<!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN" "JATS-archivearticle1-mathml3.dtd"> <article xmlns:ali="http://www.niso.org/schemas/ali/1.0/" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article" dtd-version="1.2"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn pub-type="epub" publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">75795</article-id><article-id pub-id-type="doi">10.7554/eLife.75795</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research Article</subject></subj-group><subj-group subj-group-type="heading"><subject>Evolutionary Biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Genetics and Genomics</subject></subj-group></article-categories><title-group><article-title>Unique structure and positive selection promote the rapid divergence of <italic>Drosophila</italic> Y chromosomes</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes" id="author-261762"><name><surname>Chang</surname><given-names>Ching-Ho</given-names></name><contrib-id authenticated="true" contrib-id-type="orcid">https://orcid.org/0000-0001-9361-1190</contrib-id><email>cchang2@fredhutch.org</email><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="other" rid="fund4"/><xref ref-type="other" rid="fund6"/><xref ref-type="other" rid="fund7"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-261763"><name><surname>Gregory</surname><given-names>Lauren E</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-106712"><name><surname>Gordon</surname><given-names>Kathleen E</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="fn" rid="pa2">‡</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-105932"><name><surname>Meiklejohn</surname><given-names>Colin D</given-names></name><contrib-id authenticated="true" contrib-id-type="orcid">https://orcid.org/0000-0003-2708-8316</contrib-id><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="other" rid="fund2"/><xref ref-type="other" rid="fund5"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-240375"><name><surname>Larracuente</surname><given-names>Amanda M</given-names></name><contrib-id authenticated="true" contrib-id-type="orcid">https://orcid.org/0000-0001-5944-5686</contrib-id><email>alarracu@UR.Rochester.edu</email><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="fund1"/><xref ref-type="other" rid="fund3"/><xref ref-type="other" rid="fund6"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><label>1</label><institution>Department of Biology, University of Rochester</institution><addr-line><named-content content-type="city">Rochester</named-content></addr-line><country>United States</country></aff><aff id="aff2"><label>2</label><institution>School of Biological Sciences, University of Nebraska-Lincoln</institution><addr-line><named-content content-type="city">Lincoln</named-content></addr-line><country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Matute</surname><given-names>Daniel R</given-names></name><role>Reviewing Editor</role><aff><institution>University of North Carolina, Chapel Hill</institution><country>United States</country></aff></contrib><contrib contrib-type="senior_editor"><name><surname>Wittkopp</surname><given-names>Patricia J</given-names></name><role>Senior Editor</role><aff><institution>University of Michigan</institution><country>United States</country></aff></contrib></contrib-group><author-notes><fn fn-type="present-address" id="pa1"><label>†</label><p>Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States</p></fn><fn fn-type="present-address" id="pa2"><label>‡</label><p>Department of Molecular Biology and Genetics, Field of Genetics, Genomics and Development, Cornell University, Ithaca, United States</p></fn></author-notes><pub-date date-type="publication" publication-format="electronic"><day>06</day><month>01</month><year>2022</year></pub-date><pub-date pub-type="collection"><year>2022</year></pub-date><volume>11</volume><elocation-id>e75795</elocation-id><history><date date-type="received" iso-8601-date="2021-11-24"><day>24</day><month>11</month><year>2021</year></date><date date-type="accepted" iso-8601-date="2021-12-18"><day>18</day><month>12</month><year>2021</year></date></history><permissions><copyright-statement>© 2022, Chang et al</copyright-statement><copyright-year>2022</copyright-year><copyright-holder>Chang et al</copyright-holder><ali:free_to_read/><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><ali:license_ref>http://creativecommons.org/licenses/by/4.0/</ali:license_ref><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife-75795-v2.pdf"/><self-uri content-type="figures-pdf" xlink:href="elife-75795-figures-v2.pdf"/><abstract><p>Y chromosomes across diverse species convergently evolve a gene-poor, heterochromatic organization enriched for duplicated genes, LTR retrotransposons, and satellite DNA. Sexual antagonism and a loss of recombination play major roles in the degeneration of young Y chromosomes. However, the processes shaping the evolution of mature, already degenerated Y chromosomes are less well-understood. Because Y chromosomes evolve rapidly, comparisons between closely related species are particularly useful. We generated de novo long-read assemblies complemented with cytological validation to reveal Y chromosome organization in three closely related species of the <italic>Drosophila simulans</italic> complex, which diverged only 250,000 years ago and share >98% sequence identity. We find these Y chromosomes are divergent in their organization and repetitive DNA composition and discover new Y-linked gene families whose evolution is driven by both positive selection and gene conversion. These Y chromosomes are also enriched for large deletions, suggesting that the repair of double-strand breaks on Y chromosomes may be biased toward microhomology-mediated end joining over canonical non-homologous end-joining. We propose that this repair mechanism contributes to the convergent evolution of Y chromosome organization across organisms.</p></abstract><kwd-group kwd-group-type="author-keywords"><kwd><italic>Drosophila y chromosome</italic></kwd><kwd>pacbio genome assembly</kwd><kwd>sexual conflict</kwd><kwd>convergent evolution</kwd><kwd>dna repair bias</kwd><kwd>ampliconic gene familiy</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>D. melanogaster</italic></kwd><kwd>Other</kwd></kwd-group><funding-group><award-group id="fund1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000057</institution-id><institution>National Institute of General Medical Sciences</institution></institution-wrap></funding-source><award-id>R35GM119515</award-id><principal-award-recipient><name><surname>Larracuente</surname><given-names>Amanda M</given-names></name></principal-award-recipient></award-group><award-group id="fund2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000057</institution-id><institution>National Institute of General Medical Sciences</institution></institution-wrap></funding-source><award-id>R01GM123194</award-id><principal-award-recipient><name><surname>Meiklejohn</surname><given-names>Colin D</given-names></name></principal-award-recipient></award-group><award-group id="fund3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000001</institution-id><institution>National Science Foundation</institution></institution-wrap></funding-source><award-id>MCB 1844693</award-id><principal-award-recipient><name><surname>Larracuente</surname><given-names>Amanda M</given-names></name></principal-award-recipient></award-group><award-group id="fund4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100001021</institution-id><institution>Damon Runyon Cancer Research Foundation</institution></institution-wrap></funding-source><award-id>DRG: 2438-21</award-id><principal-award-recipient><name><surname>Chang</surname><given-names>Ching-Ho</given-names></name></principal-award-recipient></award-group><award-group id="fund5"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100013235</institution-id><institution>College of Arts and Sciences, University of Nebraska-Lincoln</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Meiklejohn</surname><given-names>Colin D</given-names></name></principal-award-recipient></award-group><award-group id="fund6"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100008091</institution-id><institution>University of Rochester</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Larracuente</surname><given-names>Amanda M</given-names></name><name><surname>Chang</surname><given-names>Ching-Ho</given-names></name></principal-award-recipient></award-group><award-group id="fund7"><funding-source><institution-wrap><institution>Ministry of Education, Taiwan</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Chang</surname><given-names>Ching-Ho</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Differences in the usage of DNA repair pathways may give rise to the unique patterns of Y-linked mutations that, together with natural selection, shape rapid Y chromosome evolution.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Most sex chromosomes evolved from a pair of homologous gene-rich autosomes that acquired sex-determining factors and subsequently differentiated. Y chromosomes gradually lose most of their genes, while their X chromosome counterparts tend to retain the original autosomal complement of genes. This Y chromosome degeneration follows a suppression of recombination (<xref ref-type="bibr" rid="bib142">Rice, 1987a</xref>), which limits the efficacy of natural selection, and causes the accumulation of deleterious mutations through Muller’s ratchet, background selection, and hitchhiking effects (<xref ref-type="bibr" rid="bib9">Bachtrog, 2013</xref>; <xref ref-type="bibr" rid="bib39">Charlesworth, 1978</xref>; <xref ref-type="bibr" rid="bib143">Rice, 1987b</xref>; <xref ref-type="bibr" rid="bib40">Charlesworth et al., 1995</xref>; <xref ref-type="bibr" rid="bib41">Charlesworth and Charlesworth, 2000</xref>). As a consequence, many Y chromosomes present a seemingly hostile environment for genes, with their mutational burden, high repeat content, and abundant silent chromatin.</p><p>Genomic studies of Y chromosome evolution focus primarily on young sex chromosomes, addressing how the suppression of recombination promotes Y chromosome degeneration at both the epigenetic and genetic levels (<xref ref-type="bibr" rid="bib9">Bachtrog, 2013</xref>; <xref ref-type="bibr" rid="bib16">Bergero et al., 2015</xref>). Although sexually antagonistic selection is traditionally cited as the cause of recombination suppression on the Y chromosome, direct evidence for its role is still lacking (<xref ref-type="bibr" rid="bib17">Bergero et al., 2019</xref>) and new models propose that regulatory evolution is the initial trigger for recombination suppression (<xref ref-type="bibr" rid="bib99">Lenormand et al., 2020</xref>). Regardless of its role in initiating recombination suppression, on degenerating Y chromosomes, sexually antagonistic selection may accelerate Y-linked gene evolution to optimize male-specific functions. Indeed, Y-linked genes tend to have slightly higher rates of protein evolution than their orthologs on other chromosomes (<xref ref-type="bibr" rid="bib7">Bachtrog, 2003</xref>; <xref ref-type="bibr" rid="bib152">Singh et al., 2014</xref>). Higher rates of Y-linked gene evolution are driven by positive selection, relaxed selective constraints and male-biased mutation patterns, with most Y-linked genes evolving under at least some functional constraint (<xref ref-type="bibr" rid="bib152">Singh et al., 2014</xref>). Although there is evidence suggesting that some Y chromosomes have experienced recent selective sweeps (<xref ref-type="bibr" rid="bib93">Larracuente and Clark, 2013</xref>; <xref ref-type="bibr" rid="bib8">Bachtrog, 2004</xref>), the relative importance of positive selection in shaping Y chromosome evolution remains unclear.</p><p>Y chromosomes harbor extensive structural divergence between species, in part through the acquisition of genes from other genomic regions (<xref ref-type="bibr" rid="bib155">Soh et al., 2014</xref>; <xref ref-type="bibr" rid="bib146">Rozen et al., 2003</xref>; <xref ref-type="bibr" rid="bib75">Hughes and Page, 2015</xref>; <xref ref-type="bibr" rid="bib10">Bachtrog et al., 2019</xref>; <xref ref-type="bibr" rid="bib161">Tobler et al., 2017</xref>; <xref ref-type="bibr" rid="bib130">Peichel et al., 2019</xref>; <xref ref-type="bibr" rid="bib25">Brashear et al., 2018</xref>; <xref ref-type="bibr" rid="bib63">Hall et al., 2016</xref>). However, the functions of most Y-linked genes are unknown (<xref ref-type="bibr" rid="bib161">Tobler et al., 2017</xref>; <xref ref-type="bibr" rid="bib63">Hall et al., 2016</xref>; <xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>; <xref ref-type="bibr" rid="bib28">Carvalho et al., 2015</xref>). Some Y-linked genes are duplicated and, in extreme cases, amplified into so-called ampliconic genes—gene families with tens to hundreds of highly similar sequences. Y chromosomes of both <italic>Drosophila</italic> and mammals have independently acquired and amplified gene families, which turnover rapidly between closely related species (<xref ref-type="bibr" rid="bib155">Soh et al., 2014</xref>; <xref ref-type="bibr" rid="bib10">Bachtrog et al., 2019</xref>; <xref ref-type="bibr" rid="bib25">Brashear et al., 2018</xref>; <xref ref-type="bibr" rid="bib55">Ellison and Bachtrog, 2019</xref>; <xref ref-type="bibr" rid="bib74">Hughes et al., 2010</xref>; <xref ref-type="bibr" rid="bib122">Mueller et al., 2008</xref>). Following Y-linked gene amplification, gene conversion between gene copies may enhance the efficacy of selection on Y-linked genes in the absence of crossing over (<xref ref-type="bibr" rid="bib146">Rozen et al., 2003</xref>; <xref ref-type="bibr" rid="bib46">Connallon and Clark, 2010</xref>).</p><p>Detailed analyses of old Y chromosomes have been restricted to a few species with reference-quality assemblies, for example, mouse and human. The challenges of cloning and assembling repeat-rich regions of the genome have stymied progress towards a complete understanding of Y chromosome evolution (<xref ref-type="bibr" rid="bib27">Carlson and Brutlag, 1977</xref>; <xref ref-type="bibr" rid="bib103">Lohe and Brutlag, 1987a</xref>; <xref ref-type="bibr" rid="bib104">Lohe and Brutlag, 1987b</xref>). Recent advances in long-read sequencing make it feasible to assemble large parts of Y chromosomes (<xref ref-type="bibr" rid="bib130">Peichel et al., 2019</xref>; <xref ref-type="bibr" rid="bib63">Hall et al., 2016</xref>; <xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>; <xref ref-type="bibr" rid="bib109">Mahajan et al., 2018</xref>) enabling comparative studies of a majority of Y-linked sequences in closely related species.</p><p><italic>Drosophila melanogaster</italic> and three related species in the <italic>D. simulans</italic> clade are ideally suited to study Y chromosome evolution. These Y chromosomes are functionally divergent, contribute to hybrid sterility (<xref ref-type="bibr" rid="bib4">Araripe et al., 2016</xref>; <xref ref-type="bibr" rid="bib14">Bayes and Malik, 2009</xref>; <xref ref-type="bibr" rid="bib78">Johnson et al., 1992</xref>; <xref ref-type="bibr" rid="bib48">Coyne, 1985</xref>), and at least four X-linked meiotic drive systems likely shape Y chromosome evolution in these species (<xref ref-type="bibr" rid="bib23">Bozzetti et al., 1995</xref>; <xref ref-type="bibr" rid="bib47">Courret et al., 2019</xref>; <xref ref-type="bibr" rid="bib159">Tao et al., 2007</xref>; <xref ref-type="bibr" rid="bib158">Tao et al., 2001</xref>; <xref ref-type="bibr" rid="bib65">Helleu et al., 2019</xref>; <xref ref-type="bibr" rid="bib24">Branco et al., 2013</xref>; <xref ref-type="bibr" rid="bib119">Montchamp-Moreau et al., 2001</xref>; <xref ref-type="bibr" rid="bib114">Meiklejohn et al., 2018</xref>). Previous genetic and transcriptomic studies suggest that Y chromosome variation can impact male fitness and gene regulation (<xref ref-type="bibr" rid="bib140">Reijo et al., 1995</xref>; <xref ref-type="bibr" rid="bib162">Vogt et al., 1996</xref>; <xref ref-type="bibr" rid="bib157">Sun et al., 2000</xref>; <xref ref-type="bibr" rid="bib141">Repping et al., 2003</xref>; <xref ref-type="bibr" rid="bib120">Morgan and Pardo-Manuel de Villena, 2017</xref>; <xref ref-type="bibr" rid="bib98">Lemos et al., 2010</xref>; <xref ref-type="bibr" rid="bib165">Wang et al., 2018</xref>; <xref ref-type="bibr" rid="bib149">Sackton et al., 2011</xref>). Since there is minimal nucleotide variation and divergence in Y-linked protein-coding sequences within and between these <italic>Drosophila</italic> species (<xref ref-type="bibr" rid="bib152">Singh et al., 2014</xref>; <xref ref-type="bibr" rid="bib93">Larracuente and Clark, 2013</xref>; <xref ref-type="bibr" rid="bib65">Helleu et al., 2019</xref>), structural variation may be responsible for the majority of these effects. For example, 20–40% of <italic>D. melanogaster</italic> Y-linked regulatory variation (YRV) comes from differences in ribosomal DNA (rDNA) copy numbers (<xref ref-type="bibr" rid="bib169">Zhou et al., 2012</xref>). The chromatin on <italic>Drosophila</italic> Y chromosomes has genome-wide effects on expression level and chromatin states (<xref ref-type="bibr" rid="bib26">Brown and Bachtrog, 2017</xref>), but aside from the rDNA, the molecular basis of Y chromosome divergence and variation in these species remains elusive.</p><p>To study the factors and forces shaping the evolution of Y chromosome structure, we assembled the Y chromosomes of the three species in the <italic>D. simulans</italic> clade to reveal their structure and evolution and compared them to <italic>D. melanogaster</italic>. We find that the Y chromosomes of the <italic>D. simulans</italic> clade species have high duplication and gene conversion rates that, along with strong positive selection, shaped the evolution of two new ampliconic protein-coding gene families. We propose that, in addition to positive selection, sexual antagonism, and genetic conflict, differences in the usage of DNA repair pathways may give rise to the unique patterns of Y-linked mutations. Together these effects may drive the convergent evolution of Y chromosome structure across taxa.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Improving Y chromosome assemblies using long-read assembly and fluorescence in situ hybridization (FISH)</title><p>Long reads have enabled the assembly of many repetitive genome regions but have had limited success in assembling Y chromosomes (<xref ref-type="bibr" rid="bib10">Bachtrog et al., 2019</xref>; <xref ref-type="bibr" rid="bib130">Peichel et al., 2019</xref>; <xref ref-type="bibr" rid="bib63">Hall et al., 2016</xref>; <xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>). To improve Y chromosome assemblies for comparative genomic analyses, we applied our heterochromatin-sensitive assembly pipeline (<xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>) with long reads that we previously generated (<xref ref-type="bibr" rid="bib32">Chakraborty et al., 2021</xref>) to de novo reassemble the Y chromosome from the three species in the <italic>Drosophila simulans</italic> clade. We also resequenced male genomes using PCR-free Illumina libraries to polish these assemblies. Our heterochromatin-enriched methods improve contiguity compared to previous <italic>D. simulans</italic> clade assemblies. We recovered all known exons of the 11 canonical Y-linked genes conserved across the <italic>melanogaster</italic> group, including 58 exons missed in previous assemblies (<xref ref-type="supplementary-material" rid="supp1">Supplementary file 1</xref>; <xref ref-type="bibr" rid="bib60">Gepner and Hays, 1993</xref>; <xref ref-type="bibr" rid="bib19">Bernardo Carvalho et al., 2009</xref>). Based on the median male-to-female coverage (<xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>), we assigned 13.7–18.9 Mb of Y-linked sequences per species with N50 ranging from 0.6 to 1.2 Mb. The quality of these new <italic>D. simulans</italic> clade Y assemblies is comparable to <italic>D. melanogaster</italic> (<xref ref-type="table" rid="table1">Table 1</xref>; <xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>). We evaluated our methods by comparing our assignments for every 10 kb window of assembled sequence to its known chromosomal location. Our assignments have 96, 98, and 99% sensitivity and 5, 0, and 3% false-positive rates in <italic>D. mauritiana</italic>, <italic>D. simulans</italic>, and <italic>D. sechellia</italic>, respectively (<xref ref-type="supplementary-material" rid="supp2">Supplementary file 2</xref>). We have lower confidence in our <italic>D. mauritiana</italic> assignments, because the male and female Illumina reads are from different library construction methods. Therefore, we applied an additional criterion only in <italic>D. mauritiana</italic> based on the female-to-male total mapped reads ratio ( < 0.1), which reduces the false-positive rate from 13% to 5% in regions with known chromosomal location (<xref ref-type="supplementary-material" rid="supp2">Supplementary file 2</xref>; <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). We can detect potential misassemblies by looking for discordant assignments between 10 kb windows on the same contigs. Because we do not find any obviously discordant F/M ratios for any contigs, we make chromosome assignments based on median male-to-female coverage and the ratio of female-to-male total mapped reads across whole contigs. Based on these chromosome assignments, we find 40–44% lower PacBio coverage on Y than X chromosomes in all three species (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>; see Appendix 1).</p><table-wrap id="table1" position="float"><label>Table 1.</label><caption><title>Contiguity statistics for heterochromatin-enriched assemblies.</title></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" valign="bottom">Y chromosome assembly</th><th align="left" valign="bottom"># of contigs</th><th align="left" valign="bottom">Total length</th><th align="left" valign="bottom">Contigs N50</th></tr></thead><tbody><tr><td align="left" valign="bottom"><italic>D. melanogaster</italic><xref ref-type="table-fn" rid="table1fn1">*</xref></td><td align="char" char="." valign="bottom">80</td><td align="char" char="." valign="bottom">14,578,684</td><td align="char" char="." valign="bottom">416,887</td></tr><tr><td align="left" valign="bottom"><italic>D. mauritiana</italic><xref ref-type="table-fn" rid="table1fn2">†</xref></td><td align="char" char="." valign="bottom">55</td><td align="char" char="." valign="bottom">17,880,069</td><td align="char" char="." valign="bottom">1,628,994</td></tr><tr><td align="left" valign="bottom"><italic>D. simulans</italic><xref ref-type="table-fn" rid="table1fn2">†</xref></td><td align="char" char="." valign="bottom">38</td><td align="char" char="." valign="bottom">13,717,056</td><td align="char" char="." valign="bottom">1,031,383</td></tr><tr><td align="left" valign="bottom"><italic>D. sechellia</italic><xref ref-type="table-fn" rid="table1fn2">†</xref></td><td align="char" char="." valign="bottom">63</td><td align="char" char="." valign="bottom">14,899,148</td><td align="char" char="." valign="bottom">555,130</td></tr></tbody></table><table-wrap-foot><fn id="table1fn1"><label>*</label><p><xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>.</p></fn><fn id="table1fn2"><label>†</label><p>This paper.</p></fn></table-wrap-foot></table-wrap><p>The cytological organization of the <italic>D. simulans</italic> clade Y chromosomes is not well-described (<xref ref-type="bibr" rid="bib97">Lemeunier and Ashburner, 1984</xref>; <xref ref-type="bibr" rid="bib145">Roy et al., 2005</xref>; <xref ref-type="bibr" rid="bib18">Berloco et al., 2005</xref>). Therefore, we generated new physical maps of the Y chromosomes by combining our assemblies with cytological data. We performed FISH on mitotic chromosomes using probes for 12 Y-linked sequences (<xref ref-type="fig" rid="fig1">Figure 1</xref> and <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplements 3</xref>–<xref ref-type="fig" rid="fig1s4">4</xref>; <xref ref-type="supplementary-material" rid="supp3">Supplementary file 3</xref>) to determine Y chromosome organization at the cytological level. We also determined the location of the centromeres using immunostaining with a Cenp-C antibody (<xref ref-type="fig" rid="fig1s4">Figure 1—figure supplement 4</xref>; <xref ref-type="bibr" rid="bib56">Erhardt et al., 2008</xref>). These cytological data permit us to (1) validate our assemblies and (2) infer the overall organization of the Y chromosome by orienting our scaffolds on cytological maps. Of the 11 Y-linked genes, we successfully ordered 10, 11, and 7 genes on the cytological bands of <italic>D. simulans, D. mauritiana,</italic> and <italic>D. sechellia</italic>, respectively (<xref ref-type="fig" rid="fig1">Figure 1</xref> and <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3</xref>). Although we cannot examine the detailed organization as a complete contiguous Y-linked sequence, we did not find any discordance between our scaffolds and cytological data. We find evidence for extensive Y chromosomal structural rearrangements, including changes in satellite distribution, gene order, and centromere position. These rearrangements are dramatic even among the <italic>D. simulans</italic> clade species, which diverged less than 250 KYA (<xref ref-type="fig" rid="fig1">Figure 1</xref> and <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3</xref>). The Y chromosome centromere position appears to be the same as determined by Berloco et al. for different strains of <italic>D. simulans</italic> and <italic>D. mauritiana</italic>, but not for <italic>D. sechellia</italic> (<xref ref-type="bibr" rid="bib18">Berloco et al., 2005</xref>). One explanation for this discrepancy could be between-strain variation in <italic>D. sechellia</italic> Y chromosome centromere location. Together, our new physical maps and assemblies provide both large and fine-scale resolution on Y chromosome organization in the <italic>D. simulans</italic> clade.</p><fig-group><fig id="fig1" position="float"><label>Figure 1.</label><caption><title>Y chromosome organization in <italic>D. melanogaster</italic> and the three <italic>D. simulans</italic> clade species.</title><p>Schematics of the cytogenetic maps note the locations of Y-linked genes in <italic>D. melanogaster</italic> and <italic>D. simulans</italic> clade species. The bars show the relative placement of the scaffolds on the cytological bands based on FISH results. The solid black and dotted bars represent the scaffolds with known and unknown orientation information, respectively. The light blue and orange bars represent two new Y-linked gene families, <italic>Lhk</italic> and <italic>CK2ßtes-Y</italic> in the <italic>D. simulans</italic> clade, respectively. The arrows indicate the orientation of the genes (blue- minus strand; red- plus strand). Yellow circles denote centromere locations (cen). The blocks connecting genes between species highlight the structural rearrangements between species (purple for same, and green for inverted, orientation).</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig1.jpg"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><label>Figure 1—figure supplement 1.</label><caption><title>The distribution of female-to-male total mapped read ratio in each 10-kb window in <italic>D. mauritiana.</italic></title><p>Many non-Y regions have median male-to-female coverage 0 in our <italic>D. mauritiana</italic> data. Therefore, we applied an additional criterion based on the female-to-male total mapped reads ratio (<0.1) to reduce the false-positive rate.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig1-figsupp1.jpg"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><label>Figure 1—figure supplement 2.</label><caption><title>The low Pacbio coverage on Y chromosomes in the <italic>D. simulans</italic> clade.</title><p>We calculated the median coverage of Pacbio reads every 10-kb and plotted the histogram of depth across genomes based on their chromosome location.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig1-figsupp2.jpg"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><label>Figure 1—figure supplement 3.</label><caption><title>Summarized cytological location of satellite DNA, gene families, and conserved genes on the Y chromosome of the <italic>D. simulans</italic> clade.</title><p>We used FISH as well as our assemblies to infer the cytological location of Y-linked sequences. The bars represent the location of scaffolds or contigs, and the green bars are scaffolds or contigs without known direction. The satellites in red are sequences we cannot detect on Y chromosomes using FISH.</p><p>*Based on the repeat content from the Illumina data (<xref ref-type="supplementary-material" rid="supp6">Supplementary file 6</xref>), the AAACAT signal is probably from the AAACAAT tandem array, instead of AAACAT, in <italic>D. simulans</italic>.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig1-figsupp3.jpg"/></fig><fig id="fig1s4" position="float" specific-use="child-fig"><label>Figure 1—figure supplement 4.</label><caption><title>FISH for satellite and gene families, and conserved genes in the <italic>D. simulans</italic> clade.</title><p>We surveyed the location of 12 Y-linked sequences using FISH and immunostaining. The colors on the figure represent the probes we used for the experiments.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig1-figsupp4.jpg"/></fig><fig id="fig1s5" position="float" specific-use="child-fig"><label>Figure 1—figure supplement 5.</label><caption><title>The length of rDNA elements across chromosomes in <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade.</title><p>We surveyed the length of rDNA elements across chromosomes (A: autosomes, X: X chromosome, U: unknown location and Y: Y chromosome). The length of elements is normalized by the length of consensus from functional elements.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig1-figsupp5.jpg"/></fig></fig-group></sec><sec id="s2-2"><title>Y-linked sequence and copy number divergence across three species</title><p>Although the <italic>D. simulans</italic> clade species diverged only recently, Y chromosome introgression between pairs of species disrupts male fertility and influences patterns of genome-wide gene expression (<xref ref-type="bibr" rid="bib4">Araripe et al., 2016</xref>; <xref ref-type="bibr" rid="bib78">Johnson et al., 1992</xref>). One candidate locus that may contribute to functional divergence and possibly hybrid lethality is the Y-linked rDNA (<xref ref-type="bibr" rid="bib169">Zhou et al., 2012</xref>; <xref ref-type="bibr" rid="bib128">Paredes et al., 2011</xref>). Y-linked rDNA, specifically 28 S rDNA, were lost in <italic>D. simulans</italic> and <italic>D. sechellia</italic>, but not in <italic>D. mauritiana</italic> (<xref ref-type="bibr" rid="bib145">Roy et al., 2005</xref>; <xref ref-type="bibr" rid="bib106">Lohe and Roberts, 2000</xref>; <xref ref-type="bibr" rid="bib105">Lohe and Roberts, 1990</xref>). However, the intergenic spacer (IGS) repeats between rDNA genes, which are responsible for X-Y pairing in <italic>D. melanogaster</italic> males (<xref ref-type="bibr" rid="bib111">McKee and Karpen, 1990</xref>), are retained on both sex chromosomes in all three species (<xref ref-type="bibr" rid="bib145">Roy et al., 2005</xref>; <xref ref-type="bibr" rid="bib106">Lohe and Roberts, 2000</xref>; <xref ref-type="bibr" rid="bib105">Lohe and Roberts, 1990</xref>). Consistent with previous cytological studies (<xref ref-type="bibr" rid="bib145">Roy et al., 2005</xref>; <xref ref-type="bibr" rid="bib106">Lohe and Roberts, 2000</xref>; <xref ref-type="bibr" rid="bib105">Lohe and Roberts, 1990</xref>), we find that <italic>D. simulans</italic> and <italic>D. sechellia</italic> lost most Y-linked 18 S and 28 S rDNA sequences (<xref ref-type="fig" rid="fig1s5">Figure 1—figure supplement 5</xref>). Our assemblies indicate that, despite this loss of the rRNA coding sequences, all three species still retain IGS repeats. However, we and others do not detect Y-linked IGS repeats at the cytological level in <italic>D. sechellia</italic> (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplements 3</xref>–<xref ref-type="fig" rid="fig1s4">4</xref>; <xref ref-type="bibr" rid="bib145">Roy et al., 2005</xref>; <xref ref-type="bibr" rid="bib106">Lohe and Roberts, 2000</xref>; <xref ref-type="bibr" rid="bib105">Lohe and Roberts, 1990</xref>), suggesting that their abundance is below the level of detection by FISH in this species.</p><p>Structural variation at Y-linked genes may also contribute to functional variation and divergence in the <italic>D. simulans</italic> clade. Previous studies reported many duplications of canonical Y-linked genes in <italic>D. simulans</italic> (<xref ref-type="bibr" rid="bib65">Helleu et al., 2019</xref>; <xref ref-type="bibr" rid="bib32">Chakraborty et al., 2021</xref>; <xref ref-type="bibr" rid="bib85">Kopp et al., 2006</xref>). We find that all three species have at least one intact copy of the 11 canonical Y-linked genes, but there is also extensive copy number variation in Y-linked exons across these species (<xref ref-type="fig" rid="fig2">Figure 2</xref> and <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplements 1</xref>–<xref ref-type="fig" rid="fig2s2">2</xref>; <xref ref-type="supplementary-material" rid="supp1">Supplementary file 1</xref>; <xref ref-type="bibr" rid="bib31">Chakraborty, 2020</xref>). Using Illumina reads, we confirm the copy number variation in our assemblies and reveal some duplicated Y-linked exons, particularly in <italic>kl-3</italic>, <italic>WDY,</italic> and <italic>Ppr-Y</italic>, that are not assembled in <italic>D. sechellia</italic> (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Some duplicates may be functional because they are expressed and have complete open reading frames, (<italic>e.g</italic>. <italic>ARY</italic>, <italic>Ppr-Y1,</italic> and <italic>Ppr-Y2</italic>). The <italic>D. simulans</italic> Y chromosome has four complete copies of <italic>ARY</italic>, all of which show similar expression levels from RNA-seq data (<xref ref-type="fig" rid="fig2">Figure 2B</xref> and <xref ref-type="supplementary-material" rid="supp4">Supplementary file 4</xref>), but two copies have inverted exons 1 and 2. <italic>D. sechellia</italic> also contains at least five duplicated copies of <italic>ARY,</italic> some of which also have the inverted exons 1 and 2, but the absence of RNA-seq data from testes of this species prevents inferences regarding whether all copies of <italic>ARY</italic> are expressed. However, most duplications include only a subset of exons, and in many cases, the duplicated exons are located on the periphery of the presumed functional gene copy (<xref ref-type="fig" rid="fig2">Figure 2B</xref> and <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>, <xref ref-type="supplementary-material" rid="supp4">Supplementary file 4</xref>). For example, both <italic>D. simulans</italic> and <italic>D. mauritiana</italic> have multiple copies of exons 8–12 located at the 3’ end of <italic>kl-2</italic> (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). In <italic>D. simulans,</italic> most of these extra exons have low to no expression, while in <italic>D. mauritiana</italic>, there appears to be a substantial expression from many of the duplicated terminal exons, as well as an internal duplication of exon 5. Although the duplications of Y-linked genes can influence their expression, especially for genes with short introns (<italic>e.g</italic>. <italic>ARY</italic>, <italic>Ppr-Y1</italic> and <italic>Ppr-Y2</italic>), it is unclear what effects these duplicated exons have on the protein sequences of these fertility-essential genes.</p><fig-group><fig id="fig2" position="float"><label>Figure 2.</label><caption><title>Duplication of canonical Y-linked exons.</title><p>(<bold>A</bold>) Exon copy number is highly variable across the three <italic>D. simulans</italic> clade species and generally greater than in <italic>D. melanogaster.</italic> (<bold>B</bold>) Gene structure of <italic>kl-2</italic> and <italic>ARY</italic> inferred from assemblies and RNA-seq data. Upper bars indicate exons that are colored and numbered, with their height showing average read depth from sequenced testes RNA (<italic>D. simulans</italic> and <italic>D. mauritiana</italic> only). Lower bars indicate exon positions on the assembly and position on the Y-axis indicates coding strand. Some of the duplicated exons are expressed. For short genes (<italic>e.g., ARY</italic>), the duplicates may be functional and influence protein expression level, unlike duplicated exons of long genes (<italic>e.g.</italic>, <italic>kl-2</italic>).</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig2.jpg"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><label>Figure 2—figure supplement 1.</label><caption><title>The coverage of male Illumina DNA-seq reads in 11 canonical Y-linked genes.</title><p>To confirm the copy number of Y-linked genes across species in our assembly, we mapped the Illumina reads from males to a single of <italic>D. melanogaster</italic> Y-linked transcripts and estimated the copy number based on their coverage (black lines). For the comparison, we also simulated Illumina reads from our assemblies and mapped them to the same reference to estimate their copy number (red lines). The dotted lines separate each exon.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig2-figsupp1.jpg"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><label>Figure 2—figure supplement 2.</label><caption><title>Gene structure of 11 conserved Y-linked genes inferred from assemblies and RNA-seq data.</title><p>Upper bars indicate exons that are colored and numbered, with their height indicating average read depth from sequenced testes RNA (<italic>D. simulans</italic> and <italic>D. mauritiana</italic> only). Lower bars indicate exon positions on the assembly and position on the Y-axis indicates coding strand.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig2-figsupp2.jpg"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><label>Figure 2—figure supplement 3.</label><caption><title>The mummerplot of the <italic>ORY</italic> alignment in the <italic>D. simulans</italic> clade.</title><p>We used MUMMER to align <italic>ORY</italic> from different species and plot the figure. Purple lines and dots represent forward matches, and blue lines and dots represent reverse matches.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig2-figsupp3.jpg"/></fig></fig-group><p>All exon-intron junctions are conserved within full-length copies of the canonical Y-linked genes, but intron lengths vary between these species (<xref ref-type="fig" rid="fig3">Figure 3</xref>). The length of longer introns ( > 100 bp in any species) is more dynamic than that of short introns (<xref ref-type="fig" rid="fig3">Figure 3</xref>; <xref ref-type="supplementary-material" rid="supp5">Supplementary file 5</xref>). The dramatic size differences in most introns cannot be attributed to a single deletion or duplication (see <italic>ORY</italic> example in <xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3</xref>). Some Y-linked genes contain mega-base sized introns (<italic>i.e</italic>., mega-introns) whose transcription manifests as cytologically visible lampbrush-like loops (Y-loops) in primary spermatocytes (<xref ref-type="bibr" rid="bib21">Bonaccorsi et al., 1988</xref>; <xref ref-type="bibr" rid="bib22">Bonaccorsi et al., 1990</xref>). While Y-loops are found across the <italic>Drosophila</italic> genus (<xref ref-type="bibr" rid="bib116">Meyer, 1963</xref>; <xref ref-type="bibr" rid="bib133">Piergentili, 2007</xref>), their potential functions are unknown (<xref ref-type="bibr" rid="bib57">Fingerhut et al., 2019</xref>; <xref ref-type="bibr" rid="bib139">Redhouse et al., 2011</xref>; <xref ref-type="bibr" rid="bib136">Pisano et al., 1993</xref>; <xref ref-type="bibr" rid="bib132">Piergentili et al., 2004</xref>; <xref ref-type="bibr" rid="bib134">Piergentili and Mencarelli, 2008</xref>) and the genes/introns that produce Y-loops differs among species (<xref ref-type="bibr" rid="bib34">Chang and Larracuente, 2017</xref>). <italic>D. melanogaster</italic> has three Y-loops transcribed from introns of <italic>ORY</italic> (<italic>ks-1</italic> in previous literature), <italic>kl-3</italic>, and <italic>kl-5</italic> (<xref ref-type="bibr" rid="bib21">Bonaccorsi et al., 1988</xref>). Based on cytological evidence, <italic>D. simulans</italic> has three Y-loops, whereas <italic>D. mauritiana</italic> and <italic>D. sechellia</italic> only have two (<xref ref-type="bibr" rid="bib133">Piergentili, 2007</xref>). Of all potential loop-producing introns, we find that only the <italic>kl-3</italic> mega-intron is conserved in all four species and has the same intron structure and sequences (<italic>i.e.</italic> (AATAT)<sub>n</sub> repeats). While both <italic>kl-5</italic> and <italic>ORY</italic> produce Y-loops with (AAGAC)<sub>n</sub> repeats in <italic>D. melanogaster</italic>, (AAGAC)<sub>n</sub> is missing from the genomes of the <italic>D. simulans</italic> clade species. This observation is supported by our assemblies, the Illumina raw reads (<xref ref-type="supplementary-material" rid="supp6">Supplementary file 6</xref>), and published FISH results (<xref ref-type="bibr" rid="bib77">Jagannathan et al., 2017</xref>). In the <italic>D. simulans</italic> clade, the <italic>ORY</italic> introns do not carry any long tandem repeats. However, <italic>kl-5</italic> has introns with (AATAT)<sub>n</sub> repeats that may form a Y-loop in the <italic>D. simulans</italic> clade species. These data suggest that, while mega-introns and Y-loops may be conserved features of spermatogenesis in <italic>Drosophila</italic>, they turn over at both the sequence and gene levels over short periods of evolutionary time (<italic>i.e</italic>. ~ 2 My between <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade).</p><fig id="fig3" position="float"><label>Figure 3.</label><caption><title>Evolution of intron lengths in canonical Y-linked genes.</title><p>The intron length in canonical Y-linked genes is different between <italic>D. melanogaster</italic> and the three <italic>D.</italic> simulans clade species. Orthologous introns are connected by dotted lines. Completely assembled introns are in blue and introns with gaps in the assembly are in red, and are therefore minimum intron lengths.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig3.jpg"/></fig><p>Consistent with previous studies (<xref ref-type="bibr" rid="bib161">Tobler et al., 2017</xref>; <xref ref-type="bibr" rid="bib32">Chakraborty et al., 2021</xref>), we identify high rates of gene duplication to the <italic>D. simulans</italic> clade Y chromosome from other chromosomes. We find 49 independent duplications to the Y chromosome in our heterochromatin-enriched assemblies (<xref ref-type="fig" rid="fig4">Figure 4</xref>; <xref ref-type="supplementary-material" rid="supp7">Supplementary file 7</xref>), including eight newly discovered duplications (<xref ref-type="bibr" rid="bib161">Tobler et al., 2017</xref>; <xref ref-type="bibr" rid="bib32">Chakraborty et al., 2021</xref>). Twenty-eight duplications are DNA-based, 13 are RNA-based, and the rest are unknown due to limited sequence information (<xref ref-type="supplementary-material" rid="supp7">Supplementary file 7</xref>). The rate of transposition to the Y chromosome is about three to four times higher in the <italic>D. simulans</italic> clade compared to <italic>D. melanogaster</italic> (<xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>). We also infer that 17 duplicated genes were independently deleted from <italic>D. simulans</italic> clade Y chromosomes. Some of these Y-linked duplications, including <italic>Fdy, Mst77Y</italic> and <italic>pirate</italic>, are known to be functional and/or under purifying selection (<xref ref-type="bibr" rid="bib161">Tobler et al., 2017</xref>; <xref ref-type="bibr" rid="bib86">Krsticevic et al., 2015</xref>; <xref ref-type="bibr" rid="bib148">Russell and Kaiser, 1993</xref>; <xref ref-type="bibr" rid="bib42">Chen et al., 2021</xref>). However, based on transcriptomes from <italic>D. simulans</italic> and <italic>D. mauritiana</italic> testes, we suspect that more than half of the duplicated genes are likely pseudogenes that either show no expression in testes ( < 3 TPM) or lack open reading frames ( < 100 amino acids; <xref ref-type="supplementary-material" rid="supp7">Supplementary file 7</xref>). We also detect intrachromosomal duplications of these Y-linked pseudogenes (<xref ref-type="supplementary-material" rid="supp7">Supplementary file 7</xref>), suggesting a high duplication rate within these Y chromosomes.</p><fig id="fig4" position="float"><label>Figure 4.</label><caption><title>Turnover of new duplications to Y chromosomes in <italic>D. melanogaster</italic> and three species in the <italic>D. simulans</italic> clade.</title><p>Using phylogenetic analyses, we inferred the evolutionary histories of new Y-linked duplications. The blue and green numbers represent the number of independent duplications and deletions observed in each branch, respectively. We also detected four duplications presented in the ancestor of these four species. The deletion events that happened in the ancestor of these four species cannot be inferred without a Y chromosome assembly in the outgroup.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig4.jpg"/></fig><p>Most new Y-linked duplications in <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade are from genes with presumed functions in chromatin modification, cell division, and sexual reproduction (<xref ref-type="supplementary-material" rid="supp8">Supplementary file 8</xref>), consistent with other <italic>Drosophila</italic> species (<xref ref-type="bibr" rid="bib10">Bachtrog et al., 2019</xref>; <xref ref-type="bibr" rid="bib108">Mahajan and Bachtrog, 2017</xref>). While Y-linked duplicates of genes with these functions may be selectively beneficial, a duplication bias could also contribute to this enrichment, as genes expressed in the testes may be more likely to duplicate to the Y chromosome due to its open chromatin structure and transcriptional activity during spermatogenesis (<xref ref-type="bibr" rid="bib62">Greil and Ahmad, 2012</xref>; <xref ref-type="bibr" rid="bib107">Mahadevaraju et al., 2021</xref>; <xref ref-type="bibr" rid="bib67">Hess and Meyer, 1968</xref>).</p></sec><sec id="s2-3"><title>The evolution of new Y-linked gene families</title><p>Ampliconic gene families are found on Y chromosomes in multiple <italic>Drosophila</italic> species (<xref ref-type="bibr" rid="bib55">Ellison and Bachtrog, 2019</xref>). We discovered two new gene families that have undergone extensive amplification on <italic>D. simulans</italic> clade Y chromosomes (<xref ref-type="fig" rid="fig5">Figure 5</xref>). Both families appear to encode functional protein-coding genes with complete open reading frames and high expression in mRNA-seq data (<xref ref-type="supplementary-material" rid="supp9">Supplementary file 9</xref>) and have 36–146 copies in each species’ Y chromosome. We also confirm that >90% of the variants in our assembled Y-linked gene families are represented in Illumina DNA-seq data (Appendix 1).</p><fig id="fig5" position="float"><label>Figure 5.</label><caption><title>The history of Y-linked ampliconic genes.</title><p>(<bold>A</bold>) Schematic showing the inferred evolutionary history of <italic>SRPK-Y</italic>. <italic>SRPK</italic> duplicated to the ancestral Y chromosome in the <italic>D. simulans</italic> clade. The Y-linked copy (<italic>Lhk</italic>) retained an exon with testis-specific expression, which was lost in the parental copy on 2R. The Y-linked copy (<italic>Lhk</italic>) further duplicated and increased their expression in testes. (<bold>B</bold>) Schematic showing the inferred evolutionary history of sex-linked <italic>Ssl/CK2ßtes</italic> paralogs. In the <italic>D. melanogaster – D. simulans</italic> clade ancestor, the autosomal gene <italic>Ssl</italic>/<italic>CK2ßtes</italic> duplicated from chromosome <italic>2R</italic> to the sex chromosome and independently amplified into the multi-copy gene families <italic>CK2ßtes-like</italic> on the X chromosome and <italic>CK2ßtes-Y</italic> on the Y chromosomes (shaded orange box). The gene structures are maintained in the <italic>D. simulans</italic> clade species, but not in <italic>D. melanogaster</italic>. In the <italic>D. melanogaster</italic> lineage (shaded yellow box), <italic>CK2ßtes-Ys</italic> became pseudogenes (<italic>PCKR</italic>) and <italic>CK2ßtes-like</italic> acquired a promoter from <italic>ßNASCtes</italic> to create a chimeric gene. Subsequent duplication of the chimeric gene to the X chromosome gave rise to the X-linked <italic>Ste</italic> loci in <italic>D. melanogaster</italic>. Duplication of the chimeric gene to the Y chromosome, with a subsequent TE insertion in the promoter and amplification event, gave rise to the Y-linked <italic>Su(Ste)</italic> loci in <italic>D. melanogaster</italic>.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig5.jpg"/></fig><p>The first amplified Y-linked gene family, <italic>SR Protein Kinase</italic> (<italic>SRPK</italic>), is derived from an autosome-to-Y duplication of the sequence encoding the testis-specific isoform of the gene <italic>SR Protein Kinase (SRPK</italic>). After the duplication of <italic>SRPK</italic> to the Y chromosome, the ancestral autosomal copy subsequently lost its testis-specific exon via a deletion (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). The movement of the male-specific isoform inspired us to name the Y-linked <italic>SRPK</italic> gene family <italic>Lo-han-kha (Lhk</italic>), which is the Taiwanese term for the male vagabonds that moved from mainland China to Taiwan during the Qing dynasty. In <italic>D. melanogaster, SRPK</italic> is essential for both male and female reproduction (<xref ref-type="bibr" rid="bib102">Loh et al., 2012</xref>). We therefore hypothesize that the relocation of the testis-specific isoform to the <italic>D. simulans</italic> clade Y chromosomes may have resolved intralocus sexual antagonism over these two functions. Our phylogenetic analysis identified two subfamilies of <italic>Lhk</italic> that we designate <italic>Lhk-1</italic> and <italic>Lhk-2</italic> (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). Both subfamilies are shared by all <italic>D. simulans</italic> clade species and show a 5.5% protein divergence between species. The two subfamilies are found in different locations in our Y chromosome assemblies; consistent with this observation, we detect two to three <italic>Lhk</italic> foci on Y chromosomes in the <italic>D. simulans</italic> clade using FISH (<xref ref-type="fig" rid="fig6">Figure 6A and C</xref> and <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplements 3</xref>–<xref ref-type="fig" rid="fig1s4">4</xref>).</p><fig-group><fig id="fig6" position="float"><label>Figure 6.</label><caption><title>The rapid evolution and gene conversion of Y-linked ampliconic genes.</title><p>(<bold>A</bold>) The inferred maximum likelihood phylogeny for <italic>Lhk.</italic> Node labels indicate SH-aLRT and ultrafast bootstrap (<italic>e.g.</italic> 100/100) or rates of protein evolution from PAML with CodonFreq = 0,1, or 2 (<italic>e.g.</italic> 1.01/1.02/1.03) (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref> and <xref ref-type="fig" rid="fig6s3">Figure 6—figure supplement 3</xref>). <italic>Lhk</italic> shows evidence for positive selection (branch tests and branch-site tests with ω>1) after the duplication from 2R (<italic>SRPK</italic>) to the Y chromosome in the <italic>D. simulans</italic> clade. One <italic>Lhk</italic> subfamily (<italic>Lhk-1</italic>) is under recent purifying selection and is located close to the centromere, but the other (<italic>Lhk-2</italic>) is rapidly evolving across the species of the <italic>D. simulans</italic> clade. (<bold>B</bold>) Same as A but for <italic>CK2ßtes-Y.</italic> Both Y-linked <italic>CK2ßtes-Y</italic> and X-linked <italic>CK2ßtes-like</italic> also show positive selection. All ω values shown are statistically significant (LRT tests, P0.05; <xref ref-type="supplementary-material" rid="supp12">Supplementary file 12</xref> and <xref ref-type="supplementary-material" rid="supp14">Supplementary file 14</xref>). (<bold>C</bold>) Cytological location of Y-linked gene families detected using Immunolabeling with fluorescence in situ hybridization (immunoFISH) for the centromere (CENP-C antibody, red signal). On the Y chromosomes, <italic>Lhk</italic> FISH signals suggest that this gene family occurs in 2–3 cytological locations (green signal), with one near the centromere. <italic>CK2ßtes-Y</italic> FISH signals are only located near centromeres. Based on our analysis of sequence information, we suggest that most <italic>Lhk-1</italic> copies are located near <italic>CK2ßtes-Y</italic> and the centromere.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig6.jpg"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><label>Figure 6—figure supplement 1.</label><caption><title>The phylogeny of <italic>Lhk</italic> used in PAML analyses.</title><p>We marked the branches used in branch-model and branch-site model tests. We did all comparisons using the branch with different colors in likelihood-ratio tests. Please see detailed results in <xref ref-type="supplementary-material" rid="supp12">Supplementary file 12</xref>.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig6-figsupp1.jpg"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><label>Figure 6—figure supplement 2.</label><caption><title>The expression of different copies from <italic>Lhk</italic> and <italic>CK2ßtes-Y</italic> gene families.</title><p>(<bold>A</bold>) We quantify the frequency of each derived SNP within the genome using DNA-seq and the expression level of each allele using RNA-seq. We cataloged each SNP as synonymous, nonsynonymous or UTR. (<bold>B</bold>) We found that across three Y-linked gene families, only highly expressed <italic>Lhk-1</italic> copies have fewer nonsynonymous mutations than lowly expressed copies in <italic>D. simulans</italic>, consistent with purifying selection (<xref ref-type="supplementary-material" rid="supp13">Supplementary file 13</xref> and <xref ref-type="supplementary-material" rid="supp21">Supplementary file 21</xref>; Chi-square test’s P=0.01). We did not detect other significant changes in other comparisons (<xref ref-type="supplementary-material" rid="supp13">Supplementary file 13</xref> and <xref ref-type="supplementary-material" rid="supp21">Supplementary file 21</xref>; Chi-square test’s P > 0.01).</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig6-figsupp2.jpg"/></fig><fig id="fig6s3" position="float" specific-use="child-fig"><label>Figure 6—figure supplement 3.</label><caption><title>The phylogeny of <italic>CK2ßtes-Y</italic> used in PAML analyses.</title><p>We marked the branches used in branch-model and branch-site model tests. We did all comparisons using the branch with different colors in likelihood-ratio tests. Please see the detailed results in <xref ref-type="supplementary-material" rid="supp14">Supplementary file 14</xref>.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig6-figsupp3.jpg"/></fig></fig-group><p>The second amplified gene family comprises both X-linked and Y-linked duplicates of the <italic>Ssl</italic> gene located on chromosome 2 R; it is unclear whether the X- or Y-linked copies originated first (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). The X-linked copies are known as <italic>CK2ßtes-like</italic> in <italic>D. simulans</italic> (<xref ref-type="bibr" rid="bib82">Kogan et al., 2012</xref>). The Y-linked copies are also found in <italic>D. melanogaster</italic> but are degenerated and have little or no expression (<xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>; <xref ref-type="bibr" rid="bib50">Danilevskaya et al., 1991</xref>), leading to their designation as pseudogenes. In the <italic>D. simulans</italic> clade species, however, the Y-linked paralogs have high levels of expression ( > 50 TPM in testes, <xref ref-type="supplementary-material" rid="supp9">Supplementary file 9</xref>) and complete open reading frames, so we refer to this gene family as <italic>CK2ßtes-Y</italic>. Both <italic>CK2ßtes-like</italic> (4–9 copies) and <italic>CK2ßtes-Y</italic> (36–123 copies based on the assemblies) are amplified on the X and Y chromosome in the <italic>D. simulans</italic> clade relative to <italic>D. melanogaster</italic> (<xref ref-type="supplementary-material" rid="supp9">Supplementary file 9</xref>; <xref ref-type="bibr" rid="bib82">Kogan et al., 2012</xref>). The Y-linked copies in <italic>D. melanogaster, Su(Ste),</italic> are known to be a source of piRNAs (<xref ref-type="bibr" rid="bib6">Aravin et al., 2004</xref>). We did not detect any testis piRNAs from either gene family in two small RNA-seq datasets (SRR7410589 and SRR7410590); however, we do find some short ( < 23 nt) reads (0.003–0.005% of total mapped reads) mapped to these gene families (<xref ref-type="supplementary-material" rid="supp10">Supplementary file 10</xref>).</p><p>We inferred gene conversion rates and the strength of selection on these Y-linked gene families using phylogenetic analyses on coding sequences. We estimated the gene conversion rate in <italic>D. simulans</italic> clade Y-linked gene families based on four-gamete tests and gene similarity (<xref ref-type="bibr" rid="bib146">Rozen et al., 2003</xref>; <xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>; <xref ref-type="bibr" rid="bib125">Ohta, 1984</xref>; <xref ref-type="bibr" rid="bib12">Backström et al., 2005</xref>). In general, <italic>D. simulans</italic> clade species show similar gene conversion rates (on the order of 10<sup>–4</sup> to 10<sup>–6</sup>) in both of these families compared to our previous estimates in <italic>D. melanogaster</italic> (<xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>; <xref ref-type="supplementary-material" rid="supp11">Supplementary file 11</xref>). These higher gene conversion rates compared to the other chromosomes might be a shared feature of Y chromosomes across taxa (<xref ref-type="bibr" rid="bib146">Rozen et al., 2003</xref>).</p><p>To estimate rates of molecular evolution, we conducted branch-model and branch-site-model tests on the reconstructed ancestral sequences of <italic>Lhk-1</italic>, <italic>Lhk-2</italic>, <italic>CK2ßtes-Y,</italic> and two <italic>CK2ßtes-like</italic> using PAML (<xref ref-type="fig" rid="fig6">Figure 6A and B</xref>; <xref ref-type="table" rid="table2">Table 2</xref>; <xref ref-type="bibr" rid="bib168">Yang, 1997</xref>). We used reconstructed ancestral sequences for our analyses to avoid sequencing errors in the assemblies, which appear as singletons. We infer that after the divergence of <italic>D. simulans</italic> clade species, <italic>Lhk-1</italic> evolved under purifying selection, whereas <italic>Lhk-2</italic> evolved under positive selection (<xref ref-type="fig" rid="fig6">Figure 6A</xref>; <xref ref-type="table" rid="table2">Table 2</xref>; <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>; <xref ref-type="supplementary-material" rid="supp12">Supplementary file 12</xref>). Using transcriptome data, we observe that highly expressed <italic>Lhk-1</italic> copies have fewer nonsynonymous mutations than lowly expressed copies in <italic>D. simulans</italic>, consistent with purifying selection (Chi-square test’s p = 0.01; <xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref> and <xref ref-type="supplementary-material" rid="supp13">Supplementary file 13</xref>). Both <italic>Lhk</italic> gene families are expressed two- to seven-fold higher than the ancestral copy on 2R in the same species, and 1.9–64-fold higher than their ortholog, <italic>SRPK-RC,</italic> in <italic>D. melanogaster</italic>, suggesting that gene amplification may confer increased expression. In both <italic>D. simulans</italic> and <italic>D. mauritiana</italic>, <italic>Lhk-1</italic> is shorter due to deletions following its origin and has a higher expression level than <italic>Lhk-2</italic>. Both <italic>Lhk</italic> gene families have higher copy numbers in <italic>D. simulans</italic> than <italic>D. mauritiana,</italic> which likely contributes to their higher expression level in <italic>D. simulans</italic> (<xref ref-type="supplementary-material" rid="supp9">Supplementary file 9</xref>). For both <italic>Lhk-1</italic> and <italic>Lhk-2,</italic> copies from the same species are more similar than copies from other species—a signal of concerted evolution (<xref ref-type="bibr" rid="bib53">Dover, 1982</xref>).</p><table-wrap id="table2" position="float"><label>Table 2.</label><caption><title>PAML analyses reveal positive selection on Y-linked ampliconic gene families.</title></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="2" valign="bottom"><italic>Lhk</italic></th><th align="left" colspan="5" valign="bottom">Branch test with CodonFreq = 0</th><th align="left" valign="bottom"/><th align="left" colspan="7" valign="bottom">Branch-site test site class</th></tr><tr><th align="left" valign="bottom">ω1</th><th align="left" valign="bottom">ω2</th><th align="left" valign="bottom">ω3</th><th align="left" valign="bottom">L</th><th align="left" valign="bottom">2∆lnL</th><th align="left" valign="bottom">LRT’s P</th><th align="left" valign="bottom">ω0</th><th align="left" valign="bottom">ω1</th><th align="left" valign="bottom">ω2a</th><th align="left" valign="bottom">ω2b</th><th align="left" valign="bottom">2∆lnL</th><th align="left" valign="bottom">LRT’s P</th><th align="left" valign="bottom">Positively selected sites (BEB > 0.95)<xref ref-type="table-fn" rid="table2fn1">*</xref></th></tr></thead><tbody><tr><td align="left" valign="bottom">one ω</td><td align="char" char="." valign="bottom">0.17</td><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="char" char="." valign="bottom">–3250.74</td><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"> </td></tr><tr><td align="left" valign="bottom">two ω<xref ref-type="table-fn" rid="table2fn2">†</xref></td><td align="char" char="." valign="bottom">0.11</td><td align="char" char="." valign="bottom">1.05</td><td align="left" valign="bottom"/><td align="char" char="." valign="bottom">–3218.26</td><td align="char" char="." valign="bottom">64.94</td><td align="char" char="hyphen" valign="bottom">7.71E-16</td><td align="char" char="." valign="bottom">0.01</td><td align="char" char="." valign="bottom">1</td><td align="char" char="." valign="bottom">4.87</td><td align="char" char="." valign="bottom">4.87</td><td align="char" char="." valign="bottom">13.04</td><td align="char" char="hyphen" valign="bottom">3.05E-04</td><td align="left" valign="bottom">I4, H11, V32, V75, N99, Y100, D193, D199</td></tr><tr><td align="left" valign="bottom">three ω<xref ref-type="table-fn" rid="table2fn3">‡</xref></td><td align="char" char="." valign="bottom">0.11</td><td align="char" char="." valign="bottom">1.49</td><td align="char" char="." valign="bottom">0.43</td><td align="char" char="." valign="bottom">–3216.30</td><td align="char" char="." valign="bottom">3.92</td><td align="char" char="." valign="bottom">0.05</td><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"> </td></tr><tr><td align="left" valign="bottom"><italic>CK2ßtes</italic></td><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"> </td></tr><tr><td align="left" valign="bottom">one ω</td><td align="char" char="." valign="bottom">0.35</td><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="char" char="." valign="bottom">–3295.01</td><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"> </td></tr><tr><td align="left" valign="bottom">two ω<xref ref-type="table-fn" rid="table2fn4">§</xref></td><td align="char" char="." valign="bottom">0.25</td><td align="char" char="." valign="bottom">1.05</td><td align="left" valign="bottom"/><td align="char" char="." valign="bottom">–3272.00</td><td align="char" char="." valign="bottom">46.01</td><td align="char" char="hyphen" valign="bottom">1.18E-11</td><td align="char" char="." valign="bottom">0.05</td><td align="char" char="." valign="bottom">1</td><td align="char" char="." valign="bottom">2.21</td><td align="char" char="." valign="bottom">2.21</td><td align="char" char="." valign="bottom">6.54</td><td align="char" char="hyphen" valign="bottom">1.06E-02</td><td align="left" valign="bottom">D33, T38, K44, K100, F101, K104, M152, M155</td></tr><tr><td align="left" valign="bottom">three ω<xref ref-type="table-fn" rid="table2fn3">‡</xref></td><td align="char" char="." valign="bottom">0.20</td><td align="char" char="." valign="bottom">0.42</td><td align="char" char="." valign="bottom">1.05</td><td align="char" char="." valign="bottom">–3266.33</td><td align="char" char="." valign="bottom">11.35</td><td align="char" char="hyphen" valign="bottom">7.56E-04</td><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"/><td align="left" valign="bottom"> </td></tr></tbody></table><table-wrap-foot><fn id="table2fn1"><label>*</label><p>See <xref ref-type="supplementary-material" rid="supp12">Supplementary files 12 and 14</xref> for all sites.</p></fn><fn id="table2fn2"><label>†</label><p>Autosomal and Y lineage have protein evolution of ω1 and ω2, respectively.</p></fn><fn id="table2fn3"><label>‡</label><p>See <xref ref-type="supplementary-material" rid="supp12">Supplementary files 12 and 14</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref> and <xref ref-type="fig" rid="fig6s3">Figure 6—figure supplement 3</xref> for the assignment of lineages.</p></fn><fn id="table2fn4"><label>§</label><p>Autosomal and sex chromosomal (X and Y) have protein evolution of ω1 and ω2, respectively.</p></fn></table-wrap-foot></table-wrap><p>The ancestral <italic>Ssl</italic> gene experienced a slightly increased rate of protein evolution after it duplicated to the X and Y chromosomes (<italic>ω</italic> = 0.41 vs 0.23; p = 0.03; <xref ref-type="fig" rid="fig6">Figure 6B</xref>; <xref ref-type="table" rid="table2">Table 2</xref>; <xref ref-type="fig" rid="fig6s3">Figure 6—figure supplement 3</xref>; <xref ref-type="supplementary-material" rid="supp14">Supplementary file 14</xref>). We find that both <italic>CK2ßtes-like</italic> and <italic>CK2ßtes-Y</italic> share strong signals of positive selection, based on branch-model and branch-site-model tests (p = 8.8E-9; <xref ref-type="fig" rid="fig6">Figure 6B</xref>; <xref ref-type="table" rid="table2">Table 2</xref>; <xref ref-type="fig" rid="fig6s3">Figure 6—figure supplement 3</xref>; <xref ref-type="supplementary-material" rid="supp14">Supplementary file 14</xref>). In <italic>D. melanogaster,</italic> the overexpression of the <italic>CK2ßtes-like</italic> X-linked homolog, <italic>Stellate,</italic> can drive in the male germline by killing Y-bearing sperm and generating female-biased offspring (<xref ref-type="bibr" rid="bib110">Malone et al., 2015</xref>; <xref ref-type="bibr" rid="bib126">Palumbo et al., 1994</xref>; <xref ref-type="bibr" rid="bib117">Meyer et al., 2004</xref>). We suspect that <italic>CK2ßtes-like</italic> and <italic>CK2ßtes-Y</italic> might have similar functions and may also have a history of conflict. Therefore, the co-amplification of sex-linked genes and positive selection on their coding sequences may be a consequence of an arms race between sex chromosome drivers.</p></sec><sec id="s2-4"><title>Y chromosome evolution driven by specific mutation patterns</title><p>The specific DNA-repair mechanisms used on Y chromosomes might contribute to their high rates of intrachromosomal duplication and structural rearrangements. Because Y chromosomes lack a homolog, they must repair double-strand breaks (DSBs) by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ), which relies on short homology (usually > 2 bp) to repair DSBs (<xref ref-type="bibr" rid="bib33">Chan et al., 2010</xref>). Compared to NHEJ, MMEJ is more error-prone and can result in translocations and duplications (<xref ref-type="bibr" rid="bib113">McVey and Lee, 2008</xref>). Preferential use of MMEJ instead of NHEJ could contribute to the high duplication rate and extensive genome rearrangements that we observe on Y chromosomes. To infer the mechanisms of DSB repair on Y chromosomes, we counted indels between Y-linked duplicates and their parent genes for a set of 21 putative pseudogenes. Both NHEJ and MMEJ can generate indels, but NHEJ usually produces smaller indels (1–3 bp) compared to MMEJ ( > 3 bp) (<xref ref-type="bibr" rid="bib113">McVey and Lee, 2008</xref>; <xref ref-type="bibr" rid="bib35">Chang et al., 2017</xref>). We also cataloged short stretches of homology between each duplicate and its parent. To compare Y-linked patterns of DSB repair to other regions of the genome, we measured the size of polymorphic indels in intergenic regions and pseudogenes on the autosomes and X chromosomes from population data in <italic>D. melanogaster</italic> (DGRP; <xref ref-type="bibr" rid="bib71">Huang et al., 2014</xref>) and <italic>D. simulans</italic> (<xref ref-type="bibr" rid="bib151">Signor et al., 2018</xref>). To the extent that these indels do not experience selection, their sizes should reflect the mutation patterns on each chromosome. We observe proportionally more large deletions on Y chromosomes (25.1% of Y-linked indels are ≥10 bp deletions; <xref ref-type="supplementary-material" rid="supp15">Supplementary file 15</xref>) compared to other chromosomes in both <italic>D. melanogaster</italic> (12.8% and 15.2% of indels are ≥10 bp deletions in intergenic regions and pseudogenes) and <italic>D. simulans</italic> (7.3% of indels are ≥10 bp deletions in intergenic regions; all pairwise chi-square’s p< 1e-6; <xref ref-type="fig" rid="fig4">Figure 4A</xref>; <xref ref-type="supplementary-material" rid="supp15">Supplementary file 15</xref>). The pattern of excess large deletions is shared in the three <italic>D. simulans</italic> clade species Y chromosomes but is not obvious in <italic>D. melanogaster</italic> (<xref ref-type="fig" rid="fig7">Figure 7B</xref>). However, because most (36/41) <italic>D. melanogaster</italic> Y-linked indels in our analyses are from copies of a single pseudogene (<italic>CR43975</italic>), it is difficult to compare to the larger samples in the <italic>simulans</italic> clade species (duplicates from 17 genes). The differences in deletion sizes between the Y and other chromosomes are unlikely to be driven by heterochromatin or the lack of recombination. The non-recombining and heterochromatic dot chromosome has a deletion size profile more similar to the other autosomes in <italic>D. simulans</italic> (10.9% of indels are ≥10 bp deletions), consistent with a previous study using TE sequences across different chromatin domains (<xref ref-type="bibr" rid="bib20">Blumenstiel et al., 2002</xref>). We also found fewer large deletions (2/149 indels are ≥10 bp in 400 kb alignments; <xref ref-type="fig" rid="fig7">Figure 7A</xref>) in heterochromatic pseudogenes using 19 long-read (Pacbio or nanopore) assemblies. The enrichment of 1 bp indels (101/149; <xref ref-type="fig" rid="fig7">Figure 7A</xref>) in heterochromatic pseudogenes might represent sequencing errors in long-read assemblies (<xref ref-type="bibr" rid="bib167">Weirather et al., 2017</xref>). These results suggest that Y chromosomes may use MMEJ over NHEJ compared to other chromosomes, particularly in the simulans clade species. We also find that across the genome, larger deletions ( > 7 bp) share a similar length of microhomologies for repairing DSBs (39.5–57% deletions have ≥2 bp microhomology; Chi-square test for microhomology length between Y and other chromosomes, p > 0.24; <xref ref-type="supplementary-material" rid="supp15">Supplementary files 15–16</xref>), consistent with most being a consequence of MMEJ-mediated repair.</p><fig-group><fig id="fig7" position="float"><label>Figure 7.</label><caption><title>An excess of large deletions on Y chromosomes compared to population data suggests a preference for MMEJ.</title><p>(<bold>A</bold>) We compared the size of 223 indels on 21 recently duplicated Y-linked genes in <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade species to the indels polymorphic in the <italic>D. melanogaster</italic> and <italic>D. simulans</italic> populations. For the indels in <italic>D. melanogaster</italic> and <italic>D. simulans</italic> populations, we separated them based on their location, including autosomes (excluding dot chromosomes), X chromosomes, and dot chromosomes. We excluded the <italic>D. melanogaster</italic> dot-linked indels due to the small sample size (12). We also surveyed indel polymorphism in pseudogenes in <italic>D. melanogaster</italic> using population data. (<bold>B</bold>) We classify Y-linked indels by whether they are shared between species or specific in one species (<bold>C</bold>) The excess of large deletions (underlined) on the Y chromosomes is consistent with MMEJ between short regions of microhomology (red).</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig7.jpg"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><label>Figure 7—figure supplement 1.</label><caption><title>The abundance of repetitive elements on Y chromosomes of <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade species.</title><p>We plotted the density of 20 most enriched (by total occupying sequences) repetitive elements on Y chromosomes across four species. The colors represent the proportion of repetitive sequences in all assembled Y-linked sequences.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig7-figsupp1.jpg"/></fig><fig id="fig7s2" position="float" specific-use="child-fig"><label>Figure 7—figure supplement 2.</label><caption><title>The correlation of TE abundance between Y chromosomes and other chromosomes of <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade.</title><p>We calculated the fold changes of TE occupying sites (bp) between species by chromosomes. Each point from the figures above the diagonal represents the changes of a TE element on the Y chromosome and the other (non-Y) chromosomes. The number below the diagonal shows Spearman’s rank correlation coefficient for each comparison.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig7-figsupp2.jpg"/></fig><fig id="fig7s3" position="float" specific-use="child-fig"><label>Figure 7—figure supplement 3.</label><caption><title>The length of LTR retrotransposons between Y chromosomes and other chromosomes of <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade.</title><p>We surveyed the length of LTR retrotransposons across chromosomes (A: autosomes, X: X chromosome, U: unknown location and Y: Y chromosome). The length of elements is normalized by the length of consensus from full-length elements and represents the ages of each LTR retrotransposon.</p></caption><graphic mime-subtype="jpeg" mimetype="image" xlink:href="elife-75795.xml.media/fig7-figsupp3.jpg"/></fig></fig-group><p>The satellite sequence composition of Y chromosomes differs between species (<xref ref-type="bibr" rid="bib77">Jagannathan et al., 2017</xref>; <xref ref-type="bibr" rid="bib166">Wei et al., 2018</xref>; <xref ref-type="bibr" rid="bib29">Cechova et al., 2019</xref>). A high duplication rate may accelerate the birth and turnover of Y-linked satellite sequences. We discovered five new Y-linked satellites in our assemblies and validated their location using FISH (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplements 3</xref>–<xref ref-type="fig" rid="fig1s4">4</xref> and <xref ref-type="supplementary-material" rid="supp6">Supplementary file 6</xref>). These satellites only span a few kilobases of sequences (5,515–26,119 bp) and are homogenized. According to its flanking sequence, one new satellite, (AAACAT)<sub>n</sub>, originated from a DM412B transposable element, which has three tandem copies of AAACAT in its long terminal repeats. The AAACAT repeats expanded to 764 copies on the Y chromosome specifically in <italic>D. mauritiana</italic>. This is consistent with other reports of novel satellites arising from TEs (<xref ref-type="bibr" rid="bib51">Dias et al., 2014</xref>). The other four novel satellites are flanked by transposons ( < 50 bp) and may derive from non-repetitive sequences. The MMEJ pathway may contribute to the birth of new repeats, as this mechanism is known to generate tandem duplications via template-switching during repair (<xref ref-type="bibr" rid="bib113">McVey and Lee, 2008</xref>). Short-tandem repeats can be further amplified via saltatory replication or unequal crossing-over between sister chromatids.</p><p>Consistent with findings in other species (<xref ref-type="bibr" rid="bib130">Peichel et al., 2019</xref>; <xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>), we find an enrichment of LTR retrotransposons on the <italic>D. simulans</italic> clade Y chromosomes relative to the rest of the genome (<xref ref-type="supplementary-material" rid="supp17">Supplementary file 17</xref>). Interestingly, we find that the Y-linked LTR retrotransposons also turn over between species (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref> and <xref ref-type="supplementary-material" rid="supp18">Supplementary file 18</xref>). We find a positive correlation between the difference in Y-linked TE abundance between <italic>D. melanogaster</italic> and each of the <italic>D. simulans</italic> clade species versus the rest of the genome (rho = 0.45–0.50; <xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2</xref> and <xref ref-type="supplementary-material" rid="supp18">Supplementary file 18</xref>). This suggests that global changes in transposon activity could explain the differences in Y-linked TEs abundance between species. However, the correlations between species within the <italic>D. simulans</italic> clade are weaker (rho < 0.23; <xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2</xref> and <xref ref-type="supplementary-material" rid="supp18">Supplementary file 18</xref>), consistent with the possibility that some TEs may shift their insertion preference between chromosomes. To test this hypothesis, we estimated the ages of LTR retrotransposons by their length. We find that the recent insertions of LTR transposons are differently distributed across chromosomes between species (<xref ref-type="fig" rid="fig7s3">Figure 7—figure supplement 3</xref>), suggesting that insertion preferences towards genomic regions may differ for some TEs. For example, we detect many recent DIVER element insertions on the Y chromosome in <italic>D. simulans</italic>, but not in <italic>D. sechellia</italic> (<xref ref-type="fig" rid="fig7s3">Figure 7—figure supplement 3</xref>).</p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>Despite their independent origins, the degenerated Y chromosomes of mammals, fish, and insects have convergently evolved structural features of gene acquisition and amplification, accumulation of repetitive sequences, and gene conversion. Here, we consider the mutational processes that contribute to this structure and its consequences for Y chromosome biology. Our assemblies revealed extensive Y chromosome rearrangements between three very closely related <italic>Drosophila</italic> species (<xref ref-type="fig" rid="fig1">Figure 1</xref>). These rearrangements may be the consequence of rejoining telomeres after DSBs, as telomere-specific sequences are embedded in non-telomeric regions of <italic>Drosophila</italic> Y chromosomes (<xref ref-type="bibr" rid="bib18">Berloco et al., 2005</xref>; <xref ref-type="bibr" rid="bib1">Abad et al., 2004</xref>; <xref ref-type="bibr" rid="bib2">Agudo et al., 1999</xref>). We propose that four pieces of evidence suggest DSBs on Y chromosomes may be preferentially repaired using the MMEJ pathway. First, Y-linked sequences are generally absent from the X chromosome, precluding repair of DSBs by homologous recombination in meiosis. Second, NHEJ on Y chromosomes may be limited because the Ku complex, which is required for NHEJ (<xref ref-type="bibr" rid="bib35">Chang et al., 2017</xref>), is excluded from HP1a-rich regions of chromosomes (<xref ref-type="bibr" rid="bib44">Chiolo et al., 2011</xref>). The Ku complex also binds telomeres and might prevent telomere fusions (<xref ref-type="bibr" rid="bib115">Melnikova et al., 2005</xref>; <xref ref-type="bibr" rid="bib150">Samper et al., 2000</xref>), suggesting that a low concentration of Ku on Y chromosomes could also cause high rates of telomere rejoining. Third, the highly repetitive nature of Y chromosomes may increase the rate of DSB formation, which may also contribute to a higher rate of MMEJ (<xref ref-type="bibr" rid="bib113">McVey and Lee, 2008</xref>; <xref ref-type="bibr" rid="bib79">Katsura et al., 2007</xref>). Fourth, we show that Y chromosomes have high duplication and gene conversion rates, and larger deletion sizes than other genomic regions (<xref ref-type="fig" rid="fig7">Figure 7</xref>), consistent with a preference for MMEJ to repair Y-linked DSBs (<xref ref-type="bibr" rid="bib113">McVey and Lee, 2008</xref>).</p><p>The exclusion of the Ku complex from heterochromatin could also contribute to an excess of Y-linked duplications we observe in the <italic>D. simulans</italic> clade relative to <italic>D. melanogaster</italic> (<xref ref-type="fig" rid="fig2">Figures 2A</xref> and <xref ref-type="fig" rid="fig7">7</xref>). <italic>D. simulans</italic> clade Y chromosomes might harbor relatively more heterochromatin than the <italic>D. melanogaster</italic> Y due to the partial loss of their euchromatic rDNA repeats (<xref ref-type="bibr" rid="bib145">Roy et al., 2005</xref>; <xref ref-type="bibr" rid="bib106">Lohe and Roberts, 2000</xref>; <xref ref-type="bibr" rid="bib105">Lohe and Roberts, 1990</xref>), and <italic>D. simulans</italic> also expresses more heterochromatin-modifying factors, such as <italic>Su(var</italic>)s and <italic>E(var</italic>)s (<xref ref-type="bibr" rid="bib96">Lee and Karpen, 2017</xref>), compared to <italic>D. melanogaster</italic>. To explore these hypotheses, the distribution of the Ku complex across chromosomes in the testes of these species should be studied.</p><p>If MMEJ is preferentially used to fix DSBs on the Y chromosome, we might expect that the mutations in the MMEJ pathway would disproportionately impact Y-bearing sperm. Consistent with this prediction, a previous study showed that male <italic>D. melanogaster</italic> with a deficient MMEJ pathway (<italic>DNApol theta</italic> mutants) sire female-biased offspring (<xref ref-type="bibr" rid="bib112">McKee et al., 2000</xref>). Moreover, sperm without sex chromosomes that result from X-Y non-disjunction events are not as strongly affected by an MMEJ deficiency as Y-bearing sperm (<xref ref-type="bibr" rid="bib112">McKee et al., 2000</xref>), suggesting that sperm with Y chromosomes are more sensitive to defects in MMEJ.</p><p><italic>Drosophila</italic> Y chromosomes can act as heterochromatin sinks, sequestering heterochromatin marks from pericentromeric regions and suppressing position-effect variegation (<xref ref-type="bibr" rid="bib26">Brown and Bachtrog, 2017</xref>; <xref ref-type="bibr" rid="bib52">Dimitri and Pisano, 1989</xref>; <xref ref-type="bibr" rid="bib66">Henikoff, 1996</xref>; <xref ref-type="bibr" rid="bib59">Gatti and Pimpinelli, 1992</xref>). Therefore, retrotransposons located in heterochromatin might have higher activities in males due to the presence of Y-linked heterochromatin (<xref ref-type="bibr" rid="bib26">Brown and Bachtrog, 2017</xref>; <xref ref-type="bibr" rid="bib66">Henikoff, 1996</xref>), although the genomic distribution of heterochromatin during spermatogenesis is unknown. We find that, like <italic>D. melanogaster</italic> (<xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>), <italic>D. simulans</italic> clade Y chromosomes are enriched in retrotransposons relative to the rest of the genome; however, Y chromosomes from even the closely related <italic>D. simulans</italic> clade species harbor distinct retrotransposons (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref> and <xref ref-type="supplementary-material" rid="supp18">Supplementary file 18</xref>), indicating that some TEs may have rapidly shifted their insertion preference. This preference might benefit the TEs because Y-linked TEs might be expressed during spermatogenesis (<xref ref-type="bibr" rid="bib95">Lawlor et al., 2021</xref>). On the other hand, Y chromosomes can be a significant source of small RNAs that silence repetitive elements during spermatogenesis—for example, <italic>Su(Ste</italic>) piRNAs in <italic>D. melanogaster</italic> (<xref ref-type="bibr" rid="bib137">Quénerch’du et al., 2016</xref>; <xref ref-type="bibr" rid="bib5">Aravin et al., 2001</xref>) —and thus may also contribute to TE suppression. If Y chromosomes contribute to piRNA or siRNA production (<italic>e.g</italic>. have piRNA clusters <xref ref-type="bibr" rid="bib42">Chen et al., 2021</xref>; <xref ref-type="bibr" rid="bib5">Aravin et al., 2001</xref>), then the TE insertion preference for the Y chromosome may sometimes be beneficial for the host, as they could provide immunity against active TEs in males. In this sense, Y chromosomes may even act as “TE traps” that incidentally suppress TE activity in the male germline by producing small RNAs.</p><p>Genes may adapt to the Y chromosome after residing there for millions of years (<xref ref-type="bibr" rid="bib163">Wakimoto and Hearn, 1990</xref>; <xref ref-type="bibr" rid="bib64">Hearn et al., 1991</xref>). While most genes that move to the Y chromosome quickly degenerate (<xref ref-type="bibr" rid="bib161">Tobler et al., 2017</xref>; <xref ref-type="bibr" rid="bib28">Carvalho et al., 2015</xref>), a subset of new Y-linked genes are retained, presumably due to important roles in male fertility or sex chromosome meiotic drive. New Y-linked genes may adapt to this unique genomic environment, evolving structures and regulatory mechanisms that enable optimal expression on the heterochromatic and non-recombining Y chromosome (<xref ref-type="bibr" rid="bib54">Dupim et al., 2018</xref>). We identified many Y-linked duplicates in the ~15 Mb of Y chromosome that we surveyed in each species. Future improvements in genomic sequence data and assemblies may recover additional Y-linked duplicates among the unassembled satellite-rich sequences. Here, we describe two new Y-linked ampliconic genes specific to the <italic>D. simulans</italic> clade—<italic>Lhk</italic> and <italic>CK2ßtes-Y</italic>–that show evidence of strong positive evolution and concerted evolution, suggesting that high copy numbers and Y-Y gene conversion are often important for the adaptation of new Y-linked genes.</p><p>Many ampliconic genes are taxonomically restricted and are not maintained at high copy numbers over long periods of evolutionary time (<xref ref-type="bibr" rid="bib155">Soh et al., 2014</xref>; <xref ref-type="bibr" rid="bib10">Bachtrog et al., 2019</xref>; <xref ref-type="bibr" rid="bib25">Brashear et al., 2018</xref>; <xref ref-type="bibr" rid="bib55">Ellison and Bachtrog, 2019</xref>; <xref ref-type="bibr" rid="bib74">Hughes et al., 2010</xref>; <xref ref-type="bibr" rid="bib122">Mueller et al., 2008</xref>). Some ampliconic gene families are found on both the X and Y chromosomes (<xref ref-type="bibr" rid="bib55">Ellison and Bachtrog, 2019</xref>; <xref ref-type="bibr" rid="bib110">Malone et al., 2015</xref>; <xref ref-type="bibr" rid="bib45">Cocquet et al., 2012</xref>; <xref ref-type="bibr" rid="bib87">Kruger et al., 2019</xref>; <xref ref-type="bibr" rid="bib90">Lahn and Page, 2000</xref>). While we do not know the function of most such co-amplified gene families, the murine example of <italic>Slx/Slxl1</italic> and <italic>Sly</italic> appears to be engaged in an ongoing arms race between the sex chromosomes (<xref ref-type="bibr" rid="bib45">Cocquet et al., 2012</xref>). We propose that Y-linked gene amplification in the <italic>D. simulans</italic> clade initially occurred due to an arms race and was preserved by gene conversion.</p><p>It is intriguing that the <italic>CK2ßtes-like/CK2ßtes-Y</italic> gene family is homologous to the <italic>Ste/Su(Ste</italic>) system in <italic>D. melanogaster</italic> (<xref ref-type="bibr" rid="bib82">Kogan et al., 2012</xref>), which is also hypothesized to play a role in sex-chromosome meiotic drive (<xref ref-type="bibr" rid="bib76">Hurst, 1992</xref>). We speculate that in both the <italic>D. melanogaster</italic> and <italic>D. simulans</italic> clade lineages these gene amplifications have been driven by conflict between the sex chromosomes over transmission through meiosis, but that the conflict involves different molecular mechanisms. In the <italic>CK2ßtes-like/CK2ßtes-Y</italic> system, both X and Y-linked genes are protein-coding genes, which is reminiscent of <italic>Slx</italic>/<italic>Slxl1</italic> and <italic>Sly</italic> which compete for access to the nucleus where they regulate sex-linked gene expression (<xref ref-type="bibr" rid="bib45">Cocquet et al., 2012</xref>; <xref ref-type="bibr" rid="bib87">Kruger et al., 2019</xref>). In contrast, the Y-linked <italic>Su(Ste</italic>) copies in <italic>D. melanogaster</italic> produce small RNAs that suppress the X-linked <italic>Stellate</italic> (<xref ref-type="bibr" rid="bib6">Aravin et al., 2004</xref>). We propose that <italic>CK2ßtes-like/CK2ßtes-Y</italic> system in the <italic>D. simulans</italic> clade species may represent the ancestral state because the parental gene <italic>Ssl</italic> is a protein-coding gene. We speculate that systems arising from antagonisms between the sex chromosomes may shift from protein-coding to RNA-based over time because, with RNAi, suppression is maintained at a minimal translation cost.</p><p>Distinct Y-linked mutation patterns are described in many species (<xref ref-type="bibr" rid="bib155">Soh et al., 2014</xref>; <xref ref-type="bibr" rid="bib146">Rozen et al., 2003</xref>; <xref ref-type="bibr" rid="bib75">Hughes and Page, 2015</xref>; <xref ref-type="bibr" rid="bib10">Bachtrog et al., 2019</xref>; <xref ref-type="bibr" rid="bib161">Tobler et al., 2017</xref>; <xref ref-type="bibr" rid="bib130">Peichel et al., 2019</xref>; <xref ref-type="bibr" rid="bib25">Brashear et al., 2018</xref>; <xref ref-type="bibr" rid="bib63">Hall et al., 2016</xref>). Our analyses provide a link between Y-linked mutation patterns and Y chromosome evolution. While the lack of recombination and male-limited transmission of the Y chromosome reduces the efficacy of selection, the high gene duplication and gene conversion rates may counter these effects and help acquire and maintain new Y-linked genes. The unique Y-linked mutation patterns might be the direct consequence of the heterochromatic environment on sex chromosomes. Therefore, we predict that W chromosomes and non-recombining sex-limited chromosomes (<italic>e.g</italic>. some B chromosomes), may share similar mutation patterns with Y chromosomes. Indeed, W chromosomes of birds have ampliconic genes and are rich in tandem repeats (<xref ref-type="bibr" rid="bib12">Backström et al., 2005</xref>; <xref ref-type="bibr" rid="bib84">Komissarov et al., 2018</xref>). However, there seem to be fewer ampliconic gene families on bird W chromosomes compared to Y chromosomes in other animals, suggesting that sexual selection and intragenomic conflict in spermatogenesis are important contributors to Y-linked gene family evolution (<xref ref-type="bibr" rid="bib11">Bachtrog, 2020</xref>; <xref ref-type="bibr" rid="bib144">Rogers, 2021</xref>).</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Assembling Y chromosomes using Pacbio reads in <italic>D. simulans</italic> clade</title><p>We applied the heterochromatin-sensitive assembly pipeline from <xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>. We first extracted 229,464 reads with 2.2-Gbp in <italic>D. mauritiana</italic>, 269,483 reads with 2.3-Gbp in <italic>D. simulans</italic>, and 257,722 reads with 2.6-Gbp in <italic>D. sechellia</italic> using assemblies from <xref ref-type="bibr" rid="bib32">Chakraborty et al., 2021</xref>, respectively. We then assembled these reads using Canu v1.3 and FALCON v0.5.0 combined the parameter tuning method on two error rates, eM and eg, in bogart to optimize the assemblies. We first made the Canu assemblies using the parameters ‘genomeSize = 30 m stopOnReadQuality = false corMinCoverage = 0 corOutCoverage = 100 ovlMerSize = 31’ and ‘genomeSize = 30 m stopOnReadQuality = false’. For FALCON v0.5.0, we used the parameters ‘length_cutoff = –1; seed_coverage = 30 or 40; genome_size = 30000000; length_cutoff_pr = 1000’. We then picked the assemblies with highest contiguity and completeness without detectable misassemblies from each setting (two Canu settings and one Falcon setting).</p><p>After picking the three best assemblies for each species, we tentatively reconciled the assemblies using Quickmerge (<xref ref-type="bibr" rid="bib30">Chakraborty et al., 2016</xref>). We examined and manually curated the merged assemblies. For the <italic>D. mauritiana</italic> assembly, we merged two Canu and one FALCON assemblies, and for our <italic>D. simulans</italic> and <italic>D. sechellia</italic> assemblies, we merged one Canu and one FALCON assemblies independently. We manually curated some conserved Y-linked genes using raw reads and cDNA sequences from NCBI, including <italic>kl-3</italic> of <italic>D. mauritiana</italic>, <italic>kl-3</italic>, <italic>kl-5</italic>, and <italic>PRY</italic> of <italic>D. simulans</italic> and <italic>CCY</italic>, <italic>PRY</italic>, and <italic>Ppr-Y</italic> of <italic>D. sechellia</italic>, due to their low coverage and importance for our phylogenetic analyses. We then merged our heterochromatin restricted assemblies with contigs of the major chromosome arms from <xref ref-type="bibr" rid="bib32">Chakraborty et al., 2021</xref>. We polished the resulting assemblies once with Quiver using PacBio reads (SMRT Analysis v2.3.0; <xref ref-type="bibr" rid="bib43">Chin et al., 2013</xref>) and ten times with Pilon v1.22 (<xref ref-type="bibr" rid="bib164">Walker et al., 2014</xref>) using raw Illumina reads with parameters ‘--mindepth 3 --minmq 10 --fix bases’.</p><p>We identified misassemblies and found parts of Y-linked sequences in the contigs from major arms using our female/male coverage assays in <italic>D. sechellia</italic>. We also assembled the total reads (assuming genome size of 180 Mb) and heterochromatin-extracted reads (assuming genome size 40 Mb) using wtdbg v2.4 with parameters ‘-x rs -t24 -X 100 -e 2’ (<xref ref-type="bibr" rid="bib147">Ruan and Li, 2020</xref>) and Flye v2.4.2 (<xref ref-type="bibr" rid="bib83">Kolmogorov et al., 2019</xref>) with default parameters separately. We polished the resulting wtdbg assemblies with raw Pacbio reads using Flye v2.4.2. We then manually assembled five introns and fixed two misassemblies using sequences from wtdbg whole-genome assemblies (two introns), Flye whole-genome (two introns), and heterochromatin-enriched assemblies (one intron) in <italic>D. sechellia</italic>. We assembled one intron using sequences from wtdbg whole-genome assemblies in <italic>D. simulans</italic>.</p><p>We also extracted potential microbial reads (except for <italic>Wolbachia</italic>) that mapped to the <italic>D. sechellia</italic> microbial contigs, and assembled these reads into a 4.5 Mb contig, which represents the whole genome of a <italic>Providencia</italic> species, using Canu v 1.6 (r8426 14,520f819a1e5dd221cc16553cf5b5269227b0a3) with parameters ‘genomeSize = 5 m useGrid = false stopOnReadQuality = false corMinCoverage = 0 corOutCoverage = 100’. To detect other symbiont-derived sequences in our assemblies, we used Blast v2.7.1+ (<xref ref-type="bibr" rid="bib3">Altschul et al., 1990</xref>) with blobtools (v1.0; <xref ref-type="bibr" rid="bib89">Laetsch and Blaxter, 2017</xref>) to search the nt database (parameters ‘-task megablast -max_target_seqs 1 -max_hsps 1 -evalue 1e-25’). We estimated the Illumina coverage of each contig in males for <italic>D. mauritiana</italic>, <italic>D. simulans,</italic> and <italic>D. sechellia</italic>, respectively. We designated and removed contigs homologous to bacteria and fungi in subsequent analyses (<xref ref-type="supplementary-material" rid="supp19">Supplementary file 19</xref>).</p></sec><sec id="s4-2"><title>Generating DNA-seq from males in the <italic>D. simulans</italic> clade</title><p>We extracted DNA from 30 virgin 0-day males using DNeasy Blood & Tissue Kit and diluted it in 100 µL ddH<sub>2</sub>O. The DNA was then treated with 1 µL 10 mg/mL RNaseA (Invitrogen) at 37 °C for 1 hr and was re-diluted in 100 µL ddH<sub>2</sub>O after ethanol precipitation. The size and concentration of DNA were analyzed by gel electrophoresis, Nanodrop, Qubit and Genomic DNA ScreenTape. Finally, we constructed libraries using PCR-free standard Illumina kit and sequenced 125 bp paired-end reads with a 550 bp insert size from the libraries using Hiseq 2500 in UR Genomics Research Center. We deposited the reads in NCBI’s SRA under BioProject accession number PRJNA748438.</p></sec><sec id="s4-3"><title>Identifying Y-linked contigs</title><p>To assign contigs to the Y chromosome, we used Illumina reads from male and female PCR-free genomic libraries (except females of <italic>D. mauritiana</italic>) as described in <xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>. In short, we mapped the male and female reads separately using BWA (v0.7.15; <xref ref-type="bibr" rid="bib101">Li and Durbin, 2010</xref>) and called the coverage of uniquely mapped reads per site with samtools (v1.7; -Q 10 <xref ref-type="bibr" rid="bib100">Li et al., 2009</xref>). We further assigned contigs with the median of male-to-female coverage across contigs equal to 0 as Y-linked. We examined the sensitivity and specificity of our methods using all 10 kb regions with known location. Based on our results for 10 kb regions with known location (<xref ref-type="supplementary-material" rid="supp2">Supplementary file 2</xref>) in <italic>D. mauritiana</italic>, we set up an additional criterion for this species—‘the average of female-to-male coverage < 0.1’—to reduce the false discovery rate.</p></sec><sec id="s4-4"><title>Gene and repeat annotations</title><p>We used the same pipeline and data to annotate genomes as a previous study (<xref ref-type="bibr" rid="bib32">Chakraborty et al., 2021</xref>). We collected transcripts and translated sequences from <italic>D. melanogaster</italic> (r6.14) and transcript sequences from <italic>D. simulans</italic> <xref ref-type="bibr" rid="bib124">Nouhaud, 2018</xref> using IsoSeq3 (<xref ref-type="bibr" rid="bib61">Gordon et al., 2015</xref>). We mapped these sequences to each assembly to generate annotations using maker2 (v2.31.9; <xref ref-type="bibr" rid="bib69">Holt and Yandell, 2011</xref>). We further mapped the transcriptomes using Star 2.7.3 a 2-pass mapping with the maker2 annotation and parameters ‘-outFilterMultimapNmax 200 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 5000000 --alignMatesGapMax 5000000 --outSAMtype BAM SortedByCoordinate --readFilesCommand zcat --peOverlapNbasesMin 12 --peOverlapMMp 0.1’. We then generated the consensus annotations using Stringtie 2.0.3 from all transcriptomes (<xref ref-type="bibr" rid="bib131">Pertea et al., 2015</xref>). We further improved the mitochondria annotation using MITOS2. We assigned predicted transcripts to their homologs in <italic>D. melanogaster</italic> using BLAST v2.7.1+ (-evalue 1e-10; <xref ref-type="bibr" rid="bib3">Altschul et al., 1990</xref>).</p><p>We used RepeatMasker v4.0.5 (<xref ref-type="bibr" rid="bib153">Smit et al., 2013</xref>) with our custom library to annotate the assemblies using parameter ‘-s.’ Our custom library is modified from <xref ref-type="bibr" rid="bib32">Chakraborty et al., 2021</xref>, by adding the consensus sequence of <italic>Jockey-3</italic> from <italic>D. melanogaster</italic> to replace its homologs (<italic>G2</italic> in <italic>D. melanogaster</italic> and <italic>Jockey-3</italic> in <italic>D. simulans</italic>; <xref ref-type="bibr" rid="bib36">Chang et al., 2019</xref>). We extracted the sequences and copies of TEs and other repeats using scripts modified from <xref ref-type="bibr" rid="bib13">Bailly-Bechet et al., 2014</xref>. To annotate tandem repeats in assemblies, we used TRFinder (v4.09; <xref ref-type="bibr" rid="bib15">Benson, 1999</xref>) with parameters ‘2 7 7 80 10 100 2000 -ngs -h’. We also used kseek (<xref ref-type="bibr" rid="bib166">Wei et al., 2018</xref>) to search for tandem repeats in the male Illumina reads.</p></sec><sec id="s4-5"><title>Transcriptome analyses</title><p>We mapped the testes transcriptome to the reference genomes of <italic>D. melanogaster, D. simulans,</italic> and <italic>D. mauritiana</italic> (<xref ref-type="supplementary-material" rid="supp20">Supplementary file 20</xref>; no available transcriptome from <italic>D. sechellia</italic>). We used Stringtie 2.0.3 (<xref ref-type="bibr" rid="bib131">Pertea et al., 2015</xref>) to estimate the expression level using the annotation. However, we applied a different strategy for estimating expression levels of the Y-linked gene families due to the difficulties in precisely annotating multi-copies genes. We constructed a transcript reference using current gene annotation but replaced all transcripts from <italic>Lhk-1, Lhk-2,</italic> and <italic>CK2ßtes-Y</italic> with their species-specific reconstructed ancestral copies. We then mapped the transcriptome reads to this reference using Bowtie2 v 2.3.5.1 (<xref ref-type="bibr" rid="bib92">Langmead and Salzberg, 2012</xref>) with parameters ‘-very-sensitive -p 24 k 200 X 1000 --no-discordant --no-mixed’. We then estimated the expression level by salmon v 1.0.0 (<xref ref-type="bibr" rid="bib129">Patro et al., 2017</xref>) with parameters ‘-l A -p 24.’ We also mapped small RNA reads from <italic>D. simulans</italic> testes to our custom repeat library and reconstructed ancestral <italic>Lhk-1, Lhk-2,</italic> and <italic>CK2ßtes-Y</italic> sequences using Bowtie v 1.2.3 (<xref ref-type="bibr" rid="bib91">Langmead, 2010</xref>) with parameters ‘-v3 -q -a -m 50 --best –strata.’</p><p>To assay the specific expression of different copies, we also mapped transcriptomic and male genomic reads to the same reference using BWA (v0.7.15; <xref ref-type="bibr" rid="bib101">Li and Durbin, 2010</xref>). We used ABRA v2.22 (<xref ref-type="bibr" rid="bib121">Mose et al., 2019</xref>) to improve the alignments around the indels of these two gene families. We used samtools (v1.7; <xref ref-type="bibr" rid="bib100">Li et al., 2009</xref>) to pile up reads that mapped to reconstructed ancestral copies and estimated the frequency of derived SNPs in the reads.</p></sec><sec id="s4-6"><title>Estimating Y-linked exon copy numbers using Illumina reads</title><p>We mapped the Illumina reads from the male individuals of <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade species to a genome reference with transcripts of 11 conserved Y-linked genes and the sequences of all non-Y chromosomes (r6.14) in <italic>D. melanogaster</italic>. We called the depth using samtools depth (v1.7; <xref ref-type="bibr" rid="bib100">Li et al., 2009</xref>), and estimated the copy number of each exon using the mapped depth. We assumed most Y-linked exons are single-copy, so we divided the depth of each site by the majority of depth across all Y-linked transcripts to estimate the copy number. For the comparison, we simulated the 50 X Illumina reads from our assemblies using ART 2.5.8 with the parameter (art_illumina -ss HSXt -m 500 s 200 p -l 150 f 50; <xref ref-type="bibr" rid="bib70">Huang et al., 2012</xref>). We then mapped the simulated reads to the same reference, called the depth, and divided the depth of each site by 50.</p></sec><sec id="s4-7"><title>Immunostaining and FISH of mitotic chromosomes</title><p>We conducted FISH in brain cells following the protocol from <xref ref-type="bibr" rid="bib94">Larracuente and Ferree, 2015</xref> and immunostaining with FISH (immune-FISH) in brain cells following the protocol from <xref ref-type="bibr" rid="bib135">Pimpinelli et al., 2011</xref> and <xref ref-type="bibr" rid="bib36">Chang et al., 2019</xref>. Briefly, we dissected brains from third instar larva in 1 X PBS and treated them for 1 min in hypotonic solution (0.5% sodium citrate). Then, we fixed brain cells in 1.8% paraformaldehyde, 45% acetic acid for 6 min. We subsequently dehydrated in ethanol for the FISH experiments but not for the immune-FISH.</p><p>For immunostaining, we rehydrated the slide using PBS with 0.1% TritonX-100 after removing the coverslip using liquid nitrogen. The slides were blocked with 3% BSA and 1% goat serum/ PBS with 0.1% TritonX-100 for 30 min and hybridized with 1:500 anti-Cenp-C antibody (gift from Dr. Barbara Mellone) overnight at 4 °C. We used 1:500 secondary antibodies (Life Technologies Alexa-488, 546, or 647 conjugated, 1:500) in blocking solution with 45 min room temperature incubation to detect the signals. We fixed the slides in 4% paraformaldehyde in 4XSSC for 6 min before doing FISH.</p><p>We added probes and denatured the fixed slides at 95 °C for 5 min and then hybridized slides at 30°C overnight. For PCR amplified probes with DIG or biotin labels, we blocked the slides for 1 hr using 3% BSA/PBS with 0.1% Tween and incubated slides with 1:200 secondary antibodies (Roche) in 3% BSA/4 X SSC with 0.1% Tween and BSA at room temperature for 1 hr. We made <italic>Lhk</italic> and <italic>CK2ßtes-Y</italic> probes using PCR Nick Translation kits (Roche) and ordered oligo probes from IDT. We list probe information in <xref ref-type="supplementary-material" rid="supp3">Supplementary file 3</xref>. We mounted slides in Diamond Antifade Mountant with DAPI (Invitrogen) and visualized them on a Leica DM5500 upright fluorescence microscope, imaged with a Hamamatsu Orca R2 CCD camera and analyzed using Leica’s LAX software. We interpreted the binding patterns of Y chromosomes using the density of DAPI staining solely.</p></sec><sec id="s4-8"><title>Phylogenetic analyses of Y-linked genes</title><p>We used BLAST v2.7.1+ (<xref ref-type="bibr" rid="bib3">Altschul et al., 1990</xref>) to extract the sequences of Y-linked duplications and conserved Y-linked genes from the genome. We only used high-quality sequences polished by Pilon (--mindepth 3 --minmq 10) for our phylogenetic analyses. We aligned and manually inspected sequences with reference transcripts from Flybase using Geneious v8.1.6 (<xref ref-type="bibr" rid="bib80">Kearse et al., 2012</xref>). For most Y-linked duplications, except for the genes homologous to <italic>Lhk</italic> and <italic>CK2ßtes-Y</italic>, we constructed neighbor-joining trees using the HKY model with 1000 replicates using Geneious v8.1.6 (<xref ref-type="bibr" rid="bib80">Kearse et al., 2012</xref>) to infer their phylogenies. We also measured the length and microhomology in 223 indels from 21 Y-linked duplications using these alignments (<xref ref-type="supplementary-material" rid="supp15">Supplementary file 15</xref>). We also infer the potential mechanisms causing the indels, including tandem duplications and polymerase slippage during DNA replication. We measured the length and microhomology of polymorphic indels in <italic>D. melanogaster</italic> (DGRP <xref ref-type="bibr" rid="bib71">Huang et al., 2014</xref>) and <italic>D. simulans</italic> (<xref ref-type="bibr" rid="bib151">Signor et al., 2018</xref>) populations from <xref ref-type="bibr" rid="bib32">Chakraborty et al., 2021</xref>. For <italic>Lhk</italic> and <italic>CK2ßtes-Y</italic>, we constructed phylogeny using iqtree 1.6.12 (<xref ref-type="bibr" rid="bib123">Nguyen et al., 2015</xref>; <xref ref-type="bibr" rid="bib68">Hoang et al., 2018</xref>) using parameters “-m MFP -nt AUTO -alrt 1000 -bb 1000 -bnni”. The node labels in <xref ref-type="fig" rid="fig5">Figure 5</xref> correspond to SH-aLRT support (%) / ultrafast bootstrap support (%). The nodes with SH-aLRT ≥ 80% and ultrafast bootstrap support ≥ 95% are strongly supported. Protein evolutionary rates (with CodonFreq = 0/1/2 in PAML) of the bold branches were estimated using PAML with branch models on the reconstructed ancestor sequences (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref> and <xref ref-type="fig" rid="fig6s3">Figure 6—figure supplement 3</xref>).</p></sec><sec id="s4-9"><title>Estimating recombination and selection on Y-linked ampliconic genes</title><p>Using the phylogenetic trees from iqtree, we infer the most probable sequences for the internal nodes using MEGA 10.1.5 (<xref ref-type="bibr" rid="bib88">Kumar et al., 2018</xref>; <xref ref-type="bibr" rid="bib156">Stecher et al., 2020</xref>) using the maximal likelihood method and G + I model with GTR model. We conducted branch and branch-site models tests in PAML 4.8 using the ancestral sequences of Y-linked and X-linked ampliconic gene families with their homologs on autosomes. We plotted the tree using R package ape 5.3 (<xref ref-type="bibr" rid="bib127">Paradis et al., 2004</xref>).</p><p>We used compute 0.8.4 (<xref ref-type="bibr" rid="bib160">Thornton, 2003</xref>) to calculate Rmin and population recombination rates based on linkage disequilibrium (<xref ref-type="bibr" rid="bib73">Hudson, 1987</xref>; <xref ref-type="bibr" rid="bib72">Hudson and Kaplan, 1985</xref>) and gene similarity. We included sites with indel polymorphisms in these analyses to increase the sample size (558–1544 bp alignments). We also reanalyzed data from <xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref> to include variant information from these sites. The high similarity between Y-linked ampliconic gene copies may lead us to overestimate gene conversion based on gene similarity (<xref ref-type="bibr" rid="bib73">Hudson, 1987</xref>). We therefore also reported the lower bound on the gene conversion rate using Rmin (<xref ref-type="bibr" rid="bib72">Hudson and Kaplan, 1985</xref>).</p></sec><sec id="s4-10"><title>GO term analysis</title><p>We used PANTHER (Released 20190711; <xref ref-type="bibr" rid="bib118">Mi et al., 2019</xref>) with GO Ontology database (Released 2019-10-08) to perform Biological GO term analysis of new Y-linked duplicated genes using Fisher’s exact tests with FDR correction. We input 70 duplicated genes with any known GO terms and used all genes (13,767) in <italic>D. melanogaster</italic> as background.</p></sec><sec id="s4-11"><title>Indel analyses</title><p>We downloaded the SNP calls (vcf files) from population genomic data in North Carolina of <italic>D. melanogaster</italic> (DGRP <xref ref-type="bibr" rid="bib71">Huang et al., 2014</xref>) and California of <italic>D. simulans</italic> (<xref ref-type="bibr" rid="bib151">Signor et al., 2018</xref>). We then used vcftools (<xref ref-type="bibr" rid="bib49">Danecek et al., 2011</xref>) to remove the low-quality SNPs using parameters ‘--maf 0.1 --keep-only-indels --min-alleles 2 --max-alleles 2 --recode’. We additionally filtered out the potential mismapped regions with ‘--max-missing-count 20’ in <italic>D. melanogaster</italic> or ‘--max-missing-count 17’ in <italic>D. simulans</italic>. Lastly, we analyzed the SNPs in the specific regions using bedtools intersect (<xref ref-type="bibr" rid="bib138">Quinlan and Hall, 2010</xref>) with gene annotation files (dmel-r5.57 or dsim annotation from maker2 v2.31.9; <xref ref-type="bibr" rid="bib69">Holt and Yandell, 2011</xref>). For the heterochromatic pseudogenes, we download 18 long-read polished assemblies from NCBI (<xref ref-type="supplementary-material" rid="supp20">Supplementary file 20</xref>). We then used blastn to get sequences of pseudogenes from the population, aligned, and surveyed their indel lengths. All the alignments for our indel assignment are available in the GitHub repository (<ext-link ext-link-type="uri" xlink:href="https://github.com/LarracuenteLab/simclade_Y">https://github.com/LarracuenteLab/simclade_Y</ext-link>; <xref ref-type="bibr" rid="bib38">Chang, 2022</xref>; copy archived at <ext-link ext-link-type="uri" xlink:href="https://archive.softwareheritage.org/swh:1:dir:73ec96265042d04d5c1c7497fe2276bd83309c6b;origin=https://github.com/LarracuenteLab/simclade_Y;visit=swh:1:snp:a9c367d00c0109078ac14c44d3c97515ce040ec4;anchor=swh:1:rev:b1939db576cb1616094a59775a38014a7d61eb7f">swh:1:rev:b1939db576cb1616094a59775a38014a7d61eb7f</ext-link>) and the Dryad digital repository (<ext-link ext-link-type="uri" xlink:href="https://doi.org/10.5061/dryad.280gb5mr6">https://doi.org/10.5061/dryad.280gb5mr6</ext-link>).</p></sec></sec></body><back><sec id="s5" sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="COI-statement" id="conf1"><p>No competing interests declared</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing</p></fn><fn fn-type="con" id="con2"><p>Investigation, Validation, Writing – review and editing</p></fn><fn fn-type="con" id="con3"><p>Investigation, Writing – review and editing</p></fn><fn fn-type="con" id="con4"><p>Data curation, Formal analysis, Funding acquisition, Investigation, Validation, Visualization, Writing – review and editing</p></fn><fn fn-type="con" id="con5"><p>Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing</p></fn></fn-group></sec><sec id="s6" sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="supp1"><label>Supplementary file 1.</label><caption><title>The copy number of exons in conserved Y-linked genes.</title><p>We listed the copy number of each exon in conserved Y-linked genes based on BLAST results.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp1-v2.xlsx"/></supplementary-material><supplementary-material id="supp2"><label>Supplementary file 2.</label><caption><title>The estimates of sensitivity and specificity of our Y-linked sequence assignment methods using 10 kb regions with known chromosomal location.</title><p>We calculated the median female-over-male coverage in our Illumina data in every 10 kb region with known chromosomal location. We then estimated the sensitivity and specificity of our methods using these data.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp2-v2.xlsx"/></supplementary-material><supplementary-material id="supp3"><label>Supplementary file 3.</label><caption><title>Probe and primer information.</title></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp3-v2.xlsx"/></supplementary-material><supplementary-material id="supp4"><label>Supplementary file 4.</label><caption><title>The genomic location of duplicated exons in conserved Y-linked genes.</title><p>We listed the genomic location of each exon in conserved Y-linked genes in our assemblies based on BLAST results.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp4-v2.xlsx"/></supplementary-material><supplementary-material id="supp5"><label>Supplementary file 5.</label><caption><title>The intron length of all conserved Y-linked genes across species.</title><p>We showed the length of each Y-linked exon in all conserved Y-linked genes based on BLAST results. If there are multiple copies of an exon, we choose the copy with a complete open reading frame and the highest expression level.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp5-v2.xlsx"/></supplementary-material><supplementary-material id="supp6"><label>Supplementary file 6.</label><caption><title>The abundance of simple repeats in Illumina reads from male flies estimated with kseek and from our genome assemblies.</title><p>We used kseek to measure the relative abundance of simple repeats in our Illumina reads. We also used TRF finder to calculate repeat contents in our assemblies. We compared the two results and picked probes for our FISH experiments.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp6-v2.xlsx"/></supplementary-material><supplementary-material id="supp7"><label>Supplementary file 7.</label><caption><title>Recent Y-linked duplications in <italic>D. melanogaster</italic> and species in the <italic>D. simulans</italic> clade.</title><p>We list information on the recent Y-linked duplications and genes, including copy numbers, expression levels, phylogenies, and open reading frames. We also included some duplications from repetitive regions where we can date their origins.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp7-v2.xlsx"/></supplementary-material><supplementary-material id="supp8"><label>Supplementary file 8.</label><caption><title>Enriched GO terms in Y-linked duplicated genes in <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade.</title><p>We identified GO terms associated with genes that recently duplicated to the Y chromosome listed in <xref ref-type="supplementary-material" rid="supp7">Supplementary file 7</xref> using PANTHER (Released 20190711; [163]). We listed all GO terms significantly enriched in the duplication (FDR < 0.05).</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp8-v2.xlsx"/></supplementary-material><supplementary-material id="supp9"><label>Supplementary file 9.</label><caption><title>The summary of conserved Y-linked genes and ampliconic genes expression.</title><p>We summarized the expression level of conserved Y-linked genes and ampliconic genes. We sum up the gene expression for genes with multiple duplicated copies on Y chromosomes.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp9-v2.xlsx"/></supplementary-material><supplementary-material id="supp10"><label>Supplementary file 10.</label><caption><title>The number of small RNA reads mapped to the repetitive sequences and Y-linked gene families in the <italic>D. simulans</italic> clade.</title></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp10-v2.xlsx"/></supplementary-material><supplementary-material id="supp11"><label>Supplementary file 11.</label><caption><title>Gene conversion rates for Y-linked ampliconic genes in the <italic>D. simulans</italic> clade.</title><p>We listed the gene conversion rates and gene similarities on each Y-linked ampliconic gene family (<italic>e.g</italic>., <italic>Lhk-1, Lhk-2,</italic> and <italic>CK2ßtes-Y</italic>). We estimated gene conversion rates using both gene similarities (p) and population recombination rates (Rmin and rho).</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp11-v2.xlsx"/></supplementary-material><supplementary-material id="supp12"><label>Supplementary file 12.</label><caption><title>PAML results for branch and branch-site model analyses of <italic>Lhk</italic> in the <italic>D. simulans</italic> clade.</title><p>We showed raw results and LRT tests for branch and branch-site model analyses from PAML. We also report rates of protein evolution for each branch in each model and sites under positive selection in the branch-site model analyses.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp12-v2.xlsx"/></supplementary-material><supplementary-material id="supp13"><label>Supplementary file 13.</label><caption><title>The number of new mutations observed in highly and lowly expressed copies of Y-linked gene families.</title><p>We list the number of synonymous, nonsynonymous and UTR changes in highly and lowly expressed copies of Y-linked genes families. We suggest that highly expressed copies evolve under stronger selection (positive or purifying) than other copies. Therefore, we compared the number of synonymous changes over nonsynonymous changes in highly expressing copies to the other copies. See <xref ref-type="supplementary-material" rid="supp21">Supplementary file 21</xref> for detailed information.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp13-v2.xlsx"/></supplementary-material><supplementary-material id="supp14"><label>Supplementary file 14.</label><caption><title>PAML results for branch and branch-site model analyses of <italic>CK2ßtes-Y</italic> in the <italic>D. simulans</italic> clade.</title><p>We showed raw results and LRT tests for branch and branch-site model analyses from PAML. We also report rates of protein evolution for each branch in each model and sites under positive selection in the branch-site model analyses.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp14-v2.xlsx"/></supplementary-material><supplementary-material id="supp15"><label>Supplementary file 15.</label><caption><title>Indels in Y-linked duplications in <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade.</title><p>We listed the position and sizes of all indels we found in Y-linked duplications. We also inferred the potential microhomologies used for MHEJ repairing. We also infer other DSB repairing mechanisms, including tandem duplications and replication slippages, based on the sequence information.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp15-v2.xlsx"/></supplementary-material><supplementary-material id="supp16"><label>Supplementary file 16.</label><caption><title>Polymorphic indels in <italic>D. melanogaster</italic> and <italic>D. simulans</italic> populations.</title><p>We listed the position and sizes of polymorphic indels from <italic>D. melanogaster</italic> and <italic>D. simulans</italic> populations. We also inferred the potential microhomologies causing the deletions.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp16-v2.xlsx"/></supplementary-material><supplementary-material id="supp17"><label>Supplementary file 17.</label><caption><title>Repeat composition across chromosomes in <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade.</title><p>We list the composition of LTR retrotransposon, LINE, DNA transposons, satellite, simple repeats, rRNA, and other repeats across every chromosome in our assemblies.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp17-v2.xlsx"/></supplementary-material><supplementary-material id="supp18"><label>Supplementary file 18.</label><caption><title>The detail of repetitive sequences across chromosomes in <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade.</title><p>We list the total sequence length from each transposon or complex repeat on Y-linked contigs/scaffolds and other contigs/scaffolds in our assemblies.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp18-v2.xlsx"/></supplementary-material><supplementary-material id="supp19"><label>Supplementary file 19.</label><caption><title>The Illumina coverage and blast result for each contig in the <italic>D. simulans</italic> clade.</title><p>We used Blast v2.7.1+ [135] with blobtools (v1.0; [136]) to search the nt database (parameters “-task megablast -max_target_seqs 1 -max_hsps 1 -evalue 1e-25”). We estimated the Illumina coverage of each contig in males of <italic>D. mauritiana</italic>, <italic>D. simulans</italic> and <italic>D. sechellia</italic>, respectively.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp19-v2.xlsx"/></supplementary-material><supplementary-material id="supp20"><label>Supplementary file 20.</label><caption><title>The summary of reads data used in this study.</title></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp20-v2.xlsx"/></supplementary-material><supplementary-material id="supp21"><label>Supplementary file 21.</label><caption><title>The information and read coverage of each SNP in Y-linked gene families from Illumina reads.</title><p>We listed the coverage of each SNP in Y-linked gene from each RNA-seq replicate and DNA-seq. We also recorded their frequency in our assembly and their translated amino acid. We estimated the expression level of each variant based on the SNP frequency in the genome. We also performed Welch’s t-test to compare SNP frequency from DNA-seq and assemblies to it from RNA-seq. We further identify the SNPs associated with the allele that change more than 5 TPM compared to its estimated expression level from its frequency. The SNPs significant in the Welch’s t-test and located in lowly or highly expressing alleles are chosen to perform the Chi-square test.</p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife-75795-supp21-v2.xlsx"/></supplementary-material><supplementary-material id="transrepform"><label>Transparent reporting form</label><media mime-subtype="docx" mimetype="application" xlink:href="elife-75795-transrepform1-v2.docx"/></supplementary-material></sec><sec id="s7" sec-type="data-availability"><title>Data availability</title><p>Genomic DNA sequence reads are in NCBI's SRA under BioProject PRJNA748438. All scripts and pipelines are available in GitHub (<ext-link ext-link-type="uri" xlink:href="https://github.com/LarracuenteLab/simclade_Y">https://github.com/LarracuenteLab/simclade_Y</ext-link>; copy archived at <ext-link ext-link-type="uri" xlink:href="https://archive.softwareheritage.org/swh:1:dir:73ec96265042d04d5c1c7497fe2276bd83309c6b;origin=https://github.com/LarracuenteLab/simclade_Y;visit=swh:1:snp:a9c367d00c0109078ac14c44d3c97515ce040ec4;anchor=swh:1:rev:b1939db576cb1616094a59775a38014a7d61eb7f">swh:1:rev:b1939db576cb1616094a59775a38014a7d61eb7f</ext-link>) and the Dryad digital repository (doi:<ext-link ext-link-type="uri" xlink:href="https://doi.org/10.5061/dryad.280gb5mr6">https://doi.org/10.5061/dryad.280gb5mr6</ext-link>).</p><p>The following dataset was generated:</p><p><element-citation id="dataset1" publication-type="data" specific-use="isSupplementedBy"><person-group person-group-type="author"><name><surname>Chang</surname><given-names>C</given-names></name><name><surname>Gregory</surname><given-names>L</given-names></name><name><surname>Gordon</surname><given-names>K</given-names></name><name><surname>Meiklejohn</surname><given-names>C</given-names></name><name><surname>Larracuente</surname><given-names>A</given-names></name></person-group><year iso-8601-date="2021">2021</year><data-title>Genome sequencing of males in <italic>Drosophila</italic> simulans clade</data-title><source>NCBI BioProject</source><pub-id pub-id-type="accession" xlink:href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA748438">PRJNA748438</pub-id></element-citation></p><p><element-citation id="dataset2" publication-type="data" specific-use="isSupplementedBy"><person-group person-group-type="author"><name><surname>Chang</surname><given-names>C</given-names></name><name><surname>Gregory</surname><given-names>L</given-names></name><name><surname>Gordon</surname><given-names>K</given-names></name><name><surname>Meiklejohn</surname><given-names>CD</given-names></name><name><surname>Larracuente</surname><given-names>A</given-names></name></person-group><year iso-8601-date="2021">2021</year><data-title>Unique structure and positive selection promote the rapid divergence of <italic>Drosophila</italic> Y chromosomes</data-title><source>Dryad Digital Repository</source><pub-id pub-id-type="doi">10.5061/dryad.280gb5mr6</pub-id></element-citation></p><p>The following previously published datasets were used:</p><p><element-citation id="dataset3" publication-type="data" specific-use="references"><person-group person-group-type="author"><collab>Garrigan et al.</collab></person-group><year iso-8601-date="2012">2012</year><data-title><italic>Drosophila</italic> 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RNA-Seq</data-title><source>NCBI study</source><pub-id pub-id-type="accession" xlink:href="https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP001696">SRP001696</pub-id></element-citation></p><p><element-citation id="dataset6" publication-type="data" specific-use="references"><person-group person-group-type="author"><collab>Chakraborty et al.</collab></person-group><year iso-8601-date="2017">2017</year><data-title>DSPR Founder Genomes</data-title><source>NCBI BioProject</source><pub-id pub-id-type="accession" xlink:href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA418342/">PRJNA418342</pub-id></element-citation></p><p><element-citation id="dataset7" publication-type="data" specific-use="references"><person-group person-group-type="author"><collab>Wei et al.</collab></person-group><year iso-8601-date="2018">2018</year><data-title>D. melanogaster, D. simulans, D. sechellia, D. erecta, D. ananassae, D. pseudoobscura, D. persimilis, D mojavensis, and D. virilis Raw sequence reads</data-title><source>NCBI BioProject</source><pub-id pub-id-type="accession" xlink:href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA423291">PRJNA423291</pub-id></element-citation></p><p><element-citation id="dataset8" publication-type="data" specific-use="references"><person-group person-group-type="author"><collab>Laktionov et. al.</collab></person-group><year iso-8601-date="2018">2018</year><data-title>Genome-wide profiling of gene expression and transcription factors binding reveals new insights into the mechanisms of gene regulation during <italic>Drosophila</italic> spermatogenesis [RNA-Seq]</data-title><source>NCBI BioProject</source><pub-id pub-id-type="accession" xlink:href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA380909">PRJNA380909</pub-id></element-citation></p><p><element-citation id="dataset9" publication-type="data" specific-use="references"><person-group person-group-type="author"><collab>Lin et al.</collab></person-group><year iso-8601-date="2018">2018</year><data-title><italic>Drosophila</italic> simulans Raw sequence reads</data-title><source>NCBI BioProject</source><pub-id pub-id-type="accession" xlink:href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA477366">PRJNA477366</pub-id></element-citation></p><p><element-citation id="dataset10" publication-type="data" specific-use="references"><person-group person-group-type="author"><collab>Shah et al.</collab></person-group><year iso-8601-date="2020">2020</year><data-title>Novel quality metrics identify high-quality assemblies of piRNA clusters</data-title><source>NCBI BioProject</source><pub-id pub-id-type="accession" xlink:href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA618654/">PRJNA618654</pub-id></element-citation></p><p><element-citation id="dataset11" publication-type="data" specific-use="references"><person-group person-group-type="author"><name><surname>Kim</surname><given-names>BY</given-names></name></person-group><year iso-8601-date="2021">2021</year><data-title>Nanopore-based assembly of many drosophilid genomes</data-title><source>NCBI BioProject</source><pub-id pub-id-type="accession" xlink:href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA675888/">PRJNA675888</pub-id></element-citation></p><p><element-citation id="dataset12" publication-type="data" specific-use="references"><person-group person-group-type="author"><collab>Chakraborty et al.</collab></person-group><year iso-8601-date="2021">2021</year><data-title>Transcriptome sequencing of <italic>Drosophila</italic> simulans clade</data-title><source>NCBI BioProject</source><pub-id pub-id-type="accession" xlink:href="https://www.ncbi.nlm.nih.gov/bioproject/PRJNA541548">PRJNA541548</pub-id></element-citation></p></sec><ack id="ack"><title>Acknowledgements</title><p>This work was funded by the National Institutes of Health (NIH) (R35GM119515 to AML and R01GM123194 to CDM), National Science Foundation (NSF MCB 1844693) to AML and funding from the University of Nebraska-Lincoln to CDM. AML was supported by a Stephen Biggar and Elisabeth Asaro fellowship in Data Science. C-HC was supported by the Messersmith Fellowship from the U of Rochester, the Government Scholarship to Study Abroad from Taiwan, and the Damon Runyon fellowship (DRG: 2438–21). We thank our collaborators, Drs. JJ Emerson and Mahul Chakraborty, for generating PacBio reads in the <italic>D. simulans</italic> clade, Dr. Barbara Mellone for the antibodies, and Drs. Casey Bergman, Grace YC Lee, Kevin Wei and Anthony Geneva and Larracuente lab members for helpful discussion. 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that PacBio coverage is lower than expected on Y chromosomes and in heterochromatic regions generally (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). We found a similar bias in the <italic>D. melanogaster</italic> genome (<xref ref-type="bibr" rid="bib37">Chang and Larracuente, 2019</xref>), where the PacBio data were independently generated by a different group (<xref ref-type="bibr" rid="bib81">Kim et al., 2014</xref>). While a previous paper suggests that CsCl might contribute to this bias (<xref ref-type="bibr" rid="bib86">Krsticevic et al., 2015</xref>), we used Qiagen’s Blood and Cell culture DNA Midi Kit for DNA extraction. Heterochromatin is underreplicated in the endoreplicated cells that undergo multiple rounds of S phase but with no cell division such as those in larval salivary glands cells (<xref ref-type="bibr" rid="bib154">Smith and Orr-Weaver, 1991</xref>). Previous studies demonstrated that endoreplicated cells in the adult flies might contribute to lower coverage in Illumina sequencing data (<xref ref-type="bibr" rid="bib58">Flynn et al., 2020</xref>). Therefore, these endoreplicated cells might also contribute to the bias in Pacbio coverage.</p></sec><sec id="s9" sec-type="appendix"><title>Validation of variants in Y-linked gene families</title><p>We mapped Illumina reads from male genomic DNA and testis RNAseq to the reconstructed ancestral transcript sequences of each gene cluster (<italic>Lhk-1</italic>, <italic>Lhk-2</italic>, <italic>CK2ßtes-Y</italic>) to estimate the expression level of the different Y-linked copies. We first asked if the variants in these two gene families found in our assemblies can be consistently detected in Illumina reads from male genomes. We found that the abundance of derived variants in these two gene families in the DNA-seq data are highly correlated to the frequency of variants in our assemblies (<italic>R</italic> = 0.89 and 0.98 in <italic>D. mauritiana</italic> and <italic>D. simulans,</italic> respectively). For 559 variants in the <italic>D. simulans</italic> assembly, 33 of them (28 appear once and four appear twice) are missing from the DNA-seq data. For 446 variants in the <italic>D. mauritiana</italic> assembly, 43 of them (32 appear once and six appear twice) are missing from the DNA-seq data. Additionally, nine and eight inconsistent variants are located near ( < 100 bp) the start or end of transcripts in <italic>D. simulans</italic> and <italic>D. mauritiana</italic>, respectively. These regions at the edges of transcripts might have fewer Illumina reads coverage than more central regions.</p><p>We compared the proportion of synonymous and nonsynonymous changes between copies with high and low expression using transcriptome data to infer selection pressures on different mutations (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>; <xref ref-type="supplementary-material" rid="supp21">Supplementary file 21</xref>).</p><p>To reduce the effect of sequencing errors and simplify the phylogenetic analyses on protein evolution rates, we first reconstructed the ancestral sequences of each gene cluster (<italic>Lhk-1</italic>, <italic>Lhk-2</italic>, <italic>CK2ßtes-Y,</italic> and 2 <italic>CK2ßtes-like</italic>; see <xref ref-type="fig" rid="fig6">Figure 6</xref>). The reconstructed ancestral sequences should eliminate misassembled bases, which are typically singletons. We conducted branch-model and branch-site-model tests on the reconstructed ancestral sequence using PAML and inferred that both gene families experienced strong positive selection following their duplication to the Y chromosome (from branch model; <xref ref-type="supplementary-material" rid="supp17">Supplementary files 17 and 18</xref>, <xref ref-type="fig" rid="fig6">Figure 6</xref>). The high rate of protein evolution in the Y-linked ampliconic genes suggests that, in addition to subfunctionalization or degeneration, they may also acquire new functions and adapt to being Y-linked.</p></sec></app></app-group></back><sub-article article-type="editor-report" id="sa0"><front-stub><article-id pub-id-type="doi">10.7554/eLife.75795.sa0</article-id><title-group><article-title>Editor's evaluation</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Matute</surname><given-names>Daniel R</given-names></name><role specific-use="editor">Reviewing Editor</role><aff><institution>University of North Carolina, Chapel Hill</institution><country>United States</country></aff></contrib></contrib-group></front-stub><body><p>This manuscript by Chang et al. reports the evolutionary patterns of Y-chromosome evolution in <italic>Drosophila</italic>, providing perhaps the most comprehensive interspecific comparison of Y chromosomes available to date. They focus on four species of the <italic>melanogaster</italic> species subgroup and do extensive sequencing and assembly. The manuscript describes the pattern of divergence in these chromosomes, and uses comparative approaches to compare the drivers of evolution in flies and mammals. The authors suggest that the Y chromosome uses a different mechanism to repair double strand breaks than on autosomes. We were impressed by the novelty and rigor of the work as well as the overall presentation of the results.</p></body></sub-article><sub-article article-type="decision-letter" id="sa1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.75795.sa1</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Matute</surname><given-names>Daniel R</given-names></name><role>Reviewing Editor</role><aff><institution>University of North Carolina, Chapel Hill</institution><country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text id="box1"><p>In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.</p></boxed-text><p>[Editors' note: this paper was reviewed by <ext-link ext-link-type="uri" xlink:href="https://www.reviewcommons.org/">Review Commons</ext-link>.]</p></body></sub-article><sub-article article-type="reply" id="sa2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.75795.sa2</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><disp-quote content-type="editor-comment"><p>Reviewer #1 (Evidence, reproducibility and clarity (Required)):</p><p>Summary:</p><p>I found this an exceptionally impressive manuscript. The evolution of Y chromosomes has until recently been nearly impossible, and this research group have pioneered approaches that can yield reliable results in <italic>Drosophila</italic>. The study used an innovative heterochromatin-sensitive assembly pipeline on three D. simulans clade species, <italic>D. simulans</italic>, <italic>D. mauritiana</italic> and <italic>D. sechellia</italic>, which diverged less than 250 KYA, allowing comparisons with the group's previous results for the <italic>D. melanogaster</italic> Y.</p><p>The study is both technically impressive and extremely interesting (an highly unusual combination). It includes a rich set of interesting results about these genome regions, and furthermore the results are discussed in a well-organised way, relating both to previous observations and to understanding of the genetics and evolution of Y chromosomes, illuminating all these aspects. It is a rare pleasure to read such a study. I believe that this study will inspire and be a model for future work on these chromosomes. It shows how these difficult genome regions can be studied.</p></disp-quote><p>Thank you for the positive evaluation of our paper. While we did not make any specific revisions in response to these comments, we did attempt to improve the writing.</p><disp-quote content-type="editor-comment"><p>Major comments:</p><p>The conclusions are convincing. The methods are explained unusually clearly, and the reasoning from the results is convincing. When appropriate, the caveats, the caveats are clearly explained. The material is clearly organised and the questions studied are well related to the results. I had a few minor comments concerning the English. Even the figure (often a major problem to understand) are very clear and helpful, with proper explanations. I have very rarely read such a good manuscript, and almost never (in a long career) found a manuscript that could be published without revision being necessary.</p></disp-quote><p>Thank you for pointing out that there were minor concerns with the English. We have carefully gone through the manuscript and fixed some minor issues with the writing.</p><disp-quote content-type="editor-comment"><p>The analysis found 58 exons missed in previous assemblies (as well as all previously known exons of the 11 canonical Y-linked genes, which are present in at least one copy across the group). FISH on mitotic chromosomes using probes for 12 Y-linked sequences was used to determine the centromere locations, and to determine gene orders and relate them to the cytological chromosome bands, demonstrating changes in satellite distribution, gene order, and centromere positions between their Y chromosomes within the D. simulans clade species. It also confirmed previous results for Y-linked ribosomal DNA,genes, which are responsible for XY pairing in <italic>D. melanogaster</italic> males. Although 28S rDNA has been lost in D. simulans and <italic>D. sechellia</italic> (but not in D. mauritiana), the intergenic spacer (IGS) repeats between these repeats are retained on both sex chromosomes in all three species. Only sequencing can reliably reveal this, as their abundance is below the detection level by FISH in <italic>D. sechellia</italic>. The 11 canonical Y-linked genes' copy numbers vary between the species, and some duplicates are expressed and have complete open reading frames, and may therefore be functional because they, but most include only a subset of exons, often with duplicated exons flanking the presumed functional gene copy. Mega-introns and Y-loops were found, as already seen in <italic>Drosophila</italic> species, but this new study detects turn overs in the ~2 million years separating <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade. 49 independent duplications onto the Y chromosome were detected, including 8 not previously detected. At least half show no expression in testes, or lack open reading frames, so they are probably pseudogenes. Testis-expressed genes may be especially likely to duplicate into the Y chromosome due to its open chromatin structure and transcriptional activity during spermatogenesis, and indeed most of the new Y-linked genes in the species studied clade have likely functions in chromatin modification, cell division, and sexual reproduction.</p><p>The study discovered two new gene families that have undergone amplification on D. simulans clade Y chromosomes, reaching very high copy numbers (36-146). Both these families appear to encode functional protein-coding genes and show high expression. The paper described intriguing results that illuminate Y chromosome evolution.</p><p>First, SRPK, arose by an autosome-to-Y duplication of the sequence encoding the testis-specific isoform of the gene SR Protein Kinase (SRPK), after which the autosomal copy lost its testis-specific exon via a deletion. In <italic>D. melanogaster</italic>, SRPK is essential for both male and female reproduction, so the relocation of the testis-specific isoform to the Y chromosome in the D. simulans clade suggests that the change may have been advantageous by resolving sexual antagonism. The paper presents convincing evidence that the Y copy evolved under positive selection, and that gene amplification may confer advantageous increased expression in males. The second amplified gene family is also potentially related to an interesting function. Both Xlinked and Y-linked duplicates are found of a gene called Ssl located on chromosome 2R. In D. simulans, the X-linked copies were previously known, and called CK2ßtes-like. In <italic>D. melanogaster</italic>, degenerated Y-linked copies are also found, with little or no expression, contrasting with complete open reading frames and high expression in the D. simulans clade species in testes, consistent with the possibility of an arms race between sex chromosome meiotic drive factors.</p><p>Other interesting analyses document higher gene conversion rates compared to the other chromosomes, and evidence that these Y chromosomes may differ in the DNA-repair mechanisms (preferentially using MMEJ instead of NHEJ), perhaps contributing to their high rates of intrachromosomal duplication and structural rearrangements. The authors relate this to evidence for turnover of Y-linked satellite sequences, with the discovery of five new Y-linked satellites, whose locations were validated using FISH. The study also documented enrichment of LTR retrotransposons on the D. simulans clade Y chromosomes relative to the rest of the genome, together with turnovers between the species.</p><p>Reviewer #1 (Significance (Required)):</p><p>As described above, the advances are both, technical and conceptual for the field. The manuscript itself does an excellent job of placing the work in the context of the existing literature.</p><p>Anyone working on sex chromosomes and other non-recombining genome regions should be interested in the findings reported.</p><p>My field of expertise is the evolution of sex chromosomes, and the evolution of genome regions with suppressed recombination. I have experience of genomic analyses. I have less expertise in analyses of gene expression, but I understand enough about such approaches to evaluate the parts of this study that use them.</p><p>Reviewer #2 (Evidence, reproducibility and clarity (Required)):</p><p>The manuscript describes a thorough investigation of the Y-chromosomes of three very closely related <italic>Drosophila</italic> species (<italic>D. simulans</italic>, <italic>D. sechellia</italic>, and <italic>D. mauritiana</italic>) which in turn are closely related to <italic>D. melanogaster</italic>. The <italic>D. melanogaster</italic> Y was analysed in a previous paper by the same goup. The authors found an astonishing level of structural rearrangements (gene order, copy number, etc.), specially taking into account the short divergence time among the three species (~250 thousand years). They also suggest an explanation for this fast evolution: Y chromosome is haploid, and hence double-strand breaks cannot be repaired by homologous recombination. Instead, it must use the less precise mechanisms of NHEJ and MMEJ. They also provide circumstantial evidence that MMEJ (which is very prone to generate large rearrangements) is the preferred mechanism of repair. As far as I know this hypothesis is new, and fits nicely on the fast structural evolution described by the authors. Finally, the authors describe two intriguing Y-linked gene families in D. simulans (Lhk and CK2ßtes-Y), one of them similar to the Stellate / Suppressor of Stellate system of <italic>D. melanogaster</italic>, which seems to be evolving as part of a X-Y meiotic drive arms race. Overall, it is a very nice piece of work. I have four criticisms that, in my opinion, should be addressed before acceptance.</p></disp-quote><p>Thank you for your positive comments. We respond to your concerns point-by-point below.</p><disp-quote content-type="editor-comment"><p>The suggestion/conclusion that MMEJ is the preferential repair mechanism (over NHEJ) should be better supported and explained. At line 387, the authors stated "The pattern of excess large deletions is shared in the three D. simulans clade species Y chromosomes, but is not obvious in <italic>D. melanogaster</italic> (Figure 6B). However, because all <italic>D. melanogaster</italic> Y-linked indels in our analyses are from copies of a single pseudogene (CR43975), it is difficult to compare to the larger samples in the simulans clade species (duplicates from 16 genes). ". Given that <italic>D. melanogaster</italic> has many Y-linked pseudogenes (described by the authors and by other researchers, and listed in Table S6), there seems to be no reason to use a sample size of 1 in this species.</p></disp-quote><p>We only used pseudogenes with large alignable regions (>300 bp) to prevent the potential bias toward small indels and increase our confidence in indel calling. As a result, we excluded most of the duplicates on the <italic>D. melanogaster</italic> Y chromosome<italic>.</italic> We now include 5 additional <italic>D. melanogaster</italic> Y-linked indels in the manuscript, however, the majority of indels in this species (36/41) are still from the same gene.</p><disp-quote content-type="editor-comment"><p>Furthermore, given that <italic>D. melanogaster</italic> is THE model organism, it is the species that most likely will provide information to assess the "preferential MMEJ" hypothesis proposed by the authors.</p></disp-quote><p>A previous paper has shown that male flies deficient in MMEJ have a strong bias toward female offspring (McKee et al. 2000), suggesting that MMEJ is necessary for successfully producing Y-bearing sperm, consistent with our hypothesis. We agree with the reviewer that careful genetic and cytological experiments in <italic>D. melanogaster</italic> could further clarify the role of MMEJ in the repair of Y-linked mutations. Even more revealing would be experiments using the simulans clade species, where we hypothesize the MMEJ bias is even more pronounced on the Y chromosome. We believe, however, that these experiments are beyond the scope of this study and should merit their own papers.</p><disp-quote content-type="editor-comment"><p>Still on the suggestion/conclusion that MMEJ is the preferential repair mechanism (over NHEJ). Y chromosome in heterochromatic, haploid and non-recombining. In order to ascribe its mutational pattern to the haploid state (and the consequent impossibility of homologous recombination repair), the authors compared it to chromosome IV (the so called "dot chromosome"). This may not be the best choice: while chr IV lacks recombination in wild type flies, it is not typical heterochromatin. E.g., " results from genetic analyses, genomic studies, and biochemical investigations have revealed the dot chromosome to be unique, having a mixture of characteristics of euchromatin and of constitutive heterochromatin". Riddle and Elgin, FlyBook 2018 (https://doi.org/10.1534/genetics.118.301146). Given this, it seems appropriate to also compare the Y-linked pseudogenes with those from typical heterochromatin. In <italic>Drosophila</italic>, these are the regions around the centromeres ("centric heterochromatin"). There are pseudogenes there; e.g., the gene rolled is known to have partially duplicated exons.</p></disp-quote><p>Thank you for the suggestion. We now include the data from pericentric heterochromatin and pseudogenes in supplemental data (see Figure 7). Both data types support our conclusion that indel size is only larger on Y chromosomes, which is consistent with the comparison between the dot chromosome and pericentric heterochromatin reported by Blumenstiel et al. 2002.</p><disp-quote content-type="editor-comment"><p>In some passages of the manuscript there seems to be a confusion between new genes and pseudogenes, which should be corrected. For example, in line 261: "Most new Y-linked genes in <italic>D. melanogaster</italic> and the D. simulans clade have presumed functions in chromatin modification, cell division, and sexual reproduction (Table S7)".. Who are these "new genes"? If they are those listed in Table S6 (as other passages of the text suggest), most if not all of them are pseudogenes. If they are pseudogenes, it is not appropriate to refer to them as "new genes". The same ambiguity is present in line 263: "Y-linked duplicates of genes with these functions may be selectively beneficial, but a duplication bias could also contribute to this enrichment (…) " Pseudogenes can be selectively beneficial, but in very special cases (e.g.. gene regulation). If the authors are suggesting this, they must openly state this, and explain why. Pseudogenes are common in nearly all genomes, and should be clearly separated from genes (the later as a shortcut for functional genes). The bar for "genes" is much higher than simple sequence similarity, including expression, evidences of purifying selecion, etc., as the authors themselves applied for the two gene families they identified in D. simulans (Lhk and CK2ßtes-Y).</p></disp-quote><p>Thank you for the suggestion. We now state our criteria for calling genes based on the expression and long CDS and correct the sentences that the reviewer refers to. The protein evolution rates of many Y-linked duplicates were surveyed in Tobler et al. 2017, who found that most are not under strong purifying selection. Our study supports this previous report. We think that protein evolution rate alone may not be a good indicator for functionality. Our current study does not focus on the potential function of these genes, and we think further population studies are required to get a solid conclusion. We changed the text to clarify this point: “Most new Y-linked duplications in <italic>D. melanogaster</italic> and the <italic>D. simulans</italic> clade are from genes with presumed functions in chromatin modification, cell division, and sexual reproduction (Supplementary file 8, formerly Table S7), consistent with other <italic>Drosophila</italic> species [17, 77].” (p15 L281-284).</p><disp-quote content-type="editor-comment"><p>The authors center their analysis on "11 canonical Y-linked genes conserved across the melanogaster group ". Why did they exclude the CG41561 gene, identified by Mahajan and Bachtrog (2017) in <italic>D. melanogaster?</italic> Given that most <italic>D. melanogaster</italic> Y-linked genes were acquired before the split from the D. simulans clade (Koerich et al. Nature 2008), the same most likely is true for CG41561 (i.e., it would be Y-linked in the D. simulans clade). Indeed, computational analysis gave a strong signal of Y-linkage in D. yakuba (unpublished; I have not looked in the other species). If CG41561 is Y-linked in the simulans clade, it should be included in the present paper, for the only difference between it and the remaining "canonical genes" was that it was found later. Finally, the proper citation of the "11 canonical Y-linked genes" is Gepner and Hays PNAS 1993 and Carvalho, Koerich and Clark TIG 2009 (or the primary papers), instead of ref #55.</p></disp-quote><p>Thank you for the suggestion. <italic>CG41561</italic> is indeed a relatively young Y-linked gene because it’s not Y-linked in <italic>D. ananassae</italic> (Muller’s element E). We already have <italic>CG41561</italic> in Supplementary file 7 (formerly Table S6) and we think that it is reasonable to separate a young Y-linked gene from the others. We also fixed the reference as suggested (p5 L116).</p><disp-quote content-type="editor-comment"><p>Other points/comments/suggestions:</p><p>a. Possible reference mistake: line 88 "For example, 20-40% of <italic>D. melanogaster</italic> Y-linked regulatory variation (YRV) comes from differences in ribosomal DNA (rDNA) copy numbers [52, 53]." reference #53 is a mouse study, not <italic>Drosophila</italic>.</p></disp-quote><p>Thank you for pointing out this error, we fixed the reference (p4 L91).</p><disp-quote content-type="editor-comment"><p>b. Possible reference mistake: line 208 "and the genes/introns that produce Y-loops differs among species [75]". ref #75 is a paper on the D. pseudoobscura Y. Is it what the authors intended?</p></disp-quote><p>Yes, our previous paper (ref 75) found that Y-loops do not originate from the kl-3, kl-5, and ORY genes in <italic>D. pseudoobscura</italic> because they don’t have large introns in this species.</p><disp-quote content-type="editor-comment"><p>c. line 113. "We recovered all known exons of the 11 canonical Y-linked genes conserved across the melanogaster group, including 58 exons missed in previous assemblies (Table S1; [55])." Please show in the Table S1 which exons were missing in the previous assemblies. I guess that most if not all of these missing exons are duplicate exons (and many are likely to be pseudogenes). If they indeed are duplicate exons, the authors should made it clear in the main text, e.g., "We recovered all known exons of the 11 canonical Y-linked genes conserved across the melanogaster group, plus 58 duplicated exons missed in previous assemblies."</p></disp-quote><p>Thank you for the suggestion. However, the 58 exons did not include the duplicated exons. We are similarly surprised how much we will miss if we don’t assemble the Y chromosome carefully. We now mark these exons in red in Supplementary file 1 (formerly Table S1) to make this point clearer.</p><disp-quote content-type="editor-comment"><p>d. line 116 "Based on the median male-to-female coverage [22], we assigned 13.7 to 18.9 Mb of Y-linked sequences per species with N50 ranging from 0.6 to 1.2 Mb." The method (or a very similar one) was developed by Hall et al. BMC Genomics 2013, which should be cited in this context. (e) line 118: "We evaluated our methods by comparing our assignments for every 10-kb window of assembled sequences to its known chromosomal location. Our assignments have 96, 98, and 99% sensitivity and 5, 0, and 3% false-positive rates in <italic>D. mauritiana</italic>, <italic>D. simulans</italic>, and <italic>D. sechellia</italic>, respectively (Table S2). The procedure is unclear. Why break the contigs in 10kb intervals, instead of treating each as an unity, assignable to Y, X or A? The later is the usual procedure in computational identification of suspect Y-linked contigs (Carvalho and lark Gen Res 2013; Hall et al. BMC Genomics 2013). The only reason I can think for analyzing the contigs piecewise is a suspicion of misassemblies. If this is the case, I think it is better to explain.</p></disp-quote><p>Thank you for the suggestion. We did not break the contigs into 10kb intervals when we assigned the Y-linked contigs. As you suspect, our motivation for evaluating our methods and analyzing the contigs in 10kb intervals was to detect possible misassemblies. We rewrote the sentence to make this point clearer (p6 L129-132).</p><disp-quote content-type="editor-comment"><p>e. Figure 1. It may be interesting to put a version of Figure 1 in the SI containing only the genes and the lines connecting them among species, so we can better see the inversions etc. (like the cover of Genetics , based on the paper by Schaeffer et al. 2008).</p></disp-quote><p>Thank you for the suggestion. We would like to make a figure like that fantastic cover image you refer to, but the repetitive nature of the Y chromosome makes it difficult to illustrate rearrangements based on alignments at the contig-level. We instead opted to update Figure 1 to better highlight the rearrangements, still based on the unique protein-coding genes which are supported by the FISH experiments.</p><disp-quote content-type="editor-comment"><p>f. Table S6 (Y-linked pseudogenes). Several pseudogenes listed as new have been studied in detail before: vig2, Mocs2, Clbn, Bili (Carvalho et al. PNAS2015) Pka-R1, CG3618, Mst77F (Russel and Kaiser Genetics 1993; Krsticevic et al. G3 2015). Note also that at least two are functional (the vig2 duplication and some Mst77 duplications).</p></disp-quote><p>Thank you for the suggestion. We now include a column to indicate the potential function of Y-linked duplicates (see Supplementary file 7, formerly Table S6).</p><disp-quote content-type="editor-comment"><p>g. line 421: "one new satellite, (AAACAT)n, originated from a DM412B transposable element, which has three tandem copies of AAACAT in its long terminal repeats." The birth of satellites from TEs has been observed before, and should be cited here. Dias et al. GBE 6: 1302-1313, 2014.</p></disp-quote><p>Thank you for the suggestion. We now include a sentence to cite this reference (p27 L467-468).</p><disp-quote content-type="editor-comment"><p>h. Figure S2 shows that the coverage of PacBio reads is smaller than expected on the Y chromosome. Any explanation? This has been noticed before in <italic>D. melanogaster</italic>, and tentatively attributed to the CsCl gradient used in the DNA purification (Carvalho et al. GenRes 2016). However, it seems that the CsCl DNA purification method was not used in the simulans clade species (is it correct?). Please explain the in the manuscript, or in the SI. The issue is relevant because PacBio sequencing is widely believed to be unbiased in relation to DNA sequence composition (e.g., Ross et al. Genome Biol 2013).</p></disp-quote><p>Yes, we used Qiagen's Blood and Cell Culture DNA Midi Kit for DNA extraction. We suspect that the underrepresentation of Y-linked reads is driven by the presence of endoreplicated tissue in adults. Heterochromatin is underreplicated in endoreplicated cells, and thus there may simply be less heterochromatin in these tissues. Consistent with this idea, we find that all heterochromatin seems to be underrepresented in the reads, not just the Y chromosome (see Chakraborty et al. 2021; Flynn et al. 2020). We now include this discussion in the SI of our paper (see supplementary text p75).</p><disp-quote content-type="editor-comment"><p>i. I may have missed it, but in which public repository have the assemblies been deposited?</p></disp-quote><p>We link to the assemblies in Github (https://github.com/LarracuenteLab/simclade_Y) and they will also be in the Dryad Digital Repository (doi forthcoming).</p><disp-quote content-type="editor-comment"><p>Reviewer #3 (Evidence, reproducibility and clarity (Required)):</p><p>Due to suppressed recombination, Y chromosomes have degenerated, undergone extensive structural rearrangements, and accumulated ampliconic gene families across species. The molecular processes and selective pressures guiding dynamic Y chromosome evolution are not well understood. In this study, Chang et al. generate updated Y assemblies of three closely related species in the D. simulans complex using long-read PacBio sequencing in combination with FISH. Despite having diverged only 250,00 years ago, the authors find structural rearrangements, two newly amplified gene families and evidence of positive selection across D. simulans. The authors also suggest the high level of Y duplications and deletions may be mediated by MMEJ biased repair.</p></disp-quote><p>Our aim is to discover and understand the many different factors and processes that shape the evolution of Y chromosome organization and function. Because these Y chromosomes were largely unassembled, we needed to first generate the sequence assembly before we could ask specific questions. We prefer not to focus the manuscript solely on one specific topic such as MMEJ repair, as our other observations and analyses may be interesting to a wide range of scientists studying topics other than mutation and DNA repair. We are therefore choosing to present the more comprehensive story about Y chromosome evolution that we included in our original manuscript.</p><p>We also respectfully disagree with the comment that our paper is just a descriptive survey of Y chromosomal sequence features. On the contrary, we present thorough evolutionary analyses to test hypotheses about the forces shaping the evolution of Y chromosome organization and Y-linked genes. Specifically, we use molecular evolution and phylogenetic and comparative genomics approaches to show that multi-copy gene families experience rampant gene conversion and positive selection. We posit that one simulans clade-specific Y-linked gene family has undergone subfunctionalization, potentially resolving sexual conflict, and another may be involved in meiotic drive. We also use evolutionary genomic approaches to show that the distribution of Y-linked mutations indeed suggests that Y chromosomes disproportionately use MMEJ and we propose that this unique feature may shape the evolution of Y chromosome structural organization. This is, as far as we know, a novel hypothesis. We think that follow-up studies of either hypothesis merit different papers.</p><disp-quote content-type="editor-comment"><p>Major concerns:</p><p>1. Title: The authors use "unique structure" in the title, which is a vague point. Are not Y chromosomes, or any chromosome, "unique" in some manner? Also are there not more evolutionary processes governing the rapid divergence of the Y's.</p></disp-quote><p>Thank you for raising your concern. We believe that we are justified in referring to the Y chromosome as unique among all other chromosomes in its structural properties (e.g. combination of its hemizygosity, abundant tandem repeats, large scale rearrangements, and highly amplified testis-specific genes). Because there are many properties of Y chromosomes that we believe contribute to their rapid divergence, we opted for the general phrase ‘unique structure’ to capture all of these features. Many evolutionary processes likely shape the evolution of that unique structure (e.g. Muller’s Ratchet, background selection, Hill Robertson effects; see Charlesworth and Charlesworth 2000 for a review), and these processes are well-studied, especially on newly evolved sex chromosomes. Here our focus is on evolutionarily old Y chromosomes, which may have comparatively fewer targets of purifying selection and are more likely to be shaped by positive selection (Bachtrog 2008).</p><disp-quote content-type="editor-comment"><p>2. p.2, line 53-56: The authors claim that sexually antagonistic selection and regulatory evolution are causes of recombination suppression. Couldn't this statement be reversed? Recombination suppression via inversions or other rearrangements enable sexually antagonistic selection. This is a chicken or egg question, so it should be revised to have both possibilities be equal.</p></disp-quote><p>Thank you for the suggestion. We think that it is unlikely that recombination suppression itself is beneficial, but for sexually antagonistic selection and regulatory evolution, recombination suppression can have short-term benefits. We rephrased this sentence to be agnostic about the direction (p2 L56).</p><disp-quote content-type="editor-comment"><p>3. p.5, 118-120: Are the assemblies de novo or have they been guided based upon the D.</p><p>melanogaster Y chromosome assembly? Please clarify how the authors evaluate their methods by comparing their Y-sequence assignments to known chromosomal locations.</p></disp-quote><p>Thank you for the suggestion. We didn’t use <italic>D. melanogaster</italic> Y chromosome assembly to guide our assemblies. “All assemblies are generated de novo”, and thus we don’t think there is any potential bias. We first assigned Y-linked sequences using the presence of known Y-linked genes, and used this assignment to evaluate our methods. We now make the sentence clear (p5 L112).</p><disp-quote content-type="editor-comment"><p>4. While the gene copy number estimates are accurate, the PacBio-based genome assemblies are still not able to accurately assemble large segmental duplications (see Evan Eichler's laboratories recent primate and human genome assemblies). A statement mentioning the concerns about accuracy of the underlying sequence and genomic architecture shown should be included in the main text. FISH provides support for the location of the contigs, but not for the accuracy of the underlying genomic architecture.</p></disp-quote><p>Thank you for the suggestion. We can’t validate all Y-linked regions. We did validate the larger structural features of the assembly and only discuss the results that we are confident in. We now include sentences to address this concern (p7 L150-152).</p><disp-quote content-type="editor-comment"><p>5. The authors assigned Y-linked sequences based on median male-to-female coverage. Is this method feasible for assigning ampliconic sequence to the Y given the N50 of 0.6-1.2Mb? Are the authors potentially excluding novel Y-linked ampliconic sequence?</p></disp-quote><p>We validated our methods to assign contigs to a chromosome by comparing 10-kb intervals to the contigs with known chromosomal location, including the Y chromosome. Our assignments have high (96, 98, and 99%) sensitivity and low (5, 0, and 3%) falsepositive rates in <italic>D. mauritiana</italic>, <italic>D. simulans</italic>, and <italic>D. sechellia</italic>, respectively (see Supplementary file 2, formerly Table S2). Based on these results, we think that this method is reasonable for Y-linked contigs with N50 of 0.6-1.2Mb.</p><p>We might exclude some novel Y-linked sequences since we only assigned ~15Mb out of a total ~40 Mb Y-linked sequences. We acknowledged this possibility, and now include a sentence to address this concern (p31 L554-556).</p><disp-quote content-type="editor-comment"><p>6. Where did the rDNA sequences go in <italic>D. simulans</italic> and <italic>D. sechellia?</italic> Can they be detected on another chromosome?</p></disp-quote><p>Please see Figure S5 for detailed results. We found a few copies of rDNA on the contigs of autosomes. We assembled many copies of rDNA that can’t be confidently assigned to Y chromosomes. It’s possible that they might be located on other chromosomes. Based on our FISH data (Figure S4) and previous papers, most of these non-Y-linked rDNA copies should be on the X chromosome. However, in this study, we did not make a concerted effort to assign X-linked contigs.</p><disp-quote content-type="editor-comment"><p>7. Figure 2B is hard to follow and it is unclear what additional value it provides to part A. Why is expression level of specific exons important?</p></disp-quote><p>Exon duplication may be an important contributor to Y-linked gene evolution: most genes have duplications and our figure shows that at least some of these duplicates are expressed. The patterns we see indicate that duplication may play different roles in genes depending on their length. For example, the duplications involving short genes (e.g., ARY) may be functional and influence protein expression, whereas duplications involving large genes (e.g. kl-2) may not influence the overall protein expression level from this gene, although the expressed duplicated exons may play some other role. We revised a sentence in the main text and added a sentence to the figure 2 legend to make this point clearer.</p><disp-quote content-type="editor-comment"><p>8. Figure 3 There are many introns that contain gaps, so it is unclear how confident one can be in intron length when there are gaps.</p></disp-quote><p>Indeed, we are not confident about the length of introns with gaps. Therefore, we separated these introns and showed them in different colors.</p><disp-quote content-type="editor-comment"><p>9. Figure 4: What are the authors using as a common ancestor in this figure to infer duplications in the initial branch?</p></disp-quote><p>We used phylogenies to infer the origin of Y-linked duplicates. Any duplications that happened earlier than the divergence between four species are listed in the branch. We also edited the legend to make this point clearer.</p><disp-quote content-type="editor-comment"><p>10. p.15, paragraph 2: The authors describe a newly amplified gene, CK2Btes-Y, in D. simulans. In the first half of the paragraph the authors state that Y-linked copies are also found in <italic>D. melanogaster</italic> but have "degenerated and have little or no expression" and call them pseudogenes. Later in the paragraph, the authors state that the <italic>D. melanogaster</italic> Y-linked copies are Su(Ste), a source of piRNAs that are in conflict with X-linked Stellate. Lastly in the paragraph, the authors discuss Su(ste) as a <italic>D. melanogaster</italic> homolog of CK2Btes-Y. The logic of defining CK2Btes-Y origins is confusing. Was CK2Btes-Y independently amplified on the D. simulans Y, or were CK2BtesY and Su(Ste) amplified in a common ancestor but independently diverged?</p></disp-quote><p>The amplification of <italic>CK2Btes-Y</italic> and <italic>CK2Btes-like</italic> happened in the ancestor of <italic>D.</italic></p><disp-quote content-type="editor-comment"><p>melanogaster and D. simulans (Figure S11). However, both CK2Btes-Y and CK2Btes-like became pseudogenes (<italic>D. melanogaster</italic> CK2Btes-Y is named PCKR in a previous study) in <italic>D. melanogaster</italic>. On the other hand, Ste and Su(Ste) are only limited to D.</p></disp-quote><p><italic>melanogaster</italic> based on phylogenetic analyses (Figure 5A) and are a chimera of <italic>CK2Bteslike</italic> and <italic>NACBtes</italic>. The evolutionary history of this gene family has been detailed in other papers, except for the presence of <italic>CK2Btes-Y</italic> in the <italic>D. simulans</italic> complex, which we describe for the first time in this study. We now include a new figure (Figure 5B) a schematic of the inferred evolutionary history of sex-linked <italic>Ssl/CK2ßtes</italic> paralogs</p><disp-quote content-type="editor-comment"><p>11. Figure 5: Is each FISH signal a different gene copy?</p></disp-quote><p>Yes, based on our assemblies, <italic>Lhk-1</italic> and <italic>Lhk-2</italic> are mostly located on different contigs. Unfortunately, we are not able to design probes that can separate <italic>Lhk-1</italic> from <italic>Lhk-2</italic>.</p><disp-quote content-type="editor-comment"><p>12. The authors suggest DNA-repair on the Y chromosome is biased towards MMEJ based on indel size and microhomologies. Is there any evidence MMEJ is responsible for variable intron length in the canonical Y-linked genes or the amplification of new gene families? Since MMEJ is error-prone, it's a more tolerable repair mechanism in pseudogenes, so their findings might be biased. Rather than comparing pseudogenes to their parent genes, they should compare chrY pseudogenes to autosomal pseudogenes. Even more would be to track MMEJ on the dot chromosome which is known not recombine and is highly heterchromatic like the Y chromosome.</p></disp-quote><p>We did compare chrY pseudogenes to autosomal pseudogenes in our study. We also add new analyses to address other issues from reviewer 2, which are similar to your concern. We now include data from pericentric heterochromatin and pseudogenes (see Figure 7). Both data types support our conclusion that indel size is only larger on Y chromosomes. This is consistent with a report that the dot chromosome and pericentric heterochromatin have similar indel size distributions (Blumenstiel et al. 2002).</p><disp-quote content-type="editor-comment"><p>Reviewer #3 (Significance (Required)):</p><p>While it is a benefit to have much improved Y chromosome assemblies from the three D. simulans clade species, the gap in knowledge this manuscript is trying to address is unclear.</p><p>The manuscript is almost entirely descriptive and the figures are difficult to follow.</p></disp-quote><p>As stated above, we respectfully disagree with the comment that the manuscript is entirely descriptive, as we present thorough evolutionary analyses to test hypotheses about the forces shaping the evolution of Y chromosome organization and Y-linked genes. 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