Change of evolutionary accessibility for carB * along the evolutionary

The   production   of   the   switcher   phenotype   (opaque   and   translucent   colonies   and/or   colonies   with   opaque   sectors)   is   one   of   the   conditions   that   have   to   be   fulfilled   by   a   switcher   genotype   to   be   able   to   evolve   from   the   different   genetic   backgrounds.  Moreover,  carB*  has  to  provide  a  fitness  benefit  for  the  genotype  in   order  to  increase  in  cell  frequency  and  to  be  detectable  on  an  agar  plate  following   dilution   of   cultures   containing   109   _{cells.   The   results   of   this   study   show   that   the}  

fitness   effect   of  carB*   in   the   different   genotypes   varied   along   the   evolutionary   pathway.    

In  order  to  determine  the  effect  of  history  on  switcher  evolution  three  possibilities   were  tested:  (1)  History  has  no  effect  on  switcher  evolution  and  carB*  is  likely  to   arise  from  any  given  starting  position  due  to  an  increased  fitness  in  the  different   genotypes   (SBW25,   1s1,   1s2,   s3   and   1s4).   (2)   Every   mutation   that   occurred   throughout  the  history  in  Line  1  contributes  and  the  entire  history  is  required  for   the  evolution  of  a  switcher.  (3)  Particular  evolutionary  events  that  occurred  along   the  evolutionary  trajectory  promote  the  emergence  of  the  switcher  later  on.    

1.  Mutational  history  has  no  effect:  The  results  of  the  fitness  experiments  show  that   in  principal  the  mutational  history  is  not  relevant.  The  introduction  of  carB*  into   the  genome  of  the  ancestor  SBW25  increased  the  fitness,  indicating  that  a  switcher   based   on  carB*   already   has   a   high   probability   of   occurring   at   the   starting   point   when   none   of   the   eight   mutations   is   present.   Nevertheless   there   was   large   variation  in  the  fitness  effect  of  carB*  in  the  subsequent  genotypes.  For  example,   due  to  a  negative  fitness  of  carB*,  the  mutation  has  only  a  limited  chance  to  arise  

in  1s1  and  1s2.  The  decreased  fitness  after  carB*  was  introduced  into  1s1  and  1s2   is  likely  to  cause  the  extinction  of  the  genotype  in  the  event  of  a  carB*  mutation   and  the  evolutionary  path  would  end.  This  result  is  similar  to  outcomes  from  other   experiments   (Weinreich   et   al.,   2006;   Khan   et   al,   2011;   Meyer   et   al.,   2012).   In   genotypes  of  the  advanced  evolutionary  history,  such  as  1s3  and  1s4,  a  switcher   based  on  a  carB*  mutation  has  a  greater  chance  to  establish  within  the  population   because  it  causes  a  fitness  increase.    

2.   The   entire   mutational   history   has   an   effect:   As   mentioned   earlier   in   this   paragraph   there   was   no   stepwise   fitness   increase   of  carB*   from   the   ancestor   SBW25  across  the  subsequent  genotypes  up  to  1s4.  The  results  demonstrate  that   the  evolution  of  a  carB*  based  switcher  is  not  dependent  upon  the  combined  effect   of  all  mutations  that  occurred  previously  to  the  switcher  in  1s4  (Wichman  et  al.,   1999;  Ortlund  et  al.,  2007;  Meyer,  2012).    

3.  Few  evolutionary  events  have  an  effect:  It  was  shown  that  the  mutational  history   was   in   principal   not   needed   in   order   to   produce   a   switching   genotype.   The   ancestor  SBW25  has  already  the  capacity  to  generate  an  evolutionary  successful   switcher  based  on  carB*.  This  capacity  was  not  observed  in  1s1  and  1s2  but  again   in   1s3   and   1s4.   This   large   variation   of   the   fitness   results   from   the   different   backgrounds  indicates  that  particular  evolutionary  events  that  occurred  along  the   evolutionary  trajectory  promote  the  emergence  of  the  switcher  later  on  (Blount  et   al.,  2008;  Salverda  et  al.,  2011;  Blount  et  al.,  2012).  In  particular  the  large  fitness   increase  from  1s2  to  1s3  after  carB*  was  introduced  (Fig.  4.2.)  suggests  that  the   genotype   of   1s3   promotes   the   evolution   of   a   switcher   from   this   evolutionary   position.  

As  mentioned  earlier,  the  introduction  of  carB*  into  the  ancestral  SBW25  genome   resulted   in   a   large   fitness   increase.   This   outcome   is   not   in   agreement   with   the   findings  of  Beaumont  et  al.  (2009).  They  performed  a  similar  fitness  experiment  in   which  SBW25  competed  against  SBW25carB*  in  a  static  environment  and  found   that  no  significant  fitness  increase  could  be  attributed  to  carB*.  Gallie  (2009)  on   the  other  hand  found  that  over  the  course  of  48  hours,  carB*  can  provide  a  benefit   in   the   SBW25   genotype   under   static   conditions.   The   performance   of   the   fitness  

assay  over  the  course  of  72  hours,  however,  made  the  significant  fitness  effect  of  

carB*  disappear  due  to  other  new  types  that  emerged  as  a  result  of  progressing   evolution   (Beaumont  et   al.,   2009).   This   shows   that   the   results   of   such   fitness   experiments   are   very   responsive   to   time.   Furthermore,   population   dynamics   within  the  microcosms  are  very  sensitive  to  other  factors  as  well.  For  example  the   pH-­‐value  of  the  media  or  the  temperature  can  change  the  transition  rate  between   capsulated   and   non-­‐capsulated   cells   within   a   population   with   a   presumable   impact  on  the  outcomes  of  such  fitness  experiments  (personal  conversation  with   Jenna   Gallie).   However,   the   increased   fitness   of   SBW25   in   the   presence   of  carB*   indicates  that  in  theory  a  switcher  has  the  potential  to  evolve  from  the  ancestral   genotype  without  any  additional  mutation.  

The  carB*  mutation  had  a  negative  fitness  effect  on  1s1  and  1s2.  Even  though  the   phenotype  was  generated  by  carB*  in  both  genotypes,  the  mutation  would  not  be   able  to  increase  in  frequency  because  of  a  low  fitness.  This  indicates  strong  genetic   constraints,   perhaps   caused   by   negative   epistasis   (Khan  et   al.,   2011).   Here   two   mutations  can  be  beneficial  for  the  organism  but  when  they  occur  together  in  the   same  genotype  the  fitness  decreases  because  of  negative  interactions.    

A  significant  increase  in  fitness  was  observed  after  carB*  was  introduced  into  the   genome   of   1s3.   The   improved   performance   of   1s3   when   carB*   was   present   suggests  that  in  this  background  carB*  has  a  realistic  chance  of  occurring  and  to   increase   its   frequency.   The   genotypes   of   1s2   and   1s3   are   almost   identical.   Only   two   additional   mutations   separate   1s3   from   1s2   but   the   fitness   effects   of  carB*   were   very   different   from   each   other.   It   is   likely   that   one   of   the   two   additional   mutations   or   the   combined   effect   of   both   contribute   to   the   fitness   increase   of  

carB*   in   1s3.   Hence   the   mutations   are   perhaps   crucial   evolutionary   events   that   open  up  an  evolutionary  pathway  for  switcher  evolution  based  on  carB*.  This  will   be  further  investigated  in  Chapter  6.  

As  expected,  the  introduction  of  carB*  into  the  genome  of  the  original  immediate   ancestor   1s4   (REE;   see   Chapter   1,   sections   1.4.4   &   1.5)   resulted   in   increased   fitness  (Beaumont  et  al.,  2009).  However,  the  advantage  given  by  carB*  was  not  as   high  in  1s4  as  in  1s3.  Previous  studies  found  that  1s4,  which  evolved  in  a  shaken  

environment,   still   produced   detectable   amounts   of   a   cellulosic   polymer   (Gallie,   2009).   This   leads   to   the   ability   to   produce   a   biofilm   at   the   surface   in   a   static   environment   by   which   1s4   gains   a   significant   benefit.   The   slightly   decreased   advantage   of  carB*   in   1s4   compared   to   the   huge   advantage   in   1s3   is   therefore   perhaps   attributable   to   an   already   very   fit   1s4   type.   Nonetheless   the   carB*   switcher  is  still  fit  enough  to  arise  in  this  genotype.    

4.4.3 Impact  of  environmental  factors  on  carB*  fitness    

The   growth   curves   and   growth   rates   of   the   different   genotypes   of   Line   1   were   measured  without  and  in  the  presence  of  carB*.  Essentially  carB*  did  not  change   the  trajectory  of  the  growth  curves  of  the  different  genotypes  but  all  carB*  strains   needed  more  time  until  they  entered  the  log-­‐phase.  Similar  results  were  obtained   for   the   maximal   growth   rates.   In   general   Vmax   did   not   change   significantly   after  

carB*   was   introduced   into   the   genome   except   for   in   the   ancestral   SBW25   strain   and  1s4.  In  both  cases  Vmax  decreased  significantly.  These  findings  do  not  coincide  

with  the  results  from  the  fitness  experiments.  If  fitness  was  just  determined  by  the   cell   doubling,   one   would   expect   high   fitness   values   to   be   associated   with   high   maximal   growth   rates.   The   variation   between   the   results   from   the   fitness   experiments   and   the   maximal   growth   rates   suggest   that   factors   additional   to   growth   rates   determine   whether  carB*   provides   a   fitness   advantage   or   not.   Growth   rates   were   measured   under   shaking   conditions,   but   the   fitness   experiments   were   performed   in   static   microcosms.   It   is   highly   likely   that   ecological   interactions   not   only   between   the   competition   partners   but   also   with   the   surrounding   environment   (e.g.   the   heterogeneous   microcosm   based   on   an   oxygen   gradient)   influence   the   population   dynamics   (Meyer  et   al.,   2012)   of   the   different  types,  affecting  their  competitive  power.  To  study  ecological  dynamics  in   bacteria  in  these  microenvironments  is  challenging  but  essential  in  order  to  gain   more   insight   into   evolutionary   dynamics   based   on   random   mutation   and   deterministic  processes  in  the  context  of  environmental  conditions.