{"paper_id":"0e535cf3-dfdd-4323-9e67-a624407ba08e","body_text":"Communi t y  comp lex i ty does no t  weaken p airwise coevol ution  in  a soil ba c ter ial  1 \ncommun ity  2 \nAuthor Affilia tions  3 \nZoltan Er dos 1  (z.erdos@ex et e r.ac .uk ) ,  Daniel P adfie ld 1  (d .pa dfield @exeter . ac.uk) Elze Hes s e 1  4 \n(e.hesse @ ex eter.a c .u k ), Angus B uck li ng 1 (a.j.buckling@exeter.ac .uk) &  Me ag h a n Cas t le di ne 1  5 \n(m.castledine@exete r. ac.uk)  6 \n1 Centre for  Ecology  and C o nservati on, Faculty of Env ir onment, Scienc e and Econom y ,  7 \nUniversity  o f Exeter, Penryn, C or nw all, TR10 9F E , U .K.  8 \nKeywords: C oev olut ion, d ynamics, e v olutio n, so il bact e r ia , compet i t ion , ant agonism  9 \nCorrespo nding aut hor : Zoltan Erdo s  ( z.erdos@ex eter. ac. uk)  10 \nAuthor con tribu tions (MeR IT):  11 \nZoltan  Erdos : Inve stigati on; Co nceptualiz ation; writin g - ori gi n a l draft ; for mal  analy sis, w r i t in g  -  12 \nreview  and editing; methodolog y; Vis ualiz ation. D aniel Padfield : formal a n a lysis; revie w and 13 \nediting. Elze H es se:  Co nceptualiz at i o n; w rit i ng - r ev iew and editing ;  Superv ision. An g us 14 \nBuckling : C onceptualization; writin g -  review and editing; Supe rvision; Res ources.  Meagha n  15 \nCa stledine: Investig at i o n;  Conceptu aliza t ion; w rit i n g - or i g inal draft;  wri ting - review  and 16 \nediting; met hodolo gy .  17 \nData availability stateme n t  18 \nAll data  and cod e u s ed  in the analysi s are availabl e on Git H ub 19 \n(http s :/ /gi t hub.com/zltnerdos/ antagon istic_ coe voluti on).  20 \n  21 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\n 22 \nAbst ract  23 \nExploitativ e i nter ac t ions , such as pred ator -prey and  host-parasite interactions, are  ubiquit ous  24 \nin microbial comm unities . These int er a ct i on s  shape c om munity density and composition, 25 \nimposin g str on g  selection o n members t o evolve coun termeasures r edu ci n g  the negat iv e 26 \nimpacts o f exploitation. Ex ploitat i ve coev oluti on is oft en s tudied betw ee n species  pairs in 27 \nisolati on, w hic h may  o ver-estimate the  strengt h and relev ance of pairw ise co ev olut io n. Here w e 28 \nstudied how c om mun ity contex t i nfluences coevolut ion  between P s eudom o na s fl uorescens  29 \n(exploited) and Variovorax s p. (ex ploiter ). We evolv ed t he se species in pairw ise coculture and 30 \nembedd ed within a five-species community to i nvest igate ev olved ch anges in pairwis e 31 \ninteractions. We found ev idence for asy m metrical c oevolutio n: Variovorax  ev olved more ra pidly  32 \nthan Pseudomonas , leading to incr eased ex ploitation throug h time, while Ps eudo monas 33 \nevolv ed increased tolerance to Variov orax  with time lag. T h e  pairwise c o e volutionary dynamics 34 \nwere not aff e ct ed by the pr e sence of ot her comm unit y  members. Understanding how 35 \ncoevoluti onary  patter ns  change w ith  i n c r ea sing  communit y complexit y c an hav e important  36 \nimplication s f or c om mun ity persiste nce and fu nc t io n. 37 \nInt r oduc ti o n  38 \nExploitativ e inter a ct i o n s  -  where one s pecie s benefits at the expense of anot her – c an r e sult in 39 \nthe adaptive  evolutio n of defence , c ounter -defence or  r ec ip r ocal  evolution of these tr ai t s 40 \n(antagoni stic coevolu tio n) (B rockhurst &  Koskella, 2013; G andon et a l., 2 008). Antagon ist ic 41 \ncoevoluti on can hav e far -re aching co ns equences for e cological and evolutionary dyna m i cs of 42 \ncommunities; particularly s o in micr obial communit ies where organism s often have la r g e 43 \npopulations and short  generatio n tim es (Bar ra cloug h,  2015; G ar butt et a l.,  2011; Paterson et  44 \nal., 2010), meaning ecol ogi cal and  evoluti onary processes  often happen simultaneousl y  45 \n(Loreau et  al., 2023). Previous work int o an tagon istic  coevo l u tio n has pri marily focussed on  46 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\ninteractions  between tr ophic lev els, h osts and paras it e s (Friman & Buc k lin g, 201 3; Gómez &  47 \nBuckling, 2 011)  and predator and prey  (Friman et  a l., 2011; J ohnke  e t  al., 2017). H owe v er , it is 48 \nalso important w ith in t roph ic lev els  where interactions often occur over exploitation of  49 \nextracellula r c o mpounds (e.g. evolutio n of r es istance to  antibio tics via comp etitio n (K och et  al.,  50 \n2014), ev olut ion  of i ncreased com petitiv eness  and exploitati on in  b i of i lm s  ( Hansen et al., 200 7; 51 \nKim et  al., 201 4)).  52 \nCommuni ty co ntex t  may  have a s ign i fi cant eff e ct  on pairwi se coevoluti on (B arracloug h, 20 15;  53 \nBlazanin & Turner, 2021; Manriquez  et  al., 2021). B e ing embedded within  a community is  l ikely 54 \nto r e d uc e the occu rrence and spe ed of  p airw ise coevolution. Inter a ct i ng wi th mult iple 55 \ncommunity mem be r s  w ill po tentiall y  reduce the f r equency  of interaction f or a g iven species  56 \npair,  result in trade-offs  b etween ad a ptation to mult iple species  (A l seth et al., 2019; Friman &  57 \nBuckling, 2 013) ) and increas e the magnitude of  trade-of fs  betwe e n a biotic and b i ot ic 58 \nadapt ation (Br is coe Runqu i st et al., 2020; Gómez  &  B uck ling, 20 13; H all et al., 2018; Law r e nce  59 \net al., 2012 ;  Luján et  al . , 2022; Yin  et a l ., 2023), all of whic h w i ll reduce the s t rength  o f r e cipr oca l 60 \nselec t ion. Furt hermore, reduced pop ulat i on  siz es  w it h increas ing  commu nity  member s  wi ll 61 \nreduce t he supply  of mu t ation on wh ich s electi on a ct s  ( C astledine et  al., 2020; H art et al. ,  62 \n2019 ). Our understanding of h ow  c o mmunity complexit y  affects coevoluti onary dynamics  is 63 \nhowev er primar i ly lim ited t o s t udie s  of bacteria-virus ( bac t eriophage )  sys tems. 64 \nHere, we em ploy  a t i m e s hift (Buckl ing  & Rai ney, 2 002; G aba & Ebert, 2009)  appr oac h  to 65 \nquantify t he impact of commun it y c omplex ity o n t he coev olu tio nary dy n amics o f two f oca l 66 \nspecie s th a t  en g age in  an ex ploit ati v e interaction. V a r iov orax sp. (AB1) b e nefit s  fr om unkn ow n  67 \nmetabolites produced by  Pseudomon as fluorescens (AB1)  (h e r ea f ter re fer r e d to as Variovorax  68 \nand Pseudomonas ) ,  while the growth of Pseudomonas  i s red uced  by t he  p res ence of Va riovorax  69 \nsugg est in g t he potent i al f or stro ng sel e ct i on  for coe volut ion  (C as tledine et  al., 2024). This  so i l 70 \nmicrobial community cons ists of five  s pecies (including Pseudomo nas and Variovorax )  a nd is  71 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\ndominated by  competitive interactions (Cast ledine et al. , 2024). B riefly , unde r s imilar  72 \nexpe r imental condition s to our current work, Pseudomonas is neg ativel y im pact e d by all other  73 \nspecie s. While V a r iov orax r educes  th e fitness  of a ll other sp ec ies, it exp eriences  a growth  74 \nbenefit in t he pr es ence of three of the f our other communit y members (w it h hi ghest ex plo itatio n 75 \nagains t  Ps eudo mo nas ). Therefor e , we expec t  that communit y con text w ould r e d uc e t he extent 76 \nof pairwis e coevolution between Vario vorax  and P s eudomo na s  by dec reasing  mu tation supply  77 \nand the strength of reciprocal sele ct i on. We hypothesiz e that  s t ron g sel ect ion w ould r e sult i n  78 \nPseudomonas an d  Variovorax  evolvi ng  via  arms r ace d y namics (e.g . Pseudomonas ge t t i n g 79 \nbetter a t  defence ag ainst exploi tati on and Variovorax evolv i ng  increas e d  ex ploitation ) in 80 \ncoculture, and th a t  coev olutio n of t h e f oca l speci es w ould be weakened in a  multi species  81 \ncommunity.  82 \nMater ials an d m ethods 83 \nExperim ental e volution  84 \nExperimental evolution treat ments w er e  s et  up to s t udy  ad apt ation and pai rwise coev olut i o n  85 \nbetween Pseudomonas and V ariovora x  in different biotic condit ions (F ig ur e 1). A monoculture  86 \nevolutio n treatment  w as set up  to isolate any ef fects of co rrelate d abiotic adaptation, whi ch  87 \ncan alter competitive hierar c hies, mediate exploitativ e dynamic s, and  influence nic he 88 \npartitioni ng wit hin the c ommunit y. A coculture and community e volu tio n treatm e n t was us ed to  89 \nstudy coevolu tio n in  a pairw ise and community background r es pectivel y, and to  ass ess th e 90 \nimpact of communi ty  com ple xity  o n pairwise coev oluti on  between t w o  species.  91 \nThe community evoluti on treatment had been ca rried out  previously  and results h a ve b e en 92 \npublished in Castledine et al. ( 2020). W e  r a n domly  selected eig h t replica t e com munities  in our 93 \ncurrent study. Mono- and coculture e voluti on t reatments  were init iated wit h eig ht replica t es  94 \n(eight replicates for each species per monoculture) us ing the s ame  P s eudomo na s and 95 \nVariovorax ancestor s  and culturing proto c ol. B riefly , ba cterial isolates ob taine d  fr om s o i l 96 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\nsamples, including A c h romobact er sp. , Ochrob actr um sp . , Pseudomonas sp .,  97 \nStenotro phomonas sp. , and Va riovorax  sp. , w e r e  identified based on their distinc t colo ny 98 \nmorphologies o n Kin g' s medium B (K B) aga r. Ea ch s pecies wa s c ultivated fr om  a single colony 99 \ni n  i s o l a t i o n  f o r  t w o  d a y s  i n  6  m l  1 / 6 4 T r y p t i c  S o y  B r o t h  ( T S B )  m e d i u m  a t  2 8 ° C  i n  g l a s s  100 \nmicrocosms. Inoculated abund a nce s ( c o l o ny f orming u nit, CFU) of e ach species  w ere  101 \nestimated app roximately from optical  densities  (O D 600 ; w aveleng t h 600  nm)  after  t w o day s of  102 \ngrowth (equations for conv er ting  OD 600  to C FU /μL des cr i b e d prev iously (C as tl edine et a l., 2024) 103 \nand adjusted to: 2x 10 6 CFU/µl . R e plica t e lines  o f commun ities  (all s peci es combined), 104 \ncocultures ( Pseudomonas  and Variov or ax ) and monocultur es  ( Pseudomonas , Variovorax ) w e re  105 \nestablishe d  using a 20 μL inoculum from e ach spec ies int o fresh 6 mL 1 / 64 TSB . C ultur es  106 \nunderwent weekly  serial 10 0- fold dilu tion s (transfer of 1% i nocula in to fres h media) over ten  107 \nwee ks, w it h samples frozen every s eco nd transfer (−70 ° C in  gl ycerol, fina l concentrat ion: 2 5%). 108 \nFrozen samples from the ancestor (us ed a s inoculum above ) , 6-  and 10-wee k-old cultures w ere  109 \nplated onto KB agar and i ncubat e d  f or 2 days at 2 8 ° C . Six  clones of Pseudomonas  and 110 \nVariovorax  each pe r  commun i t y , coculture and monocult ure evolutio n lin e (replicate) w er e  111 \nisolated from each t imepoint and gro wn for 48 h our s  in 1/64 TSB before be ing combi ned and 112 \nfrozen at −70  °C  in  gl ycerol t o be u sed i n t ime-sh ift as says.  113 \nTime -shift a ssays 114 \nTo test w he t her the two focal spec ies,  Pseudomonas  and Variovorax ,  hav e c oevolv ed o ver the 115 \n10-week  period we cond uc t ed t ime- shift  assays , where o ne species  was c u l t ured w ith the  ot her 116 \nfrom a  population of th e  pas t ,  contemporary or futu re t i m e p oi n ts. T hese a ssay s were car ried  117 \nout in t he abs ence of the rest of the c o m munity. This approach allows for s ig natur e s of diffe rent 118 \ntypes of coevolutio n to be det e ct e d. Arms rac e dynamics (AR D ,  i.e.  selection of defence  and  119 \ncounter defence) is directional, with focal species h a ving a greater ab undanc e when com peting 120 \nwith t he ot her species  isolated from a past time point, a n d low er  densitie s  w hen com petin g  121 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\nagains t  com peti tors from a future t i m e  point . I f  i nstead selectio n o n specific genotypes 122 \nfluctuates throu g h t i m e  ( fluctuatin g s e lection dy namic, FS D), p erfo r mance may  be pa r ticula r ly  123 \nhigh  o r  low for  contemporaneous inter actions  ( H all et  al., 2011).  124 \nPopulations of P s eudomo nas and Va ri ov o r a x  from one of the thr e e diff e r ent timepoint s of t he 125 \nevolutio n e xperiment (ancest or, 6 week and 10  w eek) we r e r ea ssembled, res ulting  in 9  pairwis e 126 \ncombination s per tr e at ment (Figure 1B). Cultures of Pseudomo nas  a n d  Variovorax  w e r e  127 \nass em ble d us ing all 6 clones fr om th e same t r e at ment r eplicate.  Eight r epli cate mic r ocos m s  128 \nwere set up for e ach com bi n a t ion wit hin t he t hree different ev olution  ba ckgrounds except for 129 \nmonocultur e s, see below. T he culture conditions of all treatments were established as  130 \ndescribed for the initial ex periment a l e voluti on experiment, w it h a pp rox imat e l y 2x10 6  C FUs per  131 \nspecie s in oculated into  fresh microco sms. After one week , cultur e samples we r e cr y ogenically  132 \nfrozen and then pla t ed onto KB aga r . Population densities  were estimate d by  countin g the 133 \nnumber of CFUs (10 5  diluted) aft er  two day s of growt h at 28  °C. We use proportion of  134 \nPseudomonas to in terpret  coevolutionary dynamics . Pr oportion of spec ies  is often mor e 135 \ninsi g ht ful t han d e n sit ies in describi ng coev olut ion as  it  hi g hl igh ts sh i f ts in rel ative a b undance  of 136 \neach spec ies. These s hift s ar e  i ndi cative of selecti v e pr e ssure imposed by interspecies 137 \ninteraction (e.g. parasi t is m ) and is le s s  affected by f luc t u ations in tot al popu lat i on  s ize t ha t  c an 138 \nbe influenced by v ar i o us  factor s  such a s stoc hastic  variation  i n density  betw ee n  microcosms.  139 \nThree r eplica t es  in the monoculture ev olutio n line (f or both Pseudomonas and Variovorax ) w ere  140 \ncontaminated prior  t o w eek 6, therefo r e monocult ure g r ow th  assays  were ca rried out with only 141 \n5 replicates. Three repli cates of  the commu ni t y  evoluti on line w ere c on taminated during t he 142 \ntime-sh ift ass ay, and t hese w er e  removed from the analys is . The t o tal density  of pairwis e 143 \ncombination s w er e  assess ed usin g t h e total abundance (combined C FU ) of Pse u domonas and 144 \nVariovorax in each  microcosm.  145 \n 146 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\nStatisti cal analyses 147 \nAll data analys es we r e c arried out in R  v 4.3.1 (R  Core Te am, 2025). First, w e  analyse d  whether 148 \nthe dens i ty o f each species varied i n  t hei r  i nteraction  w ith  con temporary c ou nterparts o v er  149 \ntime. For  this m odel, th e  time-s hift a ssay s were subset t o retain onl y th e  bacterial densities  150 \nfrom their cont emporary assay s. The  d e nsit y of  each  species  w as  analys ed in l inear mix ed-151 \neffects models  (LMM) s eparat el y, w it h lo g 10 transformed density as  the r e sponse v ar i able,  152 \ntreatm e nt , time and their interact i on s as ex planat ory  variable and random int ercepts fitted for  153 \neach replic at e t o account  for n on- independence of observa t ion s . 154 \nOne r eplic at e  of t he  t ime- s h ift  assays  i n t he coculture treatm ent ( Pseu domonas  t ime 6, 155 \nVariovorax t i m e  10) showe d an unu sually  hig h Variov or ax  count and w as found to be an  156 \ninfluential outl ier (G rub bs -  test)  and was removed from the analys is. To test how  com munit y 157 \ncomplexi t y  affects coev olutionar y  dy namic s we used a binomial generaliz ed linear m i xed-158 \neffects models (GLMMs )  with a log it lin k function, the proportion of Pseudomonas  as combined  159 \nbinary response variabl e and  tre atm e nt  (complexity) x evoluti onary time of P s eudomo na s x 160 \nevolutio nary time of Variovorax  fitted as fixed effects, as well as t he ir  3-wa y  i n t eraction. We  161 \nincluded random inter c ept s  f or each replicate line  t o account  for non -independence of 162 \nobservations. To study coevolut io n w i th i n treatments, we tested the effect o f coev olut io nary  163 \ntime of bo t h Pseudomo nas  and Va riovorax , plus  t heir in teraction, on  the p roportion  of 164 \nPseudomonas in separate models  p er tr e at ment us ing binom ial G LMMs  w ith a log it l ink 165 \nfunction for  ea ch e voluti onary backg r ound. Total densit y  of pa ir w ise  combination s w er e 166 \nanaly sed w ith  a line ar  mixe d- effects model (LMM) wit h l og 10 tr a n s for med total dens it y as  the 167 \nresponse variable, treatment, evolutionary t i m e of P seudomonas  a n d  Va riovorax  and their 168 \ninteractions as explanatory v ariables and random intercepts fitted for ea ch r e plic at e to  account 169 \nfor non -i ndependence  of observa t ion s. T ime was included as dis cr e t e v ariable in all analy sis . 170 \nThes e analyses  employed LMMs  and GLMMs  usi ng t he ’ lme4’  pack a ge (Bat e s et al., 2015). For  171 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\nthese LMMs and G LMMs, we used t he ‘ DH A R M a ’  p ackage (Hartig, 2018)  to c heck resi du a l 172 \nbehavi o ur and mod el s imp l ification was c arried out using  likeliho od rat io test. Post- h oc 173 \nmultiple comparison test s o n the m ost parsimoni ous models  we r e carri ed out us ing  the R  174 \npackag e ‘ emm eans’ (Lenth, 2 0 23) , usi ng T ukey  adjustm e nt .  175 \nResul t s 176 \nPopu latio n den si ties chan ge acr o ss  contempo ra ry species pa i r s  evolved with in  177 \ncommun ity  178 \nWe first determined how t he r el at iv e succe ss of Variovorax  and Pseudomonas  differ ed betw een  179 \ntreatm e nt s  and through t ime.  Specifi cally , w e dete r mined the density of each species w hen  180 \ncultured with their c o ntemporary co unterparts. The effec t  of  treatment ( monocultur e , co-181 \nculture, 5 species communit y ) on P s eudom onas  densi ty  did n ot differ  thr oug h t ime (LM M: 182 \ncommunity complexit y x evolut ionary time: χ2 2  = 3.06, p = 0.22, Figure 2),  n or was  there an  183 \noverall ef fect of time  (LMM: evolution ary  time: χ2 1  = 3.14, p = 0.077). Tre at ment  did howev er 184 \nhave a significant effect on Pseudomo nas de n s ity (LMM: community complex ity: χ2 3  = 21.20, p 185 \n< 0.00 1, Figure 2). Pseudomo nas po pulations t hat had ev ol ved in c omm uni t y  (4.52 log 1 0  186 \nCF U /ml [4.43, 4.60]; Tukey H SD : es t imate = 0.21, t-ratio  = 3.1, p = 0.01 8) r e ached significantl y  187 \nlower dens ities  compared to the an cestor and tho se ev ol ved as  cocult ures ( Tukey  HSD:  188 \nestimate = 0.14, t-r atio = 2.77, p = 0.051).  Mean Pseudomonas population densi ty  in  189 \ncontemporar y  combinat i on s of mo no culture evolv ed P s eudomo na s and Va ri ov o r a x  (4.79 lo g₁₀  190 \nCF U /ml; 95% C I: [4.69, 4.88]) did not d iffer s ignificantly from that of the ancesto r P s eudom onas  191 \nand ancestor  Variovorax combination (4.73 log₁₀  CFU/ml; 95% CI: [4.62, 4.84]; T ukey HSD:  192 \nestimate = - 0.0 57, t-ratio =  -0.8 0, p = 0.85). Si m il ar ly ,  m ea n Pseudo monas densi t y  in 193 \ncontemporar y  com bi n a t ions  of cocult ure evolved lineag es (4.65 log₁₀  C FU / ml; 95% C I: [4.57, 194 \n4.73]; Tukey  HSD : estimat e  = 0.078, t -ratio = 1.19, p = 0.64) wer e not diffe r e nt to the ancestor 195 \ncombination. Evol v in g in a communi ty, and cocult ure  to a lesser ex tent,  results in low er  196 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\nPseudomonas dens it i es when  cultur ed with Variovorax , suggesting  a change in their interaction  197 \ncompare d to t he ancest ors o r  m onocu lture ev olved l ine ages.  198 \nSimilarly to Pseudomonas , the dens ity  of Variovorax w as unaf fected by an i nt eraction betw een 199 \ntreatm e nt  a nd  evolutionary t ime (LMM: commun i t y  complexit y  x evolut ionar y  time: χ2 2  =  4. 2 9,  p  200 \n= 0.12, Figure 2) or  ev olu t i o nar y  t i m e itse lf  (LM M: e volutio nary  time: χ 2 1  =  1 . 43 ,  p  =  0 . 2 3 ) ,  b u t  201 \ntreatm e nt  had a s ignificant eff e ct  on Var i ovorax density (LMM: communit y  c o mplex ity: χ2 3  =  202 \n21.14, p < 0.001, Fi gure 2). Me an c ont emporary pop ula t ion densities o f Var i ovorax in  t h e  203 \nancestor c om bina t ion ( 4.67 lo g ₁ ₀ CFU/m l ; 95% C I: [4.55, 4.80]) w er e  not differ ent to t ho s e 204 \nevolv ed in monoculture (4.70 log₁₀ CFU/ml ; 95% CI:  [4.60, 4.80];  Tukey HS D:  e stimat e = -0.024, 205 \nt-r atio = -0.30, p = 0.99) or coculture (4.83 l o g ₁ ₀  CFU/m l ; 95% CI: [4.74,  4.92]; T ukey HSD :  206 \nestimate =  -0.15,  t- ratio  = -2.00, p  = 0.21). How ever, mean Var i ovorax densit ies  in  th e 207 \ncommunity evolut io n t reatment reached si gnifican tly higher densities  than t he anc estor  in t heir  208 \nrespective  contempo rary cocultur es  (4.95 lo g₁₀ C FU /ml ; 95% CI: [4.8 6, 5.05];  Tukey  HSD:  209 \nestimate = - 0.28, t-ratio = -3.49, p = 0.007). These r es ult s  confirm that  community  co ntext has a  210 \nsig n ificant impact on t he adaptation of spe cies, and th a t  coev oluti on l e ad to a change in  211 \ninteraction  between species  ( w hen  co mpare d to ancestor  or mo noculture evo l ved li nes ).   212 \nAntagonistic coevolution evident between species pairs 213 \nTime shift assay s were used to c har ac t eris e c o e voluti on i n c o c ult ure and in a com munity by 214 \ngrowing Pseudomonas  w i t h  Variovor ax  isolated from  different time-poin t s  and v ice-ve r s a 215 \n(Figure 1).  To co ntr ol for abiot ic adaptation, pairs of mon oculture lines ( Pseudomonas and 216 \nVariovorax  cultured al o ne )  ev o l ved alongs ide the coc u l t ure and com munit y  treatment s  w ere  217 \nalso subjected t o time shift  a ssays .  218 \nThe results of the t ime sh ift a ssay s  ar e indi cat i ve of ant a g oni stic c oevoluti on betwe en  219 \nPseudomonas  and Variovorax  in bot h c ocult ure (G LMM: Pseudomonas time  x Variov or ax  time :  220 \nχ2 3  = 7.15, p = 0.067, F igure 3B)  and in t he comm uni ty  ( G LMM: P s eudom onas  tim e  x Variovorax  221 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\ntime: χ2 3  = 21.85, p < 0.0 01, Fi gur e 3C). Variovorax  s howed a tendency to i ncrease  in 222 \ncompetitivene ss  aga ins t Pseudomon as  t hrough time i n bot h th e  cocultur e and comm uni t y  223 \ntreatm e nt s .  Compared t o the ancest r al Variovorax, popul ations evolved f or  either 6-  o r 1 0-224 \nwee ks in both coculture (Figure 3B) and community (Fig ur e  3C) treatment s  reduced the  225 \npropor tion of ancestral Pseudomonas . There w a s no s ign ificant difference b etween the effect  226 \nof Variovorax  coevolved for 6- or  10-week s on the proport ion of ancestral  Pseudomonas for the  227 \ncoculture treatment (T ukey HSD coculture : es t imate = - 0.027, z - ratio = - 0.1 5, p = 0.88), h owev er 228 \nancestral P s eudom onas  propor tion was significant ly  hi gher in t he community-e volve d  229 \ntreatm e nt , when culture d w it h 10-w eek Va ri ov o r a x  comp are d to 6-w eek Variovorax  (Tukey  230 \nHSD community : es t imate = -0.50, z - ratio = -2.69, p = 0.007, Figure 3BC ) . T his latt er result could  be  231 \nattribut ed to eff ects ass ociated with o ther  s pecies in the communi ty evolut i on treatment ( e . g .  232 \nadapt ation to other species ).  233 \nPseudomonas underwent recip rocal a daptation to Variovorax .  The propor tion of P s eudom onas  234 \ncultured with 6-week  Variovorax  i n c r e a s e d  w i t h  Pseudomonas  e volut ion ary time for both 235 \ncoculture (Tukey  HS D ancestor – 6 week : e s t i m a t e  =  - 0 . 6 6 ,  z - r a t i o  =  - 3 . 45 ,  p  =  0 . 0 01 6 ;  T u k e y H S D ancestor – 236 \n10 week : estimate = -0.85, z-ratio =  -4.37, p < 0.001  , F igure 4B) and commun ity ( T ukey H SD ancestor – 10 237 \nweek : estimate = -0.79, z-rat i o = -3.07, p = 0.0061 , Figure 4C) treatments. W hil e P s eudom onas  238 \nshow ed an ove r all  increas e i n r esis t a nce through time against 6 -week Vari ovorax ,  r e s i st an ce  239 \ndeclined ag ainst ancestral Variovorax for both tr ea t ments (Fig ur e 4B C ). Resi stance to 1 0-week 240 \nVariovorax  was not diffe rent betwee n any of  the Pseudomonas evoluti on ary t i m e p oi n ts for 241 \neither th e  coc u l t ure or comm unit y  t reatment. These dynamic s ar e  cons i ste nt wit h fluct ua t in g  242 \nselec t ion ac t i ng  on P s eudomo nas , such that i t became specificall y ad apted to  evolvin g 243 \nVariovorax , while becoming maladapte d to ances tral Variov o rax .  244 \nTo contr ol for adaptation t o abiot ic conditi ons potent ially bein g i nt e r preted as coev oluti on, 245 \nmonocultur e  lines  w ere e volved alon gsi d e  the coculture and community t reat ments. We did  246 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\nnot det e ct  i n teractio ns that were s ug gesti ve of c oev olut ion  betw een P s eudomo na s and 247 \nVariovorax  e v o l v e d  i n  m o n o c u l t u r e ( G L M M:  Pseudomon a s time x Var i ovorax ti me: χ 2 3  = 4.02, p = 248 \n0.26). There was  no indica t i on of Variovorax adapting to  abiotic  condit ion s or th a t  any a biot ic 249 \nadapt ation affect e d its i n teraction  wi t h Pseudomonas  ( G LMM: Variovorax  t i m e  χ 2 2  =  0 . 8 8 ,  p  =  250 \n0.64, Fi gure 3A). We found some ev idence for  abiotic adaptation in P s e udomo na s, whi ch 251 \naffe cted interactions with  Variovorax ev olved in mo nocult ure, w ith an  increase in t he proportion  252 \nof Pseudomonas  a t  w e e k  6  ( G L M M :  P seudomonas  t i m e  χ 2 2  = 10.06, p = 0.0 07, Figure 4A), but  253 \nnot week 1 0. 254 \nCommunity complexity did not affect coevolution of species 255 \nCon trary to our ex pectation t hat e v olut io n i n a multispeci es commu n i t y  would weak en  256 \ncoevoluti on, we found n o differ ence i n coev olutio nary d y namics between t h e  coculture and 257 \ncommunity tr e at ments ( G LMM: Pseudomonas time x Variovorax time x com plex ity interactio n 258 \nχ 2 3  = 3 . 54,  p = 0.32).  Pseudomonas  p r oportion was not differentially  a ffect ed by commu ni t y  259 \ncomplexi t y  o v er ev olut i o nar y  t ime for each s pecies (G LM M: Pseudomonas time  x complexit y 260 \ninteraction  and Variovorax time  x  com plex ity i nteraction: χ 2\n2 pseudomonas  =  4. 0 1 ,  p  pseudomonas  =  0 . 1 4;  261 \nχ 2\n2 variovorax  = 4.21, p  variovorax  = 0.1 2). Howev er , selection pressures are clearly di f f erent betwe en the 262 \ntwo t reatments. Pseudomonas evolv ing in comm unit y  context di splayed lower p roport ions 263 \ncompare d to cocultur es  (Tukey HSD: estimate =  0.33, z -ratio =  5.07, p  <  0.00 1), suggest i n g that 264 \nPseudomonas adaptation to V a r iov or ax is w eakened in a community  conte xt. T h i s is  driven by 265 \nboth an increase in Variovorax  density  (Tukey  HSD: estimate = - 0.08, t-ratio  = -3.1 0, p =  0.003)  266 \nand a de cre ase  in Pseudomonas dens ity ( T ukey H SD : es t i m a t e = 0.06, t- ratio = 2.12, p = 0.037, 267 \nFigure 5C).  268 \nChanges in total density driven by Variovorax evolution 269 \nTotal dens it y (of Pseudomo nas  and Va riov or ax  cocultures) could change  o ve r ev o l u tio nary  time 270 \ndue to a change in  s pecies int eraction s (e.g. increased exploitation by  V a r iovorax could res u l t  in 271 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\na decli n e  in total density). We observ ed an increase in tot al density as a resu lt of an increase in  272 \nexploi t ation of evol ved Variovorax , an d this effect w as indep endent of evolutionary time and 273 \nbackg r ound (community complex ity)  (LMM: Pse u domonas time  x  Variovora x time  x comm unity 274 \ncomplexi t y  i nteraction: χ 2 6  =  5 . 1 1 ,  p  =  0 . 5 3 ,  F i g u r e  5 ) .  T o t a l  d e n s i t y  w a s  n o t  d i f f e r e n t i a l l y  275 \naffe cted by community complex it y ove r  ev oluti onary time for each species  (LMM: 2-way 276 \ninteraction for Pse u domonas time  x complexity and Variovorax time  x comple xity: χ 2 4 pseudomonas  =  277 \n6.58, p  pseudomonas  =  0 . 1 6 ;  χ 2\n4 variovorax  =  2 . 7 2 ,  p  vario vora x  = 0.61). Total density w as als o  no t affected by  278 \nan inter a ct i on  betwee n  the t w o s peci es evolutio nary time ( L MM: P s eudomonas  x Variovorax  279 \ntime interaction: χ2 3 = 5.90, p  = 0.12). Only  the ev olutio nary time of Variov o rax  had a sig n i ficant  280 \neffect on the d ensity  of th e  assembled  co-cultures (LMM e ff e ct  o f  V a riov or a x time: χ2 2  =  7 . 5 3 ,  p 281 \n= 0.024). This  i s driven by  an increase in Variov o rax  densit y at w eek 6 (Tukey  HSD: estimate = -282 \n0.054, t-ratio  = -2.57, p = 0.0 47) leading t o  an i ncrease in t ot al  densi ty com pa r ed t o t he ancest or 283 \n(Figure 5BC). T her e f ore , incre ase d  e xploitation of P s eudom onas  by Variovorax in cr e as ed 284 \ncommunity  densit y despite dec r eas es in P s eudom onas  den sit y.  285 \n      286 \nDis cu s sion  287 \nIn t hi s s t udy , w e soug ht  to understand the effect of  community complexity on e xploitat ion-288 \nmediated pairw ise coev olution in a  soil m i cr obia l communi ty. In o ur  model sy stem 289 \nPseudomonas (and s om e ot her members of  the community) are ex ploi ted by Variovorax  290 \nmediated by intera ctions over metabolites (Cast ledi n e  e t  al., 2024). While w e found evi d e nce  291 \nfor ant a gonis tic coe voluti on, commun ity c omplex it y  d id no t  sign ificantly  aff ect coev o l u t ionary 292 \ndynamics . This  i s despite the comm un i t y  co ntext pr oviding  m ore species for Variovorax  t o  293 \nexploi t  (ther e f ore reducing selectio n on Pseudomo nas  specifically ) ; and P s eudom onas  294 \nexpe r ie n c ing competit io n fr om more spec ies, therefor e inh ibiti ng  population  si z es and/or 295 \noffering conflictin g selec t i on press u res . T his sugge sts t ha t  reciprocal s elec t ion betwe en  296 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\nPseudomonas  and Variovorax  is  suffic iently stro ng to buff e r the ef fect of  th e  alt e r e d se lection 297 \npressures cr ea t ed by the communi ty  cont ex t. 298 \nWhile there are ex amples of wi t hin-tro phic level s t udies  of b a ct e r ia l int eractions showi ng h ow  299 \nspecie s inter a ct ions  can lead to di f fer ent evolutionary outcomes i n both coculture and  300 \nmultispecies  com munit ies  ( Chang et al., 2020; C hen & Zhang, 202 4; Pearl Miz r ahi  et  a l.,  2023; 301 \nPiccardi et  al. , 2024), to our  know led ge this is t he first study ex plicitly demonstr a t in g  w ith in-302 \ntrophic level antagoni stic c oevolut io n betw een bacteria. The mech anism s  under l ying  any  303 \nobserved ada ptations in thi s s tudy are unknown. How e ver, fr om previous w or k  w e know  that  304 \nVariovorax  benefits from  the pr es enc e a nd  metabolic activity of P s eudom onas  i n our mod e l 305 \n(Cas t le dine et al., 2024), t herefore me chanisms of Pseudomonas  r e s i s t a n c e  t o  Variov or ax  a r e  306 \nlikely  related to alteration of  metabolites, while Variovorax  becomes mor e  efficient at using 307 \nmetabolites produced  by Pseudomonas or a dapts to utilise altered  meta bolites. Analysi n g the 308 \nexa ct  na t ure of this interaction  (and evoluti onary  m e chanisms  thereof), is m ade difficult owing 309 \nto t he complex  nutr ie n t  m e d i um . 310 \nIn both pai r w ise cocultures and com mu ni t ie s, V ariov or a x became mor e  ef ficie nt  at exploit ing 311 \nPseudomonas , w ith Variovorax  reach ing hi g her  densit ies r e lat i ve to Pse u do monas  thr ough  312 \ntime. Exploitatio n of P seudomonas  b y  Variovorax  s howed an ove r all  i ncrease through  time that 313 \nis c on sis tent wi th arms race  dynamics (G andon et al., 200 8). In turn, Pseudo monas adapted by  314 \nbecoming more resistant to e xploitati on, albeit with a time  la g, t o 6-we ek e volved Variovorax . 315 \nHow ever, there was  no observed increas e over all in res istance of Pseudomonas  aga inst 316 \nVariovorax  ov er t ime, w ith t he i ncr ea se in resistance to wee k 6 Variovorax  being ac com pani ed 317 \nby a decre ase in r esi stance to ancestral V a riov orax.  This  sugges t s that fluct ua t ing  se lectio n 318 \ndynamics  (FSD ) acting  on Pseudomon as  playe d  an impor tant role  in the c oevoluti on with in t his 319 \nsy stem ( Hall et al., 2011). O ur  knowledge o f ant ag onist ic coevolut ionary dyn amics in mic r obes 320 \nis mostl y based on bacte ria-phage m odels, w here s election press ur es  an d th e  ev olut ionary 321 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\npotential ar e  diff erent t o wi t hin- trophi c coevolutio n (B uck lin g &  Rainey, 20 02; Gandon et al.,  322 \n2008; Góm e z & Buckling, 2011). Infect ion w it h lytic phage l eading to cell dea th imposes  str on g  323 \nselec t ion o n bacteria to a dapt  defenc es, w hereas comp et i t iv e i nter a ct i on  b etw een bact e r ia  –  324 \nespeci ally t ho se  in v o l v in g i nteract i on s ov er  metabolites  - mi ght  be less s pecific, leading to  325 \nwea ker s election. FS D in hos t -pathog en sys t e m s bec om es  more prevalent due to increasing 326 \ncosts of higher infecti vit y/resis t ance  for  the phag e and bacteria, resp ectively  ( Hall et al., 2011),  327 \nresulting in sel ect ion o n st a nd i ng gen etic variation. Thi s might explain w hy most work to date,  328 \nusing a bacteria-phage model sy stem r eported coevolv ing part ne r s  both exhi bit i ng ei ther ARD 329 \nor FSD. O ur  res ult s  suggest t hat th is i s  not  the cas e for  with in trop hi c- lev el  coev o l u ti on, w here  330 \ncoevolving  partners show differ e nt  coevolutio nary dynamics . Furt her w or k is  needed to ex plore 331 \nthe wider implic at i o n s  of such h igh l y a sy m metric coev olu tio nary dynamics  a nd their impact o n 332 \necology and ev o lut ion .  333 \nA common crit icism o f labor a t ory studi es of coev olut ion  is  that t hey  are overly simplis tic in t heir 334 \nconditi ons  and far removed f rom nature to of fer ins ig ht into  mor e  co mplex ev olut io nary  335 \ndynamics . Previous s t udies  have f ound t hat ev en small i ncr e a ses in com munity complexit y can  336 \nsig n ificantly affect coevoluti on (B a r raclough, 2015; Blaz ani n &  Turner, 2021; Cas t le d i ne, 337 \nSierocinski , et  a l., 2022; Manriquez  et al. , 2021). Our results instead find that pairwis e  338 \ncoevoluti on can pr edic t  coevoluti on i n community c ontext s whic h may be due to se lection  339 \npressures being sufficientl y s tro ng . Similar cases of parallel evolutionary dynamics may be 340 \nobserved i n  w ider contexts  suc h  as  ph age therapy, w here bacter i a ex perienc e strong  sel ect ion  341 \nto phage in patients and i n c losed laborator y  conditio ns ( Cas t ledi n e , P a df i el d, et  a l ., 2022). A s  342 \nPseudomonas  is  the specie s  Variovorax  derives  the stron ges t  fit ness  be n e f it  from, interaction  343 \nintens itie s  and reciprocal  selec t ion may hav e been suf ficiently stro ng for  coev olut ion des pit e 344 \nco-occurr i ng communit y members. M utation supply  rates may h a ve als o  be en non-s ig nificantl y  345 \naffe cted by oth e r  community membe rs as Var i ovorax  can generally ex ploit at least two ot her 346 \ncommunity members (o ther t han Ps eudomonas ) w h i ch may allow it  to  maintain s uff i cient  347 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\nmutation rates for  coevoluti on ( Castl edine et a l., 2024; Gandon &  Michal ak is, 200 2). Wider  348 \nwork analysing  coevo lutio nary int e r ac t ions  wi thi n trophic lev els, inc lud i n g between cro s s-349 \nfeeding mutualists and ex ploi tative int eractions, wil l g ive i ns i gh ts i nto how c oev olut ion  oc cur s 350 \nin na t ural com munity  contexts. The observ ed negative  effe ct of commun ity complexity o n 351 \nevolv ed Pse u domonas dens ities might  be explained by additional s electi on press ur e arising 352 \nfrom competition with ot h e r  community member s . Va riov o rax  be n e fi t s  fro m mos t  specie s in  353 \nthe commu ni t y , while Pseudomonas competes against them leading to str on g er s electio n on  354 \nPseudomonas in  the commun ity treatm e n t. Thi s pot ent ial ly leads to trade- offs betw een  355 \nadapt ation to Variovorax and other  c o mmunit y members.  356 \nIn thi s study, we ex plicitly demons trat e  antag o ni stic coev oluti on betw een naturally co-357 \noccurring bacteria. This a n tag on ist ic  coevoluti on leads  t o incr e ased ex ploitatio n but not  358 \nincreased res istance over the ex per im ental time and s hows an important rol e of bot h AR D and  359 \nFS D. Furthermore , we show that pairwis e coevolut ion can be robust in the face of commu ni t y  360 \ncomplexi t y . C oev oluti onary dynamics d i d not change si g n ificantly wi th i ncreased complexi t y ,  361 \ndespite finding  differences i n  dens it ie s of species s u ggest ing  a chang e i n s elective press ur es .  362 \nUnd e r s t anding  the i nterplay betwe en c o e voluti on and biot ic complex it y i s c rucial not onl y for 363 \nadvanc ing evolut ionary t heory but also for applications i n medicine, bi otechnol ogy , and 364 \necology , where microbial coevolu tio n can influence antibio tic resi s t ance ,  pathogen evoluti on, 365 \nand micro bi o me stability. Th is  w or k  contributes t o  our understanding of withi n-t rophic  leve l  366 \ncoevoluti on, wi th  implicatio ns  for  bo th  n a t ural a n d eng ineered microbial c om munities .  367 \nReferen ces 368 \n 369 \nAlseth, E. O ., Pur s ey, E., L u j án, A. M., McL eod, I., Rollie, C ., &  W es t ra, E. R.  ( 2019 ). Bacterial 370 \nbiodive r s it y  drives t he evo lutio n of CRI SPR- bas e d phage resis t anc e. Nature,  574( 777 9), 371 \n549- 552.  https://d oi .o rg/10.1038/s41 586- 0 19- 166 2-9   372 \nBarra clough, T. G . ( 201 5). H ow  Do Spe cies In t eractions Af fec t  Evolut io nary Dynamics A cross 373 \nWhole Co mmunit ies ?  Ann ual Review  o f Ecolo gy , E volutio n, and Systematics ,  374 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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Ecol ogy  L ett ers ,  2 6 (6), 942-954. 496 \nhttps://doi.org/https://doi.org/10.1111/ele.14221   497 \n 498 \nF i g u r e  1  E x p e r i m e n t a l  d e s i g n  t o  t e s t  t h e  i m p a c t  o f  c o m m u n i t y  c o m p l e x i t y  o n  c o e v o l u t i o n  b e t w e e n  f o c a l  s p e c i e s  499 \nPs e udomonas ( P ) a nd V ariovorax  (V ). Th e  ev o lution exper i m en t  consist ed o f thr e e  tr e a t m en t s : monoc u lt ure, 500 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\nco c ul t ur e  a nd community .  E ach  trea tm e n t has b een  pass a g e d ( 1% ) weekly fo r 1 0 we e k s (A). Coevol u ti o n was  501 \nasses sed  by growth ass a y s comb in in g  Ps e udo mo n as a nd Va ri ovo rax f rom d if f e re nt  evolu t ion ary ti m ep o in ts  fro m  th e  502 \nsame r e pli c at e line  wi th i n  each  tr e a tm e nt ( B). a n c  = anc e s tor,  6 =  6-week isola t es,  10  = 1 0-w eek  is o la te s . 503 \n 504 \nFi g u re  2  D e ns ity  o f  Ps e ud o monas  (A)  and V ariovorax  (B)  in  t h e ir  c o nt em p orary c o mb i na t i on s  of  a nces t o r  a nd  evolved  505 \nli ne a g e s  i n mo n o c ult ure,  cocult u r e o r  c om m uni ty c u ltu re .   Pop u la t io n densit ie s  a re  plott ed agains t evolutiona ry t i me. 506 \nBoxpl o ts sh owi n g  me di a ns , f i rs t  a nd  th i rd  q u arti les,  w h isk ers  ar e 1.5 * i n terquartil e r an g e (IQR).   I nd i vi dua l  poi n ts  507 \nrep r esen t individual tr ea t ment  r e plicates .   508 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\n 509 \nFi g u re 3 R e lati v e  proportion  of  V a r i o vo r a x  w h en c ultu r ed with  Ps eudo m o n as from  diffe r ent  ti m e -poi nts .  510 \nPseudom o na s  and  V a ri ovora x  evolved  in  (A ) m on o cu ltur e  (ea ch  sp ec i e s  in i s olatio n ), (B)  i n co c ultur e  ( P s eudo m on as  511 \nan d Va r i ovor a x )  and in  ( C )  co m m unity ( P se u do m o na s  and V a r i ovor a x  e mb e dde d wi t h 3  other b ac t eria l  speci e s )).  We 512 \nfou nd a  significan t in te raction betwe e n e v o lutionary  time  of Ps e udomonas and Va r i ovo r a x  w h en  s p ec i es  ev o l v e d  a s  513 \nco - cul tur es (B)  or with i n a  c o mmunit y  (C),  but  n ot wh e n t h es e had e v o lved i n isolatio n  (A ) . I nd ependent treat m e n t 514 \nrep li c at e s  ar e  sma ll p o ints, la rg e  p oin ts  a re  mean pr o portion of  Va r i ovor a x ,  b a r s  r e p r e s e n t  ±  S E .  T h i n  l i n e s  a r e  515 \nconn e cting the r e plicat e s  t hat a r e tracked thr ough ti me (ex cep t fo r th e a n ce s tor), thick lin e s repre s e nt th e  mean. 516 \nA n c e s t o r - a n c es t or  co m bi n at i o ns  b e t w e e n  t h e  p an el s  a r e  t h e  s a me  8  r ep l i c a t es  p r e s e nt e d  t o  a i d v i s ua l iz at i o n.  517 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\n 518 \nFi g u re 4  Re lati v e  p r opo rti on  of  Pse u domo na s  when c ultur e d with  Va r i ovor a x  fro m  diffe r en t t im e -points.  519 \nPseudom o na s  and  V a ri ovora x  evolved  in  (A ) m on o cu ltur e  (ea ch  sp ec i e s  in i s olatio n ), (B)  i n co c ultur e  ( P s eudo m on as  520 \nan d Vari ovora x )  and i n  ( C) community ( Psedu om o na s  a nd V a r i ovor a x  e mbedded with 3 other b a ct e ri al speci e s ).  521 \nPa tter ns of coevolut ion  pr esen t  for  (B )  a n d (C)  b ut not ( A) obse rved  as s i g n ifi c a nt int e r a c tion betw e e n evolut ionary  522 \ntim e of Ps eu d om o na s  and  Var i ovor ax . In d ep e nd e n t tre atm e n t r epli c a tes ar e sma ll po i nts, la rg e poin ts  a r e m e a n 523 \npro portion o f P s eu d om o na s ,  b a r s  r e pr e s e n t  ±  S E.  T h i n l i ne s  a r e  c o nn e c t i ng  t h e  r ep l i c a t es  th a t  ar e  t r ac k e d t h r o ug h 524 \ntim e (exc ep t fo r th e  a n c estor ) , thi ck lines rep r e sent the m ean. A ncestor- anc e s to r c o mbinati o ns b etwe e n the p an e ls  525 \nare t h e  sa m e  8  re plicates  pre s e nt ed to  aid visual ization.  526 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint \n\n 527 \nFi g u re  5.  T he density o f indivi dua l sp ecie s  a nd  their co m bi n ed d en s ity  wh e n a s se m ble d a s  t wo-spe c ie s  cocult u r es 528 \n(Va ri ovora x  and  Ps eudo m o n as) fol l owi n g  e v o lution in (A )  monoc u lt ur e  (each s pecies  evolved  alon e ), ( B) c o c ul t ur e 529 \n(Pse u domona s  a nd Vari o vo rax  only) , and (C )  i n a  c o m mu n i ty (wit h t h r ee  ot he r  s pe c i es). Po p ula t ion de n s ities are 530 \npl ott ed  ag ai ns t evol ut io n ary t i m e . B o x p lots  sho win g  m ed i an s , f irst  and t hi rd  q u ar tiles, w hisker s are 1.5 * int erqua r til e 531 \nran g e  (IQR) .  I ndi vi d ua l  p o ints  repr e s ent  indi vi d ua l  tr e a tm e n t  rep li c a te s .  532 \n 533 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted July 4, 2025. ; https://doi.org/10.1101/2025.07.01.662545doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}