Multiple Origins of Bioluminescence in Beetles and Evolution of Luciferase Function.

"data availability all data on the 6 beetles were submitted to ncbi (bioproject: prjna884806).; illumina short reads nanopore reads and rna reads were submitted to sra with accession numbers: srr22077043 to srr22077061 and srr12206127 (bioproject: prjna645732).; genome assemblies are available at ncbi with accession numbers: jarfj[a|b|w|x|y|z]000000000 and in the science data bank ( https://cstr cn/31253 11 sciencedb 07158 ). using nanopore long-read and illumina short-read sequencing technologies we generated high-quality genome assemblies for 6 beetle species covering luminous and nonluminous lineages within elateroidea ( table 1 ; fig 1 ; supplementary data s1 and s2 supplementary material online).; table 1 genome assembly and quality estimation of elateroidea beetles family species genome assembly genome annotation assembly size (mb) scaffold n50 (mb) complete busco ratio (%) illumina reads mapping ratio (%) rna-seq reads mapping ratio (%) repeat (% of genome) gene number complete busco ratio (%) lycidae platerodrilus igneus2595 1 5 5 94 7 95 7 91 4 72 2 25393 91 8 cantharidae lycocerus yunnanus375 1 2 7 94 9 97 3 87 4 51 4 18811 90 4 elateridae sinelater perroti 1184 7 16 7 97 8 97 9 89 7 49 6 16302 93 1 sinopyrophoridae sinopyrophorus schimmeli 275 9 2 5 96 5 98 7 93 0 51 9 12233 94 8 rhagophthalmidae menghuoius giganteus262 7 21 0 95 9 98 4 94 2 38 5 13046 93 2 lampyridae vesta saturnalis2054 2 2 8 96 3 98 8 87 5 58 1 22921 93 2 to clarify the relationships between luminous and nonluminous beetle lineages we reconstructed phylogenetic trees using concatenation-based and coalescent-based approaches across6 data matrices (supplementary data s1 supplementary material online) encompassing 568 single-copy orthologous genes ( supplementary fig s3 supplementary material online: data matrices1 to 3) 992/949 single-copy busco genes ( supplementary fig s4a and b supplementary material online: data matrices4 to 5) and whole-genome alignments (wgas; supplementary fig s5 supplementary material online: data matrix 6).; our data revealed that 83 expanded families ( p < 0 05) within the common ancestral branch of elateroidea (ab-elf) were enriched in bioluminescence-related enzymes or processes such as redox-related processes (bioluminescence oxidoreductase activity and monooxygenase activity) and lipid metabolism-related processes (fatty acid synthase long-chain acs and 4-coumarate-coa ligase [4cl]; fig 2b ; supplementary data s3 supplementary material online).; the 4cl family which belongs to the acs superfamily together with the luc and luciferase-like (ll) gene family (lll; ) was expanded in certain ancestral branches and internal branches/species of elateroidea (ab-elf ancestral branch of elateridae [ab-ela] ignelater luminosus [ilu] and ancestral branch of lampyridae [ab-lam]) but both expanded and contracted in internal species (lpj msp ess and sta; fig 2b ; supplementary data s3 supplementary material online).; we further identified rapidly evolving genes (regs; supplementary data s4 supplementary material online) and positively selected genes (psgs; supplementary data s5 supplementary material online) in the phylogenetic tree ( fig 2a ).; results indicated that the regs and psgs in most luminous species were enriched in processes associated with peroxisomal formation metabolism and oxidative stress tolerance ( fig 2c ; supplementary data s6 and s7 supplementary material online).; to resolve the evolutionary origins of bioluminescence in beetles we explored the evolutionary process of luc within the acs superfamily ( supplementary data s8 and s9 supplementary material online) given its important role in d-luciferin biosynthesis and light emission ( ) and the expansion and contraction of 4cl within elateroidea ( fig 2a and b ; supplementary data s3 supplementary material online).; phylogenetic analyses revealed a close relationship between the lll and 4cl families ( supplementary figs s12 and s13 and data s8 and s9 supplementary material online).; furthermore combining genome-wide identified luc and ll genes ( supplementary data s8 and s9 supplementary material online) with previously reported cloned luc genes in luminous beetles ( supplementary data s10 supplementary material online) we conducted phylogenetic analyses.; based on synteny analyses ( fig 3b ; supplementary fig s16 and data s11 and s12 supplementary material online) 2 copies of firefly luc genes (lampyridae: luc1 and luc2 ) exhibited a clear collinearity relationship with luc1 displaying orthology to starluc in rhagophthalmidae.; we verified the properties of the ancestral and extant lucs and their close ll proteins including luminescence intensity l-luciferin to d-luciferin transformation ability acs activity and bioluminescent spectra ( fig 3a and c ; supplementary figs s18 and s19 and data s10 supplementary material online).; regarding bioluminescent color ( fig 3a ; supplementary fig s19 and data s10 supplementary material online) spectral experiments showed that anclampherhaluc emitted orange light ( ?; results revealed distinct amino acid patterns at the lbss ("346 to 348") of lucs in different families (luminous patterns: lampyridae and elateridae: tsa; rhagophthalmidae/phengodidae: cca/csa; sinopyrophoridae: lva) and nonluminous ll genes (nonluminous pattern: tla; fig 4a ; supplementary data s10 supplementary material online).; the lbss in the reconstructed ancestral lucs were consistent with those of the corresponding extant lucs ( supplementary data s10 supplementary material online).; in this study we conducted site-directed mutagenesis for 2 extant lucs representing 2 luminous patterns ( lyuluc1 : t(s348l)a and starluc : (c343t)(c344l)a; supplementary data s10 supplementary material online).; notably through the integration of phylogeny divergence time ( fig 3a ; supplementary figs s14 and s15 and data s8 and s9 supplementary material online) genomic synteny ( fig 3b ; supplementary fig s16 and data s11 to s12 supplementary material online) and biochemical functional analyses ( fig 3c ; supplementary data s10 supplementary material online) of beetle lucs our study provides compelling evidence for at least 3 parallel origins of bioluminescence i e.; in this study we expanded our investigation beyond luc to explore the genomic underpinnings of key enzymes related to luciferin conducted within an enlarged phylogenetic framework that incorporated the genomes of crucial beetle taxa ( supplementary note s1 and figs s21 to s29 and data s13 and s14 supplementary material online).; results showed that these enzymes (i e phenoloxidase [po] atp-binding cassette protein d [abc-d] peroxisomal membrane protein 2 [pxmp2] sterol carrier protein x [scpx] luciferin-regenerating enzyme [lre] acyl-coa thioesterase [acot] sulfotransferase [st] and sulfatase [sulf]) exhibited varying copy numbers between luminescent and nonluminescent species ( supplementary data s13 and s14 supplementary material online).; we verified the pivotal role of lbss in the evolution of luc function ( fig 4 ; supplementary data s10 supplementary material online).; however ateacs6 which belonged to the same subclade as alaacs1 and lyuacs1 ( fig 3a ) emitted faint light ( fig 3c ; supplementary data s10 supplementary material online) suggesting that the ancestor of lamll and anclampherhaluc may have been bioluminescent while alaacs1 and lyuacs1 may have lost their luminescence activities.; max = 538 to 583 nm; fig 3a ; supplementary fig s19 and data s10 supplementary material online).; this diversity leads to phenotypic differentiation including bioluminescent color variation among luminous interfamily and intrafamily and even within individuals ( fig 3a ; supplementary fig s19 and data s10 supplementary material online).; six beetle species covering 6 families of elateroidea were collected from southwest china ( supplementary fig s1 and data s1 supplementary material online).; for homology-based predictions we aligned protein data sets of 5 beetles ( supplementary data s1 supplementary material online) to the repeats soft-masked genome by tblastn ( ) with an e-value <1e-5.; we generated 6 data matrices ( supplementary data s1 supplementary material online) to investigate the phylogenetic relationship among the luminous beetle lineage.; to detect the evolutionary features of the luminous beetle lineages we used 10 intra-elateroidea and 6 extra-elateroidea for comparative genomic analysis ( supplementary data s1 supplementary material online).; we also combined the lll clade 4cl clade cloned beetle luc genes and other cloned luc paralogs ( supplementary data s10 supplementary material online) to further construct the phylogenetic tree.; although posterior probabilities of several sites were low those of most sites including lbss of luc (sites346 to 348) were extremely stable ( supplementary data s15 supplementary material online). data availability all data on the 6 beetles were submitted to ncbi (bioproject: prjna884806"

"We are grateful to Wen Wang for support during this work. We thank SynBioLab for help in protein 3D structure prediction. We thank Kun Wang for his help in phylogenetic analyses. We thank Libin Wu for his help in hand-drawing the images. We sincerely thank Hao Xu for helping with the sample collection. We thank Jing-Ke Weng for his support in using the figures of luminous species. This work was supported by grants from the Yunnan Provincial Science and Technology Department (202101AS070054 and 202105AC160039) and the Chinese Academy of Sciences (Light of West China., XDPB17)."