Patterns of recombination in snakes reveal a tug-of-war between PRDM9 and promoter-like features.

In some mammals, notably humans, recombination occurs almost exclusively where the protein PRDM9 binds, whereas in vertebrates lacking an intact PRDM9, such as birds and canids, recombination rates are elevated near promoter-like features. To determine whether PRDM9 directs recombination in nonmammalian vertebrates, we focused on an exemplar species with a single, intact PRDM9 ortholog, the corn snake (Pantherophis guttatus). Analyzing historical recombination rates along the genome and crossovers in pedigrees, we found evidence that PRDM9 specifies the location of recombination events, but we also detected a separable effect of promoter-like features. These findings reveal that the uses of PRDM9 and promoter-like features need not be mutually exclusive and instead reflect a tug-of-war that is more even in some species than others.

Predatory fireflies and their toxic firefly prey have evolved distinct toxin resistance strategies.

Toxic cardiotonic steroids (CTSs) act as a defense mechanism in many firefly species (Lampyridae) by inhibiting a crucial enzyme called Na+,K+-ATPase (NKA). Although most fireflies produce these toxins internally, species of the genus Photuris acquire them from a surprising source: predation on other fireflies. The contrasting physiology of toxin exposure and sequestration between Photuris and other firefly genera suggests that distinct strategies may be required to prevent self-intoxication. Our study demonstrates that both Photuris and their firefly prey have evolved highly resistant NKAs. Using an evolutionary analysis of the specific target of CTS (ATPα) in fireflies and gene editing in Drosophila, we find that the initial steps toward resistance were shared among Photuris and other firefly lineages. However, the Photuris lineage subsequently underwent multiple rounds of gene duplication and neofunctionalization, resulting in the development of ATPα paralogs that are differentially expressed and exhibit increasing resistance to CTS. By contrast, other firefly species have maintained a single copy. Our results implicate gene duplication as a facilitator in the transition of Photuris to its distinct ecological role as a predator of toxic firefly prey.

Patterns of recombination in snakes reveal a tug of war between PRDM9 and promoter-like features.

In vertebrates, there are two known mechanisms by which meiotic recombination is directed to the genome: in humans, mice, and other mammals, recombination occurs almost exclusively where the protein PRDM9 binds, while in species lacking an intact PRDM9, such as birds and canids, recombination rates are elevated near promoter-like features. To test if PRDM9 also directs recombination in non-mammalian vertebrates, we focused on an exemplar species, the corn snake (Pantherophis guttatus). Unlike birds, this species possesses a single, intact PRDM9 ortholog. By inferring historical recombination rates along the genome from patterns of linkage disequilibrium and identifying crossovers in pedigrees, we found that PRDM9 specifies the location of recombination events outside of mammals. However, we also detected an independent effect of promoter-like features on recombination, which is more pronounced on macro- than microchromosomes. Thus, our findings reveal that the uses of PRDM9 and promoter-like features are not mutually-exclusive, and instead reflect a tug of war, which varies in strength along the genome and is more lopsided in some species than others.

Predatory fireflies and their toxic firefly prey have evolved distinct toxin resistance strategies.

Toxic cardiotonic steroids (CTS) act as a defense mechanism in many firefly species (Lampyridae) by inhibiting a crucial enzyme called Na+,K+-ATPase (NKA). While most fireflies produce these toxins internally, species of the genus Photuris acquire them from a surprising source: predation on other fireflies. The contrasting physiology of toxin exposure and sequestration between Photuris and other firefly genera suggests that distinct strategies may be required to prevent self-intoxication. Our study demonstrates that both Photuris and their firefly prey have evolved highly-resistant NKAs. Using an evolutionary analysis of the specific target of CTS (ATPα) in fireflies, and gene-editing in Drosophila, we find that the initial steps towards resistance were shared among Photuris and other firefly lineages. However, the Photuris lineage subsequently underwent multiple rounds of gene duplication and neofunctionalization, resulting in the development of ATPα paralogs that are differentially expressed and exhibit increasing resistance to CTS. In contrast, other firefly species have maintained a single copy. Our results implicate gene duplication as a facilitator in the transition of Photuris to its distinct ecological role as predator of toxic firefly prey.

Dietary stress remodels the genetic architecture of lifespan variation in outbred Drosophila.

Evolutionary theory suggests that lifespan-reducing alleles should be purged from the gene pool, and yet decades of genome-wide association and model organism studies have shown that they persist. One potential explanation is that alleles that regulate lifespan do so only in certain environmental contexts. We exposed outbred Drosophila to control and high-sugar diets and genotyped more than 10,000 adult flies to track allele frequency changes over the course of a single adult lifespan. We identified thousands of lifespan-associated alleles associated with early versus late-life trade-offs, late-onset effects and genotype-by-environment interactions. Remarkably, a third of lifespan-associated genetic variation had environmentally dependent effects on lifespan. We find that lifespan-reducing alleles are often recently derived, have stronger effects on a high-sugar diet and show signatures of selection in wild Drosophila populations, consistent with the evolutionary mismatch hypothesis. Our results provide insight into the highly polygenic and context-dependent genetic architecture of lifespan variation and the evolutionary processes that shape this key trait.

Epistatic Effects Between Amino Acid Insertions and Substitutions Mediate Toxin resistance of Vertebrate Na+,K+-ATPases.

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino acid substitutions in the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work revealed that hystricognath rodents (e.g., chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino acid insertions in the H1-H2 loop, but their functional significance was not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance in homologs of each of the two species that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla NKA unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse NKA, the amino acid insertion and substitution Q111R both contribute to an augmented CTS resistance without compromising ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific mutational effects on CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

Constraints on the evolution of toxin-resistant Na,K-ATPases have limited dependence on sequence divergence.

A growing body of theoretical and experimental evidence suggests that intramolecular epistasis is a major determinant of rates and patterns of protein evolution and imposes a substantial constraint on the evolution of novel protein functions. Here, we examine the role of intramolecular epistasis in the recurrent evolution of resistance to cardiotonic steroids (CTS) across tetrapods, which occurs via specific amino acid substitutions to the α-subunit family of Na,K-ATPases (ATP1A). After identifying a series of recurrent substitutions at two key sites of ATP1A that are predicted to confer CTS resistance in diverse tetrapods, we then performed protein engineering experiments to test the functional consequences of introducing these substitutions onto divergent species backgrounds. In line with previous results, we find that substitutions at these sites can have substantial background-dependent effects on CTS resistance. Globally, however, these substitutions also have pleiotropic effects that are consistent with additive rather than background-dependent effects. Moreover, the magnitude of a substitution's effect on activity does not depend on the overall extent of ATP1A sequence divergence between species. Our results suggest that epistatic constraints on the evolution of CTS-resistant forms of Na,K-ATPase likely depend on a small number of sites, with little dependence on overall levels of protein divergence. We propose that dependence on a limited number sites may account for the observation of convergent CTS resistance substitutions observed among taxa with highly divergent Na,K-ATPases (See S1 Text for Spanish translation).

Allele-specific knockouts reveal a role for apontic-like in the evolutionary loss of larval melanin pigmentation in the domesticated silkworm, Bombyx mori.

The domesticated silkworm, Bombyx mori, and its wild progenitor, B. mandarina, are extensively studied as a model case of the evolutionary process of domestication. A conspicuous difference between these species is the dramatic reduction in melanin pigmentation in both larval and adult B. mori. Here we evaluate the efficiency of CRISPR/Cas9-targeted knockouts of pigment-related genes as a tool to understand their potential contributions to domestication-associated melanin pigmentation loss in B. mori. To demonstrate the efficacy of targeted knockouts in B. mandarina, we generated a homozygous CRISPR/Cas9-targeted knockout of yellow-y. In yellow-y knockout mutants, black body colour became lighter throughout the larval, pupal and adult stages, confirming a role for this gene in melanin pigment formation. Further, we performed allele-specific CRISPR/Cas9-targeted knockouts of the pigment-related transcription factor, apontic-like (apt-like) in B. mori × B. mandarina F1 hybrid individuals which exhibit B. mandarina-like larval pigmentation. Knockout of the B. mandarina allele of apt-like in F1 embryos results in white patches on the dorsal integument of larvae, whereas corresponding knockouts of the B. mori allele consistently exhibit normal F1 larval pigmentation. These results demonstrate a contribution of apt-like to the evolution of reduced melanin pigmentation in B. mori. Together, our results demonstrate the feasibility of CRISPR/Cas9-targeted knockouts as a tool for understanding the genetic basis of traits associated with B. mori domestication.

WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

Genomic signatures of spatially divergent selection at clownfish range margins.

Understanding how evolutionary forces interact to drive patterns of selection and distribute genetic variation across a species' range is of great interest in ecology and evolution, especially in an era of global change. While theory predicts how and when populations at range margins are likely to undergo local adaptation, empirical evidence testing these models remains sparse. Here, we address this knowledge gap by investigating the relationship between selection, gene flow and genetic drift in the yellowtail clownfish, Amphiprion clarkii, from the core to the northern periphery of the species range. Analyses reveal low genetic diversity at the range edge, gene flow from the core to the edge and genomic signatures of local adaptation at 56 single nucleotide polymorphisms in 25 candidate genes, most of which are significantly correlated with minimum annual sea surface temperature. Several of these candidate genes play a role in functions that are upregulated during cold stress, including protein turnover, metabolism and translation. Our results illustrate how spatially divergent selection spanning the range core to the periphery can occur despite the potential for strong genetic drift at the range edge and moderate gene flow from the core populations.

Concerted evolution reveals co-adapted amino acid substitutions in Na+K+-ATPase of frogs that prey on toxic toads.

Although gene duplication is an important source of evolutionary innovation, the functional divergence of duplicates can be opposed by ongoing gene conversion between them. Here, we report on the evolution of a tandem duplication of Na+,K+-ATPase subunit α1 (ATP1A1) shared by frogs in the genus Leptodactylus, a group of species that feeds on toxic toads. One ATP1A1 paralog evolved resistance to toad toxins although the other retained ancestral susceptibility. Within species, frequent non-allelic gene conversion homogenized most of the sequence between the two copies but was counteracted by strong selection on 12 amino acid substitutions that distinguish the two paralogs. Protein-engineering experiments show that two of these substitutions substantially increase toxin resistance, whereas the additional 10 mitigate their deleterious effects on ATPase activity. Our results reveal how examination of neo-functionalized gene duplicate evolution can help pinpoint key functional substitutions and interactions with the genetic backgrounds on which they arise.

A novel family of secreted insect proteins linked to plant gall development.

In an elaborate form of inter-species exploitation, many insects hijack plant development to induce novel plant organs called galls that provide the insect with a source of nutrition and a temporary home. Galls result from dramatic reprogramming of plant cell biology driven by insect molecules, but the roles of specific insect molecules in gall development have not yet been determined. Here, we study the aphid Hormaphis cornu, which makes distinctive "cone" galls on leaves of witch hazel Hamamelis virginiana. We found that derived genetic variants in the aphid gene determinant of gall color (dgc) are associated with strong downregulation of dgc transcription in aphid salivary glands, upregulation in galls of seven genes involved in anthocyanin synthesis, and deposition of two red anthocyanins in galls. We hypothesize that aphids inject DGC protein into galls and that this results in differential expression of a small number of plant genes. dgc is a member of a large, diverse family of novel predicted secreted proteins characterized by a pair of widely spaced cysteine-tyrosine-cysteine (CYC) residues, which we named BICYCLE proteins. bicycle genes are most strongly expressed in the salivary glands specifically of galling aphid generations, suggesting that they may regulate many aspects of gall development. bicycle genes have experienced unusually frequent diversifying selection, consistent with their potential role controlling gall development in a molecular arms race between aphids and their host plants.

The Genetic Architecture of Variation in the Sexually Selected Sword Ornament and Its Evolution in Hybrid Populations.

Biologists since Darwin have been fascinated by the evolution of sexually selected ornaments, particularly those that reduce viability. Uncovering the genetic architecture of these traits is key to understanding how they evolve and are maintained. Here, we investigate the genetic architecture and evolutionary loss of a sexually selected ornament, the "sword" fin extension that characterizes many species of swordtail fish (Xiphophorus). Using sworded and swordless sister species of Xiphophorus, we generated a mapping population and show that the sword ornament is polygenic-with ancestry across the genome explaining substantial variation in the trait. After accounting for the impacts of genome-wide ancestry, we identify one major-effect quantitative trait locus (QTL) that explains ~5% of the overall variation in the trait. Using a series of approaches, we narrow this large QTL interval to several likely candidate genes, including genes involved in fin regeneration and growth. Furthermore, we find evidence of selection on ancestry at one of these candidates in four natural hybrid populations, consistent with selection against the sword in these populations.

Population genetics of the coral Acropora millepora: Toward genomic prediction of bleaching.

Although reef-building corals are declining worldwide, responses to bleaching vary within and across species and are partly heritable. Toward predicting bleaching response from genomic data, we generated a chromosome-scale genome assembly for the coral Acropora millepora We obtained whole-genome sequences for 237 phenotyped samples collected at 12 reefs along the Great Barrier Reef, among which we inferred little population structure. Scanning the genome for evidence of local adaptation, we detected signatures of long-term balancing selection in the heat-shock co-chaperone sacsin We conducted a genome-wide association study of visual bleaching score for 213 samples, incorporating the polygenic score derived from it into a predictive model for bleaching in the wild. These results set the stage for genomics-based approaches in conservation strategies.

Natural hybridization reveals incompatible alleles that cause melanoma in swordtail fish.

The establishment of reproductive barriers between populations can fuel the evolution of new species. A genetic framework for this process posits that "incompatible" interactions between genes can evolve that result in reduced survival or reproduction in hybrids. However, progress has been slow in identifying individual genes that underlie hybrid incompatibilities. We used a combination of approaches to map the genes that drive the development of an incompatibility that causes melanoma in swordtail fish hybrids. One of the genes involved in this incompatibility also causes melanoma in hybrids between distantly related species. Moreover, this melanoma reduces survival in the wild, likely because of progressive degradation of the fin. This work identifies genes underlying a vertebrate hybrid incompatibility and provides a glimpse into the action of these genes in natural hybrid populations.

Dichotomy of Dosage Compensation along the Neo Z Chromosome of the Monarch Butterfly.

Mechanisms of sex chromosome dosage compensation (SCDC) differ strikingly among animals. In Drosophila flies, chromosome-wide transcription is doubled from the single X chromosome in hemizygous (XY) males, whereas in Caenorhabditis nematodes, expression is halved for both X copies in homozygous (XX) females [1, 2]. Unlike other female-heterogametic (WZ female and ZZ male) animals, moths and butterflies exhibit sex chromosome dosage compensation patterns typically seen only in male-heterogametic species [3]. The monarch butterfly carries a newly derived Z chromosome segment that arose from an autosomal fusion with the ancestral Z [4]. Using a highly contiguous genome assembly, we show that gene expression is balanced between sexes along the entire Z chromosome but with distinct modes of compensation on the two segments. On the ancestral Z segment, depletion of H4K16ac corresponds to nearly halving of biallelic transcription in males, a pattern convergent to nematodes. Conversely, the newly derived Z segment shows a Drosophila-like mode of compensation, with enriched H4K16ac levels corresponding to doubled monoallelic transcription in females. Our work reveals that, contrary to the expectation of co-opting regulatory mechanisms readily in place, the evolution of plural modes of dosage compensation is also possible along a single sex chromosome within a species.

Adaptive substitutions underlying cardiac glycoside insensitivity in insects exhibit epistasis in vivo.

Predicting how species will respond to selection pressures requires understanding the factors that constrain their evolution. We use genome engineering of Drosophila to investigate constraints on the repeated evolution of unrelated herbivorous insects to toxic cardiac glycosides, which primarily occurs via a small subset of possible functionally-relevant substitutions to Na+,K+-ATPase. Surprisingly, we find that frequently observed adaptive substitutions at two sites, 111 and 122, are lethal when homozygous and adult heterozygotes exhibit dominant neural dysfunction. We identify a phylogenetically correlated substitution, A119S, that partially ameliorates the deleterious effects of substitutions at 111 and 122. Despite contributing little to cardiac glycoside-insensitivity in vitro, A119S, like substitutions at 111 and 122, substantially increases adult survivorship upon cardiac glycoside exposure. Our results demonstrate the importance of epistasis in constraining adaptive paths. Moreover, by revealing distinct effects of substitutions in vitro and in vivo, our results underscore the importance of evaluating the fitness of adaptive substitutions and their interactions in whole organisms.

Changes throughout a Genetic Network Mask the Contribution of Hox Gene Evolution.

Hox genes pattern the anterior-posterior axis of animals and are posited to drive animal body plan evolution, yet their precise role in evolution has been difficult to determine. Here, we identified evolutionary modifications in the Hox gene Abd-B that dramatically altered its expression along the body plan of Drosophila santomea. Abd-B is required for pigmentation in Drosophila yakuba, the sister species of D. santomea, and changes to Abd-B expression would be predicted to make large contributions to the loss of body pigmentation in D. santomea. However, manipulating Abd-B expression in current-day D. santomea does not affect pigmentation. We attribute this epistatic interaction to four other genes within the D. santomea pigmentation network, three of which have evolved expression patterns that do not respond to Abd-B. Our results demonstrate how body plans may evolve through small evolutionary steps distributed throughout Hox-regulated networks. Polygenicity and epistasis may hinder efforts to identify genes and mechanisms underlying macroevolutionary traits.

Predictability in the evolution of Orthopteran cardenolide insensitivity.

The repeated evolutionary specialization of distantly related insects to cardenolide-containing host plants provides a stunning example of parallel adaptation. Hundreds of herbivorous insect species have independently evolved insensitivity to cardenolides, which are potent inhibitors of the alpha-subunit of Na+,K+-ATPase (ATPα). Previous studies investigating ATPα-mediated cardenolide insensitivity in five insect orders have revealed remarkably high levels of parallelism in the evolution of this trait, including the frequent occurrence of parallel amino acid substitutions at two sites and recurrent episodes of duplication followed by neo-functionalization. Here we add data for a sixth insect order, Orthoptera, which includes an ancient group of highly aposematic cardenolide-sequestering grasshoppers in the family Pyrgomorphidae. We find that Orthopterans exhibit largely predictable patterns of evolution of insensitivity established by sampling other insect orders. Taken together the data lend further support to the proposal that negative pleiotropic constraints are a key determinant in the evolution of cardenolide insensitivity in insects. Furthermore, analysis of our expanded taxonomic survey implicates positive selection acting on site 111 of cardenolide-sequestering species with a single-copy of ATPα, and sites 115, 118 and 122 in lineages with neo-functionalized duplicate copies, all of which are sites of frequent parallel amino acid substitution. This article is part of the theme issue 'Convergent evolution in the genomics era: new insights and directions'.