Genetic basis of speciation and adaptation: from loci to causative mutations.

Understanding the genetic basis of reproductive isolation and adaptive traits in natural populations is one of the fundamental goals in evolutionary biology. Genome editing technologies based on CRISPR-Cas systems and site-specific recombinases have enabled us to modify a targeted genomic region as desired and thus to conduct functional analyses of target loci, genes and mutations even in non-conventional model organisms. Here, we review the technical properties of genome editing techniques by classifying them into the following applications: targeted gene knock-out for investigating causative gene functions, targeted gene knock-in of marker genes for visualizing expression patterns and protein functions, precise gene replacement for identifying causative alleles and mutations, and targeted chromosomal rearrangement for investigating the functional roles of chromosomal structural variations. We describe examples of their application to demonstrate functional analysis of naturally occurring genetic variations and discuss how these technologies can be applied to speciation and adaptation research. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

Diamondback terrapins (Malaclemys terrapin centrata) exhibit strong environmental adaptability and live in both freshwater and saltwater. However, the genetic basis of this adaptability has not been the focus of research. In this study, we successfully constructed a ∼2.21-Gb chromosome-level genome assembly for M. t. centrata using high-coverage and high-depth genomic sequencing data generated on multiple platforms. The M. t. centrata genome contains 25 chromosomes and the scaffold N50 of ∼143.75 Mb, demonstrating high continuity and accuracy. In total, 53.82% of the genome assembly was composed of repetitive sequences, and 22435 protein-coding genes were predicted. Our phylogenetic analysis indicated that M. t. centrata was closely related to the red-eared slider turtle (Trachemys scripta elegans), with divergence approximately ∼23.6 million years ago (Mya) during the early Neogene period of the Cenozoic era. The population size of M. t. centrata decreased significantly over the past ∼14 Mya during the Cenozoic era. Comparative genomic analysis indicated that 36 gene families related to ion transport were expanded and several genes (AQP3, solute carrier subfamily, and potassium channel genes) underwent specific amino acid site mutations in the M. t. centrata genome. Changes to these ion transport-related genes may have contributed to the remarkable salinity adaptability of diamondback terrapin. The results of this study not only provide a high-quality reference genome for M. t. centrata but also elucidate the possible genetic basis for salinity adaptation in this species.

BackgroundCataloguing the distribution of genes within natural bacterial populations is essential for understanding evolutionary processes and the genetic basis of adaptation. Advances in whole genome sequencing technologies have led to a vast expansion in the amount of bacterial genomes deposited in public databases. There is a pressing need for software solutions which are able to cluster, catalogue and characterise genes, or other features, in increasingly large genomic datasets.ResultsHere we present a pangenomics toolbox, PIRATE (Pangenome Iterative Refinement and Threshold Evaluation), which identifies and classifies orthologous gene families in bacterial pangenomes over a wide range of sequence similarity thresholds. PIRATE builds upon recent scalable software developments to allow for the rapid interrogation of thousands of isolates. PIRATE clusters genes (or other annotated features) over a wide range of amino acid or nucleotide identity thresholds and uses the clustering information to rapidly identify paralogous gene families and putative fission/fusion events. Furthermore, PIRATE orders the pangenome using a directed graph, provides a measure of allelic variation, and estimates sequence divergence for each gene family.ConclusionsWe demonstrate that PIRATE scales linearly with both number of samples and computation resources, allowing for analysis of large genomic datasets, and compares favorably to other popular tools. PIRATE provides a robust framework for analysing bacterial pangenomes, from largely clonal to panmictic species.

Genetic diversity is vital for the persistence and evolutionary potential of populations, but is often compromised in taxa of conservation concern. Although promoting evolutionarily resilient species is increasingly recognised as a conservation necessity, evolutionary processes are still often neglected in management strategies. Effective management strategies to promote persistence under environmental change will benefit from knowledge about the effects of environment on genetic diversity and on the neutral and adaptive processes that shape genetic diversity in natural systems. The core theme of this thesis is the application of genetics and evolutionary biology to the management of wildlife populations affected by human-driven environmental change. My thesis addresses five key general and system-specific knowledge gaps: 1) estimating evolutionary potential from genetic data in natural populations; 2) effects of environment on gene flow across landscapes; 3) effectiveness of human-assisted gene flow in conservation management; 4) effects of environment on intra-specific genetic variation across landscapes; and 5) effects of environment on adaptive processes across landscapes. To address these knowledge gaps I apply genetic approaches to several management case studies of threatened Australian passerine bird and freshwater fish species to explore the effects of environment on genetic diversity and on the neutral and adaptive processes that shape genetic diversity. In addressing key knowledge gaps, I: 1) reviewed the strengths and limitations of genomics as a tool for characterising evolutionary potential and argue that for most typical conservation scenarios, genome-wide variation represents a generalised measure of evolutionary potential that is robust to uncertainties surrounding the genetic basis of adaptation; 2) highlighted the complexities of species’ responses to habitat fragmentation, finding evidence of species- and sex- specific effects of fragmentation on genetic connectivity in natural populations of three honeyeater species; 3) used population viability analysis to demonstrate introduction of gene flow from a neighbouring closely related subspecies as a viable conservation strategy for boosting genetic diversity, reducing inbreeding and promoting population growth in a critically endangered subspecies, the helmeted honeyeater; 4) demonstrated a modelling approach that can identify environmental factors associated with range-wide intra-specific genetic variation and applied this approach to range-wide genetic data for a socially, culturally and economically important freshwater fish species, the Murray cod; and 5) detected potentially functionally relevant fixed amino acid differences between the mitochondrial genomes of inland and coastal species of Maccullochella cod, potentially as a result of adaptation to different climate and thermal regimes. Results presented in this thesis provide support for stronger integration of genetics and evolutionary processes in conservation. Effective conservation strategies that promote population persistence will be those that are informed by both broad and taxon-specific knowledge of the effects of environment on genetic diversity and on the neutral and adaptive processes that shape genetic diversity in natural systems. While challenges concerning the genetic basis of adaptation remain, the measurement of genome-wide variation has great scope for generating new insights into adaptive processes occurring across landscapes in natural populations.Awards: Winner of the Mollie Holman Doctoral Medal for Excellence, Faculty of Science, 2016.

The acquisition of floral nectar spurs is correlated with increased species diversity across multiple clades. We tested whether variation in nectar spurs influences reproductive isolation and, thus, can potentially promote species diversity using two species of Aquilegia, Aquilegia formosa and Aquilegia pubescens, which form narrow hybrid zones. Floral visitors strongly discriminated between the two species both in natural populations and at mixed-species arrays of individual flowers. Bees and hummingbirds visited flowers of A. formosa at a much greater rate than flowers of A. pubescens. Hawkmoths, however, nearly exclusively visited flowers of A. pubescens. We found that altering the orientation of A. pubescens flowers from upright to pendent, like the flowers of A. formosa, reduced hawkmoth visitation by an order of magnitude. In contrast, shortening the length of the nectar spurs of A. pubescens flowers to a length similar to A. formosa flowers did not affect hawkmoth visitation. However, pollen removal was significantly reduced in flowers with shortened nectar spurs. These data indicate that floral traits promote floral isolation between these species and that specific floral traits affect floral isolation via ethological isolation while others affect floral isolation via mechanical isolation.

By combining 7077 SNPs and 61 microsatellites, we present the first linkage map for some of the early diverged lineages of the common frog, Rana temporaria, and the densest linkage map to date for this species. We found high homology with the published linkage maps of the Eastern and Western lineages but with differences in the order of some markers. Homology was also strong with the genome of the Tibetan frog Nanorana parkeri and we found high synteny with the clawed frog Xenopus tropicalis. We confirmed marked heterochiasmy between sexes and detected nonrecombining regions in several groups of the male linkage map. Contrary to the expectations set by the male heterogamety of the common frog, we did not find male heterozygosity excess in the chromosome previously shown to be linked to sex determination. Finally, we found blocks of loci showing strong transmission ratio distortion. These distorted genomic regions might be related to genetic incompatibilities between the parental populations, and are promising candidates for further investigation into the genetic basis of speciation and adaptation in the common frog.

This paper reviews the empirical work on Chorthippus parallelus, highlighting the biogeographical history of the species group, the patterns of divergence across Europe, and the nature of the divergence across the hybrid zones. The field of speciation genetics with focus on the recent theoretical advances made in understanding hybrid incompatibilities, and the genetics of hybrid zones are also reviewed. The theoretical, quantitative and molecular genetic approaches that are currently applied to characterize the genetic basis of hybrid male sterility in C. parallelus are also outlined.

Changes in cis-regulatory regions are thought to play a major role in the genetic basis of adaptation. However, few studies have linked cis-regulatory variation with adaptation in natural populations. Here, using a combination of exome and RNA-seq data, we performed expression quantitative trait locus (eQTL) mapping and allele-specific expression analyses to study the genetic architecture of regulatory variation in wild house mice (Mus musculus domesticus) using individuals from five populations collected along a latitudinal cline in eastern North America. Mice in this transect showed clinal patterns of variation in several traits, including body mass. Mice were larger in more northern latitudes, in accordance with Bergmann's rule. We identified 17 genes where cis-eQTLs were clinal outliers and for which expression level was correlated with latitude. Among these clinal outliers, we identified two genes (Adam17 and Bcat2) with cis-eQTLs that were associated with adaptive body mass variation and for which expression is correlated with body mass both within and between populations. Finally, we performed a weighted gene co-expression network analysis (WGCNA) to identify expression modules associated with measures of body size variation in these mice. These findings demonstrate the power of combining gene expression data with scans for selection to identify genes involved in adaptive phenotypic evolution, and also provide strong evidence for cis-regulatory elements as essential loci of environmental adaptation in natural populations.

At the birth of supergenes, the genomic landscape is dramatically re-organized leading to pronounced differences in phenotypes and increased intrasexual diversity. Two of the best-studied supergenes in vertebrates are arguably the inversion polymorphisms on chromosomes 2 and 11 in the white-throated sparrow (Zonotrichia albicollis) and the ruff (Calidris pugnax), respectively. In both species, regions of suppressed recombination determine plumage coloration and social behavioural phenotypes. Despite the apparent lack of gene overlap between these two supergenes, in both cases the alternative phenotypes seem to be driven largely by alterations in steroid hormone pathways. Here, we explore the interplay between genomic architecture and steroid-related genes. Due to the highly pleiotropic effects of steroid-related genes and their universal involvement in social behaviour and transcriptomic regulation, processes favouring their linkage are likely to have substantial effects on the evolution of behavioural phenotypes, individual fitness, and life-history strategies. We propose that inversion-related differentiation and regulatory changes in steroid-related genes lie at the core of phenotypic differentiation in both of these interesting species. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

Identifying the genetic basis of adaptation is a central goal of evolutionary biology. However, identifying genes and mutations affecting fitness remains challenging because a large number of traits and variants can influence fitness. Selected phenotypes can also be difficult to know a priori, complicating top-down genetic approaches for trait mapping that involve crosses or genome-wide association studies. In such cases, experimental genetic approaches, where one maps fitness directly and attempts to infer the traits involved afterwards, can be valuable. Here, we re-analyse data from a transplant experiment involving Timema stick insects, where five physically clustered single-nucleotide polymorphisms associated with cryptic body coloration were shown to interact to affect survival. Our analysis covers a larger genomic region than past work and revealed a locus previously not identified as associated with survival. This locus resides near a gene, Punch (Pu), involved in pteridine pigments production, implying that it could be associated with an unmeasured coloration trait. However, by combining previous and newly obtained phenotypic data, we show that this trait is not eye or body coloration. We discuss the implications of our results for the discovery of traits, genes and mutations associated with fitness in other systems, as well as for supergene evolution. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

Copy number variation (CNV) can cause phenotypic changes. However, in contrast to amino acid substitutions and cis-regulatory changes, little is known about the functional categories of genes in which CNV is important for adaptation to novel environments. It is also unclear whether the same genes repeatedly change the copy numbers for adapting to similar environments. Here, we investigate CNV associated with freshwater colonization in fishes, which was observed multiple times across different lineages. Using 48 ray-finned fishes across diverse orders, we identified 23 genes whose copy number increases were associated with freshwater colonization. These genes showed enrichment for peptide receptor activity, hexosyltransferase activity and unsaturated fatty acid metabolism. We further revealed that three of the genes showed copy number increases in freshwater populations compared to marine ancestral populations of the stickleback genus Gasterosteus. These results indicate that copy number increases of genes involved in fatty acid metabolism (FADS2), immune function (PSMB8a) and thyroid hormone metabolism (UGT2) may be important for freshwater colonization at both the inter-order macroevolutionary scale and at the intra-genus microevolutionary scale. Further analysis across diverse taxa will help to understand the role of CNV in the adaptation to novel environments. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

A complete understanding of the speciation process requires the identification of genomic regions and genes that confer reproductive barriers between species. Empirical and theoretical research has revealed two important patterns in the evolution of reproductive isolation in animals: isolation typically arises as a result of disrupted epistatic interactions between multiple loci and these disruptions map disproportionately to the X chromosome. These patterns suggest that a targeted examination of natural gene flow between closely related species at X-linked markers with known positions would provide insight into the genetic basis of speciation. We take advantage of the existence of genomic data and a well-documented European zone of hybridization between two species of house mice, Mus domesticus and M. musculus, to conduct such a survey. We evaluate patterns of introgression across the hybrid zone for 13 diagnostic X-linked loci with known chromosomal positions using a maximum likelihood model. Interlocus comparisons clearly identify one locus with reduced introgression across the center of the hybrid zone, pinpointing a candidate region for reproductive isolation. Results also reveal one locus with high frequencies of M. domesticus alleles in populations on the M. musculus side of the zone, suggesting the possibility that positive selection may act to drive the spread of alleles from one species on to the genomic background of the other species. Finally, cline width and cline center are strongly positively correlated across the X chromosome, indicating that gene flow of the X chromosome may be asymmetrical. This study highlights the utility of natural populations of hybrids for mapping speciation genes and suggests that the middle of the X chromosome may be important for reproductive isolation between species of house mice.

A complete understanding of the speciation process requires the identification of genomic regions and genes that confer reproductive barriers between species. Empirical and theoretical research has revealed two important patterns in the evolution of reproductive isolation in animals: isolation typically arises as a result of disrupted epistatic interactions between multiple loci and these disruptions map disproportionately to the X chromosome. These patterns suggest that a targeted examination of natural gene flow between closely related species at X-linked markers with known positions would provide insight into the genetic basis of speciation. We take advantage of the existence of genomic data and a well-documented European zone of hybridization between two species of house mice, Mus domesticus and M. musculus, to conduct such a survey. We evaluate patterns of introgression across the hybrid zone for 13 diagnostic X-linked loci with known chromosomal positions using a maximum likelihood model. Interlocus comparisons clearly identify one locus with reduced introgression across the center of the hybrid zone, pinpointing a candidate region for reproductive isolation. Results also reveal one locus with high frequencies of M. domesticus alleles in populations on the M. musculus side of the zone, suggesting the possibility that positive selection may act to drive the spread of alleles from one species on to the genomic background of the other species. Finally, cline width and cline center are strongly positively correlated across the X chromosome, indicating that gene flow of the X chromosome may be asymmetrical. This study highlights the utility of natural populations of hybrids for mapping speciation genes and suggests that the middle of the X chromosome may be important for reproductive isolation between species of house mice.

ABSTRACTThe use of alternative methods to control cyst nematode populations has accelerated since the ban of chemical nematicides in Europe. The resistant QTL GpaVvrn, derived from the wild species Solanum vernei, is widely present in resistant European potato cultivars and provides strong protection against Globodera pallida populations although a risk of resistance breakdown has already been demonstrated in both experimental evolution studies and field populations. The wild relative S. sparsipilum, harbouring the resistant QTL GpaVspl, would be an interesting alternative source of resistance to control virulent G. pallida. The goal of the present study was to understand the genomics of adaptation of the nematode to these two colinear resistant QTLs. Starting with two natural populations, an experimental evolution approach allowed, after 10 generations on resistant potato genotypes, selecting independent nematode lineages adapted to each QTL. These virulent lineages were analysed through a combination of phenotyping and genome scans approaches. Phenotyping enabled the quantification of virulence levels and confirmed resistance breakdowns. Pool‐Seq whole genome sequencing followed by genome scan analyses identified genomic regions under selection, potentially involved in the adaptive mechanisms to each resistance factor. Candidate genes within these regions provided insights into the genetic basis of adaptation, revealing effectors known to suppress plant immunity. As genome scans highlighted distinct genomic regions for the adaptation to both resistant factors, we were able to predict and phenotypically confirm the absence of cross‐virulence between nematode lineages evolving on GpaVvrn and GpaVspl. These findings have significant implications for the design of effective and sustainable resistance management strategies.

Hybridization among diverging lineages is common in nature. Genomic data provide a special opportunity to characterize the history of hybridization and the genetic basis of speciation. We review existing methods and empirical studies to identify recent advances in the genomics of hybridization, as well as issues that need to be addressed. Notable progress has been made in the development of methods for detecting hybridization and inferring individual ancestries. However, few approaches reconstruct the magnitude and timing of gene flow, estimate the fitness of hybrids or incorporate knowledge of recombination rate. Empirical studies indicate that the genomic consequences of hybridization are complex, including a highly heterogeneous landscape of differentiation. Inferred characteristics of hybridization differ substantially among species groups. Loci showing unusual patterns - which may contribute to reproductive barriers - are usually scattered throughout the genome, with potential enrichment in sex chromosomes and regions of reduced recombination. We caution against the growing trend of interpreting genomic variation in summary statistics across genomes as evidence of differential gene flow. We argue that converting genomic patterns into useful inferences about hybridization will ultimately require models and methods that directly incorporate key ingredients of speciation, including the dynamic nature of gene flow, selection acting in hybrid populations and recombination rate variation.