Marina Telonis-Scott

Using genomics to characterize evolutionary potential for conservation of wild populations

Genomics promises exciting advances towards the important conservation goal of maximizing evolutionary potential, notwithstanding associated challenges. Here, we explore some of the complexity of adaptation genetics and discuss the strengths and limitations of genomics as a tool for characterizing evolutionary potential in the context of conservation management. Many traits are polygenic and can be strongly influenced by minor differences in regulatory networks and by epigenetic variation not visible in DNA sequence. Much of this critical complexity is difficult to detect using methods commonly used to identify adaptive variation, and this needs appropriate consideration when planning genomic screens, and when basing management decisions on genomic data. When the genomic basis of adaptation and future threats are well understood, it may be appropriate to focus management on particular adaptive traits. For more typical conservations scenarios, we argue that screening genome‐wide variation should be a sensible approach that may provide a generalized measure of evolutionary potential that accounts for the contributions of small‐effect loci and cryptic variation and is robust to uncertainty about future change and required adaptive response(s). The best conservation outcomes should be achieved when genomic estimates of evolutionary potential are used within an adaptive management framework.

Evidence for a robust sex-specific trade-off between cold resistance and starvation resistance in Drosophila melanogaster

In insects changes in lipid metabolism may underlie a trade‐off between cold resistance and starvation resistance. To test this we examined correlated responses in independent sets of Drosophila melanogaster lines selected for increased cold resistance and increased starvation resistance. The starvation lines showed correlated patterns found in other D. melanogaster populations selected for this trait, including higher lipid levels and increased resistance to desiccation, although the selected lines did not show a longer development time as found in some other studies. Consistent with the trade‐off hypothesis, selected lines with increased starvation resistance showed decreased resistance to a cold stress as measured by mortality, whereas selected lines with increased cold resistance showed a decrease in starvation resistance. To counter the possibility of inadvertent selection accounting for these patterns, selected and control lines from both selection regimes were crossed to form mass bred populations, which were left for four generations prior to establishing isofemale lines. By scoring starvation and cold resistance in these lines derived from both sets of selection regimes, we confirmed the negative association between resistance to these stresses in females but not in males. Potential implications of this trade‐off for surviving cold conditions when food resources are limiting are discussed.

A new set of laboratory-selected Drosophila melanogaster lines for the analysis of desiccation resistance: response to selection, physiology and correlated responses.

SUMMARY Artificial selection experiments provide insights into the evolutionary factors that can shape adaptive responses and have previously been utilized to examine the physiological adaptations that can improve survival to desiccation in Drosophila melanogaster. While such studies demonstrate that multiple resistance mechanisms may arise via different base populations and selection regimes, water retention emerges as a key mechanism for desiccation survival. Here, we present the physiological, correlated response and life history data for a new set of selection lines designed for the genetic dissection of desiccation resistance. After 26 generations of selection for desiccation resistance, female survival increased twofold. In contrast to previous studies, the altered resistance was associated primarily with enhanced dehydration tolerance and increased mass and less consistently with decreased rates of water loss. Life history tradeoffs and correlated selection responses were examined and overlap with previously published data. We crossed the resistant selected lines to desiccation-sensitive lines from the same control background to examine how each heterozygous resistant chromosome (excluding four) may improve desiccation resistance and observed that most of the resistance was due to genes on the third and first chromosomes, although interaction effects with the second chromosome were also detected. Results are compared to other selection responses and highlight the multiple evolutionary solutions that can arise when organisms are faced with a common selection pressure, although water loss rate remains a common mechanism in all studies.

Selection for cold resistance alters gene transcript levels in Drosophila melanogaster

Microarrays have been used to examine changes in gene expression underlying responses to selection for increased stress resistance in Drosophila melanogaster, but changes in expression patterns associated with increased resistance to cold stress have not been previously reported. Here we describe such changes in basal expression levels in replicate lines following selection for increased resistance to chill coma stress. We found significant up- or down-regulation of expression in 94 genes on the Affymetrix Genome 2.0 array. Quantitative RT-PCR was used to confirm changes in expression of six genes. Some of the identified genes had previously been associated with stress resistance but no previously identified candidate genes for cold resistance showed altered patterns of expression. Seven differentially expressed genes that form a tight chromosomal cluster and an unlinked gene AnnX may be potentially important for cold adaptation in natural populations. Artificial selection for chill coma resistance therefore altered basal patterns of gene expression, but we failed to link these changes to plastic changes in expression under cold stress or to previously identified candidate genes for components of cold resistance.

The molecular genetics of clinal variation: a case study of ebony and thoracic trident pigmentation in Drosophila melanogaster from eastern Australia

Widespread pigmentation diversity coupled with a well‐defined genetic system of melanin synthesis and patterning in Drosophila provides an excellent opportunity to study phenotypes undergoing evolutionary change. Pigmentation variation is highly correlated with different ecological variables and is thought to reflect adaptations to different environments. Several studies have linked candidate genes from Drosophila melanogaster to intra‐population variation and interspecific morphological divergence, but less clearly to variation among populations forming pigmentation clines. We characterized a new thoracic trident pigmentation cline in D. melanogaster populations from eastern Australia, and applied a candidate gene approach to explain the majority of the geographically structured phenotypic variation. More melanized populations from higher latitudes tended to express less ebony than their tropical counterparts, and an independent artificial selection experiment confirmed this association. By partitioning temperature dependent effects, we showed that the genetic differences underlying clinal patterns for trident variation at 25 °C do not explain the patterns observed at 16 °C. Changes in thoracic trident pigmentation could be a common evolutionary response to climatically mediated environmental pressures. On the Australian east coast most of the changes appear to be associated with regulatory divergence of the ebony gene but this depends on temperature.

Cross-Study Comparison Reveals Common Genomic, Network, and Functional Signatures of Desiccation Resistance in Drosophila melanogaster

Repeated attempts to map the genomic basis of complex traits often yield different outcomes because of the influence of genetic background, gene-by-environment interactions, and/or statistical limitations. However, where repeatability is low at the level of individual genes, overlap often occurs in gene ontology categories, genetic pathways, and interaction networks. Here we report on the genomic overlap for natural desiccation resistance from a Pool-genome-wide association study experiment and a selection experiment in flies collected from the same region in southeastern Australia in different years. We identified over 600 single nucleotide polymorphisms associated with desiccation resistance in flies derived from almost 1,000 wild-caught genotypes, a similar number of loci to that observed in our previous genomic study of selected lines, demonstrating the genetic complexity of this ecologically important trait. By harnessing the power of cross-study comparison, we narrowed the candidates from almost 400 genes in each study to a core set of 45 genes, enriched for stimulus, stress, and defense responses. In addition to gene-level overlap, there was higher order congruence at the network and functional levels, suggesting genetic redundancy in key stress sensing, stress response, immunity, signaling, and gene expression pathways. We also identified variants linked to different molecular aspects of desiccation physiology previously verified from functional experiments. Our approach provides insight into the genomic basis of a complex and ecologically important trait and predicts candidate genetic pathways to explore in multiple genetic backgrounds and related species within a functional framework.

Spatial analysis of gene regulation reveals new insights into the molecular basis of upper thermal limits

The cellular stress response has long been the primary model for studying the molecular basis of thermal adaptation, yet the link between gene expression, RNA metabolism and physiological responses to thermal stress remains largely unexplored. We address this by comparing the transcriptional and physiological responses of three geographically distinct populations of Drosophila melanogaster from eastern Australia in response to, and recovery from, a severe heat stress with and without a prestress hardening treatment. We focus on starvin (stv), recently identified as an important thermally responsive gene. Intriguingly, stv encodes seven transcripts from alternative transcription sites and alternative splicing, yet appears to be rapidly heat inducible. First, we show genetic differences in upper thermal limits of the populations tested. We then demonstrate that the stv locus does not ubiquitously respond to thermal stress but is expressed as three distinct thermal and temporal RNA phenotypes (isoforms). The shorter transcript isoforms are rapidly upregulated under stress in all populations and show similar molecular signatures to heat‐shock proteins. Multiple stress exposures seem to generate a reserve of pre‐mRNAs, effectively ‘priming’ the cells for subsequent stress. Remarkably, we demonstrate a bypass in the splicing blockade in these isoforms, suggesting an essential role for these transcripts under heat stress. Temporal profiles for the weakly heat responsive stv isoform subset show opposing patterns in the two most divergent populations. Innate and induced transcriptome responses to hyperthermia are complex, and warrant moving beyond gene‐level analyses.

New Levels of Transcriptome Complexity at Upper Thermal Limits in Wild Drosophila Revealed by Exon Expression Analysis

While the cellular heat-shock response has been a paradigm for studying the impact of thermal stress on RNA metabolism and gene expression, the genome-wide response to thermal stress and its connection to physiological stress resistance remain largely unexplored. Here, we address this issue using an array-based exon expression analysis to interrogate the transcriptome in recently established Drosophila melanogaster stocks during severe thermal stress and recovery. We first demonstrated the efficacy of exon-level analyses to reveal a level of thermally induced transcriptome complexity extending well beyond gene-level analyses. Next, we showed that the upper range of both the cellular and physiological thermal stress response profoundly affected message expression and processing in D. melanogaster, limiting expression to a small subset of transcripts, many that share features of known rapidly responding stress genes. As predicted from cellular heat-shock research, constitutive splicing was blocked in a set of novel genes; we did not detect changes to alternative splicing during heat stress, but rather induction of intronless isoforms of known heat-responsive genes. We observed transcriptome plasticity in the form of differential isoform expression during recovery from heat shock, mediated by multiple mechanisms including alternative transcription and alternative splicing. This affected genes involved in DNA regulation, immune response, and thermotolerance. These patterns highlight the complex nature of innate transcriptome responses under stress and potential for adaptive shifts through plasticity and evolved genetic responses at different hierarchical levels.

Isolation of a Drosophila melanogaster desiccation resistant mutant

Mutagenesis provides a powerful way of isolating genetic and physiological processes underlying complex traits, but this approach has rarely been applied to investigating water balance in insects. Here, we describe the isolation of a desiccation-resistant mutant of Drosophila melanogaster. Mutagenesis of a desiccation sensitive line resulted in the isolation of a mutant with two-fold higher resistance. The mutant was partially dominant and mapped to the second chromosome. Mutant flies showed lower rates of water loss, and had a higher water content, but showed no change in body mass, glycogen content, hemolymph volume or water content tolerated at death from desiccation. These physiological differences are contrasted to changes in lines of D. melanogaster mass selected for altered stress resistance. Isolation of this mutant provides an opportunity to identify a gene involved in water balance in insects.

Signatures of polygenic adaptation associated with climate across the range of a threatened fish species with high genetic connectivity

Adaptive differences across species’ ranges can have important implications for population persistence and conservation management decisions. Despite advances in genomic technologies, detecting adaptive variation in natural populations remains challenging. Key challenges in gene–environment association studies involve distinguishing the effects of drift from those of selection and identifying subtle signatures of polygenic adaptation. We used paired‐end restriction site‐associated DNA sequencing data (6,605 biallelic single nucleotide polymorphisms; SNPs) to examine population structure and test for signatures of adaptation across the geographic range of an iconic Australian endemic freshwater fish species, the Murray cod Maccullochella peelii. Two univariate gene–association methods identified 61 genomic regions associated with climate variation. We also tested for subtle signatures of polygenic adaptation using a multivariate method (redundancy analysis; RDA). The RDA analysis suggested that climate (temperature‐ and precipitation‐related variables) and geography had similar magnitudes of effect in shaping the distribution of SNP genotypes across the sampled range of Murray cod. Although there was poor agreement among the candidate SNPs identified by the univariate methods, the top 5% of SNPs contributing to significant RDA axes included 67% of the SNPs identified by univariate methods. We discuss the potential implications of our findings for the management of Murray cod and other species generally, particularly in relation to informing conservation actions such as translocations to improve evolutionary resilience of natural populations. Our results highlight the value of using a combination of different approaches, including polygenic methods, when testing for signatures of adaptation in landscape genomic studies.