Author response: Firefly genomes illuminate parallel origins of bioluminescence in beetles

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5 Appendix 6 Data availability References Decision letter Author response Article and author information Metrics Abstract Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles 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. To compare bioluminescent origins, 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 hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle. https://doi.org/10.7554/eLife.36495.001 eLife digest Glowing fireflies dancing in the dark are one of the most enchanting sights of a warm summer night. Their light signals are 'love messages' that help the insects find a mate – yet, they also warn a potential predator that these beetles have powerful chemical defenses. The light comes from a specialized organ of the firefly where a small molecule, luciferin, is broken down by the enzyme luciferase. Fireflies are an ancient group, with the common ancestor of the two main lineages originating over 100 million years ago. But fireflies are not the only insects that produce light: certain click beetles are also bioluminescent. Fireflies and click beetles are closely related, and they both use identical luciferin and similar luciferases to create light. This would suggest that bioluminescence was already present in the common ancestor of the two families. However, the specialized organs in which the chemical reactions take place are entirely different, which would indicate that the ability to produce light arose independently in each group. Here, Fallon, Lower et al. try to resolve this discrepancy and to find out how many times bioluminescence evolved in beetles. This required using cutting-edge DNA sequencing to carefully piece together the genomes of two species of fireflies (Photinus pyralis and Aquatica lateralis) and one species of click beetle (Ignelater luminosus). The genetic analysis revealed that, in all species, the genes for luciferases were very similar to the genetic sequences around them, which code for proteins that break down fat. This indicates that the ancestral luciferase arose from one of these metabolic genes getting duplicated, and then one of the copies evolving a new role. However, the genes for luciferase were very different between the fireflies and the click beetles. Further analyses suggested that bioluminescence evolved at least twice: once in an ancestor of fireflies, and once in the ancestor of the bioluminescent click beetles. More results came from the reconstituted genomes. For example, Fallon, Lower et al. identified the genes 'turned on' in the bioluminescent organ of the fireflies. This made it possible to list genes that may be involved in creating luciferin, and enable flies to grow brightly for long periods. In addition, the genetic information yielded sequences from bacteria that likely live inside firefly cells, and which may participate in the light-making process or the production of potent chemical defenses. Better genetic knowledge of beetle bioluminescence could bring new advances for both insects and humans. It may help researchers find and design better light-emitting molecules useful to track and quantify proteins of interest in a cell. Ultimately, it would allow a detailed understanding of firefly populations around the world, which could contribute to firefly ecotourism and help to protect these glowing insects from increasing environmental threats. https://doi.org/10.7554/eLife.36495.002 Introduction Fireflies (Coleoptera: Lampyridae) represent the best-studied case of bioluminescence. The coded language of their luminous courtship displays (Figure 1A; Video 1) has been long studied for its role in mate recognition (Lloyd, 1966; Lewis and Cratsley, 2008; Stanger-Hall and Lloyd, 2015), while non-adult bioluminescence is likely a warning signal of their unpalatable chemical defenses (De Cock and Matthysen, 1999), such as the cardiotoxic lucibufagins of Photinus fireflies (Meinwald et al., 1979). The biochemical understanding of firefly luminescence: an ATP, Mg2+, and O2-dependent luciferase-mediated oxidation of the substrate luciferin (Shimomura, 2012), along with the cloning of the luciferase gene (de Wet et al., 1985; Ow et al., 1986), led to the widespread use of luciferase as a reporter with unique applications in biomedical research and industry (Fraga, 2008). 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) (Martin et al., 2017). These four closely related families (superfamily Elateroidea) have homologous luciferases and structurally identical luciferins (Shimomura, 2012), implying a single origin of beetle bioluminescence. However, as Darwin recognized in his 'Difficulties on Theory' (Darwin, 1872), the light organs amongst the luminous beetle families are clearly distinct (Figure 1B), implying independent origins. Thus, whether beetle bioluminescence is derived from a single or multiple origin(s) remains unresolved. Figure 1 Download asset Open asset Geographic and phylogenetic context of the Big Dipper firefly, Photinus pyralis. (A) P. pyralis males emitting their characteristic swooping 'J' patrol flashes over a field in Homer Lake, Illinois. Females cue in on these species-specific flash patterns and respond with their own species-specific flash (Lloyd, 1966). Photo credit: Alex Wild. Inset: male and female P. pyralis in early stages of mating. Photo credit: Terry Priest. (B) Cladogram depicting the hypothetical phylogenetic relationship between P. pyralis and related bioluminescent and non-bioluminescent taxa with Tribolium castaneum and Drosophila melanogaster as outgroups. Numbers at nodes give approximate dates of divergence in millions of years ago (mya) (Misof et al., 2014; Mckenna et al., 2015). Right: Dorsal and ventral photos of adult male specimens. Note the well-developed ventral light organs on the true abdominal segments 6 and 7 of P. pyralis and A. lateralis. In contrast, the luminescent click beetle, I. luminosus, has paired dorsal light organs at the base of its prothorax (arrowhead) and a lantern on the anterior surface of the ventral abdomen (not visible). (C) Empirical range of P. pyralis in North America, extrapolated from 541 reported sightings (Appendix 1.2). Collection sites of individuals used for genome assembly are denoted with circles and location codes. Cross hatches represent areas which likely have P. pyralis, but were not sampled. Diagonal hashes represent Ontario, Canada. https://doi.org/10.7554/eLife.36495.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg A Photinus pyralis courtship dialogue. https://doi.org/10.7554/eLife.36495.004 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', P. pyralis (Figure 1A,C) and the Japanese 'Heike-botaru' firefly Aquatica lateralis (Figure 1B). P. pyralis was used in classic studies of firefly bioluminescent biochemistry (Bitler and McElroy, 1957) and the cloning of luciferase (de Wet et al., 1985), while A. lateralis, a species with specialized aquatic larvae, is one of the few fireflies that can be reliably cultured in the laboratory (Oba et al., 2013a). These two fireflies represent the two major firefly subfamilies, Lampyrinae and Luciolinae, which diverged from a common ancestor over 100 Mya (Figure 1B) (Misof et al., 2014; Mckenna et al., 2015). To facilitate evolutionary comparisons, we also sequenced the 'Cucubano', Ignelater luminosus (Figure 1B), a Caribbean bioluminescent click beetle, and member of the 'Pyrophorus' used by Raphaël Dubois (1849-1929) to first establish the enzymatic basis of bioluminescence in the late 1800s (Dubois, 1885; Dubois, 1886). Comparative analyses of the genomes of these three species allowed us to reconstruct the origin(s) and evolution of beetle bioluminescence. Results Sequencing and assembly of firefly and click-beetle genomes Photinus pyralis adult males were collected from the Great Smoky Mountains National Park, USA (GSMNP) and Mercer Meadows New Jersey, USA (MMNJ) (Figure 1C), and sequenced using short-insert, mate-pair, Hi-C, and long-read Pacific Biosciences (PacBio) approaches (Appendix 4—table 1). These datasets were combined in a MaSuRCA (Zimin et al., 2013) hybrid genome assembly (Appendix 1.5). The Aquatica lateralis genome was derived from an ALL-PATHs (Butler et al., 2008) assembly of short insert and mate-pair reads from a single adult female from a laboratory-reared population, whose lineage, dubbed 'Ikeya-Y90', was first collected 25 years ago from a now extinct population in Yokohama, Japan (Appendix 2.5). A single Ignelater luminosus adult male, collected in Mayagüez Puerto Rico, USA, was used to produce a high-coverage Supernova (Weisenfeld et al., 2017) linked-read draft genome (Appendix 3.5), which was further manually scaffolded using low-coverage long-read Oxford Nanopore MinION sequencing (Appendix 3.5.4). 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 beetle Tribolium castaneum (Figure 2F; Appendix 4.1). The I. luminosus genome assembly (Ilumi1.2) is less complete, but is comparable to other published insect genomes (Figure 2F; Appendix 4.1). Protein-coding genesets for our study species were produced via an EvidenceModeler-mediated combination of homology alignments, ab initio predictions, and de novo and reference-guided RNA-seq assemblies followed by manual gene curation for gene families of interest (Appendix 1.10; 2.8; 3.8). These coding gene annotation sets for P. pyralis, A. lateralis, and I. luminosus are comprised of 15,773, 14,285, and 27,557 genes containing 94.2%, 90.0%, and 91.8% of the Endopterygota Benchmarking Universal Single-Copy Orthologs (BUSCOs) (Simão et al., 2015), respectively. Protein clustering via predicted orthology indicated 77% of genes were found in orthogroups with at least one other species (Figure 2E; Appendix 4—figure 1). We found the greatest orthogroup overlap between the P. pyralis and A. lateralis genesets, as expected given the more recent phylogenetic divergence of these species. Remaining redundancy in the P. pyralis assembly and annotation, as indicated by duplicates of the BUSCOs and the assembly size (Figure 2F; Appendix 4—table 2) is likely due to the heterozygosity of the outbred input libraries (Appendix 1). The higher BUSCO completeness of the assemblies as compared to the genesets (Appendix 4—table 3), suggests that future manual curation efforts will lead to improved annotation completeness. Figure 2 Download asset Open asset Photinus pyralis genome assembly and analysis. (A) Assembled Ppyr1.3 linkage groups with annotation of the location of known luminescence-related genes, combined with Hi-C linkage density maps. Linkage group 3a (box with black arrow) corresponds to the X chromosome (Appendix 1.6.4.1). (B) Fluorescence in situ hybridization (FISH) on mitotic chromosomes of a P. pyralis larvae. The telomeric repeats TTAGG (green) localize to the ends of chromosomes stained with DAPI (blue). 20 paired chromosomes indicates that this individual was an XX female (Appendix 1.13). (C) Genome schematic of P. pyralis mitochondrial genome (mtDNA). Like other firefly mtDNAs, it has a tandem repetitive unit (TRU) (Appendix 1.8). (D) mCG is enriched across gene bodies of P. pyralis and shows methylation levels that are at least two times higher than other holometabolous insects (Appendix 1.12). (E) Orthogroup (OGs) clustering analysis of genes with Orthofinder (Emms and Kelly, 2015) shows a high degree of overlap of the P. pyralis, A. lateralis, and I. luminosus genesets with the geneset of Tribolium castaneum. Numbers within curved brackets (colored by species) represent gene count from specific species within the shared orthogroups. Numbers with square brackets (black color) represent total gene count amongst shared orthogroups. OGs = orthogroups, *=Not fully filtered to single isoform per gene. See Appendix 4.2.1 for more detail. Intermediate scripts and species-specific overlaps are available as Figure 2—source data 1. (F) Assembly statistics for presented genomes. *=Tribolium castaneum model beetle genome assembly (Tribolium Genome Sequencing Consortium et al., 2008) **=Genome size estimated by FC: flow cytometry. P. pyralis n = 5 females (SEM) I. luminosus n = 5 males (SEM), A. lateralis n = 3 technical-replicates of one female (SD). ***=Complete (C), and Duplicated (D), percentages for the Endopterygota BUSCO (Simão et al., 2015) profile (Appendix 1.4, 2.4, 3.4, 4.1). https://doi.org/10.7554/eLife.36495.005 Figure 2—source data 1 Figure 2E. Orthogroup clustering analysis. https://doi.org/10.7554/eLife.36495.006 Download elife-36495-fig2-data1-v1.zip Figure 2—source data 2 Excel file of Figure 2F table. https://doi.org/10.7554/eLife.36495.007 Download elife-36495-fig2-data2-v1.xlsx To enable the characterization of long-range genetic structure, we super-scaffolded the P. pyralis genome assembly into 11 pseudo-chromosomal linkage groups using a Hi-C proximity-ligation linkage approach (Figure 2A; Appendix 1.5.3). These linkage groups contain 95% of the assembly (448.8 Mbp). Linkage group LG3a corresponds to the X-chromosome based on expected adult XO male read coverage and gene content (Appendix 1.6.4.1) and its size (22.2 Mbp) is comparable to the expected X-chromosome size based on sex-specific genome size estimates using flow cytometry (~26 Mbp) (Lower et al., 2017). Homologs to T. castaneum X-chromosome genes were enriched on LG3a over every other linkage group, suggesting that the X-chromosomes of these distantly related beetles are homologous, and that their content has been reasonably conserved for >200 MY (Appendix 1.6.4.1) (Mckenna et al., 2015). We hypothesized that the P. pyralis orthologs of known bioluminescence genes, including the canonical luciferase Luc1 (de Wet et al., 1985) and the specialized luciferin sulfotransferase LST (Fallon et al., 2016), would be located on the same linkage group to facilitate chromosomal looping and enhancer assisted co-expression within the light organ. We, however, found these genes on separate linkage groups (Figure 2A). In addition to nuclear genome assembly and coding gene annotation, we also assembled the complete mitochondrial genomes (mtDNA) of P. pyralis (Figure 2C; Appendix 1.8) and I. luminosus (Appendix 3.10), while the mtDNA sequence of A. lateralis was recently published (Maeda et al., 2017). These mtDNA assemblies show high conservation of gene content and synteny, with the exception of the variable ~1 Kbp tandem repeat unit (TRU) found in the firefly mtDNAs. As repetitive elements are common participants and drivers of genome evolution (Feschotte and Pritham, 2007), we next sought to characterize the repeat content of our genome assemblies. Overall, 42.6%, 19.8%, and 34.1% of the P. pyralis, A. lateralis, and I. luminosus assemblies were found to be repetitive, respectively (Appendix 1.11; 2.9; 3.9). Of these repeats 66.7%, 39.4%, and 55% could not be classified as any known repetitive sequence, respectively. Helitrons, DNA transposons that transpose through rolling circle replication (Kapitonov and Jurka, 2001), are among the most abundant individual repeat elements in the P. pyralis assembly. Via in situ hybridization, we identified that P. pyralis chromosomes have canonical telomeres with telomeric repeats (TTAGG) (Figure 2B; Appendix 1.13). DNA methylation is common in eukaryotes, but varies in degree across insects, especially within Coleoptera (Bewick et al., 2017). Furthermore, the functions of DNA methylation across insects remain obscure (Bewick et al., 2017; Glastad et al., 2017). To examine firefly cytosine methylation, we characterized the methylation status of P. pyralis DNA with whole genome bisulfite sequencing (WGBS). Methylation at CpGs (mCG) was unambiguously detected at ~20% within the genic regions of P. pyralis and its methylation levels were at least twice those reported from other holometabolous insects (Figure 2D; Appendix 1.12). Molecular evolution analyses of the DNA methyltransferases (DNMTs) show that direct orthologs of both DNMT1 and DNMT3 were conserved in P. pyralis, A. lateralis, and I. luminosus (Appendix 4—figure 2; Appendix 4.2.3), implying that our three study species, and inferentially likely most firefly lineages, possess mCG. Corroborating this claim, CpG[O/E] analysis of methylation indicated our three study species had DNA methylation (Appendix 4—figure 3). The genomic context of firefly luciferase evolution Two luciferase paralogs have been previously described in fireflies (Oba et al., 2013a; Bessho-Uehara et al., 2017). P. pyralis Luc1 was the first firefly luciferase cloned (de Wet et al., 1985), and its direct orthologs have been widely identified from other fireflies (Oba, 2014). The luciferase paralog Luc2 was previously known only from a handful of Asian taxa, including A. lateralis (Oba et al., 2013a; Bessho-Uehara et al., 2017). Previous investigations of these Asian taxa have shown that Luc1 is responsible for light production from the lanterns of adults, larvae, prepupae and pupae, whereas Luc2 is responsible for the dim glow of eggs, ovaries, prepupae and the whole pupal body (Bessho-Uehara et al., 2017). From our curated genesets (Appendix 1.10; 2.8), we unequivocally identified two firefly luciferases, Luc1 and Luc2, in both the P. pyralis and A. lateralis genomes. Our RNA-Seq data further show that in both P. pyralis and A. lateralis, Luc1 and Luc2 display expression patterns consistent with previous reports. While Luc1 is the sole luciferase expressed in the lanterns of both larvae and adults, regardless of sex, Luc2 is expressed in other tissues and stages, such as eggs (Figure 3C). Notably, Luc2 expression is detected in RNA libraries derived from adult female bodies (without head or lantern), suggesting detection of ovary expression as described in previous studies (Bessho-Uehara et al., 2017). Together, these results support that since their divergence via gene duplication prior to the divergence of Lampyrinae and Luciolinae, Luc1 and Luc2 have established different, but conserved roles in bioluminescence throughout the firefly life cycle. Figure 3 Download asset Open asset A genomic view of luciferase evolution. (A) The reaction of firefly luciferase is related to that of (B) for genomic evolution of firefly from genome of luciferase in fireflies to genomic in ancestral species all genes within the same are (C) of luciferase circles gene indicate the of signal 1 indicate the per million of whole body in each to and in the prothorax or abdominal lantern to to and annotation using and and abdominal lantern expression for I. luminosus are from whole prothorax and the two most anterior abdominal = firefly luciferases, = luciferases, = (Appendix gene annotation, and expression are available as Figure data 1. (D) analysis of beetle luciferase of the A. lateralis genes closely on while 4 of the P. pyralis genes are of on with a further genes and on the same the Luc1 in P. pyralis and A. lateralis are derived from a common the of the most closely related genes have diverged between the two species. was on a separate from its most most closely related on three more distantly related genes are with In contrast, a different shows orthology to the firefly Luc1 The was produced by a manual of two (Appendix 3.5.4). with a are indicated by a dark for the genes with which represent genes an identified in the other genes are in the same in the phylogenetic and are with in and genomic regions are to = 25 from the are shown with and are with their in square ends are shown with black = = = Figure produced with et al., Figure production scripts available as Figure data Figure data 1 gene annotation, and expression for Figure Download Figure data 2 scripts for Figure Download luciferase is hypothesized to be derived from an ancestral (Figure (Oba et al., et al., We found that, in both firefly species, Luc1 is with its closely related including and which can be by the of a We also found genes (Figure that are between both species, phylogenetic analysis of the luciferases, and genes indicates that Luc1 and Luc2 two and that the and genes Luc1 three major (Figure whose common ancestor and most are and and whose common and most are Luc1 and Luc2 are conserved at the of gene are of with conserved (Appendix 4—figure Appendix 4—figure and most of and also have The and orthology of the and genes to the Luc1 remains likely due to gene divergence and (Figure Luc2 is located on a different from Luc1 in P. pyralis and on a different from Luc1 in A. lateralis, consistent with the that Luc1 and Luc2 on different chromosomes in both firefly species. or genes were found in the of Luc2 in species. These data support that tandem gene duplication in a firefly ancestor to ancestral one of which in place to the ancestral luciferase (Figure to the divergence of the firefly Lampyrinae and around 100 Mya (Appendix this duplicated, via a long-range gene duplication and then in its expression to give to Luc2, while the in place to give to Luc1 (Figure From the shared gene clustering in both fireflies, we the of the duplication one or more genes one or more genes and one or more genes (Figure of firefly and click beetle luciferase To resolve the of of luciferase and between fireflies and click beetles, we first identified the luciferase of I. luminosus luciferase and compared its genomic context to the luciferases of P. pyralis and A. lateralis (Figure other described bioluminescent which have separate luciferases expressed in the dorsal prothorax and ventral abdominal lanterns (Oba et al., we identified only a single luciferase in the I. luminosus genome which was expressed in both of the lanterns (Figure Appendix 3.8). The and of are identical to those of firefly luciferases, but the firefly luciferases which have short less than has two long (Appendix 4—figure We found genes in the I. luminosus genome which were related to and a specific to the (Figure on a Kbp containing other genes, including three related genes Figure Figure however, the genes that are most closely related to are found on a separate Kbp Figure We that the is not to the firefly Luc1 as is not with or or genes (Figure We identified a different in I. luminosus that is likely to the firefly Luc1 Figure This is based on the of A and and genes, as as and genes, both of which were found to the A. lateralis Luc1 but not the P. pyralis Luc1 (Figure the most member of was also found on (Figure This is consistent with an of duplication of the ancestor to a Overall, these genomic are consistent with independent of firefly and click beetle Figure 4 Download asset Open asset evolution of and firefly luciferase. (A) at least two gains of luciferase in bioluminescent beetles. luciferase luciferase was on firefly luciferase via or via direct The ancestral of luciferase within the ancestral nodes were then with an and a Appendix Two gains of luciferase with black and are These hypothesized gains once in a gene within the common ancestor of fireflies, and beetles, and once in a gene within the common ancestor of bioluminescent beetles. is per Numbers to nodes and available as Figure data 1 (B) Molecular analysis independent of click beetle luciferase. We the of luciferase using the for et al., 2015) (Appendix The to the

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