bioRxiv preprint doi: https://doi.org/10.1101/237586; this version posted February 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Firefly genomes illuminate parallel origins of bioluminescence in beetles Authors: Timothy R. Fallon1,2,*, Sarah E. Lower3,*, Ching-Ho Chang4, Manabu Bessho-Uehara5,6, Gavin J. Martin7, Adam J. Bewick8, Megan Behringer9, Humberto J. Debat10, Isaac Wong4, John C. Day11, Anton Suvorov7, Christian J. Silva4,12, Kathrin F. Stanger-Hall13, David W. Hall8, Robert J. Schmitz8, David R. Nelson14, Sara M. Lewis15, Shuji Shigenobu16, Seth M. Bybee7, Amanda M. Larracuente4, Yuichi Oba5, Jing-Ke Weng1,2,† Affiliations: 1Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA. 2Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 3Department of Molecular Biology & Genetics, Cornell University, Ithaca, New York 14850, USA. 4Department of Biology, University of Rochester, Rochester, New York 14627, USA. 5Department of Environmental Biology, Chubu University, Kasugai, Aichi 487-8501, Japan. 6Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464- 8601, Japan. 7Department of Biology, Brigham Young University, Provo, Utah 84602, USA. 8Department of Genetics, University of Georgia, Athens, Georgia 30602, USA. 9Biodesign Center for Mechanisms of Evolution, Arizona State University, Tempe, Arizona 85287, USA. 10Center of Agronomic Research National Institute of Agricultural Technology, Córdoba, Argentina. 11Centre for Ecology and Hydrology (CEH) Wallingford, Wallingford, Oxfordshire, UK. 12Department of Plant Sciences, University of California Davis, Davis, California, USA. 13Department of Plant Biology, University of Georgia, Athens, Georgia 30602, USA. 14Department of Microbiology Immunology and Biochemistry, University of Tennessee HSC, Memphis 38163, USA. 15Department of Biology, Tufts University, Medford, Massachusetts 02155, USA. 16NIBB Core Research Facilities, National Institute for Basic Biology, Okazaki 444-8585, Japan. †Corresponding author. Email: [email protected] (J.K.W.) *These authors contributed equally to this work. One Sentence Summary: Comparative analyses of the first linkage-group-resolution genomes of fireflies and related bioluminescent beetles address long-standing questions of the origin and evolution of bioluminescence and its associated traits. 1 bioRxiv preprint doi: https://doi.org/10.1101/237586; this version posted February 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Fireflies and their fascinating luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of their bioluminescence remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years- ago: the North American Photinus pyralis and Japanese Aquatica lateralis. We also sequenced the genome of a related click-beetle, the Caribbean Ignelater luminosus, with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing but contentious hypothesis of parallel gains of bioluminescence. Our analyses support two independent gains of bioluminescence between fireflies and click-beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle. 2 bioRxiv preprint doi: https://doi.org/10.1101/237586; this version posted February 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Fireflies (Coleoptera: Lampyridae) represent the best-studied case of bioluminescence. The coded language of their luminous courtship displays (Fig. 1A; Video S1) has been long studied for its role in mate recognition (1–3), while non-adult bioluminescence is likely a warning signal of their unpalatable chemical defenses (4), such as the cardiotoxic lucibufagins of Photinus fireflies (5). The biochemical understanding of firefly 2+ luminescence: an ATP, Mg , and O2-dependent luciferase-mediated oxidation of the substrate luciferin (6), along with the cloning of the luciferase gene (7, 8), led to its widespread use of luciferase as a reporter with unique applications in biomedical research and industry (9). With >2000 species globally, fireflies are undoubtedly the most culturally-appreciated bioluminescent group, yet there are at least three other beetle families with bioluminescent species: click beetles (Elateridae), American railroad worms (Phengodidae) and Asian starworms (Rhagophthalmidae) (10). These four closely related families (superfamily Elateroidea) have homologous luciferases and structurally identical luciferins (6), implying a single origin of beetle bioluminescence. However, as Darwin recognized in his “Difficulties on Theory” (11), the light organs of the luminous beetles are clearly distinct (Fig. 1C). Thus, whether beetle bioluminescence is derived from single or multiple origins remains unresolved. To address this long-standing question, we sequenced and analyzed the genomes of three bioluminescent beetle species. To represent the fireflies, we sequenced the widespread North American “Big Dipper Firefly”, Photinus pyralis (Fig. 1A, B) used in classic studies of firefly bioluminescent biochemistry (12) and the cloning of luciferase (7), and the Japanese “Heike-botaru” firefly Aquatica lateralis (Fig. 1C), a species with specialized aquatic larvae and one of the few fireflies that can be cultured 3 bioRxiv preprint doi: https://doi.org/10.1101/237586; this version posted February 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. in the laboratory (13). These two fireflies represent the two major firefly subfamilies, Lampyrinae and Luciolinae, which diverged from a common ancestor over 100 Mya (Fig. 1C) (14, 15). To facilitate evolutionary comparison, we also sequenced the “Cucubano”, Ignelater luminosus (Fig. 1C), a Caribbean bioluminescent click beetle, and one of the “Pyrophorus” species used by Raphaël Dubois to first establish the enzymological basis of bioluminescence in the late 1800s (16). Comparative analyses of the genomes of these three species allowed us to reconstruct the origin(s) and evolution of beetle bioluminescence. Sequencing and assembly yield high-quality genomes P. pyralis adult males were collected from the Great Smoky Mountains National Park, USA (GSMNP) and Mercer Meadows New Jersey, USA (MMNJ) (Fig. 1B), and sequenced using short-insert, mate-pair, Hi-C, and long-read Pacific Biosciences (PacBio) approaches (table S1). These datasets were combined in a MaSuRCA (17) hybrid genome assembly (Supp. Text 1.5). The A. lateralis genome was derived from an ALL-PATHs (18) assembly of short insert and mate-pair reads from a single partially- inbred laboratory-reared adult female, whose lineage was first collected 25 years ago from a now extinct population in Yokohama, Japan (Supp. Text 2.5). A single Ignelater luminosus adult male, collected in Mayagüez Puerto Rico, USA, was used to produce a high-coverage Supernova (19) linked-read draft genome (Supp. Text 3.5), which was further scaffolded using low-coverage long-read Oxford Nanopore MinION sequencing (Supp. Text 3.5.5). The gene completeness and contiguity statistics of our P. pyralis (Ppyr1.3) and A. lateralis (Alat1.3) genome assemblies are comparable to the genome of the model 4 bioRxiv preprint doi: https://doi.org/10.1101/237586; this version posted February 25, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. beetle Tribolium castaneum (Fig. 2F; Supp. Text 4.1), indicating their high quality. The I. luminosus genome assembly (Ilumi1.2) is less complete, but is comparable to other published insect genomes (Fig. 2F; Supp. Text 4.1). Our coding gene annotation sets for P. pyralis, A. lateralis, and I. luminosus are comprised of 15,770, 14,285, and 27,552 genes containing 94.2%, 90.0%, and 91.8% of the Endopterygota Benchmarking Single Copy Orthologs (BUSCOs), respectively. Protein clustering via predicted orthology (20) indicated a high similarity between the P. pyralis and A. lateralis genesets, whereas the I. luminosus geneset differed (Fig. 2E; fig. S4.2.1.1). This is as expected given the phylogenetic divergence of these species. Remaining redundancy in the P. pyralis assembly, as indicated by duplicates of the BUSCOs (21) and the assembly size (Fig 2F; Supp. Text 4.1) is likely due to the heterozygosity of the outbred input libraries (Supp. Text 1). To characterize long-range evolutionary events in the P. pyralis genome, we super-scaffolded the assembly into 11 pseudo-chromosomal linkage groups using a Hi- C proximity-ligation linkage approach (Fig. 2A; Supp. Text 1.5.3). These linkage groups contain 95% of the assembly (448.8 Mbp). Linkage group LG3a corresponds to the X- chromosome based on read coverage and gene content (Supp. Text 1.6.3) and its size (22.2 Mbp) is comparable