Systematics, biogeography and evolution of selected widespread reptile genera from the arid areas of North Africa and Arabia Margarita Metallinou ADVERTIMENT. La consulta d’aquesta tesi queda condicionada a l’acceptació de les següents condicions d'ús: La difusió d’aquesta tesi per mitjà del servei TDX (www.tdx.cat) i a través del Dipòsit Digital de la UB (diposit.ub.edu) ha estat autoritzada pels titulars dels drets de propietat intel·lectual únicament per a usos privats emmarcats en activitats d’investigació i docència. No s’autoritza la seva reproducció amb finalitats de lucre ni la seva difusió i posada a disposició des d’un lloc aliè al servei TDX ni al Dipòsit Digital de la UB. No s’autoritza la presentació del seu contingut en una finestra o marc aliè a TDX o al Dipòsit Digital de la UB (framing). Aquesta reserva de drets afecta tant al resum de presentació de la tesi com als seus continguts. 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In the using or citation of parts of the thesis it’s obliged to indicate the name of the author.                                                                                              Cover  photo:  Scenery  in  Sharjah,  U.A.E.  April,  2013                   Systematics,  biogeography  and  evolution  of  selected  widespread   reptile  genera  from  the  arid  areas  of  North  Africa  and  Arabia     Sistemàtica,  biogeografia  i  evolució  de  dos  gèneres  de  rèptils   distribuïts  per  les  zones  àrides  del  Nord  d’Àfrica  i  Aràbia  TESI  DOCTORAL………    Programa  de  Doctorat  en  Biodiversitat  Memòria  presentada  per     Margarita  Metallinou    per  a  optar  al  grau  de  doctora  per  la  Universitat  de  Barcelona     Treball  realitzat     a  l’Institut  de  Biologia  Evolutiva  (CSIC  –  Universitat  Pompeu  Fabra)        Barcelona,  2014             Doctoranda                 Margarita  Metallinou   Director  de  la  tesi                 Salvador  Carranza  Gil-­‐ Dolz  del  Castellar  Científic  Titular  Institut  de  Biologia  Evolutiva  (CSIC-­‐Universitat  Pompeu  Fabra)     Tutor  de  la  tesi                 Miguel  Angel  Arnedo   Lombarte  Professor  Titular  Universitat  de  Barcelona             Systematics,  biogeography  and  evolution  of   selected  widespread  reptile  genera  from  the   arid  areas  of  North  Africa  and  Arabia    DOCTORAL  THESIS  ……..                                         Margarita  Metallinou           Φύσις κρύπτεσθαι φιλεῖ Ηράκλειτος Conheces o nome que te deram, não conheces o nome que tens Livro das evidências, Todos os nomes José Saramago ACKNOWLEDGMENTS   AGRAÏMENTS   ΕΥΧΑΡΙΣΤΙΕΣ   This  dissertation  started  –  unofficially  –  six   years   after   I  was   almost   convinced  that   studying   Biology   was   not   a   very  good   option   for   my   future;   eight  months  after   I  had  decided  not   to  do  a  PhD   following   the   Biology   degree;   six  months  after  trying  to  contact  Salvi  –  in  vain   –   for   the   possibility   of   a  Master’s  thesis  in  his  lab  and  three  months  after  applying   for   a   fellowship   that   was  highly   improbable   to   get.   It   did   start  officially   in   November   2009,   and,   by  that   time,   I   was   more   than   convinced  that   this  was   exactly  what   I  wanted   to  do,   where   I   wanted   to   do   it   and   the  people   who   I   wanted   to   share   it   with,  inside   and   outside   the   lab.   Since   then,  but   also   long   before   that   in   some  curious  way,  many  people  have  helped  me   to   arrive   to   this   beautiful  moment,  of   being   able   to   thank   them   for  something  so  important  and  fulfilling.    So,  many  thanks  are  due  to:  my   family.   My   mother   and   father,   for  teaching  me  to  be  patient,  hardworking  and   eager   to   learn.   For   providing   me  with   everything   that   I   could   need   and  more,   for  being  persistently  present  so  as   to   make   sure   I   always   know   how  much   they   love  me.  My   sister,  my   first  and  greatest  model,  my  ally  in  learning,  my  lifelong  friend  I  have  learnt  to  cope  with  missing  so  much.   Aυτή  η  διδακτορική  διατριβή  ξεκίνησε,  άτυπα,   έξι   χρόνια   αφότου   είχα   σχεδόν   πειστεί   ότι   η   Βιολογία   δεν   ήταν   η   καλύτερη   επιλογή   για   το   μέλλον   μου,   οχτώ   μήνες   αφότου   είχα   αποφασίσει  να  μην  κάνω  διδακτορικό  μετά  το   πτυχίο  Βιολογίας,  έξι  μήνες  αφότου  –  μάταια  –   είχα  προπαθήσει  να  έρθω  σε  επαφή  με  το  Salvi   για   τη   διπλωματική   του   μεταπτυχιακού   και   τρεις  μήνες  αφότου  είχα  κάνει  αίτηση  για  μια   υποτροφία   με   ελάχιστες   πιθανότητες   επιτυχίας.   Ξεκίνησε,   όντως,   και   τυπικά   το   Νοέμβριο   του   2009,   και   τότε   ήμουν   πλέον   πεπεισμένη   ότι   αυτό   ακριβώς   ήταν   αυτό   που   ήθελα   να   κάνω,   το   μέρος   που   ήθελα   να   το   κάνω   και   αυτοί   οι   άνθρωποι,   εντός   και   εκτός   εργαστηρίου,   με   τους   οποίους   ήθελα   να   το   μοιραστώ.   Έκτοτε,   αλλά   κατά   κάποιο   ενδιαφέρον   τρόπο   και   πολύ   πριν   από   αυτό,   πολλοί   άνθρωποι   με   βοήθησαν   να   φτάσω   σε   αυτήν   την   όμορφη  στιγμή,   να  μπορώ  να   τους   ευχαριστήσω  για  κάτι  τόσο  σημαντικό.     Πολλά  ευχαριστώ,  λοιπόν,  στους:   στην   οικογένειά   μου.   Στη   μητέρα   και   τον   πατέρα  μου  που  μου  δίδαξαν  να  έχω  υπομονή,   να   δουλεύω  σκληρά   και   να  θέλω  να  μαθαίνω.   Γιατί   μου   παρέχουν   ότι   χρειάζομαι   και   παραπάνω,   γιατί   είναι   επίμονα   παρόντες   ούτως  ώστε  να  ξέρω  πάντα  πόσο  με  αγαπούν.   Στην   αδερφή   μου,   το   πρώτο   και   σημαντικότερο   παράδειγμά   μου,   τη   σύμμαχό   μου  στη  μάθηση,  την  για  πάντα  φίλη  μου·  έχω,   αναγκαστικά,   καταφέρει   να   αντιμετωπίζω   το   πόσο  πολύ  μου  λείπεις.  Salvi,   for   being   an   amazing   advisor   and   a   friend,   for   guiding,   supervising,   helping   and  commenting  on  everything,   for  believing   in  me  and  always  encouraging  me,   for  making  this  fun.  You  have  learnt  that  la  antipatía  es  un  don  and  to  never  lose  hope  when  sampling,  as  long  as  the  girls  are  around,  and  I  have  learnt  so  many  things  that  it  has  taken  me  300  pages  –  and  hopefully  more  to  come  shortly  -­‐  to  try  to  explain  it  all.   Kele,   for   his   tutorship,   generous   advice   and   nice   times   discussing   on   a   great   variety   of  subjects.  Enric,   for   the   unconditional   help   and   tremendous   patience.   For   adding   beauty   and  precision   to  my   illustrations,  criticism  to  my  thoughts,   realism  to  my  goals.   If  doing  one  dissertation  is  hard,  you  can  be  proud  of  having  done  much  more  than  that.  But  above  all,  for  the  love  and  happiness.  And  to  his  family  for  their  warm  support  in  so  many  ways.  Ναυσικά,  for  being  much  more  than  a  best  friend.  It  feels  so  nice  to  know  that  somebody  knows  you  so  well  and  to  have  shared  so  much.  I  am  lucky  to  have  you.  Κατερίνα,  Ζωζεφίνα  and  Αγγελική,   for   spending  some  of   the  most  memorable  moments  together,   from   studying,   to   chatting,   laughing,   traveling,   dancing,   dreaming,   going   away  and  getting  back  together.  I  miss  you  a  lot.  Φοίβο,   for   the   first   discussions   on   how   amazing   biology   is   and   for   some   of   best   times  spent  on  essays.  my  cousins,  for  some  of  the  best  holiday  and  other  fun  times.  And  all  my  large  family,  for  encouraging  me  through  their  interest  and  with  a  lot  of  good  food.    the  friends  in  Barcelona,  for  the  company,  excursions,  parties  and  relaxing  beer  sessions  that  have  cheered  up  all  these  years.  the  fieldtrip  companions:  Felix,  David  and  Daniel,   for  that  first  herping  overdose  back  in  2009;  Jiri,  for  working  so  hard  and  well  in  the  field,  I´m  glad  we  coincided;  Roberto,  for  his  serenity,  persistence  and  kind  smile;  Philip,  for  cheering  up  the  hard  6am  wake-­‐up  at  40C;  Ali  and  Sultan,  for  taking  care  of  us  across  the  deserts  and  the  seas  of  Oman.  the  group  members:  Raquel,   for  her   support,   sincerity  and   friendship;  Marc,   for  helping  and  caring,  in  the  lab  and  in  the  field;  Elena,  for  being  an  example  of  persistence  and  hard  work;  Josep,  for  being  my  first  lab  companion,  always  eager  to  lend  a  hand;  Joan,  for  the  methodological   and   computational   tips;   the   Portuguese   "invasion",   Catarina,   Mafalda,  Luis,  Joana,  Duarte,  and  especially  João,  and  the  students  from  all  over,  Eli,  Hernán,  Karin,  Arnaud,  Tania,  Santi,  who  have  made  these  years  diverse  and  constructive.  Άννα,   for   the   invaluable   input,   encouragement   and   understanding,   and   the   long   chats   I  missed  this  last  year.  Amparo,  Anabela,  Valeria,  David,  Rocio,  Gissela,  Cristina,  Jesús  and  more  friends  at  the  IBE  with  whom  we  have   spent   long  hours  between   labs,   corridors,  breaks,   celebrations  and  volleyball.  Rita,   Blanca,   Ana   and   Emiliano   at   the   IBE   who   have   always   helped   me   out   with  practicalities,  dreadful  BOE's  and  anything  else  I  needed  during  these  almost  6  years.  Pierre-­‐André,   for   his   useful,   overwhelming   comments   and   emails,   his   participation   and  trust;  to  Philippe,  for  generously  sharing  his  enthusiasm  and  knowledge;  to  Ze  Carlos,  for  welcoming  me  in  his  vibrant  group  and  patiently  working  with  me;  to  everyone  in  MNHN  who  helped  me  with  the  200-­‐year  old  material  and  literature.  everyone  who   contributed   tissue  material   for   the  work   in   this   dissertation.   It   has   been  very  much  appreciated  and  hopefully  the  work  carried  out  meets  your  expectations.  Ευχαριστώ  πολύ!                                                                                          This  work  was  supported  by  a  FPU  predoctoral  grant  from  the  Ministerio  de  Educación,  Cultura  y  Deporte  (AP2008-­‐01844)     TABLE  OF  CONTENTS     Chapter  1.  Introduction   Preamble   1.1.  The  study  of  Biological  Systematics  1.1.1.  Phylogenetic  reconstruction   Data  sources   Methods  for  phylogenetic  reconstruction   Tree-­‐based  inferences  1.1.2.  Classification  of  biodiversity   The  task  of  defining  species  the  species  limits   The  system  of  Zoological  Nomenclature   1.2.  The  arid  areas  of  North  Africa  and  Arabia  1.2.1.  Characteristics  and  brief  geological  and  climatic  history  of  North  Africa  and  Arabia  1.2.2.  Arid  zones  and  their  biodiversity   1.3.  The  reptile  study  groups  1.3.1.  The  use  of  reptiles  as  models  in  the  study  of  systematics,  biogeography  and  evolution  1.3.2.  The  genus  Stenodactylus  from  the  deserts  of  North  Africa  and  Arabia   Taxonomic  and  phylogenetic  background  on  Stenodactylus  1.3.3.  The  rock-­‐dwelling  genus  Ptyodactylus  from  North  Africa,  the  Sahel  and  Arabia   Taxonomic  and  phylogenetic  background  on  Ptyodactylus     Chapter  2.  Objectives  and  Structure   2.1.  Objectives   2.2.  Structure  of  the  Results     Advisor’s  reports     Chapter  3.  Results:  Systematics  of  Stenodactylus   Paper  1   Metallinou,  M.;  Arnold,  E.N.;  Crochet,  P.-­‐A.;  Geniez,  P.;  Brito,  J.C.;  Lymberakis,  P.;  Baha  El  Din,  S.;  Sindaco,  R.;  Robinson,  M.  &  Carranza,  S.,   2012.   Conquering   the   Sahara   and   Arabian   deserts:   systematics   and  biogeography   of   Stenodactylus   geckos   (Reptilia:   Gekkonidae).   BMC   Evolutionary  Biology,  12:258.     1X  3X  5x  5x  5x  6x  8x  10x  10x  14x  16x    16x  19x  21x    21x    23x  23    25x  26x    27x  29x  30x    33x    37X          41x     Paper  2   Metallinou,   M.   &   Carranza,   S.,   2013.   New   species   of   Stenodactylus   (Squamata:   Gekkonidae)   from   the   Sharqiyah   Sands   in  northeastern  Oman.  Zootaxa  3745  (4):  449-­‐468.     Chapter  4.  Results:  Nomenclature  of  Stenodactylus   Paper  3   Metallinou,   M.   &   Crochet,   P-­‐A.,   2013.   Nomenclature   of  African   species   of   the   genus   Stenodactylus   (Squamata:   Gekkonidae).   Zootaxa  3691  (3):  365-­‐376.     Chapter  5.  Results  :  Systematics  of  Ptyodactylus   Paper  4   Metallinou,   M.;   Červenka,   J.;   Crochet,   P.-­‐A.;   Kratochvíl,   L.;  Wilms,  T.;  Geniez,  P.;  Shobrak,  M.Y.;  Brito,  J.C.  &  Carranza,  S.  Species  on  the   rocks:   Systematics   and   biogeography   of   the   rock-­‐dwelling   Ptyodactylus   geckos   (Squamata:   Phyllodactylidae)   in   North   Africa   and  Arabia.  (in  preparation)     Paper  5   Metallinou,   M.;   Wilms,   T.;   Kratochvíl,   L.   &   Carranza,   S.  Multilocus  species  delimitation  in  an  old  radiation  of  North  African  and  Arabian  geckos  (Phyllodactylidae:  Ptyodactylus).  (in  preparation)       Chapter  6.  General  Discussion  and  Conclusions   6.1.  General  Discussion   Taxonomic  sampling  across  broad  areas  in  North  Africa  and  Arabia   Overview   of   molecular   data   and   reconstruction   of   phylogenetic   relationships  within  a  temporal  framework   Ecologic   remarks   and   biogeographic   patterns   in   Stenodactylus   and  Ptyodactylus  across  North  Africa  and  Arabia   Taxonomic  assessments  and  actions  in  Stenodactylus  and  Ptyodactylus   6.2.  General  Conclusions     References     Resum  en  català     Appendices   Appendix  1.  Addenda  to  Nomenclature  of  Stenodactylus   Appendix  2.  Systematics  of  Trachylepis  in  the  Socotra  Archipelago            75x    97X      101X    115.          117x        161x    199.  201x  201x    203x    205x  206x  208x    211.    225.     X  253x  263x                                 CHAPTER  1    General  Introduction       PREAMBLE  The   arid   areas   of   North   Africa   and   Arabia   cover   a   surface   of  more   than   13  million  square  kilometers  and  are  characterized  by  their  extreme  temperatures,  diversity  of  desert  habitats  and  well-­‐adapted  flora  and  fauna.  The  study  of  the  evolution  of   their  biota   sheds   light   on   the   diversification   processes   in   some   of   the   world’s   harshest  environments   and   contributes   to   our   knowledge   on   large-­‐scale   biogeographic  patterns   and   processes.   In   this   dissertation,   two   representative   reptile   genera   from  these   areas,   Stenodactylus   and   Ptyodactylus,   are   studied   from   a   systematic   and  biogeographic  perspective.  This   dissertation   compiles   three   Chapters   of   Results   (Chapters   3,   4   and   5),  including  five  papers  in  total,  either  published  or  prepared  for  publication  in  scientific  journals   in   the   fields   of   Evolution   and   Systematics.   As   a   consequence,   there   is  inevitably   some   overlap   between   the   subjects   and   background   addressed   in   the  General  Introduction  (Chapter  1)  and  the  respective  Introductions  of  the  papers,  and  accordingly   the   General   Discussion   and   Conclusions   (Chapter   6).   To   keep   this   to   a  minimum,   the   main   scope   of   the   General   Introduction   is   to   concisely   set   the  theoretical   and   methodological   framework   in   which   the   dissertation   has   been  developed.   It  avoids,  however,   repetition  of  more  specific   information  regarding   the  study   groups,  which   is   introduced,   discussed   and   updated  with   new   findings   in   the  corresponding   sections   of   the   Results.   Bibliographic   references   are   included  separately  at  the  end  of  each  paper,  while  a  common  reference  list  is  provided  for  the  General  Introduction  and  General  Discussion  at  the  end  of  the  dissertation.  Chapter  2  lists   the   aims   established   in   the   dissertation   and   how   they   are   addressed   in   the  Results.  A  report  from  the  Advisor  prior  to  Chapter  3  informs  on  the  publication  status  of  the  papers  and  details  the  participation  of  the  PhD  candidate  in  the  work  presented  herein.       3     1.1.    THE  STUDY  OF  BIOLOGICAL  SYSTEMATICS   Systematics   proposes   to   fulfill   two   major   tasks:   discover   and   describe   organic  diversity,   and   uncover   the   evolutionary   history   of   the   species   through   the  reconstruction   of   phylogenies   (Wiens,   2007;   Wiley   &   Lieberman,   2011).   With   the  revolutionary   work   of   Willi   Hennig   (1966),   the   classification   of   the   described  biodiversity   shifted   from   a   divisive   logic   to   one   that   reflects   evolutionary   relatedness   producing   only   monophyletic   groups   as   hypotheses   of   classification.  These   two   tasks   have   become   inextricably   tied   as   the   delineation   of   species   is  provided   with   assessments   of   evolutionary   relationships   through   phylogenetic  reconstructions.     1.1.1.  Phylogenetic  reconstruction  Phylogenetics,  from  the  Greek  words  φύλο  (filo)  =  tribe  and  γένεσις  (genesis)  =  origin,  focuses   on   understanding   the   relationships   between   species   (or   populations)   using  observable   characters   from   the   organisms   and   analyzing   them   with   a   variety   of  methodologies.   Data  sources  Phylogenetic  systematists  employ  a  series  of  heritable  characters  to  formulate  their  hypotheses   and   the   form   in  which   a   character   appears   in   every   organism   is   called  state.  Characters  that  can  be  ascribed  to  common  ancestry  are  homologous  and  their  comparison   is   used   for   the   phylogenetic   inference.   Systematists   usually   focus   on  characters   whose   states   vary   between   species   (but   not   too   much)   and   are   fairly  constant  within  species   (but  not   totally)   (Wiley  &  Lieberman,  2011).  The  characters  used   in   phylogenetic   reconstruction   (and   species   delimitation)   may   come   from  different  sources  such  as  morphology,  DNA  molecules,  proteins,  but  also  ecological  or  behavioral  traits  (e.g.  Wiens  2004;  Wiens  et  al.,  2010;  Caetano  &  Machado,  2013).  In   the   last  decades,  DNA  data  are  becoming   increasingly  available  and  attractive  for   phylogenetic   reconstruction   leading   to   a   great   interest   in   the   field   of  molecular  phylogenetics.   Different   types   of   DNA   data   can   nowadays   be   obtained   –   also  depending  on   the   level   of   variation  one   is   seeking   to   explore:   coding  or  non-­‐coding  mitochondrial   or   nuclear   DNA   fragments,   entire   mitochondrial   genomes,  microsatellites,  amplified   fragment   length  polymorphisms  (AFLPs),  single-­‐nucleotide  polymorphisms   (SNPs)   etc.   Although   technical   advancements   are   making   the  acquisition   of   genomic   (next-­‐generation   sequencing)   data   much   more   feasible,   the  comparability  with  data  accumulated  for  decades  and  the  pragmatic  technical  inertia  contribute  to  the  first  category  of  data  (PCR-­‐amplified  DNA  fragments)  being  still  the  prevailing  source  for  most  fields.     5 Introduction The  homologous  positions  of  the  nucleotide  sequences  obtained  need  to  be  aligned  in   order   to   create   a   data  matrix   used   in   downstream  analyses   through   a   procedure  that   turns  unequal   length  sequences   into  equal   length  character  strings  by   inserting  gaps   (Giribet   et   al.,   2002).   Sequence   alignments   are   hypotheses   of   homology,  formulated   through   the   estimation   of   essentially   unobserved   processes  (transformations,  insertions,  deletions,  etc.).  Character  transformation  models  and  the  relative   cost   of   alignment-­‐derived   gaps   play   a   central   role   in   sequence   analysis  (Wheeler,   1995),   hence   the   strong   impact   of   the   alignment   process   on   the  phylogenetic   reconstruction   (Cognato   &   Vogler,   2001;   Ogden   &   Rosenberg,   2007;  Talavera  &  Castresana,  2007).  In  the  simplest  version,  a  pairwise  sequence  alignment,  according  to  the  fundamental  method  described  by  Needleman  and  Wunsch  (1970),  is  performed  with  the  optimized  calculation  of  the  gap  penalty  and  the  change  cost.  This  process   increases   vastly   in   complexity   when   multiple   sequences   are   compared  (Sankoff   &   Cedergren,   1983),   requiring   the   use   of   heuristic   strategies   (Wheeler,  2000).   These   strategies   are   implemented   in   numerous  multiple   alignment  methods  available  nowadays,   such  as   the  Clustal   family   (Thompson  et  al.,   1994;  Larkin  et  al.,  2007),  Muscle  (Edgar,  2004),  T-­‐Coffee  (Notredame  et  al.,  2000)  and  Mafft  (Katoh  et  al.,  2002;  Katoh  &  Toh,  2008).     Methods  for  phylogenetic  reconstruction  DNA   data   matrices   are   typically   analyzed   with   character-­‐based   methods   that  undertake  a  tree  search  on  the  basis  of  an  optimality  criterion,  as  opposed  to  distance-­‐based   methods   (e.g.   UPGMA,   Neighbor   joining)   that   calculate   the   tree   based   on  distances  between  the  pairs  of  sequences  in  the  data  matrix.  Among  the  former  set  of  methodologies,   maximum   parsimony   (MP)   follows   the   principle   of   parsimony  according  to  which  the  best  phylogenetic  reconstruction   is   the  one  that   involves  the  least   number   of   evolutionary   transformations   among   the   characters.   This   was   the  prevailing  methodology  used  since  the  early  years  of  phylogenetics,  but  was  subjected  to  criticism  over  statistical   inconsistency   in  results  under  certain  cases   (Felsenstein,  1978).  Most   importantly,  development  of  newer  methods  (see  below)  permitted   the  implementation   of   explicit   mathematical   models   of   character   evolution   in   the  reconstruction   of   the   tree,   leading   to   its   gradual   replacement.   These   models   make  several  assumptions  regarding  the  probability  of  substitution  of  nucleotides  and  may  include   from   one   (Jukes   and   Cantor)   to   nine   (Generalized   time-­‐reversible,   GTR)  parameters  (Graur  &  Li,  2000).  Accounting  for  the  rate  variation  among  sites  (+G)  and  the  proportion  of  invariable  sites  (+I)  permits  the  refinement  of  the  models.  The   model-­‐based   methods   used   in   the   reconstruction   of   phylogenetic  relationships  are  those  of  maximum  likelihood  (ML)  and  Bayesian  inference  (BI)  that,   although   very   different   in   their   approach,   they   are   both   based   on   the   use   of  likelihood  calculations.  According  to  the  ML  approach  (Fisher,  1922),  the  “best”  tree  is   Introduction 6 the  one   that  has   the  highest  probability  of  producing   the  observed  data  assuming  a  model/hypothesis   that   includes   the  model   of   DNA   evolution,   the   tree   topology   and  branch   lengths   (Felsenstein,   1981;   Huelsenbeck   &   Crandall,   1997).   Given   the  immensity  of  the  tree  space  for  reconstructions  that  involve  more  than  just  a  few  tips,  ML   methods   for   phylogenetic   reconstruction   perform   heuristic   rather   than   exact  searches,  thus  rendering  it  important  to  carry  out  multiple  independent  replicates  in  the  search  for  the  optimal  tree.  On  the  other  hand,  BI  seeks  to  maximize  the  posterior  probability   of   the   tree   given   the   data   and   the   model   (Yang   &   Rannala,   1997;  Huelsenbeck  et  al.,  2001).  It  uses  the  Markov  Chain  Monte  Carlo  (MCMC)  algorithm  to  search  for  the  area(s)  of  highest  probability  density  across  the  posterior  distribution  space  of  trees  and  parameters.  Importantly,  the  output  of  a  BI  analysis  is  not  the  tree  with  the  maximum  posterior  probability,  but  rather  a  majority-­‐rule  consensus  tree  as  a  summary  of  the  posterior  distribution  of  trees.  This  way  the  posterior  probabilities  of  the  different  clades  in  a  BI  analysis  constitute  a  measure  of  tree  support,  while  in  ML   analysis   this   is   usually   obtained   through   non-­‐parametric   bootstrapping  (Felsenstein,   1985).   This   method   performs   tree   reconstructions   based   on   a   set   of  pseudoreplicate  data  matrices  originating  from  subsampling  the  original  matrix.  The  frequency  of  a  particular  node  in  the  resulting  set  of  trees  is  its  bootstrap  value.  Latest  advances   in   the   implementation   of   the   ML   and   BI   methods   focus   on   making  computation  more   efficient   for   the   increasing   amount   of   data   available   for   analysis  (Guindon  et  al.,  2010;  Ronquist  et  al.,  2012;  Stamatakis,  2014).  Although   it  was   recognized   that   increased  data   could   contribute   to  more   robust  phylogenetic   hypotheses,   following   the   idea   of   “total   evidence”   (Kluge,   1998),   the  combination  of  data   from  multiple  sources  has  not  been  straightforward  (Bull  et  al.,  1993;  Huelsenbeck  et  al.,  1996).  The  approach  that  has  been  regularly  followed  is  the   concatenation   of   (fragments   of)   different   genes   into   a   single   partitioned   matrix,  where  a  best-­‐fit  model  of  evolution  is  selected  for  each  partition,  in  order  to  account  for  heterogeneity  in  the  data.  The  resulting  trees  are  commonly  called  “concatenated  trees”  in  phylogenetic  studies  and  make  the  crucial  assumption  that  the  different  sets  of  data  (partitions)  have  congruent  underlying  phylogenetic  histories  (Wiens,  1998).  However,   this   may   not   be   true   for   all   genes   combined   into   a   matrix,   especially  when  sampling  closely  related  species  or  populations  (Hudson,  1991).  Processes  such  as   horizontal   gene   transfer,   introgression   and   recombination   can   lead   to   discord   in  the   evolutionary  histories   of   genes   (Posada  &  Crandall,   2002;  Bapteste  et   al.,   2004)  but   most   of   the   focus   has   been   placed   on   incomplete   lineage   sorting   and   the  application   of   coalescence   theory   for   dealing   with   inconsistencies   of   gene   trees  (Kingman,  1982;  Hudson,  1992;  Rosenberg,  2002;  Rannala  &  Yang,  2003;  Kubatko  &  Degnan,   2007;   Knowles   &   Carstens,   2007;   Edwards,   2009)   and   reconstructing   a   species   tree   under   the  multispecies   coalescent  model   (Degnan  &  Rosenberg,   2009;  Heled   &   Drummond,   2010).   This   approach   has   also   been   of   great   use   in   the  development  of  species-­‐delimitation  methods  (see  below).     7 Introduction Tree-­‐based  inferences  The  reconstruction  of  the  phylogenetic  relationships  between  species  is  a  first  step  to  unveiling   their   evolutionary   history.   Next,   tree-­‐based   inferences   can   be   made   to  answer  questions   related   to   the  evolution  of   characters,   the   temporal   framework  of  their  diversification  or  the  biogeographic  history  underlying  it.  In   the   study   of   the   evolutionary   history   of   the   organisms   it   may   be   of   special  interest   to   reconstruct   the   ancestral   state   of   one   or   more   of   their   traits,   like   a  morphological  feature,  a  nucleotide  deletion  or  insertion  or  an  area  of  occurrence.  The   ancestral   state   reconstruction   is   generally  performed  with  MP  or  ML/BI  methods  (Cunningham  et  al.,  1998).  As  explained  above,  the  MP  approach  will  favor  the  simpler  solution  regarding  the  total  number  of  changes  of  character  state.  On  the  other  hand,  parametric   approaches   are   able   to   incorporate   branch   length   information   and  produce  a  probability  value  for  the  state  of  a  character  at  an  ancestral  node.  The  inference  of  divergence  times,  performed  posterior  to  or  concurrently  with  the   tree   topology   estimation,   has   become   very   popular   in   phylogenetic   studies   and  other   related   fields   (Arbogast  et  al.,   2002).  This  practice   is  based  on   the   idea  of   the  molecular   clock   proposed   by   Zuckerkandl   and   Pauling   (1962)   according   to   which  changes   occur   at   an   approximately   uniform   rate   in  molecules.   Nevertheless,   during  the  following  decades,  concerns  over  the  accuracy  and  applicability  of  such  a  “strict”  model   (Kumar,   2005)   led   to   the   development   of   more   refined  models,   such   as   the  autocorrelated  and  uncorrelated  relaxed  clocks  that  permit  variation  of  evolutionary  rates   through   time   (Sanderson,   1997;   Thorne   et   al.,   1998;   Drummond   et   al.,   2006;  Rannala   &   Yang,   2007;   Lepage   et   al.,   2007).   Another   important   aspect   of   this  procedure   is   the   calibration   of   the   tree,   performed   via   the   so-­‐called   “calibration  points”.  Their  application  stems  from  the  assumption  that  the  presence  of  a  fossil  or  the   occurrence   of   an   event  may   be   reflected   in   the   node   of   a   phylogenetic   tree.   So  these  points  are  either  dated  fossils,  preferably  of  the  study  ingroup  or  closely  related  groups,  or  paleobiogeographic  events,  such  as  island  formation  continental  rifting,  etc.  (Ho,  2007;  Donoghue  &  Benton,  2007),  and  their  age  is  attributed  to  a  node  enabling  the   transformation   of   the   relative   temporal   framework   of   an   ultrametric   tree   to   an  absolute   one.   These   ages,   however,   unavoidably   bear   uncertainty   that   needs   to   be  accounted  for  by  applying  “soft”  bounds,  that  is,  prior  distributions  rather  than  point  estimates   of   the   ages   (Rannala  &   Yang,   2006;  Ho  &   Phillips,   2009).   This  way,   fossil  calibrations   are   generally   applied   as   minimum   ages   while   biogeographic   events   as  maximum  ones.   Historical   biogeography   integrates   the   evidence   obtained   within   a   temporal  framework,   such   as   the   separation   between   sister   groups   or   the   appearance   of   a  character   on   a   phylogeny,   with   the   geography   of   the   area   where   they   took   place,  enabling   to   propose   hypotheses   over   the   processes   that   have   generated   them.   A  distinction   between   ecological   and   historical   biogeography   on   the   basis   of   the   8 Introduction temporal   scale   of   study   has   been   bridged   with   the   realization   that   both   biotic  interactions  and  environmental  constraints,  and  subjects  from  geology  and  geography  are  essential  in  an  integrative  framework  for  exploration  of  the  biogeography  (Crisci   et  al.  2005;  Posadas  et  al.,  2006).  Processes   like  dispersal,  vicariance,  extinction  and  speciation   remain   at   the   epicenter   of   biogeographic   studies,   but   more   recent  approaches   to  biogeography  are   incorporating  statistical  methods   that  are  currently  shifting  the  focus  from  a  pattern-­‐based  methodology  to  an  event-­‐based  one  (Ronquist  &  Sanmartín,  2011).         9 Introduction 1.1.2.  Classification  of  biodiversity  The   identification,   description   and   classification   of   living   organisms   compose   a  challenging   enterprise   and   a   lively   field   of   science   on  which  workers   have   devoted  their   efforts   for   centuries,   probably  more   than  most   other   scientific   endeavors,   and  where  debate  on  reconciliation  of   trends  and  best  practices  persists  (Godfray,  2002;  Kuntner  &  Agnarsson,  2006;  Padial  &  de  la  Riva,  2007;  Wheeler,  2008;  Dubois  et  al.,  2013,  among  many).  The  term  taxonomy  –  from  the  Greek  words  τάξις  (taxis)  =  order  and   -­‐νομία   (-­‐nomia)   =   method,   law   –   was   first   attributed   to   this   task   in   the  seventeenth   century.   Nevertheless,   records   from   as   early   as   the   times   of   Aristotle  (384-­‐322  B.C.)  show  that  development  on  the  theory  and  practice  of  classification  of  (living)   forms   was   already   taking   place,   although   it   has   been   argued   that   this   was  rather  a  “universal”  taxonomy  (of  all  possible  objects)  than  a  biological  one  (Wilkins,  2009).   It   was   the   introduction   of   a   system   of   hierarchical   levels,   the   binomial  nomenclature,  by  Carolus  Linnaeus   in  his  Systema  Naturae   (1758)   that  set   the  basis  for   a   natural   classification   of   organisms   and   the   work   of   Darwin   On   the   Origin   of   Species   (1859)   that   led   to   the   understanding   that   such   a   classification   relies   upon  common  descent  and  biological  evolutionary  relationships.     The  task  of  defining  the  species  limits  The   basic   unit   in   biology   is   the   species   and   thus   the   importance   of   the   quest   for  delimiting  species  boundaries  is  indisputably  paramount  (Wilson,  2004).  Systematists  are  called  to  distinguish  between  intraspecific  and  interspecific  character  variation  (Wiens,   1999)   and   use   the   latter   in   order   to   delimit,   diagnose   and   differentially  describe   a   species.   They   may   use   characters   from   different   sources,   such   as  morphology,   molecules   and   behavior.   Recently,   the   application   of   ecological   niche  modeling  in  species  delimitation  has  also  seen  methodological  developments  (Rissler  &  Apodaca,  2007;  Raxworthy  et  al.,  2007).  Relevant   to   the   question   of   species   delimitation   is   also   the   concept   of   species.  Numerous   species   concepts   have   been   described   (reviewed   in   Mayden,   1997)  generating   controversy   over   the   properties   upon   which   they   are   based.   These   can  generally   be   classified   in   three   main   categories   (following   Wilkins,   2009;   Wiley   &  Lieberman,   2011):   the   evolutionary   species   concepts,   the   phylogenetic   species  concepts   and   the   reproductive   isolation   concepts   (including   the   biological   species  concept),   while   concepts   that   fall   outside   these   categories   include   the   ecological  species   concept   and   the   morphological   species   concept   among   others.   The   major  concern   of   the   debate   was   on   the   incompatibility   among   the   results   the   different  concepts  would  yield  on  the  question  of  defining  the  species  boundaries  (de  Queiroz,  2007).  The  simplifying  solution  to  this  issue  came  under  the  name  of  unified  species  concept   proposed   by   de   Queiroz   (1998,   2005,   2007)   who   unlinked   the   conceptual  problem   from   the   methodological   one.   He   argues   that   it   is   commonly   agreed   that   Introduction 10 “species  are  (segments  of)  separately  evolving  metapopulation  lineages”  and  treats  the  formerly   addressed  properties   not   as   defining   of   the   species   category  but   rather   as  evidences  of  lineage  separation.  In   the  beginning  of   the   last  decade,  Sites  and  Marshall   (2003)  claimed  that   there  were  “signs  of  a  Renaissance”  for  the  discipline  of  species  delimitation  in  the  view  of  growing   work   on   empirical   testing   of   species   limits.   Currently,   the   abundance   of  published   literature,   theoretical,   methodological   and   empirical,   on   the   subject  (reviewed  in  Camargo  &  Sites,  2013)  proved  that  they  were  right.  With  the  distinction  between  what   species   are   and   the   evidence   used   for   their   recognition   (de   Queiroz,  1998,  2007),  a  step  forward  was  made  that  facilitated  progress  on  the  subject,  with  a  special  focus  on  the  use  of  genetic  data  and  the  implementation  of  coalescence  theory  in   species   delimitation   (Wiens,   2007;   Carstens   et   al.,   2013).   There   are   nowadays  numerous  methods   at   the  disposal   of  molecular   systematists   that  permit   to   explore  species   limits   and  are   considered  especially  helpful   in   the   study  of   “cryptic”   and/or  allopatric   species   (Leavitt   et   al.,   2011;   Fujita   et   al.,   2012;   Barley   et   al.,   2013).  Nevertheless,   the   application   of   a   strictly   DNA-­‐based   approach   brings   forward   two  main  concerns,  seemingly  in  opposite  directions.  On  the  one  hand,  the  recognition  and  description   of   evolutionarily   distinct   lineages   based   on   genetic   analyses   only   as  distinct  species  should  not  overlook   the  necessity   to  provide  a  description  based  on  intrinsic  characters  of  the  species  (see  Leaché  &  Fujita,  2010  and  comment  by  Bauer   et   al.,   2010).   The   critics   claim   that   despite   the   urgent   need   for   inventorying   of   the  threatened   biodiversity   (Erwin   &   Johnson,   2000),   this   practice   could   aggravate  taxonomic  inflation,  threatening  conservation  efforts  (Isaac  et  al.,  2004).  On  the  other  hand,   only   a   small   portion   of   such   species   delimitation   studies   provide   species  description(s),   translated   either   as   lack   of   confidence   in   the   results   (Carstens  et   al.,  2013)   or   compliance   with   a   publishing   system   that   does   not   always   facilitate   the  publication   of   taxonomic   results   (Padial   &   de   la   Riva,   2007;   Carstens   et   al.,   2013).  Ultimately,  the  concern  is  common,  that  of  achieving  a  robust  taxonomy.  Among  the  currently  available  methodologies  for  DNA-­‐based  species  delimitation  (reviewed   in   Carstens   et   al.,   2013),   many   use   the   coalescent   approach.   Another  important   distinction   can   be   made   between   the   ones   that   require   an   a   priori   grouping   (validation   methods)   and   the   ones   that   can   proceed   without   initially  constraining  the  possible  assignments  (discovery  methods)  (O’Meara,  2010;  Satler  et   al.,  2013;  Edwards  &  Knowles,  2014).  Here,  I  will  briefly  elaborate  on  the  coalescent-­‐based   species   delimitation   methods   employed   in   this   dissertation,   selected   on   the  basis  of   the  needs  of  the  specific  study  and  the  evaluation  of  the  performance  of  the  methods  in  recent  literature.  The  general  mixed  Yule  coalescent  model  (GMYC)  was  introduced  by  Pons  et  al.  (2006)  as  a  method  to  delimit  independently  evolving  species  using  single-­‐locus  data.  Recently,  it  was  described  in  detail  and  evaluated  by  Fujisawa  &  Barraclough  (2013)  who   presented   a   new   version   of   the   method   with   important   reformulations.   The   11 Introduction GMYC  method  receives  as  an  input  a  reconstructed  ultrametric  gene  tree  and  does  not  require  an  a  priori  hypothesis  of  putative  species  grouping  (Fujisawa  &  Barraclough,  2013).  It  explores  the  branch  length  pattern  on  the  given  tree  in  order  to  test  species  boundaries   (Pons   et   al.,   2006).   Between-­‐species   and   within-­‐species   branch   lengths  are   related   to   different   processes:   the   former   are   determined   by   the   rates   of  speciation  and  extinction  (Nee  et  al.,  1994),  while  the  latter  by  coalescence  processes  (Hudson,   1991).   The  method   performs   a  ML   optimization   to   detect   the   shift   in   the  dynamics   of   branching   and   defines   the   transition   between   inter-­‐   and   intra-­‐specific  processes,   establishing   a   threshold   time  T   for   this   shift.   This  way   it   tests   the  model  that  the  samples  in  the  studied  clade  have  diversified  into  separate  species  versus  the  null  model  that  all   individuals  belong  to  a  single  species.  The  simplest  approach,  the  single-­‐threshold   (Pons   et   al.,   2006),   assumes   a   single   T,   placed   before   the   oldest  within-­‐species  coalescent  event   in  the  tree.  However,  as   in   large  datasets  with  many  lineages  there  may  be  multiple  divergence  levels,  Monaghan  et  al.  (2009)  developed  a  multiple-­‐threshold  method  where   the   speciation-­‐coalescent   transition   is   allowed   to  vary  across  the  tree.   Importantly,  with  the  reformulation  of  T   from  a  parameter  to  a  constraint   of   search   space,   the   two   versions   no   longer   differ   in   the   number   of  parameters   they   contain   and   thus   cannot   be   compared   by   likelihood   ratio   tests  (Fujisawa  &  Barraclough,  2013).  A  recent  evaluation  of  the  performance  of  the  GMYC  method  with   empirical   data,   showed   that   results   were   consistent   when   testing   the  effect  of  different  phylogenetic  reconstruction  methods,  high  singleton  presence  and  taxon   richness,   but   it   may   be   prone   to   oversplitting   when   sampling   is   unbalanced  across  the  geographical  range  (Talavera  et  al.,  2013).  Thus,  in  certain  systems,  it  can  represent  a  first  approach  to  roughly  assess  biodiversity  when  previous  taxonomical  knowledge  is  lacking,  and  as  such  it  is  employed  in  this  dissertation.  The   Bayesian   implementation   of   the   GMYC   method   (bGMYC)   was   proposed   by  Reid  &  Carstens  (2012)  in  order  to  mitigate  sources  of  error  related  to  uncertainty  in  model   parameters   (transition   from   speciation   to   coalescent   events)   and   in  phylogenetic   reconstruction.   Their  method   accounts   for   these   issues   by   integrating  over  uncertainty  in  topology  from  a  sample  of  multiple  trees  as  opposed  to  the  use  of  the   maximum   clade   credibility   tree   solely.   Here,   it   is   used   along   with   the   ML  approaches   of   the   method,   in   order   to   comparatively   explore   the   results   of   the  versions  of  the  GMYC  method  in  the  studied  system.  The   program   Bayesian   Phylogenetics   and   Phylogeography   (BPP)   (Yang   &  Rannala,  2010),  like  other  methods  that  implement  the  multispecies  coalescent  model  and   different   to   single-­‐locus-­‐based   methods,   can   track   the   genealogical   history   of  multiple-­‐species  samples  back  to  a  common  ancestor  modeling  gene  conflicts  due  to  lineage   sorting   (Yang   &   Rannala,   2010;   Fujita   et   al.,   2012).   BPP   calculates   the  posterior  probabilities  of  potential  species  delimitation  models/hypotheses  with   the  assumption   that  migration   ceases   as   soon   as   species   separate.   To   do   so,   it   requires  sequence  data   from  multiple   loci   and   a   fully   resolved   guide  phylogeny   (guide   tree).  Regarding   the   number   of   loci   and   individuals,   simulation   tests   showed   that   Introduction 12 performance  of  the  method  greatly   improves  increasing  the  dataset   from  one  to  five  loci   but   is   relatively   constant   further   on,   and,   most   importantly,   the   inclusion   of  multiple  sequences  (of  each  loci)  for  each  species  (more  than  one  or  two,  ideally  five  or  more)  is  an  important  factor  contributing  to  the  correct  delimitation  (Zhang  et  al.,  2011).   Moreover,   the   same   study   concluded   that   low   levels   of   gene   flow   among  species,  despite  the  assumption  of  the  method,  do  not  affect   its  performance.  On  the  other  hand,  the  role  of  the  guide  tree  on  the  inference  of  species  boundaries  is  crucial,  with  the  use  of  an  incorrect  topology  generally  resulting  in  overestimation  of  putative  species,   due   to   an   artificially   large  divergence   among  descendent   species   (Leaché  &  Fujita,   2010).   Also,   the   prior   distribution   of   parameters   such   as   the   ancestral  population  size  (θ)  and  the  root  age  (τ)  (Fig.  1)  can  have  an  impact  on  the  delimitation  process.   The   cautious   way   to   proceed,   thus,   would   be   to   test   different   prior  combinations,   to   provide   a   guide   tree   with   the   most   subdivided   possible   model   of  delimitation,  since  clusters  (species)  on  the  guide  tree  cannot  be  split  but  only  lumped  together  with  others,  and  to  analyze  multiple  distinct  guide  trees  based  on  alternative  reconstructions,  to  account  for  errors  in  the  assignment  of  individuals  in  clusters  and  topology  (Yang  &  Rannala,  2010;  Leaché  &  Fujita,  2010).  Despite  these  concerns,  BPP  has   been   shown   to   provide   very   accurate   results   compared  with   other  methods   in  empirical   studies   (Camargo   et  al.,  2012)  and  is  currently  one  of  the  most  widely  used  methods   (Fujita   et   al.,  2012),   also   employed   in  numerous   studies   on  reptile  systems  (e.g.  Leaché  &   Fujita,   2010;   Camargo   et   al.,  2012;  Gómez-­‐Díaz  et  al.,  2012;   Sistrom   et   al.,   2013;  Welton   et   al.,   2013;   Barley   et  al.,  2013,  Ahmadzadeh  et   al.,  2013).       Figure 1. A two-species model in a BPP analyses may have up to four parameters: the divergence time τ0 and three θ parameters for the three populations (adapted from Zhang et al., 2011) 13 Introduction The  system  of  Zoological  Nomenclature  The  ultimate  goal  of  the  taxonomic  procedure  is  to  identify  the  taxonomic  units  (taxa)  under   study  and,   if   pertinent,   provide  a  description  and  a  correct   name   for   a  newly  introduced  taxon.  There  is  no  set  of  rules  that  conditions  the  definition  or  ranking  of  a  taxon,  which  is  rather  left  upon  the  judgment  of  taxonomists,  but  the  designation  and  publication   of   scientific   names   (nomina)   is   regulated   by   a   series   of   nomenclatural  rules.  These  are  different  for  different  groups  of  organisms:  animals;  algae,  fungi  and  plants;   cultivated   plants;   prokaryotes;   and   viruses   (Dubois   et   al.,   2013).   In   this  dissertation,  I  will  essentially  refer  to  the  rules  established  for  the  first  group,  by  the  International   Commission   on   Zoological   Nomenclature   (ICZN),   and   when   using   the  word   taxon   I   will   for   the   most   part   to   refer   to   the   rank   of   species   (see   Box   1   for  terminology).    According   to   the   Introduction   of   the   current   fourth   edition   of   the   International   Code  of  Zoological  Nomenclature  (ICZN  1999,  the  Code  hereafter)  its  aim  is  “to  provide   the  maximum  universality  and  continuity  in  the  scientific  names  of  animals  compatible   with  the  freedom  of  scientists  to  classify  animals  according  to  taxonomic  judgments”.  At  this  end,  the  Code  provides  a  series  of  Articles  that  are  to  be  followed  for  determining  a  valid  name,  as  well  as  several  Recommendations.  Among  the  former,  is  the  need  to           Introduction 14 Figure 2. The type specimen constitutes the link between the name and the actual organism (adapted from Pyle & Michel, 2008)   provide  for  a  new  name  “a  description  or  definition  that  states  in  words  characters  that   are  purported   to  differentiate   the   taxon”   (Article  13.1.1).  However,   it   is  only   through  the   type   specimen   that   the   link   to   the   actual   organisms   is   established   (Fig.   2),  providing   an   objective   basis   for   identification   (Pyle   &   Michel,   2008).   In   case   a  taxonomist  encounters  an  irregularity  in  the  application  of  the  rules  of  nomenclature,  stemming   from   the   ignorance,   misinterpretation   or   often,   as   in   cases   of   nomina  established  centuries  ago,  non-­‐existence  of   the   rules  at   the   time,   the   inconsistencies  can  be  solved  by  direct  application  of  the  Code.  It  is  only  in  individual  nomenclatural  cases  of  uncertainty  that  the  ICZN  can  be  asked  to  rule  on  an  acceptable  solution.  Since  the  first  edition  of  the  Code   in   1961,   many   changes  have   been   made   to   meet   with  the   needs   of   an   evolving  scientific   field,   including   its  recent   amendment   (ICZN,  2012)  that  allows  for  electronic  publication   under   certain  conditions,   and   new   online   resources  are  now  available  to  taxonomists.  ZooBank,  the  official  register  of  the  ICZN,  is  an  open-­‐access  database  that  compiles   relevant   information   on   scientific   names   in   Zoology,   including   published  works,   authors   and   nomenclatural   acts   (Pyle   &   Michel,   2008).   Information   is  registered  either  via   taxonomic   journals,   like  Zootaxa  and  Zookeys,   or  by   individual  users,  permitting  thus  a  direct  contribution  of  the  broader  community  (Rosenberg  et   al.,  2012).  New  features  like  the  reconciliation  of  ZooBank  entries  with  titles  from  the  ever-­‐growing  database  of  biodiversity   literature  Biodiversity  Heritage  Library   (BHL,  2007)  are  expected  to  improve  the  utility  of  such  resources  (ZooBank  Progress  Report  Q3  2013).  These  new  tools  are  just  a  fraction  of  the  initiatives  available  nowadays  to  the   taxonomic   community   that   contribute   to   a   faster,   more   rigorous   and   more  “democratic”  process  of  the  description  and  dissemination  of  biodiversity.   15 Introduction 1.2.    THE  ARID  AREAS  OF  NORTH  AFRICA  AND  ARABIA  North  Africa  and  Arabia  comprise  an  area  of  more  than  13,000,000  square  kilometers  where  arid  conditions  prevail  and  deserts  have  a  dominant  presence.  Their  geological  and   climatic   history   is   rich   and   complex,   but   not   fully   understood,   and   it   has  essentially   conditioned   the  evolution  of   their  biota.  Although  arid   environments   are  often   perceived   as   monotonous,   North   Africa   and   Arabia   host   a   great   diversity   of  organisms  adapted  to  their  challenging  conditions.     1.2.1.  Characteristics  and  brief  geological  and  climatic  history  of  North  Africa   and  Arabia   North  Africa  is  the  historically  and  ecologically  distinct  part  of  the  African  continent  delimited   by   the   Mediterranean   Sea   in   the   north   and   the   biogeographic   transition  zone  of   the  Sahel   in   the  south  (Fig.  3).  The  Sahara,   the  world’s   largest  warm  desert,  occupies  its  greatest  part  (more  than  9  million  km2)  and  presents  a  high  diversity  of  topographic  features  and  heterogeneous  climate  with  strong  oscillations  (Brito  et  al.,  2014).   Scattered   inside   the   Sahara,   from   west   to   east,   are   found   the   mountainous  Tagant   plateau   in  Mauritania   and   the  mountain   ranges   of   the  Hoggar   and  Tassili   in  Algeria,   Aïr   in  Niger   and  Tibesti   in   Chad,   the   latter   reaching   an   altitude   of   3,445  m  (Geniez   et   al.,   2011).   Northwest   of   the   Sahara,   the  Mediterranean   ecoregion   of   the  Maghreb,   north   of   the   Atlas  Mountains,   constitutes   a   very   diverse   area   in   terms   of  elevation,   landcover   and   temperature.   The   Arabian   Peninsula   lies   in   the   east   of  North  Africa,  connected  with  it  through  the  small  Sinai  Peninsula  and  separated  from       Figure 3. Map of the study area with most relevant toponyms (Photo source: UNEP (2013). Arab Region: Atlas of Our Changing Environment. Edited) Introduction 16 it  by  the  narrow  Red  Sea.  It  belongs  to  Western  Asia,  although  geologically  it  has  been  part   of   the   Asian   continent  only   since   the   Lower  Miocene.   Rub  Al   Khali,   the   largest  sand  desert   in   the  world  with  dunes  of  up   to  250  m,   is  part  of   the  greatest  Arabian  desert   that   covers   most   of   Arabia,   its   southern   edge   reaching   the   Hadhramaut  Mountains   and   the   Dhofar   region   (Mares,   1999;   Vincent,   2008).   Along   the   Red   Sea  coast  the  mountains  become  progressively  lower,  from  the  highest  Arabian  peak,  Jabal  an-­‐Nabi  Shu'aib,   in   the  south  (3,666  m),   to   the  Asir  Mountains   in   the  center  and  the  Hejaz   in   the  north,  while   at   the   easternmost   tip  of   the  Arabian  Peninsula,   the  Hajar  Mountains  remain  isolated  between  the  sea  and  the  desert.  The  geological  history  of  these   two  major   areas   is   studied   through   the   tectonics   of   the   African   and   Arabian  Plates  (Fig.  4),  and  their  present-­‐day  aridity  is  related  to  a  climatic  history  marked  by  global-­‐scale  phenomena  that  occurred  mostly  during  the  Miocene  and  Pliocene.  The  most  relevant  geologic  episodes  for  the  processes  described  herein  started  in  the   Early   Oligocene   (~31   Ma),   when   the   African   Plate,   that   had   been   part   of   the  Pangea   (~300  Ma)   and   split   from   the   South   American   Plate   (~105  Ma),  was   found  roughly  in  its  present  position  forming  together  with  the  Arabian  Plate  a  continuous  land  surface.  The  narrowing  Western  Tethys  Ocean  lay  in  the  north,  separating  them  from  the  landmasses  that  were  the  precursors  of  Eurasia  (Rögl,  1998).  It  was  at  that  time   that   a   series   of   strong   tectonic   and  magmatic   activities   combined  with   intense  changes  in  the  global  climate  dominated  the  geological  evolution  of  the  area  (Guiraud   et  al.,  2005).  A  process  of  rifting  initiated  in  the  Gulf  of  Aden,  and  gradually  extended  to   the   Afar   region   and   southern   part   of   the   Red   Sea   (~27   Ma).   This   phenomenon  propagated   rapidly   across   the   entire   Red   Sea,   resulting   in   the   separation   and  northwards  drift  of   the  Arabian  Plate  (~24Ma),  although  posterior   land  connections  of   unknown   extent   were   prompted   by   sea   level   oscillations   during   the   Miocene  (Bosworth   et   al.,   2005).   At   about   the   same   period,   uplift   of   the   Red   Sea   shoulders  occurred  along  the  basin,  giving  rise  to  the  mountain  ranges  on  both  the  African  and  Arabian   sides.   A   subsequent   crucial   episode  was   that   of   the   formation   of   the   Afro-­‐Arabian   –   Eurasian   land   bridge,   the   Gomphotherium   bridge.   As   a   result   of   the  counterclockwise   rotation   of   the   Arabian   plate,   its   collision   with   Eurasia   was  produced  along  the  Zagros-­‐Bitlis  zone  and  a  connection  was  established  progressively  (~18-­‐15   Ma)   (Bosworth   et   al.,   2005;   Harzhauzer   et   al.,   2007).   These   events   had  significant   biogeographic   implications   for   fauna   and   flora,   with   various   exchanges  between   the   three   landmasses,   Eurasia,   Arabia   and  Africa   (Harzhauzer   et   al.,   2007;  Carranza  et  al.,  2008;  Pook  et  al.,  2009;  Thiv  et  al.,  2010;  Zhou  et  al.,  2012).   17 Introduction     Geological  evidence  for  climate  evolution  in  North  Africa  and  Arabia  related  to  the  onset   of   arid   conditions   can   be   gained   through   the   trends   observed   in   their  stratigraphic   record.   However,   this   record   is   relatively   sparse,   given   the   enormous  area,  and  its  temporal  resolution  is  generally  poor  (Swezey,  2009).  As  a  result,  major  considerations   regarding   interpretations   from   the   geological   sources   are   whether  lithologic   similarities   are   correlated   and   can   be   used   to   extrapolate   inferences   to   a  broader   area,   and  whether   climatic   conditions   persisted   ever   since   (Swezey,   2006;  Kröpelin,   2006).   Evidence   for   the   establishment   of   arid   conditions   during   the   Late  Miocene  are  found  in  northeastern  Africa  and  the  Mediterranean  basin  (11.5-­‐5.5  Ma)  (Griffin  et  al.,  2002),  in  Chad  in  central  Sahara  (~7  Ma)  (Schuster  et  al.,  2006)  and  in  the  Arabian  Peninsula  (10-­‐5.5  Ma)  (Huang  et  al.,  2007),  and  during  the  Early  Pliocene  in   southwestern   Sahara   (starting   at   4.6   Ma)   (Tiedemann   et   al.,   1989;   Le   Houérou,  1997)   and   largely   in   North   Africa   (Swezey,   2009).   Nevertheless,   there   are   also  lithologic   and   palynologic   indicators   that   humid   conditions   prevailed   in   parts   of  present-­‐day   Sahara   desert   during   the   Miocene-­‐Pliocene   transition   in   Mauritania  (Swezey,   2009),   and   as   late   as   the   Holocene   near   the   Tibesti   Mountains   (~6,000  years)   (Kröpelin,   2008).   This   climate   variability   was   the   result   of   global-­‐scale  phenomena  such  as  growth  of  the  East  Antarctic  ice  sheet,  polar  cooling,  formation  of  monsoons   and   high   pressure   belts   (Flower   &   Kennett,   1994;   Griffin   et   al.,   2002).  Importantly,  additional  evidence  for  aridification  during  the  Mid  to  Late  Miocene  can  be  obtained  from  biological  evidence,  mainly  from  hypothesized  vicariance  processes  (Douady  et  al.,  2003;  Patiny  &  Michez,  2007;  Backlund  et  al.,  2007;  Thiv  et  al.,  2010),  but  also  from  dispersal  of  well-­‐adapted  desert-­‐dwelling  groups  (Carranza  et  al.,  2008;  Lourenço  &  Duhem,  2009).   Figure 4. Map of the study area showing the limits of the tectonic plates (Map source: UNEP (2013). Arab Region: Atlas of Our Changing Environment. Edited) Introduction 18 1.2.2.  Arid  zones  and  their  biodiversity  Aridity,   the   deficit   of   moisture   in   the   environment,   is   a   complex   phenomenon   that  induces  challenging  conditions  for   living  organisms,  since  moisture   is  a   fundamental  requirement   for   many   (Thomas,   2011).   There   exists   a   great   diversity   in   arid   zone  morphology,   and  major   types   of   deserts   can   be   recognized  mainly   according   to   the  prevailing  substrate.  Some  of  the  most  common  are  sandy  deserts,  salt  flats  (sabkhas),  which   are   probably   one   the   harshest   environments   (König,   2012),   stony   and   rocky  deserts  that  cover  a  great  part  of  North  Africa  and  Arabia  (Edgell,  2006).  Despite  their  hard  conditions,  the  idea  of  arid  zones  being  bare  and  almost  devoid  of  flora  and  fauna  is  being  radically  changed  through  recent  studies   that  are  revealing  higher  diversity  and  endemism  and  interesting  biogeographic  and  diversification  patterns  (Brito  et  al.,  2014).  The  species  encountered  may  either  be  well-­‐adapted  to  the  arid  conditions,  or  relict   taxa   originating   in  more   humid   periods   and   nowadays   restricted   to  montane  refugia.    Among   plants,   some   species   present   adaptations   in   special  water-­‐storage   organs  and   deep   root   systems   that   allow   them   to   endure   long   periods   of   drought,   while  others   are   very   ephemeral,   growing   exclusively   and   rapidly   during   periods   of   less  intense  drought  (Ward,  2009).  The  flora  of   the  Sahara   includes  approximately  2,800  species  of  vascular  plants,  a  fourth  of  which  are  endemic,  and  half  of  which  are  shared  with   the   Arabian   Peninsula   (Le   Houérou,   2009).   Animals   may   follow   a   variety   of  strategies  to  deal  with  the  temperature  extremes:  evade  it  mainly  through  diel  activity  patterns,  tolerate  it,  as  seen  mostly  in  large  organisms,  or  employ  evaporative  cooling  (Willmer  et  al.,  2000).  Distributional  data  available  for  animal  taxa  in  the  Sahara  are  quite  scarce   in  relation  to  neighboring  areas,  even  in  commonly  well-­‐studied  groups  such  as  vertebrates  (Brito  et  al.,  2014),  but  reptiles  are  among  the  ones  that  have  seen  a  greater  increase  in  published  literature  in  the  last  years  (e.g.  Arnold,  1986;  Schleich   et  al.,   1996;  Bons  &  Geniez,   1996;  Geniez  et  al.,   2004;   Sindaco  &   Jeremčenko,  2008;  Sindaco  et  al.,  2013).    Despite   their   large   extension   and   the   practical   difficulties   that   limit   their  accessibility,   the   ecosystems   of   arid   areas   are   also   subject   to   human-­‐provoked  disturbances,   aggravated   by   their   inherent   sensitivity.   The   greatest   threat   to   arid  environments   is   overgrazing   that   devastates   the   sparsely   distributed   shrub   plants  that  constitute  small  centers  of  species  concentrations  (Ward,  2009;  Cox  et  al.,  2012).  Additional   threats   include   groundwater   depletion,   being   drained   for   irrigation   of  cultivated   fields   in   arid  areas,   causing  a  decline   in   the   communities  of  native  plants  and,   also,   rapid   construction   and   industrialization   occupying   unique   arid   habitats,  especially  coastal  sand  dunes  and  sabhkas  (Box  2)  (Edgell,  2006).   19 Introduction  Introduction 20 1.3.    THE  REPTILE  STUDY  GROUPS  Reptiles   are   a   major   component   of   the   global   biodiversity,   remarkable   from   an  ecological   and   evolutionary   point   of   view   (Pincheira-­‐Donoso   et   al.,   2013).   They  constitute   very   good  models   for   the   study  of   evolutionary  processes   and  have  been  used  as  such  for  many  decades,  hosting  one  of  the  best  studied  groups  in  biology,  the  iguanian   lizards  Anolis   (Losos,   2009).   In   this   dissertation,   the   selection   of   the   study  groups  Stenodactylus   Fitzinger,   1826  and  Ptyodactylus   Goldfuss,   1820  was  based  on  the   excellent   match   between   their   (known)   distribution   (Fig.   5)   and   the   area   of  interest,   and   on   the   potential   they   offer   for   studying   several   questions   related   to  phylogeny,   taxonomy,   species   delimitation   and   historical   biogeography.   Note   that  throughout   this  work   the   term  “reptiles”   refers  essentially   to   the  non-­‐avian  Reptilia  (Gauthier  et  al.,  1988).     1.3.1.  The  use  of  reptiles  as  models  in  the  study  of  systematics,  biogeography   and  evolution  Reptiles  are  distributed  across  all  continents  (except  Antarctica)  and,  with  more  than  100  species  described  during  2013,  the  number  of  known  species  is  soon  expected  to  reach   10.000   (source:   www.reptile-­‐database.org,   Uetz   &   Hošek).   Knowledge   on   the  biology   of   reptiles   and,   during   the   last   decades,   also   on   their   phylogenetic   relationships   has   been   accumulating   rapidly   through   a   series   of   fine-­‐scale   studies  and   revisionary   works   (e.g.   Pianka   &   Vitt,   2003;   Camargo   et   al.,   2010;   Aldridge   &  Sever,   2011;   Sites   et   al.,   2011;   Pyron   et   al.,   2013).   The   redefinition   of   Reptilia   as   a  monophyletic  group  with  the  inclusion  of  birds  and  the  exclusion  of  synapsid  groups  represented   an   important   philosophical   and  methodological   example   in   systematics  toward   a   phylogenetic-­‐driven   classification   (Lee   et   al.,   2004).   Furthermore,   the  reconstruction  of  robust  molecular  phylogenies  has   led   to   the  revision  of   traditional  morphology-­‐based  taxonomies  and,  together  with  them,  of  hypotheses  regarding  the  evolution   of   interesting   traits   such   as   parthenogenesis,   viviparity   and   venom   in  reptiles  (Sites  et  al.,  2011).  Nevertheless,  these  studies  present  a  certain  bias  toward  the  families  Lacertidae  and  Phrynosomatidae,  dominant  in  Europe  and  North  America,   Figure 5. Distributions of the genera Stenodactylus (a) and Ptyodactylus (b). (Illustrations from Sindaco & Jeremčenko, 2008. Edited) 21 Introduction and   an   under-­‐representation   of   the   species-­‐rich   Scincidae   and   Gekkota,   distributed  mostly   outside   these   areas   (Camargo   et   al.,   2010).   There   is   therefore   a   need   for  attention  to  be  drawn  on  the  reptile  fauna  from  less  explored  areas  that  will  improve  the   systematic   knowledge   on   it   and   contribute   to   a   better   coverage   of   the   species  diversity  in  large-­‐scale  studies.  Unlike   other   lizard   groups,   geckos   and   skinks   have   dispersed  widely   (Pianka   &  Vitt,   2003),   also   via   trans-­‐oceanic   colonization   (Carranza   et   al.,   2000;   Carranza   &  Arnold,   2003;  Gamble  et   al.,   2011;  Miralles  &  Carranza,   2010;   Sindaco  et   al.,   2012),  therefore  offering  ample  possibilities  for  studying  biogeography  at  many  levels.  The  gecko  family  Phyllodactylidae  was  established  very  recently  based  on  the  results  of  a  multi-­‐locus  phylogenetic  study  and  comprises  genera  that  were  previously  unknown  to   group   together,   presenting   a   trans-­‐Atlantic   distribution   (Gamble   et   al.,   2008).   A  later   study   including   several   Gekkota   clades   showed   that   New   World   geckos   of  different   families   have   multiple   independent   origins,   such   as   vicariance,   over-­‐seas  dispersal  and  human-­‐mediated  transport,  and  also  that  older  lineages  possess  higher  number   of   species   than   younger   ones   (Gamble   et   al.,   2011).   This   kind   of   studies  contributes   greatly   to   our   understanding   of   the   diversification   and   assembly   of   the  faunas.  Reptiles   successfully   occupy   a   huge   variety   of   habitats   worldwide   (Pincheira-­‐Donoso  et  al.,  2013)  and,  being  ectotherms,  they  are  greatly  affected  by  the  thermal   landscape  of  their  habitat  (Pianka  &  Vitt,  2003;  Shine  et  al.,  2005).  The  relevance  of  such   a   relationship   is   stressed   especially   in   cases   of   extreme-­‐temperature   arid  environments  (Pianka,  1969).  Some  desert  lizards  are  known  to  perform  at  least  some  kind  of  body  temperature  regulation  through  behavioral  adjustments,  like  basking  or  seeking  shade  (Cowles  &  Bogert,  1944;  Kearney  &  Predavec,  2000;  Labra  et  al.,  2001).  This  adds   to   the   importance  of   the  characteristics  of   their  environment,   such  as   the  substrate   and   (scarce)   vegetation   (see   above   in  1.2.2)   (Kearney  et   al.,   2009).  At   the  same   time,   there   is   also   a   phylogenetic   component   of   lizard   body   temperature   as  certain   groups   have   higher   or   lower   body   temperatures   compared   to   others,  regardless  of  where  their  members  occur  (Brattstrom,  1965;  Huey  &  Bennett,  1987).  Additionally,   it   has   been   shown   that   the   variation   in   substrate   structure   is   an  important  determinant  of  locomotor  performance  in  lizards  (Tulli  et  al.,  2012),  related  to   morphological   diversity   and   selection   on   specific   morphological   characters  (Goodman  et  al.,  2008;  Higham  &  Russel,  2010).  Temperature-­‐  and  substrate-­‐related  adaptations   are   only   few   of   the   many   aspects   of   the   biology   of   reptiles   that   offer  excellent  opportunities  to  study  evolutionary  questions.       Introduction 22 1.3.2.  The  genus  Stenodactylus  from  the  deserts  of  North  Africa  and  Arabia  The   Stenodactylus   [from   the   Greek   words   στενός  (stenos)   =   narrow   and   δάκτυλον   (daktylon)   =  finger]   are   essentially   nocturnal,   ground-­‐dwelling  geckos  of  the  family  Gekkonidae  (Box  3),  of  medium  size  (up  to  83mm  snout-­‐to-­‐vent  length  in  S.  doriae)  and   lacking   adhesive   toe-­‐pads   (Fig.   6).   They   are  widely   distributed   across   the   deserts   of   North  Africa   and   Arabia   (Arnold,   1980a;   Sindaco   &  Jeremčenko,  2008)  (Fig.  5).  According   to   the   latest  accounts,  up  to  four  species  reach  areas  of  western  Iran   (Fathinia   et   al.,   2014),   but   they   are   not   known   to   occur   further   east   in   Asia.   Stenodactylus  geckos  are  very  interesting  in  that  they  occupy  almost  all  types  of  desert  grounds,  with  different  substrates,  such  as  gravel  plains,  hard  sand,  soft  sand,  sabkhas,  having  their  characteristic  species.   23 Introduction Taxonomic  and  phylogenetic  background  on  Stenodactylus  At  the  beginning  of  this  dissertation,  at   the  end  of  2009,  no  more  than  twenty  modern-­‐time   (post-­‐1970)   published   articles   on  (species   of)   Stenodactylus   could   be  retrieved,   with   the   great   majority   of   them  on   small-­‐scale   ecological   or   behavioral  studies  and  new  distributional  records  (e.g.  Bouskila   et   al.,   1992;   Al-­‐Sirhan,   2009).  However,   a   single   comprehensive   and  thorough   work   by   Arnold   (1980a)  reviewed   the   taxonomical   knowledge   on  the   ten   species   of   Stenodactylus   known   at  the   time,   offered   morphological  descriptions   and   accounts   of   the   variation  of   all   species,   gathered   information   on   the  distributional   ranges   and   described   one  latest  addition  to  the  members  of  the  genus.  Importantly,   Arnold   also   provided   a  “provisional”   phylogeny   based   on  morphology   (Fig.   7),   although   he   noted  presence   of   homoplasy.   In   2011,   Fujita   &  Papenfuss   published   the   first   molecular  phylogenetic   study   on   Stenodactylus,   with  representatives  from  half  of  the  recognized  species.  This  study  provided  a  first  insight  on   the   genetic   variability   within   Stenodactylus,   but   the   omission   of   numerous  members   of   the   group   precluded   the   acquisition   of   robust   results   regarding   the  phylogenetic   relationships   between   the   Stenodactylus   members   or   hypotheses   over  their  biogeography.  In  relation  to  the  position  of  Stenodactylus  among  other  Gekkota,  Arnold  (1980a)  regarded   it   “undetermined”   at   the   time   based   on   morphology.   The   first   molecular  phylogenetic   study   including   a   Stenodactylus   representative   (Feng   et   al.,   2007)  indicated   its   close   relationship   with   other   Old   World   genera,   like   Tropiocolotes,   Agamura  and  Crossobamon,  and  rejected  its  sister-­‐relationship  with  Teratoscincus,  as  an   older  morphological   revision   suggested   (Kluge,   1967).   Two  more   recent   studies  restricted   the   closest   groups   to   Tropiocolotes   and  Mediodactylus,   the   former   found  across   North   Africa,   Arabia   and   one   species   in   southern   Iran,   while   the   latter  distributed   from   the   southeastern   Mediterranean   to   Pakistan   (Gamble   et   al.,   2012;  Bauer  et  al.,  2013).  The  cluster  that  they  form  is  sister  to  a  group  comprising  Agamura,   Bunopus,   Crossobamon,   Cyrtopodion   and   Tenuidactylus.   Interestingly,   all   the   above  genera  share  a  most  recent  common  toe-­‐pad-­‐less  ancestor,  a  not  so  usual  state  among  the  Gekkonidae  (Gamble  et  al.,  2012).     Figure 7. Morphology-based phylogeny of Stenodactylus in Arnold (1980) (redrawn)   Introduction 24 1.3.3.  The  rock-­‐dwelling  genus  Ptyodactylus  from  North  Africa,  the  Sahel  and   Arabia  The  etymology  of  the  generic  name  of  these  geckos  of  the  family  Phyllodactylidae  (Box  3)   makes   reference   to   their   toe   morphology   that   permits   their   unequivocal  identification:  πτύον   (ptyon)  =  winnowing-­‐shovel  and  δάκτυλον   (daktylon)  =   finger.  Their  digits   are   clawed  and  dilated  with   two  diverging   series  of   lamellae   terminally  (Schleich   et   al.,   1996)   (Fig.   8).   The   Ptyodactylus   geckos   comprise   seven   widely  accepted   species   and  are  distributed  across  Arabia,  North  Africa   and  also   the  Sahel,  where  knowledge  on   their   occurrence   is   sparser,  while   an   enigmatic   representative  from  Pakistan  is  know  from  very  few  localities  (Fig.  5)  (Sindaco  &  Jeremčenko,  2008).  They   live   on   all   kinds   of   rocky   surfaces   such   as   rocks   of   different   sizes,   cliffs,  boulders,   human-­‐made   constructions   and   in   caves   (Werner   &   Sivan,   1994;   Arnold,  1980b).  It  is  important  to  note  that  all  Ptyodactylus  species  share  the  same  structural  habitat   preferences   and  none   of   them   is   adapted   to   any   different   kind   of   substrate.  Moreover,   and   probably   related   to   this   ecological   feature,   morphological   similarity  between   the   different   species   is   conspicuous   and   has   greatly   hampered   taxonomic  work   in   the   group   (Baha   El   Din,   2006).   Ptyodactylus   geckos   are   mostly   nocturnal       25 Introduction (except  P.  puiseuxi  Boutan,  1893,  which  is  diurnal)  but  occasionally  with  some  diurnal  activity  and/or  observed  basking  (Werner  &  Sivan,  1994;  Schleich  et  al.,  1996).  One  interesting   feature   of   these   geckos   is   their   ability   to   produce   loud   click   calls,  possessing   a   wide   vocal   repertoire   (Werner   et   al.,   1978).   Although   the   intra-­‐taxon  variation   observed   could   indicate   presence   of   multiple   species,   the   lack   of   direct  evidence   on   the   role   of   these   calls   in   pair   formation   and   reproductive   isolation  renders  doubtful,  for  the  time,  the  use  of  this  character  in  taxonomy  (Werner  &  Sivan,  1994).   Taxonomic  and  phylogenetic  background  on  Ptyodactylus  As   mentioned   above,   taxonomic   work   on   Ptyodactylus   has   been   marked   by   the  difficulty  of   finding  constant  differences  among  populations  that  appear  very  similar  in   their   general   morphology.   After   several   works   that   followed   a   conservative  approach  towards  the  taxonomy  of  Ptyodactylus,  recognizing  either  one  or  two  species  with   some   “variations”   (Anderson,   1898;   Loveridge,   1947;   Kluge,   1967),   Heimes  (1987)   was   the   first   to   perform   a   comprehensive   study   using   morphological   and  electrophoresis  data,  and  he  recognized  six  taxa.  Werner  &  Sivan  (1993,  1994)  carried  out  a  thorough  work  on  morphological  and  ecological  variability  in  Ptyodactylus,  but  they   only   focused   on   the   three   species   occurring   in   the   Levant:   P.   hasselquistii  (Donndorff,   1798),   P.   guttatus   Heyden,   1827   and   P.   puiseuxi   Boutan,   1893.   Arnold  (1986)   reviewed  specimens   from   the  Arabian  Peninsula,   assigned   to  P.  hasselquistii,  and  observed  some  geographical  variation,  but  refrained  from  formally  describing   it  given   the   lack   of   sufficient   material   from   the   entire   species   range.   Ptyodactylus   siphonorhina  was  elevated  to  the  species  level  on  the  basis  of  small  morphological  and  ecological  differences  with  respect  to  P.  guttatus,  its  sister  species  (Baha  El  Din,  2006).  More   recently,   molecular   data   have   been   used   in   two   different   studies   to   assess  intraspecific   variability   in  P.   oudrii   from  Morocco   and  P.   ragazzii   from   the  western  part  of  its  distribution  along  the  Sahel,  respectively  (Perera  &  Harris,  2010;  Froufe  et   al.,   2013).  Both  works  detected  high   levels  of   genetic  variability  and  concluded   that  undescribed   diversity   is   present   in   these   groups.   Nevertheless,   despite   their   long  taxonomic  history  and  their  abundance  across  North  Africa  and  Arabia,  no  complete  phylogeny   of   Ptyodactylus   geckos,   either   morphological   or   molecular,   has   been  published  so  far.     Introduction 26                         CHAPTER  2    Objectives  and  Structure       2.1.    OBJECTIVES  The  general  aim  of  this  dissertation  is  to  investigate  the  evolution  of  the  biota  of  the  arid   areas   of   North   Africa   and   Arabia,   through   the   study   of   the   systematics   and  biogeography   of   two   representative   reptile   groups,   Stenodactylus   and   Ptyodactylus.  The  two  genera  are  distributed  across  a  large  part  of  the  study  area  occupying  most  of  the   different   habitats   and   their   study   poses   interesting   questions   regarding   their  diversity   patterns,   the   factors   that   contributed   to   their   diversification   and   their  taxonomy.  In  order  to  accomplish  this  general  aim,  several  specific  objectives  were  established:  I. Obtain   a   geographically   comprehensive   taxonomic   sampling   of   geckos   of  the   genera   Stenodactylus   and   Ptyodactylus,   with   a   special   focus   on  widespread  species  and  species  known  to  present  genetic  or  morphological  variability.  II. Generate  multilocus  molecular   data   from  both  mitochondrial   and   nuclear  markers   and   use   them   1)   to   investigate   the   inter-­‐   and   intra-­‐specific  patterns   of   genetic   diversity,   2)   to   reconstruct   the   phylogenetic  relationships   among   the   species   of   the   study   groups   and   3)   to   infer   the  temporal  framework  of  the  divergences  among  species.  III. Explore   the   biogeographic   patterns   across   the   arid   areas   of   North   Africa  and   Arabia,   using   information   from   the   time-­‐calibrated   phylogenies   of   Stenodactylus  and  Ptyodactylus,  the  ecological  features  of  the  organisms  and  the  geological  and  climatic  history  of  the  areas.  IV. Revise   the   taxonomy   and   nomenclature   of   Stenodactylus   geckos   using  molecular   and  morphological   data,   reviewing   the   available   literature   and  applying   the   International   Code   of   Zoological   Nomenclature   where  pertinent.  V. Provide  the  essential   framework  for  a  systematic  assessment  of  the  genus   Ptyodactylus   and   unravel   the   species   limits   in   the   morphologically  conserved   Ptyodactylus   hasselquistii   species   complex,   employing  multilocus,  coalescence-­‐based  species  delimitation  methods.       29 Objectives 2.2.    STRUCTURE  OF  THE  RESULTS  The  results  of   this  dissertation  are  presented   in   five  research  papers  that  have  been  organized   in   three   Chapters   (3,   4   and   5).   These   chapters,   individually   or   in  combination   with   others,   answer   the   questions   addressed   and   achieve   the   goals  proposed  above.  Specifically:  In  Chapter  3,  a  complete  taxonomic  sampling  covering  a  great  part  of  the  distribution  of  the  genus  Stenodactylus   is  obtained  and  sequences  for  two  mitochondrial  and  two  nuclear  genes  are  produced.  These  data  are  used  in  the  phylogenetic  reconstruction  of  the   relationships   between   Stenodactylus   species,   which   in   combination   with   the  estimation  of  the  divergence  timeframe,  are  used  as  the  basis  to  study  the  inter-­‐  and  intraspecific   variability   in   the   genus,   revise   its   taxonomy   and   explore   the  biogeographic  history  of   the  group.  The   impact  of   geologic   and   climatic  phenomena  that  affected  North  Africa  and  Arabia  is  discussed  in  relation  to  the  results  obtained  in   Stenodactylus   and   the   patterns   observed   in   other   groups   distributed   across   these  areas.   Following   the   discovery   of   a   divergent   lineage   in   eastern   Arabia,   additional  taxonomic  and  genetic  sampling  is  carried  out,  molecular  and  morphological  analyses  are   performed   that   result   in   the   description   of   a   new   species,   Stenodactylus   sharqiyahensis   sp.   nov.,   endemic   to  Oman.   Its  morphological   and   genetic   variability  and   its   distribution   range   are   documented   with   precision   in   relation   to   its   sister  species  in  Arabia.  (Objectives  I,  II,  III  and  IV)  This  chapter  consists  of  two  published  research  papers:   Paper  1   Metallinou,   M.;   Arnold,   E.N.;   Crochet,   P.-­‐A.;   Geniez,   P.;   Brito,   J.C.;  Lymberakis,   P.;   Baha   El   Din,   S.;   Sindaco,   R.;   Robinson,   M.   &   Carranza,   S.,   2012.  Conquering   the   Sahara   and   Arabian   deserts:   systematics   and   biogeography   of   Stenodactylus  geckos  (Reptilia:  Gekkonidae).  BMC  Evolutionary  Biology,  12:258.   Paper  2   Metallinou,   M.   &   Carranza,   S.,   2013.   New   species   of   Stenodactylus  (Squamata:   Gekkonidae)   from   the   Sharqiyah   Sands   in   northeastern   Oman.   Zootaxa  3745  (4):  449-­‐468.    In   Chapter   4,   the   complex   nomenclature   of   the   African   species   of   the   genus   Stenodactylus  is  revised  in  a  published  paper.  Bibliographical  study  and  morphological  examination   of   the   type   material   prompt   nomenclatural   actions   with   the   aim   to  ensure  stability  of  usage  of  species  names  in  Stenodactylus.  Note  that  the  application  to   the   International   Commission   of   Zoological   Nomenclature   (Case   3641)   that  complements   this   paper   has   been   published   and   is   attached   in   Appendix   1   of   this  dissertation.  (Objective  IV)   30 Structure Paper  3   Metallinou,  M.  &  Crochet,  P.-­‐A.,  2013.  Nomenclature  of  African  species  of  the  genus  Stenodactylus  (Squamata:  Gekkonidae).  Zootaxa  3691  (3):  365-­‐376.    In  Chapter  5,   a   very  broad   taxonomic   sampling   of  Ptyodactylus   geckos   is   compiled,  including   the  majority   of   the   populations   of   all   the   species   found   within   the   study  area.   The   distribution   patters   of   the  Ptyodactylus   species,   updated   herein  with   new  records,   are   discussed   in   relation   to   their   conserved   morphology   and   ecological  features.  Sequences  for  two  mitochondrial  and  four  nuclear  markers  are  obtained  and  are  used  to  produce  mitochondrial  and  multilocus  phylogenies  and  to  reconstruct  the  temporal  framework  of  the  phylogenetic  relationships.  The  results  obtained  reveal  the  unexpected  polyphyly  of  one  African  member  and  high  levels  of  genetic  variability  in  many   Ptyodactylus   species.   A   special   focus   is   placed   on   the   P.   hasselquistii   species  complex   which,   having   been   exhaustively   sampled,   is   shown   to   comprise   several  undescribed  species.  A  multilocus,  coalescence-­‐based  approach  with  the  use  of  novel  methodologies  is  followed  to  delimit  the  species  boundaries  in  this  challenging  group.  (Objectives  I,  II,  III  and  V)  This   chapter   is   structured   in   two   research   papers   that   are   in   the   final   stage   of  preparation  and  are  presented  herein  in  the  form  of  manuscripts:   Paper  4   Metallinou,   M.;   Červenka,   J.;   Crochet,   P.-­‐A.;   Kratochvíl,   L.;   Wilms,   T.;  Geniez,  P.;   Shobrak,  M.Y.;  Brito,   J.C.  &  Carranza,   S.   Species  on   the   rocks:   Systematics  and   biogeography   of   the   rock-­‐dwelling   Ptyodactylus   geckos   (Squamata:  Phyllodactylidae)  in  North  Africa  and  Arabia.   Paper  5   Metallinou,  M.;  Wilms,  T.;  Kratochvíl,  L.  &  Carranza,  S.  Multilocus  species  delimitation   in   an   old   radiation   of   North   African   and   Arabian   geckos  (Phyllodactylidae:  Ptyodactylus).     31 Structure   ADVISOR’S  REPORT  ON  THE  PUBLICATION  STATUS  OF  THE  RESULTS  AND  THE  IMPACT   FACTOR  OF  THE  PUBLISHED  PAPERS  Dr.   Salvador  Carranza,   as   advisor  of   the  PhD   thesis  of  Margarita  Metallinou  entitled  “Systematics,  biogeography  and  evolution  of  selected  widespread  reptile  genera  from  the   arid   areas   of   North   Africa   and   Arabia”,   I   hereby   present   the   following   report  regarding  the  impact  factor  of  the  published  papers  presented  in  Chapters  3  and  4  of  this  dissertation:   Paper  1   Metallinou,   M.;   Arnold,   E.N.;   Crochet,   P.-­‐A.;   Geniez,   P.;   Brito,   J.C.;  Lymberakis,   P.;   Baha   El   Din,   S.;   Sindaco,   R.;   Robinson,   M.   &   Carranza,   S.,   2012.  Conquering   the   Sahara   and   Arabian   deserts:   systematics   and   biogeography   of   Stenodactylus  geckos  (Reptilia:  Gekkonidae).  BMC  Evolutionary  Biology,  12:258.   BMC   Evolutionary   Biology   has   in   the   latest   available   edition   of   the   Journal   Citation  Reports  2012  an  impact   factor  of  3.285.  This   journal   is   in  the  second  quartile  (19  of  47)  of   the  area  “Evolutionary  Biology”.  BMC  Evolutionary  Biology   is  a  referent   in   the  field  of  evolutionary  biology  and  in  the  use  of  phylogenies  for  evolutionary  studies.       Paper  2   Metallinou,   M.   &   Carranza,   S.,   2013.   New   species   of   Stenodactylus  (Squamata:   Gekkonidae)   from   the   Sharqiyah   Sands   in   northeastern   Oman.   Zootaxa  3745  (4):  449-­‐468.   Paper  3   Metallinou,  M.  &  Crochet,  P.-­‐A.,  2013.  Nomenclature  of  African  species  of  the  genus  Stenodactylus  (Squamata:  Gekkonidae).  Zootaxa  3691  (3):  365-­‐376.   Zootaxa   has   in   the   latest   available   edition   of   the   Journal   Citation   Reports   2012   an  impact   factor   of   0.974.   This   journal   is   in   the   third   quartile   (83   of   151)   of   the   area  “Zoology”.  Zootaxa  is  mega-­‐journal  referent  for  zoological  Taxonomy  and  Systematics.  With  2,135  papers  published  in  2013  (108  monographs  of  more  than  60  pages)  and  23,373  new  taxa  published  till  the  end  of  2012  (410  of  Reptilia),  it  is  the  journal  that  is  contributing  most   to   a   better   understanding   and   cataloguing   of   the   world’s   animal  biodiversity.      Two  papers  are  presented  in  Chapter  5  in  the  form  of  manuscripts:   Paper  4   Metallinou,   M.;   Červenka,   J.;   Crochet,   P.-­‐A.;   Kratochvíl,   L.;   Wilms,   T.;  Geniez,  P.;   Shobrak,  M.Y.;  Brito,   J.C.  &  Carranza,   S.   Species  on   the   rocks:   Systematics  and   biogeography   of   the   rock-­‐dwelling   Ptyodactylus   geckos   (Squamata:  Phyllodactylidae)  in  North  Africa  and  Arabia.  The  manuscript   corresponding   to  paper  4  has  been   finished,   is  now   in   the  phase  of  friendly   review   and   it  will   be   submitted   to   the   journal  Molecular   Phylogenetics   and   33 Advisor's Reports Evolution.   This   journal   has   in   the   latest   available   edition   of   the   Journal   Citation  Reports  2012  an  impact  factor  of  4.066  and  is  in  the  second  quartile  (15  of  47)  of  the  area  “Evolutionary  Biology”.  Molecular  Phylogenetics  and  Evolution  is  a  referent  in  the  field  of  evolutionary  biology  and  in  the  use  of  phylogenies  for  evolutionary  studies.   Paper  5   Metallinou,  M.;  Wilms,  T.;  Kratochvíl,  L.  &  Carranza,  S.  Multilocus  species  delimitation   in   an   old   radiation   of   North   African   and   Arabian   geckos  (Phyllodactylidae:  Ptyodactylus).    The  manuscript  corresponding  to  paper  5  is  currently  at  the  last  stage  of  preparation  and   therefore   it   is   still   early   to   indicate   the   journal   to   which   it   will   be   submitted.  However,   it   is  planned  that  this  will  be  a   journal  of  high  impact   factor  of  the  area  of  “Evolutionary  Biology”,  preferably  one  belonging  to  the  first  or  second  quartile.    Barcelona,                        May  2014        Salvador  Carranza  Gil-­‐Dolz  del  Castellar       34 Advisor's Reports ADVISOR’S  REPORT  ON  THE  CONTRIBUTION  OF  THE  PHD  CANDIDATE  IN  THE  PAPERS  Dr.   Salvador  Carranza,   as   advisor  of   the  PhD   thesis  of  Margarita  Metallinou  entitled  “Systematics,  biogeography  and  evolution  of  selected  widespread  reptile  genera  from  the   arid   areas   of   North   Africa   and   Arabia”,   I   hereby   present   the   following   report  regarding  the  contribution  of  the  PhD  candidate  in  each  one  of  the  presented  papers.  I  guarantee   that   none   of   the   information   included   in   these   papers   will   be   used   to  elaborate  any  other  PhD  thesis.     Paper  1   SC  and  ENA  conceived  the  study.  SC  coordinated  the  study.  All  authors  collected  samples  in  the  field  and/or  provided  tissue  samples.  SC  and  MM  assembled  the   data.   MM   obtained   the   sequences,   carried   out   the   analyses   and   drafted   the  manuscript.  PAC  contributed  to  improving  the  manuscript.  MM  and  SC  wrote  the  final  manuscript.   Paper  2   MM   and   SC   conceived   the   study,   collected   samples   in   the   field   and  assembled   the   data.   MM   obtained   the   sequences   and   the   morphological   data,   and  carried  out  the  molecular  analyses.  MM  and  SC  analyzed  the  morphological  data  and  wrote  the  final  manuscript.     Paper  3   MM   and   PAC   conceived   the   study   and   performed   bibliographical  research.   MM   studied   the   material   from   museum   collections   and   drafted   the  manuscript.  MM  and  PAC  wrote  the  final  manuscript.   Paper  4   MM  and  SC  conceived   the  study.  SC  coordinated   the  study.  All  authors  collected  samples  in  the  field  and/or  provided  tissue  samples.  MM  assembled  the  data,  obtained  the  sequences,  carried  out  the  analyses  and  drafted  the  manuscript.  MM  and  SC  wrote  the  final  manuscript.     Paper  5   MM  and  SC  conceived   the  study.  SC  coordinated   the  study.  All  authors  collected   samples   in   the   field   and/or   provided   tissue   samples.   MM   obtained   the  sequences,   carried   out   the   analyses   and   wrote   the   manuscript.   SC   revised   the  manuscript.      Barcelona,                        May  2014        Salvador  Carranza  Gil-­‐Dolz  del  Castellar   35 Advisor's Reports                           CHAPTER  3    Results:  Systematics  of  Stenodactylus     Paper  1   Metallinou,  M.;  Arnold,  E.N.;  Crochet,  P.-­‐A.;  Geniez,  P.;  Brito,  J.C.;  Lymberakis,  P.;  Baha  El  Din,  S.;  Sindaco,  R.;  Robinson,  M.   &   Carranza,   S.,   2012.   Conquering   the   Sahara   and   Arabian  deserts:   systematics   and   biogeography   of   Stenodactylus   geckos  (Reptilia:  Gekkonidae).  BMC  Evolutionary  Biology,  12:258.   Paper  2   Metallinou,  M.  &   Carranza,   S.,   2013.  New   species   of   Stenodactylus  (Squamata:  Gekkonidae)  from  the  Sharqiyah  Sands  in  northeastern  Oman.  Zootaxa  3745  (4):  449-­‐468.       La  conquesta  dels  deserts  del  Sàhara  i  d'Aràbia:  sistemàtica  i   biogeografia  del  dragons  Stenodactylus  (Reptilia:  Gekkonidae)   Resum  La  història  evolutiva  de  la  biota  del  Nord  d'Àfrica  i  d'Aràbia  va  intrínsecament  lligada  a   la   complexa   història   geològica   i   als   canvis   climàtics   que   han   format   els   extensos  deserts,   actualment   dominants   en   aquestes   àrees.   Els   rèptils   constitueixen   un   grup  modèlic  per  a  l'estudi  de  les  zones  àrides  donat  l'elevat  nombre  de  representants  que  hi   estan   ben   adaptats,   com   s'ha   demostrat   en   estudis   recents   que   han   posat   al  descobert  interessants  patrons  de  diversificació  i  biogeogràfics.  En  aquest  estudi  hem  utilitzat  207   individus,   incloent-­‐hi  representants  de  totes   les  12  espècies  actualment  reconegudes  del  gènere  Stenodactylus.  Hem  inferit  filogènies  moleculars  utilitzant  dos  marcadors  mitocondrials   (12S   rRNA   i   16S   rRNA)   i   dos  marcadors   nuclears   (c-­‐mos   i   RAG2)  que  ens  han  servit  per  obtenir  una  filogènia  robusta  i  temporalment  calibrada.  Aquesta  ha  estat  utilitzada  per  investigar  les  relacions  inter-­‐  i   intraespecífiques  i  per  dilucidar   la   història   biogeogràfica   del   gènere   Stenodactylus,   repartit   extensament  inclús  en  les  àrees  àrides  i  hiper-­‐àrides  del  Nord  d'Àfrica  i  d'Aràbia.  Els   resultats   obtinguts   amb   les   anàlisis   filogenètiques   utilitzant   dades   moleculars  revelen   l'existència  de   tres  clades  principals  dins  del  gènere  Stenodactylus,   recolzats  pels   estudis   previs   basats   en   dades  morfològiques.   El   temps   de   divergència   estimat  entre   els   clades   i   sub-­‐clades   es   correlacionen   amb   els   principals   esdeveniments  geològics  de  la  regió,  amb  l'obertura  del  Mar  Roig  com  el  més  rellevant,  mentre  que  la  inestabilitat   climàtica   durant   el   Miocè   es   postula   com   a   catalitzador   de   la  diversificació.   Hem   observat   una   elevada   variabilitat   genètica   en   algunes   espècies,  suggerint  l'existència  d'espècies  no  descrites.  Manifestem  la  necessitat  d'una  profunda  revisió  taxonòmica  del  complex  d'espècies  S.  petrii  -­‐  S.  stenurus.  Hem  presentat  noves  dades  sobre  la  distribució  de  les  espècies  germanes  S.  sthenodactylus  i  S.  mauritanicus.  Amb   la   reconstrucció   filogenètica   del   gènere   Stenodactylus   presentada   en   aquest  treball  s'ha  pogut  reconstruir  la  història  biogeogràfica  d'aquests  abundants  pobladors  dels  deserts,  i  confirmar  la  importància  de  l'obertura  del  Mar  Roig  i  de  les  oscil·lacions  climàtiques  miocèniques  entre  els  principals  factors  promotors  de  la  diversificació  de  la  biota  del  Nord  d'Àfrica  i  Aràbia.  En  conjunt,  aquest  treball  ha  servit  per  a  l'estudi  de  l'evolució   d'aquest   grup   de   rèptils   profundament   especialitzats   i   àmpliament  distribuïts,   per   investigar   els   patrons   de   la   seva   elevada   diversitat   i   per   elucidar   la  seva  sistemàtica.   39 Paper 1   RESEARCH ARTICLE Conquering the Sahara an r d dr Keywords: Stenodactylus, Gekkonidae, Arabia, North Africa, Phylogeny, Biogeography, Desert, Red Sea Metallinou et al. BMC Evolutionary Biology 2012, 12:258 http://www.biomedcentral.com/1471-2148/12/258 Paper 1as well as uplifting of the newly-formed continentalBarceloneta 37-49, Barcelona 08003, Spain Full list of author information is available at the end of the articleBackground North Africa and Arabia are home to a unique fauna and flora that has been shaped by the combination of several factors including the harsh climatic conditions of the Sahara and Arabian deserts, the episodic appea- rance of humid cycles, and by the complex geological evolution of the area [1-9]. One of the most important geological phenomena of the entire Cenozoic that oc- curred in this area was the break-up of the Arabian plate from Africa. Tectonic activity started approximately 30 Ma ago at the central Gulf of Aden with the forma- tion of a rift basin in the Eritrean Red Sea and initial rif- ting at the Afar zone. A second phase of volcanism occurred 24 Ma ago, causing extension and rifting throughout the entire Red Sea, from Yemen to Egypt,* Correspondence: salvador.carranza@ibe.upf-csic.es1Institute of Evolutionary Biology (CSIC-UPF), Passeig Marítim de laPetros Lymberakis6, Sherif Baha El Din7, Roberto Sindaco8, Michael Robinson9 and Salvador Carranza1* Abstract Background: The evolutionary history of the biota of North Africa and Arabia is inextricably tied to the complex geological and climatic evolution that gave rise to the prevalent deserts of these areas. Reptiles constitute an exemplary group in the study of the arid environments with numerous well-adapted members, while recent studies using reptiles as models have unveiled interesting biogeographical and diversification patterns. In this study, we include 207 specimens belonging to all 12 recognized species of the genus Stenodactylus. Molecular phylogenies inferred using two mitochondrial (12S rRNA and 16S rRNA) and two nuclear (c-mos and RAG-2) markers are employed to obtain a robust time-calibrated phylogeny, as the base to investigate the inter- and intraspecific relationships and to elucidate the biogeographical history of Stenodactylus, a genus with a large distribution range including the arid and hyper-arid areas of North Africa and Arabia. Results: The phylogenetic analyses of molecular data reveal the existence of three major clades within the genus Stenodactylus, which is supported by previous studies based on morphology. Estimated divergence times between clades and sub-clades are shown to correlate with major geological events of the region, the most important of which is the opening of the Red Sea, while climatic instability in the Miocene is hypothesized to have triggered diversification. High genetic variability is observed in some species, suggesting the existence of some undescribed species. The S. petrii - S. stenurus species complex is in need of a thorough taxonomic revision. New data is presented on the distribution of the sister species S. sthenodactylus and S. mauritanicus. Conclusions: The phylogenetic hypothesis for the genus Stenodactylus presented in this work permits the reconstruction of the biogeographical history of these common desert dwellers and confirms the importance of the opening of the Red Sea and the climatic oscillations of the Miocene as major factors in the diversification of the biota of North Africa and Arabia. Moreover, this study traces the evolution of this widely distributed and highly specialized group, investigates the patterns of its high intraspecific diversity and elucidates its systematics.systematics and biogeog geckos (Reptilia: Gekkoni Margarita Metallinou1, Edwin Nicholas Arnold2, Pierre-An© 2012 Metallinou et al.; licensee BioMed Cen Creative Commons Attribution License (http:/ distribution, and reproduction in any medium 41Open Access d Arabian deserts: aphy of Stenodactylus ae) é Crochet3, Philippe Geniez4, José Carlos Brito5,tral Ltd. This is an Open Access article distributed under the terms of the /creativecommons.org/licenses/by/2.0), which permits unrestricted use, , provided the original work is properly cited. Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 2 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1shoulders [1]. Nevertheless, fluctuations of the sea level during the Miocene permitted the formation of transient land connections [1,10] that were subsequently lost [11]. The establishment of the Afro-Arabia - Eurasia land bridge (Gomphotherium bridge) was another crucial event with major biogeographical implications [12-14]. Following the opening of the Gulf of Aden and the Red Sea and with the counterclockwise rotation of the Arabian plate, a first connection was presumably formed between the latter and the Anatolian plate, and subsequently with Eurasia. Although the connection between the Mediterranean Sea and the Indian Ocean is hypothe- sized to have been re-established in the Upper Middle Miocene, around 15 Ma ago, it is believed that posterior to this date the land bridge has been continuously present [15]. Important faunal and floral exchanges have been attributed to the establishment of this connection ([12-14] and references therein). Although the origin of the Sahara and Arabian deserts is still hotly debated [16-19], it is generally accepted that climatic development in the late Miocene, as a result of major growth of the East Antarctic Ice Sheet and polar cooling, lead to an increase in aridification of mid- latitude continental regions [4] and that this had a pro- found effect on the diversification of faunas [20-22]. Reptiles are among the commonest inhabitants of arid areas and have long been used in biogeographic, ecological and evolutionary studies [23], constituting thus excellent models to investigate how diversity is originated and main- tained. Several cases of faunal exchanges in both directions between North Africa and Arabia have been described (e.g. [2,13,24]) showing that there is not a single pattern, but rather different hypotheses including both vicariance and dispersal, heavily dependent on the estimated time- frame of the events. Moreover, several studies have shown that climatic changes towards aridity and contraction/ expansion of the Sahara and Arabian deserts have played a decisive role in reptile species diversification [25-29]. Gekkonid lizards of the genus Stenodactylus Fitzinger, 1826 [30] are one of the most characteristic and abun- dant elements of the fauna of the arid and hyper-arid regions of Arabia and North Africa [31]. The genus comprises twelve species that are distributed in a more or less continuous range across northern Africa and Arabia, with an apparently isolated population in north- ern Kenya and extending around the Arabian Gulf to coastal southwestern Iran ([32,33]; see Figure 1). Up to three species may occur at a single locality and, where such sympatry exists, resource partitioning is largely achieved by microhabitat segregation, with species occu- pying different soil types [34]. Gravel plains, hard sand and aeolian soft sand all have their characteristic species that show specialized morphological adaptations. These include the presence of depressed and fringed toes, which 42increase the surface area and improve grip in the aeolian sand dune specialists Stenodactylus doriae (Blanford, 1874 [35]), S. petrii Anderson, 1896 [36] and S. arabicus (Haas, 1957 [37]). Extensive webbing is also observed between the fingers for efficient sand burrowing in S. arabicus [31,32,38]. When two species are regularly found on the same substrate, they greatly differ in size and there are corresponding differences in the size of prey taken [32]. Morphologically, Stenodactylus is fairly homogeneous and all species exhibit phalangeal reduction that pro- duces a formula of 2.3.3.4.3 on both fore and hind limbs and are also characterized by a very high scleral ossicle number (20–28) [31,39]. A morphology-based phylogen- etic hypothesis has been proposed by Arnold (1980) [31]. Although these two characters are also present in Pseudoceramodactylus khobarensis Haas, 1957 [37], which was widely accepted as a Stenodactylus member [31,39], a recent phylogenetic study by Fujita and Papenfuss (2011) [40] including specimens of the former and six out of the twelve species of the genus Stenodactylus proposed the resurrection of the genus Pseudoceramodactylus. This was done in order to deal with the resulting paraphyly of Stenodactylus, caused by the branching of two represen- tatives of the genus Tropiocolotes between P. khobarensis and the six Stenodactylus included in their analyses. Their molecular analyses also uncovered high levels of genetic di- vergence between the different Stenodactylus species. Ge- netic variability within some of the species, like S. arabicus and S. doriae, was also high and this could be linked to biogeographic discontinuities among some of the hyper- arid areas in Arabia. Although Stenodactylus includes a relatively low number of species compared to other gecko groups in these areas, such as Pristurus, Tarentola or Hemidactylus [26,41-46], its relatively high level of resource partitioning and habitat specialization has allowed the different species to successfully colonize almost all available habitats in the arid and hyper-arid regions of North Africa and Arabia. It constitutes, therefore, a very interesting, but still poorly studied, genus that makes an excellent model for the study of desert biodiversity and biogeography. The main objectives of the present work are: (1) to provide for the first time a complete phylogeny of the genus Stenodactylus and evaluate its concordance with previous molecular and morphology-based studies; (2) to investigate the bio- geographical and diversification patterns of Stenodactylus; and (3) to explore the interspecific relationships, the pat- terns of intraspecific diversity and the possible presence of unrecognized divergent lineages in Stenodactylus. Methods Taxon sampling, DNA extraction and sequencing A total of 207 individuals of Stenodactylus representing all twelve currently recognized species were included in Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 3 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 130°N 20°N 10°N 40°N 50°N S. pulcher S. arabicus (& cf. arabicus) S. leptocosymbotes S. affinis S. petrii S. stenurusthe present study. Whenever possible, we tried to in- clude multiple populations for each species in order to assess intraspecific variation; sampling was especially intense in the three African species with very large dis- tribution ranges. In addition, three Pseudoceramodactylus khobarensis and eight individuals representing six species of the genus Tropiocolotes were included in an attempt to further test the relationship between Stenodactylus, Pseudoceramodactylus and Tropiocolotes. Four additional specimens from other closely related genera [47-49] were used as outgroups and sixteen specimens, from several genera, were added in order to estimate divergence times (see below). Additional file 1: Table S1 lists all 238 samples used in the present work with their extraction codes, vou- cher references, localities and GenBank accession num- bers (KC190516-KC191151). Genomic DNA was extracted from ethanol-preserved tis- sue samples using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). All 222 specimens included in the phylogenetic analyses were sequenced for two mitochon- drial gene fragments: 378–388 base pairs (bp) of 12S rRNA (12S) and 498–536 bp of the 16S rRNA (16S). A subset of 106 specimens, including representatives from all inde- pendent lineages recovered by the analysis of these two 20°E10°E0°10°W20°W 0° S. doriae S. slevini S. grandiceps S. yemenensis Figure 1 Sampling localities of the Stenodactylus specimens used in t and 3 (see also Additional file 1: Table S1). The global distribution of the ge Jeremcenko, 2008). 43fragments, was also sequenced for two nuclear markers: 660 bp of the oocyte maturation factor MOS (c-mos), and 410 bp of the recombination activating gene 2 (RAG-2). Primers used for the amplification and sequencing of the 12S, 16S, c-mos and RAG-2 gene fragments as well as PCR conditions applied in the present work are listed in detail in Table 1. All amplified fragments were sequenced for both strands. Contigs were assembled in Geneious v.5.3 [50]. Phylogenetic analyses and hypothesis testing DNA sequences were aligned using the online version of MAFFT v.6 [51] with default parameters (gap opening = 1.53, offset value = 0.0) for the mitochondrial genes and with modified parameters (offset value = 0.1) for the nu- clear genes, in which long gaps are not expected. Coding gene fragments (c-mos and RAG-2) were translated into amino acids and no stop codons were observed. Uncor- rected p-distances were calculated in MEGA v.5 [52]. Phylogenetic analyses of the combined dataset were done employing maximum likelihood (ML) and Bayesian (BI) methods. Separate ML analyses were also performed on 12S, 16S, c-mos and RAG-2 to test for conflicting sig- nal among genes. Best-fitting nucleotide substitution mo- dels were selected for each partition under the Akaike 50°E40°E30°E 60°E his study. Colors and locality numbers refer to specimens in Figures 2 nus is seen on the upper right (data from Sindaco and TC C th Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 4 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1information criterion [53] using jModelTest v.0.1.1 [54]. The GTR + I + G model was independently estimated for each of the 12S, 16S, RAG-2 partitions and the GTR +G model for the c-mos partition. Alignment gaps were trea- ted as missing data and the nuclear gene sequences were not phased. Hemidactylus frenatus was used for rooting Table 1 Primers used in this study Gene fragment Primer name Or.1 Sequence (50- 30) 12S 12Sa F AAACTGGGATTAGATACCCCACTA 12Sb R GAGGGTGACGGGCGGTGTGT L1.STENO F GGATTAGATACCCCACTATGC H1.STENO1 R TGACGGGCGGTGTGTACG 16S 16Sa F CGCCTGTTTATCAAAAACAT 16Sb R CCGGTCTGAACTCAGATCACGT 16SaST F ATCAAAAACATCGCCTTTAGC 16SbST R CTGAACTCAGATCACGTAGGAC C-mos FUF F TTTGGTTCKGTCTACAAGGCTAC FUR R AGGGAACATCCAAAGTCTCCAAT G73_STENO F GCTGTAAAGCAGGTGAAGAAATG G74_STENO R GAACATCCAAAGTCTCCAATCTTG G73.5_STENO F GCATTTGGACTTAAAACCTG G708 R GCTACATCAGCTCTCCARCA RAG-2 RAG2-PY1-F F CCCTGAGTTTGGATGCTGTACTT RAG2-PY1-R R AACTGCCTRTTGTCCCCTGGTAT List of primers used in the amplification and sequencing of gene fragments, with 1Orientation.the tree, based on published evidence [47,48]. A Bayesian analysis of the combined dataset was per- formed in MrBayes 3.1.2 [55,56] with best fitting models applied to each partition (gene) and all parameters unlinked across partitions. Analyses ran for 107 genera- tions, with sampling intervals of 1000 generations, produ- cing 10000 trees. Convergence and appropriate sampling were confirmed examining the standard deviation of the split frequencies between the two simultaneous runs and the Potential Scale Reduction Factor (PSRF) diagnostic. Burn-in was performed discarding the first 2500 trees of each run and a majority-rule consensus tree was gener- ated from the remaining trees. ML analyses were per- formed in RAxML v.7.0.3 [57]. A GTR + I + G model was used and parameters were estimated independently for each partition. Node support was assessed by bootstrap analysis [58] including 1000 replications. Haplotype networks were constructed for the two nu- clear markers c-mos and RAG-2. The software PHASE v.2.1.1 [59,60] was used to resolve the haplotypes where more than one heterozygote position was present. Input files were prepared using Seqphase [61]. In order to in- clude as much information as possible for the better resolution of the haplotypes, the alignment of all full- length sequences of each marker was used. Phase 44probabilities parameter was set at 0.7 and all other set- tings were set by default. The network of the resulting haplotypes was calculated with TCS v.1.21 [62] applying default settings (probability of parsimony cutoff: 95%). Topological constraints to test alternative topologies were constructed by hand and compared to the uncon- Reference PCR conditions Kocher et al. (1989) 94º (5'); 94º (45"), 51º (45"), 72º (80") × 35; 72 (5') Kocher et al. (1989) This study 94º (5'); 94º (45"), 52º (45")’, 72º (90") × 35; 72º (5') This study Palumbi (1996) 94º (5'); 94º (45"), 51 (45"), 72 (80") × 35; 72º (5') Palumbi (1996) This study 94º (5'); 94º (45"), 57º (45"), 72º (70") × 35; 72º (5') This study Gamble et al. (2008) 94º (5'); 94º (30"), 55º (45"), 72º (70") × 35; 72º (10') Gamble et al. (2008) This study 94º (5'); 94º (45"), 56º (45"), 72º (80") × 35; 72º (5') This study This study Hugall et al. (2008) Gamble et al. (2008) 94º (5'); 94º (45"), 55º (45"), 72º (70") × 35; 72º (5') Gamble et al. (2008) e corresponding source and PCR conditions.strained (best) tree using the Approximately-Unbiased (AU) [63] and Shimodaira-Hasegawa (SH) [64] tests. Per-site log likelihoods were estimated in RAxML 7.0.3 [57] and P-values were calculated using CONSEL [65]. Tests were also run in a Bayesian framework, where the relative support of competing hypotheses given the data was quantified using the Bayes factor (BF) [66]. Topolo- gies were constrained in analyses run in BEAST v.1.6.1 [67], the marginal likelihood for each topology was esti- mated using the harmonic mean estimator and the Bayes factors were calculated by taking the ratios, as estimated in Tracer v.1.5 [68]. Estimation of divergence times A Bayesian approach was used to estimate divergence times as implemented in the software BEAST v.1.6.1. The dataset comprised sequences from all four partitions (the nuclear genes c-mos and RAG-2 unphased). An ar- bitrarily pruned phylogeny was used in order to include only one representative from each species or main lineage uncovered with the concatenated analysis (45 specimens in total; see Additional file 1: Table S1). This method excludes closely related terminal taxa because the Yule tree prior does not include a model of coalescence, which can complicate rate estimation for closely related Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 5 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1sequences [69]. Additionally, several taxa belonging to other gecko genera were added for the calibration process (see below). Two individual runs were performed for 4 × 107 genera- tions with a sampling frequency of 4000 and the results were combined to infer the ultrametric tree after discard- ing 10% of the samples from each run. Models and prior specifications applied were as follows (otherwise by de- fault): GTR + I + G (12S, 16S), GTR + I (c-mos), HKY + I (RAG-2); Relaxed Uncorrelated Lognormal Clock (esti- mate); Yule process of speciation; random starting tree; alpha Uniform (0, 10); yule.birthRate (0, 1000). Parameter values both for clock and substitution models were unlinked across partitions. Unfortunately, no fossils belonging to Stenodactylus, Pseudoceramodactylus or Tropiocolotes are known, pre- cluding the direct estimation of the time of the clado- genetic events within our study group. Consequently, the estimation was based on well-known calibration points published in recent literature [70,71] related to members of the families Phyllodactylidae and Sphaero- dactylidae (see Additional file 1: Table S1). Three fossil and biogeographical calibration points were applied as “soft” priors, in order to account for uncertainty in the date of the corresponding nodes: (1) the minimum age for the divergence between Euleptes and its sister clade was set to 22.5 Ma ago using the approximate age of a fossil Euleptes [72,73] (Lognormal distribution: median 22.5, 97.5% 36.55); (2) the split between Teratoscincus scincus - Teratoscincus roborowskii caused by the Tien Shan-Pamir uplift 10 Ma ago [74-76] (Lognormal distribu- tion: median 10.08, 97.5% 12.96); (3) the age of El Hierro island [77] at 1.12 Ma ago, assuming that divergence be- tween Tarentola boettgeri hierrensis and Tarentola boettgeri bischoffi began soon after its appearance [26,44] (Uniform distribution: lower 1, upper 1.12). In order to cross-check the results, the posterior mean rates of the mitochondrial gene fragments of our analysis were compared to the rates calculated for well-known and well-studied reptile groups from the Canary Islands (the geckos of the genus Tarentola, the lacertid lizards of the endemic genus Gallotia and the skinks of the genus Chalcides), for which robust calibrated phylogenies have been produced in se- veral independent analyses ([26,45,78-80], among others), and evolutionary rates for the 12S gene have been obtained using BEAST [44]. Ancestral area reconstruction MacClade v. 4.08 [81] was used to reconstruct the ancestral areas for the Stenodactylus species in a parsimony frame- work, using both delayed transformation (DELTRAN) and accelerated transformation (ACCTRAN). Additionally, in order to incorporate branch-length information, ML was used as implemented in the Mesquite software package 45[82]. Both Markov k-state 1-parameter and Asymmetrical Markov k-state 2-parameter models were applied and a likelihood ratio test was used to choose the best reconstruc- tion. Two states, Arabia and Africa, were identified in the extant species depending on the present distribution of the species [33] and were used with both methodologies. Results Phylogenetic analyses and topological tests Two datasets were used to infer the phylogenetic rela- tionships of the genus Stenodactylus: a mitochondrial one for building the preliminary phylogeny and analy- zing the divergence patters, and a multi-locus one for producing a more robust phylogeny (TreeBASE ID: 13567). The first dataset consisted of an alignment of 974 bp of mitochondrial DNA (415 bp of 12S and 559 bp of 16S, of which 270 in both cases were variable positions) for 222 terminals including 207 Stenodactylus. The results of the ML and BI of this dataset were very similar and are summarized in Supplementary Figure 1 (Additional file 2: Figure S1). In order to improve our phylogenetic hypothesis applying a multi-locus approach, a second dataset was assembled with a selection of 106 terminals, including 91 Stenodactylus (see Additional file 1: Table S1) for which two extra nuclear genes were sequenced. The aligned dataset consisted of 2092 bp (419 bp of 12S, 560 bp of 16S, 703 bp of c-mos and 410 bp of RAG-2, of which 262, 269, 109 and 99 positions were variable, respectively). The result of the phylogenetic analyses of the concatenated align- ment of four genes is shown in Figure 2. Well-supported relationships in the independent gene trees were congruent among partitions, but at this level not all markers offered sufficient resolution to differentiate par- ticularly between S. sthenodactylus and S. mauritanicus (data not shown but see below). The networks con- structed for the phased haplotypes of the nuclear markers are presented in Figure 3. Not all ambiguities were resolved. Both ML and Bayesian analyses of the concatenated alignment of four gene fragments (Figure 2) gave almost identical results to the mtDNA tree from the Additional file 2: Figure S1 There is low support over the relationships between the genera Stenodactylus, Pseudoceramodactylus and Tropiocolotes. According to the results, the North African T. algericus and T. tripolitanus branch first and P. khobarensis is sister to a poorly supported clade formed by two reciprocally monophyletic groups: one in- cluding T. scorteccii, T. steudneri,T. nubicus and the Middle Eastern T. nattereri and the other one including all 12 spe- cies of the genus Stenodactylus. In order to further investi- gate these relationships, three topological tests were carried out: (1) Stenodactylus + Pseudoceramodactylus were forced us t s al s al Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 6 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1Bunop Tropiocolote Tropiocolote Agamura persica84 99 100 100 100monophyletic; (2) Tropiocolotes was forced monophyletic; and (3) Stenodactylus + Pseudoceramodactylus were forced monophyletic and Tropiocolotes was forced monophy- letic on the same constraint tree. The resulting con- strained topologies were compared to our optimal topology T Tropiocolo 100 9 Tropio Tro T100 97 90 36.7 Ma (25.8-48.5) 100 17.3 Ma (11.3-23.6) 6.4 Ma (3.9-9.3) 29.5 Ma (20.7-39.2) 7.0 Ma (4.2-10.1) 78 100 99 91 95 96 11.0 Ma (7.4-15.0) 6.7 Ma (4.1-9.3) 21.8 Ma (15.4-29.1) 19.3 Ma (13.1-25.5) 15.4 Ma (10.5-20.8) 10.0 Ma (6.6-13.7) 0.3 82 Figure 2 BI tree of the genus Stenodactylus inferred using 12S, 16S m the nodes indicate posterior probability values above 0.95 in the Bayesian support of the Maximum Likelihood analysis (only values above 70 are sho arrow, with the corresponding age range in brackets. The tree was rooted specimens code refer to localities in Figure 1. Information on the samples i not submitted to precise relative scaling. 46uberculatus Crossobamon orientalis gericus - 1 - W. Sahara gericus - 2 - W. Saharafrom Figure 2 under both ML and Bayesian frameworks (see Table 2). The results of the topological tests indicate that our dataset cannot reject the alternative hypothesis of monophyly of Stenodactylus + Pseudoceramodactylus (AU:0.461, SH:0.839, BF:0.647), monophyly of Tropiocolotes ropiocolotes tripolitanus - 1 - W. Sahara tes tripolitanus - 2 - Egypt Pseudoceramodactylus khobarensis - 3 - Oman Pseudoceramodactylus khobarensis - 2 - Kuwait Pseudoceramodactylus khobarensis - 1 - Kuwait9 colotes nattereri - Jordan piocolotes scorteccii - Oman ropiocolotes steudneri - Egypt Tropiocolotes nubicus - Egypt S. pulcher - 1 - Yemen [47] S. pulcher - 2 - Yemen [48] S. cf. arabicus - 3 - Oman [6] S. cf. arabicus - 1 S. cf. arabicus - 2 S. arabicus - 7 S. arabicus - 8 Oman [5] Kuwait [9] 100 100 99 92 100 100 71 S. arabicus - 6 S. arabicus - 5 Oman [8] S. arabicus - 9 - U.A.E. [10] S. arabicus - 4 - Qatar [7] S. leptocosymbotes - 2 - Oman [21] S. leptocosymbotes - 3 - Oman [22] S. leptocosymbotes - 4 - Oman [23] S. leptocosymbotes - 1 - Oman [20] S. leptocosymbotes - 5 - U.A.E. [24] 100 S. doriae - 5 - Kuwait [14] 100 S. doriae - 15 - Yemen [136]S. doriae - 6 - Israel [15] S. doriae - 4 - Jordan [13] 73 99 S. doriae - 14 - Yemen [136] S. doriae - 3 - U.A.E. [12] S. doriae - 1 - Oman [11] S. doriae - 2 - Oman [6] 100 100 100 100 S. slevini - 1 - Jordan [49] S. slevini - 2 S. slevini - 3 S. slevini - 4 - Kuwait [50] S. slevini - 6 - U.A.E. [52] S. slevini - 5 - Qatar [51] Jordan [13] 100 100 98 S. grandiceps - 3 - Jordan [17] S. grandiceps - 2 S. grandiceps - 1 Jordan [16] S. grandiceps - 9 S. grandiceps - 10 Jordan [19] S. affinis - 2 - Iran [2] S. affinis - 1 - Iran [1] S. affinis - 5 S. affinis - 4 S. affinis - 3 - Iran [3] Kuwait [4] S. petrii - 1 - Egypt [35] S. petrii - 2 - Egypt [36] S. petrii - 3 - Israel [37] S. stenurus - Tunisia [137] S. petrii - 33 - Tunisia [53] S. petrii - 34 S. petrii - 35 Tunisia [54] 100 100 83 64 79 91 98 96 100 100 100 96 S. petrii - 5 - Morocco [39] S. petrii - 4 - Mauritania [38] S. petrii - 6 - Algeria [40] S. petrii - 8 - Mauritania [42] S. petrii - 7 - Algeria [41] S. petrii - 9 - W. Sahara [43] S. petrii - 10 - Mauritania [44] S. petrii - 12 S. petrii - 13 S. petrii - 11 - W. Sahara [45] W. Sahara [46] S. yemenensis - 1 - Yemen [71] S. yemenensis - 2 - Yemen [72] S. yemenensis - 7 - Yemen [139] S. yemenensis - 3 - Yemen [138] 99 99 72 75 76 93 100 100 89 99 96 91 81 S. mauritanicus - 5 - Libya [29] S. mauritanicus - 1 - Egypt [25] S. mauritanicus - 2 - Tunisia [26] S. mauritanicus - 3 - Tunisia [27] S. mauritanicus - 4 - Tunisia [28] S. mauritanicus - 6 - Morocco [30] S. mauritanicus - 11 - Morocco [34] S. mauritanicus - 9 - W. Sahara [32] S. mauritanicus - 10 - W. Sahara [33] S. mauritanicus - 7 S. mauritanicus - 8 W. Sahara [31] S. mauritanicus - 30 - Morocco [92] S. sthenodactylus - 1 - Egypt [25] S. sthenodactylus - 8 - Israel [60] S. sthenodactylus - 7 - Israel [37] S. sthenodactylus - 9 - Jordan [61] S. sthenodactylus - 2 - Egypt [55] S. sthenodactylus - 3 - Egypt [56] S. sthenodactylus - 18 - Mauritania [70] S. sthenodactylus - 11 - Libya [63] S. sthenodactylus - 12 - Algeria [64] S. sthenodactylus - 13 - Mauritania [65] S. sthenodactylus - 17 - W. Sahara [69] S. sthenodactylus - 16 - W. Sahara [68] S. sthenodactylus - 15 - W. Sahara [67] S. sthenodactylus - 14 - Mauritania [66] S. sth. zavattari - 1 - Kenya [62] S. sthenodactylus - 4 - Egypt [57] S. sthenodactylus - 5 - Egypt [58] S. sthenodactylus - 6 - Egypt [59] 100 87 72 6.1 Ma (3.9-8.6) 6.6 Ma (4.0-9.5) 4.8 Ma (2.8-6.9) 96 93 100 74 C A B B1 B2 C1 C2 C3 tDNA and c-mos, RAG-2 nuclear gene fragments. Black circles on Inference analysis. Numbers next to the nodes indicate bootstrap wn). Ages of the nodes estimated with BEAST are indicated with an using Hemidactylus frenatus. Numbers in square brackets next to ncluded is shown in Additional file 1: Table S1. Species' pictures were M171a M70a M101a M73a M172a M193a,b M136a M157b M157a M80a,b M177a,b M176a,b M111b M112b M141a,b M87a,b M175a M174a,b M173a M175b M173b M22b M22a M192a,b M171b M161a,b M70b M101b M73b M172b M136b M117a,b M118a,b E1505331a E1505332b M4b E1505316b M4a E1505316a M165a,b E1505 332a M92a,b E1505331b M78a M6a M2a M2b M180a,b M182a,b M178a,b M6b M107b M105a,b M107a M78b M213a,b M206a,b M126a M128a M126b M128b M184a,b M187b M19b M19a M187a M8bM32b M7a,b M8a M32a M191a,b M14a,b M13a,b M203a,b M204a,b M25a,b M26a,b M18a,b M28a M211a M30a M185a,b M210a,b M28b M211b E150538b M31a,b M169a,b M30b E150 538a M130a,b M122b M122a M129b M9b M129a M9aM10b M10a M36a,b M166a,b M111a M112a M199a,b c-mos S. pulcher S. arabicus (& cf. arabicus) S. leptocosymbotes S. doriae S. slevini S. grandiceps S. affinis S. petrii S. stenurus S. yemenensis S. mauritanicus S. sthenodactylus M22a,b M70b M161a M192a,b M176b M115b M88a M110a M88b M110b E1505353b M111b M112b E1505339 a,b M117a,b M87a,b M173a M175a,b M174a M173b M174b M161b M171a M171b M101a,b M70a M136a M193a,b M136b M165a E1505316a E1505332a,b E1505331a,b M78a,b E1505326a,b M180a,b M178a,b M182a,b M6a,b M2a,b E1505316b M4a,b M165b M213a,b M206a,b M126a,b M128a,b M203b M204b M191a,b M14a,b M13a,b M203a M204a M187a,b M19a,b M20a,b E1505334a,b M18b M23b M28b M211b E150 538b M131a M7a,b M8a,b M32a,b M131b M130a,b M122a M122b M36a,b M10a,b M34a E150536a M129a,b M33a,b M35a M34b M9a,b M35b E150 536b M166a,b M157a,b E1505353a M112a M111a M141a,b M80a,b M177a,b M115a M176a M73a,b M123a,b M172a,b M199a,b M28a M211a M30a,b M185a,b M210a,b E150538a M23a M25a,b M26a,b M18a RAG-2 Figure 3 Haplotype networks of the nuclear markers c-mos and RAG-2. Only full-length sequences were used and phase probabilities were set as≥ 0.7. Information on the samples included is shown in Additional file 1: Table S1. Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 7 of 17 http://www.biomedcentral.com/1471-2148/12/258 47 Paper 1 (AU:0.161, SH:0.495, BF:-0.530) or both concurrently (AU:0.153, SH:0.492, BF:1.589). Within Stenodactylus, three well supported clades are revealed (see Figure 2): (i) clade A, formed by the Arabian species S. pulcher Anderson, 1896 [36], S. arabicus and the divergent lineage S. cf. arabicus, (ii) clade B, that includes five Arabian species (S. leptocosymbotes Leviton and Anderson, 1967 [83], S. doriae, S. slevini Haas, 1957 [37], S. grandiceps Haas, 1952 [84] and S. affinis (Murray, 1884) [85] grouped in 2 sub-clades, and (iii) clade C, formed by the four African species (S. petrii, S. stenurus Werner, 1899 [86], S. mauritanicus Guichenot, 1850 [87] and S. sthenodactylus (Lichtenstein, 1823) and the south- Finally, clade C consists of three sub-clades, two Afri- can and one Arabian. The North African sub-clade C1 braches first, and the Arabian S. yemenensis is sister to sub-clade C3 formed by the two North African species S. mauritanicus and S. sthenodactylus, making the group of North African Stenodactylus species paraphyletic. Topological constraint analyses indicate that the alterna- tive hypothesis of monophyly of the North African species is rejected by the AU and BF tests (AU:0.029, SH:0.123, BF:7.221) (Table 2). In sub-clade C1, S. stenurus is nested within S. petrii, rendering the latter paraphyletic. The results of the topological constraint analysis in which S. petrii was en -lo 1 1 1 1 1 1 d in SH: Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 8 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1west Arabian endemic S. yemenensis Arnold, 1980 [31]. Clade A is sister to the remaining species of the genus and includes the two morphologically similar but highly divergent species S. pulcher and S. arabicus (p-distance 12S: 12.5% and 16S: 14.5%) (Additional file 3: Table S2a). Genetic variability within S. arabicus is very high and includes two reciprocally monophyletic deep lineages (p-distance 12S: 7.7% and 16S: 5.0%) (Additional file 3: Table S2c), one of them restricted to the Sharqiya Sands (formerly Wahiba Sands) in Oman, hereafter referred to as S. cf. arabicus, and the other one covering the rest of the distribution range of the species. Network analysis of the nuclear gene fragments c-mos and RAG-2 shows that for the former all alleles are unique for each lineage and all but one for the latter (Figure 3). Clade B is well supported and groups S. doriae and S. leptocosymbotes in sub-clade B1, while S. slevini, S. grandiceps and S. affinis in B2. Phylogenetic relation- ships are not completely resolved in the latter. Genetic distances between these five species are among the lowest in the genus (Additional file 3: Table S2a). Nuclear net- work analyses (Figure 3) reveal only unique alleles in the c-mos gene fragment for all five species, while there is some allele sharing in RAG-2 between S. doriae and S. leptocosymbotes. Table 2 Statistical support for alternative hypotheses on St Tree Unconstrained tree Monophyly of Stenodactylus+Pseudoceramodactylus Monophyly of Tropiocolotes Monophyly of Stenodactylus+Pseudoceramodactylus and Tropiocolotes Monophyly of African species Monophyly of S. petrii All topological tests are done versus the unconstrained (best) tree. Values in bol 1ML: Maximum likelihood; AU: Approximately Unbiased test (Shimodaira, 2002); are significantly different. 2HME: The harmonic mean of sampled likelihoods as estimated by Tracer. BF: Bayes significant difference between solutions. 48forced monophyletic show that this hypothesis is rejected by both AU and BF tests (AU:0.036, SH:0.210, BF:2.578) (Table 2). Network analysis shows that S. stenurus lacks unique alleles in both nuclear markers (Figure 3). The level of intraspecific genetic variability within S. petrii (Additional file 3: Table S2b) is very high: the uncorrected p-distances between specimens from Egypt and Israel, and the remaining S. petrii specimens sampled for this study is 7.2% and 6.0% for the 12S and 16S mitochon- drial markers, respectively (Additional file 3: Table S2c). Nuclear networks indicate that all six c-mos and four out of six RAG-2 alleles investigated are unique to this former lineage of S. petrii (Figure 3). In sub-clade C3, the two North African species S. sthe- nodactylus and S. mauritanicus are reciprocally mono- phyletic and highly divergent (p-distance 12S: 10.9% and 16S: 7.2%) (Additional file 3: Table S2a). The former is highly variable (p-distance: 12S 4.7% and 16S 3.2%) (Additional file 3: Table S2b) and presents three deep lineages that follow a clear geographical pattern (Figures 1 and 2), grouping animals from: 1.- northern Egypt, Israel and Jordan; 2.- south, southeast Egypt and Kenya; 3.- all the animals from Libya, Algeria, Tunisia, Western Sahara and Mauritania, although a single specimen from NE Egypt (loc. 127 in Figure 1, Siwa Oasis) is also part of this odactylus phylogeny ML framework1 Bayesian framework2 g likelihood AU P SH P HME log10 BF 5180.095955 −15175.4907 5180.877129 0.461 0.839 −15175.0633 0.647 5183.765511 0.161 0.495 −15175.6215 −0.530 5183.696084 0.153 0.492 −15177.9132 1.589 5192.711115 0.029 0.123 −15190.3457 7.221 5189.220967 0.036 0.210 −15181.4116 2.578 dicate statistically significant results. Shimodaira & Hasegawa (1999) test. P < 0.05 suggests that the two solutionsFactor. A log10 Bayes factor > 2 indicates decisive evidence for statistically Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 9 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1latter clade. The results of the network analyses also show a differentiation between these three lineages. In c-mos (Figure 3), in the first lineage 7 out of 10 alleles are unique, in the second lineage 2 out of 6 and in the third lineage 9 out of 16, while in RAG-2, 3 out of 10, 2 out of 8 and 10 out of 12 are unique, respectively (Figure 3). Genetic variability within S. mauritanicus is slightly higher than in S. sthenodactylus (p-distance: 12S 4.7% and 16S 4.3%) (Additional file 3: Table S2b) and six differ- ent mitochondrial lineages with geographic structure are found: 1.- easternmost part of Libya and Egypt; 2.- central Libya; 3.- Tunisia; 4.- Northern Morocco; 5.- two very divergent samples from southeastern Morocco; and 6.- all the southern Morocco plus Western Sahara samples (see Figure 1 and Additional file 2: Figure S1). Estimation of divergence times Convergence was confirmed examining the likelihood and posterior trace plots of the two runs with Tracer v.1.5. Effective sample sizes of the parameters were above 200, indicating a good representation of independ- ent samples in the posterior. The estimated divergence times are illustrated in Figure 2 and the chronogram can be seen in Supplementary Figure 2 (Additional file 4: Figure S2). Diversification within Stenodactylus initiated 29.5 Ma ago (95% HPD: 20.7-39.2). In clade A, the split between S. pulcher and S. arabicus is dated back to 17.3 Ma (95% HPD: 11.3-23.6). The separation between the ancestors of clades B and C dates back to 21.8 Ma (95% HPD: 15.4-29.1), while diversification within these two clades started 11.0 Ma (95% HPD: 7.4-15.0) and 19.3 Ma (95% HPD: 13.1-25.5) ago, respectively. Posterior mean rates for the 12S and 16S mitochon- drial gene fragments were estimated at 0.00701 and 0.00642 substitutions per lineage per million years, re- spectively (or divergence rate: 1.402% and 1.284%). The posterior rates for the nuclear fragments, c-mos and RAG-2, were 0.00052 and 0.00060 respectively, more than 10 times lower than the mitochondrial ones. The 12S mitochondrial rate concords extremely well with the average rate for the same mitochondrial gene for three Canary Island reptile groups (Gallotia, Tarentola and Chalcides; 0.00755 for the 12S gene) as estimated by Carranza and Arnold (2012) [44]. Ancestral area reconstruction Reconstruction of the ancestral areas of Stenodactylus species was done in a parsimony framework based on the topology of the phylogeny presented in Figure 2. The analysis indicates that the reconstruction of the area for some of the ancestors is equivocal (see Figure 4). These are the common ancestor of clade C, formed by all North African species and S. yemenensis, and the ancestor of the latter and the sister species S. sthenodactylus/ 49S. mauritanicus. Reconstructions using accelerated transformation (ACCTRAN) or delayed transform- ation (DELTRAN) optimizations support an identical number of events involving Arabia and Africa, but the direction of events is different. ML-based reconstruc- tion, considering branch-length information, with the best-fit Markov k-state 1-parameter model also provided results with fairly similar probabilities for the two states in the aforementioned nodes (Figure 4). Discussion This constitutes the first phylogenetic study using a complete sampling of Stenodactylus taxa and including 207 specimens from across the entire distribution range of North Africa and Arabia (Figure 1). This has enabled a robust phylogenetic reconstruction (see Figure 2 and Additional file 2: Figure S1), the uncovering of intraspeci- fic diversity and, in some cases, the unveiling of interesting distribution patterns (see below). The phylogenetic results show a high level of support in most of the nodes and a striking agreement with the phylogenetic analyses of Stenodactylus by Arnold (1980) [31], based on morpho- logical data, increasing our confidence that the recovered topology represents the true evolutionary history of the genus. Monophyly of Stenodactylus Despite the general concordance between morpho- logical and phylogenetic conclusions, one important discrepancy is observed: while morphology supports the inclusion of P. khobarensis in the genus Stenodactylus, the results of our molecular analyses indicate that Pseudoceramodactylus and Stenodactylus are not even sister genera (Figure 2). Kluge (1967) [39] transferred P. khobarensis to the genus Stenodactylus based on a “large number of external (meristic and mensural) and in- ternal morphological similarities”, including relevant cha- racters like the phalangeal reduction to a formula of 2.3.3.4.3 on both fore and hind limbs and a very high scleral ossicle number (20–28). Arnold (1980) [31], despite poin- ting out some unique scale characters of P. khobarensis, retained it in Stenodactylus and considered the scalation characters as “convincing pointers to holophyly”. However, according to a recent molecular analysis of the group by Fujita and Papenfuss (2011) [40] based on independent samples and sequences of different mitochondrial and nu- clear regions, two representatives of Tropiocolotes branched between P. khobarensis and the six species of Stenodactylus included in the analysis (see Figure 1 of [40]). In order to deal with the non-monophyly of Stenodactylus, the genus Pseudoceramodactylus was resurrected. This pattern is repeated and further investigated in our study, with a complete taxon sampling of Stenodactylus and the inclu- sion of a greater number of representatives of Tropiocolotes, Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 10 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 10.95 0.05 0.75 0.25 1.00 0.00 0.64 0.36resulting in the splitting of the latter genus into two groups, a surprising but not strongly supported, albeit consistent, result. We performed a series of constraint analyses in which Stenodactylus and Pseudoceramodactylus were forced to form a monophyletic group. Results clearly show that our dataset cannot reject the alternative hypothesis of a monophyletic Stenodactylus + Pseudoceramodactylus group (Table 2). In order to further investigate this, the dataset of Fujita and Papenfuss (2011) [40] was subjected to the same ML topological tests, but also could not reject the alternative hypothesis of mono- phyly of Stenodactylus + Pseudoceramodactylus (AU P = 0.074; SH P = 0.092). In view of the confusing molecu- lar evidence and taking into account the morphological data, we think that the resurrection of Pseudoceramodac- tylus was precipitated, but in the meanwhile, this change accommodates for both the paraphyly reported by Fujita and Papenfuss and confirmed here, and the hypothesis of 0.48 0.52 0.43 0.57 0.03 0.97 0.00 1.00 Arabia Africa Figure 4 Ancestral area reconstruction. The tree figure illustrates the pa correspond to ML probabilities for character states. Black and white colors equivocal nodes. 50S. pulcher S. cf. arabicus S. arabicus S. leptocosymbotes S. doriae S. slevini S. grandiceps S. affinis S. petrii (E)monophyly of Stenodactylus + Pseudoceramodactylus. We recommend not performing any further changes at the generic level before an in-depth revision clarifies the evo- lutionary relationships between the genera Stenodactylus, Pseudoceramodactylus and Tropiocolotes. Systematics and evolution The well-supported clade A is formed by the morpho- logically similar S. pulcher, S. arabicus and the lineage S. cf. arabicus and, according to the inferred dates, the split between the former and the two latter spe- cies dates back to approximately 17 Ma ago (95% HPD: 11.3-23.6) (Figure 2). On the one hand, variabil- ity within S. pulcher is very low, probably as a result of the two specimens analyzed being from very close localities. On the other hand, the S. cf. arabicus lineage from the Sharqiya Sands (formerly Wahiba Sands), as already highlighted by Fujita and Papenfuss (2011) [40], is genetically very distinct from all other S. stenurus S. petrii (W) S. yemenensis S. mauritanicus (E) S. mauritanicus (W) S. sthenodactylus (E) S. sthenodactylus (W) rsimony reconstruction, while numbers above and below nodes correspond to Africa and Arabia respectively, and grey color indicates Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 11 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1populations of S. arabicus included in our study in both mitochondrial and nuclear markers (Additional file 3: Table S2c and Figure 2), where almost all alleles are lineage-specific (see Results and Figure 3). This sup- ports the idea that the Sharqiya Sands are isolated and surrounded by some areas of unsuitable habitat for sand dune specialists like this species [88-90]. Further morpho- logical and molecular studies including more specimens from putative contact zones and faster nuclear markers are expected to give S. cf. arabicus formal recognition. Clade B is well-supported (ML 100%, BI 1.0) and was also recovered by the morphological analysis of Arnold (1980) [34]. Stenodactylus doriae and S. leptocosymbotes are reciprocally monophyletic and form the relatively well-supported sub-clade B1 (Figure 2). Our molecular results agree with the results of the morphological ana- lysis by Arnold (1980) [31], who also recovered the two species as sister taxa based on three synapomorphies. The two species diverged approximately 7.0 Ma ago (95% HPD: 4.2-10.1) (Figure 2) and, like the two North African sister species S. sthenodactylus and S. mauritanicus, they are ecologically distinct. Stenodactylus leptocosymbotes is an arid-adapted species that lives on relatively hard, although usually sandy, substrates being replaced by its sister species, S. doriae, on soft, wind-blown sand [34,91]. Thanks to its morphological and physiological adapta- tions, the latter is able to live in hyper-arid sand dune environments like for example the Eastern Rub al Khali [92], one of the largest and driest sand deserts in the world [93]. Given the clear morphological and ecological differences between these two species and the apparent absence of morphologically intermediate individuals [31,34], it seems reasonable to deduce that allele sharing in RAG-2 (see Results), which is limited to the ancestral allele, is the result of incomplete lineage sorting rather than ongoing gene flow between the two species. Varia- bility within S. leptocosymbotes is rather low (Additional file 3: Table S2b) and the number of samples included per- mit to observe only moderate geographical structuring (Figures 1 and 2, Additional file 2: Figure S1). In contrast, S. doriae, shows a higher level of genetic differentiation, with the Sharqiya Sands lineage being quite divergent (Additional file 3: Table S2c and Figure 2), as already men- tioned by Fujita and Papenfuss (2011) [40]. Sister to sub-clade B1 is a group composed by S. slevini, S. grandiceps and S. affinis, for which support is relatively low (ML 62, BI = 0.95). The topology within this sub-clade differs from the morphological hypothesis of Arnold (1980) [34], which supported the following relationship: (S. grandiceps (S. affinis (S. slevini (S. leptocosymbotes, S. doriae)))). Stenodactylus slevini is the only member of the group with two divergent lineages, one limited to Jordan and the other with representatives from East Arabia. Although the divergence based on mi- 51tochondrial data is clear (Additional file 2: Figure S1), there is no supporting nuclear data available (Figure 3), and no obvious morphological differences (pers. obs.). With the only exception of the soft wind-blown sand spe- cialist S. doriae, all remaining representatives of clade B plus two other species, the African S. sthenodactylus and the Arabian S. yemenensis, appear to occupy rather similar spatial niches. These six species are adapted to living on relatively hard ground, coarse sandy planes, large wadis and sandy substrates and, based on their head dimensions, probably feed on similar-sized prey [31,32,34]. As a conse- quence of that, these species rarely coexist and have largely allopatric distribution ranges, while in places where they coincide they are not syntopic [31,33,34]. The ana- lysis of the nuclear allele networks (Figure 3) indicate that the morphologically and ecologically similar and phylo- genetically closely related S. leptocosymbotes, S. slevini, S. grandiceps and S. affinis do not share a single allele in the c-mos and RAG-2 genes analyzed, even though the results of the calibration analyses suggest that S. grandiceps and S. affinis diverged later (6.7 Ma ago; 95% HPD: 4.1- 9.3) than other lineages for which extensive allele sharing in the RAG-2 has been identified (S. doriae and S. leptocosymbotes; see above and Results). These differences of the level of lineage sorting in some of the morphologically well-recognized species may also be the result of differences in effective population sizes, which affect the lineage coalescence time [94]. In sub-clade C1, S. petrii is grouped together with the North African endemic S. stenurus that branches inside it (Figure 2). As a result, S. petrii is paraphyletic and constitutes the only exception among the otherwise monophyletic Stenodactylus species. The results of the topological tests (Table 2) indicate that our data- set most probably rejects the monophyly of this species (AU:0.036, SH:0.210, BF:2.578). Stenodactylus stenurus was described by Werner (1899) [86] and synonymized ten years later by the same author [95]. It remained in syn- onymy until Kratochvil et al. (2001) [96] recognized it as a valid species, based on a multivariate analysis of several metric and scalation characters. It is noteworthy that the representative of S. stenurus included in our analysis is one of the specimens used by Kratochvil et al. (2001) [96] in their study. The highly divergent lineage that includes specimens from Egypt and Israel (see Results) is estimated to have split from specimens further west in Algeria, Morocco, Western Sahara and Mauritania approximately 6.1 Ma ago (95% HPD: 3.9-8.6) (Figure 2). In fact, the northern Sinai populations of S. petrii have been reported to be morphologically distinct and, as a result of that, were considered a different species (S. elimensis) by Barbour (1914) [97], now under the synonymy of S. petrii [31,98]. Yet, specimens from this area included in our analyses Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 12 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1do not present considerable genetic differences with the rest of the Egyptian and Israeli specimens (Figures 1 and 2, Additional file 2: Figure S1). It should be pointed out that the type locality of S. petrii is Egypt and, thus, this lineage represents the 'true' S. petrii. The pattern in the nuclear genes, with numerous unique alleles for this lineage (Figure 3), contrasts with the situation in S. stenurus that lacks unique alleles. This suggests that further analyses and a thorough taxonomic revi- sion including more samples of S. petrii, especially from not sampled areas of Algeria and Libya, and mainly S. stenurus will be necessary in order to evalu- ate the status of the populations assigned to the two species. With this evidence it will be possible to dif- ferentiate between a single species with high genetic variability (petrii), two species (petrii in the East and stenurus in the West) or three species, if stenurus proves to be distinct from the more western forms. The two North African species of sub-clade C3, S. sthenodactylus and S. mauritanicus, are shown to be reciprocally monophyletic and highly divergent (Additional file 3: Table S2a), while their separation dates back to ap- proximately 10.0 Ma (95% HPD: 6.6-13.7) (Figure 2). These results help to clarify the status of these two taxonomically controversial taxa that were treated as two different subspe- cies by Loveridge (1947) [99] and Sindaco and Jeremcenko (2008) [33], as the same monotypic species by Arnold (1980) [31] and that were finally considered as full species by Baha el Din (2006) [98], who found them in sympatry at particular localities in northern Egypt. As observed by Baha el Din (2006) [98], although these two sister species can be morphologically similar and share similar habits, they are ecologically different. Stenodactylus mauritanicus is restricted to fairly mesic coastal semi-desert under the in- fluence of the Mediterranean (see Figure 1), where it inha- bits flat rock-strewn sand and gravel plains with fairly good vegetation cover. On the contrary, S. sthenodactylus inha- bits areas of the Sahara that are far more arid and inhospit- able than the ones of its sister species (see Figure 1), being the only vertebrate to be readily found in some parts of the Western Desert of Egypt [98]. It prefers gravelly and coarse sandy plains and large wadis and, although the species is typical of hard coarse substrates, it sometimes penetrates some dune areas [98]. The distributions of these two species, as introduced by the present study, give insights into the controversial taxo- nomic status and frequent misidentification of the two forms [99]. Our analysis concludes that S. sthenodactylus extends west from the Middle East and Egypt, previously thought to be its eastern limit, across the Sahara and into Mauritania (Figure 1). Stenodactylus mauritanicus is con- firmed to be present in Egypt [98] and has a wide, almost continuous distribution roughly along the northern margin of the Sahara desert. The two species are found in 52sympatry or in close proximity in Egypt and coastal Mauritania, yet retain distinct mtDNA lineages and exhibit only limited allele sharing in the nuclear markers, most of which is due to sharing of ancestral alleles and hence is likely to represent incomplete lineage sorting (see Figure 3). Stenodactylus sthenodactylus presents high variability, both at genetic (see Results) and morphological levels [31]. Its three deep lineages are estimated to have diverged approximately 4.8 Ma ago (95% HPD: 2.8-6.9) (Figure 2). According to Baha el Din (2006) [98], some morphological characters appear to correlate with envi- ronmental factors, with populations from hyper-arid places showing a very slender body, less contrasting pattern and tubular nostrils, while populations from more mesic areas being usually more robust, with thick limbs, big heads and marked pattern [31,36,98]. The populations from coastal regions in southeast Egypt are especially distinct and, according to Baha el Din (2006) [98], they resemble speci- mens of S. s. zavattarii from Kenya, which Loveridge (1957) [100] synonymized with S. sthenodactylus. Two spe- cimens of this form were included in our phylogenetic ana- lyses (see Figure 2 and Additional file 2: Figure S1), and indeed they belong to a clade with samples from south and southeast Egypt. These results suggest that some of the morphological variability between popula- tions of S. sthenodactylus may also be supported by mo- lecular data. A nomenclatural revision of North African Stenodactylus (work in progress) is essential for stability before any changes are performed, while further work focused on the contact zones between the three lineages and combining detailed morphological analyses with add- itional nuclear data is needed in order to determine if they deserve formal recognition. On the other hand, the high genetic variability within S. mauritanicus (Figure 2 and Additional file 3: Table S2b) does not seem to correlate with differences in morphology. This species is fairly uniform morphologically, with populations from the West being a bit larger than Egyptian ones but generally maintaining the same propor- tions, pattern and scalation across most of its distribution range [98]. Nevertheless, the intra-specific divergence is estimated to date back to 6.6 Ma ago (95% HPD: 4.0-9.5) and the six mitochondrial lineages present a clear geo- graphical pattern (Figure 1 and Additional file 2: Figure S1). The relationship between these lineages, however, is not clear and neither is any structure observed in the nuclear alleles (Figure 3), both facts being mirrored in the low- supported nodes of the concatenated phylogeny (Figure 2). Origin, biogeography and diversification of Stenodactylus Reconstruction of ancestral areas with both parsimony and ML methods (Figure 4) suggests that the genus Stenodactylus originated in Arabia approximately Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 13 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 130 Ma ago (95% HPD: 20.7-39.2) (Figure 2), a time of high geological instability as a result of the onset of major seismic and volcanic events in the general area of Ethiopia, northeast Sudan and southwest Yemen [101]. These major volcanic and tectonic events, cen- tered over the Afar region, marked the onset of the formation of some of the most relevant and complex physiographical features in the contact zone between Africa and Arabia, like the Gulf of Aden, the Red Sea and the elevation of the Afro-Arabian rift-flanks to heights above 3600 m [1,101,102]. The tempo and mode of the deep splits in Stenodactylus bear a striking resemblance to the basal splits that oc- curred in the African-Eurasian snake genus Echis [13,103], which suggests a common biogeographical pattern for both groups. The distribution of the members of Arabian clade B (S. doriae, S. leptocosymbotes, S. slevini, S. grandiceps, S. affinis) and the mainly African clade C (S. petrii, S. stenurus, S. yemenensis, S. mauritanicus, S. sthenodactylus) (Figures 1 and 2) extend primarily on the opposite sides of the Red Sea, mimicking the situ- ation of the sister taxa E. coloratus (mainly Arabian) and E. pyramidum (mainly African). The split between these two Stenodactylus groups dates back to 21.8 Ma ago (95% HPD: 15.4-29.1) (Figure 2), which roughly coincides with the split between E. coloratus and E. pyramidum calcu- lated at approximately 19.4 Ma ago. The dates of these phylogenetic events follow a well-studied phase of volcan- ism and strong rifting initiated at approximately 24 Ma ago, that appeared in an almost synchronous way through- out the entire Red Sea [1]. Therefore, it is possible that the formation of the Red Sea acted as a vicariant event separa- ting the aforementioned clades of Stenodactylus, as also suggested by Pook et al. (2009) [13] for the genus Echis. The agamid lizards of the genus Uromastyx [25] is yet an- other group that could have been affected by such an event, although in this case the split between the Arabian and African clades seems to have happened later, at 11–15 Ma ago. Amer and Kumazawa (2005) [25] attributed this split to a dispersal event from Arabia into North Africa, coinciding with climatic changes towards aridity in this latter area, rather than to vicariance. How- ever, since earlier dates had also been calculated for the split between African and Arabian Uromastyx that coin- cide with the inferred dates for Stenodactylus and Echis (18 Ma ago; [104]), a reassessment of the calibration dates of Uromastyx using relaxed clock methods like the ones applied by Pook et al. (2009) [13] and in the present study seems necessary (work in progress). The split between the Arabian S. yemenensis and the an- cestor of the African S. mauritanicus and S. sthenodactylus on either sides of the Red Sea also parallels the splits between Arabian and African sister clades of the E. pyramidum complex [13] and Uromastyx ocellata and 53U. ornata [25]. Although the divergence time estimate for the Stenodactylus members (15.4 Ma ago (95% HPD: 10.5-20.8), Figure 2) predates the ones of the other two groups by almost 7 Ma, the split between African and Arabian lineages might be explained by the complex geo- logy of the Red Sea. Several recurrent episodes during the Miocene caused the desiccation and refilling of this tec- tonically active rifting area [1,105] and provoked the seve- ring of the land bridges that had existed after the initial formation of the Red Sea in the early Miocene. So, the separation between S. yemenensis and the ancestor of S. mauritanicus and S. sthenodactylus was probably also the result of vicariance, similarly to Echis and Uromastyx. After this event, S. yemenensis would have remained isolated at the coastal side of the southern Arabian highlands (Figures 1 and 2). In Arabia, an example of a similar biogeographical pat- tern caused by a different biogeographical process is the case of the ecologically similar sister species of clade A, S. pulcher and S. arabicus (including S. cf. arabicus), which, according to the results (Figure 2) and the geo- logical data available, are hypothesized to result from vicariance caused by the uplift of the Yemen Mountains approximately 18 Ma ago [1,101,102]. The splits within clade B, however, seem more difficult to interpret, as little information is available on the geological and climatic his- tory of the interior of Arabia. A general pattern could be proposed with a first North–South split between the ancestors of S. doriae, S. leptocosymbotes and S. slevini, S. grandiceps, S. affinis, respectively, followed by the pos- terior range expansion of some of these species. Interest- ingly, in Arabia, even though evidence exists for an increase in aridification [106], it has been hypothesized that at the same time an important river system, as evidenced by the fluvial sediments, could characterize the interior of the pe- ninsula [93,107]. Such dynamic scenery could be respon- sible for the rapid diversification within clade B, having caused fragmentation of the distribution range of the ances- tor(s) and the different lineages to split allopatrically. The onset of diversification in clade B coincides in time with the split between the African S. mauritanicus and S. sthenodactylus in sub-clade C3 (Figure 2). These speciation events match very closely the estimates of the formation, in the late Miocene, of a major east-Antarctic ice sheet with its associated polar cooling, which triggered the aridification of mid-latitude continental regions and a shift in North Africa from forest to dry open woodlands and savannahs [4,20,108]. The two North African forms, S. mauritanicus and S. sthenodactylus, seem to have diverged in ecological niche, with one form adapted to mesic environments and the other occupying much dryer areas, respectively. It has been proposed that the gradual increase in aridity that took place in northern Africa during the late Miocene accelerated the diversification refilling of the Red Sea, during the Miocene. An interest- ing distribution pattern is revealed for the sister species the faunal interchanges between North Africa and Arabia Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 14 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1process in reptile faunas [21]. The estimated diver- gence times of the North African Stenodactylus seem to corroborate a common emerging pattern among European biota, according to which the speciation events in many reptile and amphibian groups do not coincide with the accentuated environmental instability during the Pleistocene, but rather date back into the Miocene and proceeding through the Quaternary, when many species and populations originated [109,110]. It has been suggested that 18 Ma ago, Africa con- nected with Eurasia through the closure of the Eastern Mediterranean seaway (the Gomphotherium land bridge) [15]. This land bridge later became disconnected temporar- ily but it has been continuously present since approximately 15 Ma ago. It is interesting to notice that, despite the exist- ence of a continuous passage between Arabia and Eurasia, our phylogeny suggests that colonization of Eurasia by members of the genus Stenodactylus occurred much later and was very restricted geographically. In fact, only two Stenodactylus species extend their ranges into Eurasia (S. affinis and S. doriae). From these two, only samples of S. affinis from Eurasia (Iran) were available, while for the other species a specimen from neighboring Kuwait was included. In both species, however, the low intraspecific genetic variability suggests that the colonization of Eurasia was a very recent event (Figure 2 and Additional file 3: Table S2b). One possible explanation of this biogeographical pattern may be the existence of ecologically and morpho- logically very similar forms in Iran like Crossobamon (formerly a member of Stenodactylus [39]) and Agamura, which may compete with Stenodactylus and therefore may have not allowed it to expand further outside the narrow coastal strip in southwestern Iran (Arnold, 1980). This situ- ation is completely different than the one in North Africa, where no ecological analogs to Stenodactylus seem to exist and therefore several of its species are found across an area of more than 10 million Km2 [31,33,98,111,112]. Conclusions The analyses presented in this study, based on a multi- locus dataset that derives from a complete sampling of the 12 species of the genus Stenodactylus, reveal the existence of three clades with deep divergences within Stenodactylus and high intraspecific variability in some species, while the estimation of divergence times allows for biogeographical interpretations. The geckos Stenodactylus originated in Arabia 30 Ma ago. In clade A, the split be- tween the two species is hypothesized to have resulted from vicariance caused by the uplift of the Yemen Moun- tains approximately 18 Ma ago. Stenodactylus cf. arabicus from the Sharqiya Sands constitutes a genetically and mor- phologically distinct lineage. In clade B, rapid diversifica- tion seems to relate to climatic and geological instability in the late Miocene, but this hinders the reconstruction of 54and the evolutionary processes in these arid areas. Additional files Additional file 1: Table S1. Information on the specimens used in the phylogenetic analyses. Additional file 2: Figure S1. BI tree of the genus Stenodactylus inferred using 12S and 16S mtDNA gene fragments. Description of data: Posterior probability values above 0.95 in the Bayesian Inference analysis are indicated next to the nodes with an asterisk, while numbers correspond to bootstrap support of the Maximum Likelihood analysis (only values above 70 are shown). The tree was rooted using Hemidactylus frenatus. Numbers in square brackets next to specimen code refer to Figure 1. Information on the samples included is shown in Additional file 1: Table S1. Additional file 3: Table S2. Uncorrected p-distances (pairwise deletion). Additional file 4: Figure S2. Chronogram obtained with BEAST inferred using all markers and 3 calibration points. Description of data: Chronogram obtained with relaxed uncorrelated lognormal clock and Yule model of speciation. Filled numbered circles correspond to calibration points described in Materials and Methods. The x axis is in million years and the bars indicate 95% HPD intervals. Information on the samples included is shown in Additional file 1: Table S1. Abbreviations rRNA: ribosomal ribonucleic acid; c-mos: oocyte maturation factor Mos; RAG-2: Recombination activating gene 2; PCR: Polymerase chain reaction; ML: Maximum likelihood; BI: Bayesian Inference; AU: Approximately unbiased; SH: Shimodaira-Hasegawa; BF: Bayes factor; Ma: Megaannum; HPD: Highest posterior density.S. sthenodactylus and S. mauritanicus, differing greatly from what was previously thought. Several speciation events in Stenodactylus are estimated to date back to the late Miocene, indicating that this was an impor- tant period for reptile diversification in this area. The split between clades B and C is attributed to the opening of the Red Sea in the Upper Miocene, acting as a vicariant agent. On the other hand, the forma- tion of the connection between Africa and Eurasia seems to have had little effect on Stenodactylus, pro- bably because of the existence of ecological analogs. On a taxonomic level, further studies are expected to re- solve the systematics of the S. petrii - S. stenurus complex. Validity of the specific status of S. mauritanicus is con- firmed with mitochondrial and nuclear data. Overall, this work unveils the evolutionary history of Stenodactylus geckos and highlights their use as a model in the study ofrobust phylogenetic relationships between some species. The Sharqiya Sands host yet another divergent lineage, that of the species S. doriae. In clade C, the split between S. yemenensis and sub-clade C3 is hypothesized to re- late to the recurrent episodes of the desiccation andCompeting interests The authors declare that they have no competing interests. M Charpentier, D Donaire, D Escoriza, T Gamble, J Harris, Y Hingrat, H in den 2. Carranza S, Arnold EN, Geniez P, Roca J, Mateo J: Radiation, multiple Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 15 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 1dispersal and parallelism in the skinks, Chalcides and Sphenops (Squamata: Scincidae), with comments on Scincus and Scincopus and the age of the Sahara desert. Mol Phylogenet Evol 2008, 46:1071–1094. 3. Dean WRJ: Nomadic Desert Birds. Berlin, Heidelberg, New York: Springer Verlag; 2004. 4. Flower BP, Kennett JP: The middle Miocene climatic transition: East Antarctic ice sheet development, deep ocean circulation and global carbon cycling. Palaeogeogr Palaeoclimatol Palaeoecol 1994, 108:537–555.Bosch. D Modry, J Padial, O Peyre, JM Pleguezuelos, C Rato, J Renoult, B Shacham, J Smid, J Viglione. We are especially grateful to Jiri Moravec and Lukas Kratochvil for providing the S. stenurus tissue sample and the photograph of the species. We would also like to thank Elena Gómez-Diaz for providing helpful comments and Enric Planas for fruitful discussion and help with the figures. We are indebted to Ali Alkiyumii and the other members of the Ministry of Environment and Climate Affairs of the Sultanate of Oman for their help and support and for issuing collecting permits (Refs: 08/2005; 16/2008; 38/2010; 12/2011). This work was supported by grants CGL2009-11663⁄BOS from the Ministerio de Economía y Competitividad, Spain, Fondos FEDER - EU, and 2012RU0055 from the Consejo de Investigaciones Cientificas (CSIC) and the Russian Foundation for Basic Research (RFBR). SC and MM are members of the Grup de Recerca Emergent of the Generalitat de Catalunya: 2009SGR1462; MM is supported by a FPU predoctoral grant from the Ministerio de Educación, Cultura y Deporte, Spain (AP2008-01844). Some phylogenetic analyses were run in the cluster facility of the IBE funded by the Spanish National Bioinformatics Institute (http:// www.inab.org) and in the CIPRES Science Gateway web portal. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). We thank two anonymous reviewers for helpful comments on an earlier version of the manuscript. Author details 1Institute of Evolutionary Biology (CSIC-UPF), Passeig Marítim de la Barceloneta 37-49, Barcelona 08003, Spain. 2The Natural History Museum, Cromwell Road, London SW7 5BD, UK. 3CNRS-UMR 5175 Centre d'Ecologie Fontionnelle et Evolutive, 1919 Route de Mende, 34293, Montpellier cedex 5, France. 4EPHE-UMR, Centre d'Ecologie Fontionnelle et Evolutive, 1919 Route de Mende, 34293, Montpellier cedex 5, France. 5CIBIO/InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do Porto, Instituto de Ciências Agrárias de Vairão, R. Padre Armando Quintas 4485-661, Vairão, Portugal. 6Natural History Museum of Crete, University of Crete, Knosou Av, P.O. Box 220871409, Heraklion, Greece. 7Nature Conservation Sector, Egyptian Environmental Affairs Agency, 3 Abdalla El Katib, Apt. 3, Cairo, Dokki, Egypt. 8Museo Civico de Storia Naturale, via San Francesco di Sales 188, Carmagnola I-10022, Italy. 9Sultan Qaboos University, Department of Biology, College of Science, P.O. Box 36Al-Khod, Muscat, Sultanate of Oman. Received: 20 September 2012 Accepted: 3 December 2012 Published: 31 December 2012 References 1. Bosworth W, Huchon P, McClay K: The Red Sea and Gulf of Aden Basins. J Afr Earth Sci 2005, 43:334–378.Authors’ contributions SC and ENA conceived the study. SC coordinated the study. All authors collected samples in the field and/or provided tissue samples. SC and MM assembled the data. MM obtained the sequences, carried out the analyses and drafted the manuscript. PAC contributed to improving the manuscript. MM and SC wrote the final manuscript. All authors read and approved the final manuscript. Acknowledgements The authors are grateful to people who donated samples for this study or helped in the field: F Ahmadzadeh, F Amat, A Bouskila, A Cluchier, O Chaline,5. Griffin DL: Aridity and humidity: two aspects of the late Miocene climate of North Africa and the Mediterranean. Palaeogeogr Palaeoclimatol Palaeoecol 2002, 182:65–91. 556. Guiraud R, Bosworth W, Thierry J, Delplanque A: Phanerozoic geological evolution of Northern and Central Africa: an overview. J Afr Earth Sci 2005, 43:83–143. 7. Lourenço W, Duhem B: Saharo-Sindian buthid scorpions; description of two new genera and species from Occidental Sahara and Afghanistan. ZooKeys 2009, 14:37–54. 8. Quezel P: Analysis of the flora of Mediterranean and Saharan Africa. Ann Mo Bot Gard 1978, 65:479–534. 9. Yom-Tov Y: Character displacement in the Psammophile Gerbillidae of Israel. Oikos 1991, 60:173–179. 10. Haq BU, Hardenbol J, Vail PR: Chronology of fluctuating sea levels since the Triassic. Science 1987, 235:1156–1167. 11. Fernandes CA, Rohling EJ, Siddall M: Absence of post-Miocene Red Sea land bridges: biogeographic implications. J Biogeogr 2006, 33:961–966. 12. Harzhauser M, Kroh A, Mandic O, Piller WE, Göhlich U, Reuter M, Berning B: Biogeographic responses to geodynamics: a key study all around the Oligo-Miocene Tethyan Seaway. Zoologischer Anzeiger-A Journal of Comparative Zoology 2007, 246:241–256. 13. Pook CE, Joger U, Stümpel N, Wüster W: When continents collide: phylogeny, historical biogeography and systematics of the medically important viper genus Echis (Squamata: Serpentes: Viperidae). Mol Phylogenet Evol 2009, 53:792–807. 14. Zhou L, Su YCF, Thomas DC, Saunders RMK: 'Out-of-Africa' dispersal of tropical floras during the Miocene climatic optimum: evidence from Uvaria (Annonaceae). J Biogeogr 2012, 39:322–335. 15. Rögl F: Paleogeographic Considerations For Mediterranean And Paratethys Seaways (Oligocene And Miocene). Wien: Annalen des Naturhistorischen Museums in; 1998. 99A: 279-331. 16. Kroepelin S: Revisiting the age of the Sahara desert. Science 2006, 312:1138–1139. 17. Schuster M: Revisiting the age of the Sahara Desert. Science 2006, 312:1138–1139. 18. Schuster M, Duringer P, Ghienne J-F, Vignaud P, Mackaye HT, Likius A, Brunet M: The age of the Sahara desert. Science 2006, 311:821. 19. Swezey CS: Revisiting the age of the Sahara desert. Science 2006, 312:1138–1139. 20. Douady CJ, Catzeflis F, Raman J, Springer MS, Stanhope MJ: The Sahara as a vicariant agent, and the role of Miocene climatic events, in the diversification of the mammalian order Macroscelidea (elephant shrews). Proc Natl Acad Sci 2003, 100:8325–8330. 21. Fu J: Toward the phylogeny of the family Lacertidae–Why 4708 base pairs of mtDNA sequences cannot draw the picture. Biol J Linn Soc 2000, 71:203–217. 22. Guillaumet A, Crochet PA, Pons JM: Climate-driven diversification in two widespread Galerida larks. BMC Evol Biol 2008, 8:32. 23. Camargo A, Sinervo B, Sites JW Jr: Lizards as model organisms for linking phylogeographic and speciation studies. Mol Ecol 2010, 19:3250–3270. 24. Kapli P, Lymberakis P, Poulakakis N, Mantziou G, Parmakelis A, Mylonas M: Molecular phylogeny of three Mesalina (Reptilia: Lacertidae) species (M. guttulata, M. brevirostris and M. bahaeldini) from North Africa and the Middle East: another case of paraphyly? Mol Phylogenet Evol 2008, 49:102–110. 25. Amer SAM, Kumazawa Y: Mitochondrial DNA sequences of the Afro- Arabian spiny-tailed lizards (genus Uromastyx; family Agamidae): phylogenetic analyses and evolution of gene arrangements. Biol J Linn Soc 2005, 85:247–260. 26. Carranza S, Arnold EN, Mateo JA, Geniez P: Relationships and evolution of the North African geckos, Geckonia and Tarentola (Reptilia: Gekkonidae), based on mitochondrial and nuclear DNA sequences. Mol Phylogenet Evol 2002, 23:244–256. 27. Carranza S, Arnold EN, Pleguezuelos JM: Phylogeny, biogeography, and evolution of two Mediterranean snakes, Malpolon monspessulanus and Hemorrhois hippocrepis (Squamata, Colubridae), using mtDNA sequences. Mol Phylogenet Evol 2006, 40:532–546. 28. Fonseca MM, Brito JC, Rebelo H, Kalboussi M, Larbes S, Carretero MA, Harris DJ: Genetic variation among spiny-footed lizards in the Acanthodactylus pardalis group from North Africa. African Zoology 2008, 43:8–15. 29. Gonçalves DV, Brito JC, Crochet PA, Geniez P, Padial JM, Harris DJ: Phylogeny of North African Agama lizards (Reptilia: Agamidae) and the role of the Sahara desert in vertebrate speciation. Mol Phylogenet Evol 2012, 64:582–591. Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 16 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 130. Fitzinger LJ: Neue Classification Der Reptilien Nach Ihren Natürlichen Verwandtschaften: Nebst Einer Verwandtschafts-Tafel Und Einem Verzeichnisse Der Reptilien-Sammlung Des KK Zoologischen Museum's Zu Wien. Wien: JG Heubner; 1826. 31. Arnold EN: Reptiles of Saudi Arabia: a review of the lizard genus Stenodactylus (Reptilia: Gekkonidae). Fauna of Saudia Arabia 1980, 2:368–404. 32. Arnold EN: Little-known geckoes (Reptilia: Gekkonidae) from Arabia with descriptions of two new species from the Sultanate of Oman. The Scientific Results of the Oman Flora and Fauna Survey 1975, 1977:81–110. 33. Sindaco R, Jeremcenko VK: The Reptiles Of The Western Palearctic. Latina (Italy): Edizioni Belvedere; 2008. 34. Arnold EN: Ecology of lowland lizards in the eastern United Arab Emirates. J Zool 1984, 204:329–354. 35. Blanford WT: Descriptions of new reptilia and amphibia from Persia and Baluchistan. The Annals and Magazine of Natural History, London 1874, 4:31–35. 36. Anderson J: A Contribution To The Herpetology Of Arabia: With A Preliminary List Of The Reptiles And Batrachians Of Egypt. London: RH Porter; 1896. 37. Haas G: Some amphibians and reptiles from Arabia. Proc Calif Acad Sci 1957, 29:47–86. 38. Bauer AM, Russell AP: Pedal specialisations in dune-dwelling geckos. J Arid Environ 1991, 20:43–62. 39. Kluge AG: Higher taxonomic categories of gekkonid lizards and their evolution. Bull Am Mus Nat Hist 1967, 135:1–60. 40. Fujita MK, Papenfuss TJ: Molecular systematics of Stenodactylus (Gekkonidae), an Afro-Arabian gecko species complex. Mol Phylogenet Evol 2011, 58:71–75. 41. Arnold EN: Relationships, evolution and biogeography of Semaphore geckos, Pristurus (Squamata, Sphaerodactylidae) based on morphology. Zootaxa 2009, 2060:1–21. 42. Arnold EN, Vasconcelos R, Harris DJ, Mateo JA, Carranza S: Systematics, biogeography and evolution of the endemic Hemidactylus geckos (Reptilia, Squamata, Gekkonidae) of the Cape Verde Islands: based on morphology and mitochondrial and nuclear DNA sequences. Zoologica Scripta 2008, 37:619–636. 43. Carranza S, Arnold EN: Systematics, biogeography, and evolution of Hemidactylus geckos (Reptilia: Gekkonidae) elucidated using mitochondrial DNA sequences. Mol Phylogenet Evol 2006, 38:531–545. 44. Carranza S, Arnold EN: A review of the geckos of the genus Hemidactylus (Squamata: Gekkonidae) from Oman based on morphology, mitochondrial and nuclear data, with descriptions of eight new species. Zootaxa 2012, 3378:1–95. 45. Carranza S, Arnold EN, Mateo JA, López-Jurado LF: Long-distance colonization and radiation in gekkonid lizards, Tarentola (Reptilia: Gekkonidae), revealed by mitochondrial DNA sequences. Proc R Soc London, Ser B 2000, 267:637. 46. Gamble T, Bauer AM, Colli GR, Greenbaum E, Jackman TR, Vitt LJ, Simons AM: Coming to America: multiple origins of New World geckos. J Evol Biol 2011, 24:231–244. 47. Feng J, Han D, Bauer AM, Zhou K: Interrelationships among Gekkonid Geckos inferred from mitochondrial and nuclear gene sequences. Zoolog Sci 2007, 24:656–665. 48. Gamble T, Bauer AM, Greenbaum E, Jackman TR: Out of the blue: a novel, trans-Atlantic clade of geckos (Gekkota, Squamata). Zoologica Scripta 2008, 37:355–366. 49. Han D, Zhou K, Bauer AM: Phylogenetic relationships among gekkotan lizards inferred from C-mos nuclear DNA sequences and a new classification of the Gekkota. Biol J Linn Soc 2004, 83:353–368. 50. Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Heled J, Kearse M, Moir R, Stones-Havas S, Sturrock S: Geneious v5. 1. 2010. Available from www.geneious.com. 51. Katoh K, Toh H: Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 2008, 9:286–298. 52. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011, 28:2731–2739.53. Akaike H: Information theory and an extension of the maximum likelihood principle. In Second International Symposium on Information Theory. Edited by Petrov BN, Csaki F. Budapest (Hungary): Akademiai Kiado; 1973:267–281. 5654. Posada D: jModelTest: phylogenetic model averaging. Mol Biol Evol 2008, 25:1253. 55. Huelsenbeck JP, Ronquist F: MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 2001, 17:754–755. 56. Ronquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003, 19:1572–1574. 57. Stamatakis A: RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 2006, 22:2688. 58. Felsenstein J: Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985, 39:783–791. 59. Stephens M, Scheet P: Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. Am J Hum Genet 2005, 76:449–462. 60. Stephens M, Smith NJ, Donnelly P: A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001, 68:978–989. 61. Flot JF: Seqphase: a web tool for interconverting phase input/output files and fasta sequence alignments. Mol Ecol Resour 2010, 10:162–166. 62. Clement M, Posada D, Crandall KA: TCS: a computer program to estimate gene genealogies. Mol Ecol 2000, 9:1657–1659. 63. Shimodaira H: An approximately unbiased test of phylogenetic tree selection. Syst Biol 2002, 51:492. 64. Shimodaira H, Hasegawa M: Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol 1999, 16:1114–1116. 65. Shimodaira H, Hasegawa M: CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 2001, 17:1246. 66. Suchard MA, Weiss RE, Sinsheimer JS: Models for estimating bayes factors with applications to phylogeny and tests of monophyly. Biometrics 2005, 61:665–673. 67. Drummond AJ, Rambaut A: BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 2007, 7:214. 68. Rambaut A, Drummond AJ: Tracer v1. 4, 2007 [http://beast.bio.ed.ac.uk/ Tracer]. 69. Ho SYW, Phillips MJ, Cooper A, Drummond AJ: Time dependency of molecular rate estimates and systematic overestimation of recent divergence times. Mol Biol Evol 2005, 22:1561–1568. 70. Gamble T, Bauer AM, Greenbaum E, Jackman TR: Evidence for Gondwanan vicariance in an ancient clade of gecko lizards. J Biogeogr 2008, 35:88–104. 71. Vasconcelos R, Carranza S, Harris DJ: Insight into an island radiation: the Tarentola geckos of the Cape Verde archipelago. J Biogeogr 2010, 37:1047–1060. 72. Agustí J, Cabrera L, Garcés M, Krijgsman W, Oms O, Parés JM: A calibrated mammal scale for the Neogene of Western Europe. State of the art. Earth- Science Reviews 2001, 52:247–260. 73. Müller J: A new fossil species of Euleptes from the early Miocene of Montaigu, France (Reptilia, Gekkonidae). Amphibia-Reptilia 2001, 22:341–348. 74. Abdrakhmatov KY, Aldazhanov SA, Hager BH, Hamburger MW, Herring TA, Kalabaev KB, Makarov VI, Molnar P, Panasyuk SV, Prilepin MT, et al: Relatively recent construction of the Tien Shan inferred from GPS measurements of present-day crustal deformation rates. Nature 1996, 384:450–453. 75. Macey JR, Wang Y, Ananjeva NB, Larson A, Papenfuss TJ: Vicariant patterns of fragmentation among Gekkonid lizards of the Genus Teratoscincus produced by the Indian collision: a molecular phylogenetic perspective and an area cladogram for Central Asia. Mol Phylogenet Evol 1999, 12:320–332. 76. Tapponnier P, Mattauer M, Proust F, Cassaigneau C: Mesozoic ophiolites, sutures, and large-scale tectonic movements in Afghanistan. Earth Planet Sci Lett 1981, 52:355–371. 77. Guillou H, Carracedo JC, Torrado FP, Badiola ER: K-Ar ages and magnetic stratigraphy of a hotspot-induced, fast grown oceanic island: El Hierro, Canary Islands. J Volcanol Geotherm Res 1996, 73:141–155. 78. Arnold EN, Arribas O, Carranza S: Systematics of the Palaearctic and Oriental lizard tribe Lacertini (Squamata: Lacertidae: Lacertinae), with descriptions of eight new genera. Zootaxa 2007, 1430:1–86. 79. Brown RP, Yang Z: Bayesian dating of shallow phylogenies with a relaxed clock. Syst Biol 2010, 59:119. 80. Cox SC, Carranza S, Brown RP: Divergence times and colonization of the Canary Islands by Gallotia lizards. Mol Phylogenet Evol 2010, 56:747–757. 81. Maddison DR, Maddison WP: MacClade 4.0. Sunderland, Massachusetts: Sinauer; 2000. 1996. doi:10.1186/1471-2148-12-258 Cite this article as: Metallinou et al.: Conquering the Sahara and Arabian deserts: systematics and biogeography of Stenodactylus geckos (Reptilia: Gekkonidae). BMC Evolutionary Biology 2012 12:258. Metallinou et al. BMC Evolutionary Biology 2012, 12:258 Page 17 of 17 http://www.biomedcentral.com/1471-2148/12/258 Paper 182. Maddison WP, Maddison DR: Mesquite: A Modular System For Evolutionary Analysis. Version 2.73. http://mesquiteproject.org. 83. Leviton AE, Anderson SC: Survey of the reptiles of the Sheikhdom of Abu Dhabi, Arabian Peninsula. Part II. Systematic account of the collection of reptiles made in the Sheikhdom of Abu Dhabi by John Gasperetti. Proc Calif Acad Sci 1967, 35:157–192. 84. Haas G: Two collections of Reptiles from Iraq, with descriptions of two new forms. Copeia 1952, 1952:20–22. 85. Murray JA: Additions to the present knowledge of the vertebrate Zoology of Persia. The Annals and Magazine of Natural History 1884, 14:97–106. 86. Werner F: Allerlei aus dem Kriechtierleben im Käfig. II. Zoologischer Garten, Frankfurt am Main 1899, 40:12–24. 87. Guichenot AA: Histoire Naturelle Des Reptiles Et Des Poissons. Paris: Imprimerie nationale; 1850. 88. Gardner RAM: Aeolianites and marine deposits of the Wahiba Sands: character and palaeoenvironments. The Journal of Oman Studies 1988, 3:1985–1987. 89. Preusser F, Radies D, Driehorst F, Matter A: Late Quaternary history of the coastal Wahiba Sands, Sultanate of Oman. J Quat Sci 2005, 20:395–405. 90. Preusser F, Radies D, Matter A: A 160,000-year record of Dune development and atmospheric circulation in Southern Arabia. Science 2002, 296:2018–2020. 91. Gallagher MD, Arnold EN: Reptiles and amphibians from the Wahiba Sands, Oman. J Oman Stud, Spec Rep 1988, 3:405–413. 92. Blanford WT: Descriptions of new lizards from Persia and Baluchistan. Ann Mag Nat Hist 1874, 13:453–455. 93. Garzanti E, Andò S, Vezzoli G, Dell'era D: From rifted margins to foreland basins: investigating provenance and sediment dispersal across desert Arabia (Oman, U.A.E.). J Sediment Res 2003, 73:572–588. 94. Knowles LL, Carstens BC: Delimiting species without monophyletic gene trees. Syst Biol 2007, 56:887–895. 95. Werner F: Reptilien, Batrachier und Fische von Tripolis und Barka. Zoologische Jahrbucher Abteilung fur Systematik, Geographie und Biologie der Tiere 1909, 27:595–646. 96. Kratochvil L, Frynta D, Moravec J: Third Stenodactylus in Africa: return of the forgotten form Stenodactylus stenurus. Israel Journal of Zoology 2001, 47:99–110. 97. Barbour T: Notes on some reptiles from Sinai and Syria. Proceedings of the New England Zoological Club 1914, 5:73–92. 98. Baha El Din S: A Guide to the Reptiles and Amphibians of Egypt. Cairo and New York: The American University in Cairo Press, xvi; 2006. 99. Loveridge A: Revision of the African lizards of the family Gekkonidae. Bulletin of the Mus Comp Zool, Harvard 1947, 98:1–469. 100. Loveridge A: Checklist of the reptiles and amphibians of East Africa. Bulletin of The Museum of Comparative Zoology 1957, 117:151–362. 101. Menzies MA, Baker J, Bosence D, Dart C, Davison I, Hurford A, Al'Kadasi M, McClay K, Nichols G, Al'Subbary A, Yelland A: The timing of magmatism, uplift and crustal extension: preliminary observations from Yemen. Geological Society, London, Special Publications 1992, 68:293–304. 102. Autin J, Leroy S, Beslier MO, DíAcremont E, Razin P, Ribodetti A, Bellahsen N, Robin C, Al Toubi K: Continental break up history of a deep magma poor margin based on seismic reflection data (northeastern Gulf of Aden margin, offshore Oman). Geophys J Int 2010, 180:501–519. 103. Arnold EN, Robinson MD, Carranza S: A preliminary analysis of phylogenetic relationships and biogeography of the dangerously venomous Carpet Vipers, Echis (Squamata, Serpentes, Viperidae) based on mitochondrial DNA sequences. Amphibia-Reptilia 2009, 30:273–282. 104. Joger U: Phylogenetic analysis of Uromastyx lizards, based on albumin immunological distances. In Studies in Herpetology. Edited by Rocek Z. Bonn, Germany: Societas Europaea Herpetologica; 1986:187–192. 105. Girdler RW: The Afro-Arabian rift system - an overview. Tectonophysics 1991, 197:139–153. 106. Huang Y, Clemens SC, Liu W, Wang Y, Prell WL: Large-scale hydrological change drove the late Miocene C4 plant expansion in the Himalayan foreland and Arabian Peninsula. Geology 2007, 35:531–534. 107. Friend PF: Rivers of the Lower Baynunah Formation, Emirate of Abu Dhabi, United Arab Emirates. In Fossil Vertebrates Of Arabia, With Emphasis On The Late Miocene Faunas, Geology, And Palaeoenvironments Of The Emirate Of Abu Dhabi, United Arab Emirates. New Haven, Connecticut: Yale University Press; 1999:38–49. 57Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution108. Zachos J, Pagani M, Sloan L, Thomas E, Billups K: Trends, rhythms, and aberrations in global climate 65 Ma to Present. Science 2001, 292:686–693. 109. Paulo OS, Dias C, Bruford MW, Jordan WC, Nichols RA: The persistence of Pliocene populations through the Pleistocene climatic cycles: evidence from the phylogeography of an Iberian lizard. Proc R Soc London, Ser B 2001, 268:1625–1630. 110. Tzedakis PC, Lawson IT, Frogley MR, Hewitt GM, Preece RC: Buffered tree population changes in a quaternary refugium: evolutionary implications. Science 2002, 297:2044–2047. 111. Bons J, Geniez P: Amphibians And Reptiles Of Morocco. Barcelona: Asociación herpetológica Española; 1996. 112. Schleich HH, Kästle W, Kabisch K: Amphibians And Reptiles Of North Africa: Biology, Systematics, Field Guide. Königstein, Germany: Koeltz Scientific Books;Submit your manuscript at www.biomedcentral.com/submit A dd iti on al fi le 1 T ab le S 1. In fo rm at io n on th e sp ec im en s u se d in th e ph yl og en et ic a na ly se s L oc al ity C od e Sp ec ie s D N A E xt ra ct io n C od e V ou ch er (o r T is su e) C od e C ou nt ry o r R eg io n L oc al ity G en ba nk a cc es si on c od es (1 2S /1 6S /c -m os /R A G -2 ) 1 St en od ac ty lu s a ffi ni s - 1 M 13 1 Ir an H el ed N at io na l P ar k K C 19 06 79 / K C 19 08 73 / - / K C 19 01 05 6 2 St en od ac ty lu s a ffi ni s - 2 M 19 8 Ir an B an da r e K ha m ir, se m id es er t K C 19 06 77 / K C 19 08 71 / - / - 3 St en od ac ty lu s a ffi ni s - 3 ** M 7 B EV .1 00 36 K uw ai t 50 0 m W o f S ul ai bi kh at R es er ve , W . K uw ai t C ity K C 19 06 75 / K C 19 08 69 / K C 19 09 46 / K C 19 10 54 4 St en od ac ty lu s a ffi ni s - 4 M 8 B EV .1 00 95 K uw ai t M in a Sa id K C 19 06 76 / K C 19 08 70 / K C 19 09 47 / K C 19 10 55 4 St en od ac ty lu s a ffi ni s - 5 M 32 B EV .1 00 96 K uw ai t M in a Sa id K C 19 06 74 / K C 19 08 68 / K C 19 09 45 / K C 19 10 53 73 St en od ac ty lu s a ffi ni s - 6 M 13 2 Ir an - / K C 19 09 13 73 St en od ac ty lu s a ffi ni s - 7 M 13 3 Ir an K C 19 06 78 / K C 19 08 72 5 St en od ac ty lu s c f. ar ab ic us - 1* * M 10 O m an S of A l M in tri b K C 19 06 96 / K C 19 08 90 / K C 19 09 98 / K C 19 11 16 5 St en od ac ty lu s c f. ar ab ic us - 2 M 36 O m an S of A l M in tri b K C 19 06 97 / K C 19 08 91 / K C 19 09 99 / K C 19 11 17 6 St en od ac ty lu s c f. ar ab ic us - 3 M 16 6 O m an A l M in tri b, S ha rq iy a Sa nd s K C 19 06 99 / K C 19 08 93 / K C 19 10 00 / K C 19 11 18 7 St en od ac ty lu s a ra bi cu s - 4 M 12 9 Q at ar 15 k m N E of A s S al w a K C 19 06 94 / K C 19 08 88 / K C 19 09 96 / K C 19 11 15 8 St en od ac ty lu s a ra bi cu s - 5 M 9 O m an Sa nd d un es N o f S hi su r K C 19 06 95 / K C 19 08 89 / K C 19 09 97 / K C 19 11 12 8 St en od ac ty lu s a ra bi cu s - 6 M 33 O m an Sa nd d un es N o f S hi su r K C 19 06 92 / K C 19 08 86 / K C 19 10 01 / K C 19 11 13 9 St en od ac ty lu s a ra bi cu s - 7 ** M 34 B EV .1 00 42 K uw ai t W af ra h Fa rm s, 20 k m E o f W af ra h K C 19 06 90 / K C 19 08 84 / K C 19 09 94 / K C 19 11 10 9 St en od ac ty lu s a ra bi cu s - 8 M 35 B EV .1 00 43 K uw ai t W af ra h Fa rm s, 20 k m E o f W af ra h K C 19 06 91 / K C 19 08 85 / K C 19 09 95 / K C 19 11 11 10 St en od ac ty lu s a ra bi cu s - 9 E1 50 53 6 U A E D ha fr a B ea ch H ot el K C 19 06 93 / K C 19 08 87 / K C 19 10 11 / K C 19 11 14 6 St en od ac ty lu s c f. ar ab ic us - 10 M 16 7 O m an A l M in tri b, S ha rq iy a Sa nd s K C 19 06 98 / K C 19 08 92 11 St en od ac ty lu s d or ia e - 1 M 31 O m an A l-A re es h D es er t C am p, S ha rq iy a Sa nd s K C 19 06 43 / K C 19 08 37 / K C 19 09 36 / K C 19 10 44 6 St en od ac ty lu s d or ia e - 2 M 16 9 O m an A l M in tri b, S ha rq iy a Sa nd s K C 19 06 44 / K C 19 08 38 / K C 19 09 37 / K C 19 11 21 58 Paper 1 12 St en od ac ty lu s d or ia e - 3 ** E1 50 53 8 U A E Sa nd d un es a t A l A in K C 19 06 53 / K C 19 08 47 / K C 19 09 39 / K C 19 10 50 13 St en od ac ty lu s d or ia e - 4 M 18 5 B EV .T 37 59 Jo rd an B et w ee n A qa ba a nd W ad i R um K C 19 06 50 / K C 19 08 44 / K C 19 09 41 / K C 19 10 47 14 St en od ac ty lu s d or ia e - 5 M 28 B EV .1 00 37 K uw ai t 13 k m N -N E of Ja hr a K C 19 06 46 / K C 19 08 40 / K C 19 09 38 / K C 19 10 45 15 St en od ac ty lu s d or ia e - 6 M 30 B EV .8 48 3 Is ra el 14 k m N o f E ilo t K C 19 06 49 / K C 19 08 43 / K C 19 09 40 / K C 19 10 46 6 St en od ac ty lu s d or ia e - 7 M 17 0 O m an A l M in tri b, S ha rq iy a Sa nd s K C 19 06 45 / K C 19 08 39 74 St en od ac ty lu s d or ia e - 8 M 12 O m an A l-A re es h D es er t C am p, S ha rq iy a Sa nd s K C 19 06 42 / K C 19 08 36 75 St en od ac ty lu s d or ia e - 9 E1 50 53 7 U A E A l A ya , A bu D ha bi K C 19 06 52 / K C 19 08 46 18 St en od ac ty lu s d or ia e - 1 0 M 11 B EV .1 00 38 K uw ai t W af ra h Fa rm s, 20 k m E o f W af ra h K C 19 06 47 / K C 19 08 41 13 St en od ac ty lu s d or ia e - 1 1 M 18 6 B EV .T 37 60 Jo rd an B et w ee n A qa ba a nd W ad i R um K C 19 06 51 / K C 19 08 45 15 St en od ac ty lu s d or ia e - 1 2 M 29 B EV .8 48 2 Is ra el 14 k m N o f E ilo t K C 19 06 48 / K C 19 08 42 76 St en od ac ty lu s d or ia e - 1 3 M 12 0 Q at ar K C 19 06 54 / K C 19 08 48 13 6 St en od ac ty lu s d or ia e - 1 4 M 21 0 Y em en M a'r ib K C 19 07 20 / K C 19 09 22 / K C 19 10 30 / K C 19 11 38 13 6 St en od ac ty lu s d or ia e - 1 5 M 21 1 Y em en M a'r ib K C 19 07 21 / K C 19 09 23 / K C 19 10 31 / K C 19 11 39 13 6 St en od ac ty lu s d or ia e - 1 6 M 21 2 Y em en M a'r ib K C 19 07 22 / K C 19 09 24 16 St en od ac ty lu s g ra nd ic ep s - 1 ** M 13 Jo rd an W ad i B ay ir K C 19 06 68 / K C 19 08 62 / K C 19 09 52 / K C 19 10 62 16 St en od ac ty lu s g ra nd ic ep s - 2 M 14 Jo rd an W ad i B ay ir K C 19 06 67 / K C 19 08 61 / K C 19 09 51 / K C 19 10 61 17 St en od ac ty lu s g ra nd ic ep s - 3 M 19 1 B EV .1 09 11 Jo rd an 6 km E o f S ha w ba k K C 19 06 73 / K C 19 08 67 / K C 19 09 53 / K C 19 10 63 77 St en od ac ty lu s g ra nd ic ep s - 4 M 15 8 N H M C 80 .3 .1 07 .6 Jo rd an 4k m N o f A l M an sh iy ya K C 19 06 69 / K C 19 08 63 77 St en od ac ty lu s g ra nd ic ep s - 5 M 15 9 N H M C 80 .3 .1 07 .2 Jo rd an 4k m N o f A l M an sh iy ya K C 19 06 70 / K C 19 08 64 17 St en od ac ty lu s g ra nd ic ep s - 6 M 18 9 B EV .1 09 09 Jo rd an 6 km E o f S ha w ba k K C 19 06 71 / K C 19 08 65 17 St en od ac ty lu s g ra nd ic ep s - 7 M 19 0 B EV .1 09 10 Jo rd an 6 km E o f S ha w ba k K C 19 06 72 / K C 19 08 66 78 St en od ac ty lu s g ra nd ic ep s - 8 M 16 0 N H M C 80 .3 .1 07 .1 Jo rd an 30 k m W o f A zr aq - / K C 19 09 12 19 St en od ac ty lu s g ra nd ic ep s - 9 M 20 3 Jo rd an A zr aq K C 19 07 14 / K C 19 09 15 / K C 19 10 27 / K C 19 11 35 19 St en od ac ty lu s g ra nd ic ep s - 1 0 M 20 4 Jo rd an A zr aq K C 19 07 15 / K C 19 09 16 / K C 19 10 28 / K C 19 11 36 19 St en od ac ty lu s g ra nd ic ep s - 1 1 M 20 5 Jo rd an A zr aq K C 19 07 16 / K C 19 09 17 20 St en od ac ty lu s l ep to co sy m bo te s - 1 M 18 O m an Th um ra it K C 19 06 62 / K C 19 08 56 / K C 19 09 43 / K C 19 10 52 21 St en od ac ty lu s l ep to co sy m bo te s - 2 M 23 O m an 4k m S W A l M aa ym ir K C 19 06 55 / K C 19 08 49 / K C 19 10 12 / K C 19 10 51 22 St en od ac ty lu s l ep to co sy m bo te s - 3 M 25 O m an 2. 5 km S E A r R um ay liy ah K C 19 06 57 / K C 19 08 51 / K C 19 09 42 / K C 19 10 48 59 Paper 1 23 St en od ac ty lu s l ep to co sy m bo te s - 4 ** M 26 O m an W ad i M aa hd i, M as ira h is la nd K C 19 06 66 / K C 19 08 60 / K C 19 09 44 / K C 19 10 49 24 St en od ac ty lu s l ep to co sy m bo te s - 5 E1 50 53 13 U A E A l A in K C 19 06 64 / K C 19 08 58 / K C 19 10 13 / K C 19 11 05 20 St en od ac ty lu s l ep to co sy m bo te s - 6 e9 03 61 1 O m an Th um ra it K C 19 06 60 / K C 19 08 54 20 St en od ac ty lu s l ep to co sy m bo te s - 7 e9 03 61 2 O m an Th um ra it K C 19 06 61 / K C 19 08 55 79 St en od ac ty lu s l ep to co sy m bo te s - 8 M 24 O m an K C 19 06 58 / K C 19 08 52 21 St en od ac ty lu s l ep to co sy m bo te s - 9 e9 03 61 O m an K C 19 06 56 / K C 19 08 50 21 St en od ac ty lu s l ep to co sy m bo te s - 1 0 e9 03 63 O m an K C 19 06 59 / K C 19 08 53 24 St en od ac ty lu s l ep to co sy m bo te s - 1 1 e9 03 66 U A E A l A in K C 19 06 63 / K C 19 08 57 24 St en od ac ty lu s l ep to co sy m bo te s - 1 2 e9 03 68 U A E A l A in K C 19 06 65 / K C 19 08 59 25 St en od ac ty lu s m au ri ta ni cu s - 1 E1 50 53 39 Eg yp t El O m ay ed p ro te ct ed a re a K C 19 05 77 / - / K C 19 10 18 / K C 19 10 83 26 St en od ac ty lu s m au ri ta ni cu s - 2 E1 50 53 53 Tu ni si a O ue d sh ili , 3 1 km N E of T oz eu r K C 19 05 66 / K C 19 07 70 / K C 19 10 16 / K C 19 10 77 27 St en od ac ty lu s m au ri ta ni cu s - 3 M 11 1 Tu ni si a C ro ss ro ad to Jb el C ha m bi K C 19 05 67 / K C 19 07 71 / K C 19 09 76 / K C 19 11 08 28 St en od ac ty lu s m au ri ta ni cu s - 4 M 11 2 Tu ni si a Jb el T am es m id a K C 19 05 68 / K C 19 07 72 / K C 19 09 77 / K C 19 10 78 29 St en od ac ty lu s m au ri ta ni cu s - 5 ** M 15 7 N H M C 80 .3 .1 52 .1 Li by a 30 k m S o f M is ira ta K C 19 05 79 / K C 19 07 82 / K C 19 09 81 / K C 19 10 74 30 St en od ac ty lu s m au ri ta ni cu s - 6 ** M 80 M or oc co 44 k m S -S W o f S id i I fn i K C 19 05 90 / K C 19 07 91 / K C 19 09 78 / K C 19 10 72 31 St en od ac ty lu s m au ri ta ni cu s - 7 M 88 W . S ah ar a, M or oc co 84 k m N o f B ou dj ou r K C 19 05 95 / K C 19 07 96 / K C 19 10 17 / K C 19 10 85 31 St en od ac ty lu s m au ri ta ni cu s - 8 M 11 0 W . S ah ar a, M or oc co 5 km N E of L em si d, K C 19 05 96 / K C 19 07 97 / K C 19 10 08 / K C 19 10 86 32 St en od ac ty lu s m au ri ta ni cu s - 9 M 11 5 W . S ah ar a, M or oc co 10 0 km S o f B ou jd ou r K C 19 05 97 / K C 19 07 98 / K C 19 10 09 / K C 19 10 79 33 St en od ac ty lu s m au ri ta ni cu s - 1 0 M 17 6 B EV .1 08 35 W . S ah ar a, M or oc co O ue d Lc ra a, 1 70 k m S -S W o f B ou jd ou r K C 19 05 99 / K C 19 08 00 / K C 19 09 80 / K C 19 10 80 34 St en od ac ty lu s m au ri ta ni cu s - 1 1 M 17 7 B EV .1 08 36 M or oc co 12 k m S -S W T an T an K C 19 05 87 / K C 19 07 89 / K C 19 09 79 / K C 19 10 73 80 St en od ac ty lu s m au ri ta ni cu s - 1 2 M 55 B EV .9 15 7 M au rit an ia C ap B la nc K C 19 06 00 / K C 19 08 01 32 St en od ac ty lu s m au ri ta ni cu s - 1 3 M 11 6 W . S ah ar a, M or oc co 10 0 km S o f B ou jd ou r K C 19 05 98 / K C 19 07 99 43 St en od ac ty lu s m au ri ta ni cu s - 1 4 M 74 W . S ah ar a, M or oc co 25 k m W o f S m ar a K C 19 05 83 / K C 19 07 86 43 St en od ac ty lu s m au ri ta ni cu s - 1 5 M 75 W . S ah ar a, M or oc co 25 k m W o f S m ar a K C 19 05 84 / K C 19 07 87 81 St en od ac ty lu s m au ri ta ni cu s - 1 6 M 49 B EV .2 38 7 M or oc co B et w ee n K hn ifi ss a nd T an ta n K C 19 05 88 / - 81 St en od ac ty lu s m au ri ta ni cu s - 1 7 M 50 B EV .2 38 8 M or oc co B et w ee n K hn ifi ss a nd T an ta n K C 19 05 86 / - 82 St en od ac ty lu s m au ri ta ni cu s - 1 8 M 54 B EV .7 88 9 M or oc co 4 km N w of T an ta n K C 19 05 85 / K C 19 07 88 83 St en od ac ty lu s m au ri ta ni cu s - 1 9 M 82 M or oc co S of S id i I fn i K C 19 05 92 / K C 19 07 93 84 St en od ac ty lu s m au ri ta ni cu s - 2 0 M 83 M or oc co S of S id i I fn i K C 19 05 93 / K C 19 07 94 85 St en od ac ty lu s m au ri ta ni cu s - 2 1 M 81 M or oc co S of S id i I fn i K C 19 05 91 / K C 19 07 92 60 Paper 1 86 St en od ac ty lu s m au ri ta ni cu s - 2 2 M 84 M or oc co S of S id i I fn i K C 19 05 94 / K C 19 07 95 87 St en od ac ty lu s m au ri ta ni cu s - 2 3 M 79 M or oc co S of S id i I fn i K C 19 05 89 / K C 19 07 90 88 St en od ac ty lu s m au ri ta ni cu s - 2 4 M 14 2 N H M C 80 .3 .8 8. 40 M or oc co 14 6 km E o f O ua rz az at e K C 19 06 02 / K C 19 08 03 89 St en od ac ty lu s m au ri ta ni cu s - 2 5 M 13 7 N H M C 80 .3 .8 8. 45 Li by a 55 k m S o f M is ra ta h K C 19 05 78 / K C 19 07 81 90 St en od ac ty lu s m au ri ta ni cu s - 2 6 M 15 3 N H M C 80 .3 .8 8. 1 Li by a Li by a - E gy pt b or de rs - / K C 19 09 11 91 St en od ac ty lu s m au ri ta ni cu s - 2 7 M 13 5 N H M C 80 .3 .8 8. 47 Li by a Q am in is , 1 20 k m N E of A dj ed ab ia K C 19 05 71 / K C 19 07 75 91 St en od ac ty lu s m au ri ta ni cu s - 2 8 M 15 4 N H M C 80 .3 .1 53 .2 Li by a Q am in is , 1 20 k m N E of A dj ed ab ia K C 19 05 72 / K C 19 07 76 91 St en od ac ty lu s m au ri ta ni cu s - 2 9 M 15 5 N H M C 80 .3 .1 53 .1 Li by a Q am in is , 1 20 k m N E of A dj ed ab ia K C 19 05 75 / K C 19 07 79 92 St en od ac ty lu s m au ri ta ni cu s - 3 0 M 14 1 N H M C 80 .3 .8 8. 41 M or oc co 30 k m N E of G ou lm in a K C 19 06 01 / K C 19 08 02 / K C 19 09 88 / K C 19 10 75 93 St en od ac ty lu s m au ri ta ni cu s - 3 1 M 14 0 N H M C 80 .3 .8 8. 42 Li by a K ik la K C 19 05 80 / K C 19 07 83 94 St en od ac ty lu s m au ri ta ni cu s - 3 2 M 15 6 N H M C 80 .3 .1 52 .2 Li by a A in T ag ni t K C 19 05 81 / K C 19 07 84 95 St en od ac ty lu s m au ri ta ni cu s - 3 3 M 15 1 N H M C 80 .3 .8 8. 29 Li by a 75 k m W o f T ob ru k - / K C 19 09 10 96 St en od ac ty lu s m au ri ta ni cu s - 3 4 M 13 4 N H M C 80 .3 .8 8. 9 Li by a Ig de id a se m id es er t K C 19 05 74 / K C 19 07 78 96 St en od ac ty lu s m au ri ta ni cu s - 3 5 M 15 2 N H M C 80 .3 .8 8. 28 Li by a Ig de id a se m id es er t K C 19 05 73 / K C 19 07 77 97 St en od ac ty lu s m au ri ta ni cu s - 3 6 M 13 8 N H M C 80 .3 .8 8. 44 Li by a A l M ab ne K C 19 05 76 / K C 19 07 80 98 St en od ac ty lu s m au ri ta ni cu s - 3 7 E1 50 53 49 M or oc co 17 K m N or tw es t o f M is so ur K C 19 05 58 / - 99 St en od ac ty lu s m au ri ta ni cu s - 3 8 M 56 B EV .9 98 5 M or oc co A l B at en p la in K C 19 05 64 / - 99 St en od ac ty lu s m au ri ta ni cu s - 3 9 M 57 B EV .9 98 6 M or oc co A l B at en p la in K C 19 05 61 / - 99 St en od ac ty lu s m au ri ta ni cu s - 4 0 M 58 B EV .9 99 0 M or oc co A l B at en p la in K C 19 05 60 / - 10 0 St en od ac ty lu s m au ri ta ni cu s - 4 1 M 98 M C C R 11 60 -1 M or oc co B et w ee n D eb do u an d A in B en im at ha r K C 19 05 62 / K C 19 07 69 10 1 St en od ac ty lu s m au ri ta ni cu s - 4 2 M 10 3 M or oc co N o f M so un , T az a K C 19 05 65 / - 10 1 St en od ac ty lu s m au ri ta ni cu s - 4 3 M 76 M or oc co N o f M so un , T az a K C 19 05 59 / K C 19 07 68 10 2 St en od ac ty lu s m au ri ta ni cu s - 4 4 E1 50 53 50 M or oc co 19 K m W o f E l A io um K C 19 05 63 / - 10 3 St en od ac ty lu s m au ri ta ni cu s - 4 5 M 14 8 N H M C 80 .3 .8 8. 34 Tu ni si a 2 km N o f O ul ed M on ac eu r - / K C 19 09 09 28 St en od ac ty lu s m au ri ta ni cu s - 4 6 M 11 3 Tu ni si a Jb el T am es m id a, b as e of K C 19 05 96 / K C 19 07 73 28 St en od ac ty lu s m au ri ta ni cu s - 4 7 M 11 4 Tu ni si a Jb el T am es m id a, b as e of K C 19 05 70 / K C 19 07 74 10 4 St en od ac ty lu s m au ri ta ni cu s - 4 8 M 12 4 Li by a K C 19 05 82 / K C 19 07 85 35 St en od ac ty lu s p et ri i - 1 ** M 4 B EV .8 98 7 Eg yp t W ad i E l N at ru n K C 19 06 03 / K C 19 08 04 / K C 19 09 54 / K C 19 10 93 36 St en od ac ty lu s p et ri i - 2 E1 50 53 16 Eg yp t E of A l A ris h, S in ai K C 19 06 10 / K C 19 08 11 / K C 19 09 55 / K C 19 10 94 37 St en od ac ty lu s p et ri i - 3 M 16 5 H U JR -2 37 76 Is ra el N W N eg ev sa nd s, O vi tz fi el d H al uz za K C 19 06 11 / K C 19 08 12 / K C 19 09 56 / K C 19 11 07 38 St en od ac ty lu s p et ri i - 4 M 6 B EV .9 13 1 M au rit an ia El B ey ed K C 19 06 12 / K C 19 08 13 / K C 19 09 57 / K C 19 11 02 39 St en od ac ty lu s p et ri i - 5 M 78 M or oc co Er g C he bb i K C 19 06 17 / K C 19 08 18 / K C 19 09 60 / K C 19 10 95 40 St en od ac ty lu s p et ri i - 6 M 10 5 B EV .1 01 57 A lg er ia Ta ss ili N 'A je r K C 19 06 13 / K C 19 08 14 / K C 19 09 58 / K C 19 11 06 41 St en od ac ty lu s p et ri i - 7 M 10 7 B EV .1 01 81 A lg er ia Ti sr as K C 19 06 19 / K C 19 08 19 / K C 19 09 59 / - 61 Paper 1 42 St en od ac ty lu s p et ri i - 8 M 11 9 M au rit an ia Ta rf T az az m ou t K C 19 06 15 / K C 19 08 16 / K C 19 10 07 / K C 19 11 33 43 St en od ac ty lu s p et ri i - 9 ** M 2 W . S ah ar a, M or oc co 25 k m W o f S m ar a K C 19 06 26 / K C 19 08 26 / K C 19 09 64 / K C 19 11 03 44 St en od ac ty lu s p et ri i - 1 0 E1 50 53 26 M au rit an ia O ue d C ho um , A dr ar K C 19 06 31 / K C 19 08 29 / K C 19 10 19 / K C 19 10 96 45 St en od ac ty lu s p et ri i - 1 1 M 17 8 B EV .T 36 92 W . S ah ar a, M or oc co 90 k m N W o f A ou ss er d K C 19 06 21 / K C 19 08 21 / K C 19 09 65 / K C 19 10 97 46 St en od ac ty lu s p et ri i - 1 2 M 18 0 B EV .1 08 42 W . S ah ar a, M or oc co 22 k m N W o f A ou ss er d K C 19 06 23 / K C 19 08 23 / K C 19 09 66 / K C 19 10 98 46 St en od ac ty lu s p et ri i - 1 3 M 18 2 B EV .1 08 44 W . S ah ar a, M or oc co 22 k m N W o f A ou ss er d K C 19 06 25 / K C 19 08 25 / K C 19 09 67 / K C 19 10 99 10 5 St en od ac ty lu s p et ri i - 1 4 E1 50 53 21 M au rit an ia 35 K m S o f B en ni ch ch ab K C 19 06 32 / - 10 5 St en od ac ty lu s p et ri i - 1 5 E1 50 53 22 M au rit an ia 35 K m S o f B en ni ch ch ab K C 19 06 33 / - 10 5 St en od ac ty lu s p et ri i - 1 6 E1 50 53 23 M au rit an ia 35 K m S o f B en ni ch ch ab K C 19 06 29 / - 10 6 St en od ac ty lu s p et ri i - 1 7 E1 50 53 24 M au rit an ia B en A m ira (A dr ar ) K C 19 06 20 / K C 19 08 20 10 7 St en od ac ty lu s p et ri i - 1 8 E1 50 53 29 M au rit an ia 20 K m W o f T m ei m ic ha t K C 19 06 28 / K C 19 08 28 44 St en od ac ty lu s p et ri i - 1 9 E1 50 53 25 M au rit an ia O ue d C ho um (A dr ar ) K C 19 06 30 / - 44 St en od ac ty lu s p et ri i - 2 0 E1 50 53 27 M au rit an ia O ue d C ho um (A dr ar ) K C 19 06 27 / K C 19 08 27 46 St en od ac ty lu s p et ri i - 2 1 M 17 9 B EV .1 08 41 W . S ah ar a, M or oc co 22 k m N W o f A ou ss er d K C 19 06 22 / K C 19 08 22 46 St en od ac ty lu s p et ri i - 2 2 M 18 1 B EV .1 08 43 W . S ah ar a, M or oc co 22 k m N W o f A ou ss er d K C 19 06 24 / K C 19 08 24 10 8 St en od ac ty lu s p et ri i - 2 3 E1 50 53 19 M au rit an ia 30 K m N o f Z ou er at K C 19 06 34 / - 10 9 St en od ac ty lu s p et ri i - 2 4 M 10 6 B EV .1 01 80 A lg er ia K C 19 06 14 / K C 19 08 15 11 0 St en od ac ty lu s p et ri i - 2 5 E1 50 53 28 W . S ah ar a, M or oc co Ti ff ar ity K C 19 09 16 / K C 19 08 17 11 1 St en od ac ty lu s p et ri i - 2 6 M 93 M C C 14 81 Eg yp t N S in ai , Z ar an ik N at io na l P ar k K C 19 06 08 / K C 19 08 09 35 St en od ac ty lu s p et ri i - 2 7 M 3 B EV .8 98 6 Eg yp t W ad i E l N at ru n K C 19 06 07 / K C 19 08 08 35 St en od ac ty lu s p et ri i - 2 8 M 5 B EV .8 98 8 Eg yp t W ad i E l N at ru n K C 19 06 04 / K C 19 08 05 25 St en od ac ty lu s p et ri i - 2 9 E1 50 53 17 Eg yp t El O m ay ed p ro te ct ed a re a K C 19 06 06 / K C 19 08 07 11 2 St en od ac ty lu s p et ri i - 3 0 M 94 M C C 14 80 -1 Eg yp t N S in ai , n ea r B ir el A bd K C 19 06 09 / K C 19 08 10 11 3 St en od ac ty lu s p et ri i - 3 1 E1 50 53 18 Eg yp t N S in ai , Z ar an ik N at io na l P ar k K C 19 06 05 / K C 19 08 06 11 4 St en od ac ty lu s p et ri i - 3 2 M 10 2 M or oc co 15 K m S o f E rf ou d K C 19 06 18 / - 53 St en od ac ty lu s p et ri i - 3 3 M 92 M C C 13 29 Tu ni si a 6 km W o f N ef ta K C 19 06 36 / K C 19 08 30 / K C 19 09 61 / K C 19 11 04 54 St en od ac ty lu s p et ri i - 3 4 E1 50 53 31 Tu ni si a 6 km W o f N ef ta K C 19 06 39 / K C 19 08 33 / K C 19 09 62 / K C 19 11 00 54 St en od ac ty lu s p et ri i - 3 5 E1 50 53 32 Tu ni si a 6 km W o f N ef ta K C 19 06 37 / K C 19 08 31 / K C 19 09 63 / K C 19 11 01 11 7 St en od ac ty lu s p et ri i - 3 6 M 97 M C C 13 35 Tu ni si a 34 k m S o f H az ou a K C 19 06 38 / K C 19 08 32 54 St en od ac ty lu s p et ri i - 3 7 M 1 Tu ni si a 6 K m w es t o f N ef ta K C 19 06 35 / - 62 Paper 1 47 St en od ac ty lu s p ul ch er - 1* * M 12 2 Y em en M uk ka lla K C 19 07 00 / K C 19 08 94 / K C 19 10 02 / K C 19 11 19 48 St en od ac ty lu s p ul ch er - 2 M 13 0 Y em en A l R ay an K C 19 07 01 / K C 19 08 95 / K C 19 10 03 / K C 19 11 20 49 St en od ac ty lu s s le vi ni - 1* * M 19 Jo rd an A ba r a l H az im K C 19 06 80 / K C 19 08 74 / K C 19 09 50 / K C 19 10 57 13 St en od ac ty lu s s le vi ni - 2 M 18 4 B EV .1 08 84 Jo rd an B et w ee n A qa ba a nd W ad i R um K C 19 06 82 / K C 19 08 76 / K C 19 09 48 / K C 19 11 32 13 St en od ac ty lu s s le vi ni - 3 M 18 7 Jo rd an B et w ee n A qa ba a nd W ad i R um K C 19 06 83 / K C 19 08 77 / K C 19 09 49 / K C 19 10 58 50 St en od ac ty lu s s le vi ni - 4 M 20 B EV .1 00 65 K uw ai t Sa ba h A l-A hm ed N at ur al R es er ve K C 19 06 85 / K C 19 08 79 / K C 19 10 14 / K C 19 10 59 51 St en od ac ty lu s s le vi ni - 5 M 12 7 Q at ar 26 k m S E of A s S al w a K C 19 06 89 / K C 19 08 83 / - / - 52 St en od ac ty lu s s le vi ni - 6 E1 50 53 34 U A E Ta ba l D an i K C 19 06 87 / K C 19 08 81 / K C 19 09 93 / K C 19 10 60 11 5 St en od ac ty lu s s le vi ni - 7 E1 50 53 35 U A E Ta ba l D an na h (U A E) K C 19 06 88 / K C 19 08 82 13 St en od ac ty lu s s le vi ni - 8 M 18 3 B EV .1 08 83 Jo rd an B et w ee n A qa ba a nd W ad i R um K C 19 06 81 / K C 19 08 75 13 St en od ac ty lu s s le vi ni - 9 M 18 8 B EV .T 37 62 Jo rd an B et w ee n A qa ba a nd W ad i R um K C 19 06 84 / K C 19 08 78 11 6 St en od ac ty lu s s le vi ni - 10 M 38 B EV .T 15 01 K uw ai t R at qa , K uw ai t-I ra k bo rd er s K C 19 06 86 / K C 19 08 80 13 7 St en od ac ty lu s s te nu ru s* * M 19 9 C U P\ R EP T\ LI B \1 43 Tu ni si a C ha ff ar K C 19 07 13 / K C 19 09 14 / K C 19 10 26 / K C 19 11 34 25 St en od ac ty lu s s th en od ac ty lu s - 1 ** M 22 Eg yp t El O m ay ed p ro te ct ed a re a K C 19 05 52 / K C 19 07 62 / K C 19 09 90 / K C 19 10 70 55 St en od ac ty lu s s th en od ac ty lu s - 2 M 64 B EV .7 21 9 Eg yp t 10 k m N o f H ur gh ad a, E l G ou na K C 19 05 24 / K C 19 07 37 / K C 19 10 15 / - 56 St en od ac ty lu s s th en od ac ty lu s - 3 M 70 B EV .8 99 0 Eg yp t W ad i G ha ra nd al , S in ai K C 19 05 26 / K C 19 07 39 / K C 19 09 68 / K C 19 10 66 57 St en od ac ty lu s s th en od ac ty lu s - 4 M 73 B EV .9 02 7 Eg yp t 26 k m S -S W o f B ee r A br aq K C 19 05 43 / K C 19 07 54 / K C 19 09 71 / K C 19 10 67 58 St en od ac ty lu s s th en od ac ty lu s - 5 M 12 3 Eg yp t Fa ra fr a O as is K C 19 05 50 / K C 19 07 60 / K C 19 10 10 / K C 19 10 68 59 St en od ac ty lu s s th en od ac ty lu s - 6 M 17 2 B EV .1 03 70 Eg yp t A bu S im be l K C 19 05 48 / K C 19 07 58 / K C 19 09 72 / K C 19 10 69 37 St en od ac ty lu s s th en od ac ty lu s - 7 M 16 1 H U JR -2 37 93 Is ra el N W N eg ev sa nd s, O vi tz fi el d H al uz za K C 19 05 22 / K C 19 07 35 / K C 19 09 89 / K C 19 10 76 60 St en od ac ty lu s s th en od ac ty lu s - 8 M 17 1 B EV .1 01 99 Is ra el 5 km S -S W o f B ok er , N eg ev K C 19 05 29 / K C 19 07 42 / K C 19 09 82 / K C 19 10 84 61 St en od ac ty lu s s th en od ac ty lu s - 9 M 19 2 B EV .T 39 51 Jo rd an 8 km o f A d- D ur a, Sa ud i A ra bi a bo rd er s K C 19 05 55 / K C 19 07 65 / K C 19 09 83 / K C 19 10 71 63 St en od ac ty lu s s th en od ac ty lu s - 1 1 M 13 6 N H M C 80 .3 .8 8. 46 Li by a 20 k m W o f D er j o as is K C 19 05 41 / K C 19 07 52 / K C 19 09 73 / K C 19 10 81 64 St en od ac ty lu s s th en od ac ty lu s - 1 2 M 19 3 B EV .T 41 35 A lg er ia O ue d D id er , A gu el m an e A ss ar K C 19 05 37 / K C 19 07 49 / K C 19 09 69 / K C 19 10 82 65 St en od ac ty lu s s th en od ac ty lu s - 1 3 M 11 7 M au rit an ia 18 k m N W o f B ai e d' A rg ui n K C 19 05 40 / K C 19 07 51 / K C 19 09 75 / K C 19 10 90 63 Paper 1 66 St en od ac ty lu s s th en od ac ty lu s - 1 4 M 11 8 M au rit an ia 5 km S o f T îg ja fâ t K C 19 05 34 / K C 19 07 47 / K C 19 09 74 / K C 19 11 09 67 St en od ac ty lu s s th en od ac ty lu s - 1 5 M 17 3 B EV .1 08 32 W . S ah ar a, M or oc co 11 8 km N W o f A ou ss er d K C 19 05 54 / K C 19 07 64 / K C 19 09 87 / K C 19 10 91 68 St en od ac ty lu s s th en od ac ty lu s - 1 6 M 17 4 B EV .1 08 33 W . S ah ar a, M or oc co 42 k m N W o f A ou ss er d K C 19 05 33 / K C 19 07 46 / K C 19 09 86 / K C 19 10 89 69 St en od ac ty lu s s th en od ac ty lu s - 1 7* * M 17 5 B EV .1 08 34 W . S ah ar a, M or oc co 59 k m N W o f A ou ss er d K C 19 05 31 / K C 19 07 44 / K C 19 09 85 / K C 19 10 88 70 St en od ac ty lu s s th en od ac ty lu s - 1 8 M 87 M au rit an ia 17 0 km E o f B ou L en oi r K C 19 05 32 / K C 19 07 45 / K C 19 09 84 / K C 19 10 87 11 8 St en od ac ty lu s s th en od ac ty lu s - 1 9 M 60 B EV .2 41 0 M au rit an ia 26 k m S o f C ho tt B ou l K C 19 05 38 / - 11 9 St en od ac ty lu s s th en od ac ty lu s - 2 0 M 53 B EV .2 41 1 M au rit an ia 70 k m E -N E A kj ou jt K C 19 05 30 / K C 19 07 43 70 St en od ac ty lu s s th en od ac ty lu s - 2 1 M 10 9 M au rit an ia In êl , 5 km S o f D ak hl et -N ou âd hi bo u - / K C 19 09 06 12 0 St en od ac ty lu s s th en od ac ty lu s - 2 2 M 86 M au rit an ia K C 19 05 39 / K C 19 07 50 12 0 St en od ac ty lu s s th en od ac ty lu s - 2 3 M 10 8 M au rit an ia B oû L an ou âr , 1 7k m E o f D ak hl et - N ou âd hi bo u - / K C 19 09 07 12 1 St en od ac ty lu s s th en od ac ty lu s - 2 4 E1 50 53 36 Eg yp t N o f G eb el E lb a K C 19 05 42 / K C 19 07 53 12 2 St en od ac ty lu s s th en od ac ty lu s - 2 5 M 72 B EV .9 00 4 Eg yp t 7 km N E of A bu S im be l K C 19 05 49 / K C 19 07 59 12 3 St en od ac ty lu s s th en od ac ty lu s - 2 6 M 66 B EV .7 22 2 Eg yp t A bu S im be l K C 19 05 46 / K C 19 07 56 12 3 St en od ac ty lu s s th en od ac ty lu s - 2 7 M 68 B EV .7 24 2 Eg yp t A bu S im be l K C 19 05 47 / K C 19 07 57 12 4 St en od ac ty lu s s th en od ac ty lu s - 2 8 E1 50 53 45 Eg yp t R ed S ea C oa st , E gy pt K C 19 05 44 / - 12 5 St en od ac ty lu s s th en od ac ty lu s - 2 9 E1 50 53 47 Eg yp t W ad i ‘ A db a l M al ik K C 19 05 45 / K C 19 07 55 55 St en od ac ty lu s s th en od ac ty lu s - 3 0 M 65 B EV .7 22 0 Eg yp t 10 k m N o f H ur gh ad a, E l G ou na K C 19 05 25 / K C 19 07 38 12 6 St en od ac ty lu s s th en od ac ty lu s - 3 1 M 62 B EV .7 21 5 Eg yp t Fe ira n O as is , S in ai K C 19 05 28 / K C 19 07 41 56 St en od ac ty lu s s th en od ac ty lu s - 3 2 M 71 B EV .8 99 1 Eg yp t W ad i G ha ra nd al , S in ai K C 19 05 27 / K C 19 07 40 12 7 St en od ac ty lu s s th en od ac ty lu s - 3 3 E1 50 53 46 Eg yp t Si w a O as is K C 19 05 36 / - 12 8 St en od ac ty lu s s th en od ac ty lu s - 3 4 M 14 6 N H M C 80 .3 .8 8. 37 Eg yp t W ad i S ud r, 10 k m S E of Q a'l at e l J un di - / K C 19 09 08 12 9 St en od ac ty lu s s th en od ac ty lu s - 3 5 M 63 B EV .7 21 6 Eg yp t 10 k m N o f A ïn S uk hn a K C 19 05 19 / K C 19 07 32 13 0 St en od ac ty lu s s th en od ac ty lu s - 3 6 M 61 B EV .7 21 4 Eg yp t 25 k m W o f S ue z K C 19 05 17 / K C 19 07 30 13 0 St en od ac ty lu s s th en od ac ty lu s - 3 7 M 67 B EV .7 24 1 Eg yp t 25 k m W o f S ue z K C 19 05 18 / K C 19 07 31 11 1 St en od ac ty lu s s th en od ac ty lu s - 3 8 M 90 M C C 14 79 Eg yp t N S in ai , Z ar an ik N at io na l P ar k K C 19 05 21 / K C 19 07 34 13 1 St en od ac ty lu s s th en od ac ty lu s - 3 9 M 89 M C C 14 49 Li by a G ha da m es K C 19 05 35 / K C 19 07 48 13 2 St en od ac ty lu s s th en od ac ty lu s - 4 0 E1 50 53 37 Eg yp t 30 k m N E of C ai ro K C 19 05 16 / K C 19 07 29 13 3 St en od ac ty lu s s th en od ac ty lu s - 4 1 M 69 B EV .8 98 9 Eg yp t W ad i E l N at ru n K C 19 05 20 / K C 19 07 33 13 4 St en od ac ty lu s s th en od ac ty lu s - 4 2 M 12 5 Eg yp t K C 19 05 23 / K C 19 07 36 25 St en od ac ty lu s s th en od ac ty lu s - 4 3 E1 50 53 40 Eg yp t El O m ay ed p ro te ct ed a re a K C 19 05 53 / K C 19 07 63 25 St en od ac ty lu s s th en od ac ty lu s - 4 4 E1 50 53 42 Eg yp t El O m ay ed p ro te ct ed a re a K C 19 05 51 / K C 19 07 61 62 St en od ac ty lu s s th . z av at ta ri i - 1 M 10 1 M C C R 13 72 K en ya B et w ee n G at ab a nd S ou th H or r, Sa m bu ru d is tr. K C 19 05 57 / K C 19 07 67 / K C 19 09 70 / K C 19 10 92 13 5 St en od ac ty lu s s th . z av at ta ri i - 2 M 95 M C C 12 97 -1 K en ya N or th H or r K C 19 05 56 / K C 19 07 66 64 Paper 1 71 St en od ac ty lu s y em en en si s - 1 ** M 12 6 Y em en M ok ka K C 19 06 40 / K C 19 08 34 / K C 19 09 91 / K C 19 10 64 72 St en od ac ty lu s y em en en si s - 2 M 12 8 Y em en N . Y uk ht al K C 19 06 41 / K C 19 08 35 / K C 19 09 92 / K C 19 10 65 13 8 St en od ac ty lu s y em en en si s - 3 M 20 6 Y em en N o f A de n K C 19 07 17 / K C 19 09 18 / K C 19 10 29 / K C 19 11 37 13 8 St en od ac ty lu s y em en en si s - 4 M 20 7 Y em en N o f A de n K C 19 07 18 / K C 19 09 19 13 8 St en od ac ty lu s y em en en si s - 5 M 20 8 Y em en N o f A de n K C 19 07 19 / K C 19 09 20 13 8 St en od ac ty lu s y em en en si s - 6 M 20 9 Y em en N o f A de n - / K C 19 09 21 13 9 St en od ac ty lu s y em en en si s - 7 M 21 3 Y em en N o f L ah j, W ad i T ub an K C 19 07 23 / K C 19 09 25 / K C 19 10 32 / K C 19 11 40 13 9 St en od ac ty lu s y em en en si s - 8 M 21 4 Y em en N o f L ah j, W ad i T ub an K C 19 07 24 / K C 19 09 26 O ut gr ou ps Ag am ur a pe rs ic a* * E1 80 11 01 7 Ir an D Q 85 27 26 / - / K C 19 10 25 / - Ar is te lli ge r g eo rg ee ns is * E2 80 51 1 B el iz e K C 19 09 34 / K C 19 09 27 / K C 19 10 43 / K C 19 11 41 Bu no pu s t ub er cu la tu s* * B un tu b K uw ai t EU 58 91 60 / - / A F1 48 70 6 / - C ro ss ob am on o ri en ta lis ** C ro or i In di a H M 92 11 59 / H M 04 09 44 / D Q 85 27 30 / - Eu le pt es e ur op ae a* E2 60 67 1 Ita ly K C 19 09 35 / - / K C 19 10 42 / K C 19 11 42 G ek ko g ec ko * G ek ge c C hi na /In do ne si a N C 00 76 27 / N C 00 76 27 / EF 53 49 39 / EF 53 49 81 G ek ko v itt at us * G ek vi t N C 00 87 72 / N C 00 87 72 / - / - H em id ac ty lu s f re na tu s* * H em fr e In do ne si a G Q 24 59 70 / G Q 24 59 70 / - / E F5 34 98 2 Ps eu do ce ra m od ac ty lu s k ho ba re ns is - 1* * M 16 B EV .1 00 39 K uw ai t W af ra h Fa rm s, 20 k m E o f W af ra h K C 19 07 03 / K C 19 08 97 / K C 19 10 05 / K C 19 11 23 Ps eu do ce ra m od ac ty lu s k ho ba re ns is - 2 M 37 B EV .1 00 40 K uw ai t W af ra h Fa rm s, 20 k m E o f W af ra h K C 19 07 02 / K C 19 08 96 / K C 19 10 04 / K C 19 11 22 Ps eu do ce ra m od ac ty lu s k ho ba re ns is - 3 M 19 6 O m an B ar r A l-H ic km an K C 19 07 04 / K C 19 08 98 / K C 19 10 06 / K C 19 11 24 Sa ur od ac ty lu s b ro ss et i* Sa ub ro M or oc co EU 01 43 00 / EF 56 40 06 / EF 53 49 28 / EF 53 49 70 Ta re nt ol a m au ri ta ni ca * Ta rm au Sp ai n/ Eg yp t N C 01 23 66 / N C 01 23 66 / EU 29 36 86 / EU 29 37 31 Ta re nt ol a de la la nd ii* M ta rd eT C an ar y Is la nd s Te ne rif e A F1 86 13 1 / K C 19 09 28 / K C 19 10 33 / K C 19 11 43 Ta re nt ol a de la la nd ii* M ta rd eP C an ar y Is la nd s La P al m a A F1 86 13 0 / K C 19 09 29 / K C 19 10 34 / K C 19 11 44 Ta re nt ol a bo et tg er i b oe ttg er i* E1 01 13 11 C an ar y Is la nd s G ra n C an ar ia A F1 86 12 5 / K C 19 09 30 / K C 19 10 35 / K C 19 11 45 Ta re nt ol a bo et tg er i b is ch of fi* E1 01 13 8 M ad ei ra Se lv ag en s A F1 86 12 8 / - / - / - Ta re nt ol a bo et tg er i h ie rr en si s* E1 01 13 10 C an ar y Is la nd s El H ie rr o K C 19 07 25 / - / K C 19 10 36 / K C 19 11 46 Ta re nt ol a bo et tg er i b oe ttg er i* E1 01 13 13 C an ar y Is la nd s G ra n C an ar ia A F1 86 12 3 / - / K C 19 10 37 / K C 19 11 47 65 Paper 1 Ta re nt ol a bo et tg er i b oe ttg er i* E1 01 13 14 C an ar y Is la nd s G ra n C an ar ia A F1 86 12 4 / - / K C 19 10 38 / K C 19 11 48 Te ra to sc in cu s s ci nc us * M te r1 JF B M 14 25 2 Tu rk m en is ta n K C 19 07 26 / K C 19 09 31 / K C 19 10 39 / K C 19 11 49 Te ra to sc in cu s r ob or ow sk ii* M te r2 C hi na K C 19 07 27 / K C 19 09 32 / K C 19 10 40 / K C 19 11 50 Te ra to sc in cu s m ic ro le pi s* M te r3 Pa ki st an K C 19 07 28 / K C 19 09 33 / K C 19 10 41 / K C 19 11 51 Tr op io co lo te s a lg er ic us - 1* * M T5 B EV .1 08 62 W . S ah ar a, M or oc co 17 5 km N W o f A ou ss er d K C 19 07 09 / K C 19 09 02 / - / - Tr op io co lo te s a lg er ic us - 2* * M T6 B EV .1 08 60 W . S ah ar a, M or oc co 11 0 km N W o f B ou dj ou r K C 19 07 10 / K C 19 09 03 / K C 19 10 23 / K C 19 11 29 Tr op io co lo te s t ri po lit an us - 1* * M T7 B EV .1 08 63 W . S ah ar a, M or oc co A ou ss er d K C 19 07 11 / K C 19 09 04 / K C 19 10 24 / K C 19 11 30 Tr op io co lo te s t ri po lit an us - 2* * M T8 B EV .9 02 5 Eg yp t W ad i E l N at ru n K C 19 07 12 / K C 19 09 05 / - / K C 19 00 31 Tr op io co lo te s n ub ic us ** M T9 B EV .9 02 0 Eg yp t A bu S im be l K C 19 07 06 / K C 19 09 00 / K C 19 10 20 / K C 19 11 25 Tr op io co lo te s s te ud ne ri ** M T1 0 B EV .1 03 67 Eg yp t 17 5 km S o f S af ag a K C 19 07 05 / K C 19 08 99 / K C 19 10 21 / K C 19 11 26 Tr op io co lo te s n at te re ri ** M T1 1 B EV .1 08 86 Jo rd an W ad i R um K C 19 07 08 / K C 19 09 01 / - / K C 19 00 28 Tr op io co lo te s s co rt ec ci i* * M T1 2 O m an C oa st , 2 50 k m E o f S al al ah K C 19 07 07 / - / K C 19 10 22 / K C 19 11 27 Sp ec im en s a re li st ed in a lp ha be tic al o rd er , w ith th e co rr es po nd in g G en B an k ac ce ss io n nu m be rs . S pe ci m en s i nd ic at ed w ith a n as te ris k (* ) w er e in cl ud ed in th e di ve rg en ce ti m e an al ys is , s pe ci m en s i nd ic at ed w ith a d ou bl e as te ris k (* *) w er e in cl ud ed b ot h in th e ph yl og en et ic a nd d iv er ge nc e tim e an al ys es , a nd sp ec im en s w ith no a st er is k w er e in cl ud ed in th e ph yl og en et ic a na ly se fs o nl y. L oc al ity c od es re fe r t o Fi gu re 1 . V ou ch er c od es o f s pe ci m en s a va ila bl e in c ol le ct io ns re fe r t o th e fo llo w in g co lle ct io ns : B EV .[X ]: La bo ra to ire d e B io gé og ra ph ie e t É co lo gi e de s V er té br és d e l'É co le P ra tiq ue d es H au te s E tu de s, M on tp el lie r, Fr an ce ; N H M C .[X ]: N at ur al H is to ry M us eu m o f C re te , G re ec e; M C C [X ]: M us eo C iv ic o di S to ria N at ur al e di C ar m ag no la , I ta ly ; H U JR -[ X ]: N at io na l N at ur al H is to ry C ol le ct io ns o f t he H eb re w U ni ve rs ity o f J er us al em , I sr ae l; C U P[ X ]: C ha rle s U ni ve rs ity , P ra gu e; JF B M [X ]: Ja m es F or d B el l M us eu m , A m ph ib ia n an d R ep til e C ol le ct io n, U ni ve rs ity of M in ne so ta . C od es B EV .T [X ] r ef er to th e tis su e co lle ct io n of L ab or at oi re d e B io gé og ra ph ie e t É co lo gi e de s V er té br és d e l'É co le P ra tiq ue d es H au te s E tu de s, M on tp el lie r, Fr an ce . 66 Paper 1 S. pulcher S. arabicus S. leptocosymbotes S. doriae S. slevini S. grandiceps S. affinis S. petrii iirtep .S S. yemenensis sucinatiruam .S sulytcadonehts .S S. stenurus 100/* 96/< 93/* 83/* 95/* 99/* 74/* 100/* 100/* 100/* 100/* 100/* 100/* 99/* 99/* 95/* 86/* 87/* 100/* 100/* 100/* 100/* 100/* 100/* 100/* 100/* 100/* 100/* 100/* 100/* 79/* 74/* 98/* 75/* 90/* 87/* 2>5>1, relative length of toes 4>3>5>2>1; 4th finger length (from inser- tion of 3rd finger, claw included) = 9.5 mm; fourth toe VGS 65 62 SL l ⁄ r 7 ⁄ 7 8 ⁄ 8 LF4 l ⁄ r 17 ⁄ 16 17 ⁄ 17 LT4 l ⁄ r 22 ⁄ 25 22 ⁄ 23 Dorsal keels 2–3 2–3 Supranasals arrangement (C) (C) Prefrontals arrangement C C Supraocul. ent. frontal I–II I–II ⁄ I–II-(III) Tarsal scales B B Palmar scales B B Foot lamellae S S Hand lamellae S WK Morphobank images M85683–M85688 M85867–M85872 SVL, snout-vent length; TotL, total length; HL1, head length from tip of snout to posterior border of ear opening; HL, head length from tip of snout to posterior border of parietals; HW, head width at level of ears; EST, distance between anterior margin of eye to tip of snout; EED, distance between posterior margin of eye to anterior margin of ear; MSR, number of midbody scale rows; SC, number of subcaudals; SL, number of supralabials; LF4 l ⁄ r, number of subdigital lamellae under 4th finger (left ⁄ right); LT4 l ⁄ r, number of subdigital lamellae under 4th toe (left ⁄ right); Dorsal keels: number of dorsal keels; Supranasals arrangement: arrangement of supranasals; S, separated; (C), in short contact; C, in broad contact; Prefrontal arrangement: arrangement of prefrontals; S, separated; (C), in short contact; C, in broad contact; Supraocul. ent. frontal: supraoculars entering the frontal scale; Tarsal scales: shape of tarsal scales (foot); F, flat; G, granular; B, blunt tubercles; P, pointed tubercles; S, spinose (mucronated) tubercles; (S), slightly spinose; Palmar scales: shape of palmar scales (hand); F, flat; G, granular; P, pointed tubercles; S, spinose (mucronated) tubercles; (S), slightly spinose; Foot lamellae: shape of lamellae under the 4th toe; Hand lamellae: number of lamellae under the 4th finger. Morphobank images: Morphobank codes (project P461) for the pictures of the T. cristinae specimens. The Trachylepis skinks of the Socotra Archipelago d Sindaco et al. 356is, the parietal overlaps the upper anterior temporal. pair of almost smooth nuchal shields (the right almost ed), each with three rows of cycloid scales homologue uchal ones; any secondary nuchals present (sensuMabuya socotrana – Ro¨sler & Wranik 2004: 524 (par- tim): ‘Abd Al Kuri Island: 1212¢N 5215¢E, and slope Jabal Salih, 1210¢N 5215¢E, 100–450 m, 17- 19.II.1999¢. Etymology. The species epithet is a genitive Latin noun to honour Cristina Grieco, who found the holotype in Abd Al Kuri. Diagnosis. A member of the Trachylepis brevicollis complex characterized by high number of scale rows at midtrunk (38), palmar and tarsal scales never pointed nor spinose, subdigital lamellae smooth or only weakly keeled; color characteristic (see description); SVL of adults up to 114 mm. Description of the holotype specimen. (Fig. 2b–c; Appendix S6 and S7 a–c; Table 2; Morphobank pictures M85683– M85688). Measurements and scale counts are presented in Table 2. Adult specimen (a male) with robust and slightly depressed body; snout-vent length = 114 mm. Head equally longer than broad (ratio 1.05), slightly depressed, relatively pointed, slightly broader than the neck region at the level of the ear openings. Lower eyelid with a distinct transparent oval window. Ear openings not covered by enlarged scales, the left vertically oval and the right a verti- cal slit (an abnormality). Snout slightly convex in profile, with a relatively large nostril sunk into the rear edge of the nasal; a small postnasal, separated from the nostril by the rim of the nasal. Supranasals with a short median contact suture. Internasal (frontonasal) distinctly pointed ante- riorly. Prefrontals pentagonal, distinctly pointed poste- riorly, with a median contact suture long 1 ⁄2 of their length. Two undivided loreals, of which the larger, posterior one, borders the first loreal, the prefrontal, two supralabials (3rd and 4th), the first supraciliar, and a preocular; the latter is followed by a single pre-subocular. Supraciliaries five, the second partly coalescent with the first supraocular. Pretem- poral two, both contacted by parietal. Sub-postocular two, upper contacting lower pretemporal. Frontal nearly as long as the distance between its anterior tip and tip of snout, laterally in contact with two supraoculars (1st and 2nd). Frontoparietal shields in contact between them and with 2nd, 3rd (the left also with the 4th) supraoculars. Parietal shields completely separated by a very elongated interparie- tal shield, without visible parietal eye. Primitive condition of overlap pattern between parietal scale and upper anteriorª 2012 The Authors d Zoologica S 275Table 2 Measurements and scale counts of the type specimens of Trachylepis cristinae, sp. nov. (all measurements in mm) MCCI-R1585 NMW-9396 Sex M F SVL 114 110 TotL 220 175 HL 20.6 20.6 HW 19.5 18.2 Ratio HL ⁄ HW 1.05 1.13 EST 12.0 10.6 EED 11.3 10.0 MSR 38 38cripta ª 2012 The Norwegian Academy of Science and Letters, 41, 4, July 2012, pp 346–362 Sindaco et al. d The Trachylepis skinks of the Socotra Archipelagolength (from insertion of 5th toe, claw inclu- ded) = 14.5 mm; subdigital lamellae under the fourth toe 22 ⁄25 (left ⁄ right), flat and smooth. Subdigital lamellae un- keeled. Palmar scales blunt, tarsal scales are blunt or obtuse. Raised tubercles never pointed. Dorsal and ventral scales are clearly imbricating. Thirty-eight rows of scales at midbody. Sixty-five smooth gulars + ventrals from postmentals to vent. Scales at the back and upper flanks each bearing two or three distinct keels. Scales and shields of the head, scales of the under- surfaces of head, belly, most of tail (except proximal dorsal tail scales) as well as those of the lower flanks unkeeled. The scales of the dorsum of the fore and hind limbs are weakly bi- or tricarinate, or almost smooth. Tail with a 81-mm-long regenerated tip with total length (106 mm) slightly shorter than SVL, its basal part rectangular (height = 12 mm, width = 18 mm), and the distal half clearly laterally compressed. Coloration in life. Dorsal parts brownish with indistinct black spots, attributed to the dark ground color of the scales, blackish with light brown along the posterior mar- gin. Upper parts of the head brownish, with blackish nuances, particularly along sutures of the head plates and the tip of the snout. Upper parts of the head brownish, with margins of the plates irregularly edged with black, and tip of the snouth (rostral, nasals and supranasals) black. Sides of the head brownish, with temporals, post- suboculars and posterior supralabials mostly black, as well as the neck (roughly under a line connecting the ear ope- ning with the eye). Underparts of the head bicolor: black anteriorly (chin, infralabials and anterior gular shields) and on the sides, reddish (with black spots) posteriorly. Flanks, neck and sides of the original tail with rather distinct irregular black vertical bands (13–14 between axilla and groin), 2–3 scales wide, sometimes fused, alternating with narrow indistinct whitish-orange bands; the black bands are more irregular on the sides of the neck. Belly, under- parts of limbs, and ventral side of the original tail reddish; regenerated tail uniformly blackish. Iris pink-beige, with black mottling; pupil black, highly irregularly shaped. Weight in life: 39.5 g. (paratype) (Appendix S7 d–f; Table 2; Morphobank: M85867-M85872). Variation in sca- lation and body measurements of the NMW specimen is reported in Table 2; it differs from the type specimen by the following variations. Relative length of fingers 4>3>2>5>1. Tail with a 65-mm-long regenerated tip, its distal half not clearly compressed. Ratio between length and width of the head = 1.37. Ear opening oval, about 2.5 times as high as wide, with one small anterior rounded lobule. The first lor- eal on the right side is divided vertically. Frontal laterally in contact with 1st and 2nd supraoculars (right side) and inª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 27short contact with the 3rd (left). Interparietal with visible parietal eye. Nuchals undivided. The postmental plate con- tacts in a point the third left infralabial. Subdigital lamellae weakly keeled. Pattern and color in this one century old pre- served specimen are similar to the type, but slightly faded; the belly is whitish. Comparison with other species. For comparison with Trachylepis socotrana and T. brevicollis see Fig. 2e-l; Appen- dix S2; and Morphobank project P461. Although not clo- sely related to it, in Appendix S8, we list the main differences between T. cristinae sp. nov. and other Arabian, East and Southern African species of Trachylepis. Informa- tion on the morphology of these species was obtained from Lanza & Carfi (1968); Largen & Spawls (2006); Arnold (1986); Spawls et al. (2002); Broadley & Howell (1991); Gu¨nther et al. (2005). Trachylepis socotrana, the only other Trachylepis of the Socotra Archipelago, is easily distinguishable from T. cristinae by its smaller size (SVL up to 97 mm, but usually less in adult individuals, with a mean of 84.4 ± 7.6; N = 20; Appendix S1 and unpublished field data), by having prefrontals separated or in short contact (largely in contact in T. cristinae), frontal scale entering the supraoculars (I)-II-III (only the supraoculars I and II in T. cristinae), internasal (frontonasal) scale pointed posteriorly (truncated posteriorly); frontoparietals in contact with the supraocu- lars III and IV [in contact with supraoculars II-III-(IV) in T. cristinae] (Fig. 2); most dorsal scales with 3–5 keels (2 or 3 in T. cristinae), 30–34 scales around midtrunk (data from Appendix S2 and from Boulenger 1903: 85) (38 in T. cristinae). Moreover, T. socotrana has a completely different color pattern, usually uniformly brownish-grey; some adults have orange head, and some specimens bear scattered dark spots on the throat. Some specimens from Darsa Island have a striped pattern of five longitudinal blackish stripes (one vertebral, two dorsolateral and two lateral), alterna- ting with two beige broader paravertebral ones and two evident withish dorsolateral ones (MCCI-R1583). Trachylepis cristinae sp. nov. differs from members of the T. brevicollis complex in the following characteristics: 38 scales at midtrunk (between axilla and groin), versus 30–37 (usually 32–34; mean 33.4 ± 1.71, N = 33) recorded in the Arabian populations of T. brevicollis (Tornier 1905: 386; Anderson 1895: 648; Fritz & Schu¨tte 1988: 44–49; original data) and 29–35 (usually 30–33, depending on populations) in Africa (Lanza & Carfi 1968: 214-216, Fritz & Schu¨tte 1988: 44–49, original data). The subdigital lamellae under the toes, which are smooth or only weakly keeled in T. cristinae but always keeled in both Arabian and African specimens, seem to be a good character. The number of subdigital lamellae is higher in the two known specimens41, 4, July 2012, pp 346–362 357 6 The Trachylepis skinks of the Socotra Archipelago d Sindaco et al.of T. cristinae (4th finger = 16–17; 4th toe = 22–25) and in Arabian T. brevicollis (4th finger = 13–17, mean = 15.24 ± 1.07; 4th toe = 16–23, mean = 19.71 ± 1.55, N = 34) than in African T. brevicollis (4th fin- ger = 12–15, mean = 13.54 ± 1,14; 4th toe = 15–19, mean = 17.11 ± 1,05; N = 27). The appearance of the palmar and tarsal scales, which are smooth in Abd Al Kuri speci- mens, is diagnostic only with respect to African specimens (spinose in almost all African specimens examined; N = 27), whereas they are smooth in most of the Arabian specimens studied (in 100% of specimens from Oman and the Hadra- maut; N = 9). As in most Arabian specimens (65%; N = 34), the supranasals are in close contact in T. cristinae, whereas in African specimens they are mostly separated (40.1%), in contact over only a small portion of the scale (22.2%) or, more rarely, in close contact (37%) in the specimens studied (N = 27). Furthermore, the prefrontal scales are in close contact in T. cristinae, whereas they are usually separated (52.9%) or have a narrow suture (20.6%) in Arabian T. brevicollis (N = 34); similar frequencies also occur in African spe- cimens, where the prefrontals are separated in 48.1%, in slight contact in 25.9% and in close contact in 25.1% of the specimens examined (N = 27). Other head scalation charac- teristics (i.e. supraoculars entering the frontal scale), the shape and size of the ear opening and the number and shape of the ear opening lobules, appear to be very variable amongst individuals and populations. The coloration pattern seems to be very distinct as all African T. brevicollis specimens examined are characterized by a light color, with or without dark blotches, ocelli on the back, or white spots on the head and trunk. The dis- tinctive lateral dark sidebands found in T. cristinae sp. nov. have also been observed in two specimens from Gardo, Somalia (MZUF 10138, 10154), although all NE African specimens studied lack the extensive dark coloration on the head and throat. Arabian specimens also exhibit con- siderable variation. Thus, according to Arnold (1980: 313) ‘even in Arabia there are differences from place to place as well as individual variation; in Dhofar a broad, light, dor- solateral band is present and females have a series of dark blotches on the dorsum; males are uniform but have black- ish heads with small light spots on the body’, and in some areas, animals are often very dark. The specimens exam- ined often show whitish spots, dark blotches and ocelli, and their throats are light, more or less distinctly striped longitudinally or even mottled. The head shape of T. cristinae is also characteristic: the snout is longer and more slender than in T. brevicollis in both sexes when viewed from above, with the sides slightly concave between the eye and nostril; this feature is even more evident in the jaw when observed from below (Appendix S7 c, f).358 ª 2012 The Authors d Zoologica S 277Discussion As first suggested by Mausfeld & Schmitz (2003) and Carranza & Arnold (2003) and confirmed by the results presented here (see Fig. 3), the phylogeny of Mabuya sensu lato includes five well-differentiated lineages that are clas- sified into four different genera. While Mausfeld & Sch- mitz (2003) proposed a monophyletic Trachylepis, which would include both the Afro-Malagasy as well as the Mid- dle East species, they also already suggested that the Mid- dle East species may still represent a distinct radiation and thus would require a separate genus to be erected. Although Mausfeld & Schmitz (2003) recovered the monophyly of Trachylepis in their ML tree (Fig 2, Maus- feld & Schmitz 2003), the bootstrap support for this assemblage was very low (54%), and the tree only included two of four genera of Mabuya sensu lato (Eutropis and Trachylepis) and a very limited taxon sampling within Trachylepis (three species from the Middle East clade and seven from the Afro-Malagasy clade). The phylogenetic tree presented in Fig. 3 has been inferred with the most complete dataset of Mabuya sensu lato assembled to date (Morphobank Doc1), including a complete taxon sampling at the generic level and all the representatives of Trachylepis from GenBank for which the 12S and 16S mtDNA regions were available (see Table 1). Although the results suggest that Trachylepis is polyphy- letic (Fig. 3), the constraint analyses (see results section) do not rule out a monophyletic Trachylepis including both the Middle East and Afro-Malagasy clades. Pending fur- ther data, we also preliminary consider the Middle East species to be part of the Trachylepis radiation. It seems clear from Fig. 3 that the two mtDNA regions used in the present analyses cannot resolve the phylogenetic relation- ships at the base of the Mabuya sensu lato tree, thus mean- ing that until more data, including nuclear genes, are added, it will not be possible to satisfactorily resolve the taxonomy and the deep level evolutionary relationships of this complex. The present study shows that the Socotra Archipelago was independently colonized by members of the Trachylepis brevicollis complex twice (see Fig. 3) and that these pro- cesses gave rise to two independent species: T. socotrana, which inhabits the islands of Socotra, Darsa and Samha, and a second species, T. cristinae sp. nov., which is ende- mic to Abd Al Kuri. According to our phylogenetic analy- ses, these two colonization events took place at different times, approximately 10.5 (7.3–13.8) mya for T. socotrana, and 2.9 (1.5–4.5) mya for T. cristinae. These events are posterior to the late Oligocene ⁄ early Miocene, the time that has been suggested for the onset of the drifting of the Socotra Archipelago from its original position in the Dhofar region, Southern Oman (see introduction).cripta ª 2012 The Norwegian Academy of Science and Letters, 41, 4, July 2012, pp 346–362 Sindaco et al. d The Trachylepis skinks of the Socotra ArchipelagoThe geological origin of the archipelago, together with the calibrated phylogenetic tree presented in Fig. 3, there- fore suggest that, contrary to other Socotran reptiles (Nagy et al. 2003; unpublished data), the origin of the two Trachylepis species is not the result of vicariance. According to several authors (Laughton 1966; Laughton et al. 1970; Samuel et al. 1997; Fleitmann et al. 2004; Bosworth et al. 2005; Autin et al. 2010; amongst others), by the time of the inferred colonization events of the Socotra Archipelago by Trachylepis, the islands had already drifted close to their actual position in the Arabian Sea, thus meaning that the two colonization events must have been the result of dis- persal. Long-distance transmarine colonization events are not rare in reptiles (Carranza et al. 2000; see de Queiroz 2005 for a review), and, together with some gecko groups, the skinks of the Mabuya sensu lato complex are amongst the best dispersers. For instance, they have naturally crossed the Atlantic Ocean on two occasions (Mausfeld et al. 2002; Carranza & Arnold 2003; Miralles & Carranza 2010) and colonized multiple archipelagoes across their large distribution range, including the Cape Verde islands (Carranza et al. 2001; Miralles et al. 2011) and the islands of the Gulf of Guinea (Jesus et al. 2005a,b). Although the island of Abd Al Kuri is closer to mainland Africa than to Arabia, Trachylepis cristinae seems to be phy- logenetically and morphologically more closely related to T. brevicollis from Oman than to any African Trachylepis (Fig. 3). In fact, the two T. brevicollis specimens from Africa included in our analyses appear to be more closely related to the recently described species T. dichroma from Tanzania (Gu¨nther et al. 2005) than to the single Omani T. brevicollis included in Fig. 3. These results suggest that T. brevicollis might be polyphyletic, with Arabian and African popula- tions being unrelated, a hypothesis that has already been proposed by other authors on the basis of morphological evidence (Lanza 1983; Fritz & Schu¨tte 1988; Scha¨tti & Gasperetti 1994). A constrained phylogeny in which T. brevicollis was forced to be monophyletic was not rejected by either the AU or the SH tests (see results), thus indicating that, at least from a molecular point of view, the monophyly of T. brevicollis cannot be rejected and that more data are therefore needed. However, our results sug- gest that the taxonomic status of the Arabian and African T. brevicollis needs to be reassessed (Scha¨tti & Gasperetti 1994) as current data are insufficient to establish whether more than one species is present in Arabia (Fritz & Schu¨tte 1988) and whether animals genetically similar to African T. brevicollis also occur on the Arabian side of the Red Sea. Many names are available for both the Arabian and African populations: Euprepes brevicollis Wiegmann, 1837 (type locality ‘Abyssinia’ = Ethiopia), Euprepes pyrrhocepha- lus Wiegmann, 1837 (type locality: ‘in Aschik, insula marisª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 27rubri’ [=Ashik Island, Red Sea, Saudi Arabia]), Tiliqua bur- toni Blyth, 1856 (type locality: ‘Somali Country’), Mabuya chanleri Stejneger, 1893 (type locality: ‘Tana River, East Africa’ [now Kenya]), Mabuia (sic) Rotschildi Mocquard, 1905 (type locality: ‘Endessa (Abyssinie)’), Mabuya pulchra Matschie, 1893 (type locality: ‘Scadi prope Lahadsch’ [=‘Scadi’, close to Lahej, north of Aden]), Mabuya somalica Calabresi, 1915 (type locality ‘Barde`ra [and] Gorie`i’, Somalia). As stated by Scha¨tti & Gasperetti (1994): ‘the uncertainty surrounding these names cannot be elucidated without a study of the type material; finally, it cannot be ruled out that the type locality of M. brevicollis (‘Abys- sinia’) might be in error’. Owing to the morphological plasticity of members of the T. brevicollis complex, a genetic analysis of specimens coming from the type locali- ties of all of the described taxa would appear to be neces- sary to solve the systematics of the group and draw species boundaries. With the description of T. cristinae sp. nov., all five spe- cies of terrestrial reptiles present on Abd Al Kuri can now be considered to be endemic, thus highlighting the impor- tance of this small island from a biodiversity point of view. Although Abd Al Kuri lies just 66 km to the west of Samha and 105 km from Socotra (Fig. 1), our analyses clearly show that the ancestor of T. cristinae is more closely related to T. brevicollis from Southern Arabia (more than 350 km to the north) than to T. socotrana from these neighbouring islands. This biogeographical pattern is also found in other groups, especially Mesalina, with M. kuri from Abd Al Kuri being more closely related to mainland Arabian Mesalina than to the neighbouring Mesalina balfouri from Samha and Socotra (Joger & Mayer 2002), in the two Hemidactylus (H. forbesi and H. oxyrhinus, unpublished data), and in Pristu- rus abdelkuri (Papenfuss et al. 2009), although in this latter case it is not clear whether Abd Al Kuri was colonized from mainland Arabia or from Africa. At present, there are two annual monsoons that affect the Socotra Archipelago: the south-west monsoon, which blows from early June to early October, and the north-east monsoon, which blows from April to May. These two monsoons also affect the direction of the oceanic currents, which change direction depending on the monsoons. During winter, the flow of the upper ocean is directed westwards from near the Indonesian Archipelago to the Arabian Sea. During summer, the direc- tion reverses, with eastwards flow extending from Somalia into the Bay of Bengal (Shankar et al. 2002). According to the results of the present work and what has been inferred from other groups such as Mesalina and Hemidactylus (Joger & Mayer 2002; unpublished results), it is suggested that the westwards winter monsoon current may have played a very important role in the transmarine colonization of the Soco- tra Archipelago by several groups.41, 4, July 2012, pp 346–362 359 8 The Trachylepis skinks of the Socotra Archipelago d Sindaco et al.Acknowledgements Heinz Grillitsch, Richard Gemel and Silke Schweiger (Na- turhistorisches Museum, Wien) kindly helped us to exam- ine the collections of the Museum of Vienna; Annamaria Nistri and Stefano Vanni have made available the collec- tions of the Museo Zoologico ‘La Specola’ in Florence. Stefano Scali (Museo Civico di Storia naturale di Milano) provided pictures of specimens of T. brevicollis held in the Milan Museum. This research was accomplished within the framework of the programmes ‘Socotra Conservation and Development’ by United Nations Development Program, and ‘Capacity Development for Soqotra Archipelago Con- servation’ by the Italian Cooperation. Thanks are due to PROGES s.r.l. and to the Environment Protection Agency of Socotra for their support during field surveys and for the collecting permits. We would also like to thank the Minister Abd al-Rahman Fadhl al-Iriyani (Ministry of Water and Environment of Yemen) for his support and interest in the project. DNA work was funded by grant CGL2009-11663 ⁄BOS from the Ministerio de Educacio´n y Ciencia, Spain. S.C. and M.M. are members of the Grup de Recerca Emergent of the Generalitat de Catalunya: 2009SGR1462; M.M. is supported by a FPU predoctoral grant from the Ministerio de Ciencia e Innovacio´n, Spain (AP2008-01844). Research work by S.C. at the BMNH received support from the SYNTHESIS project GB-TAF- 270, which is financed by the European Community Research Infrastructure under the FP7 ‘Structuring the European Research Area’ Program. Some phylogenetic analyses were run in the cluster facility of the IBE funded by the Spanish National Bioinformatics Institute (http:// www.inab.org). Special thanks to Cristina Grieco, who dis- covered the holotype in Abd Al Kuri and collaborated in the study of museum specimens and in preparing the draw- ings, Elisa Riservato, who participated in the expedition to Abd Al Kuri, and Francesca Pella for logistic support. We are grateful to the comments by two anonymous reviewers, which helped to improve the manuscript. References Akaike, H. (1973). Information theory and an extension of the maximum likelihood principle. In B. N. Petrov & F. Csaki (Eds) Second International Symposium on Information Theory (pp. 267–281). Budapest, Hungary: Akademiai Kiado. Arnold, E. N. (1986). New species of semaphore gecko (Pristurus: Gekkonidae) from Arabia and Socotra. Fauna of Saudi Arabia, 8, 352–377. Austin, J. J., Arnold, E. & Jones, C. (2009). Interrelationships and history of the slit-eared skinks (Gongylomorphus, Scincidae) of the Mascarene Islands, based on mitochondrial DNA and nuclear gene sequences. Zootaxa, 2153, 55–68. Autin, J., Leroy, S., Beslier, M. O., Dı´Acremont, E., Razin, P., Ribodetti, A., Bellahsen, N., Robin, C. & Al Toubi, K. (2010).360 ª 2012 The Authors d Zoologica S 279Continental break up history of a deep magma poor margin based on seismic reflection data (Northeastern Gulf of Aden margin, offshore Oman). Geophysical Journal International, 180, 501–519. Battistuzzi, F. U., Filipski, A., Hedges, S. B. & Kumar, S. (2010). Performance of Relaxed-Clock Methods in Estimating Evolutionary Divergence Times and Their Credibility Intervals. Molecular Biology and Evolution, 27, 1289–1300. Bosworth, W., Huchon, P. & McClay, K. (2005). The Red Sea and Gulf of Aden basins. Journal of African Earth Sciences, 43, 334–378. Brandley, M. C., Schmitz, A. & Reeder, T. W. (2005). Partitioned Bayesian analyses, partition choice, and the phylogenetic relationships of scincid lizards. Systematic biology, 54, 373. Broadley, D. G. & Howell, K. (1991). A checklist of the reptiles of Tanzania, with synoptic keys. Harare and Bulawayo, Zimbabwe: National Museum and Monuments of Zimbabwe. Brown, R. & Pestano, J. (1998). Phylogeography of skinks (Chalcides) in the Canary Islands inferred from mitochondrial DNA sequences. Molecular Ecology, 7, 1183–1191. Brown, R. P. & Yang, Z. (2010). Bayesian dating of shallow phylogenies with a relaxed clock. Systematic biology, 59, 119. Carranza, S. & Arnold, E. (2003). Investigating the origin of transoceanic distributions: mtDNA shows Mabuya lizards (Reptilia, Scincidae) crossed the Atlantic twice. Systematics and Biodiversity, 1, 275–282. Carranza, S. & Arnold, E. (2006). Systematics, biogeography, and evolution of Hemidactylus geckos (Reptilia: Gekkonidae) elucidated using mitochondrial DNA sequences. Molecular phylogenetics and evolution, 38, 531–545. Carranza, S., Arnold, E. N., Mateo, J. & Lo´pez-Jurado, L. (2000). Long-distance colonization and radiation in gekkonid lizards, Tarentola (Reptilia: Gekkonidae), revealed by mitochondrial DNA sequences. Proceedings of the Royal Society of London. Series B: Biological Sciences, 267, 637. Carranza, S., Arnold, E., Mateo, J. & Lo´pez-Jurado, L. (2001). Parallel gigantism and complex colonization patterns in the Cape Verde scincid lizards Mabuya and Macroscincus (Reptilia: Scincidae) revealed by mitochondrial DNA sequences. Proceedings of the Royal Society of London. Series B: Biological Sciences, 268, 1595. Carranza, S., Arnold, E., Geniez, P., Roca, J. & Mateo, J. (2008). Radiation, multiple dispersal and parallelism in the skinks, Chalcides and Sphenops (Squamata: Scincidae), with comments on Scincus and Scincopus and the age of the Sahara Desert. Molecular phylogenetics and evolution, 46, 1071–1094. Cheung, C. & DeVantier, L. (2006). Socotra - a natural history of the islands and their people. Hong Kong: Odyssey Books and Guides, Airphoto International Ltd. Drummond, A. J. & Rambaut, A. (2007). BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7, 214. Drummond, A. J., Ho, S. Y. W., Phillips, M. J. & Rambaut, A. (2006). Relaxed phylogenetics and dating with confidence. PLoS Biology, 4, e88. Drummond, A., Ashton, B., Buxton, S., Cheung, M., Cooper, A., Heled, J., Kearse, M., Moir, R., Stones-Havas, S. & Sturrock, S. (2010). Geneious v5.1. Available via http://www.geneious. com.cripta ª 2012 The Norwegian Academy of Science and Letters, 41, 4, July 2012, pp 346–362 Sindaco et al. d The Trachylepis skinks of the Socotra ArchipelagoFelsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783–791. Fleitmann, D., Matter, A., Burns, S. J., Al-Subbary, A. & Al- Aowah, M. A. (2004). Geology and Quaternary climate history of Socotra. Fauna of Arabia, 20, 27–44. Fritz, J. & Schu¨tte, F. (1988). Skinke aus der Arabischen Republik Jemen (Sauria: Scincidae) Les scinques de la Re´publique Arabe du Ye´men (Sauria: Scincidae) Skinks of the Arabian Republic of Yemen (Sauria: Scincidae). Salamandra, 24, 41–52. Graur, D. & Martin, W. (2004). Reading the entrails of chickens: molecular timescales of evolution and the illusion of precision. Trends in Genetics, 20, 80–86. Greer, A. E. & Nussbaum, R. A. (2000). New character useful in the systematics of the scincid lizard genus Mabuya. Copeia, 2000, 615–618. Gu¨nther, R., Whiting, A. & Bauer, A. (2005). Description of a new species of African skink of the genus Trachylepis. Herpetozoa, 18, 11–24. Ho, S. Y. W. & Philips, M. J. (2009). Accounting for Calibration Uncertainty in Phylogenetic Estimation of Evolutionary Divergence Times. Systematic Biology, 58, 367–380. Huelsenbeck, J. P. & Ronquist, F. (2001). MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics, 17, 754–755. Jesus, J., Brehm, A. & Harris, D. J. (2005a). Relationships of scincid lizards (Mabuya spp.) from the islands of the Gulf of Guinea based on mtDNA sequence data. Amphibia-Reptilia, 26, 467–473. Jesus, J., Harris, D. J. & Brehm, A. (2005b). Phylogeography of Mabuya maculilabris (Reptilia) from Sa˜o Tome´ island (Gulf of Guinea) inferred from mtDNA sequences. Molecular phylogenetics and evolution, 37, 503–510. Joger, U. & Mayer, W. (2002). A new species of Mesalina (Reptilia: Lacertidae) from Abd Al-Kuri, Socotra archipelago, Yemen, and a preliminary molecular phylogeny for the genus Mesalina. Fauna of Arabia, 19, 497–506. Kapli, P., Lymberakis, P., Poulakakis, N., Mantziou, G., Parmakelis, A. & Mylonas, M. (2008). Molecular phylogeny of three Mesalina (Reptilia: Lacertidae) species (M. guttulata, M. brevirostris and M. bahaeldini) from North Africa and the Middle East: another case of paraphyly? Molecular phylogenetics and evolution, 49, 102–110. Katoh, K., Misawa, K., Kuma, K. & Miyata, T. (2002). MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic acids research, 30, 3059. Kocher, T. D., Thomas, W. K., Meyer, A., Edwards, S. V., Pa¨a¨bo, S., Villablanca, F. X. & Wilson, A. C. (1989). Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proceedings of the National Academy of Sciences, USA, 86, 6196. Lanza, B. (1978). On some new or interesting East African amphibians and reptiles. Monitore zoologico italiano NS Supplemento, 14, 229–297. Lanza, B. (1983). A list of the Somali amphibians and reptiles. Monitore zoologico italiano, 8, 193–247. Lanza, B. & Carfi, S. (1968). Gli scincidi della somalia (reptilia, squamata). Monitore Zoologico Italiano, Supplemento, 2, 207–260. Largen, M. & Spawls, S. (2006). Lizards of ethiopia (reptilia sauria): an annotated checklist, bibliography, gazetteer and identification key. Tropical Zoology, 19, 21–109.ª 2012 The Authors d Zoologica Scripta ª 2012 The Norwegian Academy of Science and Letters, 28Laughton, A. (1966). The Gulf of Aden. Philosophical Transactions of the Royal Society of London. Series A, Mathematical and Physical Sciences, 259, 150. Laughton, A., Whitmarsh, R., Jones, M. & Habicht, J. (1970). The evolution of the Gulf of Aden [and Discussion]. Philosophical Transactions of the Royal Society of London. Series A, Mathematical and Physical Sciences, 267, 227. Macey, J. R., Kuehl, J. V., Larson, A., Robinson, M. D., Ugurtas, I. H., Ananjeva, N. B., Rahman, H., Javed, H. I., Osman, R. M. & Doumma, A. (2008). Socotra Island the forgotten fragment of Gondwana: unmasking chameleon lizard history with complete mitochondrial genomic data. Molecular phylogenetics and evolution, 49, 1015. Mausfeld, P. & Schmitz, A. (2003). Molecular phylogeography, intraspecific variation and speciation of the Asian scincid lizard genus Eutropis Fitzinger, 1843 (Squamata: Reptilia: Scincidae): taxonomic and biogeographic implications. Organisms Diversity & Evolution, 3, 161–171. Mausfeld, P., Schmitz, A., Bo¨hme, W., Misof, B., Vrcibradic, D. & Rocha, C. F. D. (2002). Phylogenetic affinities of Mabuya atlantica Schmidt, 1945, endemic to the atlantic ocean archipelago of Fernando de Noronha (Brazil): necessity of partitioning the genus Mabuya Fitzinger, 1826 (Scincidae: Lygosominae). Zoologischer Anzeiger-A Journal of Comparative Zoology, 241, 281–293. Mausfeld, P., Vences, M., Schmitz, A. & Veith, M. (2000). First data on the molecular phylogeny of scincid lizards of the genus Mabuya. Molecular phylogenetics and evolution, 17, 11–14. Mausfeld-Lafdhiya, P., Schmitz, A., Ineich, I. & Chirio, L. (2004). Genetic variation in teo African Euprepis species (Reptilia, Scincidae), based on maximum-likelihood and Bayesian analyses: taxonomic and biogeographic conclusions. Bonner zooligische Beitrage, 52, 159–177. Miralles, A. (2006). A new species of Mabuya (Reptilia, Squamata, Scincidae) from the Caribbean island of San Andre´s, with a new interpretation of nuchal scales: a character of taxonomic importance. Herpetological Journal, 16, 1–7. Miralles, A. & Carranza, S. (2010). Systematics and biogeography of the Neotropical genus Mabuya, with special emphasis on the Amazonian skink Mabuya nigropunctata (Reptilia, Scincidae). Molecular phylogenetics and evolution, 54, 857–869. Miralles, A., Chaparro, J. C. & Harvey, M. B. (2009a). Three rare and enigmatic South American skinks. Zootaxa, 2012, 47–68. Miralles, A., Fuenmayor, G. R., Bonillo, C., Schargel, W. E., Barros, T., Garcı´a Perez, J. E. & Barrio Amoro´s, C. L. (2009b). Molecular systematics of Caribbean skinks of the genus Mabuya (Reptilia, Scincidae), with descriptions of two new species from Venezuela. Zoological Journal of the Linnean Society, 156, 598– 616. Miralles, A., Vasconcelos, R., Perera, A., Harris, D. J. & Carranza, S. (2011). An integrative taxonomic revision of the Cape Verdean skinks (Squamata, Scincidae). Zoologica Scripta, 40, 16–44. Nagy, Z. T., Joger, U., Wink, M., Glaw, F. & Vences, M. (2003). Multiple colonization of Madagascar and Socotra by colubrid snakes: evidence from nuclear and mitochondrial gene phylogenies. Proceedings of the Royal Society of London. Series B: Biological Sciences, 270, 2613. Palumbi, S. (1996). Nucleic acids II: the polymerase chain reaction. In D. Hillis, C. Moritz & B. K. Mable (Eds) Molecular41, 4, July 2012, pp 346–362 361 0 systematics (pp. 205–247). Sunderland, Massachusetts: Sinauer Associates. Stamatakis, A. (2006). RaxML-VI-HPC: maximum likelihood- based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22, 2688. The Trachylepis skinks of the Socotra Archipelago d Sindaco et al.Papenfuss, T. J., Jackman, T., Bauer, A., Stuart, B. L., Robinson, M. D. & Parham, J. F. (2009). Phylogenetic relationships among species in the sphaerodactylid lizard genus Pristurus. Proceedings of the California Academy of Sciences, 60, 675–681. Posada, D. (2008). jModelTest: phylogenetic model averaging. Molecular Biology and Evolution, 25, 1253. de Queiroz, A. (2005). The resurrection of oceanic dispersal in historical biogeography. Trends in Ecology & Evolution, 20, 68–73. Rambaut, A. & Drummond A. J. (2007). Tracer v1.4. Available from the BEAST site: http://beast.bio.ed.ac.uk/. Razzetti, E., Sindaco, R., Grieco, C., Pella, F., Ziliani, U., Pupin, F., Riservato, E., Pellitteri-Rosa, D., Suleiman, A. S., Al-Aseily, B. A., Carugati, C., Boncompagni, E. & Fasola, M.. (2011). The herpetofauna of the Socotran Archipelago, Yemen (Reptilia). Zootaxa, 2826, 1–44. Robinson-Rechavi, M. & Huchon, D. (2000). RRTree: relative- rate tests between groups of sequences on a phylogenetic tree. Bioinformatics, 16, 296. Ro¨sler, H. & Wranik, W. (2000). Beitra¨ge zur herpetologie der Republik Jemen. 6. Erste u¨bersicht zur herpetofauna der Insel Darsa (Reptilia: Sauria et Serpentes). Faunistische Abhandlungen Staatliches Museum fu¨r Tierkunde Dresden, 22, 85–94. Ro¨sler, H. & Wranik, W. (2004). A key and annotated checklist to the reptiles of the Socotra Archipelago. Fauna of Arabia, 20, 505–534. Samuel, M. A., Harbury, N., Bott, R. & Manan Thabet, A. (1997). Field observations from the Socotran platform: their interpretation and correlation to Southern Oman. Marine and petroleum geology, 14, 661–673. Scha¨tti, B. & Desvoignes, A. (1999). The herpetofauna of Southern Yemen and the Sokotra Archipelago (pp. 1–178). Gene´ve: Instrumenta Biodiversitatis IV, Muse`um d’Histoire Naturelle. Scha¨tti, B. & Gasperetti, J. (1994). A contribution to the herpetofauna of Southwest Arabia. Fauna of Saudi Arabia, 14, 348–423. Shankar, D., Vinayachandran, P. N. & Unnikrishnan, A. S. (2002). The monsoon currents in the north Indian Ocean. Progress In Oceanography, 52, 63–120. Shimodaira, H. (2002). An approximately unbiased test of phylogenetic tree selection. Systematic biology, 51, 492. Shimodaira, H. & Hasegawa, M. (1999). Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Molecular Biology and Evolution, 16, 1114–1116. Shimodaira, H. & Hasegawa, M. (2001). CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics, 17, 1246. Sindaco, R., Ziliani, U., Razzetti, E., Carugati, C., Grieco, C., Pupin, F., Al-Aseily, B. A., Pella, F. & Fasola, M. (2009). A misunderstood new gecko of the genus Hemidactylus from Socotra Island, Yemen (Reptilia: Squamata: Gekkonidae). Acta Herpetologica, 4, 83–98. Spawls, S., Howell, K., Drewes, R. & Ashe, J. (2002). A field guide to the reptiles of East Africa. San Diego, CA, USA: Princeton University Press.362 ª 2012 The Authors d Zoologica S 281Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731–2739. Van Damme, K. (2009). Socotra Archipelago. In R. G. Gillespie & D. A. Clague (Eds) Encyclopedia of Islands (p. 1111). Berkeley CA: University of California Press. Whiting, A. S., Sites, J. W., Jr, Pellegrino, K. & Rodrigues, M. T. (2006). Comparing alignment methods for inferring the history of the new world lizard genus Mabuya (Squamata: Scincidae). Molecular phylogenetics and evolution, 38, 719–730. Wranik, W. (1998a). Faunistic notes on Soqotra island. In: H. Dumont (Ed.), Proceedings of the First International Symposium on Soqotra island: present & future, Aden, 1996 (pp. 135–198). New York: United Nations Development Programme. Wranik, W. (1998b). Contributions to the herpetology of the Republic of Yemen. 4. Sokotra Island and southern Yemen mainland. Zoologische Abhandlungen Staatliches Museum fu¨r Tierkunde Dresden, 21, 163–179. Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Measurements and scale counts of the specimens of Trachylepis brevicollis (all measurements in mm). Appendix S2. Specimens used in estimation of diver- gence times and RRT tests. Appendix S3: Results of the BEAST analysis including two partitions (12S and 16S) performed using the software BEAST v.1.6.1. Appendix S4: Results of the ML (A) and Bayesian (B) analyses. The alignment of the Mabuya sensu lato dataset included a total of 904 base pairs (bp), of which 392 corre- sponded to the 12S and 512 to the 16S. Appendix S5- Uncorrected p-distances based on gene fragment 12S above the diagonal and 16S below the diagonal. Appendix S6. Living holotype of Trachylepis cristinae (MCCI-R1585). Appendix S7. Type specimens of Trachylepis cristinae sp. nov. (a, b, c) – holotype, MCCI-R1585; (d, e, f) – paratype, NMW 9396. Appendix S8. Comparison with other Trachylepis from Eastern and Southern Africa, and Arabia. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials sup- plied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.cripta ª 2012 The Norwegian Academy of Science and Letters, 41, 4, July 2012, pp 346–362 A pp en di x S1 : M ea su re m en ts a nd s ca le c ou nt s of th e sp ec im en s of T ra ch yl ep is b re vi co lli s (a ll m ea su re m en ts in m m ). C at al og ue : B M = B rit is h M us eu m , M ZU F = M us eu m o f Zo ol og y, U ni ve rs ity o f F lo re nc e; L oc al ity : O M = O m an , S A = S au di A ra bi a, S O = S om al ia , Y E = Y em en ; S V L: s no ut -v en t l en gt h; H L: h ea d le ng th fr om ti p of s no ut to p os te rio r b or de r o f p ar ie ta ls ; H W : h ea d w id th a t l ev el o f e ar o pe ni ng s; ra tio H L/ H W ; E ST : d is ta nc e be tw ee n an te rio r m ar gi n of e ye to ti p of sn ou t; EE D : d is ta nc e be tw ee n po st er io r m ar gi n of e ye to a nt er io r m ar gi n of e ar ; M SR : n um be r o f m id bo dy s ca le ro w s; c ou nt s fo r s pe ci m en s in di ca te d w ith (* ) w er e pu bl is he d by L an za & C ar fì (1 96 8) , t og et he r w ith a dd iti on al s pe ci m en s no t s tu di ed b y us ; S L l/r : n um be r of s up ra la bi al s (le ft / rig ht ); LF 4 r: nu m be r of l am el la e un de r th e 4t h rig ht f in ge r; LT 4 r: nu m be r of l am el la e un de r th e 4t h rig ht t oe ; D or sa l ke el s: n um be r of d or sa l ke el s; S up ra na sa ls ar ra ng em en t: S = se pa ra te d; ( C ) = in s ho rt co nt ac t; C = i n br oa d co nt ac t; Pr ef ro nt al a rr an ge m en t: S = se pa ra te d; ( C ) = in s ho rt co nt ac t; C = i n br oa d co nt ac t; Su pr ao cu l. en t. fr on ta l: su pr ao cu la rs e nt er in g th e fr on ta l s ca le , * th ird p la te f ra gm en te d; T ar sa l s ca le s: s ha pe o f ta rs al s ca le s (f oo t); F = f la t; G = g ra nu la r; B = b lu nt tu be rc le s; P = p oi nt ed tu be rc le s; S = s pi no se (m uc ro na te d) tu be rc le s; (S ) = s lig ht ly s pi no se ; P al m ar s ca le s: s ha pe o f p al m ar s ca le s (h an d) ; F = fl at ; G = g ra nu la r; P = po in te d tu be rc le s; S = s pi no se (m uc ro na te d) tu be rc le s; (S ) = s lig ht ly sp in os e; F oo t l am el la e: s ha pe o f l am el la e un de r t he 4 th to e; H an d la m el la e: n um be r o f l am el la e un de r t he 4 th fi ng er ; M or ph ob an k im ag es : M or ph ob an k co de s (p ro je ct P 46 1) fo r t he p ic tu re s of th e T. b re vi co lli s a nd T . s oc ot ra na sp ec im en s. Sp ec ie s C at al og ue L oc al ity Sex SVL HL HW ratio HL/HW EST EED MSR SL l/r LF4 r LT4 r Dorsal keels Supranasals arrangement Prefrontals arrangement Supraocul. ent. frontal Tarsal scales Palmar scales Foot lamellae Hand lamellae Morphobank images T. b re vi co lli s B M 19 75 .1 38 5 O M , D ho fa r, Sa la la h ? 12 0 20 .9 18 .3 1. 14 10 .1 9. 7 34 5/ 5 16 20 2 (C ) (C ) I- II -I II / I- II -I II F F k sc M 86 87 5- M 86 88 6 T. b re vi co lli s B M 19 75 .1 38 6 O M , D ho fa r, Sa la la h ? 11 3 19 .5 17 .8 1. 10 9. 5 9. 3 34 5/ 5 15 18 2 (C ) S I- II / I- II -I II S F w k s M 86 88 8- M 86 90 0 T. b re vi co lli s B M 19 78 .7 77 O M , D ho fa r, R ay su t ? 13 5 21 .4 19 .3 1. 11 11 .3 10 .9 34 5/ 5 16 19 2 S S I- II / I- II -I II F F k s M 86 90 1- M 86 91 4 T. b re vi co lli s B M 19 77 .1 17 7 O M , D ho fa r, Je be l Q am ai r ? 18 0 23 .5 20 .7 1. 14 12 .5 10 .2 32 5/ 5 13 16 2 S S I- II -I II / I- II -I II F F w k s M 86 91 5- M 86 93 0 T. b re vi co lli s B M 19 77 .1 18 0 O M , D ho fa r, Sa la la h ? 14 0 20 .8 19 .0 1. 09 11 .2 10 .4 37 5/ 5 15 21 2 S S I- II -I II / I- II -I II F F k s M 86 93 1- M 86 94 4 T. b re vi co lli s B M 19 85 .6 18 O M , D ho fa r, K ho rta w t ? 16 6 24 .4 21 .5 1. 13 12 .8 10 .8 34 5/ 5 16 20 2 (C ) S I- II -I II / I- II -I II F F w k w k M 86 94 5- M 86 95 7 T. b re vi co lli s B M 97 .3 .1 1. 90 .9 3A Y E, H ad ra m au t M 12 8 21 .7 19 .7 1. 10 10 .9 10 .1 33 5/ 5 17 21 2 C C I- II / br ok en S F w k s M 86 98 8- M 87 00 1 T. b re vi co lli s B M 97 .3 .1 1. 90 .9 3B Y E, H ad ra m au t M 12 1 21 .7 20 .1 1. 08 10 .8 9. 3 32 5/ 5 13 17 2 C C I- II -I II / I- II -I II F F w k w k M 87 00 2- M 87 01 5 T. b re vi co lli s B M 97 .3 .1 1. 90 .9 3C Y E, H ad ra m au t ? 86 14 .9 12 .0 1. 24 7. 8 5. 1 34 5/ 5 15 17 2 S C I- II -I II / I- II -I II S F k w k M 87 01 6- M 87 16 7 T. b re vi co lli s M ZU F- 28 90 2 A ra bi a ? 13 5 24 .5 20 .9 1. 17 9. 3 9. 8 37 5/ 5 16 22 2 (3 ) S (C ) I- II -I II / I- II -I II P G - w k M 85 85 2- M 85 86 0 T. b re vi co lli s M ZU F- 28 90 3 A ra bi a ? 13 8 25 .6 21 .9 1. 17 11 .5 9. 5 37 5/ 5 16 23 2 (3 ) (C ) S I- II -I II / I- II -I II P G - w k M 85 86 1- M 85 86 6 T. b re vi co lli s M ZU F- 28 65 1 A ra bi a ? 10 9 18 .8 18 .1 1. 04 10 .0 10 .3 35 5/ 5 16 20 2 C S I- II / I- II P P k s M 85 84 5- M 85 85 1 T. b re vi co lli s M ZU F- 28 61 3 A ra bi a ? 11 7 18 .8 16 .7 1. 13 9. 8 8. 3 30 5/ 5 15 21 2 (3 ) C S I- II -I II / I- II -I II P P k w k M 85 80 9- M 85 81 4 T. b re vi co lli s M ZU F- 28 61 2 A ra bi a ? 12 1 19 .4 19 .4 1. 00 13 .0 9. 3 32 5/ 5 15 20 2 C S I- II -I II / I- II P P k w k M 85 79 4- M 85 80 8 T. b re vi co lli s M ZU F- 28 61 5 A ra bi a M 10 3 19 .4 19 .6 0. 99 13 .0 9. 2 32 5/ 5 16 19 2 C S I- II -I II / I- II -I II P P k k M 85 83 0- M 85 84 4 T. b re vi co lli s M ZU F- 28 61 1 A ra bi a M 98 17 .6 15 .7 1. 12 9. 2 8. 1 32 5/ 5 16 21 2 (3 ) C S I- II -I II / I- II -I II S S k w k M 85 78 5- M 85 79 3 T. b re vi co lli s M ZU F- 28 61 4 A ra bi a im m 79 13 .9 11 .2 1. 24 7. 2 6. 3 32 5/ 5 15 18 ? C (C ) I- II -I II / I- II -I II P P k k M 85 81 5- M 85 82 9 T. b re vi co lli s B M 19 82 .1 16 2 Y E, a b M ar aw ag ha ? 12 6 22 .6 18 .9 1. 20 11 .6 10 .1 35 5/ 5 15 20 2 S S I- II -I II / I- II -I II F F w k w k M 87 16 7- M 87 18 2 T. b re vi co lli s B M 19 88 .3 12 Y E, W ad i W ar az an ? 14 6 23 .6 21 .3 1. 11 12 .2 10 .7 34 5/ 5 15 20 3 C (C ) I- II / I- II -I II B F w k s M 87 18 3- M 87 19 8 T. b re vi co lli s B M 99 .1 2. 13 .7 0 Y E, A bi an H ill C ou nt ry F 11 4 19 .4 16 .3 1. 19 9. 7 8. 2 32 5/ 5 17 21 3 C C I- II -I II / I- II -I II S F k s M 87 19 9- M 87 21 3 T. b re vi co lli s B M 99 .1 2. 13 .7 4 Y E, A bi an C ou nt ry ? 12 7 20 .7 19 .6 1. 06 10 .7 9. 6 32 5/ 5 15 21 2 C C I- II -I II / I- II -I II S F k s M 87 56 7- M 87 58 0 T. b re vi co lli s B M 95 .5 .2 3. 74 Y E, S he ik h O sm an M 12 2 21 .0 18 .2 1. 15 9. 9 8. 9 31 5/ 5 15 21 3 C C I- II -I II / I- II -I II S F k s M 87 58 1- M 87 59 3 T. b re vi co lli s B M 95 .5 .2 3. 75 Y E, S he ik h O sm an F 12 8 20 .5 16 .3 1. 26 10 .3 8. 3 32 5/ 5 14 19 3 C C I- II -I II / I- II -I II S S w k w k M 87 59 4- M 87 60 7 T. b re vi co lli s B M 95 .5 .2 3. 71 Y E, A de n F 14 5 24 .1 23 .0 1. 05 12 .9 10 .2 32 6/ 5 16 21 3 C C I- II -I II / I- II -I II S F w k s M 87 60 8- M 87 61 9 T. b re vi co lli s B M 95 .5 .2 3. 70 Y E, A de n M 14 0 22 .4 20 .2 1. 11 11 .3 10 .6 32 5/ 5 15 22 3 C (C ) I- II -I II / I- II -I II S S k s M 89 77 4- M 89 78 7 T. b re vi co lli s B M 19 03 .3 .6 .4 5 SA , A zr ah i R av in e M 12 0 22 .3 20 .1 1. 11 11 .2 10 .6 35 5/ 5 16 20 3 C S I- II -I II / I- II -I II S F w k s M 87 62 1- M 87 63 5 T. b re vi co lli s B M 19 03 .3 .6 .4 6 SA , G er ba ? 11 7 19 .0 15 .0 1. 27 10 .2 8. 9 34 5/ 5 15 19 3 C S I- II -I II / I- II -I II F F k w k M 87 63 6- M 87 64 7 T. b re vi co lli s B M 19 03 .3 .6 .1 6 SA , e l K ub ar ? 13 4 21 .7 19 .7 1. 10 11 .4 7. 3 34 5/ 5 16 20 2- 3 C S I- II -I II / I- II -I II F F w k s M 87 64 9- M 87 66 2 T. b re vi co lli s B M 19 03 .3 .6 .1 5 SA , e l K ub ar ? 12 1 20 .1 17 .5 1. 15 10 .7 9. 0 34 5/ 5 14 19 2 C S I- II -I II / I- II -I II B F k w k M 87 66 4- M 87 67 6 T. b re vi co lli s B M 19 03 .6 .2 6. 16 SA , e l K ub ar ? 11 9 20 .0 17 .6 1. 14 10 .7 8. 8 34 ?? ? 16 19 2- 3 S (C ) I- II / I- II F F k w k M 87 67 8- M 87 69 0 T. b re vi co lli s B M 19 79 .9 75 SA , a l J am m um ? 12 1 19 .0 16 .0 1. 19 9. 3 8. 8 32 5/ 4 16 19 2 (C ) (C ) I- II -I II / I- II S S k w k M 87 69 1- M 87 70 3 T. b re vi co lli s B M 19 64 .1 44 SA , W ad i L ah si ba ? 96 16 .7 14 .0 1. 19 9. 3 6. 6 ? 5/ 4 13 18 2 C S I- II -I II / I- II F F k w k M 87 70 4- M 87 71 5 T. b re vi co lli s B M 19 80 .6 0 SA , 3 0 km S E A bh a ? 13 0 21 .9 19 .8 1. 11 11 .3 10 35 5/ 5 13 20 2 C S I- II -I II / I- II -I II F F w k s M 87 71 6- M 87 73 0 T. b re vi co lli s B M 19 80 .5 9 SA , A l J em un ? 11 4 19 .2 16 .3 1. 18 9. 6 8. 1 33 5/ 5 16 18 2 C C I- II -I II / I- II -I II S F k w k M 87 73 1- M 87 74 4 34 34 34 34 34 34 33 33 34 34 12 3. 7 20 .6 18 .2 1. 13 10 .6 9. 1 33 .3 4. 9 15 .2 19 .7 18 0 25 .6 23 1. 27 13 10 .9 37 5 17 23 79 13 .9 11 .2 0. 99 7. 2 5. 1 30 4 13 16 Su m m ar y St at is tic s N um be r o f I nd iv id ua ls (N ) M ea n M ax im um M in im um St an da rd E rr or M ea n 3. 41 0. 44 0. 45 0. 01 1 0. 23 0. 23 0. 29 0. 02 0. 18 0. 26 282 Sp ec ie s C at al og ue L oc al ity Sex SVL HL HW ratio HL/HW EST EED MSR SL l/r LF4 r LT4 r Dorsal keels Supranasals arrangement Prefrontals arrangement Supraocul. ent. frontal Tarsal scales Palmar scales Foot lamellae Hand lamellae Morphobank images T. b re vi co lli s M ZU F- 17 08 SO , D in so r ? 12 2 19 .5 22 .0 0. 89 9. 3 9. 0 (* ) 5/ 4 15 17 2 (3 ) S S I- II -( II I) / I- II S S k k M 85 71 8- M 85 72 0 T. b re vi co lli s M ZU F- 15 99 SO , D in so r ? 12 2 20 .6 20 .7 1. 00 10 .9 10 .0 (* ) 4/ 5 13 16 2 C S I- II -I II / I- II S S k w k M 85 71 5- M 85 71 7 T. b re vi co lli s M ZU F- 17 59 SO , D in so r M 13 9 22 .5 23 .7 0. 95 11 .6 11 .0 (* ) 5/ 4 13 18 2 S S (I )- II / I- II S S k k M 85 73 4- M 85 73 6 T. b re vi co lli s M ZU F- 17 56 SO , D in so r M 12 4 20 .2 20 .9 0. 97 10 .9 11 .0 (* ) 5/ 5 14 16 3 (C ) (C ) I- II / I- II S S k w k M 85 72 8- M 85 73 0 T. b re vi co lli s M ZU F- 17 57 SO , D in so r ? 13 0 20 .9 19 .7 1. 06 11 .3 11 .3 (* ) 5/ 5 15 17 2/ 3 (C ) (C ) I- II -I II / I- II -I II S S k w k M 85 73 1M 85 73 3 T. b re vi co lli s M ZU F- 17 09 SO , D in so r ? 13 6 22 .1 23 .0 0. 96 12 .0 10 .4 (* ) 5/ 5 12 17 2/ 3 S S I- II -I II / I- II S (S ) k w k M 85 72 1- M 85 72 3 T. b re vi co lli s M ZU F- 17 55 SO , D in so r im m 11 4 18 .8 16 .5 1. 14 - - (* ) 5/ 5 14 17 2 (C ) S I- II -( II I) / I- II S S k k M 85 72 4- M 85 72 7 T. b re vi co lli s M ZU F- 17 51 SO , D in so r im m 10 2 16 .5 17 .1 0. 96 - - (* ) 5/ 5 13 18 2 (C ) S I- II / I- II S S k k - T. b re vi co lli s M ZU F- 10 13 1 SO , G ar do ? 11 8 19 .5 22 .5 0. 87 11 .6 10 .3 (* ) 5/ 5 14 19 2 (3 ) (C ) S (I )- II / (I )- II -I II S S k k M 85 75 0- M 85 75 3 T. b re vi co lli s M ZU F- 10 12 9 SO , G ar do ? 12 5 21 .1 22 .5 0. 94 11 .2 10 .9 (* ) 5/ 5 15 18 2 (3 ) (C ) S I- II -( II I) / I- II -( II I) S S k k M 85 74 3- M 85 74 5 T. b re vi co lli s M ZU F- 10 13 7 SO , G ar do ? 12 1 19 .9 21 .0 0. 95 11 .2 10 .5 (* ) 5/ 5 13 17 2 (3 ) S S II / II -( II I) S S k k M 85 76 8- M 85 77 2 T. b re vi co lli s M ZU F- 10 13 2 SO , G ar do ? 11 5 20 .5 21 .8 0. 94 11 .7 11 .0 (* ) 5/ 5 15 19 2 (3 ) S (C ) I- II -I II / (I )- II -I II S S k k M 85 75 4- M 85 75 6 T. b re vi co lli s M ZU F- 10 13 8 SO , G ar do ? 12 5 20 .3 20 .3 1. 00 11 .6 10 .9 (* ) 5/ 5 13 17 2 (3 ) S (C ) (I )- II / I- II S S k k M 85 77 3- M 85 77 5 T. b re vi co lli s M ZU F- 10 13 4 SO , G ar do ? 11 8 19 .8 20 .0 0. 99 11 .4 11 .2 (* ) 5/ 6 14 17 2 (3 ) C S I- II -I II / I- II -I II S S k k M 85 76 1- M 85 76 4 T. b re vi co lli s M ZU F- 10 13 0 SO , G ar do ? 11 7 19 .0 18 .2 1. 04 10 .4 10 .1 (* ) 5/ 4 - 18 2 S (C ) I- II -( II I) / I- II -( II I) S S k k M 85 74 6- M 85 74 9 T. b re vi co lli s M ZU F- 10 13 3 SO , G ar do ? 11 4 20 .0 21 .4 0. 93 10 .9 10 .5 (* ) 5/ 4 13 19 3 (2 ) C S I- II / I- II -I II S S k w k M 85 75 7- M 85 76 0 T. b re vi co lli s M ZU F- 10 15 4 SO , G ar do ? 10 1 17 .4 16 .9 1. 03 9. 7 9. 6 (* ) 5/ 4 15 17 2 (3 ) S (C ) I- II / I- II -I II S S k w k M 85 77 6- M 85 77 8 T. b re vi co lli s M ZU F- 10 13 6 SO , G ar do ? 94 17 .7 17 .4 1. 02 9. 6 9. 2 (* ) 5/ 5 13 18 2 S (C ) I- II / II S S k k M 85 76 5- M 85 76 7 T. b re vi co lli s B M 19 37 .1 2. 5. 66 9 SO , B oh od le ? 12 2 18 .5 18 .3 1. 01 10 .1 8. 2 32 5/ 5 12 16 2 C C II -I II / II -I II S F k w k M 87 74 5- M 87 75 8 T. b re vi co lli s B M 19 37 .1 2. 5. 67 5 SO , H au d ? 12 3 19 .9 18 .6 1. 07 11 .6 8. 0 33 5/ 5 15 16 2 C C II -I II / II -I II S F k w k M 87 75 9- M 87 77 4 T. b re vi co lli s B M 19 37 .1 2. 5. 67 1 SO , H or u Fo di ? 11 0 19 .0 17 .3 1. 10 9. 7 9. 0 33 5/ 5 12 16 2 C C II -I II / II -I II B F k w k M 87 77 5- M 87 78 9 T. b re vi co lli s B M 19 68 .1 24 9 SO , D in so r M 13 0 21 .1 19 .3 1. 09 10 .6 9. 2 33 5/ 5 12 16 2- 3 S S I- II / I- II S F k w k M 87 79 0- M 87 80 3 T. b re vi co lli s B M 19 94 .5 26 SO , A rg ei sa ? 13 5 22 .6 21 .5 1. 05 11 .2 11 .1 32 4/ 4 14 17 2 C C I- II -I II / I- II S S w k w k M 87 80 4- M 87 81 7 Su m m ar y St at is tic s N um be r o f I nd iv id ua ls (N ) 23 23 23 23 21 21 5 23 22 23 M ea n 11 9. 8 19 .8 20 0. 99 10 .8 10 .1 32 .6 4. 8 13 .6 17 .2 M ax im um 13 9 22 .6 23 .7 1. 14 12 11 .3 33 5. 5 15 19 M in im um 94 16 .5 16 .5 0. 87 9. 3 8 32 4 12 16 St an da rd E rr or M ea n 2. 31 0. 31 0. 44 0. 01 4 0. 17 0. 21 0. 24 0. 06 0. 23 0. 2 Sp ec ie s C at al og ue L oc al ity Sex SVL HL HW ratio HL/HW EST EED MSR SL l/r LF4 r LT4 r Dorsal keels Supranasals arrangement Prefrontals arrangement Supraocul. ent. frontal Tarsal scales Palmar scales Foot lamellae Hand lamellae Morphobank images T. so co tr an a N M W 93 97 Y E. S oc ot ra ? 97 15 .9 14 .5 1. 10 8. 2 11 .7 33 4/ 4 14 23 3 (5 ) C S II II I / II II I - - - - M 89 78 8- M 89 79 3 T. so co tr an a N M W 93 93 (1 ) Y E. S oc ot ra ? 87 12 .8 9. 4 - 7. 4 6. 3 32 4/ 4 14 19 3 C S II * / I I I II bl un t bl un t w k s M 89 79 4- M 89 80 2 T. so co tr an a N M W 93 93 (2 ) Y E. S oc ot ra ? 78 13 .1 11 .2 1. 17 6. 4 5. 2 33 4/ 4 15 21 3 C S II . I II / (I ) I I I II bl un t bl un t k s M 89 80 6- M 89 81 4 T. so co tr an a N M W 93 95 (1 ) Y E. S oc ot ra ? 72 12 .4 10 .8 1. 15 6. 1 5. 5 32 4/ 4 15 19 3 C S II / II (I II ) bl un t bl un t w k s M 89 81 5- M 89 82 1 T. so co tr an a N M W 93 95 (2 ) Y E. S oc ot ra ? 75 12 .3 11 1. 12 6. 2 5. 5 34 4/ 4 14 21 3 C S II II I / II II I w k bl un t k s M 89 78 8- M 89 79 3 T. so co tr an a N M W 93 95 (3 ) Y E. S oc ot ra ? 78 13 .1 11 .6 1. 13 6. 8 5. 6 32 4/ 4 14 18 3 C S II II I / II II I bl un t bl un t w k s M 89 83 0- M 89 83 6 T. so co tr an a N M W 93 98 (1 ) Y E. S oc ot ra ? 90 14 .3 13 .5 1. 06 7. 5 6. 8 32 4/ 4 14 20 5 C S II II I / II II I bl un t bl un t w k s M 89 83 7- M 89 84 4 T. so co tr an a N M W 93 98 (2 ) Y E. S oc ot ra ? 81 13 .3 11 .2 1. 19 6. 8 6. 5 32 4/ 4 14 21 3 C S II II I / II II I bl un t bl un t s s M 89 85 4- M 89 89 0 T. so co tr an a N M W 93 94 (1 ) Y E. S oc ot ra ? 85 13 .3 11 .9 1. 12 6. 9 6. 1 33 4/ 4 14 21 3 (5 ) C S (I ) I I I II / II II I bl un t bl un t w k s M 89 89 1- M 89 89 7 T. so co tr an a N M W 93 94 (2 ) Y E. S oc ot ra ? 83 13 .3 12 1. 11 6. 8 6. 2 32 4/ 4 13 19 3 (5 ) C S II II I / II II I bl un t bl un t w k s M 89 89 8- M 89 90 4 Su m m ar y St at is tic s N um be r o f I nd iv id ua ls (N ) 10 10 10 9 10 10 10 10 10 10 M ea n 82 .6 13 .3 11 .7 1. 12 6. 9 6. 5 32 .5 4 14 .1 20 .2 M ax im um 97 15 .9 14 .5 1. 19 8. 2 11 .7 34 4 15 23 M in im um 72 12 .3 9. 4 1. 06 6. 1 5. 2 32 4 13 18 St an da rd E rr or M ea n 2. 36 0. 33 0. 45 0. 01 2 0. 20 0. 59 0. 22 0. 00 0. 17 0. 46 283 A pp en di x S2 Sp ec im en s u se d in e st im at io n of d iv er ge nc e tim es a nd R R T te st s Ta xa Lo ca lit y G en B an k A cc es si on N um be rs 1 2S /1 6S C od e Fi g. X R ef er en ce C or dy lu s w ar re ni N C _0 05 96 2 C or w ar K um az aw a 20 04 G er rh os au ru s v al id us Li m po po , R SA H Q 16 71 35 /H Q 16 72 46 G er va l St an le y et a l. 20 11 Eu m ec es sc hn ei de ri sc hn ei de ri Eg yp t EU 27 80 04 /E U 27 80 71 Eu m sc h1 C ar ra nz a et a l. 20 08 Eu m ec es sc hn ei de ri a lg er ie ns is M as sa , M or oc co EU 27 80 21 /E U 27 80 86 Eu m al g1 C ar ra nz a et a l. 20 08 C ha lc id es b ou le ng er i O ue d Sh ili , T un is ia EU 27 79 24 /E U 27 80 45 C ha bo u1 C ar ra nz a et a l. 20 08 C ha lc id es se ps oi de s (E gy pt EU 27 79 25 /E U 27 80 46 C ha se p1 C ar ra nz a et a l. 20 08 C ha lc id es sp he no ps ifo rm is M as sa , M or oc co EU 27 78 75 /E U 27 80 31 C ha sp hM O C ar ra nz a et a l. 20 08 C ha lc id es m au ri ta ni cu s R as E l M a, M or oc co EU 27 79 71 /E U 27 80 60 C ha m au 1 C ar ra nz a et a l. 20 08 C ha lc id es g ue nt he ri N ah al Q et al av , I sr ae l EU 27 80 01 /- C ha gu e C ar ra nz a et a l. 20 08 C ha lc id es m in ut us 1 D eb do u, M or oc co EU 27 79 72 /E U 27 80 61 C ha m in 1 C ar ra nz a et a l. 20 08 C ha lc id es m in ut us 2 Je be l B ou Ib la ne , M or oc co EU 27 79 73 /E U 27 80 62 C ha m in 2 C ar ra nz a et a l. 20 08 C ha lc id es m er te ns i A in S ol ta ne , T un is ia EU 27 79 75 /E U 27 80 64 C ha m er 2 C ar ra nz a et a l. 20 08 C ha lc id es c ha lc id es c ha lc id es G ig lio Is la nd , I ta ly EU 27 79 79 /- C ha ch ac 3 C ar ra nz a et a l. 20 08 C ha lc id es c ha lc id es v itt at us Tu ni s, Tu ni si a EU 27 79 84 /E U 27 80 65 C ha ch av 1 C ar ra nz a et a l. 20 08 C ha lc id es p se ud os tr ia tu s Sk hi ra t, M or oc co EU 27 79 85 /E U 27 80 66 C ha ps e1 C ar ra nz a et a l. 20 08 C ha lc id es st ri at us Sa la m an ca , S pa in EU 27 79 95 /E U 27 80 67 C ha st r1 1 C ar ra nz a et a l. 20 08 C ha lc id es c ol os ii M ok ris se t, M or oc co EU 27 79 30 /E U 27 80 49 C ha co l1 C ar ra nz a et a l. 20 08 C ha lc id es p ar al le lu s R as E l M a, M or oc co EU 27 79 21 /E U 27 80 43 C ha pa r C ar ra nz a et a l. 20 08 C ha lc id es la nz ai A zr ou , M or oc co EU 27 79 20 /E U 27 80 42 C ha la n1 C ar ra nz a et a l. 20 08 C ha lc id es b ed ri ag ai b ed ri ag ai Ja en , S pa in EU 27 79 16 /E U 27 80 41 C ha be db 2 C ar ra nz a et a l. 20 08 C ha lc id es o ce lla tu s o ce lla tu s Ta ta , M or oc co EU 27 79 38 /E U 27 80 52 C ha oc o1 C ar ra nz a et a l. 20 08 C ha lc id es o ce lla tu s t ili gu gu A lg ie rs , A lg er ia EU 27 79 37 /E U 27 80 51 C ha oc t3 C ar ra nz a et a l. 20 08 C ha lc id es o ce lla tu s o ce lla tu s N eg ev D es er t, Is ra el EU 27 79 51 /E U 27 80 55 C ha oc o1 3 C ar ra nz a et a l. 20 08 C ha lc id es o ce lla tu s o ce lla tu s C yp ru s EU 27 79 43 /E U 27 80 53 C ha oc o1 8 C ar ra nz a et a l. 20 08 C ha lc id es o ce lla tu s t ili gu gu A in D ra ha m , T un is ia EU 27 79 58 /E U 27 80 57 C ha oc t3 3 C ar ra nz a et a l. 20 08 C ha lc id es v ir id an us Te ne rif e, C an ar y Is la nd s, Sp ai n EU 27 78 85 /E U 27 80 36 C ha vi rT E C ar ra nz a et a l. 20 08 C ha lc id es se xl in ea tu s s ex lin ea tu s G ra n C an ar ia , C an ar y Is la nd s, Sp ai n EU 27 78 80 /E U 27 80 34 C ha ss G C C ar ra nz a et a l. 20 08 C ha lc id es se xl in ea tu s b is tr ia tu s G ra n C an ar ia , C an ar y Is la nd s, Sp ai n A F0 54 53 0/ A F0 54 54 4 C ha sb 4G C B ro w n & P es ta no 1 99 8 C ha lc id es se xl in ea tu s b is tr ia tu s G ra n C an ar ia , C an ar y Is la nd s, Sp ai n EU 27 78 79 /E U 27 80 33 C ha sb 6G C C ar ra nz a et a l. 20 08 C ha lc id es c oe ru le op un ct at us La G om er a, C an ar y Is la nd s, Sp ai n EU 27 78 92 /E U 27 80 38 C ha co 1G O C ar ra nz a et a l. 20 08 284 C ha lc id es c oe ru le op un ct at us El H ie rr o, C an ar y Is la nd s, Sp ai n EU 27 78 91 /E U 27 80 37 C ha co 4H I C ar ra nz a et a l. 20 08 C ha lc id es si m on yi Fu er te ve nt ur a, C an ar y Is la nd s, Sp ai n EU 27 78 72 /E U 27 80 30 C ha si m C ar ra nz a et a l. 20 08 C ha lc id es m io ne ct on tr ifa sc ia tu s Si di If ni , M or oc co EU 27 78 70 /E U 27 80 29 C ha m io t1 C ar ra nz a et a l. 20 08 C ha lc id es m io ne ct on m io ne ct on Es sa ou ira , M or oc co EU 27 78 68 /E U 27 80 28 C ha m io m 2 C ar ra nz a et a l. 20 08 C ha lc id es m an ue li Si di If ni , M or oc co EU 27 78 57 /E U 27 80 23 C ha m an 1 C ar ra nz a et a l. 20 08 C ha lc id es p ol yl ep is M ar ra ke ch , M or oc co EU 27 78 59 /E U 27 80 24 C ha po l3 C ar ra nz a et a l. 20 08 C ha lc id es p ol yl ep is M or oc co EU 27 78 63 /E U 27 80 26 C ha po l6 C ar ra nz a et a l. 20 08 285 Appendix S3: Results of the BEAST analysis including two partitions (12S and 16S) performed using the software BEAST v.1.6.1 (Drummond & Rambaut 2007) with the following model and prior specifications (otherwise, by default): GTR+G+I, Relaxed Uncorrelated Lognormal Clock (rates 0.01305 and 0.008, respectively), Yule process of speciation, random starting tree, alpha Uniform (0,10), yule.birthRate (0,1000), nucleotide substitution rates Uniform (1,100) initial value=1. See Materials & Methods for more details 286 Appendix S4: Results of the ML (A) and Bayesian (B) analyses. The alignment of the Mabuya sensu lato dataset included a total of 904 base pairs (bp), of which 392 corresponded to the 12S and 512 to the 16S. The alignment matrix is available from Morphobank (Doc1) and the codes used in the matrix can be deciphered with Table 1. Of the total of 392 bp of the aligned 12S sequences 294 were variable and 169 parsimony-informative, while the respective sites for the 512 bp long 16S fragment were 202 and 195. A 287 B 288 A pp en di x S5 - U nc or re ct ed p -d is ta nc es b as ed o n ge ne fr ag m en t 1 2S a bo ve th e di ag on al a nd 1 6S b el ow th e di ag on al . 1 2 3 4 5 6 7 8 9 10 11 1. - T . b re vi co lli s 1 0. 04 21 0. 03 68 0. 05 00 0. 05 00 0. 03 68 0. 07 63 0. 07 63 0. 07 63 0. 07 37 0. 07 37 2. - T . b re vi co lli s 2 0. 04 34 0. 02 11 0. 03 95 0. 03 95 0. 04 21 0. 08 95 0. 08 95 0. 08 95 0. 08 68 0. 08 68 3. - T . b re vi co lli s 3 0. 04 56 0. 02 17 0. 03 95 0. 03 95 0. 04 21 0. 08 42 0. 08 42 0. 08 42 0. 08 68 0. 08 16 4. - T . d ic hr om a 2 0. 02 82 0. 03 90 0. 04 12 0. 00 00 0. 05 00 0. 07 63 0. 07 63 0. 07 63 0. 07 37 0. 07 37 5. - T . d ic hr om a 1 0. 02 82 0. 03 90 0. 04 12 0. 00 00 0. 05 00 0. 07 63 0. 07 63 0. 07 63 0. 07 37 0. 07 37 6. - T . c ris tin ae sp .n . 0. 02 39 0. 04 56 0. 04 34 0. 03 25 0. 03 25 0. 07 89 0. 07 89 0. 07 89 0. 07 63 0. 07 63 7. - T . s oc ot ra na 4 0. 07 16 0. 07 38 0. 06 72 0. 06 94 0. 06 94 0. 06 94 0. 00 00 0. 00 00 0. 00 26 0. 00 53 8. - T . s oc ot ra na 3 0. 07 16 0. 07 38 0. 06 72 0. 06 94 0. 06 94 0. 06 94 0. 00 00 0. 00 00 0. 00 26 0. 00 53 9. - T . s oc ot ra na 5 0. 08 46 0. 08 68 0. 08 03 0. 08 24 0. 08 24 0. 08 24 0. 01 30 0. 01 30 0. 00 26 0. 00 53 10 .- T. so co tra na 1 0. 07 16 0. 07 38 0. 06 72 0. 06 94 0. 06 94 0. 06 94 0. 00 00 0. 00 00 0. 01 30 0. 00 79 11 .- T. so co tra na 2 0. 06 94 0. 07 16 0. 06 51 0. 06 72 0. 06 72 0. 06 72 0. 00 22 0. 00 22 0. 01 52 0. 00 22 289 290 291 Appendix S8 Comparison with other Trachylepis from Eastern and Southern Africa, and Arabia. T. cristinae differs from other Arabian, East- and Southern African Trachylepis by the following characters, according to Lanza and Carfì (1968); Largen and Spawls (2006); Arnold (1986); Spawls, Howell et al. (2002); Broadley and Howell (1991); Günther, Whiting et al. (2005); in square brackets are indicated the state of the same characters in T. cristinae. T. bayoni: frontoprietals fused [not fused]; lower margin of the subocular clearly shorter than its upper margin [not clearly shorter], sometimes excluded by the lip [reaching the lip]; E-African highland endemic. T. boulengeri: 28-32 midbody scales [38]; build slender [robust]; scales with (3) 7-9 (11) keels [2-3]. T. brauni: small (Ltot to 13 cm) [large, Ltot = 22 cm]; subocular clearly shorter than its upper margin (less than a third of upper border) [not clearly shorter]; dorsals usually with 2 keels, or with a poorly defined median keel [2-3 keels]; distinct pale vertebral and dorsolateral stripes [different pattern]; scales on soles of feet keeled and spinose [smooth and not spinose]; a mountain endemic of S-Tanzania. T. dichroma: dorsal and lateral scales with 2 keels [2-3]; different finger 3>4>2>5>1 [3≈4>2>5>1] and toe 3>4>2>5>1 [4>3>5>2>1] formulae; frontal in contact with three supraoculars [only 2]; frontoparietals not in contact [in contact]; subdigital lamellae keeled [smooth]; different colouration. T. hemmingi: dorsals unkeeled in the anterior part of the body [keeled], with 3 barely visible keels posteriorly. T. hildebrantii: auricular lobes conspicuously long and pointed [almost absent]; adult males uniform pale brown above with a series of large dark blotches in a longitudinal row behind the ear [different pattern], body scales sharply 3-keeled, in 32 series at midbody. T. irregularis: subocular with upper margin clearly shorter than its upper margin (less than a third of upper border) [not clearly shorter]; dorsals with 2-5 keels [2-3 292 keels]; a distinct vertebral double-stripe and dorsolateral stripes [different pattern]; a mountain endemic of Kenya. T. isselii: frontoparietal shields fused [not fused]; mid-body scale rows 30-34 [38], the dorsals tricarinate [2-3 carinated]; a dark lateral band from eye to groin with a pale dorsolateral stripe above and pale lateral stripe beneath [different pattern]. T. maculilabris: dorsal scales with 5-8 keels [2-3]; mid-body scale rows 30-38 (but rarely more than 34) [38]; pattern usually with distinct white, black-speckled lips [different head pattern], but occasionally some individuals have dark vertical flank bars. T. margaritifer: mid-body scale rows 38-52 [38]; adult males uniform above with a series of large dark blotches in a longitudinal row behind the ear, young and female striped [different pattern]. T. megalura: slim build [robust] with little limbs [well developed] and very long tail [not particularly elongated]; mid-body scale rows 22-28 [38]; dorsal scales smooth [keeled]; striped pattern [different pattern]. T. planifrons: mid-body scale rows 26-32 [38]; dorsal scales with 3 (rarely 4-5) keels [2-3 keels]; supranasals generally in broad contact [in narrow contact]; dark lateral band strongly demarcated from the pale lower flanks [different pattern]. T. quinquetaeniata: adult males uniform above with a series of large dark blotches in a longitudinal row behind the ear, young and female striped [different pattern]; mid- body scale rows 32-46 (most commonly 34-40) [38]; dorsal scales with 3 (rarely 4 or 5) distinct keels [2-3 keels]; T. striata: subocular scale usually excluded from the edge of the lip [entering the lip]; a dark lateral band from eye to groin and broad dorsolateral pale stripes [different pattern]; scales on soles of feet keeled and spinose [unkeeled and not spinose]. T. varia: lower margin of the subocular clearly shorter than its upper margin [not clearly shorter]; a dark lateral band from eye to groin bordered by conspicuous pale dorsolateral and lateral stripes [different pattern]; small (SVL ~ 60 mm) [SVL = 114 mm]; scales on soles of feet keeled and spinose [unkeeled and not spinose]. T. wingati: a very prominent pale line with distinct dark margins running from beneath the eye, along the lower flank to the groin [different pattern]; mid-body scale rows 30-32 [38]; dorsal scales with 3 distinct keels [2-3]. T. septemtaeniata: 4 sopraocular scales in front of the subocular [5]; typically a pattern of dark stripes on foreparts and light dorsolateral streaks along body [different 293 pattern]; 32-38 midbody scales [38]; third supraocular shield in contact with the frontal shield [not in contact]. T. tessellata: back scales virtually smooth [keeled]; frontal scale does not contact the 1st supraocular [frontal entering the 1st sopraocular]; 4 sopralabial scales in front of the subocular [5]; colour usually uniform [pattern different]; 29-34 scaled around midbody [38]. According to FitzSimons (1943), Branch (1992) and Laurent (1964), among other southern African species, T. capensis, T. chimbana, T. occidentalis and T. variegata have scales on sole of feet keeled and usually spinose, and subdigital lamellae sharply uni- or tri-carinated, as well T. acutilabris, T. damarana, T. lacertiformis, T. sulcata, T. spilogaster, and T. punctulata that have also the subocular distinctly narrowed below or not reaching the lip. T. homalocephala (including the taxa peringueyi, smithii and depressa) is characterized by a lower number of scales around midbody (28-30). T. binotata has the subocular narrowed below, the lower border from ½ to 2/3 length of upper, and a broad black band on either side of neck. T. hoeschi and T. laevis have 29- 33 midbody scales; moreover T. laevis has smooth dorsal scales. 294