Nature has devised many ways of producing males and females. Here, we report on a previously undescribed mechanism for Lepidoptera that functions without a female-specific gene. The number of alleles or allele heterozygosity in a single Z-linked gene (BaMasc) is the primary sex-determining switch in Bicyclus anynana butterflies. Embryos carrying a single BaMasc allele develop into WZ (or Z0) females, those carrying two distinct alleles develop into ZZ males, while (ZZ) homozygotes initiate female development, have mismatched dosage compensation, and die as embryos. Consequently, selection against homozygotes has favored the evolution of spectacular allelic diversity: 205 different coding sequences of BaMasc were detected in a sample of 246 females. The structural similarity of a hypervariable region (HVR) in BaMasc to the HVR in Apis mellifera csd suggests molecular convergence between deeply diverged insect lineages. Our discovery of this primary switch highlights the fascinating diversity of sex-determining mechanisms and underlying evolutionary drivers.

Biology Centre of the Czech Academy of Sciences Institute of Entomology 370 05 České Budějovice Czech Republic

Department of Biological Sciences National University of Singapore Singapore 117543 Singapore

Department of Evolution Ecology and Behaviour University of Liverpool Liverpool L69 7ZB UK

Department of Zoology University of Cambridge Cambridge CB2 3EJ UK

Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Netherlands eScience Center Science Park 402 1098 XH Amsterdam Netherlands

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Bachtrog D., Mank J. E., Peichel C. L., Kirkpatrick M., Otto S. P., Ashman T.-L., Hahn M. W., Kitano J., Mayrose I., Ming R., Perrin N., Ross L., Valenzuela N., Vamosi J. C.; Tree of Sex Consortium , Sex determination: Why so many ways of doing it? PLOS Biol. 12, e1001899 (2014). PubMed PMC

L. W. Beukeboom, N. Perrin, The Evolution of Sex Determination (Oxford University Press, 2014).

Cline T. W., The Drosophila sex determination signal: How do flies count to two? Trends Genet. 9, 385–390 (1993). PubMed

Beye M., Hasselmann M., Fondrk M. K., Page R. E., Omholt S. W., The gene csd is the primary signal for sexual development in the honeybee and encodes an SR-type protein. Cell 114, 419–429 (2003). PubMed

Kiuchi T., Koga H., Kawamoto M., Shoji K., Sakai H., Arai Y., Ishihara G., Kawaoka S., Sugano S., Shimada T., Suzuki Y., Suzuki M. G., Katsuma S., A single female-specific piRNA is the primary determiner of sex in the silkworm. Nature 509, 633–636 (2014). PubMed

Hall A. B., Basu S., Jiang X., Qi Y., Timoshevskiy V. A., Biedler J. K., Sharakhova M. V., Elahi R., Anderson M. A. E., Chen X.-G., Sharakhov I. V., Adelman Z. N., Tu Z., A male-determining factor in the mosquito Aedes aegypti. Science 348, 1268–1270 (2015). PubMed PMC

Sharma A., Heinze S. D., Wu Y., Kohlbrenner T., Morilla I., Brunner C., Wimmer E. A., van de Zande L., Robinson M. D., Beukeboom L. W., Bopp D., Male sex in houseflies is determined by Mdmd, a paralog of the generic splice factor gene CWC22. Science 356, 642–645 (2017). PubMed

Meccariello A., Salvemini M., Primo P., Hall B., Koskinioti P., Dalíková M., Gravina A., Gucciardino M. A., Forlenza F., Gregoriou M. E., Ippolito D., Monti S. M., Petrella V., Perrotta M. M., Schmeing S., Ruggiero A., Scolari F., Giordano E., Tsoumani K. T., Marec F., Windbichler N., Arunkumar K. P., Bourtzis K., Mathiopoulos K. D., Ragoussis J., Vitagliano L., Tu Z., Papathanos P. A., Robinson M. D., Saccone G., Maleness-on-the-Y (MoY) orchestrates male sex determination in major agricultural fruit fly pests. Science 365, 1457–1460 (2019). PubMed

Zou Y., Geuverink E., Beukeboom L. W., Verhulst E. C., van de Zande L., A chimeric gene paternally instructs female sex determination in the haplodiploid wasp Nasonia. Science 370, 1115–1118 (2020). PubMed

Traut W., Sahara K., Marec F., Sex chromosomes and sex determination in Lepidoptera. Sex. Dev. 1, 332–346 (2007). PubMed

Saccheri I. J., Whiteford S., Yung C. J., Van't Hof A. E., Recessive Z-linked lethals and the retention of haplotype diversity in a captive butterfly population. Heredity 125, 28–39 (2020). PubMed PMC

Jiggins F. M., Hurst G. D. D., Schulenburg J. H. G. V. D., Majerus M. E. N., Two male-killing Wolbachia strains coexist within a population of the butterfly Acraea encedon. Heredity 86, 161–166 (2001). PubMed

Charlat S., Hornett E. A., Dyson E. A., Ho P. P., Loc N. T., Schilthuizen M., Davies N., Roderick G. K., Hurst G. D., Prevalence and penetrance variation of male-killing Wolbachia across Indo-Pacific populations of the butterfly Hypolimnas bolina. Mol. Ecol. 14, 3525–3530 (2005). PubMed

Sugimoto T. N., Kayukawa T., Shinoda T., Ishikawa Y., Tsuchida T., Misdirection of dosage compensation underlies bidirectional sex-specific death in Wolbachia-infected Ostrinia scapulalis. Insect Biochem. Mol. Biol. 66, 72–76 (2015). PubMed

Fukui T., Kawamoto M., Shoji K., Kiuchi T., Sugano S., Shimada T., Suzuki Y., Katsuma S., The endosymbiotic bacterium Wolbachia selectively kills male hosts by targeting the masculinizing gene. PLOS Pathog. 11, e1005048 (2015). PubMed PMC

Verhulst E. C., Van de Zande L., Double nexus — Doublesex is the connecting element in sex determination. Brief. Funct. Genomics 14, 396–406 (2015). PubMed PMC

Fukui T., Kiuchi T., Shoji K., Kawamoto M., Shimada T., Katsuma S., In vivo masculinizing function of the Ostrinia furnacalis Masculinizer gene. Biochem. Biophys. Res. Commun. 503, 1768–1772 (2018). PubMed

Wang X.-Y., Zheng Z.-Z., Song H.-S., Xu Y.-Z., Conserved RNA cis-elements regulate alternative splicing of Lepidopteran doublesex. Insect Biochem. Mol. Biol. 44, 1–11 (2014). PubMed

Suzuki M. G., Ohbayashi F., Mita K., Shimada T., The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori. Insect Biochem. Mol. Biol. 31, 1201–1211 (2001). PubMed

Gu L., Reilly P. F., Lewis J. J., Reed R. D., Andolfatto P., Walters J. R., Dichotomy of dosage compensation along the neo Z chromosome of the monarch butterfly. Curr. Biol. 29, 4071–4077.e3 (2019). PubMed PMC

Katsuma S., Kiuchi T., Kawamoto M., Fujimoto T., Sahara K., Unique sex determination system in the silkworm, Bombyx mori: Current status and beyond. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 94, 205–216 (2018). PubMed PMC

Pospíšilová K., Van't Hof A. E., Yoshido A., Kružíková R., Visser S., Zrzavá M., Bobryshava K., Dalíková M., Marec F., Masculinizer gene controls male sex determination in the codling moth, Cydia pomonella. Insect Biochem. Mol. Biol. 160, 103991 (2023). PubMed

Sainudiin R., Durrett R. T., Aquadro C. F., Nielsen R., Microsatellite mutation models: Insights from a comparison of humans and chimpanzees. Genetics 168, 383–395 (2004). PubMed PMC

Katsuma S., Sugano Y., Kiuchi T., Shimada T., Two conserved cysteine residues are required for the masculinizing activity of the silkworm Masc protein. J. Biol. Chem. 290, 26114–26124 (2015). PubMed PMC

Kiuchi T., Sugano Y., Shimada T., Katsuma S., Two CCCH-type zinc finger domains in the Masc protein are dispensable for masculinization and dosage compensation in Bombyx mori. Insect Biochem. Mol. Biol. 104, 30–38 (2019). PubMed

Sugano Y., Kokusho R., Ueda M., Fujimoto M., Tsutsumi N., Shimada T., Kiuchi T., Katsuma S., Identification of a bipartite nuclear localization signal in the silkworm Masc protein. FEBS Lett. 590, 2256–2261 (2016). PubMed

Chiu C. H., Wang Y. T., Walther B. A., Chao A., An improved nonparametric lower bound of species richness via a modified Good-Turing frequency formula. Biometrics 70, 671–682 (2014). PubMed

Hasselmann M., Beye M., Signatures of selection among sex-determining alleles of the honey bee. Proc. Natl. Acad. Sci. U.S.A. 101, 4888–4893 (2004). PubMed PMC

Muirhead C. A., Consequences of population structure on genes under balancing selection. Evolution 55, 1532–1541 (2001). PubMed

Schierup M. H., Vekemans X., Charlesworth D., The effect of subdivision on variation at multi-allelic loci under balancing selection. Genet. Res. 76, 51–62 (2000). PubMed

de Jong M. A., Wahlberg N., van Eijk M., Brakefield P. M., Zwaan B. J., Mitochondrial DNA signature for range-wide populations of Bicyclus anynana suggests a rapid expansion from recent refugia. PLOS ONE 6, e21385 (2011). PubMed PMC

Zareba J., Blazej P., Laszkiewicz A., Sniezewski L., Majkowski M., Janik S., Cebrat M., Uneven distribution of complementary sex determiner (csd) alleles in Apis mellifera population. Sci. Rep. 7, 2317 (2017). PubMed PMC

Ding G., Hasselmann M., Huang J., Roberts J., Oldroyd B. P., Gloag R., Global allele polymorphism indicates a high rate of allele genesis at a locus under balancing selection. Heredity 126, 163–177 (2021). PubMed PMC

Beye M., Seelmann C., Gempe T., Hasselmann M., Vekemans X., Fondrk M. K., Page R. E. Jr., Gradual molecular evolution of a sex determination switch through incomplete penetrance of femaleness. Curr. Biol. 23, 2559–2564 (2013). PubMed

Van't Hof A. E., Marec F., Saccheri I. J., Brakefield P. M., Zwaan B. J., Cytogenetic characterization and AFLP-based genetic linkage mapping for the butterfly Bicyclus anynana, covering all 28 karyotyped chromosomes. PLOS ONE 3, e3882 (2008). PubMed PMC

Nowell R. W., Elsworth B., Oostra V., Zwaan B. J., Wheat C. W., Saastamoinen M., Saccheri I. J., Van't Hof A. E., Wasik B. R., Connahs H., Aslam M. L., Kumar S., Challis R. J., Monteiro A., Brakefield P. M., Blaxter M., A high-coverage draft genome of the mycalesine butterfly Bicyclus anynana. Gigascience 6, 1–7 (2017). PubMed PMC

Otte M., Netschitailo O., Weidtkamp-Peters S., Seidel C. A. M., Beye M., Recognition of polymorphic Csd proteins determines sex in the honeybee. Sci. Adv. 9, eadg4239 (2023). PubMed PMC

Leducq J. B., Llaurens V., Castric V., Saumitou-Laprade P., Hardy O. J., Vekemans X., Effect of balancing selection on spatial genetic structure within populations: Theoretical investigations on the self-incompatibility locus and empirical studies in Arabidopsis halleri. Heredity 106, 319–329 (2011). PubMed PMC

Hasselmann M., Vekemans X., Pflugfelder J., Koeniger N., Koeniger G., Tingek S., Beye M., Evidence for convergent nucleotide evolution and high allelic turnover rates at the complementary sex determiner gene of Western and Asian honeybees. Mol. Biol. Evol. 25, 696–708 (2008). PubMed

Charlesworth D., Balancing selection and its effects on sequences in nearby genome regions. PLOS Genet. 2, e64 (2006). PubMed PMC

Duplouy A., Brattström O., Wolbachia in the genus Bicyclus: A forgotten player. Microb. Ecol. 75, 255–263 (2018). PubMed PMC

Dyson E. A., Kamath M. K., Hurst G. D. D., Wolbachia infection associated with all-female broods in Hypolimnas bolina (Lepidoptera: Nymphalidae): Evidence for horizontal transmission of a butterfly male killer. Heredity 88, 166–171 (2002). PubMed

Sugimoto T. N., Ishikawa Y., A male-killing Wolbachia carries a feminizing factor and is associated with degradation of the sex-determining system of its host. Biol. Lett. 8, 412–415 (2012). PubMed PMC

Kageyama D., Ohno M., Sasaki T., Yoshido A., Konagaya T., Jouraku A., Kuwazaki S., Kanamori H., Katayose Y., Narita S., Miyata M., Riegler M., Sahara K., Feminizing Wolbachia endosymbiont disrupts maternal sex chromosome inheritance in a butterfly species. Evol. Lett. 1, 232–244 (2017). PubMed PMC

Katsuma S., Hirota K., Matsuda-Imai N., Fukui T., Muro T., Nishino K., Kosako H., Shoji K., Takanashi H., Fujii T., Arimura S. I., Kiuchi T., A Wolbachia factor for male killing in lepidopteran insects. Nat. Commun. 13, 6764 (2022). PubMed PMC

Hornett E. A., Moran B., Reynolds L. A., Charlat S., Tazzyman S., Wedell N., Jiggins C. D., Hurst G. D. D., The evolution of sex ratio distorter suppression affects a 25 cM genomic region in the butterfly Hypolimnas bolina. PLOS Genet. 10, e1004822 (2014). PubMed PMC

Hornett E. A., Kageyama D., Hurst G. D. D., Sex determination systems as the interface between male-killing bacteria and their hosts. Proc. R. Soc. B 289, 20212781 (2022). PubMed PMC

Hedrick P. W., What is the evidence for heterozygote advantage selection? Trends Ecol. Evol. 27, 698–704 (2012). PubMed

Prakash A., Monteiro A., Doublesex mediates the development of sex-specific pheromone organs in Bicyclus butterflies via multiple mechanisms. Mol. Biol. Evol. 37, 1694–1707 (2020). PubMed PMC

Frey U. H., Bachmann H. S., Peters J., Siffert W., PCR-amplification of GC-rich regions: 'Slowdown PCR'. Nat. Protoc. 3, 1312–1317 (2008). PubMed

Chen S., Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta 2, e107 (2023). PubMed PMC

Nei M., Gojobori T., Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3, 418–426 (1986). PubMed

Tamura K., Stecher G., Kumar S., MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 38, 3022–3027 (2021). PubMed PMC

A. Chao, C.-H. Chiu, "Species Richness: Estimation and Comparison" in Wiley StatsRef: Statistics Reference Online, pp. 1–26.

A. Chao, K. H. Ma, T. C. Hsieh, C. H. Chiu, SpadeR (Species-richness Prediction And Diversity Estimation in R): An R package in CRAN. (2016).

R Core Team. (R Foundation for Statistical Computing, Vienna, Austria, 2022).

Ranwez V., Harispe S., Delsuc F., Douzery E. J. P., MACSE: Multiple Alignment of Coding SEquences accounting for frameshifts and stop codons. PLOS ONE 6, e22594 (2011). PubMed PMC

Lücking R., Hodkinson B. P., Stamatakis A., Cartwright R. A., PICS-Ord: Unlimited coding of ambiguous regions by pairwise identity and cost scores ordination. BMC Bioinform. 12, 10 (2011). PubMed PMC

Cartwright R. A., Ngila: Global pairwise alignments with logarithmic and affine gap costs. Bioinformatics 23, 1427–1428 (2007). PubMed PMC

Kozlov A. M., Darriba D., Flouri T., Morel B., Stamatakis A., RAxML-NG: A fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics 35, 4453–4455 (2019). PubMed PMC

Kamvar Z. N., Tabima J. F., Grünwald N. J., Poppr: An R package for genetic analysis of populations with clonal, partially clonal, and/or sexual reproduction. PeerJ 2, e281 (2014). PubMed PMC

Patro R., Duggal G., Love M. I., Irizarry R. A., Kingsford C., Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017). PubMed PMC

Pertea M., Pertea G. M., Antonescu C. M., Chang T. C., Mendell J. T., Salzberg S. L., StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 33, 290–295 (2015). PubMed PMC

Harrison P. W., Mank J. E., Wedell N., Incomplete sex chromosome dosage compensation in the Indian meal moth, Plodia interpunctella, based on de novo transcriptome assembly. Genome Biol. Evol. 4, 1118–1126 (2012). PubMed PMC

Love M. I., Huber W., Anders S., Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). PubMed PMC

Soneson C., Love M. I., Robinson M. D., Differential analyses for RNA-seq: Transcript-level estimates improve gene-level inferences. F1000Res 4, 1521 (2015). PubMed PMC

R. J. Challis, S. Kumar, K. K. Dasmahapatra, C. D. Jiggins, M. Blaxter, Lepbase: The lepidopteran genome database. bioRxiv 10.1101/056994 (2016).

Priyam A., Woodcroft B. J., Rai V., Moghul I., Munagala A., Ter F., Chowdhary H., Pieniak I., Maynard L. J., Gibbins M. A., Moon H., Davis-Richardson A., Uludag M., Watson-Haigh N. S., Challis R., Nakamura H., Favreau E., Gómez E. A., Pluskal T., Leonard G., Rumpf W., Wurm Y., Sequenceserver: A modern graphical user interface for custom BLAST databases. Mol. Biol. Evol. 36, 2922–2924 (2019). PubMed PMC

Banerjee T. D., Monteiro A., CRISPR-Cas9 mediated genome editing in Bicyclus anynana butterflies. Methods Protoc 1, 16 (2018). PubMed PMC

Šíchová J., Nguyen P., Dalíková M., Marec F., Chromosomal evolution in tortricid moths: Conserved karyotypes with diverged features. PLOS ONE 8, e64520 (2013). PubMed PMC

Traut W., Marec F., Sex chromatin in Lepidoptera. Q. Rev. Biol. 71, 239–256 (1996). PubMed

Fuková I., Traut W., Vítková M., Nguyen P., Kubíčková S., Marec F., Probing the W chromosome of the codling moth, Cydia pomonella, with sequences from microdissected sex chromatin. Chromosoma 116, 135–145 (2007). PubMed

Zrzavá M., Hladová I., Dalíková M., Šíchová J., Õunap E., Kubíčková S., Marec F., Sex chromosomes of the iconic moth Abraxas grossulariata (Lepidoptera, Geometridae) and its congener A. sylvata. Genes 9, 279 (2018). PubMed PMC

Kubickova S., Cernohorska H., Musilova P., Rubes J., The use of laser microdissection for the preparation of chromosome-specific painting probes in farm animals. Chromosome Res. 10, 571–577 (2002). PubMed

Beldade P., Saenko S. V., Pul N., Long A. D., A gene-based linkage map for Bicyclus anynana butterflies allows for a comprehensive analysis of synteny with the lepidopteran reference genome. PLOS Genet. 5, e1000366 (2009). PubMed PMC

Traut W., Sahara K., Otto T. D., Marec F., Molecular differentiation of sex chromosomes probed by comparative genomic hybridization. Chromosoma 108, 173–180 (1999). PubMed

A. Yoshido, K. Sahara, Y. Yasukochi, "Silk moths (Lepidoptera)" in Protocols for Cytogenetic Mapping of Arthropod Genomes, I. V. Sharakhov, Ed. (CRC Press, 2014), pp. 219–256.

Nguyen P., Sýkorová M., Šíchová J., Kůta V., Dalíková M., Čapková Frydrychová R., Neven L. G., Sahara K., Marec F., Neo-sex chromosomes and adaptive potential in tortricid pests. Proc. Nat. Acad. Sci. U. S. A. 110, 6931–6936 (2013). PubMed PMC

Yoshido A., Šíchová J., Pospíšilová K., Nguyen P., Voleníková A., Šafář J., Provazník J., Vila R., Marec F., Evolution of multiple sex-chromosomes associated with dynamic genome reshuffling in Leptidea wood-white butterflies. Heredity 125, 138–154 (2020). PubMed PMC

Pfaffl M. W., A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001). PubMed PMC

Van't Hof A. E., Zwaan B. J., Saccheri I. J., Daly D., Bot A. N. M., Brakefield P. M., Characterization of 28 microsatellite loci for the butterflyBicyclus anynana. Mol. Ecol. Notes 5, 169–172 (2005).