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Figure   13:   portion   of   the   distance  matrix  used  for  the   selection   of   mates   for   the   F1xF1  breeding  plan.    

Distances   are   estimated   using  the  cumulative  profile   of   each   family   at   24   loci.  

Red   cells   correspond   to   d<0,35   and   cannot   be   selected,   while   green   cells   are   the   recommended   crosses.    

4.5.3  Sustainability  of  the  breeding  plan  

Initially,   to   assess   the   sustainability   of   this   theoretical   breeding   plan,   an   R-­‐script  

;͞ŽƐƚƐƌĞĞĚŝŶŐ^ƚƵƌŐĞŽŶƐ͟)   was   compiled   to   estimate   how   many   mates   per   year   could   be   performed   in   a   given   hatchery,   based   on   the   features   of   a   given   plant,   providing   also   an   estimation  of  the  years  required  to  accomplish  the  whole  plan.  The  software  estimates  the   costs  for  the  different  grow  phases  in  order  to  calculate  the  required  financial  support  per   year;  moreover,  it  also  provides  an  outline  of  the  releases  that  can  be  made  per  year  under   different  economical  and  logistical  constraints.  

The  variables  used  in  the  simulation  are  referred  to  a  hatchery  located  in  Cassolnovo,  where   the   ͞breeding   units͟   will   be   reared   in   the   near   future.   Detailed   information   about   the   outputs  of  the  script  are  described  in  the  manuscript  (Annex  D).    

A  second  R-­‐ƐĐƌŝƉƚ;͞BreedingPlanSturgeons͟ͿǁĂƐƵƐĞd  to  assess  the  recommended  number   of   individuals   to   breed   per   family   and   to   estimate   the   expected   fraction   of   alleles   of   the   parental   generation   that   are   successful   inherited   in   the   F2   virtual   progeny   under   different   strategies  of  family  selection  (reported  in  detailed  in  the  Annex  D).  

4.

C

Several   restocking   activities   have   been   conducted   in   the   last   20   years   with   the   aim   of   avoiding  the  extinction  of  the  Adriatic  sturgeon.   Nevertheless,  in  the  same  period  no  clear   evidences   of   natural   reproductions   were   recorded.   The   establishment   of   self-­‐sustaining   natural  populations  is  still  a  distant  goal  and  the  future  of  this  species  presently  depends  on   the  careful  management  of  the  last  captive  stocks.  Accordingly,  the  mail  goal  of  the  present   study   was   to   propose   a   breeding   program   that,   if   followed,   will   allow   a   long-­‐term   preservation  of  most  residual  genetic  diversity  of  the  Adriatic  sturgeon.    

To   this   purpose   several   preliminary   investigations   were   performed   in   order   to   i)   complete   the   genetic   characterization   of   the   main   stocks   reared   in   Italy,   ii)   assess   the   inheritance   pattern   of   this   species   and   optimize   a   parental   allocation   procedure,   iii)   reconstruct   the   pedigree   information   for   all   stocks,   iv)   optimize   the   first   species   and   hybrids   identification   procedure   based   on   a   single   nuclear   marker   and   consequently   v)   check   the   stocks   for   the   presence  of  alien  species  or  interspecific  hybrids.    

All  the  above  preliminary  actions  allowed  to  gain  relevant  information  for  the  development   of  a  complete  breeding  plan  articulated  in  two  phases:  the  first  involving  the  last  13  animals   of  wild  origin  and  the  second  focusing  on  the  about  30  families  of  F1,  trying  to  maximise  the   fraction  of  genetic  diversity  transmitted  to  the  future  captive  generations  of  breeders  as  well   as  to  the  animals  that  will  be  released  in  the  wild.  Moreover,  an  assessment  of  sustainability   of  the  plan  considering  economically  and  managerially  constraints  is  estimated  to  ensure  its   feasibility.  

In  general,  this  plan  represents  the  first  active  effort  towards  a  responsible  management  of   captive  stocks  of  this  species.  The  strategy  here  proposed  includes  also  releases  in  the  wild   of   such   animals   that   cannot   be   retained   in   captivity   due   to   spatial   limitations.   Despite   the   released  animals  are  large  enough  to  reduce  the  chances  of  predation,  the  expected  success   in   terms   of   survival   and   fitness   is   not   optimal   mainly     because   the   environment   is   not   yet   suitable  for  these  animals.    

The  limited  success  of  re-­‐introductions  is  the  main  problem  of  several  breeding  plans  and  it   is  well  documented  in  literature  (>LJŶĐŚĂŶĚK͛,ĞůLJ͕ϮϬϬϭ͖Woodworth  et  al.,  2002;  Williams   and   Hoffman,   2009;   Lyon   et   al.,   2012).   As   example,   Beck   et   al.,   (1994)  have   reviewed   that   only  11%  of  re-­‐introduction  plans  had  success,  while  for  Fisher  and  Lindenmayer  (2000)  this   value   increased   up   to   13%.   This   might   be   due   to   different   reasons   in   part   related   to   the   quality  of  the  animals  released  and  in  part  to  habitat  degradation.    

Moreover,  the  animals  released  might  not  be  fit  for  survival  in  the  wild.    In  the  past  decades,   Adriatic  sturgeon  recovery  programmes  have  adopted  standard  aquaculture  technologies  for   the   production   of   fingerlings   to   be   released,   ignoring   the   need   for   producing   animals   acclimated  or  trained  to  survive  in  the  wild.   Avoiding  crosses  between  related  families  and   equalizing  the  F1s  family  contribution  in  the  F2  generation,  our  strategy  could  be  considered   as   a   starting   point   that   tries   to   maximize   the   level   of   diversity   of   stocks   produced   to   be   released.  However,  the  establishment  of  a  rearing  protocol  for  the  production  and  training   of  animals,  according  to  the  criteria  of  the  fitness  for  survival  is  strongly  recommended.    

Concerning  the  habitat  degradation,  one  of  the  main  causes  for  the  imperilled  status  of  the   Adriatic  sturgeon  is  the  fragmentation  of  the  main  rivers  with  dams,  that  prevents  the  access   to  spawning  sites.  For  this  reason,  constructing  adequate  fish  passages  specifically  projected   for  these  big  anadromous  fishes  represents  a  priority.    

In   general,  all   strategies,   methods   and   recommendations  proposed   in   this   work   are   in   line   with   the   international   FAO   guidelines   for   sturgeon   management   and   release.   The   present   work  represents  the  first  breeding  plan  for  the  Adriatic  sturgeon  based  on  a  detailed  genetic   characterization  and  can  be  considered  as  a  reference  guideline  for  all  conservation  actions   based  on  controlled  reproductions  of  this  species.  

Moreover,  this  is  the  first  attempt  to  propose  a  coordinated  strategy  for  the  safeguard  of  the   captive  critically  endangered  Adriatic  sturgeon,  aimed  to  preserve  its  genetic  diversity.  Given   the  critical  status  of  this  species,  the  actions  here  proposed   should  be  realized  in  the  near   future  and  should  be  complemented  by    activities  at  different  levels  such  as  the  restoration   of   natural   environments   or   the   implementation   of     releasing   protocols   aimed   to   maximize   the  fitness  of  the  animals.    

Finally,  this  breeding  plan  and  the  related  approaches,  may  represent  a  useful  reference  also   for  other  imperilled  tetraploid  sturgeon  species.