SummaryPlant specialized 1,4-naphthoquinones present a remarkable case of convergent evolution. Species across multiple discrete orders of vascular plants produce diverse 1,4-naphthoquinones via one of several pathways using different metabolic precursors. Evolution of these pathways was preceded by events of metabolic innovation and many appear to share connections with biosynthesis of photosynthetic or respiratory quinones. Here, we sought to shed light on the metabolic connections linking shikonin biosynthesis with its precursor pathways and on the origins of shiknoin metabolic genes. Downregulation of Lithospermum erythrorhizon geranyl diphosphate synthase (LeGPPS), recently shown to have been recruited from a cytoplasmic farnesyl diphosphate synthase (FPPS), resulted in reduced shikonin production and a decrease in expression of mevalonic acid and phenylpropanoid pathway genes. Next, we used LeGPPS and other known shikonin pathway genes to build a coexpression network model for identifying new gene connections to shikonin metabolism. Integrative in silico analyses of network genes revealed candidates for biochemical steps in the shikonin pathway arising from Boraginales-specific gene family expansion. Multiple genes in the shikonin coexpression network were also discovered to have originated from duplication of ubiquinone pathway genes. Taken together, our study provides evidence for transcriptional crosstalk between shikonin biosynthesis and its precursor pathways, identifies several shikonin pathway gene candidates and their evolutionary histories, and establishes additional evolutionary links between shikonin and ubiquinone metabolism. Moreover, we demonstrate that global coexpression analysis using limited transcriptomic data obtained from targeted experiments is effective for identifying gene connections within a defined metabolic network.

Global, large-scale surveys of phylogenetically diverse plant and lichen hosts have revealed an extremely high richness of endophytes in the Xylariales, one of the largest clades of filamentous fungi and a significant source of novel secondary metabolites (SMs). Endophytes may produce host protective antimicrobial or insecticidal SMs, as well as compounds that facilitate symbiotic establishment through suppression or degradation of host immune response, but the ecological roles of most SMs are unknown. Here we characterized metabolic gene clusters in 96 genomes of endophytes and closely related saprotrophs and pathogens in two clades of Xylariales (Xylariaceae s.l. and Hypoxylaceae). Hundreds of genes appear horizontally transferred to xylarialean fungi from distantly related fungi and bacteria, including numerous genes in secondary metabolite gene clusters (SMGCs). Although all xylarialean genomes contain hyperabundant SMGCs, we show that increased gene duplications, horizontal gene transfers (HGTs), and SMGC content in Xylariaceae s.l. taxa are linked to greater phylogenetic host breadth, larger biogeographic distributions, and increased capacity for lignocellulose decomposition compared to Hypoxylaceae taxa. Overall, our results suggest that xylarialean endophytes capable of dual ecological modes (symbiotic and saprotrophic) experience greater selection to diversify SMGCs to both increase competitiveness within microbial communities and facilitate diverse symbiotic interactions.

23Fructophily is a rare trait that consists in the preference for fructose over other carbon sources. 24Here we show that in a yeast lineage (the Wickerhamiella/Starmerella, W/S clade) formed by 25 fructophilic species thriving in the floral niche, the acquisition of fructophily is part of a wider 26 process of adaptation of central carbon metabolism to the high sugar environment. Coupling 27 comparative genomics with biochemical and genetic approaches, we show that the alcoholic 28 fermentation pathway was profoundly remodeled in the W/S clade, as genes required for 29 alcoholic fermentation were lost and subsequently re-acquired from bacteria through horizontal 30 gene transfer. We further show that the reinstated fermentative pathway is functional and that an 31 enzyme required for sucrose assimilation is also of bacterial origin, reinforcing the adaptive 32 nature of the genetic novelties identified in the W/S clade. This work shows how even central 33 carbon metabolism can be remodeled by a surge of HGT events.

Cytolethal distending toxins (CDTs) are tripartite eukaryotic genotoxins encoded in diverse bacterial and phage genomes. The cdtB subunit is a DNAse that causes eukaryotic cell cycle arrest and apoptosis, and in one context, is associated with resistance against parasitoid wasp infections. Here we report the discovery of functional cdtB copies in the nuclear genomes 5 of insect species from two distantly related insect orders, including fruit flies (Diptera: Drosophilidae) and aphids (Hemiptera: Aphididae). Insect cdtB copies are most closely related to bacteriophage copies, were horizontally transferred to insect genomes > 40 million years ago and encode a protein that retains ancestral DNase activity. This phage-derived toxin has been domesticated by diverse insects and we hypothesize that it is used as a defensive weapon against 10 parasitoid wasps. One Sentence Summary:We report horizontal transfer of the gene cytolethal distending toxin B, which encodes a DNase, into eukaryotic genomes from bacteriophage. 15 Significance: Cytolethal distending toxins (CDTs) are secreted by diverse pathogenic bacterial species to kill animal cells. The cdtB subunit enters cell nuclei, damaging the DNA and leading to mitotic arrest and apoptosis. In the pea aphid, a bacterial endosymbiont provides protection against wasp attack, possibly via cdtB. We discovered that this same endosymbiont-encoded lineage of cdtB was transferred to the genomes of Diptera and Hemiptera species and retains 20 ancestral DNase activity. This is the first report of cdtB outside of bacteria or phages. A toxin that first evolved to kill eukaryotic cells has been co-opted by insects, potentially to their benefit. 3 Main TextCytolethal distending toxins (CDTs) are widespread intracellular-acting eukaryotic genotoxins encoded by a gene family restricted to Actinobacteria, Proteobacteria and bacteriophage genomes (1). CDTs are found in diverse pathogens, including Campylobacter jejuni, Escherichia coli, Salmonella enterica, and Yersinia pestis and may be a cause of irritable bowel syndrome 5 (1). CDT holotoxin is an AB2 toxin typically encoded in a three-gene operon (cdtA, cdtB, and cdtC) (2) and cdtB is the catalytic subunit necessary for DNase activity (3, 4). CdtB nicking leads to DNA damage in eukaryotic cells followed by cell cycle arrest, cellular distention and death (5).Although cdtB is a eukaryotic genotoxin, in one context it is associated with increased 10 fitness of eukaryotes. Some strains of the bacterium Candidatus Hamiltonella defensa, a secondary endosymbiont of the pea aphid (Acyrthosiphon pisum), are infected with strains of the lysogenic bacteriophage APSE (6, 7). APSE-positive Ca. H defensa strains confer protection from attack by parasitoid braconid wasps that insert eggs into aphids (8). Comparative genomic studies point to cdtB, which is encoded in the genome of phage strain APSE-2, as a likely 15 candidate underlying this protective effect (6-8).We used a sequence similarity-based screen (9) to identify a cdtB homolog as a horizon...