Experimental evolution of pseudogenization and gene loss in a plant RNA virus

A icle Expe imen al E olu ion o Pseudogeniza ion and Gene Loss in a Plan RNA Vi us Ma k P. Zwa ,* ,1 Anouk Willemsen, 1 Jose ´-An onio Da o `s, 1 and San iago F. Elena 1,2 1 Ins i u o de Biologı ´a Molecula y Celula de Plan as, Consejo Supe io de In es igaciones Cien ı ´ icas-UPV, Vale `ncia, Spain 2 The San a Fe Ins i u e *Co esponding au ho : E-mail: ma [email protected] .es. Associa e edi o : Howa d Ochman Abs ac Vi uses ha e e ol ed highly s eamlined genomes and a a ie y o mechanisms o comp ess hem, sugges ing ha genome size is unde s ong selec ion. Ho izon al gene ans e has, on he o he hand, played an impo an ole in i us e olu ion. Howe e , e olu ion canno in eg a e ini ially non unc ional sequences in o he i al genome i hey a e apidly pu ged by selec ion. He e we epo on he expe imen al e olu ion o pseudogeniza ion in i us genomes using a plan RNA i us exp essing a he e ologous gene. When long 9-week passages we e pe o med, he added gene was los in all lineages, whe eas i uses wi h la ge genomic dele ions we e ixed in only wo ou o en 3-week lineages and none in 1-week lineages. Illumina nex -gene a ion sequencing e ealed conside able con e gen e olu ion in he 9- and 3-week lineages wi h genomic dele ions. Genome size was co ela ed o wi hin-hos compe i i e i ness, al hough he e was no co ela ion wi h i us accumula ion o i ulence. Wi hin-hos compe i i e i ness o he 3-week i us lineages wi hou genomic dele ions was highe han o he 1-week lineages. Ou esul s show ha he s eng h o selec ion o a educed genome size and he a e o pseudogeniza ion depend on demog aphic condi ions. Mo eo e , o he 3-week passage condi ion, we obse ed inc eases in wi hin-hos i ness, whe eas selec ion was no s ong enough o quickly emo e he non unc ional he e ologous gene. These esul s sugges a demog aphically de e mined “swee spo ” migh exis , whe e he e ologous inse ions a e no immedia ely los while e olu ion can ac o in eg a e hem in o he i al genome. Key wo ds: genome e olu ion, plan i us, ho izon al gene ans e , pseudogeniza ion, i ness, nex -gene a ion sequencing. In oduc ion Vi us genomes a e highly s eamlined. Compa ed wi h mo e complex o ganisms, i uses end o ha e small genomes wi h 1) a high pe cen age o coding sequences, 2) none o li le in onic sequences, and 3) only sho s e ches o in e genic sequences (Lynch 2006;Belshaw e al. 2007). Mo eo e , i uses ha e e ol ed s a egies o u he comp ess hei genomes, such ameshi s and o e lapping open eading ames (ORFs) (e.g., Belshaw e al. 2007;Chung e al. 2008). Field obse a ions sugges ha genome sh inkage some imes occu s du ing epidemic sp ead and migh be linked o in- c eased wi hin-hos i ness and be adap i e o whi e spo synd ome i us (WSSV), a la ge DNA i us (Ma ks e al. 2005; Zwa , Dieu, e al. 2010). Mo eo e , i appea s o be a e y gene al obse a ion ha i uses exp essing he e ologous genes end o be uns able (Chapman e al. 1992;Dolja e al. 1993;Guo e al. 1998;Pijlman e al. 2001;Chung e al. 2007; Paa e al. 2007). Fu he mo e, unde condi ions maximizing selec ion o compe i i e i ness—exempli ied by undilu ed se ial passage in cul u ed cells— i uses end o apidly e ol e de ec i e in e e ing pa icles (DIPs): i uses wi h la ge genomic dele ions a e unable o eplica e au onomously bu wi h a eplica i e ad an age a high mul iplici ies o in ec ion (Huang 1973;Simon e al. 2004;Zwa e al. 2008; Pa hak and Nagy 2009). All hese obse a ions sugges ha genome size is unde s ong selec ion o i uses and ha ha ing unnecessa y genomic sequences has i ness cos s. By con as , s iking cases o genome sh inkage ha e been ound o obliga e hos -dependen species o bac e ia, bu his sh inkage appea s o be he esul o a mu a ional bias owa d dele ions and gene ic d i (Ochman and Da alos 2006;Kuo and Ochman 2009). Vi uses play an impo an e olu iona y ole as ec o s o ho izon al gene ans e (HGT) in he genomes o hei hos s (Canchaya e al. 2003;Belshaw e al. 2004;Rou h e al. 2012). I is mo eo e becoming inc easingly appa en ha HGT is also widesp ead in mos i uses and is a key mechanism in hei e olu ion (Hughes and F iedman 2005;Dolja and Koonin 2011;Koonin and Dolja 2012;Liu e al. 2011,2012; Yu in and Koonin 2012). S iking inno a ions such as he DNA-RNA i us hyb id (Dieme and S edman 2012)and he coop ion o an en i e hos immuni y mechanism by a phage (Seed e al. 2013) exempli y how HGT can empowe he e olu iona y p ocess. Howe e , s ong selec ion o genome size would in p inciple be an impedimen o HGT. In o de o a he e ologous sequence o be bene icial o he ecipien i us,i mus beaccommoda edin o he i us genome, ansc ip ome, and p o eome. Mu a ion and selec- ion mus he e o e ac on newly ans e ed he e ologous sequences, bu o do so, hese sequences mus no be pu ged igh upon acquisi ion because o selec ion o a s eamlined genome. The mu a ional supply may a o he dele ion o ßThe Au ho 2013. Published by Ox o d Uni e si y P ess on behal o he Socie y o Molecula Biology and E olu ion. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion Non-Comme cial License (h p:// c ea i ecommons.o g/licenses/by-nc/3.0/), which pe mi s non-comme cial e-use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. Fo comme cial e-use, please con ac jou nals.pe miss[email p o ec ed] Open Access Mol. Biol. E ol. 31(1):121–134 doi:10.1093/molbe /ms 175 Ad ance Access publica ion Oc obe 8, 2013 121 by gues on Janua y 3, 2014h p://mbe.ox o djou nals.o g/Downloaded om he e ologous sequences; dele iono asequenceby ecombi- na ion p obably has a g ea e likelihood han he occu ence o bene icial mu a ions unc ionally in eg a ing his elemen in he i us. I he e ec s o dele ions o he e ologous sequences a e bene icial on he sho e m, how can HGT be common in i uses? One possible answe o his ques ion is ha selec ion o genome size is no a e y s ong o ce. Fi s , i is no a all clea ha he me abolic cos o addi ional gene ic ma e ial is su icien o accoun o he expec ed i ness cos s (Lynch 2007). Second, expe imen al esul s on he ela ionship be ween genome size and eplica i e i ness a e ambiguous. When compa ing phages wi h di e en genome o ganiza- ions adap ed o as eplica ion, he expec ed ela ionship was no ound (Bull e al. 2004). When exp essing di e en - size ma ke p o eins in Sendai i us, an in e se ela ionship be ween inse size and eplica ion was ound in cul u ed cells (Sakai e al. 1999). Howe e , his ela ionship was no obse ed in i o (Sakai e al. 1999) and, u he mo e, he use o sequences coding o di e en ma ke p o eins makes he compa ison oublesome (Maje e al. 2013). Mo eo e , ins abili y o i uses exp essing he e ologous sequences (Chapman e al. 1992;Dolja e al. 1993;Guo e al. 1998;Chung e al. 2007) may depend on many en i on- men al ac o s (Paa e al. 2007), and e en sub le ies o he he e ologous sequence, such as guanine-u acil con en (Lee e al. 2002). Finally, expe imen s co obo a ing he ela ion- ship be ween genome size and i ness o WSSV we e pe - o med wi h ield isola es (Ma ks e al. 2005;Zwa , Dieu, e al. 2010), and hence, o he gene ic a ia ion could be a con- ounding ac o . In conside ing whe he HGT is eally implau- sible, we he e o e need o ask whe he inc eases in genome size eally ha e app eciable i ness cos s and wha i ness componen s migh be a ec ed. He e we explo e he p ocess o pseudogeniza ion in i us genomes by means o expe imen al e olu ion. As a model sys em, we conside a plan RNA i us exp essing a non oxic he e ologous gene, whose exp ession has been enginee ed o minimally dis up he i al genome. We i s looked o condi ions unde which he he e ologous gene would be main ained in he genome. I has been shown ha he ime pe iod be ween wo consecu i e ansmission e en s, ha is, he ime a i al popula ion has o expand be ween wo consecu i e bo lenecks, can be ins umen al in de e mining genome s abili y in plan RNA i uses (Dolja e al. 1993). Howe e , in his s udy, he he e ologous gene was exp essed as a usion wi h one o he i al cis ons and, gi en he s ong e ec s on i al accumula ion (Dolja e al. 1993), mus he e o e be seen as a dele e ious, a he han a me ely non unc ional addi ion. We hen conside whe he he dele ion o he he e ologous gene was adap i e and wha i us cha ac e is ics i modi ies. Finally, we conside whe he he e a e condi ions ha ul ill wo equi emen s: 1) he he e ologous gene has a high p obabili y o being main ained in he e ol ed i us popula ion and 2) he e is e idence ha he i us popula ion is unde posi i e selec ion and expe iences inc eases in i ness. We hink he combina- ion o condi ions is ele an o he con ex o HGT in i uses. I hese wo condi ions a e ul illed, hen in p inciple, a he e ologous sequence can pe sis o long pe iods o ime in he i us popula ion, while inc eases in i ness imply ha na u al selec ion is ac ing on he popula ion and could “ inke ” wi h he he e ologous gene, sequences egula ing i s exp ession, and o he loci in e ac ing wi h he he e olo- gous gene, po en ially and unc ionally in eg a ing i in o he i al genome. On he o he hand, a he e ologous gene may no be los in a i us popula ion subjec o high le els o gene ic d i , bu i is hen also unlikely ha na u al selec ion ac s o unc ionally in eg a e i . Simila ly, a i us popula ion unde s ong posi i e selec ion in which he he e ologous gene is los is also a dead end o HGT. Howe e , simul aneously ha ing main enance o he he e ol- ogous gene and inc eases in i al i ness sugges s he occu - ence o a “swee spo ” ha could help explain how HGT occu s in i uses. Resul s and Discussion Resul s o Se ial Passage Expe imen s As a model sys em o i us exp essing a he e ologous gene wi hou app eciable oxici y o dis up ion o i al eplica ion, we used a a ian o Tobacco e ch i us (TEV; genus Po y i us, amily Po y i idae). TEV is a posi i e-sense single-s anded RNA i us ha encodes a polyp o ein au oca aly ically clea ed in o en ma u e p o eins (Riechmann e al. 1992) and a pa ially o e lapping ORF wi h a +2 ameshi (Chung e al. 2008). The TEV a ian we used exp esses enhanced g een luo escen p o ein (eGFP) as a sepa a e cis- on be ween P1 and HC-P o by in oducing a second NIa-P o p o eoly ic si e downs eam o he eGFP sequence while e- aining he exis ing C- e minal si e in P1 ( ig. 1)(Zwa e al. 2011). E olu ion expe imen s we e pe o med in Nico iana abacum L. c . Xan hi plan s. In b ie , 4-week-old plan s we e inocula ed wi h high i us doses, and passages las ing ei he 1 week ( o a o al o 27 consecu i e passages), 3 weeks (nine passages) o 9 weeks ( h ee passages) we e pe o med. Each “passage” is he in ec ion o a single plan and ha es ing o issues a he designa ed ime (i.e., he “passage du a ion”). The e o e, al hough he passage du a ion a ied among FIG.1. Scheme o TEV-eGFP. Lines ep esen he i al 50-and3 0-un ansla ed egions (50-UTR and 30-UTR), he g ay box ep esen s eGFP, open boxes ep esen he i al cis ons P1, HC-P o, P3, 6K1, CI, 6K2, VPg, NIa-P o, NIb, and CP, whe eas P3N-PIPO is indica ed by he lowe box. 122 Zwa e al. .doi:10.1093/molbe /ms 175 MBE by gues on Janua y 3, 2014h p://mbe.ox o djou nals.o g/Downloaded om ea men s, each lineage e ol ed o he same o al ime (27 weeks) in N. abacum. A he end o a passage, all he lea es abo e he inocula ed lea we e collec ed, pooled, and used o ob ain he inoculum o he nex ound o se ial passaging. Ten independen lineages we e gene a ed and main ained o each passage du a ion (1, 3, o 9 weeks). An o e iew o he expe imen al se up used is gi en in igu e 2,and u he de- ails a e gi en in he Ma e ials and Me hods. eGFP exp ession was eadily appa en in in ec ed plan s ( ig. 3A) and was used as a i s indica ion o whe he he he e ologous gene was in ac . Pa ial losses o luo escence ( ig. 3B) almos always p eceded comple e losses o luo es- cence ( ig. 3C). One ou o en 1-week lineages showed a pa ial loss o luo escence, i s obse ed a passage 7 and main ained un il passage 27 ( ig. 3Eand F). Two ou o en 3-week lineages showed a loss o luo escence. All bu one 9-week passage showed dec eased le els o luo escence a e a single passage, and all lineages showed a comple e loss o luo escence a e wo passages. Re e se ansc ip ion poly- me ase chain eac ion (RT-PCR) wi h p ime s lanking eGFP (Ma e ials and Me hods) con i med he occu ence o geno- mic dele ions in all lineages wi h a pa ial o comple e loss o luo escence ( ig. 3G). These esul s a e cong uen wi h p e- ious wo k wi h TEV (Dolja e al. 1993), excep ha he de- le ion o he he e ologous gene occu s much mo e slowly he e, as an icipa ed. We hen inocula ed N. abacum wi h TEV-eGFP and, a 9 weeks o in ec ion, ha es ed e e y i h lea up he s em. Because he in ec ion p og esses linea ly as he plan g ows, hese lea es enable us o moni o aquali a i e imecou seo e olu ion wi hin he plan . All plan s had by hen eached he 45-lea s age, excep o one ha had only 40 lea es. We pe o med RT-qPCR on indi idual lea es o asce ain a wha lea le el dele ions occu ed, and i hey we e subse- quen ly main ained in he popula ion. This analysis can be pe o med since he i us mo es mainly upwa d in he plan (Dolja e al. 1992). In mos cases, once a dele ion was de ec ed in one lea , i was main ained and ixed in he supe io lea es ( ig. 4). This obse a ion sugges s ha selec- ion o dele ion a ian s is e y s ong in his expe imen , being a s onge e olu iona y o ce han gene ic d i wi hin he hos . The i s lea in which a no el dele ion was de ec ed was no uni o mly dis ibu ed o e all es ed lea es (lea es 5– 45; one-sample Kolmogo o –Smi no es : n= 13; P= 0.019); new dele ion a ian s we e usually i s de ec ed in highe lea es (mean ±s anda d de ia ion [SD] = 32.31 ±9.92). This esul sugges s ha passage du a ion is impo an o he dy- namics o he e ologous gene dele ion because i egula es he amoun o expansion ha occu s be ween bo lenecks. Du ing i us in ec ion o mechanically inocula ed plan s, gene ic bo lenecks in he i us popula ion can occu du ing p ima y in ec ion o he inocula ed lea and he subsequen en y in o sys emically in ec ed lea es (Hall e al. 2001; Sac is a ´n e al. 2003;Gu ie ´ ez e al. 2012). Fo TEV in ec ion o obacco plan s, he numbe o p ima y in ec ion oci in he inocula ed lea is a good es ima o o he numbe o ounde s, whe eas he subsequen bo lenecks du ing en y in o sys emically in ec ed lea es do no appea o be se e e (Zwa e al. 2011). We he e o e inocula ed eigh plan s wi h TEV-eGFP using he same p ocedu e and condi ions, as du ing se ial passaging (see Ma e ials and Me hods; homogenized issue o plan s in ec ed wi h TEV-eGFP was used as an inoculum), and coun ed he numbe o p ima y in ec ion oci (Zwa e al. 2011). The mean numbe o oci obse ed ±SD was 417 ±140, sugges ing ha al hough he e is a bo leneck a he s a o in ec ion, i is no oo se e e. Ne e heless, his bo leneck could emo e a ia ion gene - a ed de no o du ing he p e ious passage and he eby limi he a ia ion upon which selec ion can ac . Longe passage du a ion would allow bene icial a ia ion o inc ease in equency, hus making i less likely o lose such a ian s due o gene ic d i a he nex ansmission e en . The oc- cu ence o gene ic bo lenecks he e o e ein o ces he idea ha passage du a ion migh be impo an o he e olu iona y dynamics in his sys em. Genome Sequences o E ol ed Lines wi h Dele ions All e ol ed lineages in which dele ions had been de ec ed by RT-PCR we e ully sequenced by Illumina nex -gene a ion sequencing (NGS). We de eloped an app oach (Ma e ials and Me hods) o mapping la ge genomic dele ions (i.e., de- le ions la ge han he ead size). We consis en ly saw pseu- dogeniza ion o comple e loss o eGFP in all hese lineages ( ig. 5A). None o hese dele ions included he C- e minus o P1, while o 7 ou o 13 lineages, hese dele ions included N- e minal egions o HC-P o, simila o he p e ious esul s (Dolja e al. 1993). The N- e minal egion o HC-P o is no essen ial o eplica ion and mo emen (Dolja e al. 1993; C onin e al. 1995) bu has been implica ed in ec o -bo ne ansmission (Tho nbu y e al. 1990;A eya e al. 1992), which is no a selec i e o ce in ou mechanical ansmission passage expe imen s. In he se en lineages wi h dele ions ex ending in o HC-P o, he emains o eGFP we e used wi h HC-P o, while he p o eoly ic si e be ween P1 and eGFP emained in ac . The s a o genomic dele ion (50end) was no uni- o mly dis ibu ed (one-sample Kolmogo o –Smi no es : n= 13; P= 0.006), and he e is clus e ing a he 50o he eGFP cis on ( ig. 5Band C), sugges ing he exis ence o a ho spo o ecombina ion o he un iabili y o any dele ions in he P1-eGFP p o eoly ic si e. On he o he hand, he 30end o he genomic dele ion was uni o mly dis ibu ed (one- sample Kolmogo o –Smi no es : n= 13; P= 0.130). We pe o med addi ional analyses o de ec mino i y a ian s wi h di e en dele ion sizes in sequenced lineages (Ma e ials and Me hods). Al hough mino i y a ian s we e some imes de ec ed in he 3- and 9-week lineages, hese we e always p esen a low equencies (<1.5%). Only in he case o he 1-week lineage wi h a pa ial eGFP loss was a mino i y a ian p esen : 47.1% o he popula ion was composed o a a ian wi h in ac eGFP. The dele ion in he majo i y a ian ex ends beyond he HC-P o N- e minal egions nonessen ial o eplica ion and mo emen (C onin e al. 1995), sugges ing his majo i y a ian is no able o eplica e wi hou being complemen ed by he ull-leng h a ian . When plan s we e inocula ed wi h low i us doses 123 Expe imen al E olu ion o Pseudogeniza ion .doi:10.1093/molbe /ms 175 MBE by gues on Janua y 3, 2014h p://mbe.ox o djou nals.o g/Downloaded om FIG.2. O e iew o he expe imen al se up employed in he s udy. A he s a o he se ial passage expe imen , 4-week-old N. abacum plan s we e mechanically inocula ed wi h TEV-eGFP in he hi d ue lea (indica ed in ed abo e). A he end o he designa ed passage du a ion (1, 3, o 9 weeks), all lea es abo e he inocula ed lea , which a e indica ed in g een abo e, we e collec ed and s o ed a 80 C. The ozen issue was hen homogenized, and a sample o he homogenized issue was g ound o a ine powde . Fo inocula ion o subsequen passages, powde was esuspended in inocula ion bu e and new N. abacum plan s we e inocula ed. Al hough he du a ion o he passages a ied (1, 3, and 9 weeks), he numbe o passages was se so ha he o al ime each lineage was p esen in plan s was he same, being 27 weeks o all lineages. Fo each passage du a ion used, en independen lineages we e aken. No e ha he igu e is only schema ic: he dis ance be ween lea laye s has been exagge a ed, and a e 9 weeks o in ec ion, N. abacum plan s a e in eali y ela i ely alle han depic ed he e. 124 Zwa e al. .doi:10.1093/molbe /ms 175 MBE by gues on Janua y 3, 2014h p://mbe.ox o djou nals.o g/Downloaded om FIG.3. Loss o eGFP luo escence and sequence du ing se ial passages: (A)AnN. abacum lea ha is comple ely symp oma ic, indica ing hea y i us in ec ion, showed 1 week pos inocula ion wi h an e ol ed TEV-eGFP lineage wi h no loss o luo escence. (B) A comple ely symp oma ic lea showed 1 week pos inocula ion wi h an e ol ed TEV-eGFP lineage wi h a pa ial loss o luo escence (1-week passage lineage 7). (C) A comple ely symp oma ic lea showed 1 week pos inocula ion wi h an e ol ed TEV-GFP lineage wi h a comple e loss o luo escence. (D)Obse ed luo escence du ing se ial passage o TEV-eGFP o 1-week passages. Fo panels D–E, g een squa es indica e no loss o eGFP luo escence (as in panel A), ha ched g een squa es indica e a pa ial loss o luo escence (as in panel B) in pa s o all o he plan , and whi e squa es indica e no luo escence was obse ed in he whole plan (as in panel C). (E) Obse ed luo escence o 3-week passages. (F) Obse ed luo escence o 9-week passages. (G)Anaga osegelwi hRT-PCR p oduc s o dele ions in he eGFP locus o TEV-eGFP is shown o he i s passage o he 9-week lineages. The alue 1 kb +indica es he lane wi h a 1kb+DNA ladde , NTC is he non empla e con ol, and NVC is he non i us con ol, a mock-inocula ed heal hy plan . TEV-eGFP, he ances al i us o he e olu ion expe imen s, and TEV, he wild- ype i us wi hou he he e ologous gene inse ed, a e included o compa ison. No e ha in each e ol ed lineage, dele ions o eGFP a e isible, al hough hei equency appea s o a y. In lineage 1, he band co esponding o he ances al i us is s ill e y s ong, whe eas o lineage 4, i is no longe isible. FIG.4. A single 9-week passage o TEV-eGFP was pe o med in eigh plan s, and he smalles obse ed inse size a he eGFP locus (o dina e) was measu ed e e y i h lea (abscissa). Do ed lines indica e he ull-leng h eGFP sequence was s ill de ec ed, solid lines indica e i is no longe de ec ed, and a ci cle indica es he poin a which he ull-leng h sequence is i s no longe de ec ed, and each eplica e has a di e en colo . Dele ions we e ixedin ou ou o eigh eplica es, and only in a single eplica e we e no dele ions de ec ed h oughou in ec ion (yellow). Only in one case (pink) was a dele ion no main ained a e i has i s been obse ed (a dele ion is obse ed in lea 40 bu no in lea 45). 125 Expe imen al E olu ion o Pseudogeniza ion .doi:10.1093/molbe /ms 175 MBE by gues on Janua y 3, 2014h p://mbe.ox o djou nals.o g/Downloaded om FIG.5. Genome sequences o e ol ed lineages: (A) NGS da a o he e ol ed lineages wi h dele ions om he se ial passage expe imen ( ig. 3Dand F) a e gi en. The names on he le iden i y lineages (e.g., 9W L1 is he inal popula ion o 9-week passage, lineage 1). G ay boxes indica e genomic dele ions in he majo i y a ian . Full ci cles and open ci cles a e nonsynonymous and synonymous subs i u ions, espec i ely. Black subs i u ions occu in only one lineage, whe eas colo -coded subs i u ions a e epea ed in wo o mo e lineages. Fo lineage 1W L7, only a single sequence is ep esen ed. Howe e , no e ha in his lineage, ano he a ian wi h he ull-leng h TEV-eGFP genome is p esen a a equency 47.1%. None o he single-nucleo ide mu a ions in 1W L7 we e ixed, sugges ing hey occu ed a e he genomic dele ion and a e p esen in only one o he wo a ian s p esen . Howe e , he sequence a ia ion is minimal and he wo a ian s a e p esen a app oxima ely same equencies in his lineage. The single-nucleo ide mu a ions can he e o e no be assigned o he ull-leng h o he dele ed a ian , al hough hey a e ep esen ed on he dele ion a ian in he igu e. In all o he lineages, he ull-leng h i us was no de ec ed and a ian s wi h o he genomic dele ions we e p esen only a e y low equencies (<1.5%). (B) His og am o he posi ion o he s a o genomic dele ions in he e ol ed lines. Fo panels Band C, da k lines indica e egions in he eGFP cis on, whi e lines indica e egions in he i al genome, and ha ched lines indica e he egions encompassing wo cis ons (i.e., P1 and eGFP in panel B). (C) His og am o he posi ion o he end o he genomic dele ion in e ol ed lines. Table 1. In Vi o Cloning o 1-Week Passage Lineage 7 (1W L7). Expe imen Mean Foci Plan s Unin ec ed eGFP Only Mix u e No eGFP 1 1.022 9 (0.360) 12 (0.480) 4 (0.160) 0 (0.000) 2 1.514 18 (0.243) 36 (0.486) 20 (0.270) 0 (0.000) NOTE.–Two eplica e expe imen s we e pe o med in which Nico iana abacum plan s we e in ec ed wi h a 1:1,000 dilu ion o in ec ious sap o he inal e ol ed popula ion o 1W L7. This popula ion had appa en ly ha bo ed a i us a ian ha had los luo escence ( ig. 3B), al hough his a ian ne e wen o ixa ion ( ig. 3D). E en a low doses, all in ec ed plan s (as de e mined by symp oms 2 weeks a e inocula ion) con ained only he eGFP-exp essing i us (“eGFP only”; luo escence pa e ns simila o ig. 3A) o bo h i us a ian s (“Mix u e”; ig. 3B). A symp oma ic plan wi hou eGFP exp ession was ne e obse ed (“No eGFP”; ig. 3C), al hough he low dose esul ed in a low mean numbe o p ima y in ec ion oci o TEV-eGFP (“mean oci”) and many unin ec edplan s.RT-PCRwaspe o medon i eplan sjudgedasbeing unin ec ed, in ec ed only wi h he eGFP a ian , o in ec ed wi h a mix u e o he wo i uses, and he RT-PCR esul s we e always cong uen wi h mic oscopic obse a ions. We he e o e conclude he i us a ian in he popula ion ha does no exp ess eGFP, has he e o e p obably los in ec i i y, o i has a e y low in ec i i y. This a ian is ne e heless su p isingly s able. The 1W L7 popula ion was pu h ough h ee 1-week passages o one 3-week passage, wi h en eplica es each. The p esence o he i us a ian no exp essing eGFP could always be deduced om eGFP exp ession pa e ns (i.e., ig. 3B). 126 Zwa e al. .doi:10.1093/molbe /ms 175 MBE by gues on Janua y 3, 2014h p://mbe.ox o djou nals.o g/Downloaded om o his e ol ed lineage, we we e indeed unable o in i o clone he majo i y a ian wi hou in ac eGFP ( able 1). When single-base subs i u ions we e de ec ed in se- quenced lineages, he e appea ed o be con e gen e olu ion in he 3- and 9-week lineages ( ig. 5A). All hese lineages con ained a leas one subs i u ion p esen in ano he lineage, wo subs i u ions we e p esen in 8 ou o 12 lineages, and one subs i u ion was p esen in 9 ou o 12 lineages. O e all, mo e han hal o he subs i u ions ound we e p esen in o he lineages, and some lineages con ained only subs i u- ions also p esen in o he lineages (9-week lineages 1 and 2). In he single 1-week passage sequenced, none o he epea ed subs i u ions we e p esen , sugges ing ha con e gen e olu ion did no occu unde hese condi ions. O he subs i u ions ound he e, 28 we e nonsynonymous and 53 we e synonymous. Ele en nonsynonymous subs i u- ions we e con e gen , while 29 synonymous subs i u ions we e con e gen . Synonymous subs i u ions we e he e o e mo e common han nonsynonymous subs i u ions, al hough bo h we e equally likely among cases o con e gence (Fishe ’s exac es P= 0.244). Con e gen , nonsynonymous subs i u- ions we e always ound in he P1 cis on (A872G, N!Sin9 ou o 13 lineages; all posi ions gi en ela i e o he o iginal TEV-eGFP genome, GenBank KC918545), o he emaining 50end o he eGFP cis on (A1085U, E!V in 2 lineages). Con e gen synonymous subs i u ions we e ound in P1 (C795U in six lineages), HC-P o (C1927A in i e lineages), NIa-P o (U7092C in wo lineages and A7479C in eigh line- ages), and NIb cis ons (A8253C in eigh lineages). The A7479C and A8253C subs i u ions always occu ed oge he , sugges ing syne gis ic epis asis o possibly e en ecip ocal sign epis asis, whe eas he A7479C and A8253C ne e occu ed oge he wi h U7092C, sugges ing an agonis ic epis asis. Howe e , nei he o hese wo e ec s was signi ican gi en he numbe o obse a ions ( able 2). No a single mu a ion was de ec ed in he coa p o ein (CP). The o e all d N /d S a io was signi ican ly smalle han 1 (mean ±SD = 0.058 ±0.002; z- es P<0.001), sugges ing he polyp o ein sequence is unde pu i ying selec ion. Wi hin-popula ion single nucleo- ide polymo phisms (SNPs) we e analyzed o e e y e ol ed lineage agains hei co esponding consensus sequence. O he SNPs ound, en we e synonymous and six we e non- synonymous. Fi e ou o 13 e ol ed lineages con ained he same synonymous SNP in he HC-P o cis on (C1879A). None o he o he synonymous and nonsynonymous SNPs we e epea ed in he e ol ed lineages. P e ious e olu ion expe imen s wi h TEV ha e also shown no genomic con e gences o 1-week passages in N. abacum: a e 15 weeks o e olu ion, no subs i u ions we e epea ed in di e en lineages (Bedhomme e al. 2012). These obse a ions sugges ha li le adap i e e olu ion migh occu when sho 1-week se ial passages a e pe o med. Fu he mo e, he speci ic con e gen mu a ions obse ed he e we e no obse ed in o he 1-week passage expe imen s (Bedhomme e al. 2012; T omas N, Zwa MP, Elena SF, unpublished manusc ip ). This sugges s ha hese con e gen mu a ions may be linked o he inse ion o he eGFP gene. To es his possibili y, we conside ed whe he he mos common mu a- ions (C795U, A872G, A7479C, and A8253C) occu ed in lin- eages o he wild- ype TEV pu h ough h ee 9-week passages in N. abacum (Ma e ials and Me hods). In none o such lineages we e any o hese mu a ions ound, s ongly sugges - ing hey a e linked o he p esence o eGFP. Al hough we sequenced only a small pa o hese e ol ed TEV genomes, we did ind one mu a ion epea ed in 3 ou o 10 lineages (A6806G, K!E), sugges ing he e is a leas some con e gen e olu ion when he wild- ype TEV is pu h ough long passages. Accumula ion, Vi ulence, and Wi hin-Hos Compe i i e Fi ness o E ol ed Lineages We biologically cha ac e ized all e ol ed lineages, in e ms o hei i ulence and i al accumula ion, and measu ed wi hin-hos compe i i e i ness (W; Ma e ials and Me hods). The e was no e ec o passage du a ion on ei he i al accumula ion a 7 days pos inocula ion (dpi) o i ulence ( ig. 6Aand B; able 3). On he o he hand, he e was a highly signi ican e ec o passage du a ion on wi hin-hos compe i i e i ness, which inc eased signi ican ly wi h passage du a ion ( ig. 6Cand able 3). Fo he cu en expe imen al se up, we a p io i expec ha wi hin-hos compe i i e i ness will be unde selec ion, be- cause we a e passaging a i us wi hin a single hos a high inocula ion doses. Those i us a ian s ha exis a he high- es equency a he end o in ec ion a e he e o e mos likely o be ans e ed, i espec i e o accumula ion le els o he en i e popula ion. We did no expec i ulence o change, because we do no expec i o be unde selec ion and i is p obably no linked o wi hin-hos i ness in ou model sys em (Ca asco, de la Iglesia, e al. 2007). Gi en ha high inocula ion doses a e used, i us accumula ion is no likely o be e y impo an ei he , so long as i is enough o main ain in ec ion in he nex ound o passaging. No e ha a 1,000- old dilu ion o an inoculum s ill causes mode a e le els o in ec ion, as shown by he in i o cloning esul s ( able 1). Published da a (Ca asco, de la Iglesia, e al. 2007;Lalic ´e al. Table 2. Cooccu ence o Single-Nucleo ide Subs i u ions. Subs i u ion Combina ion P AðÞP BðÞ¼P A BðÞ.Obs A BðÞ.PValue A7479C A8253C. (8/12)(8/12) = 0.444 8/12 = 0.667 0.1501 A7479C A8253CðÞ U7092C. (8/12)(2/12) = 0.111 0/12 = 0 0.3841 NOTE.–He e we es whe he he cooccu ence, o lack he eo , is s a is ically signi ican o wo g oups o single-nucleo ide subs i u ion (i.e., subs i u ion combina ion). P AðÞP BðÞ¼P A BðÞis he p oduc o he equency o occu ence o he subs i u ions, he expec ed equency a which we expec o see bo h subs i u ions in he absence o any epis a ic in e ac ions. We only conside ed he 3- and 9-week passages, because we do no hink selec ion is ac ing on he 1-week lineage.Obs A BðÞgi es he equency a which he combina ion was obse ed, and P alue is he signi icance as de e mined by compa ison o p edic ed and obse ed alues wi h an exac binomial es . 127 Expe imen al E olu ion o Pseudogeniza ion .doi:10.1093/molbe /ms 175 MBE by gues on Janua y 3, 2014h p://mbe.ox o djou nals.o g/Downloaded om FIG.6. Vi ulence, accumula ion, and wi hin-hos i ness o e ol ed s ains a e gi en. (A) The heigh o con ol plan s (NVC), and plan s in ec ed wi h TEV, TEV-eGFP ( he ances al i us o e ol ed lineages), and all lineages o e ol ed i uses a e gi en. We conside he in e se o plan heigh as a p oxy o i ulence, hough in he absence o signi ican di e ences in he da a, we simply p esen he aw da a. Fo all panels, g een columns indica e no dele ions in he he e ologous gene (eGFP) we e de ec ed, o ange indica es pa o all o he eGFP was no p esen , and ed indica es pa o eGFP and he i al HC-P o cis on a e no p esen . The 1W L7 popula ion is ma ked magen a because i con ains a la ge dele ion esul ing in a i us appa en ly unable o in ec on i s own. (B) Vi us accumula ion, as measu ed by RT-qPCR. (C) The eplica i e ad an age (W) o he es ed i us wi h espec o a common compe i o , TEV-mChe y, is gi en, as de e mined by compe i ion expe imen s and RT-qPCR (Ma e ials and Me hods). We conside Was an indica o o he wi hin-hos compe i i e i ness. Table 3. Nes ed ANOVAs on Plan Heigh , Accumula ion, and Wi hin-Hos Fi ness o E ol ed Lineages. T ai Sou ce o Va ia ion SS d MS FP Plan heigh T ea men 66.040 2 33.020 0.791 0.463 Lineage wi hin ea men 1126.600 27 41.726 7.120 <0.001 E o 703.200 120 5.826 Accumula ion T ea men 41.440 2 20.720 0.391 0.680 Lineage wi hin ea men 1431.520 27 53.019 6.998 <0.001 E o 909.200 120 7.577 Fi ness a T ea men 1.881 2 0.940 48.534 <0.001 Lineage wi hin ea men 0.523 27 0.019 3.267 <0.001 E o 0.356 60 0.006 Fi ness b T ea men 0.100 1 0.100 7.868 0.013 Lineage wi hin ea men 0.203 16 0.013 4.096 <0.001 E o 0.112 36 0.003 a Fi ness is a compa ison o all lineages. b Fi ness compa es only hose lineages ha ha e no ixed genomic dele ions, en o which a e om he 1-week ea men and eigh o which a e om he 3-week ea men (see ig. 5). ANOVA was used o his compa ison o wo g oups o allow lineage o be nes ed wi hin ea men . T ea men is he passage du a ion (1, 3, o 9 weeks). 128 Zwa e al. .doi:10.1093/molbe /ms 175 MBE by gues on Janua y 3, 2014h p://mbe.ox o djou nals.o g/Downloaded om 2011) show ha he mu a ional e ec s on wi hin-hos i ness and i us accumula ion a 7 dpi, exp essed as he Mal husian g ow h a e pe day (Lalic ´e al. 2011), a e no co ela ed (Spea man co ela ion: =0.034, 19 d ,P= 0.884; see also supplemen a y ig. S1,Supplemen a y Ma e ial online). The e o e, accumula ion was no expec ed o inc ease as a pleio opic e ec o inc eases in wi hin-hos i ness ei he . Accumula ion will be an impo an pa ame e when each i us lineage is e ol ed in mul iple hos o ganisms, as highe accumula ion can hen lead o a highe equency o a i us a ian in he inal popula ion (e.g., Zwa , Van de We , e al. 2010). Fo a plan i us, i is plausible ha wi hin-hos i ness and accumula ion a e la gely decoupled. Local in ec ion by cell- o- cell mo emen can be achie ed by a small numbe o i ions anspo ed o an adjacen cell (Miyashi a and Kishino 2010). The e o e, hose i us a ian s ha sp ead apidly need no necessa ily accumula e a high numbe o i ions pe cell. Exclusion is mo eo e hough o play an impo an ole in in ec ion (Die ich and Maiss 2003,Folimono a 2012), po en ially allowing i uses ha sp ead quickly o each a high equency and ye ha e ela i ely low accumula ion. Fu he mo e, i should be no ed ha e en i wo i us a ian s ha e he same le el o accumula ion la e in in ec ion (e.g., 7 dpi), i uses wi h a high wi hin-hos compe i i e i ness may each highe le els o accumula ion ea ly in in ec ion (e.g., 3 dpi). When a i us apidly exi s he inocula ed lea , his can lead o highe le els o in ec ion be o e in ec ion le els sa u a e (La o gue e al. 2012;Zwa e al. 2012). Rapid eplica ion and mo emen migh he e o e be he mechanism by which wi hin-hos i ness is inc eased in he e ol ed lineages, especially i genome size was di ec ly linked o eplica ion. Ne e heless, he key ai o measu e om an e olu iona y pe spec i e—because i is expec ed o be unde selec ion in his expe imen al se up—is compe i i e wi hin-hos i ness. We hen conside ed he ela ionship be ween genome size and wi hin-hos i ness o he e ol ed lineages ( ig. 7) and also ound a highly signi ican ela ionship (Spea man co ela ion: =0.877, 28 d ,P<0.001). The mean i ness o e ol ed lineages wi h genomic dele ions was highe han ha o he ances al i us wi hou he he e ologous gene (TEV) o 10 ou o 12 lineages. Howe e , when we pe o med pai wise compa isons be ween TEV and he e ol ed s ains ha ixed dele ions, no signi ican di e ences we e ound ( - es wi h Holm–Bon e oni co ec ion on he log- ans o med W alues). S a is ical powe is low when compa ing indi idual lineages because indi idual-plan le el a ia ion is high, a limi a ion o ou expe imen al sys em. We mus he e o e conclude ha i ness o he wild- ype TEV and e ol ed s ains is simila . This esul is, howe e , cong uen wi h he obse a ion ha he con e gen single-nucleo ide mu a ions obse ed in he e ol ed TEV- eGFP lineages we e no obse ed in wild- ype TEV in his s udy and o he s (Bedhomme e al. 2012;T omasN, Zwa MP, Elena SF, unpublished manusc ip ); i sup- po s he sugges ion ha hese mu a ions a e speci ic o accommoda ing changes in he TEV-eGFP backg ound and will p obably no be bene icial in he wild- ype i us backg ound. We hen conside ed he wi hin-hos compe i i e i ness o hose lineages wi hou genomic dele ions ( ig. 6C). We ound ha 3-week lineages wi hou genomic dele ions had a signi ican ly highe i ness han he 1-week lineages ( able 3). The 3-week lineages appea o be a he swee spo whe e he he e ologous gene is main ained in many i al lineages, while he e a e concomi an ly signi ican inc eases in i al i ness. This obse a ion sugges s ha demog aphy can play an impo an ole in modula ing he e olu iona y ou come o HGT. Al hough we ha e shown simul aneous main enance o he he e ologous gene and inc eases in i ness, he inc eases in FIG.7. The ela ionship be ween genome size (abscissa) and wi hin-hos compe i i e i ness (o dina e) is gi en. G een da a poin s indica e no dele ions in he he e ologous gene (eGFP) we e de ec ed, o ange indica es pa o all o he eGFP was no p esen , and ed indica es pa o eGFP and o he i al HC-P o cis on a e no p esen . The da a poin s o he ances al TEV-eGFP and TEV ha e been illed, and TEV-eGFP has been shi ed o he igh so ha i can be easily iden i ied, e en hough i s genome size is he same as o he lineages wi hou dele ions. A linea eg ession line has been added only o emphasize he end in he da a. No e ha mos o he e ol ed i uses wi h dele ions ha e a highe i ness han TEV, implica ing he obse ed subs i u ions wi h inc eased i ness. 129 Expe imen al E olu ion o Pseudogeniza ion .doi:10.1093/molbe /ms 175 MBE by gues on Janua y 3, 2014h p://mbe.ox o djou nals.o g/Downloaded om