Rapid evolution of Wolbachia in cherry fruit flies

Rapid evolution of Wolbachia in cherry fruit flies | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Rapid evolution of Wolbachia in cherry fruit flies View ORCID Profile Daniel J. Bruzzese , Hannes Schuler , View ORCID Profile Wee L. Yee , View ORCID Profile Aurel Holzschuh , Jeffrey L. Feder doi: https://doi.org/10.1101/2025.01.13.632697 Daniel J. Bruzzese 1 Department of Biological Sciences, University of Notre Dame , Notre Dame, Indiana, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel J. Bruzzese For correspondence: daniel.bruzzese{at}yale.edu Hannes Schuler 2 Faculty of Science and Technology, Free University of Bozen-Bolzano , Bozen-Bolzano, Italy 3 Competence Centre for Plant Health, Free University of Bozen-Bolzano , Bozen-Bolzano, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site Wee L. Yee 4 United States Department of Agriculture, Temperate Tree Fruit & Vegetable Research Unit, Agricultural Research Service , Wapato, Washington, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Wee L. Yee Aurel Holzschuh 1 Department of Biological Sciences, University of Notre Dame , Notre Dame, Indiana, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aurel Holzschuh Jeffrey L. Feder 1 Department of Biological Sciences, University of Notre Dame , Notre Dame, Indiana, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abstract Full Text Info/History Metrics Preview PDF Abstract Wolbachia is a widespread bacterial endosymbiont in arthropods known for its ability to manipulate host reproduction through cytoplasmic incompatibility (CI), thereby promoting its spread. However, Wolbachia harbors an array of mobile genetic elements (MGEs) that can rapidly alter its genomic structure and content, including the cif loci responsible for CI. At the phylogenetic scale, Wolbachia genomes are shown to be dynamic with MGEs causing widespread genome rearrangements. But on ecological time scales, the evolution of Wolbachia remains largely unknown. In this study, we leverage the natural history of Wolbachia in cherry-infesting fruit flies in the Rhagoletis cingulata sibling species group. Members of this species group share a common Wolbachia strain, w Cin2, and their divergence spans from 1,000 to 150,000 years ago. We utilized Nanopore sequencing to characterize w Cin2 Wolbachia genome divergence across recent to more distant evolutionary timescales. We report rapid evolution of population-level differences in gene content (including cif loci and MGEs) and genome structure, with differentiation increasing with time since host divergence. Notably, structural variants were the first to appear both between and among w Cin2 populations. Our results also indicated that the CI phenotype previously attributed to a distinct Wolbachia strain, w Cin3, that coinfects flies in the southwestern USA and Mexico, may instead be caused by cif genes that were horizontally transferred into w Cin2. Finally, we discovered a novel Wolbachia strain, w Ind, which appears to have been recently horizontally acquired by cherry fly populations in the Pacific Northwest of the USA. Our findings underscore the fluidity and rapid genome evolution of Wolbachia , with significant implications for cif gene dynamics and potential impacts on host evolutionary trajectories. Author Summary In our study, we explore the genome evolution of Wolbachia , a common bacterial endosymbiont found in many insects that can manipulate its hosts’ reproduction to help it spread more effectively. Wolbachia’s genome is shown to change dramatically over long evolutionary periods due to mobile genetic elements. However, little is known about how Wolbachia’s genome evolves over shorter ecological timeframes. Here, we utilized cherry-infesting fruit flies and their Wolbachia strain known as w Cin2, both of which have spread across North America for the past 150,000 years. Using long-read sequencing, we assembled multiple w Cin2 Wolbachia genomes from across North America and made comparisons between w Cin2 Wolbachia genomes that have recently diverged and comparisons to those that have been isolated for over 150,000 years. Our findings reveal that Wolbachia’s genome can evolve swiftly, with differences in gene content and most prominently genome structure arising between recently separated Wolbachia populations and increasing as populations diverge over time. Interestingly, these changes also impact the key genes responsible for Wolbachia’s reproductive manipulation. This research highlights the rapid and dynamic nature of Wolbachia genome evolution across different evolutionary timescales and offers insights into endosymbiont-host relationships as well as their potential impact on host speciation and vector control. Introduction The Alphaproteobacteria Wolbachia is one of the most common intracellular endosymbionts, infecting over 50% of all terrestrial arthropod species [ 1 ], as well as nematodes [ 2 ]. Primarily passed vertically from mother to offspring and occasionally horizontally between different host species [ 3 , 4 ], Wolbachia teeters between conflict and cooperation with its hosts to aid its spread. In some instances, Wolbachia hastens its spread within host populations by positively impacting host fitness via nutrient supplementation [ 5 – 7 ] or disease resistance [ 8 – 10 ]. Conversely, many Wolbachia strains selfishly manipulate host reproduction to invade new populations [ 11 ]. Wolbachia’s reproductive manipulation ranges from male killing, parthenogenesis, feminization, and, most commonly, cytoplasmic incompatibility (CI) [ 12 , 13 ]. CI occurs when male hosts infected with a Wolbachia strain mate with uninfected females or females infected with a different, incompatible strain, resulting in the embryonic death of offspring. As a result, selection acts in a frequency dependent manner on CI-inducing Wolbachia . Above a critical threshold frequency (usually about 10%), CI-inducing Wolbachia can rapidly sweep through a host population [ 14 , 15 ]. Yet, below this threshold, the endosymbiont does not increase in frequency and can be eliminated from host populations [ 14 , 15 ]. CI-inducing Wolbachia can potentially contribute to speciation by causing postmating reproductive isolation between hosts infected with different incompatible strains [ 16 – 18 ]. It is also possible that CI can select for increased premating reproductive isolation in its host through reinforcement, by favoring uninfected females that avoid mating with infected males [ 19 , 20 ]. Two Wolbachia genes, cifA and cifB that are associated with the Wolbachia prophage WO control the induction and rescue of CI [ 21 – 25 ]. Expression of cifA alone in females is sufficient to rescue CI in crosses with infected males [ 25 , 26 ]. The mechanism(s) behind CI induction, however, can vary between strains, with some systems requiring co-expression of cifA and cifB in male testes to induce CI [ 13 , 23 , 25 ], while in other systems, the expression of cifB alone suffices [ 27 ]. Phylogenetic analysis has identified five types (I-V) of cifA and cifB homologs that have co-diversified [ 28 , 29 ]. Each cif type shares common protein domains and the ability to rescue CI if induced by cif genes of the same type [ 13 ]. Adding further complexity, cif gene expression can also be influenced by the genetic background of the endosymbiont host, environmental conditions, and pseudogenization—all of which can impact the strength of CI [ 30 – 32 ]. The dynamic nature of Wolbachia genomes plays an important role in mediating both the transfer and expression of cif genes. Wolbachia genomes contain many active mobile genetic elements (MGEs), including prophages, transposable elements (TEs), and plasmids [ 33 – 37 ]. Active MGEs can transfer both prophage DNA, as well as nearby cif gene pairs, between Wolbachia strains [ 28 , 29 , 38 , 39 ]. In addition to horizontal gene transfer between Wolbachia strains, internal rearrangements within the Wolbachia genome itself may also alter the expression of cif genes. Normally, cifA and cifB genes are transcribed in tandem, with the cifA locus upstream of cifB [ 23 ]. MGEs can cause translocations or inversions that can separate tandem copies of cif genes or insertions/deletions that disrupt cif gene expression [ 40 ]. Finally, point mutations unrelated to MGEs can also accumulate in cif genes and cause their pseudogenization [ 29 , 41 , 42 ]. Over long-term, phylogenetic scales, Wolbachia genomes both accrue genomic rearrangements and exchange their cif gene modules, the latter of which is often independent of Wolbachia and host phylogenies [ 39 ]. There are considerably fewer studies on the short-term evolution of Wolbachia genomes on ecological timeframes, however, limited evidence does suggest that Wolbachia genomes can evolve rapidly over tens of years [ 43 – 45 ]. Cherry-infesting fruit flies from the Rhagoletis cingulata (Diptera: Tephritidae) sibling species group and their associated Wolbachia strain, w Cin2, provide an excellent system to study Wolbachia genome evolution across ecological to phylogenetic evolutionary timescales. Rhagoletis indifferens Curran, is endemic to the Pacific Northwest (PNW) region and Northern California of N. America and infests the fruit of bitter cherry ( Prunus emarginata ) [ 46 ] ( Fig 1A ). Rhagoletis cingulata (Loew), attacks black cherry ( P. serotina ) in eastern North America (ENA), the southwestern USA (SW), and in the Sierra Madre Oriental Mountains (SMO) and the Eje Volcánico Transversal de Mexicano (EVTM) regions of Mexico ( Fig 1A ) [ 47 – 50 ]. Genetic surveys of microsatellite loci revealed a pattern of clinal geographic variation among cherry flies across N. America, suggesting populations in the PNW, ENA, SW, SMO, and EVTM became isolated from one another due to the onset of warmer and drier conditions at the end of the Pleistocene and beginning of the Holocene ∼15,079 ya (95% credible interval 7,143 to 31,270 years) ( Fig 1B ) [ 49 ]. In contrast, mtDNA haplotypes display a disjunct geographic divide that differentiates cherry fly populations ( Fig 1B ). In this regard, R. cingulata in the SW, SMO, and EVTM possess a distinct mtDNA haplotype that differs from that shared between R. indifferens in the PNW and R. cingulata in ENA populations, with an estimated divergence time of ∼100,000 to 157,000 years [ 50 ]. Download figure Open in new tab Fig 1. Cherry fly populations and their genotypes. (A) Map of R. cingulata and R. indifferens populations from Eastern N. America (ENA), the Southwest (SW), Sierra Madre Oriental Mountains (SMO), the Eje Volcánico Transversal de Mexicano (EVTM), and two populations from the Pacific Northwest (PNW) (S1 Table). (B) A neighbor-joining tree of R. cingulata and R. indifferens from Doellman et al. (2020) [ 50 ] superimposed with mtDNA haplotypes (haplotype 1 in blue and haplotype 2 in red) and known associated Wolbachia strains. The disjunct geographic distribution of mtDNA haplotypes in cherry flies coincides with a difference in Wolbachia strain composition. All cherry fly individuals across N. America are infected by the same group A Wolbachia strain, w Cin2 [ 18 , 51 , 52 ]. However, Multi Locus Sequence Typing (MLST) indicates that R. cingulata flies in the SW, SMO, and EVTM are also coinfected by a second, highly diverged group B strain, w Cin3 [ 18 ]. Crosses between doubly infected SW to singly infected PNW and ENA cherry flies show a pattern consistent with w Cin3 inducing unidirectional CI [ 18 , 48 ]. It has therefore been hypothesized that a cherry fly population in Mexico became coinfected with the CI-inducing w Cin3 strain from a yet to be identified source 100,000 to 157,000 years ago, resulting in its association with the derived mtDNA haplotype. Subsequently, w Cin3 spread northward carrying the disjunct haplotype but was halted before it could reach the PNW or ENA due to climate change in the Holocene [ 18 ]. Hitchhiking of a mitochondrial haplotype with a spreading Wolbachia strain is not uncommon and is found in several other insect systems [ 53 , 54 ]. Given their recent Holocene separation across N. America and the more distant mtDNA divergence associated with the acquisition and sweep of w Cin3, the R. cingulata species group presents a model system for studying Wolbachia genome dynamics across different evolutionary timescales. Here, we leverage this natural history to investigate the dynamics of genome evolution for the universal w Cin2 Wolbachia strain that diverged with its cherry fly hosts across three timeframes: (1) Recent: between two populations of R. indifferens infesting bitter cherry, isolated by distance in the PNW by < 1,000 years; (2) Intermediate: between populations of R. indifferens in the PNW and R. cingulata infesting black cherry in the ENA, estimated to have an early Holocene divergence; and (3) Distant: between the R. cingulata populations in the SW versus R. indifferens in the PNW and R. cingulata in the ENA, where strains have been separated by > 100,000 years. Using Nanopore sequencing, we assembled complete Wolbachia genomes from these host fly populations and assessed them for differences in gene sequence content, cif genes, MGEs, and genome structure to characterize Wolbachia genome evolution over varying temporal scales. While earlier MLST analyses suggested that w Cin2 strains from the ENA and PNW were identical, with w Cin2 from the SW differing by only a single SNP [ 18 ], our whole genome approach found that w Cin2 strains are far more diverse than previously thought. The assembled w Cin2 genomes revealed differences in sequence identity, gene content, cif genes, MGEs, synteny, and structural rearrangements, with levels of differentiation increasing with time since separation. Crucially, our findings indicate that Wolbachia genomes, influenced by MGEs, can evolve rapidly and impact cif genes and subsequent host diversification via CI. Results Long-read sequencing generated six closed Wolbachia genomes Wolbachia genomes were assembled with 25X to 50X Oxford Nanopore Technologies (ONT) (Oxford, UK) long reads generated from high molecular weight (HMW) DNA extracted from individual flies from the PNW1, PNW2, and SW populations ( Fig 1 and S1 Table). Additionally, we included the completed 1.53 Mb w Cin2-ENA genome (GCF_017604245.1 [ 52 ]) in our analyses. All Wolbachia genomes assembled into single circular contigs with Benchmarking Universal Single-Copy Orthologs (BUSCO) scores greater than 98.70% based on the Rickettsiales dataset ( Fig 2 and S2 Table). For the PNW1 population, a single 1.53 Mb w Cin2-PNW1 strain was assembled. For the PNW2 population, two coinfecting Wolbachia strains were assembled: a 1.53 Mb w Cin2-PNW2 stain and a new, previously undescribed, 1.24 Mb Wolbachia strain, designated w Ind ( Fig 2 ). For the SW population, two coinfecting Wolbachia strains were also assembled: a 1.56 Mb w Cin2-SW strain and a 1.52 Mb w Cin3 strain. In silico annotations show that MGEs, including transposases, recombinases, and resolvases, make up a sizable portion of these assembled genomes, from 10.74% to 12.39% (S3 Table). We identified eight prophage regions in w Cin2-ENA, w Cin2-PNW1, and w Cin2-PNW2, but only three prophage regions in w Cin2-SW ( Fig 2 ). Additionally, the w Cin3 strain coinfecting flies in the SW contained eight prophage regions, while the w Ind strain coinfecting PNW2 flies contained two prophage regions ( Fig 2 ). Download figure Open in new tab Fig 2. Six closed Wolbachia genomes were assembled from four cherry fly populations. The inner ring represents forward and reverse coding sequences. The middle ring highlights predicted prophage regions. The outermost ring depicts functional and pseudogenized cif genes. See S2 Table for additional assembly statistics and for NCBI accession numbers. 33 pairs of cif genes were identified To catalogue the cif gene repertoire of the assembled cherry fly Wolbachia genomes, we used protein BLAST (BLASTp) searches using reference cif homologues (S4 Table). These searches identified 33 cif gene pairs representing all five major cif gene types in the assembled cherry fly Wolbachia genomes ( Fig 2 and S5 Table). Many functional and several pseudogenized cif genes were found, the latter indicated by the presence of missense frameshift mutations and stop codons. In general, cifB genes were more likely to be pseudogenized than cifA genes, agreeing that selection acts to maintain CI rescue functions but not CI induction [ 29 , 41 , 55 ]. Out of the 14 possible pseudogenized cif genes identified, only two were cifA genes compared to 12 cifB genes. Strains w Cin2-ENA, w Cin2-PNW1, and w Cin2-PNW2 shared five pairs of identical cif genes, comprising one pseudogenized pair of cif [T1] , one pair of cif [T3] , and three pairs of cif [T5] genes, with one of the three pairs of cif [T5] genes predicted to be pseudogenized ( Fig 2 , S5 Table, S1 Fig, and S2 Fig). The pair of cif [T1] genes had high identities (99%) to the Drosophila cif wMel[T1] gene but likely do not induce CI as the cifB [T1] genes were truncated and split into two fragments, with the second fragment containing an internal stop codon. Furthermore, cifB [T1] in w Cin2-ENA was upstream of cifA [T1] , preventing joint transcription of cifA and cifB . In w Cin2-PNW1 and w Cin2-PNW2, the cifB [T1] was translocated several hundred kilobases away from its paired cifA [T1]. The pair of cif [T3] genes had high identities (99% and 97%) to cif wNol[T3] , are likely functional, and are in an inversion in the w Cin2-PNW1 and w Cin2-PNW2 strains compared to w Cin2-ENA ( Fig 2 and S5 Table). Two pairs of cif [T5] genes were also predicted to be functional with identities closest to cif wTri-2[T5] and cif wStri-1[T5] , respectively. Finally, one pair of cif [T5] genes with closest identities to cif wStri-2[T5] were pseudogenized with stop codons found in both the cifA and cifB genes. Only the cifA wStri-2[T5] gene from w Cin2-ENA appeared functional. Strain w Cin2-SW had 11 pairs of cif genes, possessing: two cif [T1] pairs, one cif [T2] pair, three cif [T4] pairs, and five cif [T5] pairs ( Fig 2 , S5 Table, S1 Fig, and S2 Fig). The first cif [T1] pair had high identities (99%) to cif wMel[T1] , but a stop codon pseudogenized the cifB [T1] gene. The second pair cif [T1] pair was derived with low identities (65% and 61%) to cif wMel[T1] but appeared functional. The cif [T2] pair were also derived with low identities (58% and 56%) to cif wRi[T2] and was predicted to be functional. The three cif [T4] pairs had identities ranging from 97% to 52% to cif wAlbB[T4] . However, only one of the three cif [T4] pairs was functional, with the other two pairs containing internal stop codons in the cifB [T4] gene. All five cif [T5] pairs had identities ranging from 84% to 56% to cif wTri-2[T5] , cif Stri-1[T5] , cif wStri-2[T5] , or cif wStri-1[T5] and appeared functional ( Fig 2 and S5 Table). Strain w Ind infecting R. indifferens in the PNW2 population had a pair of syntenic cif [T1] genes displaying 61% sequence identity to cifA wMel[T1] and 60% identity to cifB wMel[T1] , respectively ( Fig 2 , S5 Table, S1 Fig, and S2 Fig). Strain w Cin3 infecting R. cingulata in the SW had six pairs of cif [T5] genes, with identities ranging from 93% to 43% identity to cif wTri-2[T5] , cif Stri-1[T5] , cif wStri-2[T5] or cif wStri-1[T5] . While all the cifA [T5] genes were functional, only one of six were paired with a functional cifB gene. The remaining five cifB genes were pseudogenized containing stop codons and/or frameshift mutations ( Fig 2 and S5 Table). w Cin3 and w Ind are highly diverged from w Cin2 To place the newly assembled w Cin3 and w Ind Wolbachia strains in a larger phylogenetic context, we constructed a RAxML tree based on 208 single-copy orthologous loci shared among all our sequenced strains and other representative A and B supergroup Wolbachia strains ( Fig 3 ). Strain w Cin3, coinfecting R. cingulata flies from the SW and Mexico, was inferred to be within the Wolbachia B supergroup. The closest related strain to w Cin3 was w AlbB, a Wolbachia strain that infects Aedes albopictus . Strain w Ind was genetically diverged from the other w Cin2 strains and placed at the base of the Wolbachia A supergroup. The closest relatives to w Ind are the Wolbachia strain w Ano62 that infects ants [ 56 ] and w Orie and w Neo, that infect Drosophila species [ 29 ]. All the w Cin2 strains clustered with the w Mel clade within the A supergroup. Download figure Open in new tab Figure 3. Wolbachia RAxML tree shows w Cin3 and w Ind are diverged from w Cin2. The A and B supergroup Wolbachia RAxML tree was derived from a set of 208 single-copy orthologous shared genes. Bootstrap support values are given for nodes based on 1,000 replicates. The cherry fly infecting Wolbachia strains are highlighted with different colors. Metadata for the A and B supergroup reference genomes are found in the S6 Table. w Cin2-SW is diverged from other w Cin2 strains To resolve the evolutionary relationships among w Cin2 strains, a RAxML tree was constructed that was limited to A supergroup w Mel-like Wolbachia and cherry fly w Cin2 strains using a shared set of 975 single-copy orthologous genes ( Fig 4A ). Here, all w Cin2 strains in R. cingulata and R. indifferens clustered and were sister to w Mel strains from Drosophila ( Fig 4A ). In addition, strain w Cin2-SW was genetically distinct from the w Cin2-ENA, w Cin2-PNW1, and w Cin2-PNW2 strains ( Fig 4A ). Mean sequence similarity between the w Cin2-ENA, w Cin2-PNW1, and w Cin2-PNW2 strains was very high (>99.85%), resulting in a polytomy in the RAxML tree ( Fig 4B ). Pairwise comparisons showed the highest sequence similarity between w Cin2-ENA and w Cin2-PNW2 strains (99.92%) and between w Cin2-PNW1 and w Cin2-PNW2 strains (99.91%). Sequence similarity was lower for all pairwise comparisons involving the ENA and PNW strains to w Cin2-SW (>98.21%). The RAxML tree for w Cin2 therefore concurred with the disjunct pattern displayed by cherry fly mtDNA haplotypes and differed from the clinal pattern shown by host microsatellites, with a genetic break seen for w Cin2 between the ENA and PNW versus the SW [ 50 ]. Download figure Open in new tab Figure 4. w Cin2-SW is diverged from the other w Cin2 strains. (A) RAxML tree for the clade of w Mel-like Wolbachia strains based on 975 single-copy shared orthologous genes with 1,000 bootstraps. The w Cin2 strains are highlighted with different colors. (B) Pairwise percent identity for the four w Cin2 genomes that infect cherry flies. (C) Venn diagram detailing the number of unique genes for each w Cin2 strain taken from the 1,486 genes in the w Cin2 pangenome. (D) Venn diagram showing 17 unique genes shared between the co-infecting Wolbachia strains w Cin3 and w Cin2-SW and 54 unique genes shared between w Cin3 and wCn2-ENA/PNW1, not found in w Cin2-SW. (E) Venn diagram showing 0 unique genes shared between w Ind and w Cin2-PNW2 as well as the other w Cin2 strains. Analysis of a set of 1,486 loci shared amongst w Cin2 strains indicated that the genome of w Cin2-SW not only differed in sequence content but also differed significantly in gene content from the other strains ( Fig 4C ). Overall, 1,100 genes were shared among the four w Cin2 strains. There were no unique genes identified in the genome of w Cin2-PNW1, one unique gene was found in w Cin2-PNW2, and 10 unique genes were detected in w Cin2-ENA. In contrast, 153 unique genes were identified in w Cin2-SW. Furthermore, w Cin2 from the ENA, PNW1, and PNW2 shared 215 unique genes not found in the w Cin2-SW strain. Most of the unique genes from each strain had annotations related to MGEs (S3 Fig). For example, of the 10 genes unique to w Cin2-ENA, five were in silico annotated and four of these were classified as MGEs (S7 Table). Of the 153 unique genes from w Cin2-SW, 40 of the 64 annotated genes were classified as MGEs (S8 Table). Finally, of the 215 unique genes shared among w Cin2-ENA, w Cin2-PNW1 and w Cin2-PNW2, 59 of the 101 annotated genes were classified as MGEs (S9 Table). Comparisons of shared and unique genes between the w Cin2 strains and w Cin3 yielded surprising results that suggest the possibility of past genetic exchange between the coinfecting strains. A total of 17 genes were shared only between w Cin3 and w Cin2-SW ( Fig 4D and S10 Table). Notably, cifA wStri-2[T5] and a cifB wStri-2[T5] genes were identified as being unique to both strains, clustering together in their respective cifA and cifB RAxML trees (Figs S1 and S2). We also identified 54 genes shared only between w Cin3, w Cin2-ENA, and w Cin2-PNW1, not found in w Cin2-SW ( Fig 4D ). Again, most of these shared genes were classified as MGEs and related to prophages (S11 Table). In contrast, we found no unique gene shared between coinfecting strains w Ind and w Cin2-PNW2 and no unique gene shared between w Ind and w Cin2-PNW1 and w Cin2-ENA ( Fig 4E ). Structural variation differentiates closely related w Cin2 strains Despite having near-identical gene content and sequence similarity, we detected unique structural variation between w Cin2-ENA, w Cin2-PNW1, and w Cin2-PNW2 ( Fig 5 ). Two major genomic translocations and a large inversion differentiated the w Cin2-ENA from the PNW strains. The two PNW w Cin2 stains, w Cin2-PNW1 and w Cin2-PNW2, also differed from each other by a large 344 kb inversion ( Fig 5 ). To determine the genes associated with these structural variants, we manually searched for annotated genes contained within the inversions and translocations. Each inversion and translocation contained MGEs, including prophages and transposases, as well as cif genes. One of the translocations between w Cin2-ENA and the PNW w Cin2 contained a complete prophage and a pair of cif [T5] genes. The second, smaller translocation between w Cin2-ENA and PNW w Cin2 contained a cifA [T1] gene. The inversion between w Cin2-ENA and PNW w Cin2 strains contained a pair of cif [T5] genes and an unpaired cifB [T1] . The large inversion between w Cin2-PNW1 and w Cin2-PNW2 contained a pair of cif [T5], genes, an unpaired cifA [T1] , and a complete prophage. Whole genome alignments were also performed between the w Cin2-ENA, w Cin2-PNW2, and w Cin2-PNW1 strains versus w Cin2-SW, resulting in the identification of over 51 rearrangements between them (S4 Fig). Download figure Open in new tab Figure 5. Synteny plots detail structural differences between the closely related w Cin2 strains. Whole genome alignments found that two translocations and an inversion differentiate the w Cin2-ENA and the w Cin2 strains from the PNW. The w Cin2-PNW1 and w Cin2-PNW2 strains are differentiated by a large 344 kb inversion. Within population differentiation of Wolbachia strains To investigate potential intra-strain variability, we conducted nanopore sequencing on two to three additional samples from each population and performed structural variant scans. Our analysis revealed that the major translocations and inversions identified above, which distinguish w Cin2 strains, were also present in the additional sequenced individuals and thus appear fixed at the population level. Besides these fixed inversions and translocations, we identified several indels segregating among the w Cin2 samples within each population. Most of the detected indels were less than 50 bp, although indels greater than 50 bp were present in nine out of ten samples (S12 Table). Consequently, while each sequenced w Cin2 sample shared population-specific structural variants, each also exhibited a unique set of indels. Discussion In this study, we characterized the evolution of the Wolbachia strain, w Cin2, present in all cherry-infesting populations of Rhagoletis in the R. cingulata sibling species group. We utilized nanopore sequencing to assemble closed, circular, genomes for Wolbachia from flies from four different populations across N. America that diverged from less than 1,000 to more than 100,000 years ago. This approach enabled comparative genomic analysis of Wolbachia sequence divergence, gene content, synteny, and structure across evolutionary timescales ranging from the recent to the more distant past. While earlier MLST analyses suggested that w Cin2 strains from the ENA and PNW were identical, with w Cin2 from the SW differing by only a single SNP [ 18 ], our whole genome approach revealed that w Cin2 strains are far more diverse than previously thought. The assembled w Cin2 genomes exhibited substantial differences in sequence identity, gene content, cif genes, MGEs, and structural rearrangements, with levels of differentiation increasing with time since separation. Most importantly, our findings show that Wolbachia genome structure can rapidly evolve. On a recent timescale of less than 1,000 years, genomic sequences for w Cin2 strains from the two cherry fly populations in the Pacific Northwest (PNW1 & PNW2) were near-identical. However, a notable structural variation was observed where w Cin2-PNW1 and w Cin2-PNW2 differ by a 344 kb inversion, flipping two prophage regions and a cif gene pair. At an intermediate timescale of several thousand years separating cherry flies from the PNW and ENA, genome sequences were also near-identical for the w Cin2 strains. Nevertheless, small differences in gene content were observed between the w Cin2 from the ENA and PNW, including 10 unique genes in w Cin2-ENA and a pseudogenized cifA [T5] in the PNW, the latter of which appears functional in w Cin2-ENA. Most significantly, two translocations and a small inversion further distinguished w Cin2 strains from the ENA and PNW populations. With additional nanopore sequencing of two to three additional individuals per population, we confirmed that the major structural variants noted between the w Cin2 genomes appear to be fixed at the population level, at least in the samples we assessed. These results are consistent with other recent studies suggesting that population structure may be common in some Wolbachia strains [ 36 , 57 ]. In addition to the fixed structural rearrangements distinguishing w Cin2 strains between populations, we identified polymorphic deletions and insertions in w Cin2 genomes within populations, most of which were under 1000 bp. Indeed, within a fly population, every sequenced host fly exhibited a unique set of indels in its associated w Cin2 genome. Intra-population structural variants have recently been identified as mutants in lab populations of w Mel and w Pip [ 43 – 45 ], but our results show that intra-population structural variants are likely prevalent in wild populations too. Our findings highlight the dynamic evolution of Wolbachia , and rather than a fixed strain, Wolbachia invasions could be made of up of a group of highly similar variants. Population-level sampling will be important to fully understand Wolbachia strain diversity in the future. The implications of rapid structural evolution of Wolbachia genomes over short to modest time periods are even more pronounced when considering strains that diverged over much longer time scales. In this regard, our results align with previous studies, demonstrating that once Wolbachia strains exhibit measurable levels of sequence divergence, their gene content and genome structure are typically already highly differentiated [ 29 , 37 , 39 , 42 , 58 , 59 ]. The w Cin2 strain from the SW, which became associated with a unique mtDNA haplotype over 100,000 years ago, differs by an estimated 51 translocations and inversions from wCin2 genomes in the PNW and ENA, with very few regions showing synteny. Gene content also rapidly diversified in w Cin2-SW, with the strain containing 153 unique genes, a reduction in the number of prophage regions from eight to three, and the presence of 11 pairs of cif genes — five more pairs than any other w Cin2 strain. Notably, by our estimates the 11 pairs of cif genes in w Cin2-SW are the highest number identified in a single Wolbachia strain to date [ 29 , 37 ]. These findings in cherry flies thus echo the broader pattern exhibited in other systems, where over longer evolutionary time periods, the gain and loss of cif genes decouples them from Wolbachia and host divergence patterns, often leading to the creation of new CI phenotypes [ 38 , 41 , 42 ]. Our findings provide further evidence that MGEs constitute a substantial portion of the Wolbachia genome and significantly contribute to variation in gene content and structure among closely related strains [ 36 , 52 , 60 – 65 ]. In our assembled Wolbachia genomes, more than 10% of genes were annotated as transposases, and each genome contained between two and eight predicted active prophages. Additionally, MGEs can be disruptive force and lead to the pseudogenization of Wolbachia genes [ 66 , 67 ]. In our study, all major structural rearrangements detected in w Cin2 involved MGEs, either containing prophages or transposons. We also identified several instances of pseudogenized cif genes resulting from MGE associated translocations and inversions, including a cifB gene removed from its cif A partner. Furthermore, MGEs often move between different Wolbachia strains and can facilitate the transfer of nearby genes, most notably cif genes [ 29 , 38 , 68 ]. We found evidence for gene transfer events between w Cin3 and w Cin2-SW, with 17 unique genes shared between them, most of which were prophage related. The high number of cif genes found in w Cin2-SW could partly be attributed to transfers from the coinfecting w Cin3 strain, or perhaps another unknown, previous coinfecting strain. Specifically, we discovered that both the w Cin2-SW and the coinfecting strain w Cin3 possess a cif [wDacT5] gene pair not found in any of the other w Cin2 strains from the ENA or PNW. Finally, we observed that most cif gene pairs in w Cin2 were flanked by transposases, likely aiding in their intra- and inter-genomic transfers [ 38 ]. These findings underscore the significant role MGEs play in shaping Wolbachia genomes, driving genetic diversity and adaptability through rapid structural evolution and gene transfer events. Previous work suggested that the unidirectional reduction in egg hatch rate observed in cherry fly crosses between SW males to ENA and PNW females was due to CI induced by w Cin3, a supergroup B strain coinfecting SW flies along with w Cin2 [ 18 , 48 ]. However, our genomic results suggest a possible alternative scenario, where w Cin2-SW may also play a role in causing CI. The genome of w Cin3 currently contains six pairs of cif [T5] genes, with only a single cif [T5] gene pair appearing functional. While cif [T5] homologs have been shown to induce CI [ 29 ], they are present in all the w Cin2 strains, including those infecting cherry flies from the ENA and PNW. Given that related cif homologs can generally rescue CI [ 13 , 63 ], this implies that w Cin3 alone could not be responsible for causing the observed CI in crosses between male SW cherry flies to PNW and ENA females [ 18 ]. It remains possible, however, that CI in cherry flies is caused by strains infected with cif [T5] genes from different clades. Martinez et al . (2020) showed that the cif [T5] clades are as diverse as the cif [T1] , cif [T2] , cif [T3] , and cif [T4] clades, and perhaps like these clades, cif [T5] clades may be able to induce and rescue their own specific type of CI. Notably, we found that strain w Cin2-SW and the coinfecting strain w Cin3 both possess a cif [wDacT5] gene pair not found in the other w Cin2 strains. This suggests that cif [wDacT5] may have been acquired in w Cin2-SW through inter-strain transfer from w Cin3, and together both strains could now be contributing to CI. Alternatively, w Cin2-SW alone could be responsible for the observed CI. Strain w Cin2-SW harbors 11 pairs of cif genes, eight of which are putatively functional. Two types of cif genes present in w Cin2-SW, cif [T2] and cif [T4] , are absent in other w Cin2 strains from the PNW1, PNW2, and ENA. One pair of cif [T4] and one pair of cif [T2] homologs are particularly significant because in silico annotations predict them to be fully functional, with no evidence of pseudogenization. Genome comparisons also revealed that w Cin2-ENA, w Cin2-PNW1, and w Cin2-PNW2 all share a unique functional pair of cif [T3] homologs not found in w Cin2-SW or w Cin3. It is possible that CI may also be induced by cif [T3] genes from w Cin2-ENA, w Cin2-PNW1, and w Cin2-PNW2. Empirical evidence, however, does not support this scenario, as crosses between ENA and PNW males to SW females show no evidence for reduced egg hatch [ 18 ]. This discrepancy suggests that CI induction by the cif [T3] homologs might be rescued by one of the other cif homologs in w Cin2-SW or w Cin3, or is due to some other factor such as cif transcript levels [ 31 ] or other Wolbachia cif- associated genes [ 63 ]. In summary, our examination of the Wolbachia strain w Cin2, which infests cherry fly populations ranging in divergence times from the recent to the more distant past, has provided significant insights into the rapid and complex evolutionary dynamics of Wolbachia genomes. Our findings demonstrate that Wolbachia genomes, driven by MGEs, can evolve swiftly, leading to substantial differences in sequence identity, gene content (including cif genes), and most prominently structural rearrangements. This work highlights the value of using long-read sequencing to assemble completed genomes needed to unravel the intricate and often subtle genomic changes that occur within Wolbachia strains and populations. Future research should focus on further characterizing the functional roles of the identified cif genes in w Cin2 and w Cin3 to better understand how CI is induced by SW R. cingulata males, potentially using techniques such as antisense RNA to selectively silence specific cif genes [ 69 ]. By continuing to compare Wolbachia strains and cif gene dynamics across different evolutionary timescales and host populations, we can deepen our understanding of the evolutionary processes that govern endosymbiont-host relationships, with broader implications for adaptation and speciation. Methods Fly collection and ONT sequencing Specimens of R. cingulata and R. indifferens were collected from 2018 to 2021 as larvae feeding in infested P. serotina fruit from the eastern USA (ENA), southwest USA (SW), and from P. emarginata fruit from two sites in the Pacific Northwest USA (PNW): Hood River, Oregon (PNW1) and Cle Elum, Washington (PNW2) ( Fig 1A ; S1 Table). Larvae were reared to adulthood following standard Rhagoletis husbandry techniques, with a 6–8 month overwintering treatment at 4 °C [ 18 , 48 , 70 , 71 ]. Newly eclosed adults were isolated by sex and matured for a minimum of seven days in cages with food (1-part autolyzed yeast to 3-parts honey) and water. Mature females were frozen and stored at -80 °C until sequencing. High molecular weight DNA was extracted from individual flies following Wolfe et al. [ 52 ]. ONT LSK109 ligation sequencing libraries were prepared according to manufacturer’s instructions from induvial flies and sequenced on R9.4.1 flow cells using a MinION Mk1B device. For cherry fly populations from PNW1, PNW2, and SW (the w Cin2 from ENA has already been published: GCF_017604245.1 [ 52 ]), we first sequenced one female fly at high coverage (25X-50X Wolbachia reads) to generate closed Wolbachia genomes. After this, two additional flies were sequenced from each population at a low coverage (∼10X Wolbachia reads) to search for any missed structural variants at the population level (S1 Table). Finally, 150 bp paired-end Illumina libraries using the same DNA from the high coverage individuals were prepared by the Notre Dame Genomics Core and sequenced on a HiSeq X Ten platform at BGI Genomics to polish final Wolbachia assemblies. Wolbachia genome assembly ONT signal data were basecalled using Guppy v6.01 with the SUP model and gently quality filtered (>Q8 and >500 bp) with nanoq [ 72 ]. For the high coverage sequencing runs used for genome assembly, reads were mapped with minimap2 [ 73 ] to the w Cin2 reference genome (GCF_017604245.1) and assembled using Flye v2.9.1 [ 74 ]. Assemblies were first polished using Racon v1.4.13 [ 75 ] followed by medaka 1.5.0. Short reads, quality filtered (>Q20 and >80 bp) with fastp [ 76 ], were used to further polish ONT assemblies with four iterations of Pilon [ 77 ]. Circlator [ 78 ] synchronized the start site for all the completed assemblies to dnaA . Assemblies were evaluated for completeness using BUSCO v4 with the Rickettsiales dataset [ 79 ]. Annotation of Wolbachia genomes and cif genes Completed Wolbachia assemblies were first annotated with Prokka v1.14.6 [ 80 ] using Pfam, PGAP, and HAMAP protein databases [ 81 – 83 ]. Phage regions were annotated using PHASTEST [ 84 ]. Finally, cif genes were identified using representative cifA and cifB genes from each of the five phylogenetic types as query sequences for BLASTp searches [ 28 , 29 ] (S4 Table). Positive BLASTp hits were accepted if E-values were close to 0, identity was greater than 40%, and the positive hit was greater than 40% of the queries length. Putative cif candidates were evaluated for pseudogenization using Artemis [ 85 ] to manually check for insertions, premature stop codons, or frameshift mutations and to evaluate if the cifA and cifB genes were in a complete module, with cifB immediately downstream from cifA . The above gene, phage, and cif gene annotations were visualized with pyCirclize [ 86 ]. cif gene phylogenies Predicted cifA and cifB genes identified from the above BLASTp searches and the BLASTp cif references (S4 Table) were aligned with MAFFT [ 87 ]. Both the derived cifA and cifB pseudogenes and the derived w Bor reference cif genes were excluded to improve phylogenetic signal. The cifA and cifB amino acid alignments were used to generate cifA and cifB RAxML-NG v1.1 [ 88 ] trees using the LG+G8+F substitution model with 25 random and 25 parsimony-based starting trees and 1,000 bootstrap replicates. Wolbachia phylogenetic analyses Phylogenetic analysis was performed at two scales, involving (1) both A and B supergroup Wolbachia strains; and (2) considering only w Mel-like A supergroup strains. Panaroo v1.3 [ 89 ] found sets of orthologous single-copy core genes using the annotations produced by Prokka from the genomes assembled above and a group of reference genomes (S6 Table). The data set for A and B supergroups together included 208 single-copy orthologous genes (387,932 bp) and the dataset for A group w Mel-like Wolbachia alone included 975 orthologous single-copy genes (924,342 bp). For each dataset, gene sequences were concatenated and aligned using MAFFT [ 87 ] and RAxML-NG v1.1 [ 88 ] constructed phylogenetic trees using the GTR+G substitution model, with 50 random and 50 parsimony-based starting trees, and with 1,000 bootstrap replicates. Wolbachia strain w Cin2 genome comparisons Shared and unique genes for w Cin2 strains were determined from a set of single-copy orthologous genes. Using the previously generated Prokka annotations, Panaroo generated a pangenome for all the 1,486 protein coding genes for all four w Cin2 strains. Using this dataset, R was used to determine the unique and shared genes between each strain [ 90 ]. Pairwise whole genome comparisons were performed for all w Cin2 genomes using MUMmer4 [ 91 ] to estimate average percent gene identity. MUMmer4 was also used to generate whole-genome alignments between strains that had the highest identity to one another to detect structural rearrangements (PNW1 to PNW2 and ENA to PNW2). Synteny and any rearrangements detected between these alignments were annotated with SyRI [ 92 ] and plotted with Plotsr [ 93 ]. SyRI annotations were validated against results produced by Mauve [ 94 ]. We were unable to use SyRI for comparisons to the w Cin2-SW genome because of its higher divergence, and instead used Mauve [ 94 ]. To verify whether the identified structural variations were representative of each w Cin2 population, we checked for additional structural variants from two to three additional samples sequenced at low coverage. We used CuteSV2, shown to have high accuracy at estimating structural variants (SVs) with low coverage data [ 95 ], to scan the ONT reads for any additional synteny not captured in our assembled reference genomes. For each population, ONT reads were aligned to their reference genome using minimap2 and SVs were called using cuteSV2 with settings optimized for ONT data with a minimum of five reads needed to call a breakpoint. Supporting information S1 Table. Collection metadata for sequenced flies. Populations are shown in Fig. 1 . Flies were sampled from 2018-2021 from the ENA, the SW, and two populations in the PNW – one from Hood River, Oregon (PNW1) and one from Cle Elum, Washington State (PNW2). Samples sequenced at high coverage (∼30-50X Wolbachia reads) were used for genome assembly and samples sequenced at low coverage (∼10X Wolbachia reads) were used for structural variant scans. GCF_017604245.1 is the ENA-HC sample and the w Cin2 reference genome. S2 Table. Genome assembly stats for cherry fly associated Wolbachia strains. BUSCO scores from the Rickettsiales dataset. Assembly stats are for all assembled Wolbachia genomes, including the reference w Cin2 genome (GCF_017604245.1) from the ENA. S3 Table. MGEs are highly prevalent in the assembled Wolbachia genomes. Counts of the number of in silico Bakta annotations that are classified of MGEs compared to the total number of coding genes in the genome S4 Table. Representative cif genes for each type (I-V) were used for reference sequences for BLASTp searches of the assembled Wolbachia genomes. Listed are the Wolbachia strains associated with each cif gene type and associated NCBI gene ID. S5 Table. BLASTp hit table of putative cif homologous for all assembled Wolbachia strains. Representative cif genes from S1 Table were used as reference sequences. Hit start and Hit end denote the start and stop coordinates of the cif genes in assembled genomes, direction describes the coding direction of the cif genes, and e-val quantifies the significance of the BLASTp hits. Gene identities quantify similarity between the reference cif gene and the cif BLASTp hits. S6 Table. Reference genomes used for phylogenetic analyses. The table lists the Wolbachia strain, its supergroup, the RefSeq assembly ID, genome size, number contigs, and associated references. S7 Table. Annotations for the 10 unique genes for w Cin2-ENA. All genes for w Cin2-ENA were annotated with Prokka using the Pfam, PGAP, and HAMAP protein databases. Then using R, the genes unique to w Cin2-ENA were sorted. S8 Table. Annotations for the 153 unique genes for w Cin2-SW. All genes for w Cin2-SW were annotated with Prokka using the Pfam, PGAP, and HAMAP protein databases. Then using R, the genes unique to w Cin2-SW were sorted. S9 Table. Annotations for the 215 unique genes shared between w Cin2-ENA, w Cin2-PNW1, and w Cin2-PNW2. All genes within the shared gene set were annotated with Prokka using the Pfam, PGAP, and HAMAP protein databases. Then using R, the genes shared between w Cin2-ENA, w Cin2-PNW1, and w Cin2-PNW2 were sorted. AS10 Table. Annotations for the 17 unique genes shared between w Cin2-SW and w Cin3. All genes within the shared gene set were annotated with Prokka using the Pfam, PGAP, and HAMAP protein databases. Most annotated genes are MGEs. S11 Table. Annotations for the 54 unique genes shared between w Cin3. w Cin2-ENA, and w Cin2-PNW1. All genes within the shared gene set were annotated with Prokka using the Pfam, PGAP, and HAMAP protein databases. Most annotated genes are MGEs. S12 Table. Several small SVs are found at the population level for the w Cin2 strains. Low coverage SV scans using CuteSV against the assembled wCin2 genomes identified several SVs less than 1000 bp and four SVs between 1000 and 10,000 bp S1 Fig. The cherry fly cifA gene RAxML phylogeny highlights cifA gene diversity. The RAxML phylogeny was made using a MAFFT amino acid alignment with 1000 bootstraps. Derived pseudogenes were removed before constructing the phylogeny. Strain w Cin2-SW has the most cifA genes with 11 copies, w Cin3 has six copies, w Cin2-ENA has five copies, and the w Cin2 strains from the PNW have four functional copies. See S5 Table for more detailed cif gene information. S2 Fig. The cherry fly cifB gene RAxML phylogeny highlights cifB gene diversity. The RAxML phylogeny was made using a MAFFT amino acid alignment with 1000 bootstraps. Derived pseudogenes were removed before constructing the phylogeny, those that remain are denoted with **. Strain w Cin2-SW has the most cifB functional copies with eight, followed by the ENA and PNW strains with two each, and further followed still by w Cin3 and w Ind with one copy. See S5 Table for more detailed cif gene information. S3 Fig. Annotated genes show enrichment for MGE genes in w Cin2 strains. Most annotated genes unique to either the w Cin2-ENA, w Cin2-PNW1/PNW2/ENA, or w Cin2-SW strains are MGEs. S4 Fig. Synteny comparisons between the w Cin2 strains show w Cin2-SW is highly diverged. Structural genome rearrangements were identified with Mauve. Strain w Cin2-SW has over 51 structural rearrangements compared to w Cin2-ENA. The PNW w Cin2 strains only have four and three rearrangements compared to w Cin2-ENA. Acknowledgements We would like to thank Jaqueline Lopez and the Note Dame Genomics and Bioinformatics core facility for the preparation of Illumina libraries and DNA quality control, as well as Mike Pfrender and Stuart Jones for insightful comments on early drafts. We also thank Robert B Goughnour, Glen Ray Hood, Meredith M. Doellman, and Cheyenne Tait for fly collection and/or husbandry for samples sequenced in the study. References 1. ↵ Weinert LA , Araujo-Jnr EV , Ahmed MZ , Welch JJ . The incidence of bacterial endosymbionts in terrestrial arthropods . Proc Biol Sci . 2015 ; 282 : 20150249 . doi: 10.1098/rspb.2015.0249 OpenUrl CrossRef PubMed 2. ↵ Taylor MJ , Bandi C , Hoerauf A . Wolbachia bacterial endosymbionts of filarial nematodes . Adv Parasitol . 2005 ; 60 : 245 – 284 . doi: 10.1016/S0065-308X(05)60004-8 OpenUrl CrossRef PubMed Web of Science 3. ↵ Baldo L , Ayoub NA , Hayashi CY , Russell JA , Stahlhut JK , Werren JH . Insight into the routes of Wolbachia invasion: high levels of horizontal transfer in the spider genus Agelenopsis revealed by Wolbachia strain and mitochondrial DNA diversity . Mol Ecol . 2008 ; 17 : 557 – 569 . doi: 10.1111/j.1365-294X.2007.03608.x OpenUrl CrossRef PubMed Web of Science 4. ↵ Sanaei E , Charlat S , Engelstädter J . Wolbachia host shifts: routes, mechanisms, constraints and evolutionary consequences . Biol Rev Camb Philos Soc . 2021 ; 96 : 433 – 453 . doi: 10.1111/brv.12663 OpenUrl CrossRef PubMed 5. ↵ Dedeine F , Vavre F , Fleury F , Loppin B , Hochberg ME , Boulétreau M . Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp . Proc Natl Acad Sci U S A . 2001 ; 98 : 6247 – 6252 . doi: 10.1073/pnas.101304298 OpenUrl Abstract / FREE Full Text 6. Hosokawa T , Koga R , Kikuchi Y , Meng X-Y , Fukatsu T . Wolbachia as a bacteriocyte-associated nutritional mutualist . Proc Natl Acad Sci U S A . 2010 ; 107 : 769 – 774 . doi: 10.1073/pnas.0911476107 OpenUrl Abstract / FREE Full Text 7. ↵ Newton ILG , Rice DW . The jekyll and hyde symbiont: could Wolbachia be a nutritional mutualist? J Bacteriol . 2020 ; 202 : e00589 – 19 . doi: 10.1128/JB.00589-19 OpenUrl CrossRef PubMed 8. ↵ Hedges LM , Brownlie JC , O’Neill SL , Johnson KN . Wolbachia and virus protection in insects . Science . 2008 ; 322 : 702 – 702 . doi: 10.1126/science.1162418 OpenUrl Abstract / FREE Full Text 9. Teixeira L , Ferreira Á , Ashburner M . The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster . PLoS Biol . 2008 ; 6 : e1000002 . doi: 10.1371/journal.pbio.1000002 OpenUrl CrossRef PubMed 10. ↵ Cogni R , Ding SD , Pimentel AC , Day JP , Jiggins FM . Wolbachia reduces virus infection in a natural population of Drosophila . Commun Biol . 2021 ; 4 : 1 – 7 . doi: 10.1038/s42003-021-02838-z OpenUrl CrossRef PubMed 11. ↵ Kaur R , Shropshire JD , Cross KL , Leigh B , Mansueto AJ , Stewart V , et al. Living in the endosymbiotic world of Wolbachia : A centennial review . Cell Host Microbe . 2021 ; 29 : 879 – 893 . doi: 10.1016/j.chom.2021.03.006 OpenUrl CrossRef PubMed 12. ↵ Correa CC , Ballard JWO . Wolbachia associations with insects: winning or losing against a master manipulator . Front Ecol Evol . 2016 ; 3 : 153 . doi: 10.3389/fevo.2015.00153 OpenUrl CrossRef 13. ↵ Shropshire JD , Leigh B , Bordenstein SR . Symbiont-mediated cytoplasmic incompatibility: What have we learned in 50 years? eLife . 2020 ; 9 : e61989 . doi: 10.7554/eLife.61989 OpenUrl CrossRef PubMed 14. ↵ Telschow A , Hammerstein P , Werren JH . The effect of Wolbachia on genetic divergence between populations: models with two-way migration . Am Nat . 2002 ; 160 : S54 – S66 . doi: 10.1086/342153 OpenUrl CrossRef PubMed Web of Science 15. ↵ Flor M , Hammerstein P , Telschow A . Wolbachia -induced unidirectional cytoplasmic incompatibility and the stability of infection polymorphism in parapatric host populations . J Evol Biol . 2007 ; 20 : 696 – 706 . doi: 10.1111/j.1420-9101.2006.01252.x OpenUrl CrossRef PubMed 16. ↵ Brucker RM , Bordenstein SR . Speciation by symbiosis . Trends Ecol Evol . 2012 ; 27 : 443 – 451 . doi: 10.1016/j.tree.2012.03.011 OpenUrl CrossRef PubMed Web of Science 17. Cruz MA , Magalhães S , Sucena É , Zélé F . Wolbachia and host intrinsic reproductive barriers contribute additively to postmating isolation in spider mites . Evolution . 2021 ; 75 : 2085 – 2101 . doi: 10.1111/evo.14286 OpenUrl CrossRef PubMed 18. ↵ Bruzzese DJ , Schuler H , Wolfe TM , Glover MM , Mastroni JV , Doellman MM , et al. Testing the potential contribution of Wolbachia to speciation when cytoplasmic incompatibility becomes associated with host-related reproductive isolation . Mol Ecol . 2022 ; 31 : 2935 – 2950 . doi: 10.1111/mec.16157 OpenUrl CrossRef 19. ↵ Telschow A , Hammerstein P , Werren JH . The effect of Wolbachia versus genetic incompatibilities on reinforcement and speciation . Evolution . 2005 ; 59 : 1607 – 1619 . doi: 10.1111/j.0014-3820.2005.tb01812.x OpenUrl CrossRef PubMed Web of Science 20. ↵ Jaenike J , Dyer KA , Cornish C , Minhas MS . Asymmetrical reinforcement and Wolbachia infection in Drosophila . PLoS Biol . 2006 ; 4 : e325 . doi: 10.1371/journal.pbio.0040325 OpenUrl CrossRef PubMed 21. ↵ Beckmann JF , Ronau JA , Hochstrasser M . A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility . Nat Microbiol . 2017 ; 2 : 17007 . doi: 10.1038/nmicrobiol.2017.7 OpenUrl CrossRef PubMed 22. Chen H , Ronau JA , Beckmann JF , Hochstrasser M . A Wolbachia nuclease and its binding partner provide a distinct mechanism for cytoplasmic incompatibility . Proc Natl Acad Sci U S A . 2019 ; 116 : 22314 – 22321 . doi: 10.1073/pnas.1914571116 OpenUrl Abstract / FREE Full Text 23. ↵ LePage DP , Metcalf JA , Bordenstein SR , On J , Perlmutter JI , Shropshire JD , et al. Prophage WO genes recapitulate and enhance Wolbachia -induced cytoplasmic incompatibility . Nature . 2017 ; 543 : 243 – 247 . doi: 10.1038/nature21391 OpenUrl CrossRef PubMed 24. McNamara CJ , Ant TH , Harvey-Samuel T , White-Cooper H , Martinez J , Alphey L , et al. Transgenic expression of cif genes from Wolbachia strain w AlbB recapitulates cytoplasmic incompatibility in Aedes aegypti . Nat Commun . 2024 ; 15 : 869 . doi: 10.1038/s41467-024-45238-7 OpenUrl CrossRef PubMed 25. ↵ Shropshire JD , Bordenstein SR . Two-By-One model of cytoplasmic incompatibility: Synthetic recapitulation by transgenic expression of cifA and cifB in Drosophila . PLoS Genet . 2019 ; 15 : e1008221 . doi: 10.1371/journal.pgen.1008221 OpenUrl CrossRef PubMed 26. ↵ Shropshire JD , On J , Layton EM , Zhou H , Bordenstein SR . One prophage WO gene rescues cytoplasmic incompatibility in Drosophila melanogaster . Proc Natl Acad Sci U S A . 2018 ; 115 : 4987 – 4991 . doi: 10.1073/pnas.1800650115 OpenUrl Abstract / FREE Full Text 27. ↵ Adams KL , Abernathy DG , Willett BC , Selland EK , Itoe MA , Catteruccia F . Wolbachia cifB induces cytoplasmic incompatibility in the malaria mosquito vector . Nat Microbiol . 2021 ; 6 : 1575 – 1582 . doi: 10.1038/s41564-021-00998-6 OpenUrl CrossRef PubMed 28. ↵ Lindsey ARI , Rice DW , Bordenstein SR , Brooks AW , Bordenstein SR , Newton ILG . Evolutionary genetics of cytoplasmic incompatibility genes cifA and cifB in Prophage WO of Wolbachia . Genome Biol Evol . 2018 ; 10 : 434 – 451 . doi: 10.1093/gbe/evy012 OpenUrl CrossRef PubMed 29. ↵ Martinez J , Klasson L , Welch JJ , Jiggins FM . Life and death of selfish genes: comparative genomics reveals the dynamic evolution of cytoplasmic incompatibility . Mol Biol Evol . 2021 ; 38 : 2 – 15 . doi: 10.1093/molbev/msaa209 OpenUrl CrossRef PubMed 30. ↵ Reynolds KT , Thomson LJ , Hoffmann AA . The effects of host age, host nuclear background and temperature on phenotypic effects of the virulent Wolbachia strain popcorn in Drosophila melanogaster . Genetics . 2003 ; 164 : 1027 – 1034 . doi: 10.1093/genetics/164.3.1027 OpenUrl Abstract / FREE Full Text 31. ↵ Shropshire JD , Hamant E , Conner WR , Cooper BS . cifB -transcript levels largely explain cytoplasmic incompatibility variation across divergent Wolbachia . PNAS Nexus . 2022 ; 1 : pgac099 . doi: 10.1093/pnasnexus/pgac099 OpenUrl CrossRef 32. ↵ Wybouw N , Mortier F , Bonte D . Interacting host modifier systems control Wolbachia -induced cytoplasmic incompatibility in a haplodiploid mite . Evol Lett . 2022 ; 6 : 255 – 265 . doi: 10.1002/evl3.282 OpenUrl CrossRef PubMed 33. ↵ Klasson L , Westberg J , Sapountzis P , Näslund K , Lutnaes Y , Darby AC , et al. The mosaic genome structure of the Wolbachia w Ri strain infecting Drosophila simulans . Proc Natl Acad Sci U S A . 2009 ; 106 : 5725 – 5730 . doi: 10.1073/pnas.0810753106 OpenUrl Abstract / FREE Full Text 34. Tanaka K , Furukawa S , Nikoh N , Sasaki T , Fukatsu T . Complete WO phage sequences reveal their dynamic evolutionary trajectories and putative functional elements required for integration into the Wolbachia genome . Appl Environ Microbiol . 2009 ; 75 : 5676 – 5686 . doi: 10.1128/AEM.01172-09 OpenUrl Abstract / FREE Full Text 35. Kent BN , Bordenstein SR . Phage WO of Wolbachia : lambda of the endosymbiont world . Trends Microbiol . 2010 ; 18 : 173 – 181 . doi: 10.1016/j.tim.2009.12.011 OpenUrl CrossRef PubMed Web of Science 36. ↵ Hill T , Unckless RL , Perlmutter JI . Positive selection and horizontal gene transfer in the genome of a male-killing Wolbachia . Mol Biol Evol . 2022 ; 39 : msab303 . doi: 10.1093/molbev/msab303 OpenUrl CrossRef PubMed 37. ↵ Martinez J , Ant TH , Murdochy SM , Tong L , Filipe A da S , Sinkins SP . Genome sequencing and comparative analysis of Wolbachia strain w AlbA reveals Wolbachia -associated plasmids are common . PLoS Genet . 2022 ; 18 : e1010406 . doi: 10.1371/journal.pgen.1010406 OpenUrl CrossRef 38. ↵ Cooper BS , Vanderpool D , Conner WR , Matute DR , Turelli M . Wolbachia acquisition by Drosophila yakuba -clade hosts and transfer of incompatibility loci between distantly related Wolbachia . Genetics . 2019 ; 212 : 1399 – 1419 . doi: 10.1534/genetics.119.302349 OpenUrl Abstract / FREE Full Text 39. ↵ Scholz M , Albanese D , Tuohy K , Donati C , Segata N , Rota-Stabelli O . Large scale genome reconstructions illuminate Wolbachia evolution . Nat Commun . 2020 ; 11 : 5235 . doi: 10.1038/s41467-020-19016-0 OpenUrl CrossRef PubMed 40. ↵ Collier LS , Largaespada DA . Transposable elements and the dynamic somatic genome . Genome Biol . 2007 ; 8 : S5 . doi: 10.1186/gb-2007-8-s1-s5 OpenUrl CrossRef PubMed 41. ↵ Turelli M , Cooper BS , Richardson KM , Ginsberg PS , Peckenpaugh B , Antelope CX , et al. Rapid global spread of w Ri-like Wolbachia across multiple Drosophila . Curr Biol . 2018 ; 28 : 963 – 971 .e8. doi: 10.1016/j.cub.2018.02.015 OpenUrl CrossRef PubMed 42. ↵ Meany MK , Conner WR , Richter SV , Bailey JA , Turelli M , Cooper BS . Loss of cytoplasmic incompatibility and minimal fecundity effects explain relatively low Wolbachia frequencies in Drosophila mauritiana . Evolution . 2019 ; 73 : 1278 – 1295 . doi: 10.1111/evo.13745 OpenUrl CrossRef 43. ↵ Duarte EH , Carvalho A , López-Madrigal S , Costa J , Teixeira L . Forward genetics in Wolbachia : Regulation of Wolbachia proliferation by the amplification and deletion of an addictive genomic island . PLoS Genet . 2021 ; 17 : e1009612 . doi: 10.1371/journal.pgen.1009612 OpenUrl CrossRef PubMed 44. Chrostek E , Teixeira L . Mutualism Breakdown by Amplification of Wolbachia Genes . PLoS Biol . 2015 ; 13 : e1002065 . doi: 10.1371/journal.pbio.1002065 OpenUrl CrossRef PubMed 45. ↵ Namias A , Ngaku A , Makoundou P , Unal S , Sicard M , Weill M . Intra-lineage microevolution of Wolbachia leads to the emergence of new cytoplasmic incompatibility patterns . PLoS Biol . 2024 ; 22 : e3002493 . doi: 10.1371/journal.pbio.3002493 OpenUrl CrossRef PubMed 46. ↵ Bush GL . The taxonomy, cytology, and evolution of the genus Rhagoletis in North America (Diptera , Tephritidae). Bull Mus Comp Zool . 1966 ; 134 : 431 – 562 . OpenUrl 47. ↵ Bush GL . Sympatric host race formation and speciation in frugivorous flies of the genus Rhagoletis (Diptera, Tephritidae) . Evolution . 1969 ; 23 : 237 – 251 . doi: 10.2307/2406788 OpenUrl CrossRef PubMed Web of Science 48. ↵ Tadeo E , Feder JL , Egan SP , Schuler H , Aluja M , Rull J . Divergence and evolution of reproductive barriers among three allopatric populations of Rhagoletis cingulata across eastern North America and Mexico . Entomol Exp Appl . 2015 ; 156 : 301 – 311 . doi: 10.1111/eea.12331 OpenUrl CrossRef 49. ↵ Doellman MM , Schuler H , Jean GS , Hood GR , Egan SP , Powell THQ , et al. Geographic and ecological dimensions of host plant-associated genetic differentiation and speciation in the Rhagoletis cingulata (Diptera: Tephritidae) sibling species group . Insects . 2019 ; 10 : 275 . doi: 10.3390/insects10090275 OpenUrl CrossRef 50. ↵ Doellman MM , Jean GS , Egan SP , Powell THQ , Hood GR , Schuler H , et al. Evidence for spatial clines and mixed geographic modes of speciation for North American cherry-infesting Rhagoletis (Diptera: Tephritidae) flies . Ecol Evol . 2020 ; 10 : 12727 – 12744 . doi: 10.1002/ece3.6667 OpenUrl CrossRef PubMed 51. ↵ Schuler H , Bertheau C , Egan SP , Feder JL , Riegler M , Schlick-Steiner BC , et al. Evidence for a recent horizontal transmission and spatial spread of Wolbachia from endemic Rhagoletis cerasi (Diptera: Tephritidae) to invasive Rhagoletis cingulata in Europe . Mol Ecol . 2013 ; 22 : 4101 – 4111 . doi: 10.1111/mec.12362 OpenUrl CrossRef 52. ↵ Wolfe TM , Bruzzese DJ , Klasson L , Corretto E , Lečić S , Stauffer C , et al. Comparative genome sequencing reveals insights into the dynamics of Wolbachia in native and invasive cherry fruit flies . Mol Ecol . 2021 ; 30 : 6259 – 6272 . doi: 10.1111/mec.15923 OpenUrl CrossRef 53. ↵ Hurst GDD , Jiggins FM . Problems with mitochondrial DNA as a marker in population, phylogeographic and phylogenetic studies: the effects of inherited symbionts . Proc R Soc Lond B Biol Sci . 2005 ; 272 : 1525 – 1534 . doi: 10.1098/rspb.2005.3056 OpenUrl CrossRef PubMed Web of Science 54. ↵ Schuler H , Köppler K , Daxböck-Horvath S , Rasool B , Krumböck S , Schwarz D , et al. The hitchhiker’s guide to Europe: the infection dynamics of an ongoing Wolbachia invasion and mitochondrial selective sweep in Rhagoletis cerasi . Mol Ecol . 2016 ; 25 : 1595 – 1609 . doi: 10.1111/mec.13571 OpenUrl CrossRef 55. ↵ Beckmann JF , Vaerenberghe KV , Akwa DE , Cooper BS . A single mutation weakens symbiont-induced reproductive manipulation through reductions in deubiquitylation efficiency . Proc Natl Acad Sci U S A . 2021 ; 118 . doi: 10.1073/pnas.2113271118 OpenUrl Abstract / FREE Full Text 56. ↵ Lee C-C , Lin C-Y , Tseng S-P , Matsuura K , Yang C-CS . Ongoing coevolution of Wolbachia and a widespread invasive ant, Anoplolepis gracilipes . Microorganisms . 2020 ; 8 : 1569 . doi: 10.3390/microorganisms8101569 OpenUrl CrossRef PubMed 57. ↵ Kaur R , Siozios S , Miller WJ , Rota-Stabelli O . Insertion sequence polymorphism and genomic rearrangements uncover hidden Wolbachia diversity in Drosophila suzukii and D. subpulchrella . Sci Rep . 2017 ; 7 : 14815 . doi: 10.1038/s41598-017-13808-z OpenUrl CrossRef PubMed 58. ↵ Vancaester E , Blaxter M . Phylogenomic analysis of Wolbachia genomes from the Darwin Tree of Life biodiversity genomics project . PLoS Biol . 2023 ; 21 : e3001972 . doi: 10.1371/journal.pbio.3001972 OpenUrl CrossRef PubMed 59. ↵ Gao S , Ren Y-S , Su C-Y , Zhu D-H . High levels of multiple phage WO infections and its evolutionary dynamics associated with Wolbachia -infected butterflies . Front Microbiol . 2022 ; 13 : 865227 . doi: 10.3389/fmicb.2022.865227 OpenUrl CrossRef PubMed 60. ↵ Ishmael N , Hotopp JCD , Ioannidis P , Biber S , Sakamoto J , Siozios S , et al. Extensive genomic diversity of closely related Wolbachia strains . Microbiology . 2009 ; 155 : 2211 – 2222 . doi: 10.1099/mic.0.027581-0 OpenUrl CrossRef PubMed Web of Science 61. Ellegaard KM , Klasson L , Näslund K , Bourtzis K , Andersson SGE . Comparative genomics of Wolbachia and the bacterial species concept . PLoS Genet . 2013 ; 9 : e1003381 . doi: 10.1371/journal.pgen.1003381 OpenUrl CrossRef PubMed 62. Gerth M , Bleidorn C . Comparative genomics provides a timeframe for Wolbachia evolution and exposes a recent biotin synthesis operon transfer . Nat Microbiol . 2016 ; 2 : 1 – 7 . doi: 10.1038/nmicrobiol.2016.241 OpenUrl CrossRef 63. ↵ Baião GC , Janice J , Galinou M , Klasson L . Comparative genomics reveals factors associated with phenotypic expression of Wolbachia . Genome Biol Evol . 2021 ; 13 : evab111 . doi: 10.1093/gbe/evab111 OpenUrl CrossRef PubMed 64. Wu M , Sun LV , Vamathevan J , Riegler M , Deboy R , Brownlie JC , et al. Phylogenomics of the reproductive parasite Wolbachia pipientis w Mel: a streamlined genome overrun by mobile genetic elements . PLoS Biol . 2004 ; 2 : e69 . doi: 10.1371/journal.pbio.0020069 OpenUrl CrossRef PubMed 65. ↵ Duplouy A , Iturbe-Ormaetxe I , Beatson SA , Szubert JM , Brownlie JC , McMeniman CJ , et al. Draft genome sequence of the male-killing Wolbachia strain w Bol1 reveals recent horizontal gene transfers from diverse sources . BMC Genomics . 2013 ; 14 : 20 . doi: 10.1186/1471-2164-14-20 OpenUrl CrossRef PubMed 66. ↵ Sanogo YO , Dobson SL , Bordenstein SR , Novak RJ . Disruption of the Wolbachia surface protein gene wspB by a transposable element in mosquitoes of the Culex pipiens complex (Diptera , Culicidae). Insect Mol Biol . 2007 ; 16 : 143 – 154 . doi: 10.1111/j.1365-2583.2006.00707.x OpenUrl CrossRef PubMed Web of Science 67. ↵ Queffelec J , Postma A , Allison JD , Slippers B . Remnants of horizontal transfers of Wolbachia genes in a Wolbachia -free woodwasp . BMC Ecol Evo . 2022 ; 22 : 36 . doi: 10.1186/s12862-022-01995-x OpenUrl CrossRef 68. ↵ Wang GH , Sun BF , Xiong TL , Wang YK , Murfin KE , Xiao JH , et al. Bacteriophage WO can mediate horizontal gene transfer in endosymbiotic Wolbachia genomes . Front Microbiol . 2016 ; 7 : 1867 . doi: 10.3389/fmicb.2016.01867 OpenUrl CrossRef PubMed 69. ↵ Hussain M , Zhang G , Leitner M , Hedges LM , Asgari S . Wolbachia RNase HI contributes to virus blocking in the mosquito Aedes aegypti . iScience . 2022 ; 26 : 105836 . doi: 10.1016/j.isci.2022.105836 OpenUrl CrossRef PubMed 70. ↵ Feder JL , Chilcote CA , Bush GL . Inheritance and linkage relationships of allozymes in the apple maggot fly . J Hered . 1989 ; 80 : 277 – 283 . doi: 10.1093/oxfordjournals.jhered.a110854 OpenUrl CrossRef Web of Science 71. ↵ Yee WL , Goughnour RB , Hood GR , Forbes AA , Feder JL . Chilling and host plant/site-associated eclosion times of Western cherry fruit fly (Diptera: Tephritidae) and a host-specific parasitoid . Environ Entomol . 2015 ; 44 : 1029 – 1042 . doi: 10.1093/ee/nvv097 OpenUrl CrossRef PubMed 72. ↵ Steinig E , Coin L . Nanoq: ultra-fast quality control for nanopore reads . J Open Source Softw . 2022 ; 7 : 2991 . doi: 10.21105/joss.02991 OpenUrl CrossRef 73. ↵ Li H . Minimap2: pairwise alignment for nucleotide sequences . Bioinformatics . 2018 ; 34 : 3094 – 3100 . doi: 10.1093/bioinformatics/bty191 OpenUrl CrossRef PubMed 74. ↵ Kolmogorov M , Yuan J , Lin Y , Pevzner PA . Assembly of long, error-prone reads using repeat graphs . Nat Biotechnol . 2019 ; 37 : 540 – 546 . doi: 10.1038/s41587-019-0072-8 OpenUrl CrossRef PubMed 75. ↵ Vaser R , Sovic I , Nagarajan N , Sikic M . Fast and accurate de novo genome assembly from long uncorrected reads . Genome Res . 2017 ; gr.214270.116 . doi: 10.1101/gr.214270.116 OpenUrl Abstract / FREE Full Text 76. ↵ Chen S , Zhou Y , Chen Y , Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor . Bioinformatics . 2018 ; 34 : i884 – i890 . doi: 10.1093/bioinformatics/bty560 OpenUrl CrossRef PubMed 77. ↵ Walker BJ , Abeel T , Shea T , Priest M , Abouelliel A , Sakthikumar S , et al. Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement . PLoS One . 2014 ; 9 : e112963 . doi: 10.1371/journal.pone.0112963 OpenUrl CrossRef PubMed 78. ↵ Hunt M , Silva ND , Otto TD , Parkhill J , Keane JA , Harris SR . Circlator: automated circularization of genome assemblies using long sequencing reads . Genome Biol . 2015 ; 16 : 294 . doi: 10.1186/s13059-015-0849-0 OpenUrl CrossRef PubMed 79. ↵ Manni M , Berkeley MR , Seppey M , Simão FA , Zdobnov EM . BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for ccoring of Eukaryotic, Prokaryotic, and viral genomes . Mol Biol Evol . 2021 ; 38 : 4647 – 4654 . doi: 10.1093/molbev/msab199 OpenUrl CrossRef PubMed 80. ↵ Seemann T . Prokka: rapid prokaryotic genome annotation . Bioinformatics . 2014 ; 30 : 2068 – 2069 . doi: 10.1093/bioinformatics/btu153 OpenUrl CrossRef PubMed Web of Science 81. ↵ Pedruzzi I , Rivoire C , Auchincloss AH , Coudert E , Keller G , de Castro E , et al. HAMAP in 2015: updates to the protein family classification and annotation system . Nucleic Acids Res . 2015 ; 43 : D1064 – D1070 . doi: 10.1093/nar/gku1002 OpenUrl CrossRef PubMed 82. Tatusova T , DiCuccio M , Badretdin A , Chetvernin V , Nawrocki EP , Zaslavsky L , et al. NCBI prokaryotic genome annotation pipeline . Nucleic Acids Res . 2016 ; 44 : 6614 – 6624 . doi: 10.1093/nar/gkw569 OpenUrl CrossRef PubMed 83. ↵ Mistry J , Chuguransky S , Williams L , Qureshi M , Salazar GA , Sonnhammer ELL , et al. Pfam: The protein families database in 2021 . Nucleic Acids Res . 2021 ; 49 : D412 – D419 . doi: 10.1093/nar/gkaa913 OpenUrl CrossRef PubMed 84. ↵ Wishart DS , Han S , Saha S , Oler E , Peters H , Grant JR , et al. PHASTEST: faster than PHASTER, better than PHAST . Nucleic Acids Res . 2023 ; 51 : W443 – W450 . doi: 10.1093/nar/gkad382 OpenUrl CrossRef PubMed 85. ↵ Carver T , Harris SR , Berriman M , Parkhill J , McQuillan JA . Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data . Bioinformatics . 2012 ; 28 : 464 – 469 . doi: 10.1093/bioinformatics/btr703 OpenUrl CrossRef PubMed Web of Science 86. ↵ Shimoyama Y. pyCirclize: Circular visualization in Python . 2022 . Available: https://github.com/moshi4/pyCirclize 87. ↵ Katoh K , Misawa K , Kuma K , Miyata T . MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform . Nucleic Acids Res . 2002 ; 30 : 3059 – 3066 . doi: 10.1093/nar/gkf436 OpenUrl CrossRef PubMed Web of Science 88. ↵ Kozlov AM , Darriba D , Flouri T , Morel B , Stamatakis A . RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference . Bioinformatics . 2019 ; 35 : 4453 – 4455 . doi: 10.1093/bioinformatics/btz305 OpenUrl CrossRef PubMed 89. ↵ Tonkin-Hill G , MacAlasdair N , Ruis C , Weimann A , Horesh G , Lees JA , et al. Producing polished prokaryotic pangenomes with the Panaroo pipeline . Genome Biol . 2020 ; 21 : 180 . doi: 10.1186/s13059-020-02090-4 OpenUrl CrossRef PubMed 90. ↵ R Core Team . R: a language and environment for statistical computing . Vienna, Austria : R Foundation for Statistical Computing ; 2019 . Available: https://www.R-project.org/ 91. ↵ Marçais G , Delcher AL , Phillippy AM , Coston R , Salzberg SL , Zimin A . MUMmer4: A fast and versatile genome alignment system . PLoS Comput Biol . 2018 ; 14 : e1005944 . doi: 10.1371/journal.pcbi.1005944 OpenUrl CrossRef PubMed 92. ↵ Goel M , Sun H , Jiao W-B , Schneeberger K . SyRI: finding genomic rearrangements and local sequence differences from whole-genome assemblies . Genome Biol . 2019 ; 20 : 277 . doi: 10.1186/s13059-019-1911-0 OpenUrl CrossRef PubMed 93. ↵ Goel M , Schneeberger K. plotsr: visualizing structural similarities and rearrangements between multiple genomes . Bioinformatics . 2022 ; 38 : 2922 – 2926 . doi: 10.1093/bioinformatics/btac196 OpenUrl CrossRef PubMed 94. ↵ Darling ACE , Mau B , Blattner FR , Perna NT . Mauve: multiple alignment of conserved genomic sequence with rearrangements . Genome Res . 2004 ; 14 : 1394 – 1403 . doi: 10.1101/gr.2289704 OpenUrl Abstract / FREE Full Text 95. ↵ Jiang T , Liu Y , Jiang Y , Li J , Gao Y , Cui Z , et al. Long-read-based human genomic structural variation detection with cuteSV . Genome Biol . 2020 ; 21 : 189 . doi: 10.1186/s13059-020-02107-y OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted January 13, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Rapid evolution of Wolbachia in cherry fruit flies Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Rapid evolution of Wolbachia in cherry fruit flies Daniel J. Bruzzese , Hannes Schuler , Wee L. Yee , Aurel Holzschuh , Jeffrey L. Feder bioRxiv 2025.01.13.632697; doi: https://doi.org/10.1101/2025.01.13.632697 Share This Article: Copy Citation Tools Rapid evolution of Wolbachia in cherry fruit flies Daniel J. Bruzzese , Hannes Schuler , Wee L. Yee , Aurel Holzschuh , Jeffrey L. Feder bioRxiv 2025.01.13.632697; doi: https://doi.org/10.1101/2025.01.13.632697 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Evolutionary Biology Subject Areas All Articles Animal Behavior and Cognition (7617) Biochemistry (17633) Bioengineering (13856) Bioinformatics (41841) Biophysics (21399) Cancer Biology (18529) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24281) Genetics (15582) Genomics (22461) Immunology (17700) Microbiology (40293) Molecular Biology (17140) Neuroscience (88413) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4813) Physiology (7632) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)