A draft reference genome assembly of California Pipevine, Aristolochia californica Torr.

The California Pipevine, Aristolochia californica Torr., is the only endemic California species within the cosmopolitan birthwort family Aristolochiaceae. It occurs as an understory vine in riparian and chaparral areas and in forest edges and windrows. The geographic range of this plant species almost entirely overlaps with that of its major specialized herbivore, the California Pipevine Swallowtail Butterfly Battus philenor hirsuta. While this species pair is a useful, ecologically well-understood system to study co-evolution, until recently, genomic resources for both have been lacking. Here, we report a new, chromosome-level assembly of A. californica as part of the California Conservation Genomics Project (CCGP). Following the sequencing and assembly strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin proximity sequencing technology to produce a de novo assembled genome. Our genome assembly, the first for any species in the genus, contains 531 scaffolds spanning 661 megabase (Mb) pairs, with a contig N50 of 6.53 Mb, a scaffold N50 of 42.2 Mb, and BUSCO complete score of 98%. In combination with the recently published B. philenor hirsuta reference genome assembly, the A. californica reference genome assembly will be a powerful tool for studying co-evolution in a rapidly changing California landscape.

Taste evolution in an herbivorous drosophilid.

Plant secondary metabolites pose a challenge for generalist herbivorous insects because they are not only potentially toxic, they also may trigger aversion. On the contrary, some highly specialized herbivorous insects evolved to use these same compounds as 'token stimuli' for unambiguous determination of their host plants. Two questions that emerge from these observations are how recently derived herbivores evolve to overcome this aversion to plant secondary metabolites and the extent to which they evolve increased attraction to these same compounds. In this study, we addressed these questions by focusing on the evolution of bitter taste preferences in the herbivorous drosophilid Scaptomyza flava, which is phylogenetically nested deep in the paraphyletic Drosophila. We measured behavioral and neural responses of S. flava and a set of non-herbivorous species representing a phylogenetic gradient (S. pallida, S. hsui, and D. melanogaster) towards host- and non-host derived bitter plant compounds. We observed that S. flava evolved a shift in bitter detection, rather than a narrow shift towards glucosinolates, the precursors of mustard-specific defense compounds. In a dye-based consumption assay, S. flava exhibited shifts in aversion toward the non-mustard bitter, plant-produced alkaloids caffeine and lobeline, and reduced aversion towards glucosinolates, whereas the non-herbivorous species each showed strong aversion to all bitter compounds tested. We then examined whether these changes in bitter preferences of S. flava could be explained by changes in sensitivity in the peripheral nervous system and compared electrophysiological responses from the labellar sensilla of S. flava, S. pallida, and D. melanogaster. Using scanning electron microscopy, we also created a map of labellar sensilla in S. flava and S. pallida. We assigned each sensillum to a functional sensilla class based on their morphology and initial response profiles to bitter and sweet compounds. Despite a high degree of conservation in the morphology and spatial placement of sensilla between S. flava and S. pallida, electrophysiological studies revealed that S. flava had reduced sensitivity to glucosinolates to varying degrees. We found this reduction only in I type sensilla. Finally, we speculate on the potential role that evolutionary genetic changes in gustatory receptors between S. pallida and S. flava may play in driving these patterns. Specifically, we hypothesize that the evolution of bitter receptors expressed in I type sensilla may have driven the reduced sensitivity observed in S. flava, and ultimately, its reduced bitter aversion. The S. flava system showcases the importance of reduced aversion to bitter defense compounds in relatively young herbivorous lineages, and how this may be achieved at the molecular and physiological level.

Chromosome-level reference genomes of two imperiled desert fishes: spikedace (Meda fulgida) and loach minnow (Tiaroga cobitis).

North American minnows (Cypriniformes: Leuciscidae) comprise a diverse taxonomic group, but many members, particularly those inhabiting deserts, face elevated extinction risks. Despite conservation concerns, leuciscids remain under sampled for reference assemblies relative to other groups of freshwater fishes. Here, we present 2 chromosome-scale reference genome assemblies spikedace (Meda fulgida) and loach minnow (Tiaroga cobitis) using PacBio, Illumina and Omni-C technologies. The complete assembly for spikedace was 882.1 Mb in total length comprised of 83 scaffolds with N50 = 34.8 Mb, L50 = 11, N75 = 32.3 Mb, and L75 = 18. The complete assembly for loach minnow was 1.3 Gb in total length comprised of 550 scaffolds with N50 = 48.6 Mb, L50 = 13, N75 = 42.3 Mb, and L75 = 20. Completeness assessed via Benchmarking Universal Single-Copy Orthologues (BUSCO) metrics using the Actinopterygii BUSCO database showed ∼97% for spikedace and ∼98% for loach minnow of complete BUSCO proportions. Annotation revealed approximately 32.58 and 29.04% of spikedace and loach minnow total genome lengths to be comprised of protein-coding genes, respectively. Comparative genomic analyses of these endangered and co-distributed fishes revealed widespread structural variants, gene family expansions, and evidence of positive selection in both genomes.

A draft reference genome assembly of the Pipevine Swallowtail butterfly, Battus philenor hirsuta.

The California Pipevine Swallowtail Butterfly, Battus philenor hirsuta, and its host plant, the California Pipevine or Dutchman's Pipe, Aristolochia californica Torr., are an important California endemic species pair. While this species pair is an ideal system to study co-evolution, genomic resources for both are lacking. Here, we report a new, chromosome-level assembly of B. philenor hirsuta as part of the California Conservation Genomics Project (CCGP). Following the sequencing and assembly strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin proximity sequencing technology to produce a de novo assembled genome. Our genome assembly, the first for any species in the genus, contains 109 scaffolds spanning 443 mega base (Mb) pairs, with a contig N50 of 14.6 Mb, a scaffold N50 of 15.2 Mb, and BUSCO complete score of 98.9%. In combination with the forthcoming A. californica reference genome, the B. philenor hirsuta genome will be a powerful tool for documenting landscape genomic diversity and plant-insect co-evolution in a rapidly changing California landscape.

Convergent resistance to GABA receptor neurotoxins through plant-insect coevolution.

The molecular mechanisms of coevolution between plants and insects remain elusive. GABA receptors are targets of many neurotoxic terpenoids, which represent the most diverse array of natural products known. Over deep evolutionary time, as plant terpene synthases diversified in plants, so did plant terpenoid defence repertoires. Here we show that herbivorous insects and their predators evolved convergent amino acid changing substitutions in duplicated copies of the Resistance to dieldrin (Rdl) gene that encodes the GABA receptor, and that the evolution of duplicated Rdl and terpenoid-resistant GABA receptors is associated with the diversification of moths and butterflies. These same substitutions also evolved in pests exposed to synthetic insecticides that target the GABA receptor. We used in vivo genome editing in Drosophila melanogaster to evaluate the fitness effects of each putative resistance mutation and found that pleiotropy both facilitates and constrains the evolution of GABA receptor resistance. The same genetic changes that confer resistance to terpenoids across 300 Myr of insect evolution have re-evolved in response to synthetic analogues over one human lifespan.

Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genomic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families-genes directly mediating interactions with plant chemical defenses-underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many insect lineages are ancient (>150 million years ago (mya)), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several nonherbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza has among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant-binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on living plants (bitter or electrophilic phytotoxins) or their ancestral diet (fermenting plant volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight gene candidates that have also been linked to other dietary transitions in Drosophila.

Mistletoes.

Whiteman introduces mistletoes, above-ground parasitic plants.

Evolution of insect innate immunity through domestication of bacterial toxins.

Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. Here, we show how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. We discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, we focused on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. We found that cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and we observed that D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were more susceptible to parasitoids than the wild type. We conclude that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.

Evolution of chemosensory and detoxification gene families across herbivorous Drosophilidae.

Herbivorous insects are exceptionally diverse, accounting for a quarter of all known eukaryotic species, but the genetic basis of adaptations that enabled this dietary transition remains poorly understood. Many studies have suggested that expansions and contractions of chemosensory and detoxification gene families - genes directly mediating interactions with plant chemical defenses - underlie successful plant colonization. However, this hypothesis has been challenging to test because the origins of herbivory in many lineages are ancient (>150 million years ago [mya]), obscuring genomic evolutionary patterns. Here, we characterized chemosensory and detoxification gene family evolution across Scaptomyza, a genus nested within Drosophila that includes a recently derived (<15 mya) herbivore lineage of mustard (Brassicales) specialists and carnation (Caryophyllaceae) specialists, and several non-herbivorous species. Comparative genomic analyses revealed that herbivorous Scaptomyza have among the smallest chemosensory and detoxification gene repertoires across 12 drosophilid species surveyed. Rates of gene turnover averaged across the herbivore clade were significantly higher than background rates in over half of the surveyed gene families. However, gene turnover was more limited along the ancestral herbivore branch, with only gustatory receptors and odorant binding proteins experiencing strong losses. The genes most significantly impacted by gene loss, duplication, or changes in selective constraint were those involved in detecting compounds associated with feeding on plants (bitter or electrophilic phytotoxins) or their ancestral diet (yeast and fruit volatiles). These results provide insight into the molecular and evolutionary mechanisms of plant-feeding adaptations and highlight strong gene candidates that have also been linked to other dietary transitions in Drosophila .

Evolution and genomic basis of the plant-penetrating ovipositor: a key morphological trait in herbivorous Drosophilidae.

Herbivorous insects are extraordinarily diverse, yet are found in only one-third of insect orders. This skew may result from barriers to plant colonization, coupled with phylogenetic constraint on plant-colonizing adaptations. The plant-penetrating ovipositor, however, is one trait that surmounts host plant physical defences and may be evolutionarily labile. Ovipositors densely lined with hard bristles have evolved repeatedly in herbivorous lineages, including within the Drosophilidae. However, the evolution and genetic basis of this innovation has not been well studied. Here, we focused on the evolution of this trait in Scaptomyza, a genus sister to Hawaiian Drosophila, that contains a herbivorous clade. Our phylogenetic approach revealed that ovipositor bristle number increased as herbivory evolved in the Scaptomyza lineage. Through a genome-wide association study, we then dissected the genomic architecture of variation in ovipositor bristle number within S. flava. Top-associated variants were enriched for transcriptional repressors, and the strongest associations included genes contributing to peripheral nervous system development. Individual genotyping supported the association at a variant upstream of Gαi, a neural development gene, contributing to a gain of 0.58 bristles/major allele. These results suggest that regulatory variation involving conserved developmental genes contributes to this key morphological trait involved in plant colonization.

A Reference Genome Assembly of Hybrid-Derived California Wild Radish (Raphanus sativus × raphanistrum).

For agriculturally important plants, pollination and herbivory are 2 ecological factors that play into the success of crop yields. Each is also important in natural environments where invasive plants and their effect on species interactions may alter the native ecology. The California Wild Radish (Raphanus sativus × raphanistrum), a hybrid derived from an agriculturally important crop and a nonnative cultivar, is common in California. Remarkably, it has recently replaced wild populations of both progenitor species. Experiments on phenotypic variation for petal color and antiherbivore defenses suggest both pairs of polymorphisms are maintained as a result of pollinator- and herbivore-mediated natural selection. This species provides an opportunity to understand how natural selection shapes the evolution of ecologically important traits when traits are constrained by 2 opposing forces. Here we provide the genome assembly of the California Wild Radish displaying improvement to currently existing genomes for agronomically important crucifers. This genome sequence provides the tools to dissect the genomic architecture of traits related to herbivory and pollination using natural variation in the wild as well as the ability to infer demographic and selective history in the context of hybridization. Study systems like these will improve our understanding and predictions of evolutionary change for correlated traits.

The problem of interaction.

As we look to the next 75 years, I recount how the sociopolitical milieu in which the Society for the Study of Evolution (SSE) and the journal Evolution were formed in 1947 was a "problem of interaction."

Evolution in small steps and giant leaps.

The first Editor of Evolution was Ernst Mayr. His foreword to the first issue of Evolution published in 1947 framed evolution as a "problem of interaction" that was just beginning to be studied in this broad context. First, I explore progress and prospects on understanding the subsidiary interactions identified by Mayr, including interactions between parts of organisms, between individuals and populations, between species, and between the organism and its abiotic environment. Mayr's overall "problem of interaction" framework is examined in the context of coevolution within and among levels of biological organization. This leads to a comparison in the relative roles of biotic versus abiotic agents of selection and fluctuating versus directional selection, followed by stabilizing selection in shaping the genomic architecture of adaptation. Oligogenic architectures may be typical for traits shaped more by fluctuating selection and biotic selection. Conversely, polygenic architectures may be typical for traits shaped more by directional followed by stabilizing selection and abiotic selection. The distribution of effect sizes and turnover dynamics of adaptive alleles in these scenarios deserves further study. Second, I review two case studies on the evolution of acquired toxicity in animals, one involving cardiac glycosides obtained from plants and one involving bacterial virulence factors horizontally transferred to animals. The approaches used in these studies and the results gained directly flow from Mayr's vision of an evolutionary biology that revolves around the "problem of interaction."

Evolution of Olfactory Receptors Tuned to Mustard Oils in Herbivorous Drosophilidae.

The diversity of herbivorous insects is attributed to their propensity to specialize on toxic plants. In an evolutionary twist, toxins betray the identity of their bearers when herbivores coopt them as cues for host-plant finding, but the evolutionary mechanisms underlying this phenomenon are poorly understood. We focused on Scaptomyza flava, an herbivorous drosophilid specialized on isothiocyanate (ITC)-producing (Brassicales) plants, and identified Or67b paralogs that were triplicated as mustard-specific herbivory evolved. Using in vivo heterologous systems for the expression of olfactory receptors, we found that S. flava Or67bs, but not the homologs from microbe-feeding relatives, responded selectively to ITCs, each paralog detecting different ITC subsets. Consistent with this, S. flava was attracted to ITCs, as was Drosophila melanogaster expressing S. flava Or67b3 in the homologous Or67b olfactory circuit. ITCs were likely coopted as olfactory attractants through gene duplication and functional specialization (neofunctionalization and subfunctionalization) in S. flava, a recently derived herbivore.

Convergent evolution of cardiac-glycoside resistance in predators and parasites of milkweed herbivores.

The community of plant-feeding insects (herbivores) that specialize on milkweeds (Apocynaceae) form a remarkable example of convergent evolution across levels of biological organization1. In response to toxic cardiac glycosides produced by these plants, the monarch butterfly (Danaus plexippus) and other specialist herbivores have evolved parallel substitutions in the alpha subunit (ATPA) of the Na+/K+-ATPase. These substitutions render the pump insensitive to cardiac glycosides2,3, allowing the monarch and other specialists, from aphids to beetles, to sequester cardiac glycosides, which in turn provide defense against attacks by enemies from the third trophic level4. The evolution of 'target-site-insensitivity' substitutions in these herbivores poses a fundamental biological question: have predators and parasitoids that feed on cardiac-glycoside-sequestering insects also evolved Na+/K+-ATPases that are similarly insensitive to cardiac glycosides (as predicted by Whiteman and Mooney)5? In other words, can plant toxins cause evolutionary cascades that reach the third trophic level? Here we show that at least four enemies of the monarch and other milkweed herbivores have indeed evolved amino-acid substitutions associated with target-site insensitivity to cardiac glycosides. These attackers represent four major animal clades, implicating cardiac glycosides as keystone molecules6 and establishing ATPalpha, which encodes ATPA, as a keystone gene with effects that reverberate within ecological communities7.

Ecological basis and genetic architecture of crypsis polymorphism in the desert clicker grasshopper (Ligurotettix coquilletti).

Color polymorphic species can offer exceptional insight into the ecology and genetics of adaptation. Although the genetic architecture of animal coloration is diverse, many color polymorphisms are associated with large structural variants and maintained by biotic interactions. Grasshoppers are notably polymorphic in both color and karyotype, which makes them excellent models for understanding the ecological drivers and genetic underpinnings of color variation. Banded and uniform morphs of the desert clicker grasshopper (Ligurotettix coquilletti) are found across the western deserts of North America. To address the hypothesis that predation maintains local color polymorphism and shapes regional crypsis variation, we surveyed morph frequencies and tested for covariation with two predation environments. Morphs coexisted at intermediate frequencies at most sites, consistent with local balancing selection. Morph frequencies covaried with the appearance of desert substrate-an environment used only by females-suggesting that ground-foraging predators are major agents of selection on crypsis. We next addressed the hypothesized link between morph variation and genome structure. To do so, we designed an approach for detecting inversions and indels using only RADseq data. The banded morph was perfectly correlated with a large putative indel. Remarkably, indel dominance differed among populations, a rare example of dominance evolution in nature.

Horizontal Transfer of Microbial Toxin Genes to Gall Midge Genomes.

A growing body of evidence has underscored the role of horizontal gene transfer (HGT) in animal evolution. Previously, we discovered the horizontal transfer of the gene encoding the eukaryotic genotoxin cytolethal distending toxin B (cdtB) from the pea aphid Acyrthosiphon pisum secondary endosymbiont (APSE) phages to drosophilid and aphid nuclear genomes. Here, we report cdtB in the nuclear genome of the gall-forming "swede midge" Contarinia nasturtii (Diptera: Cecidomyiidae) via HGT. We searched all available gall midge genome sequences for evidence of APSE-to-insect HGT events and found five toxin genes (aip56, cdtB, lysozyme, rhs, and sltxB) transferred horizontally to cecidomyiid nuclear genomes. Surprisingly, phylogenetic analyses of HGT candidates indicated APSE phages were often not the ancestral donor lineage of the toxin gene to cecidomyiids. We used a phylogenetic signal statistic to test a transfer-by-proximity hypothesis for animal HGT, which suggested that microbe-to-insect HGT was more likely between taxa that share environments than those from different environments. Many of the toxins we found in midge genomes target eukaryotic cells, and catalytic residues important for toxin function are conserved in insect copies. This class of horizontally transferred, eukaryotic cell-targeting genes is potentially important in insect adaptation.

Whiteflies weaponize a plant defense via horizontal gene transfer.

Co-opting enemy weapons is a proven strategy in warfare. The war of nature is no different. In this issue of Cell, Xia and colleagues show how a major crop pest stole a plant phenolic glucoside malonyltransferase gene, allowing neutralization of a large class of plant defense compounds.