO categories enriched for such outliers pertained to gene regulation, much as we had observed in another recent population genomic analysis.

Of course that’s how we normally think of natural selection’s influence on genetic variation – specific beneficial mutations leading to selective sweeps (whether hard or soft, partial or complete), each one influencing diversity on a limited genomic scale.  And at least in
species with large outbreeding populations like Drosophila, recurrent hitchhiking may be common enough to affect diversity at random sites in the genome (e.g. 1, 2, 3).  So we weren’t surprised to find sweep signals.  The bigger surprise to us was finding evidence that specific episodes of natural selection had affected genetic variation on the scale of whole chromosome arms or the entire genome.

The first major surprise concerned genomic patterns of non-African admixture in African D. melanogaster populations.  The occurrence of such introgression had been documented before, and there were previous findings that non-African genotypes were associated with urban environments in Africa, and that admixture levels could vary within the genome. We developed a hidden Markov model approach to detect admixed chromosomal regions (based simply on the reduced diversity found in populations outside sub-Saharan Africa).  Whereas we tend to think of admixture as a selectively neutral force, the genomic patterns of admixture we observed did not seem consistent with passive gene flow.  Non-African genotypes had displaced large portions of the gene pool of presumably quite large African populations, and this had occurred within a very short time (judging by the megabase scale of admixture tracts).  Levels of admixture across the genome showed both broad-scale heterogeneity (chromosomal differences) and relatively narrow “spikes” of admixture.  These peaks of admixture quite often overlapped with outliers for high FST between Africa and Europe, as would be expected if these regions contained functional differences between populations for which introgressing non-African alleles may now be favored in some African environments (e.g. modernizing cities).  

The second surprise came as we documented population genetic patterns associated with polymorphic inversions (as further analyzed in a forthcoming paper by Russ Corbett-Detig and Dan Hartl).  It was already known that inversions tend to differ in frequency between D. melanogaster populations, but theory and most empirical data suggested that only diversity around the inversion breakpoints should be affected.  Instead, we observed some African populations in which elevated inversion frequencies were associated with notable reductions in diversity for entire chromosome arms (and ultimately affecting genome-wide average diversity), consistent with directional selection on rearrangements or linked loci.  Perhaps more surprisingly, mostinversions found in the non-African sample (France) served to substantially increase diversity across whole chromosome arms (by up to 29% in the case of inversions on arm 3R), and by 12% genome-wide.  Here, we can only suggest that selection may have acted to favor inverted chromosomes that recently originated from a more genetically diverse (e.g. African or African-admixed) population.  Accounting for these inversions substantially alters chromosomal diversity ratios between African and European populations.

Hence, we may have the curious situation of natural selection driving introgression in both directions across the sub-Saharan/cosmopolitan population genetic divide in D. melanogaster.

You can find our draft manuscript here, supplemental items here, and the data here.

 I’m definitely glad we were able to post a draft at arXiv – it was time to communicate our findings to the research community (especially to facilitate our colleagues’ analysis and publication plans for this data set), and there’s really no downside to us as authors.  I also appreciate the chance to post here at Haldane’s Sieve, and it would be great to discuss any aspect of our draft.

John Pool

Our paper: Population genomics of the Wolbachia endosymbiont in Drosophila melanogaster

Casey Bergman [@caseybergman and @bergmanlab] kindly wrote a post about his recently arXived paper:
Population genomics of the Wolbachia endosymbiont in Drosophila melanogaster
ArXived here.
As part of the Drosophila 12 Genome Project, Steve Salzberg and colleagues’ published a pioneering paper in 2005 showing that complete genomes of the bacterial endosymbiont Wolbachia pipientis can be extracted from the whole-genome shotgun sequence assemblies of Drosophila species. This paper always left an impression on me as a very clever use of extracting new biology from existing genomic data, and when the era of resequencing multiple strains of D. melanogaster kicked off a few years ago, it seemed like a natural extension to ask if this approach could be adapted to a next-generation sequencing data to study the co-evolution of Wolbachia and Drosophila using whole genome data.

In the current work, we used short-read next generation sequencing data from two major resequencing efforts in D. melanogaster — the Drosophila Genetic Reference Panel (DGRP) and Drosophila Population Genomics Project (DPGP) — together with the reference Wolbachia genome published by Wu et al. (2005) and extracted over 175 complete Wolbachia genomes and nearly 300 complete mitochondrial genomes. Readers can find the main results in the paper, which is currently in review. I’d like to discuss here the social context of the project and some of the reasons we submitted to arXiv.

This project started out as summer project for a masters student, Mark Richardson, in 2010 who did an amazing job developing the initial pipeline made most of the initial discoveries in the paper. Mark and I started a collaboration with Frank Jiggins and Mike McGwire shortly after to verify that our in silico genotyping results were making sense, who suggested to bring in Lucy Weinart and John Welch to help with the more sophisticated Bayesian phylogenetic analysis. Another PhD student in my lab, Raquel Linheiro, adapted her transposable element detection pipeline to identify particular Wolbachia sublineages which was crucial to linking our data with previous results. This was a great collaboration, where everyone made significant contributions, and I would collaborate with everyone again (and I hope to!).

At the time (summer 2010), we only had access to the North American strains from the DGRP sample; knowing that North American D. melanogaster are derived populations, we were cautious about the impact that population structure had on our results. We planned in early 2011 to publish on only the DGRP dataset since Mark was going off to do a PhD in Australia and I didn’t have anyone else in the group working on this project. In the summer of 2011, the African DPGP data came online and I decided to take a peek and run the pipeline on the African strains as well. This led to a major overhaul of the project and set us back a year, since all the data had to be reanalyzed again together and the interpretation of the biogeography results was substantially altered. This was in some ways lucky because our initial interpretation of evidence for a selective sweep on one of the cytoplasmic lineages was probably wrong, and it saved us from having to back peddle on this misinterpretation in a later publication.

As we plugged away at trying to finish this project, we had inquiries about the status of the project from several other groups working in the Wolbachia field. Honestly this stressed me out quite a bit, since some of the inquiries were coming from post-docs in big labs. But instead of just sitting on the data, after we finalized the dataset we decided to release these data openly on our lab blog in April 2012. We decided on an open release as a way to help these teams (and others we didn’t know about), but also to get some priority in this area by providing the “gold standard” that other groups could use (and cite!). For the record, I will note that we asked two teams who contacted us about our project if they would reciprocate by sharing unpublished genomic data or in one case published genomic data that was not submitted to GenBank; both declined.

After making the decision to release the data pre-publication, it was a natural step to submit the manuscript to arXiv. I’m an open science advocate and used the Nature Preprint server occasionally in the past. I never really liked the Nature Preprint server, though, since I thought people posted there to give their manuscript the stink of being “Nature (in prep)” on their CV. And I never posted to arXiv in the past, since I always thought it was for more hardcore computational or mathematical biology. But recently, I was convinced by Rosie Redfield, Leonid Kruglyak and colleagues putting their Arsenic Life paper on arXiv that more empirical work in quantitative biology was arXiv-able. And just as with releasing our data early, it seemed like the best way to prevent being scooped was to get our results out as quickly as possible and letting people know about it.

So we went for it. And I have to say the experience has been thoroughly rewarding. Submitting was a piece of cake, easier than any journal I’ve ever submitted to. Having a URL to point to allowed me to tweet about it, which got some exposure to the paper and some new colleagues on twitter. It also allowed me to send a submitted manuscript around to colleagues for informal review, without cluttering up their inboxes with big attachments or providing a moral dilemma about who they can share the manuscript with. And somehow submitting to arXiv pushed the “it’s submitted” button in my brain, which made me a whole lot less stressed about the possibility of being scooped and I’ve been more relaxed throughout the formal submission process. Finally, I know that the pre-publication release of the data and posting of the manuscript has led to a group in Russia using these sequences into their work, and I’ve just gotten a manuscript to review from this group citing our arXiv manuscript and extending our results before our paper is even published! This is what research is all about, right: doing science, getting it out, and letting others build on it. I’ll definitely submit to arXiv for all my papers from my lab, and look forward to the Haldane’s Sieve readership giving us a hard time about our manuscripts while they evolve into formal publications.

Casey Bergman

Population genomics of sub-Saharan Drosophila melanogaster: African diversity and non-African admixture

Population genomics of sub-Saharan Drosophila melanogaster: African diversity and non-African admixture
John E. Pool, Russell B. Corbett-Detig, Ryuichi P. Sugino, Kristian A. Stevens, Charis M. Cardeno, Marc W. Crepeau, Pablo Duchen, J. J. Emerson, Perot Saelao, David J. Begun, Charles H. Langley
(Submitted on 23 Aug 2012)

(ABRIDGED) We report the genome sequencing of 139 wild-derived strains of D. melanogaster, representing 22 population samples from the sub-Saharan ancestral range of this species, along with one European population. Most genomes were sequenced above 25X depth from haploid embryos. Results indicated a pervasive influence of non-African admixture in many African populations, motivating the development and application of a novel admixture detection method. Admixture proportions varied among populations, with greater admixture in urban locations. Admixture levels also varied across the genome, with localized peaks and valleys suggestive of a non-neutral introgression process. Genomes from the same location differed starkly in ancestry, suggesting that isolation mechanisms may exist within African populations. After removing putatively admixed genomic segments, the greatest genetic diversity was observed in southern Africa (e.g. Zambia), while diversity in other populations was largely consistent with a geographic expansion from this potentially ancestral region. The European population showed different levels of diversity reduction on each chromosome arm, and some African populations displayed chromosome arm-specific diversity reductions. Inversions in the European sample were associated with strong elevations in diversity across chromosome arms. Genomic scans were conducted to identify loci that may represent targets of positive selection. A disproportionate number of candidate selective sweep regions were located near genes with varied roles in gene regulation. Outliers for Europe-Africa FST were found to be enriched in genomic regions of locally elevated cosmopolitan admixture, possibly reflecting a role for some of these loci in driving the introgression of non-African alleles into African populations.