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,
. Our integrative genomic analyses thus show that it is possible to connect individual base-pair changes to the overall selection response.
Abstract
Northern sand lance (Ammodytes dubius) are essential forage fish in most offshore, temperate-to-polar waters on the Northwest Atlantic shelf (NWA), but their population structure and genetic ...separation from the American sand lance (A. americanus) remain unresolved. We assembled a reference genome for A. dubius (first in the Ammodytidae) and then used low-coverage whole genome sequencing on 262 specimens collected across the species distribution (Mid-Atlantic Bight to Greenland) to quantify genetic differentiation between geographic regions based on single nucleotide polymorphisms. We found strong separation between A. dubius from locations north and south of the Scotian Shelf, largely due to massive genetic differentiation spanning most of chromosomes 21 and 24. Genetic distance increased with geographic distance in the smaller southern cluster but not in the larger northern cluster, where genetic homogeneity appeared across large geographic distances (>103 km). The two genetic clusters coincide with a clear break in winter sea surface temperature, suggesting that differential offspring survival, rather than limited transport, causes a break in realized connectivity. Nuclear and mitochondrial DNA both clearly delineated A. dubius from A. americanus, thereby confirming a species boundary through spatial niche partitioning into inshore (A. americanus) and offshore (A. dubius) sand lance species on the NWA.
The role of calcium, but not of other intracellular signaling molecules, in the release of pituitary hormones by exocytosis is well established. Here, we analyzed the contribution of ...phosphatidylinositol kinases (PIKs) to calcium-driven prolactin (PRL) release in pituitary lactotrophs: PI4Ks - which control PI4P production, PIP5Ks - which synthesize PI(4, 5)P2 by phosphorylating the D-5 position of the inositol ring of PI4P, and PI3KCs - which phosphorylate PI(4, 5)P
to generate PI(3, 4, 5)P
. We used common and PIK-specific inhibitors to evaluate the strength of calcium-secretion coupling in rat lactotrophs. Gene expression was analyzed by single-cell RNA sequencing and qRT-PCR analysis; intracellular and released hormones were assessed by radioimmunoassay and ELISA; and single-cell calcium signaling was recorded by Fura 2 imaging. Single-cell RNA sequencing revealed the expression of
and
, as well as
and
, in lactotrophs. Wortmannin, a PI3K and PI4K inhibitor, but not LY294002, a PI3K inhibitor, blocked spontaneous action potential driven PRL release with a half-time of ~20 min when applied in 10 µM concentration, leading to accumulation of intracellular PRL content. Wortmannin also inhibited increase in PRL release by high potassium, the calcium channel agonist Bay K8644, and calcium mobilizing thyrotropin-releasing hormone without affecting accompanying calcium signaling. GSK-A1, a specific inhibitor of PI4KA, also inhibited calcium-driven PRL secretion without affecting calcium signaling and
expression. In contrast, PIK93, a specific inhibitor of PI4KB, and ISA2011B and UNC3230, specific inhibitors of PIP5K1A and PIP5K1C, respectively, did not affect PRL release. These experiments revealed a key role of PI4KA in calcium-secretion coupling in pituitary lactotrophs downstream of voltage-gated and PI(4, 5)P2-dependent calcium signaling.
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.
Significance
A defining goal in genetics is linking variation in DNA sequence to trait evolution between populations and, ultimately, species. Genome sequencing efficiently captures such variation ...but typically in millions of tiny fragments that omit haplotype or linkage information. We present “haplotagging,” a simple, rapid linked-read sequencing technique that allows high-throughput sequencing without sacrificing haplotype information. We validated this affordable approach for whole-genome haplotyping in large populations. We used haplotagging to investigate the rise of a novel hybrid morph in parallel hybrid zones of two comimetic
Heliconius
butterfly species in Ecuador. Our results reveal that strikingly parallel divergences in their genomes produced coordinated shifts in haplotype frequencies across the hybrid zone, giving rise to comimetic hybrid morphs in each 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.
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 F
ST. 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.
Abstract
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.
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.
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 F
<0.086.
Our study demonstrates the feasibility of ddRAD-seq in Apodemus and provides the first insights into the population genomics of the species.
11β-Hydroxysteroid dehydrogenase type 1 (11HSD1) is an enzyme that interconverts active 11-hydroxy glucocorticoids (cortisol, corticosterone) and their inactive 11-oxo derivatives (cortisone, ...11-dehydrocorticosterone). Although bidirectional, it is considered to operate in vivo as an 11-reductase that regenerates active glucocorticoids and thus amplifies their local activity in mammals. Here we report the cloning, characterization and tissue distribution of chicken 11HSD1 (ch11HSD1). Its cDNA predicts a protein of 300 amino acids that share 51–56% sequence identity with known mammalian 11HSD1 proteins, while in contrast to most mammals, ch11HSD1 contains only one N-linked glycosylation site. Analysis of the tissue distribution pattern by RT-PCR revealed that ch11HSD1 is expressed in a large variety of tissues, with high expression in the liver, kidney and intestine, and weak in the gonads, brain and heart. 11-Reductase activity has been found in the liver, kidney, intestine and gonads with low or almost zero activity in the brain and heart. These results provide evidence for a role of 11HSD1 as a tissue-specific regulator of glucocorticoid action in non-mammalian vertebrates and may serve as a suitable model for further analysis of 11HSD1 evolution in vertebrates.
NAD
+-dependent 11β-hydroxysteroid dehydrogenase (11HSD2) converts glucocorticoids to 11-oxo derivatives and thus decreases their local concentration and prevents them from activating corticosteroid ...receptors. In this paper we report the partial cloning, characterization and tissue distribution of chicken 11HSD2. A cDNA of 991
bp was cloned from kidney mRNA by reverse transcription and polymerase chain reaction. At the amino acid level, the sequence of PCR product had 56–59% homology with mammalian and 46–48% with fish 11HSD2. The consensus sequences of the short-chain dehydrogenase/reductase superfamily such as the catalytic activity motif Tyr-X-X-X-Lys and cosubstrate-binding motif Gly-X-X-X-Gly-X-Gly, were found in the cloned cDNA. Analysis of the tissue expression of chicken 11HSD2 mRNA and NAD
+-dependent 11β-oxidase activity showed a similar tissue distribution pattern in the majority of tissues. High levels of expression and activity were found in kidney, small intestine, colon and oviduct; low in ovary and almost zero in brain, liver and testis.
PDF
