Valoración y estigmatización de la mujer operada

from  the  model.      

3.3.1 The  SREE  from  genotypically  different  founder  populations    

3.3.1.1 Average  total  switcher  production  in  1s4,  3s4,  6s4  and  12s4  

The  plot  in  Figure  3.3  displays  the  average  number  of  switchers  that  were  found  in   a   total   of   56   replicate   microcosms   during   the   SREE   of   each   lineage   in   three   experimental   blocks   (N  =   3).   The   most   switchers   were   found   in   1s4,   which   is   shown  in  the  first  bar  plot  (Fig.  3.3).  Here  on  average  seven  out  of  20  microcosms   produced  a  switcher.  Replaying  evolution  from  the  genotypes  3s4  and  6s4  led  on   average  to  two  switchers  in  both  backgrounds.  No  switcher  was  detected  in  12s4   (Fig.  3.3).  Most  of  the  observed  variance  was  explained  by  the  lineage  factor  (F3,8  =  

22.3,  P  <  0.001,  R2  _{=  0.89)  and  a  Tukey-­‐Kramer  posthoc  test  over  all  four  strains}  

identified   1s4   as   different   from   3s4   (P   =   0.002),   6s4   (P   =   0.002)   and   12s4   (P   <   0.001)  in  its  average  switcher  occurrence.  The  lineages  3s4,  6s4  and  12s4  showed   similar  behaviour.  

Figure   3.3:   Average   number   of   replicates   of   1s4,   3s4,   6s4   and   12s4  that  evolved  switchers.  The   switchers   evolved   within   three   days   under   static   conditions.   Displayed  are  SEM  based  on  N  =  3.  

3.3.1.2 Time  taken  to  detect  switchers  in  each  strain    

The  four  lineages  1s4,  3s4,  6s4  and  12s4  differed  in  the  time  needed  for  a  switcher   genotype  to  arise.  All  re-­‐evolved  switchers  in  6s4  occurred  within  the  first  three   days   of   the   experiment.   In   3s4   one   transfer   at   maximum   was   necessary   to   see   a   switcher  on  the  agar  plates.  Both  strains  showed  the  new  phenotype  rather  quickly   in   comparison   with   1s4   (Fig.   3.4).   In   1s4   it   took   up   to   five   transfers   for   a   phenotypic  switcher  to  be  detected.  In  addition,  in  1s4  the  number  of  microcosms   that   had   a   switcher   increased   with   the   number   of   transfers.   Again   re-­‐evolution   from  12s4  did  not  result  in  a  detectable  phenotypic  switcher  (Fig.  3.4).  If  any  kind   of  new  type  was  found  on  an  agar  plate  the  replicate  was  terminated.    

Figure  3.4:  Average  number  of  microcosms  of  1s4,  3s4,  6s4  and  12s4  with  switchers   after   each   transfer.  The   first   transfer   was   performed   after   the   initial   three   days   of   bacterial   growth   in   a   static   environment   (zero   transfer)   if   no   new   type   was   detected   on  

agar   plates.   The   new   microcosms   were   then   incubated   for   a   further   three   days   under   static   conditions   and   plated   and   checked   for   new   types   and   switchers.   The   bacterial   cultures  were  transferred  until  a  new  type  was  found.  Displayed  are  SEM  based  on  N  =  3.   Vertical  dotted  lines  separate  different  transfers.  

3.3.1.3 Time  taken  to  detect  new  phenotypes  in  each  strain    

The  strains  3s4,  6s4  and  12s4  showed  the  highest  average  number  of  microcosms   with  newly  evolved  types  within  the  first  three  days  in  a  static  environment  (75%   of  the  microcosms).  Only  a  few  replicates  of  3s4  needed  one  transfer  for  a  novel   colony   type   to   appear.   Some   replicates   of   6s4   and   12s4   were   transferred   twice   before  a  type  with  new  colony  morphology  was  detected  (Fig  3.5).    

Figure   3.5:   Average   number   of   microcosms   of   1s4,   3s4,   6s4   and   12s4   with   a   novel   phenotype   after   each   transfer.  Every  three  days  the  cultures  were  transferred  to  new   microcosms,  and  screened  for  new  colony  morphologies.  This  process  was  repeated  until  a   new  type  was  found.  Illustrated  are  the  average  numbers  of  microcosms  that  had  a  novel  

type  after  each  transfer.  Displayed  are  SEM  based  on  N  =  3.  Vertical  dotted  lines  separate   different  transfers.  

Re-­‐evolution  of  novel  types  from  1s4  started  on  average  with  a  small  proportion  of   the   microcosms   (less   then   10%),   which   stayed   on   average   below   five   during   the   entire  experiment.  All  replicates  of  1s4  showed  a  new  type  after  the  fifth  transfer   (Fig.  3.5).  

3.3.2 Genotypic  analysis  of  re-­‐evolved  switcher  genotypes    

Next  it  was  checked  whether  the  detected  switcher  colony  morphology  of  the  re-­‐ evolved  switchers  were  caused  by  a  mutation  within  the  carB  gene.  The  carB  gene   was  chosen  because  carB*  was  the  underlying  mutation  of  the  switcher  genotype   that   evolved   during   the   REE   (1w4)   and   previous   replay   experiments   from   1s4   showed  a  tendency  of  repeated  switcher  evolution  based  on  mutations  in  the  carB  

gene   (Gallie,   2009).   But   the   results   were   less   powerful   due   to   a   small   replicate   number   (N   =   36).   Therefore,   the   carB   gene   of   all   re-­‐evolved   switchers   was   amplified,  sequenced,  and  compared  to  the  ancestral  carB  sequence.  

The   results   of   the   SREE   from   1s4,   3s4,   6s4   and   12s4   showed   that   from   22   phenotypic  switchers  that  were  found  in  1s4,  only  two  had  a  mutation  in  the  carB   gene.  In  6s4,  none  of  the  six  observed  switchers  had  a  mutation  in  the  carB  gene,   and   in   3s4   two   out   of   the   six   switchers   had   a   mutation   in   the   carB   gene.   No   phenotypic  switcher  was  found  in  12s4.  

3.3.3 Phenotypic  characterization  of  12s4carB*      

Genetic   analysis   and   fitness   assays   were   used   to   gain   information   as   to   whether   Line  12,  where  no  switcher  was  observed,  had  the  capacity  for  a  switcher  type  to   arise   in   this   genotype.   A   mutation   in   the  carB   gene   (carB*)   was   causal   for   the