Transposable Element Insertions Are Associated with Batesian Mimicry in the Pantropical Butterfly Hypolimnas misippus.

Hypolimnas misippus is a Batesian mimic of the toxic African Queen butterfly (Danaus chrysippus). Female H. misippus butterflies use two major wing patterning loci (M and A) to imitate three color morphs of D. chrysippus found in different regions of Africa. In this study, we examine the evolution of the M locus and identify it as an example of adaptive atavism. This phenomenon involves a morphological reversion to an ancestral character that results in an adaptive phenotype. We show that H. misippus has re-evolved an ancestral wing pattern present in other Hypolimnas species, repurposing it for Batesian mimicry of a D. chrysippus morph. Using haplotagging, a linked-read sequencing technology, and our new analytical tool, Wrath, we discover two large transposable element insertions located at the M locus and establish that these insertions are present in the dominant allele responsible for producing mimetic phenotype. By conducting a comparative analysis involving additional Hypolimnas species, we demonstrate that the dominant allele is derived. This suggests that, in the derived allele, the transposable elements disrupt a cis-regulatory element, leading to the reversion to an ancestral phenotype that is then utilized for Batesian mimicry of a distinct model, a different morph of D. chrysippus. Our findings present a compelling instance of convergent evolution and adaptive atavism, in which the same pattern element has independently evolved multiple times in Hypolimnas butterflies, repeatedly playing a role in Batesian mimicry of diverse model species.

On the origin and structure of haplotype blocks.

The term "haplotype block" is commonly used in the developing field of haplotype-based inference methods. We argue that the term should be defined based on the structure of the Ancestral Recombination Graph (ARG), which contains complete information on the ancestry of a sample. We use simulated examples to demonstrate key features of the relationship between haplotype blocks and ancestral structure, emphasizing the stochasticity of the processes that generate them. Even the simplest cases of neutrality or of a "hard" selective sweep produce a rich structure, often missed by commonly used statistics. We highlight a number of novel methods for inferring haplotype structure, based on the full ARG, or on a sequence of trees, and illustrate how they can be used to define haplotype blocks using an empirical data set. While the advent of new, computationally efficient methods makes it possible to apply these concepts broadly, they (and additional new methods) could benefit from adding features to explore haplotype blocks, as we define them. Understanding and applying the concept of the haplotype block will be essential to fully exploit long and linked-read sequencing technologies.

Repeated genetic adaptation to altitude in two tropical butterflies.

Repeated evolution can provide insight into the mechanisms that facilitate adaptation to novel or changing environments. Here we study adaptation to altitude in two tropical butterflies, Heliconius erato and H. melpomene, which have repeatedly and independently adapted to montane habitats on either side of the Andes. We sequenced 518 whole genomes from altitudinal transects and found many regions differentiated between highland (~ 1200 m) and lowland (~ 200 m) populations. We show repeated genetic differentiation across replicate populations within species, including allopatric comparisons. In contrast, there is little molecular parallelism between the two species. By sampling five close relatives, we find that a large proportion of divergent regions identified within species have arisen from standing variation and putative adaptive introgression from high-altitude specialist species. Taken together our study supports a role for both standing genetic variation and gene flow from independently adapted species in promoting parallel local adaptation to the environment.

Genetic studies of human-chimpanzee divergence using stem cell fusions.

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee autotetraploids and allotetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or autotetraploid iPSCs. Analysis of gene expression patterns in interspecific allotetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allotetraploid cells. We successfully generated derivative cells with nested deletions or interspecific recombination on the X chromosome. These studies confirm an important role for the X chromosome in trans regulation of expression differences between species and illustrate the potential of this system for more detailed cis and trans mapping of the molecular basis of human and chimpanzee evolution.

Genomics of altitude-associated wing shape in two tropical butterflies.

Understanding how organisms adapt to their local environment is central to evolution. With new whole-genome sequencing technologies and the explosion of data, deciphering the genomic basis of complex traits that are ecologically relevant is becoming increasingly feasible. Here, we studied the genomic basis of wing shape in two Neotropical butterflies that inhabit large geographical ranges. Heliconius butterflies at high elevations have been shown to generally have rounder wings than those in the lowlands. We reared over 1,100 butterflies from 71 broods of H. erato and H. melpomene in common-garden conditions and showed that wing aspect ratio, that is, elongatedness, is highly heritable in both species and that elevation-associated wing aspect ratio differences are maintained. Genome-wide associations with a published data set of 666 whole genomes from across a hybrid zone, uncovered a highly polygenic basis to wing aspect ratio variation in the wild. We identified several genes that have roles in wing morphogenesis or wing aspect ratio variation in Drosophila flies, making them promising candidates for future studies. There was little evidence for molecular parallelism in the two species, with only one shared candidate gene, nor for a role of the four known colour pattern loci, except for optix in H. erato. Thus, we present the first insights into the heritability and genomic basis of within-species wing aspect ratio in two Heliconius species, adding to a growing body of evidence that polygenic adaptation may underlie many ecologically relevant traits.

Haplotype tagging reveals parallel formation of hybrid races in two butterfly species.

Genetic variation segregates as linked sets of variants or haplotypes. Haplotypes and linkage are central to genetics and underpin virtually all genetic and selection analysis. Yet, genomic data often omit haplotype information due to constraints in sequencing technologies. Here, we present "haplotagging," a simple, low-cost linked-read sequencing technique that allows sequencing of hundreds of individuals while retaining linkage information. We apply haplotagging to construct megabase-size haplotypes for over 600 individual butterflies (Heliconius erato and H. melpomene), which form overlapping hybrid zones across an elevational gradient in Ecuador. Haplotagging identifies loci controlling distinctive high- and lowland wing color patterns. Divergent haplotypes are found at the same major loci in both species, while chromosome rearrangements show no parallelism. Remarkably, in both species, the geographic clines for the major wing-pattern loci are displaced by 18 km, leading to the rise of a novel hybrid morph in the center of the hybrid zone. We propose that shared warning signaling (Müllerian mimicry) may couple the cline shifts seen in both species and facilitate the parallel coemergence of a novel hybrid morph in both comimetic species. Our results show the power of efficient haplotyping methods when combined with large-scale sequencing data from natural populations.

Rapid genotype imputation from sequence with reference panels.

Inexpensive genotyping methods are essential to modern genomics. Here we present QUILT, which performs diploid genotype imputation using low-coverage whole-genome sequence data. QUILT employs Gibbs sampling to partition reads into maternal and paternal sets, facilitating rapid haploid imputation using large reference panels. We show this partitioning to be accurate over many megabases, enabling highly accurate imputation close to theoretical limits and outperforming existing methods. Moreover, QUILT can impute accurately using diverse technologies, including long reads from Oxford Nanopore Technologies, and a new form of low-cost barcoded Illumina sequencing called haplotagging, with the latter showing improved accuracy at low coverages. Relative to DNA genotyping microarrays, QUILT offers improved accuracy at reduced cost, particularly for diverse populations that are traditionally underserved in modern genomic analyses, with accuracy nearly doubling at rare SNPs. Finally, QUILT can accurately impute (four-digit) human leukocyte antigen types, the first such method from low-coverage sequence data.

Independent evolution toward larger body size in the distinctive Faroe Island mice.

Most phenotypic traits in nature involve the collective action of many genes. Traits that evolve repeatedly are particularly useful for understanding how selection may act on changing trait values. In mice, large body size has evolved repeatedly on islands and under artificial selection in the laboratory. Identifying the loci and genes involved in this process may shed light on the evolution of complex, polygenic traits. Here, we have mapped the genetic basis of body size variation by making a genetic cross between mice from the Faroe Islands, which are among the largest and most distinctive natural populations of mice in the world, and a laboratory mouse strain selected for small body size, SM/J. Using this F2 intercross of 841 animals, we have identified 111 loci controlling various aspects of body size, weight and growth hormone levels. By comparing against other studies, including the use of a joint meta-analysis, we found that the loci involved in the evolution of large size in the Faroese mice were largely independent from those of a different island population or other laboratory strains. We hypothesize that colonization bottleneck, historical hybridization, or the redundancy between multiple loci have resulted in the Faroese mice achieving an outwardly similar phenotype through a distinct evolutionary path.

Population structure of Apodemus flavicollis and comparison to Apodemus sylvaticus in northern Poland based on RAD-seq.

BACKGROUND: Mice of the genus Apodemus are one the most common mammals in the Palaearctic region. Despite their broad range and long history of ecological observations, there are no whole-genome data available for Apodemus, hindering our ability to further exploit the genus in evolutionary and ecological genomics context. RESULTS: Here we present results from the double-digest restriction site-associated DNA sequencing (ddRAD-seq) on 72 individuals of A. flavicollis and 10 A. sylvaticus from four populations, sampled across 500 km distance in northern Poland. Our data present clear genetic divergence of the two species, with average p-distance, based on 21377 common loci, of 1.51% and a mutation rate of 0.0011 - 0.0019 substitutions per site per million years. We provide a catalogue of 117 highly divergent loci that enable genetic differentiation of the two species in Poland and to a large degree of 20 unrelated samples from several European countries and Tunisia. We also show evidence of admixture between the three A. flavicollis populations but demonstrate that they have negligible average population structure, with largest pairwise FST<0.086. CONCLUSION: Our study demonstrates the feasibility of ddRAD-seq in Apodemus and provides the first insights into the population genomics of the species.

A major locus controls a biologically active pheromone component in Heliconius melpomene.

Understanding the production, response, and genetics of signals used in mate choice can inform our understanding of the evolution of both intraspecific mate choice and reproductive isolation. Sex pheromones are important for courtship and mate choice in many insects, but we know relatively little of their role in butterflies. The butterfly Heliconius melpomene uses a complex blend of wing androconial compounds during courtship. Electroantennography in H. melpomene and its close relative Heliconius cydno showed that responses to androconial extracts were not species specific. Females of both species responded equally strongly to extracts of both species, suggesting conservation of peripheral nervous system elements across the two species. Individual blend components provoked little to no response, with the exception of octadecanal, a major component of the H. melpomene blend. Supplementing octadecanal on the wings of octadecanal-rich H. melpomene males led to an increase in the time until mating, demonstrating the bioactivity of octadecanal in Heliconius. Using quantitative trait locus (QTL) mapping, we identified a single locus on chromosome 20 responsible for 41% of the parental species' difference in octadecanal production. This QTL does not overlap with any of the major wing color or mate choice loci, nor does it overlap with known regions of elevated or reduced FST . A set of 16 candidate fatty acid biosynthesis genes lies underneath the QTL. Pheromones in Heliconius carry information relevant for mate choice and are under simple genetic control, suggesting they could be important during speciation.

Genomic Structure of Hstx2 Modifier of Prdm9-Dependent Hybrid Male Sterility in Mice.

F1 hybrids between mouse inbred strains PWD and C57BL/6 represent the most thoroughly genetically defined model of hybrid sterility in vertebrates. Hybrid male sterility can be fully reconstituted from three components of this model, the Prdm9 gene, intersubspecific homeology of Mus musculus musculus and Mus musculus domesticus autosomes, and the X-linked Hstx2 locus. Hstx2 modulates the extent of Prdm9-dependent meiotic arrest and harbors two additional factors responsible for intersubspecific introgression-induced oligospermia (Hstx1) and meiotic recombination rate (Meir1). To facilitate positional cloning and to overcome the recombination suppression within the 4.3 Mb encompassing the Hstx2 locus, we designed Hstx2-CRISPR and SPO11/Cas9 transgenes aimed to induce DNA double-strand breaks specifically within the Hstx2 locus. The resulting recombinant reduced the Hstx2 locus to 2.70 Mb (chromosome X: 66.51-69.21 Mb). The newly defined Hstx2 locus still operates as the major X-linked factor of the F1 hybrid sterility, and controls meiotic chromosome synapsis and meiotic recombination rate. Despite extensive further crosses, the 2.70 Mb Hstx2 interval behaved as a recombination cold spot with reduced PRDM9-mediated H3K4me3 hotspots and absence of DMC1-defined DNA double-strand-break hotspots. To search for structural anomalies as a possible cause of recombination suppression, we used optical mapping and observed high incidence of subspecies-specific structural variants along the X chromosome, with a striking copy number polymorphism of the microRNA Mir465 cluster. This observation together with the absence of a strong sterility phenotype in Fmr1 neighbor (Fmr1nb) null mutants support the role of microRNA as a likely candidate for Hstx2.

An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice.

Evolutionary studies are often limited by missing data that are critical to understanding the history of selection. Selection experiments, which reproduce rapid evolution under controlled conditions, are excellent tools to study how genomes evolve under selection. Here we present a genomic dissection of the Longshanks selection experiment, in which mice were selectively bred over 20 generations for longer tibiae relative to body mass, resulting in 13% longer tibiae in two replicates. We synthesized evolutionary theory, genome sequences and molecular genetics to understand the selection response and found that it involved both polygenic adaptation and discrete loci of major effect, with the strongest loci tending to be selected in parallel between replicates. We show that selection may favor de-repression of bone growth through inactivating two limb enhancers of an inhibitor, Nkx3-2. Our integrative genomic analyses thus show that it is possible to connect individual base-pair changes to the overall selection response.

Characterization of a non-sexual population of Strongyloides stercoralis with hybrid 18S rDNA haplotypes in Guangxi, Southern China.

Strongyloidiasis is a much-neglected but sometimes fatal soil born helminthiasis. The causing agent, the small intestinal parasitic nematode Strongyloides stercoralis can reproduce sexually through the indirect/heterogonic life cycle, or asexually through the auto-infective or the direct/homogonic life cycles. Usually, among the progeny of the parasitic females both, parthenogenetic parasitic (females only) and sexual free-living (females and males) individuals, are present simultaneously. We isolated S. stercoralis from people living in a village with a high incidence of parasitic helminths, in particular liver flukes (Clonorchis sinensis) and hookworms, in the southern Chinese province Guangxi. We determined nuclear and mitochondrial DNA sequences of individual S. stercoralis isolated from this village and from close by hospitals and we compared these S. stercoralis among themselves and with selected published S. stercoralis from other geographic locations. For comparison, we also analyzed the hookworms present in the same location. We found that, compared to earlier studies of S. stercoralis populations in South East Asia, all S. stercoralis sampled in our study area were very closely related, suggesting a recent common source of infection for all patients. In contrast, the hookworms from the same location, while all belonging to the species Necator americanus, showed rather extensive genetic diversity even within host individuals. Different from earlier studies conducted in other geographic locations, almost all S. stercoralis in this study appeared heterozygous for different sequence variants of the 18S rDNA hypervariable regions (HVR) I and IV. In contrast to earlier investigations, except for three males, all S. stercoralis we isolated in this study were infective larvae, suggesting that the sampled population reproduces predominantly, if not exclusively through the clonal life cycles. Consistently, whole genome sequencing of individual worms revealed higher heterozygosity than reported earlier for likely sexual populations of S. stercoralis. Elevated heterozygosity is frequently associated with asexual clonal reproduction.

A novel enhancer near the Pitx1 gene influences development and evolution of pelvic appendages in vertebrates.

Vertebrate pelvic reduction is a classic example of repeated evolution. Recurrent loss of pelvic appendages in sticklebacks has previously been linked to natural mutations in a pelvic enhancer that maps upstream of Pitx1. The sequence of this upstream PelA enhancer is not conserved to mammals, so we have surveyed a large region surrounding the mouse Pitx1 gene for other possible hind limb control sequences. Here we identify a new pelvic enhancer, PelB, that maps downstream rather than upstream of Pitx1. PelB drives expression in the posterior portion of the developing hind limb, and deleting the sequence from mice alters the size of several hind limb structures. PelB sequences are broadly conserved from fish to mammals. A wild stickleback population lacking the pelvis has an insertion/deletion mutation that disrupts the structure and function of PelB, suggesting that changes in this ancient enhancer contribute to evolutionary modification of pelvic appendages in nature.

Genetic mapping of species differences via in vitro crosses in mouse embryonic stem cells.

Discovering the genetic changes underlying species differences is a central goal in evolutionary genetics. However, hybrid crosses between species in mammals often suffer from hybrid sterility, greatly complicating genetic mapping of trait variation across species. Here, we describe a simple, robust, and transgene-free technique to generate "in vitro crosses" in hybrid mouse embryonic stem (ES) cells by inducing random mitotic cross-overs with the drug ML216, which inhibits the DNA helicase Bloom syndrome (BLM). Starting with an interspecific F1 hybrid ES cell line between the Mus musculus laboratory mouse and Mus spretus (∼1.5 million years of divergence), we mapped the genetic basis of drug resistance to the antimetabolite tioguanine to a single region containing hypoxanthine-guanine phosphoribosyltransferase (Hprt) in as few as 21 d through "flow mapping" by coupling in vitro crosses with fluorescence-activated cell sorting (FACS). We also show how our platform can enable direct study of developmental variation by rederiving embryos with contribution from the recombinant ES cell lines. We demonstrate how in vitro crosses can overcome major bottlenecks in mouse complex trait genetics and address fundamental questions in evolutionary biology that are otherwise intractable through traditional breeding due to high cost, small litter sizes, and/or hybrid sterility. In doing so, we describe an experimental platform toward studying evolutionary systems biology in mouse and potentially in human and other mammals, including cross-species hybrids.

Genetic Coupling of Female Mate Choice with Polygenic Ecological Divergence Facilitates Stickleback Speciation.

Ecological speciation with gene flow is widespread in nature [1], but it presents a conundrum: how are associations between traits under divergent natural selection and traits that contribute to assortative mating maintained? Theoretical models suggest that genetic mechanisms inhibiting free recombination between loci underlying these two types of traits (hereafter, "genetic coupling") can facilitate speciation [2-4]. Here, we perform a direct test for genetic coupling by mapping both divergent traits and female mate choice in a classic model of ecological speciation: sympatric benthic and limnetic threespine stickleback (Gasterosteus aculeatus). By measuring mate choice in F2 hybrid females, we allowed for recombination between loci underlying assortative mating and those under divergent ecological selection. In semi-natural mating arenas in which females had access to both benthic and limnetic males, we found that F2 females mated with males similar to themselves in body size and shape. In addition, we found two quantitative trait loci (QTLs) associated with female mate choice that also predicted female morphology along the benthic-limnetic trait axis. Furthermore, a polygenic genetic model that explains adaptation to contrasting benthic and limnetic feeding niches [5] also predicted F2 female mate choice. Together, these results provide empirical evidence that genetic coupling of assortative mating with traits under divergent ecological selection helps maintain species in the face of gene flow, despite a polygenic basis for adaptation to divergent environments.

A Step-by-Step Guide to Assemble a Reptilian Genome.

Multiple technologies and software are now available facilitating the de novo sequencing and assembly of any vertebrate genome. Yet the quality of most available sequenced genomes is substantially poorer than that of the golden standard in the field: the human genome. Here, we present a step-by-step protocol for the successful sequencing and assembly of a high-quality snake genome that can be applied to any other reptilian or avian species. We combine the great sequencing depth and accuracy of short reads with the use of different insert size libraries for extended scaffolding followed by optical mapping. We show that this procedure improved the corn snake scaffold N50 from 3.7 kbp to 1.4 Mbp, currently making it one of the snake genomes with the longest scaffolds. Short guidelines are also given on the extraction of long DNA molecules from reptilian blood and the necessary modifications in DNA extraction protocols. This chapter is accompanied by a website ( www.reptilomics.org/stepbystep.html ), where we provide links to the suggested software, examples of input and output files, and running parameters.

Evolution: two (very long) legs to stand on.

How do the legs of jerboas get so long? A comprehensive study of the Dipodidae family of two-legged rodents reveals many evolutionary refinements in toe numbers, bone structures and proportions. Clearly, this adorable emerging developmental model system has legs.

Extent of QTL Reuse During Repeated Phenotypic Divergence of Sympatric Threespine Stickleback.

How predictable is the genetic basis of phenotypic adaptation? Answering this question begins by estimating the repeatability of adaptation at the genetic level. Here, we provide a comprehensive estimate of the repeatability of the genetic basis of adaptive phenotypic evolution in a natural system. We used quantitative trait locus (QTL) mapping to discover genomic regions controlling a large number of morphological traits that have diverged in parallel between pairs of threespine stickleback (Gasterosteus aculeatus species complex) in Paxton and Priest lakes, British Columbia. We found that nearly half of QTL affected the same traits in the same direction in both species pairs. Another 40% influenced a parallel phenotypic trait in one lake but not the other. The remaining 10% of QTL had phenotypic effects in opposite directions in the two species pairs. Similarity in the proportional contributions of all QTL to parallel trait differences was about 0.4. Surprisingly, QTL reuse was unrelated to phenotypic effect size. Our results indicate that repeated use of the same genomic regions is a pervasive feature of parallel phenotypic adaptation, at least in sticklebacks. Identifying the causes of this pattern would aid prediction of the genetic basis of phenotypic evolution.

Genetics of ecological divergence during speciation.

Ecological differences often evolve early in speciation as divergent natural selection drives adaptation to distinct ecological niches, leading ultimately to reproductive isolation. Although this process is a major generator of biodiversity, its genetic basis is still poorly understood. Here we investigate the genetic architecture of niche differentiation in a sympatric species pair of threespine stickleback fish by mapping the environment-dependent effects of phenotypic traits on hybrid feeding and performance under semi-natural conditions. We show that multiple, unlinked loci act largely additively to determine position along the major niche axis separating these recently diverged species. We also find that functional mismatch between phenotypic traits reduces the growth of some stickleback hybrids beyond that expected from an intermediate phenotype, suggesting a role for epistasis between the underlying genes. This functional mismatch might lead to hybrid incompatibilities that are analogous to those underlying intrinsic reproductive isolation but depend on the ecological context.