Applied Landscape Genetics to Chimpanzee Conservation In Guinea- Bissau. USFWS, Final report
64
1 U. S. Fish & Wildlife Service 2 Great Ape Conservation Fund Applied Landscape Genetics to Chimpanzee Conservation In Guinea2 Bissau Ref. GA20678 November 2013 © Joana Roque de Pinho FINAL REPORT O r g a n e b a u a ´ r A m g y l c h e d d & ONE
Transcript of Applied Landscape Genetics to Chimpanzee Conservation In Guinea- Bissau. USFWS, Final report
! 1!
U.#S.#Fish#&#Wildlife#Service#2#Great#Ape#Conservation#Fund#!!
!!
Applied#Landscape#Genetics#to#Chimpanzee#Conservation#In#Guinea2
Bissau#Ref.#GA20678#
#November#2013#
#
©!Joana!Roque!de!Pinho!
#FINAL#REPORT#
##
!
O r g a n e b a u a´r A m
g y l c h e d d &ONE
! 2!
Applied#Landscape#Genetics#to#Chimpanzee#Conservation#In#Guinea2
Bissau##
Ref.#GA20678#
#November#2013#
#
FINAL#REPORT###
Edited#by:#Sá,#R.;#Minhos,#T.;#Ferreira#da#Silva,#M.;#Sousa,#C.;#Bruford,#M.#
##
A#conservation#research#project#in#cooperation#with:#
#Supported#by#
###
##
Funded#by##
O r g a n e b a u a´r A m g y l c h e d d &
ONE
! 3!
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
General# Organization:# Organisms! and! Environment! Division,! Cardiff! School! of!
Biosciences,!UK!
!
Financial#support#provided#by:!Great!Ape!Fund,!U.S.!Fish!and!Wildlife!Service!
!
Institutional# support# provided# by:! Instituto! da! Biodiversidade! e! Áreas! Protegidas,!
GuineaIBissau;!Projecto!Dari,!Portugal;!ADIAcção!para!o!Desenvolvimento,!GuineaI
Bissau;!Direcção!Geral!de!Florestas!e!Fauna.!
!!Sá,# R.#M.;#Minhos,# T.;# Ferreira# da# Silva,#M.;# Sousa,# C.;# Bruford,#M.! 2013.!Applied!landscape!genetics!for!chimpanzee!conservation!in!GuineaIBissau.!Final!Report.!U.S.!Fish!and!Wildlife!Service,!Cardiff,!UK.!! !
! 4!
ACKNOWLEDGEMENTS#!
!
We! thank! the! Instituto! da! Biodiversidade! e! das!Áreas! Protegidas! of!GuineaIBissau!
and!Direcção!Geral! de! Florestas! e! Caça! for! providing! licences! for! the! collection! of!
samples!and!logistical!support.!We!thank!all!the!Cantanhez!National!Park!and!Cufada!
Lagoons! Natural! Park! rangers! and! guides! for! their! invaluable! support.! We! thank!
Chimbo! Foundation! and! Daridibo,! particularly! to! Joost! Van! Schinjdel! for! support!
during!fieldwork!in!the!Boé!sector,!and!to!ADIAcção!para!o!Desenvolvimento,!for!the!
help!in!Cantanhez.!!To!INEP!(Instituto!Nacional!de!Estudos!e!Pesquisa)!for!sharing!the!
GIS! shaple! files.!We! thank! Catarina! Casanova,!Marta! Carmo,! André! Barata,! Joana!
Roque!de!Pinho!and!Fernando!Sousa!and!to!all!Dari!Project!members!for!assistance!
with!the!field!work.!!We!also!thank!Kathejine!Koops!from!the!Leverhulme!Center!for!
Human! Evolutionary! Studies,! Cambridge,! UK! for! the! cooperation! and! sampling! in!
Nimba,!Republic!of!Guinea.!Finally!we!thank!to!the!U.S.F.W.S.—!Great!Ape!Fund!for!
funding!this!project.!!!
! !
! 5!
EXECUTIVE#SUMMARY#!
Chimpanzees!are!disappearing!at!an!alarming!rate!and!it!is!imperative!that!strategies!
should! be! applied! towards! their! conservation.! All! subspecies! of! the! common!
chimpanzee! Pan$ troglodytes,! including! the! western! chimpanzee,! P.$ t.$ verus,! are!
categorized!in!the!Red!List!of!Threatened!Species!as!Endangered!by!the!International!
Union! for! Conservation! of! nature! (IUCN)!meaning! that! they! are! currently! facing! a!
high!extinction!risk!(Carlsen!et!al.!2012).!!
!
West!African!chimpanzees!are!more!threatened!than!in!any!other!region!across!the!
continent! (Kormos! and! Boesch! 2003).! It! is! estimated! that! there! are! currently! less!
than! 38,000!western! chimpanzees! (Butynski! 2003)! and! only! between! 600! to! 1000!
individuals!(Gippoliti!et!al.!2003)! in!the!Republic!of!GuineaIBissau,!according!to!the!
estimate! of! UNEPIGRASP! (United! Nations! Environment! Programme! –! Great! Apes!
Survival!Project,!Caldecott!and!Miles!2005).!In!fact,!they!have!even!been!considered!
extinct! in! this!almost!unknown!country! (Lee!et$al.! 1988).!The! scientific! community!
agrees!unanimously,!that!tropical!forests!are!disappearing!at!an!excessive!speed!and!
with!them!the!last!populations!of!great!apes!(Laurance!et!al.!2012).!
!
The! evolutionary! history! of! West! African! chimpanzees! remains! ambiguous! and!
controversial.! Chimpanzees! in! GuineaIBissau! live! at! the!most!western! limit! of! the!
species!distribution!and!no! studies! so! far!have! included! individuals! from! this!area.!
Little! is! known! about! their! genetic! structure! and! phylogeographic! structure,!
important!info!for!!
!
The!main!goal!of!this!project!was!to!contribute!to!the!preservation!of!the!longIterm!
survival! of! the! chimpanzees! in! GuineaIBissau! by! creating! a! genetic! management!
plan.!The!project!objectives!include!the!assessment!of!the!geographical!distribution!
of!the!chimpanzee!genetic!diversity!within!the!country!and!the!appraisal!of!possible!
anthropogenic! barriers! to! their! dispersal.! The!major! outcome! of! this! project! is! to!
! 6!
integrate!the!results!obtained!within!the!framework!of!the!National!Action!Plan!for!
chimpanzee!conservation!in!GuineaIBissau.!!
!
The!phylogeography!of!West!African!chimpanzees!has!been! intensively!studied!yet!
remarkably! none! have! included! the! relict! and! peripheral! population! of! GuineaI
Bissau,!with!known!populations!in!the!Quínara,!Tombali!and!Gabú!regions.!!
!
Genetic!diversity,!phylogeographic!structure!and!demographic!history!of!this!country!
were!investigated!by!analyzing!it!alongside!existing!Pan$troglodytes$verus!data!from!
the! hypervariable! mitochondrial! region.! 334! individuals! from! 28! locations! were!
included!in!this!study,!including!chimpanzees!from!Guinea!Bissau!(n!=!189)!and!the!
Nimba!Mountains!(Guinea,!n!=!145).!From!the!51!haplotypes!detected,!haplotype!(h)!
and! nucleotide! diversity! was! high! (94%! and! 0.05325! ±0.00116)! and! phylogenetic!
analyses!revealed!the!existence!of!two!deep!evolutionary!lineages.!I!
!
In! addition,! eleven! subclades! are! described,! where! subclade! B1! presented! a! star!
shaped! pattern! that! was! observed! in! the! median! joining! network! suggesting! a!
demographic!expansion.!The!bimodal!mismatch!distribution!and!the!neutrality!tests!
imply!that!an!expansion!has!occurred!promoting!secondary!contact!between!the!two!
lineages.! Analysis! of! molecular! variance! revealed! 74%! of! the! genetic! variation! is!
partitioned!within!individuals!( ST=0.25956,!p<0.0001).!!
!
Barrier! analysis! identified! three! major! genetic! discontinuities! and! the! estimated!
divergence! times! point! to! vicariance! events! in! the! Middle! Pleistocene! and! the!
recurrence!of!past!climatic!oscillations!in!the!African!forests.!!
!
Chimpanzees! from! Guinea! Bissau! showed! overall! relatively! high! levels! of! genetic!
diversity!with!microsatellite! data! for! the! four! sampling! locations! exhibiting! similar!
levels!of!diversity.!The!observed!heterozygosity!ranged!from!0,630!–!0,797!and!the!
unbiased!expected!heterozigosity!from!0,694!to!0,768.!Cantanhez!is!the!area!where!
chimpanzees! showed! higher! number! of! effective! alleles.! The! observed!
heterozygosity! was! highest! in! Catio! and! Cantanhez! is! the! location! with! higher!
! 7!
unbiased! expected! heterozygosity.! The! analysis! of! population! structure! using!
STRUCTURE! revealed! the!existence!of! three!distinct! genetic! clusters.!However,!we!
did! not! find! a! correspondence! between! different! genetic! clusters! and! different!
geographic!locations.!The!AMOVA!analysis!for!the!nuclear!markers!revealed!that!the!
majority! of! the! genetic! diversity! was! represented! within! each! of! the! geographic!
locations!and!only!4%!of!the!total!genetic!variance!is!explained!by!differences!among!
locations.!!
!
Mantel!tests!demonstrated!the!existence!of!isolation!by!distance!pattern!suggesting!
that! geographic! distance! is! the! main! factor! shaping! the! genetic! structure! of! the!
Guinea!Bissau!chimpanzees!in!concordance!with!mtDNA!analysis.!Chimpanzees!from!
Guinea! Bissau! are! overall! unrelated! (R=I0,0086).! Catio! showed! the! highest! mean!
pairwise! relatedness! and! Boe! is! the! location! where! individuals! are! less! related!
among!each!other.!!
!
The! analysis! of! genetic! discontinuities! revealed! for! both! molecular! markers! the!
existence!of!three!barriers!to!chimpanzee!dispersal,!and!these!vicariant!barriers!are!
coincident!with!major!rivers.!!
!
It! is!crucial!for!the!maintenance!of!the!population!that!the!gene!flow!is!maintained!
and! as! the! Population! viability! Analysis! showed! that! with! only! around! 1000!
individuals!the!population!will!most!likely!not!be!viable!long!term.!
!
Two! phylogeographic! groups! should! be! considered! as! distinct! management! units,!
and! the! populations! of! MadinaICanglode,! Cadique;! Lautchande;! MejoIAmidara;!
Briame,! Empada;! Cufada,! Vendu! Leidi,! Aicum! and! Béli,! indicate! priority! areas! for!
conservation! based! on! mtDNA! genetic! diversity! of! the! populations.! More!
importantly,!it!is!crucial!for!the!survival!of!the!population!that!gene!flow!is!promoted!
with! trans! frontier! populations! (e.g.! Republic! of! Guinea! and! Senegal)! in! order! to!
increase!the!population!size.!!
!
!
! 8!
To!address!all! these! issues! this! report! first! introduces! the!current!major! threats! to!
chimpanzees!in!GuineaIBissau.!The!adopted!methodology!is!summarized!in!Section!2!
as!well!as!the!scientific!findings!of!the!project.!This!section!is!organized!by!objectives!
and!associated!activities.!Moreover,!the!report!is!divided!in!two!parts!(Performance!
Report!and!Financial!status!report).!The!first!part!encompasses!sections!1I2,!and!the!
second! part! presents! the! financial! balance! of! the! project! and! total! expenditure.!
Finally,! a! list! of! publications! and! international! meetings! communications! is!
presented!in!appendix.!
! !
! 9!
Table#of#Contents#!
ACKNOWLEDGEMENTS......................................................................................................4!
EXECUTIVE.SUMMARY.......................................................................................................5!
Part.A....................................................................................................................................10!
1..INTRODUCTION............................................................................................................10!
1.1>.Project.Objectives:............................................................................................................12!
2..METHODS.AND.RESULTS...........................................................................................14!
Objective.1:.To.assess.the.geographical.distribution.of.the.genetic.diversity.
within.GB......................................................................................................................................14!
Activity!1:!DNA!sampling!..................................................................................................................!14!
Activity!2:!!Laboratory!procedures!...............................................................................................!16!
Activity!3:!!Assessment!of!levels!of!genetic!diversity!and!genetic!structure!..............!21!
Objective.2:..To.assess.anthropogenic.barriers.to.dispersal......................................40!
Activity!4:!Design!of!a!GIS!Model!with!information!of!anthropogenic!density!and!
impact!........................................................................................................................................................!40!
Activity!5:!Identification!of!barriers!to!gene!flow!..................................................................!42!
Objective.3:.Viability.Assessment.(PHVA).modeling.exercise.and.to.propose.
specific.actions.for.conservation.........................................................................................47!
Activity!6:!!Carry!out!a!Population!Viability!Assessment!modeling!exercise!.............!47!
Activity!7:!Choice!of!subOpopulations!to!apply!measures!of!conservation!..................!51!
Activity!9:!Disseminate!results!and!main!conclusions!to!partners!and!local!
communities!...........................................................................................................................................!53!
Objective.4:..Integration.of.results.with.the.National.Action.Plan.framework....55!
Activity!10:!Combine!the!information!of!chimpanzee!population!specific!needs!with!
the!requirements!of!local!human!populations.!........................................................................!55!
REFERENCES.......................................................................................................................57!
PART.B..................................................................................................................................62!
FINANCIAL.STATEMENT.................................................................................................62!
APPENDIX............................................................................................................................64!
!
!
! 10!
Part.A.
1.#INTRODUCTION#!
GuineaIBissau! represents! the! westernImost! limit! of! the! endangered!West! African!
Chimpanzee! Pan$ troglodytes$ verus! (Sousa! et$ al.,! 2005).! During! the! 1980s,!
Chimpanzees! were! erroneously! considered! extinct! in! the! country! due! to! a! total!
absence!of!information!owing!largely!to!political!and!civil!unrests!(Lee!et$al.,!1988).!!
In!1994,!a!preliminary!survey!was!conducted!and!the!presence!of!Chimpanzees!was!
reconfirmed! (Gippoliti! and! Dell’! Omo,! 1995;! 1996).! ! More! recently,! research! has!
been!carried!out! in! coIoperation!with!national! and! local! authorities,! establishing!a!
system!for!the!systematic!monitoring!and!management!of!this!great!ape!(Casanova!
and!Sousa,!2007).!!Within!the!country,!Chimpanzees!are!distributed!across!the!south!
of!the!Corubal!River.!!Their!presence!is!confirmed!in!two!protected!areas!Cantanhez!
National! Park! (CNP)! and! Cufada! Lagoons!Natural! Park! (CLNP)! in! the! southwestern!
region,!and!in!the!eastern!region!of!Boé!(Casanova!and!Sousa!2007;!Brugiere!et$al.,!
2009).!
!
Due!to!high!levels!of!exploitation,!loss!of!habitat!as!a!result!of!human!activities,!this!
subspecies!is!estimated!to!have!experienced!a!significant!population!reduction!in!the!
past!20!to!30!years!(IUCN,!2011).!!However,!no!recent!data!are!available!to!allow!for!
an!estimation!of! rates!of! decline! (IUCN,! 2011).! ! The!most! recent! figures! available,!
from! 1996! (Gippoliti! et$ al.,! 2003),! estimate! that! the! number! of! chimpanzees! in!
GuineaIBissau! ranges! from!between!600! and!1000! individuals.! It! is! estimated! that!
chimpanzee! density! in! the! southern! area! of! CNP! is! of! 2.34! nest! builders/km2! in! a!
total!area!of!17.225!km2!corresponding!to!40!individuals!(Sousa!et$al.,!2011),!while!in!
the! neighbouring! east! area! of! Gadamael,! just! outside! the! CNP! area,! this! value!
decreases!to!0.89!nest!builders/km2!in!a!total!area!of!36.513!km2,!which!corresponds!
to!33!individuals!(Sousa!et$al.,!2009).!!However,!the!exact!number!of!individuals!and!
communities!for!the!whole!CNP!and!the!rest!of!the!country!remain!unclear.!
!
! 11!
Anthropogenic! disturbances! such! as! habitat! loss! and! fragmentation! (e.g.! logging!
activities! and! shifting! land! occupation! for! the! purposes! of! agriculture! and! food!
production,! e.g.! cashew!nuts,! the!hunting!of! infant! animals! for! the!pet! trade,! and!
casual! deaths! from! crop! raiding! allied! to! extrinsic! factors! such! as! disease! are! the!
main! threats,! not! only! to! chimpanzees! but! to! all! nonIhuman! primates! in! GuineaI
Bissau!(Gippoliti!et$al.,!2003;!Casanova!and!Sousa,!2007;!Brugiere!et$al.,!2009).!!The!
species!is!classified!by!IUCN!as!Endangered,!and!listed!in!CITES!Appendix!I,!being!also!
protected!at!national!level.!Even!though!most!primate!species!in!GuineaIBissau!are!
traded! for! meat! consumption,! there! is! no! evidence! that! this! is! the! case! for!
Chimpanzees!(Minhos!et$al.,!2013).!Nevertheless,!chimpanzee!body!parts!are!being!
traded!for!medicinal!and!animistic!purposes!at!a!transnational!scale!in!GuineaIBissau!
and!this!fact!constitute!an!additional!threat!that!can’t!be!neglected!(Sá!et$al.!2012).!
!
!
Fig.# 1:#AnimalIderived!products! for!human! traditional!purposes! in!Bandim!market,!
Bissau,!GuineaIBissau,!including!a!chimpanzee!skin!!(2)!(Sá!et!al.!2012).!
!
! 12!
Considerable!efforts!have!been!made!by!several!organizations!towards!chimpanzee!
conservation! in! GuineaIBissau.! However,! knowledge! of! the! population’! genetic!
diversity!and!demographic!structure!remains!lacking,!and!this! information!is!crucial!
for!the!longIterm!survival!of!this!ape.!!!
!
The!major!goals!of! this!project!were:! i)I!determine!genetic!management!units! that!
correspond!to!real!chimpanzee!conservation!needs;! ii)I!develop!strategies! that!can!
increase!demographic!connectivity!between!forest!patches!and!iii)I!using!Population!
Viability!Assessment!tools,!create!a!genetic!management!plan!that!can!be!integrated!
into!the!GuineaIBissau!National!Action!Plan!for!Chimpanzees.!
!
1.12#Project#Objectives:#
!
We!predicted!that!there!is!a!correlation!between!the!geographical!distribution!of!the!
genetic! diversity! and! structure! of! the! Guinea! Bissau! chimpanzees’! and! the!
fragmented!landscape!in!which!they!live.!!
!
To!accomplish!the!project!goals,!an!extensive!nonIinvasive!survey!across!the!species!
distribution! in! the! country! was! conducted.! The! objectives! of! the! project! and!
respective!associated!activities!were:!
!
I.#To#assess#the#geographical#distribution#of#chimpanzee’s#genetic#diversity#within#GB#!!Activities!for!objective!1:!!
1. DNA!sampling;!
2. DNA!extraction!and!amplification!of!an!mtDNA#fragment!and!9!autosomal!microsatellite!loci!
3. Assessment!of!genetic!structure!and!levels!of!genetic!diversity!!
!
#
! 13!
II.#To#assess#anthropogenic#barriers#to#dispersal!
Activities!for!objective!2:!!
4. Design!of!a!GIS!Model!with!information!of!anthropogenic!density!and!impact!
5. Identification!of!barriers!to!gene!flow!
!
III.#Viability#Assessment#(PHVA)#modeling#exercise#and#to#propose#specific#actions#for#conservation#!
Activities!for!objective!3:!!
6. Carry!out!a!Population!Viability!Assessment!modeling!exercise!
7. Choice!of!subpopulations!to!apply!measures!of!conservation!
8. Design!of!specific!actions!for!conservation!
9. Disseminate!results!and!main!conclusions!to!partners!and!local!communities!
!
IV.##Integration#of#results#with#the#National#Action#Plan#framework.#
!
Activity!for!objective!4:!!
10.!Combine!the!information!of!chimpanzee!population!specific!needs!with!the!requirements!of!local!human!populations.!
! !
! 14!
2.#METHODS#AND#RESULTS#!
!
Objective#1:#To#assess#the#geographical#distribution#of#the#genetic#diversity#
within#GB#
!
Activity#1:#DNA#sampling##
!Data!were!collected!from!five!wild!populations!of!P.$troglodytes$verus$across!GuineaI
Bissau.!From!May!to!August!2010,!500!nonIinvasive!samples!(Table!1)!were!collected!
from:!Buba,!Empada,!Catió,!Bedanda,!and!Boé!sectors!(Fig.!2).!These!samples!were!
taken! from! free! ranging! chimpanzees! from! 22! communities,! but! not! all! group!
members!were! sampled.! Cantanhez! region!was! sampled! extensively.! Communities!
ranged!in!size!from!2!to!23!animals!when!it!was!possible!to!see!and!count!them.!For!
each! sample! collected,! and! individual! code,! region,!place,!GPS! coordinates,! status,!
sex!and!age!if!possible!and!morphometric!measures!were!recorded.!Approximately!
5g!of!the!outer!layer!of!fecal!material!were!stored!at!room!temperature!in!absolute!
ethanol!for!24h!and!then!transferred!to!a!second!tube!with!silica!gel!type!III!(Sigma!
Alrich)!and!maintained!at!room!temperature!until!DNA!was!extracted!according!with!
“Two!steps”!protocol!suggested!by!Roeder!et!al.!(2004).!
Permits!to!transport!samples!were!obtained!near!the!official!institutions!of!GuineaI
Bissau,!Portugal!and!UK!(IBAP,!ICNB!and!DEFRA).!
!
!
!
!
!
!
!
!
!
! 15!
!
Table#1:!Chimpanzee!(Pan$troglodytes$verus)!2010!sampling,!GuineaIBissau!!
!Region# Sampling#Site# N# Lat.# Lon.# Sample#site#
Number#Cantanhez! Madina! 25! 11°12'47.!64"N! !15°!2'12.72"W! 1!Cantanhez! Farim! 20! 11°12'31.!02"N! 15°!4'25.80"W! 2!Cantanhez! Caiquéne! 25! 11°13'30.!93"N! 15°!5'47.73"W! 3!Cantanhez! Canghode! 27! 11°!9'40.!68"N! !15°!7'35.46"W! 4!Cantanhez! Catomboi! 15! 11°11'38.28"N! !15°!4'36.36"W! 5!Cantanhez! Amidara! 15! 11°16'52.38"N! !14°58'34.02"W! 6!Cantanhez! Cambéque! 10! 11°11'30.18"N! !15°!0'49.74"W! 7!Cantanhez! São!Francisco! 15! 11°19'18.96"N! !14°59'29.22"W! 8!Cantanhez! Ponta!Nova! 15! 11°18'41.64"N! !14°58'51.36"W! 9!Cantanhez! Cadique! 60! 11°14'19.92"N! !15°6'51.30"W! 10!Cantanhez! Muna! 25! 11°10'2.46"N! !15°8'36.00"W! 11!Cantanhez! Mejo! 15! 11°22'48.45"N! !14°54'51.00"W! 12!Cantanhez! Quebo!Sutuba! 10! 11°9'34.51"N! !14°54'41.12"W! 13!Cantanhez! Lautchande! 25! 11°14'55.14"N! !15°4'20.40"W! 14!Cantanhez! Cambafre! 15! !11°18'1.74"N! !14°56'22.20"W! 15!Cantanhez! Cancira! 11! 11°22'48.45"N! !14°54'51.00"W! 16!
Cantanhez! Bagriel! 4! 11°20'22.44"N! !15°!0'50.94"W! 17!Cufada! Buba!Itchinque! 27! 11°45'11.58"N! !15°!5'18.36"W! 18!Cufada! Tira!Camisa! 5! 11°41'25.56"N! !15°!4'2.94"W! 19!Cufada! !Bani! 5! 11°43'42.54"N! !15°!5'31.20"W! 20!Cufada! Injassane! 5! 11°44'29.88"N! !14°59'4.74"W! 21!Cufada! NhalaIMolha!Pé! 10! 11°37'45.72"N! 14°53'55.08"W! 22!Cufada! Buba!Tumbo! 5! 11°38'37.92"N! 15°!0'19.80"W! 23!Empada! Dois!Rios! 20! 11°37'56.22"N! 15°!9'18.66"W! 24!Catió! Briame! 18! 11°17'19.92"N! 15°14'28.86"W! 25!Boé! Dinguirai! 12! 11°45'18.72"N! 13°51'55.38"W! 26!Boé! Pataque! 8! 11°53'04.9''!N!! 13°57'32.5''!W! 27!Boé! Aicum! 6! 11°56'29.22"N! 13°52'37.38"W! 28!Boé! Béli!North! 18! 11°!52'04.7''!N! 13°!57'!31.9''!W! 29!Boé! Béli! 14! 11°51'10.80"N! 13°54'39.66"W! 30!Boé! Vendu!Leidi! 10! 11°44'33.00"N! 13°43'22.44"W! 31!Boé! Munhini! 5! 11°45'9.84"N! 13°55'27.66"W! 32!
!! !
! 16!
!!Fig.#2:!GuineaIBissau!sampling!sites!for!this!study.!
!!
Activity#2:##Laboratory#procedures###
DNA$extraction$and$PCR$amplification$!
DNA!was!extracted!from!all!samples!using!QIAGEN’s!QIAamp!Stool!Mini!Kit®#with!the!
following!modifications:!~2g!of!fecal!material!was!added!to!a!2ml!tube!and!1.4ml!ASL!
was!added,!vortexed!and!let!soak!overnight!in!an!orbital!shaker.!All!centrifuge!times!
were!increased!3!minutes.!After!the!incubation!period!for!2h!or!longer!in!AL!buffer,!5!
μl!of!1350!μg/ml!POLYIA!carrier!RNA!was!added! to!promote!a!better!DNA!binding!
before!the!ethanol!precipitation!step.!A!final!elution!with!150!μl!AE!buffer!was!used!
instead!of!the!200!μl!recommended!to!assure!a!better!DNA!quality!concentration.!All!
extractions!were!performed!in!a!Class!II!fume!hood!with!ultraviolet!sterilization.!All!
! 17!
material!used!(pipettes,!tubes,!tips,!etc.)!were!autoclaved!and!sterilized!for!15!min!
with!UV! light!prior! to!DNA!extraction.!Surface!of! the!hood!and!bench!was!cleaned!
with!90%!bleach!solution!and!DNA!Away®!decontaminant!solution.!Pipette!tips!with!
aerosolIbarrier!filters!were!purchased!preIsterilized.!Two!blank!extraction!negatives!
were!included!to!monitor!possible!contaminations!and!several!for!PCR’s.!
!
To! test! the! extraction! success,! a! mtDNA! HVRI! PCR! was! performed! on! a! Applied!
Biosystems!Gene!Amp®#PCR!System!9700!using!200!μl!PCR!tubes!in!a!total!volume!of!
20! μL! PCR! reaction! mixture! consisting! of:! 9! μL! of! QIAGEN! Multiplex! PCR! kit!
containing!buffer,!DNTP’s,! Taq!polymerase! and!3!mM!MgCl2! (final! concentration),!
0.1!μL!1x!BSA,!0.1!μL!1x!Q!solution,!6.8!μL!ultrapure!water!and!0.2!μM!primers!and!2!
μL!DNA!template.!PCR!profile!were!as!follows:!95_C!for!15!min,!then!amplified!for!45!
cycles!of!94_C!for!30!s,!51_C!for!60!s,!and!72_C!for!60!s,!and!finally!extended!at!72_C!
for!10!min.!!
!
Primers! L15926! (5’ITAC! ACT! GGT! CTT! GTA! AAC! CI3’,! corresponding! to! positions!
15326I15344! of! the! complete! chimpanzee! mtDNA! genome! [EMBL/GenBank!
accession! No.! D38113)! and! H16555! (5’ITGA! TCC! ATC! GTG! ATG! TCT! TAI3’,!
corresponding!to!positions!15971I15990!of!D38113)!were!used!because!they!amplify!
the!HVRI!mtDNA!region!of!chimpanzees.!!
!
These!primers!have!been!reported!to!reliably!amplify!a!600!bp!of!the!mitochondrial!
control! region! fragment! (also! known! as! DIloop)! in! chimpanzees! (Shimada! et! al.!
2007).!Reactions!were!visualized!on!a!2%!agarose!gel!stained!with!ethidium!bromide!
and!DNA!was!quantified!based!on!the!intensity!of!the!band!when!compared!to!the!
ladder!scale!intensity!(Fig.!3).!Samples!with!less!than!5ng/!μL!were!not!used!because!
they!rarely!work!both!for!sequencing!and!microsatellite!analyses.!!
!
!
!
!
!
! 18!
DNA$sequencing$!
PCR! products! were! purified! with! Exonuclease! I! and! Shrimp! Alkaline! Phosphatase!
(SAP)!enzymes!(New!England!Biolabs,!UK)!at!37_C!for!60!min!and!inactivated!at!80_C!
for! 15! min! and! send! for! sequencing! to! MACROGEN! EUROPE! using! the! same!
amplification!primers! (Fig.!3).!Chromatograms!were! inspected!and!sequences!were!
edited,! assembled! and! aligned! using! the! program! SEQUENCHER! (Gene! Codes!
Corporation).! The! consensus! sequences! were! BLASTed! to! confirm! chimpanzee!
species.!
$
Fig.# 3:!mtDNA! gel! picture.! Positive! amplifications! indicated! by! bright! band! around!
700bp.!+!positive!control;!I!DNA!isolation!controls;!b!blank!PCR!control.!
!
Microsatellite$analysis$$
We!started!to!test!9!microsatellite!loci!initially!developed!for!humans!but!that!cross!
amplify! great! apes.! All! primers! were! previously! used! in! chimpanzee! population!
genetic! studies! by! Gusmão! et! al! (2002)! and! Roeder! et! al! (2006).! The! 9! loci! were!
designed,! optimized! and! combined! in! two! multiplexes! by! using! the! software!
Multiplex! Manager! 1.0! (Holleley! and! Geerts! 2009):! M1! (Amelogenin,! D16S2624,!
D1S550,! D10S1432,! and! D2S1326)! and! M2! (D5S1457,! HUMFIBRA,! D4S1627,! and!
DYS439).!Multiplex!1!includes!the!amelogenin!sexing!system!that!allows!an!accurate!
DNAIbased!sex!identification!of!chimpanzees!using!nonIinvasive!samples!(Bradley!et!
al.! 2001).! PCR!amplification!mixture!was! carried!using!a!QIAGEN!Multiplex!PCR!kit!
! 19!
according! to! the! manufacturer’s! instructions.! Final! reaction! volume! was! 8! μL.!
Primers!were!5’Ifluorolabelled!and!their!final!concentration!in!the!PCR!mixture!was!
0.2! μM.! PCR! profile! on! a! Applied! Biosystems! Gene! Amp®#PCR! System! 9700! were:!
95_C!for!15!min,!45!cycles!of!94_C!for!30!s,!57_C!(M1)!or!58_C!(M2)!for!1,!40!min,!
72_C!for!1,!30!min!and!a!final!extension!of!72_C!for!20!min.!!Loci!used,!ranges,!dyes,!
primer! sequences! and!multiplex! combination! are! shown! in! Table! 3.! Samples!were!
sent! to! MACROGEN! KOREA! where! they! were! electrophoresed! on! an! automated!
genetic! analyzer! using! 1! μL! PCR! product! and! the! internal! size! standard! ROX! 400.!
Alleles!were!scored!using!GeneMapper!v.!3.2!software!(Applied!Biosystems).!
!
From! these! multiplexes! (M1! and! M2)! tree! loci! are! not! working! properly! and!
therefore!a!new!multiplex!combination!(M3)!with!5!additionally!loci!was!created!and!
optimized!(Table!4).!!!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
! 20!
Table#3:!Multiplex!composition,!primer!information!and!allelic!size!ranges!
!
Locus# Multiplex# Label# Allele#size#range#
(bp)#
Primers#
Amelogenin! !
!
M1!
FAM! 104I110! FI5’I!CCTGGGCTCTGTAAAGAATAGTGI3’!
RI5’IATCAGAGCTTAAACTGGGAAGCTGI3’!
D16S2624! NED! 119I139! FI!5’I!TGAGGCAATTTGTTACAGAGCI3’!RI!5’ITAATGTACCTGGTACCAAAAACAI3’!
D10S1432! FAM! 162I182! FI!5’IAGACAGTCAAGAATAACTGCCCI3’!RI5’I!CTGTGGCTCAAAAGCTGAATI3’!
D5S1457! !
!
M2!
FAM! 101I133! FI!5’ITAGGTTCTGGGCATGTCTGTI3’!RI!5’ITGCTTGGCACACTTCAGGI3’!
HUMFIBRA! HEX! 171I203! FI5’IGCCCCATAGGTTTTGAACTCAI3’!RI5’ITGATTTGTCTGTAATTGCCAGCI3’!
D4S1627! FAM! 214I250! FI5’AGCATTAGCATTTGTCCTGGI3’!RI5’IGACTAACCTGACTCCCCCTCI3’!
!!
#
#
Table#4:!Multiplex!3!composition,!primer!information!and!allelic!size!ranges.!
!!
Locus! Multiplex! Label! Allele#size#range#(bp)!
Primers!
DQCAR! !!
!!
M3!
FAM! 99I119! FI!5’IGAAACATATATTAACAGAGACAGACAAAI3’!RI!5’ICATTTCTCTTCCTTATCACTTCATAI3’!
D1S207! HEX! 128I160! FI!5’ICACTTCTCCTTGAATCGCTTI3’!RI!5’IGCAAGTCCTGTTCCAAGTCTI3’!
D13S159! TAMRA! 154I190! FI!5’IAGGCTGTGACTTTTAGGCCAI3’!RI!5’I!CCAGGCCACTTTTGATCTGTI3’!
D14S306! FAM! 196I248! FI!5’I!AAAGCTACATCCAAATTAGGTAGGI3’!RI!5’I!TGACAAAGAAACTAAAATGTCCCI3’!
D6S311! HEX! 208I248! FI5’I!ATGTCCTCATTGGTGTTGTGI3’!RI!5’I!GATTCAGAGCCCAGGAAGATI3’!
#
! 21!
Activity#3:##Assessment#of#levels#of#genetic#diversity#and#genetic#structure##
Mitochondrial$DNA$$
The!haplotypes!were!defined!by!analyzing!the!sequences! in! the!program!DNASP!v.!
4.01! (Rozas! et! al.! 2003).! The!haplotype!diversity! (h)! nucleotide! (π)! and!Watterson!
(θW)!were!estimated!in!the!program!ARLEQUIN!v.3.01!(Excoffier!et!al.!2005)!as!well!
as! the! estimator! FST! in! the! analysis! of! their! genetic! differentiation.! The! haplotype!
richness! (Hr)! was! calculated! using! the! method! implemented! in! the! software!
CONTRIB!v.1.02!(Petit!et!al.!1998).!The!analysis!of!molecular!variance!(AMOVA)!using!
the! pairwise! differences! were! also! held! in! the! program! ARLEQUIN! and! the! most!
likely!pattern!of!population! subdivision!was!estimated!using! the! spatial! analysis!of!
molecular!variance!in!the!program!SAMOVA!1.0!(Dupanloup!et!al.!2002).!
!
In!order!to!evaluate!a!possible!pattern!of!insolation!by!distance!a!Mantel!test!and!a!
RMA!were!applied!in!the!program!IBDWS!v!3.16!(Jensen!et!al.!2005).!!
!
The! phylogenetic! reconstruction! of! the! Network! was! made! in! the! program!
NETWORK!v.!4.6,!which!uses!the!algorithm!of!medianIjoining!(Bandelt!et!al.!1999).!
!
For! the! phylogenetic! analysis,! the! best! model! of! evolution! and! replacement! of!
nucleotides! was! asseed! through! the! Akaike! Information! Criterion! implemented! in!
the! program! jMODELTEST! v.0.1.1! (Posada! 2008).! Using! the! methods! of! neighbor!
joining! and! the! Bayesian! inference! program! MRBAYES! v.! 3.1! (Ronquist! and!
Huelsenbeck!2003)!for!phylogenetic!trees!reconstruction.!The!posterior!probabilities!
were! inferred! using! the!MetropolisICoupled!Markov! Chain!Monte! Carlo! algorithm!
(MCIMCMC)!from!one!million!generations,!with!sampling!every!100,!4!chains,!2!runs!
and!an! initial!burnIin!25%.!Stationarity!of! the!chains!was!established! from!ρ!<0.01!
split!frequencies.!The!convergence!of!the!MCMC!Bayesian!inferences!was!evaluated!
in!the!program!Tracer!v.1.5!(Rambaut!and!Drummond!2007).!All!trees!obtained!were!
viewed!and!edited! in! Figtree! software!v.1.3.1! (Rambaut!2006)! and! in! the!program!
TREEGraph!v.!2.0!(Stöver!and!Müller!2010).!
! 22!
!
With! the! aim!of! analyzing! their! divergence! times,! a! relaxed!molecular! clock! and! a!
coalescent! approach,! implemented! in! the! program! BEAST! v.! 1.6.2,! was! used!
(Drummond!and!Rambaut!2007).!Three!independent!runs!were!performed!using!the!
same!mutation! rate;! the! same!optimized!parameters! and! the!model! suggested!by!
jMODELTEST.! The! convergence! of! the! runs! was! also! evaluated! in! the! program!
TRACER! after! the! combining! of! logs! and! trees! in! LogCombiner! and! TreeAnnotator!
respectively!(Drummond!and!Rambaut!2007).!
!
Finally,!the!analysis!of!demographic!history!and!the!chance!of!sudden!expansion!and!
space!were!tested!using:!mismatch!distributions!(Rogers!and!Harpending!1992)!and!
tests!of!neutrality!(Tajima!D!and!Fu!Fs)!and!the!program!ARLEQUIN;!tests!Fu!and!Li!F!
*!and!D!*!in!the!program!DNASP!v.4.01.!
$
MTDNA$diversity$!
In!order!to!compare!the!evolutionary!history!of!the!chimpanzees!We!obtained!a!261!
bp! sequence! for! the!mitochondrial! HVR1! from!334!west! African! chimpanzee! fecal!
samples!(189!from!GuineaIBissau,!and!145!from!the!Nimba!Mountains,!Guinea).!We!
found!55!polymorphic!sites!comprising!52!transitions!(ti)!and!three!transversions!(tv)!
with!an!overall!ti/tv!ratio!of!31.04!following!the!maximum!likelihood!estimate.!The!
nucleotide! composition! was! as! follows:!A! (37.67%),! T! (17.10%),! C! (6.73%),! and!G!
(38.51%).!
#
FiftyIone!haplotypes!were!identified,!found!in!1!I!3!populations!(Table!3.2)!with!19!
shared,! including! the! haplotypes!H1,! H13! and!H35! that!were! distributed! between!
the! two! countries.! Nimba! possessed! the! highest! number! of! exclusive! haplotypes!
(n=12),!followed!by!Cadique!(n=!8)!and!Vendu!Leidi!(n=8)!in!GuineaIBissau.!H13!was!
the!most!common!haplotype,!present! in!46! individuals.!H3!and!H4!occured!only! in!
GuineaIBissau!and!were!widespread!across!all!study!areas.!Cantanhez!possessed!the!
highest! number! of! private! haplotypes! (n=! 11)! followed!by!Nimba! and!Boé! (with! 9!
! 23!
each),!Cufada! (n=5),!Catió! (n=!3)!and!Empada!with!one! (H5).!Three!haplotypes!are!
communal! between! CNP! and! CLNP! (i.e.! H2,! H6,! H9),! and! five! between! Boé! and!
Cantanhez! (i.e.! H14,! H18,! H19,! H21,! H24).!Within! CNP,! H15! is! the!most! frequent!
haplotype!occurring!in!seven!localities!(n=!21).!!
#
The!haplotype!diversity!(h)!for!each!study!area!ranged!from!0.836!in!Nimba!to!0.929!
in!Gabú.!The!highest!values!of!the!nucleotide!diversity!(π)!also!occurred!in!Gabú!(π=!
0.05516)!and!the! lowest! in!Nimba!(π=!0.0429).!The!total!haplotype!and!nucleotide!
diversities!were!h=!0.935!and!π=!0.05325,!respectively.!An!alternative!measure!to!π!
is! Watterson’s! theta! estimator! (θW).! The! average! high! θW! for! all! population! was!
0.03058! with! a! variation! between! 0.02804! and! 0.03620! in! Nimba! and! Quínara,!
respectively.!Estimates!of!the!molecular!diversity!indexes!are!given!in!Table!5.!
!
The! P.$ troglodytes$ verus! populations! contributed! differently! to! the! total! genetic!
diversity!and!differentiation!inferred!for!the!study!areas.!The!contributions!from!the!
diversity! (CS! and! CRS)! and! differentiation! (CD! and! CRD)! components! of! each!
population! are! shown! in! Fig.! 4.! The! highest! CS! and! CRS! values! were! observed! in!
Aicum,! Béli,! Béli! North! and! Vendu! Leidi! populations,! all! belonging! to! the! Gabú!
region.!In!contrast,!the!highest!CD!and!CRD!values!were!seen!in!Bani,!and!Injassane!
from! Quínara! region! and! in! general! from! the! Cantanhez! populations.! These!
contributions! to! genetic! differentiation!were!probably!due! to!exclusive!haplotypes!
found!in!medium!to!high!frequencies!in!this!region.!
!
The!medianIjoining!phylogenetic!network!confirmed!the!results!of!the!phylogenetic!
tree! analysis,! but! it! provided! more! resolution! on! relationships! among! haplotypes!
and!within!subclades!where!it!was!possible!to!observe!a!clear!starIshape!pattern!in!
Lineage!B!(Fig.!5).!
! 24!
!
Table&5:&Summary!statistics!and!demographic!expansion!tests!observed!in!the!study!areas!and!in!all!populations,!including!the!mean!number!of!
pairwise!differences!(k),!and!the!theta!per$site!from!S!(θW)!according!to!Watterson!(1975).!
!
Parameters! Tombali! Quínara! Gabú! Nimba! All!populations!
Sample!size! 124! 30! 35! 145! 334!
Number!of!populations! 16! 6! 5! 1! 28!
Number!of!polymorphic!sites! 45! 39! 42! 44! 55!
Number!of!haplotypes! 26! 12! 18! 12! 51!
Haplotype!diversity!(SD)! 0.9251!(±0.100)! 0.897!(±0.031)! 0.929!(±0.021)! 0.836!(±0.012)! 0.935!(±0.005)!
Nucleotide!diversity!(SD)! 0.05355!(±0.00129)! 0.04839!(±0.00322)! 0.05516!(±0.00311)! 0.04082!(±0.00307)! 0.05325!(±0.00116)!
θW! 0.02958! 0.03620! 0.03510! 0.02804! 0.03058!
k! 13.761! 12.485! 14.121! 10.490! 13.633!
Tajima’s!D! 2.36727!! 1.17622!! 1.89567!! 1.36985!! 1.93048!!
Fu!and!Li’s!F’! 2.14021**& 1.73893*& 2.23514**& 1.98212*& 2.53128**&
Fu!and!Li’s!D’! 1.29726!! 1.59058**& 1.81072**& 1.84959**& 2.08803**&
Fu’s!Fs! 3.62642!! 3.30846!! 0.64391!! 13.64252!! Z93294&&
Mismatch!distribution! Bimodal! Bimodal! Unimodal! Multimodal! Bimodal!
*!P<0.05;!**&P<0.02!!
! 25!
!
!
!!!!!!!!!!!!!!!!!!&
&
&
Fig.&4:&The!contribution!to!the!total!haplotype!diversity!(CT)!and!haplotypic!richness!(CTR)!of!each!population!of!chimpanzees!using!the!HVR1!
haplotypes.! The! blue! and! red! bars! represent! the! contribution! of! diversity! (CS! and! CRS)! and! differentiation! (CD! and! CRD),! respectively.
! 26!
!
Fig.& 5:& Haplotype! network! recovered! by! median! joining! analysis.! Haplotypes! are! represented! by! circles! with! size! proportional! to! their!
frequencies!and!coloured!according!to!subclades!recovered!in!the!phylogenetic!analyses.!Numbers!outside!the!circles!represent!the!mutational!
steps,!and!inside!the!circles!the!number!of!individuals!for!each!haplotype.!Grey!circles!represent!missing!haplotypes,!extinct!or!not!sampled.&
! 27!
Phylogeographic-structure-!
Pairwise! FST! values! calculated! from! haplotype! frequencies! of! the! 28! populations!
sampled! indicate! significant! differentiation! (Table! 6).! However,! when! the!
populations! were! grouped! together! the! FST! values! varied! from! 0.00334! (TombaliG
Quínara)!to!0.21095!(QuínaraGNimba)!and!only!the!Nimba!differed!significantly!from!
zero! (Table! 3.4).! Pairwise! PhiST! calculated! from! Kimura’s! two! parameter! genetic!
distances! revealed! similar! results! demonstrating! that! the! three! regions! within!
GuineaGBissau!are!not!significantly!differentiated.!!
!
In! order! to! reveal! the! partitioning! of! genetic! diversity! a! hierarchical! AMOVA! was!
conducted!showing!a!strong!differentiation!among!all!chimpanzee!populations!(ϕST!
=! 0.25956,! p<0.0001).! AMOVA! revealed! 74.04%! of! overall! genetic! variance!
partitioned! among! individuals! within! populations! whereas! differences! among!
populations! within! regions! accounted! for! 23.74%! (ϕSC!=! 0.24274,! p<0.0001).! Only!
2.22%! of! the! genetic! variation! is! partitioned! among! regions! and! this! was! not!
significant!(AMOVA!ϕCT!=0.2221,!p>0.05)!(Table!7).!
!
The!SAMOVA!demonstrated!that!the!strongest!partitioning!of!genetic!diversity!was!
obtained!when!samples!were!assigned!to!2!G!10!groups.!The!best!grouping!scheme!
divided! the! distribution! range! of!P.- troglodytes- verus- in! GuineaGBissau! and!Nimba!
into! two! main! geographical! groups! (K=2;! FCT! =! 0.34253,! p<0.0001).! When! the!
number!of! groups! exceeded! three,! SAMOVA! showed! lower! FCT! values.!With!K=2! a!
partition!between!the!south!Cantanhez!populations!and!all!the!others!corresponded!
to! the! two!main!HVR1!haplotype! lineages.! The! populations! from! south!Cantanhez!
(i.e.! Farim,! Catomboi,! Cafatche! and! Canghode)! comprised! only! haplotypes! from!
Lineage! A! while! the! rest! of! the! populations! in! Guinea! Bissau! and! Nimba! have!
haplotypes! from!both! lineages.! Table! 8! summarizes! the! SAMOVA!analysis! and! the!
components!of!genetic!variance!explained!for!each!cluster.!
!
! 28!
Mantel!tests!performed!to!assess!isolation!by!distance!revealed!no!correlation!when!
all! the! populations! were! considered! (Z=! 5.999555.85,! r=G! 0.0822,! one! sided! p=!
0.8370!from!30!000!permutations).!However,!when!considering!Cantanhez!only!(i.e.!
excluding!Nimba!and!Gabú)!to!take!into!account!the!longGterm!historical!divergence!
among! populations! as! recommended! by! Telles! and! DinizGFilho,! (2005)! this!
correlation!was!significant!(Z=159.8819,!r=!0.5661,!oneGsided!p=!0.0080!from!30!000!
permutations).! The! isolation! by! distance! among! Cantanhez! populations! was!
confirmed!by!the!RMA!(y=G0.08010!x=0.03709,!r2=0.320).!
!
Table&6:&Pairwise!FST!values!between!chimpanzee!study!areas! in!GuineaGBissau!and!
Nimba!Mountains.&&
&
! Tombali! Quínara! Gabú! Nimba!
Tombali! —! ! ! !
Quínara! 0.00334! —! ! !
Gabú! 0.01178! 0.00984! —! !
Nimba! 0.19211& 0.21095& 0.19992& —!
P<0.05&
&
&
Table&7:&Hierarchical!analysis!of!molecular!variance!(AMOVA)!for!chimpanzee!HVR1!
mtDNA!sequences!from!GuineaGBissau!and!Nimba.!
!
Variance&component& Variance& %&
Total&
d.f.& SSD& &statistics&
& P&
Among!regions! ! 0.17410! 2.22! 5! 298.867!0.02221!
! 0.52590!
Among!populations!
within!regions! !
1.86041! 23.74! 22! 394.057!0.24274!
! <0.0001!
Within!populations!
!
5.80365! 74.04! 307! 1781.721!0.25956!
! <0.0001!
! 29!
&
Table& 8:& Results! from! SAMOVA! analysis! with! different! clusters! defined! a! priori.!
Number!of!groups!(K),!group!composition!and!fixation!indexes!are!reported.!!
&
K& Group&composition& FCT*& FSC*& FST*&
2! (2,!4,!5,!6)!(1,!3,!7,!8,!9,!10,!11,!12,!13,!14,!15,!16,!17,!
18,!19,!20,!21,!22,!23,!24,!25,!26,!27,!28)!
0.34253! 0.17112! 0.45503!
3! (2,!4,!5,!6)!(24)!(1,!3,!7,!8,!9,!10,!11,!12,!13,!14,!15,!16,!
17,!18,!19,!20,!21,!22,!23,!25,!26,!27,!28)!
0.33889! 0.16451! 0.44765!
4! (1)!(2,!4,!5,!6)!(7,!8,!9,!10,!11,!12,!13,!14,!15,!16,!17,!
18,!19,!20,!21,!22,!23,!25,!27,!28)!(24,!26)!
0.33661! 0.14285! 0.43137!
5! (7,!8,!9,!10,!11,!12,!13,!14,!15,!16,!17,!19,!20,!21,!22,!
23,!25,!27,!28)!(1,!2,!3,!4,!5)!(26)!(24)!
0.33159! 0.14037! 0.42542!
6! (2,!3,!4,!5)!(24)!(1)!(18)!(7,!8,!9,!10,!11,!12,!13,!14,!15,!
16,!17,!19,!20,!21,!22,!23,!25,!27,!28)!(26)!
0.32997! 0.14092! 0.42439!
7! (1)!(!5)!(7,!8,!9,!10,!11,!12,!13,!14,!15,!17,!19,!20,!21,!
22,!23,!25,!27,!28)!(16,!26)!(18)!(2,!3,!4)!(24)!
0.31821! 0.12368! 0.40253!
8! (1)!(16,!26)!(5)!(2,!3,!4)!(24)!(19)!(18)!(7,!8,!9,!10,!11,!
12,!13,!14,!15,!17,!20,!21,!22,!23,!25,!27,!28)!
0.30858! 0.12287! 0.39353!
9! (1)!(16)!(2,!3,!4)!(7,!8,!9,!10,!11,!12,!13,!14,!15,!17,!19,!
20,!21,!22,!23,!25,!27,!28)!(5)!(24)!(19)!(18)!(26)!
0.29791! 0.12851! 0.38814!
10! (8)!(1)!(24,!26)!(2)!(3,!4)!(5)!(21)!(7,!9,!10,!11,!12,!13,!
14,!15,!17,!19,!20,!22,!23,!25,!27,!28)!(18)!(19)!
0.28449! 0.11777! 0.36876!
*P<0.0001&
&
! !
! 30!
Demographic-history-and-divergence-time-!
Historical! demography!was! analyzed! by! estimating! parameters! for! neutrality! tests!
and!by!conducting!mismatch!distribution!analyses.!Tajima’s!D!tests!were!positive!but!
not! statistically! significant! for!each! region!or! for! all! populations!grouped! together.!
Similarly,!the!Fu’s!Fs!values!were!not!significant,!although!presenting!a!negative!value!
for! all! populations! (Fu’s! Fs!=G93294).! The! test! Fu! and! Li’s! and!D’! presented!positive!
values!and!were!statistical!significant!in!each!region!and!in!all!populations,!with!the!
exception!of! the! test!Fu!and!Li’s!D’! for! the!Tombali! region! that!was!not!significant!
suggesting!a!deviation!from!neutrality!(see!Table!5).!
!
Considered!altogether,!the!mismatch!distributions!for!all!populations!were!bimodal!
and! they! fitted! with! their! distributions! under! the! sudden! and! spatial! expansion!
models!and!this!may!be!due!to!their!peripheral!nature.!The!tests!for!the!goodnessG
ofGfit! confirmed! both! demographic! models! (SSD=! 0.00621,! p>0.05;! SSD=! 0.00710,!
p>0.05!for!the!sudden!and!spatial!expansion!models,!respectively).!However,!when!
considering! only! the! sequences! from! Lineage! A! and! Lineage! B! defined! by! the!
phylogenetic! and! network! inferences,! the! patterns!were! unimodal! fitting!with! the!
distributions! expected! under! the! sudden! and! spatial! expansion! models.!
Furthermore,!the!model!parameters!Θ0!and!Θ1!calculated!for!Linage!A!and!Lineage!B!
separately,! showed! values! expected! under! a!model! of! rapid! growth! in! both! cases!
(Fig.! 6).! Tests! for! the! goodnessGofGfit! for! the! observed! data! support! similarly! the!
expansion!model!revealed!by!the!SSD!values!for!both!lineages!(SSD=0.037,!P=!0.046;!
and! SSD=! 0.0155,! P=! 0.192,! respectively).! Additionally,! the! small! raggedness! index!
values!supported!a!smooth!distribution!(RGIndex=!0.044;!P=!0.237;!RGIndex=!0.0189,!
P=!0.055).!
!
Considering!a!divergence!rate!of!22.5%!per!million!years,!and!a!split! time!between!
humans! and! chimpanzees! of! 7G5!mya,! and! between! chimpanzees! and! bonobos! of!
2.1G1.5!mya9,!the!two!major!mitochondrial!lineages!are!estimated!to!have!diverged!
at!1!mya!(95%!HPD:!0.34G2.11).!The!most!recent!common!ancestor!(mrca)!of!Lineage!
A!is!estimated!at!0.67!mya,!while!the!TMRCA!of!Lineage!B!is!estimated!at!0.43!mya!
! 31!
(95%!HPD:!0.34!to!2.11!mya).!The!divergence!time!among!the!Lineage!A!haplogroups!
varied!between!0.32!mya!(95%!HPD:!0.03!to!0.95)!for!haplogroup!A3!and!0.47!mya!
(95%!HPD:!0.9!to!1.22)!and!between!0.07!mya!(95%!HPD:!0G47G0.97)!for!haplogroup!
B2!and!0.29!mya!(95%!HPD:!0.04!to!0.81)!for!haplogroup!B2.!!
!
!
!&
Fig.& 6:!Mismatch! distributions! for! the! GuineaGBissau! and! Nimba! chimpanzees! and!
respective!associated!parameters.!Mismatch!distribution!according!with!the!sudden!
expansion!model!and!with!the!spatial!expansion!model!for!Lineage!A!(A)!and!Lineage!
B! (B),! respectively.! Mean! pairwise! differences! between! pairs! of! haplotypes! (k),!
mismatch! derived! parameters! (Θ0,! τ,! Θ1).! Sum! of! squared! differences! (SSD)! and!
Harpending!Raggedness!Index!with!respective!P!values.!Number!of!female!migrants!
(M).!
!
!
!
!
! 32!
Nuclear-DNA-!
For!genotyping!criteria!the!“multiGtubes”!approach!(Taberlet!et!al.!1996)!was!initially!
considered! (up! to! seven!positive! PCR! repeats! to! confirm!homozygosity).!However,!
due! to! the! high! number! of! extracted!DNA! samples,! time! and! financial! constraints!
prevented! that!each! individual!was!genotyped!as!many! times!as! recommended!by!
the!protocol.!The!number!of!replicates!was!therefore!estimated!by!using!a!maximum!
likelihood! approach! implemented! in! the! software! GEMINI! v.! 1.4.1! (Valière! et! al.!
2002)! that! uses! a! priori! information! about! the! error! rates! (i.e.! allelic! dropout! and!
false!alleles),!which!were!determined! in! the! software!PEDANT!v.! 1.0! (Johnson!and!
Haydon! 2007,! 2009).! General! parameters! in! the! software! GEMINI! included! 100!
simulation!replicates!and!the!range!of!repetition!number!was!set!from!2G12.!This!test!
revealed!that!four!PCR!repetitions!were!appropriate!to!achieve!a!high!probability!of!
identity! assuring! a!95%!confidence!of! the! genotypes,! and! the!per! locus! consensus!
threshold!revealed!that!for!a!sample!to!be!included!in!the!final!dataset!must!have!at!
least!two!positive!PCR!results!for!nine!of!the!14!loci.!For!molecular!sexing!the!result!
was!considered!as!true!when!observed!three!times!out!of!the!four!repetitions.!
!
Allelic!dropout,!null!alleles!and!stutter!peaks!were!assessed!using!MICROCHECKER,!v.!
2.2.3! (van! Oosterhout! et! al.! 2004).!Moreover,! a! quality! index! (QI)! for! all! loci! was!
determined!according!to!Miquel!et!al.!(2006)!and!samples!with!a!QI!below!0.50!were!
removed!from!the!dataset,!as!they!are!genotypes!with!poor!quality.!
!
To!detect!repeated!individuals!the!software!GIMLET!v.!1.3.3!(Valière!2002)!was!used,!
and!duplicated!genotypes!were! removed.!This! software!was!also!used! to!calculate!
the!Probability!of!Identity!(PI)!and!the!Probability!of!Identity!between!siblings!(PIsibs)!
(Waits!et!al.!2001).!Finally,!GenAlEx!v.!6.4.1!(Peakall!and!Smouse!2006)!was!used!to!
conduct!all!standard!population!genetic!analyses.!!
!
Genetic! diversity! was! analyzed! for! each! locus! and! across! all! samples! through! the!
number! of! alleles! (Na),! number! of! effective! alleles! (Ne),! observed! (Ho),! expected!
heterozygosity! (He),! unbiased! expected! heterozygosity! (UHe)- using! GenAlEx! 6.41!
! 33!
(Peakall!and!Smouse,!2006).!Same!analyses!were!also!carried!out!for!each!sampling!
location! and! across! all! samples.! Analysis! of! molecular! variance! (AMOVA),! using! a!
codominant! genotypic! distance! matrix! to! calculate! ΦPT,! was! also! implemented! in!
GenAlEx! 6.41! (Peakall! and! Smouse,! 2006)! to! understand! how! diversity! was!
partitioned!within!and!among!sampling! locations.!Significant! tests!were!performed!
through!9999!permutations.!!
-
We! investigated! population! genetic! structure! using! two! Bayesian! modelGbased!
approaches! and! compared! the! consistency! of! results.! Since! these! approaches! use!
multiGlocus! genotypes! to! cluster! individuals! into! populations! that!minimize! HardyG
Weinberg!(HW)!and!linkage!disequilibrium!(LD),!any!departure!from!random!mating!
leads! to! subdivision! of! the! data! into! subGpopulations! (Pritchard! et! al.,! 2000;!
Beaumont!&!Rannala,!2004).!We!first!used!STRUCTURE!2.2! (Pritchard!et!al.,!2000),!
without! a! spatial! prior,! to! detect! the! optimal! number! of! genetic! clusters! (K).!We!
allowed! K! to! range! from! one! to! ten,! using! five! independent! runs,! with! 1,000,000!
Markov!chain!Monte!Carlo!(MCMC)!iterations,!after!a!100,000!burnGin!period.!Runs!
were!performed!under!the!admixture!module!and!allele!frequencies!were!assumed!
to!be!correlated.!!
!
The!most!appropriate!K!was!chosen!following!the!summary!statistic!ΔK!described!by!
Evanno!et!al.! (2005),!which! is!based!on!the!rate!of!change! in!the! log!probability!of!
the! data! between! successive! K! values.! Secondly,! we! used! a! spatially! explicit!
approach! using! the! GENELAND! v3.3.0! (Guillot! et! al.! 2005b).! We! performed! five!
independent! runs,! assuming! K! to! vary! between! one! and! ten,! assuming! nonG
correlated! allele! frequencies! and! accounting! for! the! absence!of! null! alleles.! In! the!
advanced!options,! the!maximum!number!of!nuclei!was!set!to!408!(three!times!the!
samples! number)! and! the!maximum! rate! of! the! Poisson! process!was! fixed! at! 136!
(corresponding! to! the! number! of! genotypes).! Each! run! used! 1,000,000! iterations,!
with! a! thinning! value! of! 10,000.!We! repeated! the! analysis! for! several! degrees! of!
Geographic!Positioning!System!(GPS)!uncertainty,!varying!between!0!and!4!km!(the!
maximum! distance! for! which! we! could! assure! independence! of! social! units)! and!
found!no!effect!for!the!results!for!nonGcorrelated!alleles!frequencies.!Therefore,!we!
! 34!
only!show!the!analysis!using!4!km!as!GPS!uncertainty.!
!
Mean!pairwise!relatedness!was!estimated!using!Kingroup!v2_101202!(Konovalov!et!
al.,!2004).!The!relatedness!estimator!of!Queller!and!Goodnight!(1989)!was!used!for!
all!possible!dyads!in!each!sampling!location.!
!
FineGscale! spatial! genetic! structure! was! investigated! by! analysing! isolationGbyG
distance.!GenAlEx!6.41! (Peakall! and!Smouse,!2006)!was!used! to!perform!a!Mantel!
test!to!estimate!the!correlation!between!the!Nei!genetic!distance!and!geographical!
distance!between!all!pairs!of!individuals.!The!significance!of!results!was!assessed!by!
9999!permutations.!136!individuals!constitute!the!final!dataset.!
!
Nuclear-DNA-diversity--
Chimpanzees! from! Guinea! Bissau! showed! overall! relatively! high! levels! of! genetic!
diversity!with!the!four!sampling!locations!exhibiting!similar!levels!of!diversity!(Table!
9!and!10).!The!number!of!effective!alleles!across!loci!(Ne)!ranged!from!3,236!–!4,584,!
the!observed!heterozygosity!ranged!from!0,630!–!0,797!and!the!unbiased!expected!
heterozigosity! from! 0,694! to! 0,768.! Cantanhez! is! the! area! where! chimpanzees!
showed!higher!number!of!effective!alleles.!The!observed!heterozygosity!was!highest!!
in!Catio!and!Cantanhez!is!the!location!with!higher!unbiased!expected!heterozygosity!
(Table!9!and!10).!
! 35!
Table&9:!Indices!of!genetic!diversity!for!each!locus!and!across!all!loci.!Na!–!number!of!alleles;!Ne!–number!of!effective!alleles;!Ho!–!observed!
heterozygosity;!He!–!expected!heterozygosity;!UHe!–!unbiased!expected!heterozygosity!
!
!
!!! Na& Ne& Ho& He& UHe&Locus& Mean& SE& Mean& SE& Mean& SE& Mean& SE& Mean& SE&&D16S2624&& 6,500! 0,866! 3,952! 0,286! 0,843! 0,063! 0,743! 0,018! 0,758! 0,015!D10S1432&& 5,750! 1,031! 2,944! 0,407! 0,550! 0,089! 0,633! 0,067! 0,646! 0,067!&D5S1457&& 5,750! 1,031! 3,284! 0,275! 0,758! 0,025! 0,689! 0,027! 0,702! 0,026!&HUMFIBRA&& 4,500! 0,645! 2,698! 0,262! 0,612! 0,059! 0,618! 0,040! 0,630! 0,038!&D4S1627&& 7,000! 1,080! 4,325! 0,714! 0,842! 0,023! 0,752! 0,035! 0,768! 0,032!&DQCAR&& 7,000! 1,581! 3,163! 0,424! 0,597! 0,168! 0,663! 0,052! 0,678! 0,054!&D14S306&& 6,250! 0,479! 3,884! 0,266! 0,788! 0,058! 0,739! 0,018! 0,755! 0,014!&D1S207&& 8,750! 1,436! 5,017! 0,616! 0,958! 0,020! 0,791! 0,026! 0,809! 0,022!&D6S311&& 5,250! 0,750! 2,794! 0,207! 0,503! 0,082! 0,637! 0,025! 0,651! 0,024!&D13S159&& 10,500! 1,500! 6,476! 0,906! 0,850! 0,017! 0,833! 0,030! 0,851! 0,028!Overall& 6,725! 0,405! 3,854! 0,225! 0,730! 0,031! 0,710! 0,015! 0,725! 0,015!
!
&&&&&&
! 36!
&Table&10:!Indices!of!genetic!diversity!for!each!sampling!location!anda!cross!all!samples.!Na!–!number!of!alleles;!Ne!–number!of!effective!alleles;!
Ho!–!observed!heterozygosity;!He!–!expected!heterozygosity;!UHe!–!unbiased!expected!heterozygosity!
!!!
Location& && Na& Ne& Ho& He& UHe&Cantanhez& Mean& 9,100! 4,584! 0,713! 0,762! 0,768!!! SE& 0,795! 0,488! 0,048! 0,021! 0,022!Boe& Mean& 6,800! 4,131! 0,630! 0,719! 0,731!!! SE& 0,786! 0,555! 0,086! 0,035! 0,036!Cufada& Mean& 5,900! 3,236! 0,780! 0,674! 0,694!!! SE& 0,504! 0,214! 0,056! 0,030! 0,031!Catio& Mean& 5,100! 3,464! 0,797! 0,684! 0,706!!! SE& 0,586! 0,397! 0,045! 0,029! 0,030!Overall& Mean& 6,725! 3,854! 0,730! 0,710! 0,725!!! SE& 0,405! 0,225! 0,031! 0,015! 0,015!
!!
! 37!
Analysis(of(population(structure(!
The! analysis! of! population! structure! using! STRUCTURE! revealed! the! existence! of!
three!distinct!genetic! clusters! (Fig.!7).!However,!we!did!not! find!a! correspondence!
between! different! genetic! clusters! and! different! geographic! locations.! Individuals!
from! two!genetic! clusters! (represented! in! red!and!green)!are! found! in!all! sampled!
locations.!Only!the!individuals!belonging!mainly!to!the!blue!genetic!cluster!were!not!
found!in!Boe!and!Cufada.!!
!
The! little! genetic! structure! showed! by! STRUCTURE!was! also! seen! for! the! AMOVA!
results!(Fig,!8).!!We!found!that!the!majority!of!the!genetic!diversity!was!represented!
within!each!of!the!geographic!locations!and!only!4%!of!the!total!genetic!variance!is!
explained! by! differences! among! locations.! Despite! the! fact! that! the! chimpanzees!
from! Guinea! Bissau! showed! some! level! of! genetic! differentiation! (ΦPT! =! 0.04,! P<!
0.001),!this!seems!to!be!weak!with!STRUCTURE!results!supporting!this!evidence.!
!
!!Fig.7:!Bayesian!population!genetic!clustering!of!the!chimpanzee!from!Guinea!Bissau!
using!SRUCTURE!software.!Different!colors! represent!different!genetic!clusters!and!
the! names! represent! different! sampling! locations.! Each! column! represents! a!
different! individual! and! Q! corresponds! to! the! percentage! of!membership! to! each!
genetic!cluster.!!
!
!
! 38!
!
!Fig.'8:!Graphical!representation!of!the!Analysis!of!Molecular!Variance!(AMOVA).!P[!
value!=!0,001!(based!on!9999!permutations).!!
!!!Pairwise! ΦPT! suggested! the! highest! level! of! differentiation! between! Catio! and!
Cufada,! despite! being! adjacent! locations.! Additionally,! Cantanhez! is! less!
differentiated! from! Boe! than! it! is! from! Cufada! and! Catio,! despite! being!
geographically!more!distant!from!the!former!(Table!11).!!
!!Mantel!tests!demonstrated!the!existence!of!isolation!by!distance!pattern!(Fig.!9,!p!=!
0,021),! suggesting! that! geographic! distance! is! the!main! factor! shaping! the! genetic!
structure!of!the!Guinea!Bissau!chimpanzees.!!!Table' 11:! !Genetic!differentiation!between!sampling! locations.!PhiPT!Values!below!
diagonal.!Probability!values!based!on!9999!permutations!are!shown!above!diagonal.!
!!! Cantanhez' Boe' Cufada' Catio'Cantanhez' 0,000! 0,001! 0,000! 0,002!Boe' 0,025! 0,000! 0,000! 0,000!Cufada' 0,051! 0,078! 0,000! 0,000!Catio' 0,036! 0,061! 0,089! 0,000!!
Among&Pops&4%&
Within&Pops&96%&
Percentages*of*Molecular*Variance*
! 39!
!
!
!
!
!
!
!
!
!
!
Fig.'9:!Correlation!(r)!between!Nei!genetic!distance!and!geographic!distance!(Km).!
Each!dot!represents!a!dyad!of!individuals.!Mantel!Test!significance!0,021.!
!
!
We!found!that!chimpanzees!from!Guinea!Bissau!are!overall!unrelated.!Catio!showed!
the!highest!mean!pairwise!relatedness!and!Boe!is!the!location!where!individuals!are!
less!related!among!each!other!(Table!12).!Despite!the!differences!between!locations,!
none!showed!significant!levels!of!relatedness.!!
!
!
Table' 12:! Mean! pairwise! relatedness! (Queller! and! Goodnght! estimator)! between!
individuals!from!each!of!the!sampling!locations.!
!Population' Relatedness'Boe! [0,004!Cantanhez! 0,0281!Catio! 0,1597!Cufada! 0,0912!Overall! [0,0086!
!! !
y"="0,0089x"+"0,4924"R²"="0,00576"
0,000"0,200"0,400"0,600"0,800"1,000"1,200"1,400"1,600"1,800"
0,000" 5,000" 10,000"15,000"20,000"25,000"30,000"35,000"40,000"
Geograph
ic+Distan
ce+
Gene0c+Distance+
Y"
Linear"(Y)"
! 40!
Objective'2:''To'assess'anthropogenic'barriers'to'dispersal'
Activity'4:'Design'of'a'GIS'Model'with'information'of'anthropogenic'density'and'
impact'
!
A!collaboration!was!established!with! INEP! (Instituto!Nacional!de!Estudos!e!Pesquisas! [!
National!Institute!of!studies!and!research,!http://www.inep[bissau.org/).!This!institute!is!
the!only!public!organization!with!a!research!remit!in!the!country!and!it!was!founded!to!
promote! research! in! many! areas,! including! environmental! studies! (http://www.inep[
bissau.org/).! As! part! of! previous! work,! this! institute! holds! GIS! maps! of! Cufada! and!
Cantanhez!parks!produced!in!2004,!based!in!satellite!maps!produced!by!a!French!team.!
Additionally,! we! used! spatial! data! compiled! by! CS! during! research! project! FCT!
POCI/ANT/57434/2004!and!PPCDT/57434/2004.!!
!
Quantum!GIS!(QGIS)!version!1.8.0[Lisboa!was!used!to!design!a!map!with!information!on!
habitat! type! and! anthropogenic! features.!We! used! shapefiles! describing! soil! use! and!
location!of!villages!within! the!parks!of!Cufada!and!Cantanhez! (facilitated!by! INEP)!and!
data! on! location! of! cities,! villages! located! south! the! Corubal! river! and! outside! the!
protected! areas,! main! roads,! main! rivers! and! smaller! watercourses! (facilitated! by!
Cláudia!Sousa[!Dári!Project).!As!soil!use!data!was!composed!of!more!than!60!categories!
of!vegetation,!we!simplified!the!information!by!joining!together!all!categories!of!forest!
(bright! green),!mangrove! (pastel! green),! savanna! (yellow)! and! crops! (dark!purple)! (Fig!
XX).!We!also!used!data!collected!in!Cantanhez!and!Cufada!during!fieldwork!on!location!
of!villages!and!crop!areas.!For!the!purpose!of!clarity,!we!present!the!spatial!information!
compiled!by!this!project! in!two!different!maps:!one!with!natural!features!(Fig.!10)!and!
one! with! anthropogenic! features! of! the! landscape! (Fig.! 11).! In! order! to! accomplish!
activity!5!all!information!together!was!set!together.!
!
!
!
!
! 41!
!
Fig.' 10:!Map! of! soil! use! and! hydrographic! information! for! southern!Guinea[Bissau!
created!using!QGIS!and!based!in!spatial!data!facilitated!by!INEP!and!CS.!
!
We!compiled!all!the!data!on!location!of!villages!and!cities.!
!
Fig.'11:!Map!showing!the!location!of!cities,!villages,!main!and!secondary!roads!across!
southern!Guinea[Bissau.!
!
! 42!
Activity'5:'Identification'of'barriers'to'gene'flow'''
To! assess! whether! there! are! vicariant! barriers! that! may! prevent! gene! flow,! the!
program! BARRIER! v.! 2.2! was! used! (Manni! et( al.! 2004)! both! for! mtDNA! and!
microsatellite! data.! This! software! take! advantage! of! the! maximum! difference!
algorithm! of!Monmonier.! This! algorithm! identifies! boundaries! or! areas!where! the!
differences!between!pairs!of!populations!are!larger.!These!barriers!are!areas!where!
there! is! an! abrupt! change! in! the! pattern! of! genetic! variation! due! to! physical,!
ecological!or!geographical!factors.!!
MTDNA(Barriers(
The!Monmonier!algorithm!(BARRIER!software)!suggested!three!main!barriers! (α,!β,!
γ)! to! gene! flow! in! the! Guinea[Bissau! chimpanzee! distribution! range:! (α)! the!most!
significant!barrier!is!located!approximately!in!the!south!of!the!CNP!in!the!Cubucaré!
Peninsula,! thus! isolating! the! populations! of! Catomboi,! Cafatche! and! Canghode!
(11°11'19.26"N[!15°!6'32.49"W);!(β)!another!barrier!located!south!of!the!Rio!Grande!
de!Buba!in!the!Quínara!region!causing!genetic!discontinuity!between!the!CLNP!and!
the! Empada! and! Catió! chimpanzees! (11°37'47.19"N[! 15°10'23.11"W),! and! (γ)! one!
barrier!that!separates!Aicum!and!Vendu!Leidi!populations!from!the!others!in!the!Boé!
sector!and! in! the!Tombali! region,! located! in! the!southeast!of! the!country!between!
the! geographic! coordinates! (11°44'19.79"N[! 13°44'3.80"W)! and! (11°56'39.19"N[!
13°53'0.34"W).!These!barriers!are!represented!in!Figure!12.!!!
!
Nuclear(Barriers(
!
The! two! spatial! analyses! of! genetic! discontinuities! (Geneland! and! Barrier)! are!
concordant! in! finding! barriers! isolating! some! individuals! from! Boe! and! some!
individuals!from!Cantanhez!(Figure!13!and!14).!The!analysis!from!Geneland!divided!
Boe!chimpanzees! in! two!distinct!clusters.!One!completely! isolated! from!the!others!
and! the! second! grouping! together! with! Cufada! and! Catio! chimpanzes.! On! the!
contrary,! BARRIER! did! not! detect! any! barrier! within! Boe! but! instead! isolated! Boe!
from! the! extant! locations.! Both! approaches! are! concordant! when! separating!
! 43!
Cantanhez! from! the! other! locations! but! disagree! regarding! Cufada! and! Catio.!
Geneland!considered!that!both!locations!belong!to!the!same!genetic!cluster!whereas!
BARRIER!detected!a!weak!but!existent!barrier!between!those!chimpanzees.!!
After!superimposing!the!map!with!anthropogenic!features!on!the!spatial!distribution!
of!genetic!structure!it!is!clear!that:!
Using!BARRIER:!
1!–!The!barrier! that!BARRIER! found!between!Cufada!and!Catio! corresponds! to!Rio!
Grande!de!Buda!and/or! several! human!villages.! ! There! is! no! abrupt! change! in! the!
habitat!type.!
2!–!The!barrier!between!Cufada!and!Cantanhez!is!most!likely!due!to!crop!areas!and!
multiple!anthropogenic!features!(e.g.!roads,!villages,!etc…)!that!severely!fragmented!
the! habitat! between! these! regions.! Rio! Cumbidja!might! also! greatly! contribute! to!
this!differentiation.!
3! –! The! slight! barrier! found! between! Cantanhez! and! Catio! is! probably! a! result! of!
human!settlements!and!the!Cumbidja!river.!
4!–!The!second!slight!barrier! is!between!Boe!and!all!other!is!most!likely!due!to!the!
geographic!distance!as!the!areas!that!separate!Boe!from!the!other!locations!are!not!
so!human!impacted.!!
!
Using!Geneland:!
1!–!The!barrier!that!separate!Boe!chimpanzees!in!two!sub[populations!corresponds!
to! the!Fefine!river.!Boe! is!not!a!very!human!populated!area! (contrary! to! the!other!
locations)!and!there!is!no!habitat!type!change!within!Boe!region.!
2! –! The! barrier! found! within! Cantanhez! is! probably! a! result! of! the! severe! forest!
fragmentation! in! this! region! (forest! transformed! in! crop!areas!where! chimpanzees!
are!chased)!that!isolates!the!most!southern!groups.!
!
The!few!differences!found!between!BARRIER!and!Geneland!can!be!explained!by!the!
fact! that! Geneland! used! individual! GPS! coordinates! from! each! sampling! locations,!
while!BARRIER!grouped!all!GPS!coordinates!from!each!location.!!
!
! !
! 44!
!!!!!!!!!!!!!!!!!!!!!!!!!!!
Fig.! 12:!Map! showing! the! geographic! distribution! of! the!HVR1!haplotypes! of! the!P.# troglodytes# verus! populations! in!Guinea;Bissau.! The!
haplotypes!are!identified!in!the!legend.!The!broken!lines!in!red!indicate!the!genetic!barriers!(α,!β,!γ)!as!defined!by!the!Monmonier!algorithm.!
!!!
! 45!
!!!!!!!!!!!!!!!!!!!!!!!!!!
!
Fig.! 13:!Map!of!nuclear!genetic!discontinuity!of! the!Guinea;Bissau! chimpanzees!provided!by!BARRIER! software.!Voronoi! tessellation!and!
virtual!points!are!indicated!in!blue,!Delaunay!triangulation!in!green.!Numbers!represent!the!populations!sampled:!1;!Cantanhez,!2;!Boé,!3;!
Cufada,!and!4;!Catió.!Extent!of! the!3!genetic!barriers!computed! from!the!matrix!of!Nei’s!genetic!distance!and!application!of!Monmonier!
algorithm!are!indicated!in!red!with!100!replications!of!the!genetic!distance!matrix.!
! 46!
!
!
!
!
!
Fig.% 14:! !Spatial!structure!as! inferred!by!GENELAND.!The!maps!show!the!estimated!
population!membership!inferred!and!the!probability!of!samples!in!location!belonging!
to!cluster!1,!cluster!2,!and!cluster!3.!!
! !
11.0 11.5 12.0
-15.2
-15.0
-14.8
-14.6
-14.4
-14.2
-14.0
-13.8
x coordinates
y co
ordi
nate
s
Map of posterior probability to belong to cluster 3
!"#$%$&
'()&
!$*+$*,)-&
!$.(&
!/"0+)1&4&11.0 11.5 12.0
-15.2
-15.0
-14.8
-14.6
-14.4
-14.2
-14.0
-13.8
x coordinates
y co
ordi
nate
s
Map of posterior probability to belong to cluster 1
!"#$%$&
'()&
!$*+$*,)-&
!$.(&
!/"0+)1&2&
11.0 11.5 12.0
-15.2
-15.0
-14.8
-14.6
-14.4
-14.2
-14.0
-13.8
x coordinates
y co
ordi
nate
s
Map of posterior probability to belong to cluster 2
!"#$%$&
'()&
!$*+$*,)-&
!$.(&
!/"0+)1&3&
! 47!
%
Objective%3:%Viability%Assessment%(PHVA)%modeling%exercise%and%to%propose%
specific%actions%for%conservation%
!
Activity%6:%%Carry%out%a%Population%Viability%Assessment%modeling%exercise%%%Computer!modeling! can!be!a! valuable! instrument! for! assessing! risk!of!decline!and!
extinction! of! wildlife! populations,! and! such! assessment! of! population! persistence!
under! current! and! varying! conditions! is! commonly! referred! to! as! a! population!
viability!analysis!(PVA)!(Carlsen!et!al.!2012).!
!
The!simulation!software!Vortex!(v9.99)!(Lacy!1993b,!2000)!was!used!to!examine!the!
viability! of! GuineaMBissau! chimpanzee! populations! under! a! variety! of! conditions.!
Information!from!the!following!sources!was!used!in!the!Vortex!model:!Nishida!et!al.!
2003;!Pusey!et!al.!2007;!Yoshikawa!et!al.!2008!and!Carlsen!et!al.!2012).!The!model!
and! sensitivity! analysis!was! based! and! developed! for! the! life! history! of!wild!West!
African!chimpanzees!of!Sierra!Leone!(Carlsen!et!al.!2013).!
!
Base!Model!Parameters!
!
_Simulations!were!run!for!1000!iteractions!over!a!period!of!100!years;!
_Initial! population! size! was! set! for! 1000! individuals,! except! for! a! unique! scenario!
where!we!considered!unlikely!possibility!of!N=!5000;!
_Reproductive! parameters:! polygynous!mating! system,!with! all! adult!males! in! the!
potential!breeding!pool.!All!births!were!single!(no!twins),!with!a!sex!ratio!at!birth!of!
40%!of!males;!
_Age!at!first!reproduction:!set!to!13!years!(females);!15!years!(males);!
_Percent!of!adult!females!breeding!per!year:!set!to!22%;!
_Mortality!rates:!the!maximum!age!was!set!to!43!years!(males)!and!53!(females);!
! 48!
_Annual!mortality! rates:!20%!(males)!15%!(females)!with!age!0M1!and! :!9%! (males)!
8%! (females)! for! adults;! For! the! scenario! of! low! mortality,! adult! mortality! was!
reduced!to!2%!
!
_Environmental!variation:!was!set!relatively!low!for!survival!(SD!=!20%!of!the!mean)!
and!at!a!moderate!level!for!reproduction!(SD!=!36%!of!the!mean);!
_Carrying! capacity! (K)!was! set! for! 2000! individuals! for!most! scenarios,! except! the!
ones!indicating!K!4000,!with!no!future!change!in!K.!!
_Some!scenarios!included!1!catastrophe!(forest!fragmentation)!that!occurred!with!a!
periodicity!of!every!3!years!reducing!20%!of!the!reproduction!and!survival;!
_Harvest!was!also!considered!for!some!scenarios.!We!considered!harvest!to!start!on!
the! first! year!and! finish! ten!years! later,!with!harvest!occurring! in!every!other!year!
within!this!10Myear!period.!We!considered!that!two!males!and!two!females!of!age!0M
1!and!two!adult!males!and!two!adult! females!were!harvested!from!the!population!
every!year.!!
!
Guinea!Bissau!chimpanzees!PVA!!
We!simulated!a!total!of!nine!different!scenarios!to!understand!the!evolution!of!the!
population!of!chimpanzees!from!Guinea!Bissau!during!the!next!100!years.!In!all!but!
one!scenario!(scenario!7)!we!used!1000!individuals!as!the!initial!population!size.!This!
number!was!taken!from!the!Chimpanzee!Action!Plan!for!Guinea!Bissau!(Casanova!&!
Sousa,!2008)!that!estimated!that!there!were!between!600!and!1000!chimpanzees!in!
Guinea! Bissau.! ! In! both! scenarios!where!we! considered! Forest! Fragmentation! and!
Harvest! to! occur! (1! and! 2),! which! is! the!most! likely! scenario! for! this! chimpanzee!
population,!the!population!will!most! likely!be!extinct! in!the!next!couple!of!years.! If!
we! remove! the! threats! but!maintain! all! other! parameters! (Scenario! 3)! or! increase!
the!carrying!capacity!to!4000!individuals!(scenario!4),!the!probability!of!extinction!is!
still!very!high!(above!0,9).!The!biggest!difference!from!scenarios!1!and!2!is!that!the!
time! of! extinction! increases! to! around! 70! years.! The! only! parameter! that! greatly!
influences!the!decrease!of!the!probability!of!extinction!(to!around!0,3)! is!when!we!
! 49!
reduce! the!natural!mortality!of! the!adult! females! (from!8! females! to!2!each!year).!!
The! second! lowest! probability! of! extinction! is! for! scenario! 7,! where! we! also!
increased!the!initial!population!size!to!5000!individuals,!which!is!very!unlikely!to!be!
real!at!this!point!in!time.!Even!if!for!scenarios!5!to!8,!the!probability!of!extinction!is!
low,!the!expected!number!of!remaining!individuals!in!the!population!will!be!very!low!
(around!30!individuals!for!the!best!case!scenario!7).!This!being!true!means!that,!even!
if!the!population!of!chimpanzees!from!Guinea!Bissau!survives!for!the!next!100!years,!
it!will!experience!a!great!population! loss! that!will!most! likely!make! the!population!
unviable!longMterm.!The!only!scenario!where!the!population!would!increase!its!initial!
size!was! for!scenario!9!where!we!completely!removed!the!natural!mortality!of! the!
population,!which!is!completely!unrealistic!for!a!natural!population.!!
! !
! 50!
Table&13:!!Demographic!parameters!resultant!from!Vortex!simulations!after!100!years!under!different!ecological!and!demographic!scenarios.!
Stoc;r!!;!Growth!rate;!Prob!Extintion!–!Probability!of!extinction;!N!–!ext!–!population!size!after!100!years;!GeneDiv!–!Genetic!diversity!after!100!
years;!Median!TE!–!Median!time!of!extinction;!Mean!TE!–!Mean!time!of!extinction!
!
!
Scenarios& Stoc2r& SD(r)&Prob&
Extition& N&2&ext& SD(Next)& GeneDiv& SD(GD)&Median&
TE& MeanTE&1!;!Forest!Fragmentation!and!Harvest! ;0,078! 0,045! 1! 0! 0! 0! 0! 2! 3,2!2!;!Forest!Fragmentation!and!Harvest!;!Low!Female!Mortality! ;0,051! 0,048! 1! 0! 0! 0! 0! 2! 3,4!3!;!No!Threats! ;0,081! 0,097! 0,973! 3,63! 1,84! 0,7117! 0,1099! 72! 71,8!4!;!No!Threats!;!K4000! ;0,081! 0,098! 0,977! 3,17! 1,47! 0,6958! 0,0907! 71! 71,4!5!;!No!Threats!;!Low!Female!Mortality! ;0,054! 0,095! 0,375! 9,82! 7,75! 0,8582! 0,0832! 0! 86,7!6!;!No!Threats!;!K4000!and!!Low!Female!Mortality! ;0,053! 0,094! 0,371! 10,44! 8,43! 0,8667! 0,0789! 0! 88,1!7!;!No!Threats!!;!K4000!Low!Female!mortality!!and!N5000! ;0,053! 0,088! 0,063! 30,91! 26,06! 0,9466! 0,045! 0! 90!8!;!No!Threats!;!Low!Mortality!for!Both!Sexes! ;0,053! 0,105! 0,339! 10,1! 8,53! 0,8706! 0,0826! 0! 88!9!;!No!Threats!;!!No!Mortality!for!Both!Sexes! 0,041! 0,105! 0! 1722,83! 180,7! 0,9977! 0! 0! 0!
!!
! 51!
Activity'7:'Choice'of'sub2populations'to'apply'measures'of'conservation''
Based&on&the&mtDNA&analyses&the&two&phylogeographic&groups&should&be&considered&
as& distinct&management& units,& and& the& populations& of&Madina<Canglode,& Cadique;&
Lautchande;&Mejo<Amidara;&Briame,&Empada;&Cufada,&Vendu&Leidi,&Aicum&and&Béli,&
indicate& priority& areas& for& conservation.& Based& on& the& microsatellite& analyses&
chimpanzees&from&GB&appear&to&be&part&of&the&same&population&but&showing&a&weak&
but&significant&population&structure.&However&gene&flow&is&still&possible&between&all&
regions.& It& is& crucial& for& the& maintenance& of& the& population& that& the& gene& flow& is&
maintained&and&as&vortex&analysis&showed&that&with&only&around&1000&individuals&the&
population&will&most&likely¬&be&viable&long&term.&
'
'Activity!8:!Design!specific!actions!for!conservation!'
The&recommendations&proposed&by&Casanova&and&Sousa&(2007)&should&be&addressed&
seriously& by& the& Guinea<Bissau& national& agencies& and& local& NGO’s.& Biodiversity&
management& authorities& in& Guinea<Bissau& (IBAP%and&Direcção%Geral% de% Florestas% e%
Fauna)%have& introduced&new&laws&to®ulate&the&trade& in&wild&meat&(e.g.&recently,&
the& hunting& of& primates& throughout& the& country& was& prohibited& (Anon.,& 2011).&
However,& limited& resources&and& lack&of&awareness&of&management&authorities&and&
politicians& is& hindering& law& enforcement& in& the& country.& At& the& international& level,&
conservation& agencies& should& re<examine& their& strategies& to& mitigate& the& illegal&
chimpanzee&body&parts&trade,&and,&at&the&national&level,&specific&programs&should&be&
designed& and& applied& to& empower& all& actors& involved& (e.g.& park& rangers,& Customs&
officers,& the&military,&police,&etc.),&complemented&at& the&same&time&by&provision&of&
environmental& education& for& the& local& communities.& The& political& instability& in& the&
country& has& neglecting& CITES,& that& was& never& ratified,& thus& the& government& and&
political&authorities&need&to&rethink&their&position.&
&
The&chimpanzee&pet&trade&within&the&country&is&frequent&and&usually&the&animals&live&
in& miserable& conditions.& The& local& authorities& should& not& facilitate& this& kind& of&
! 52!
practice&by& confiscating& the&animals& and& releasing& them& into& sanctuaries&or& rescue&
centers&(Casanova&and&Sousa&2007).&Although&this&kind&of&facilities&do¬&exist&in&the&
country,&with&international&help&it&will&be&possible&to&secure&them&abroad.&&
&
The&creation&of& the&national&parks&by& IBAP&was&a&good&conservation&strategy,&as& in&
principle& they& can& protect& the& genetic& diversity& of& the& chimpanzee& populations,&
however,& in& reality,& the& Parks& are& lacking,& infrastructure,& monitoring& and& seizure&
resources.&&
&
The&guards&should&be&empowered&with&field&equipment&and&adequate&material,&and&
they&should&receive,& in&a®ular&basis,&adequate&payments&for&their&services&either&
from& the& official& agencies& and& local& NGO’s,& or& from& research&members& as& well& as&
adequate&training.&
&
Based& on& the& genetic& approach& two& chimpanzee& phylogeographical& clusters& were&
identified:& one& south& of& Cantanhez,& and& a& second& encompassing& the& remaining&
regions.&The&first&group&(constituted&by&Farim,&Catomboi,&Cafatche&and&Canghode)&in&
South&Cantanhez& is& in&eminent& risk&of&becoming&extinct,& as& they&are&under& intense&
anthropogenic&pressure.&
&
&Significant& conservation& efforts& should& be& directed& towards& them.& In& the& other&
group,&three&major&priority&areas&should&be&taking& into&consideration:&the&northern&
area&of&Cantanhez&constituted&by:&Cadique,&Lautchande,&Amidara&and&Mejo&and&the&
communities&of&Buba& Intchingue&and& Injassane& in&Cufada& (i.e.&Quínara& region);& the&
area&of&Catió<Empada&area&where&they&will&become&isolated&if&the&corridor&of&Nhala&
disappear&and&the&Boé®ion:&especially&the&populations&of&Béli,&Aicum,&Vendu&Leidi&
and&Dinguirai.&&
&
Moreover& the& forest& connectivity& between& all& regions& based& on& theµsatellite&
results& must& be& reinforced,& as& the& gene& flow& is& ongoing.& & More& importantly,& it& is&
crucial& for& the& survival& of& the& population& that& gene& flow& is& promoted& with& trans&
! 53!
frontier&populations& (e.g.&Republic&of&Guinea&and&Senegal)& in&order& to& increase& the&
population&size.&&
&
Chimpanzee& gastrointestinal& surveys& should& be& implemented& on& a& regular& basis,&
every& two& years& in& order& to& monitor& their& health& status,& pathogen& status,& and&
epidemiological& surveillance.& Baseline& information&was& established&with& this& study.&
However,& it& is&necessary& that& further& studies&proceed& the&monitoring,&as& infectious&
diseases&in&wild&great&apes&are&one&of&the&major&threats&to&their&survival.&Research&of&
chimpanzee& infections& is& of& importance& for& the& understanding& of& the& role& and&
circulation&of&neglected&and&emergent& infectious&diseases,¬&only&for&the&species,&
but&also&for&other&species,&including&humans.&&
&
Finally,& local& people& must& be& empowered& economically& with& sustainable& activities&
(i.e.& local& handicraft,& regional& products:& honey,& jams,& beverages,& etc.)& to& sell& at& a&
national& and& eventually& at& an& international& level,& where& the& producers& should& be&
paid& adequately& in& a& fair& trade& basis.& Effective& conservation& projects& must& be&
implemented& and& developed& with& the& full& engagement& of& local& and& national&
stakeholders& (Ancrenaz& et& al.& 2007).& Chimpanzees& have& great& potential& value& for&
attracting&ecotourism&and&this&opportunity&can&be&explored&across&Guinea<Bissau.&&
Activity'9:'Disseminate'results'and'main'conclusions'to'partners'and'local'
communities'
!The&local&communities&were&informed&about&the&results&of&the&project&during&March&
2013& both& at& Cantanhez& National& Park& and& Cufada& Lagoons& Natural& Park&with& the&
presence& of& all& ecoguides& and& park& rangers& as& well& as& the& presence& of& traditional&
elder& authorities& (Fig.& 15).& The& scientific& outputs& of& the& project& were& put& in& an&
accessible&language&in&order&to&promote&the&engagement&of&the&community.&&
&
A& fruitful&discussion&was& followed&where&many&members&of& the&community& realize&
the& importance&of&the&study&and&mentioned&the&fact& it&was&the&first&time&that&they&
were&called¬&only&to&work&actively&with&the&researchers&but&also&to&obtain&those&
! 54!
results&that&now&they&can&use&to&promote&their®ion.&The&discussion&also&include&a&
section&where&the&local&participants&reflected&on&their&priority&development&needs&
&
&
&
&
&
&
&
&
&
%
'
Fig.'15:&Results&restitution&to&the&community,&March&2013.&&&
& &
! 55!
Objective'4:''Integration'of'results'with'the'National'Action'Plan'framework'
&
Activity'10:'Combine'the'information'of'chimpanzee'population'specific'needs'
with'the'requirements'of'local'human'populations.'
'
A&formal&presentation&of&the&results&of&the&project&was&given&to&IBAP,&in&Bissau&by&Rui&
Sá& in& 9th& May& 2013.& IBAP& director,& the& director& of& Direcção& Geral& de& Florestas& e&
Fauna,&IUCN<Guinea<Bissau&representatives&and&other&members&of&the&staff&including&
national& directors& of& the& protected& areas& and&members& of& other& foreign& research&
teams&attended& this&meeting.& & In& this&meeting&besides& the&presentation&of& results,&
many&members& engaged& in& the& discussion& that& followed& in& order& to& create& better&
strategies& to& implement& the& suggested& recommendations.& It& was& agreed& between&
IBAP& Director& and& National& Parks& directors& that& both& a& copy& of& the& Chimpanzee&
National& Action& Plan& and& this& study& should& be& available& in& each& park& where&
chimpanzees& live& for& public& consultation.& In& this& context& it& was& also& discussed& the&
impact&of&erratic&logging&that&was&occurring&in&the&country&at&during&that&period.&&
&
In& order& to& negotiate& mitigation& strategies& and& compensation& plans& the& directors&
agree&that&both&local&human&communities&and&chimpanzee&ecological&needs&must&be&
taken& into& account& when& government& concedes& new& forest& concessions& for&
resources&exploitation.&&&
&
&
&
&
&
&
&
&
! 56!
Rui&Sá&also&had&meetings&with&Acção¶&o&Desenvolvimento&e&Chimbo&Foundation&
both&with&directors&and&technical&staff&in&May&2013&in&order&these&organisations&can&
use& the& results& of& this& project& directly& in& chimpanzee& conservation& in& their&
operational,&particularly&in&Cantanhez&and&in&the&Boé§or&respectively&(Fig.&16).&&
&
&
&
&
&
&
&
&
&
&
&
Fig.'16:&Results&restitution&to&the&AD&eco<guides,&May&2013.&
! 57!
REFERENCES'!!Ancrenaz&M,&Dabek&L,&O’Neil&S.&2007.&The&costs&of&exclusion:& recognizing&a& role& for&local&communities&in&biodiversity&conservation.&PLoS&Biology&5,&e289.&'Anon.& 2011.& Decreto<Lei& 5/2011& de& 22& Fevereiro.& Boletim& Oficial& da& República& da&Guiné<Bissau&8,&1–13.&&Bandelt& H,& Forster& P,& Röhl& H.& 1999.& Median<joining& networks& for& inferring&intraspecifics&phylogenies.&Mol&Biol&Evol&16:&37<&48.&&Bradley,&B.;&Chambers,&K.;&Vigilant,&L.&2001.&Accurate&DNA<based&sex&identification&of&apes&using&non<invasive&samples.&Conservation&Genetics&2:&179<181.&&&&Brugiere,&D.,&Badjinca,&I.,&Silva,&C.,&and&Serra,&A.&(2009).&&Distribution&of&chimpanzees&and& interactions&with& humans& in& Guinea<Bissau& and&Western& Guinea,&West& Africa.&Folia%Primatologica&80:353–358.&&Butynski&T.&2003.&The& robust&chimpanzee& (Pan& troglodytes:& taxonomy,&distribution,&abundance&and&conservation&status.&In&Kormos&R,&Boesch&C,&Bakarr&M&and&Butynski&T&(Eds.).&West&african&chimpanzees:&status&survey&and&conservation&action&plan.&IUCN,&Gland,&Switzerland.&&Caldecott&J,&and&Kapos&V.&2005&Great&ape&habitats:&tropical&moist&forests&of&the&Old&World.& In&Caldecott& J,&and&Miles,& L.& (Eds.)&The&World&Atlas&of&Great&Apes&and& their&Conservation.& pp.& 31<42.& California& University& Press& (Berkeley,& California& and&London).&&Carlsen,& F.,& Leus,& K.,& Traylor<Holzer,& K.,& McKenna,& A.& (Editors).& 2012.& Western&Chimpanzee& Population& and& Habitat& Viability& Assessment& for& Sierra& Leone:& Final&Report.& IUCN/SSC&Conservation&Breeding&Specialist&Group&–&Europe&(CBSG&Europe),&Copenhagen,&Denmark.&&Casanova,&C.,&and&Sousa,&C.&(2007).&Action%Plan%for%the%Conservation%of%Chimpanzees,&red%western%colobus%and%king%colobus%in%GuineaCBissau%Republic.&IBAP,&Guiné<Bissau.&&Corander,& J.&and&Marttinen,&P.&and&Sirén,& J.&and&Tang,& J.&2008a.&Enhanced&Bayesian&modelling& in& BAPS& software& for& learning& genetic& structures& of& populations.& BMC&Bioinformatics&9,&p.&539.&&&Drummond&A,&Rambaud&A.&2007.&BEAST:&Bayesian&evolutionary&analysis&by&sampling&trees.&BMC&Evol&Biol&7:214.&&Dupanloup& I,& Schneider& S,& Excoffier& L.& 2002.& A& simulated& annealing& approach& to&
! 58!
define&the&genetic&structure&of&populations.&Mol&Ecol&11:&2571<2581.&&Evanno,&G.&and&Regnaut,&S.&and&Goudet,&J.&2005.&Detecting&the&number&of&clusters&of&individuals&using&the&software&STRUCTURE:&a&simulation&study.&Molecular&Ecology&14&pp.&2611&<&2620.&&&Excoffier& L,& Laval&G,& Schneider& S.& 2005.&ARLEQUIN&ver.&3.0:&An& integrated& software&package&for&population&genetics&data&analysis.&Evolutionary&Bioinformatics&Online&1:&47<50.&&Gippoliti,& S.,& and& Dell'Omo,& G.& (1995).& Status& and& conservation& of& the& chimpanzee&Pan%troglodytes%verus&in&Guinea<Bissau.&African%Primates&1:3–5.&&Gippoliti,& S.,& Embalo,& D.,& and& Sousa,& C.& (2003).& Guinea<Bissau.& In:& West% African%chimpanzees:% Status% Survey% and% Conservation% Action% Plan.& Kormos,& R.,& Boesch,& C.,&Bakarr,&M.,&and&Butynski,&T.&(Eds),&IUCN,&Gland,&Switzerland.'Pp.55–61.&&Guillot&G& and&Mortier& F& and& Estoup&A& 2005b.&GENELAND:& a& computer& package& for&landscape&genetics.&Molecular&Ecology&Notes&5,&pp.&712<715.&&Gusmão&L,&Gonzalez<Neira&A,&Alves&C,&et&al.&2002.&Genetic&diversity&of&Y<specific&STRs&in&chimpanzees&(Pan%troglodytes).&American&Journal&of&Primatology&57:&21<29.&IUCN& (2011).& IUCN% Red% List% of% Threatened% Species.& Version& 2010.4.&www.iucnredlist.org.&Viewed&on&21&April&2011.&&Jensen&J,&Bohonak&J,&Kelley&S.&2005.&Isolation&by&distance,&web&service&BMC&Genetics&6:13.&&Johnson&PC,&Haydon&DT.&2009.&Software&for&quantifying&and&simulatingµsatellite&genotyping&error.&Bioinformatics&and&Biology&Insights,1:&71<75.&&Konovalov&DA,&Manning&C,&Henshaw&MT.&2004.&KINGROUP:&a&program&for&pedigree&relationship& reconstruction& and& kin& group& assignments& using& genetic&markers.&Mol&Ecol&Notes&4:779–782.&&Kormos& R.& and& Boesch& C.& 2003.& Regional& Action& Plan& for& the& Conservation& of&Chimpanzees&in&West&Africa,&IUCN.&&Lacy,&R.C.& 2000.& Structure&of& the&VORTEX& simulation&model& for&population& viability&analysis.&Ecological&Bulletins&48:191<203.&&Lacy,& R.C.& 1993b.& Impacts& of& inbreeding& in& natural& and& captive& populations& of&vertebrates:& implications& for& conservation.& Perspectives& in& Biology& and& Medicine&36:480<496.&&
! 59!
Laurance&WF,&Useche&DC,& Rendeiro& J,& et& al.& 2012.& Averting& biodiversity& collapse& in&
tropical&forest&protected&areas.&Nature&489:&290<294.&
&
Lee,& P.C.,& Thornback,& J.& and& Bennet,& E.L.& (1988).& Threatened% Primates% of% Africa.&Gland,&Switzerland.&
&
Manni& F,&Guérard& E,&Heyer& E.& 2004.&Geographic& patterns&of& (genetic,&morphologic,&
linguistic)& variation:& how&barriers& can& be& detected& by& ‘Monmonier& algorithm.&Hum&
Biol&76:&173<190.&
&
Minhos,& T.;&Wallace,& E.;& Ferreira&da&Silva&M.;& Sá,&R.&M.;&Carmo,&M.;&Barata,&A.;& and&
Bruford&M.& 2013.& DNA& identification& of& primate& bushmeat& from& urban&markets& in&
Guinea<Bissau&and&its&implications&for&conservation.&Biological&Conservation,&167:&43<
49.&
&
Miquel&C,&Bellemain&E,&Poillot&C,&Bessiére&J,&Durand&A&and&Taberlet&P.&2006.&Quality&
indexes& to&assess& the&reliability&of&genotypes& in&studies&using&noninvasive&sampling&
and&multiple<tube&approach.&Molecular&Ecology&Notes%6:&985<988.&&
Nishida,&T.,&N.&Corp,&M.&Hamai,&T.&Hasegawa,&M.&Hiraiwa<Hasegawa,&K.&Hosaka,&K.D.&
Hunt,& N.& Itoh,& K.& Kawanaka,& A.& Matsumoto<Oda,& J.C.& Mitani,& M.& Nakamura,& K.&
Norikoshi,& T.& Sakamaki,& L.& Turner,& S.& Uehara,& and& K.& Zamma.& 2003.& Demography,&
female& life& history,& and& reproductive& profiles& among& the& chimpanzees& of& Mahale.&
American&Journal&of&Primatology&59:&99<121.&
&
Peakall& R,& and& Smouse&PE.& 2006.&GENALEX&6:&Genetic& analysis& in& Excel.& Population&
genetic&software&for&teaching&and&research.&Molecular&Ecolology&Notes&6:288–295.&
&
Petit&R,&Mousadik&E,&Pons&O.&1998.& Identifying&populations& for&conservation&on& the&
basis&of&genetic&markers.&Conserv&Biol&12:&844<855.&
&
Posada&D.&2008.&jMODELTEST:&Phylogenetic&model&averaging.&Mol&Biol&Evol&25:&1253<
1256.&
&
Pritchard&J&K&and&Stephens&M&and&Donnelly&P&2000.&Inference&of&Population&Structure&
Using&Multilocus&Genotype&Data.&Genetics&155,&pp.&945<959.&
&
Pusey,& A.E.,& L.& Pintea,& M.L.& Wilson,& S.& Kamenya,& and& J.& Goodall.& 2007.& The&
contribution& of& long<term& research& at& Gombe& National& Park& to& chimpanzee&
conservation.&Conservation&Biology&21(3):&623<634&
&
Queller,& D.& C.& and& Goodnight,& K.& F.& 1989.& Estimating& relatedness& using& genetic&
markers.&Evolution&43,&pp.&258–275.&
&
Rambaud& A.& 2006.& FiggTree& v.1.3.1.& Available& from&
http://tree.bio.ed.ac.uk/software/figgtree.&
&
! 60!
Rambaud& A,& Drummond& A.& 2007.& TRACER& v.1.5.& Available& from&
http://www.beast.bio.edu.ac.uk/Tracer.&
&
Roeder&A,&Archer&F,&et&al.&2004.&A&novel&method& for&collection&and&preservation&of&
faeces&for&genetic&studies."Molecular&Ecology&4:&761<764.&
&
Ronquist&F,&Huelsenbeck&J.&2003.&MRBAYES&3:&Bayesian&phylogenetic&inference&under&
mixed&models.&Bioinformatics&19:&1572<1574.&
&
Roeder&A,& Jeffery&K,& et& al.& 2006.&A&universalµsatellite&multiplex& kit& for& genetic&
analysis&of&great&apes.&Folia&Primatologica&77:&240<245.&
&
Rogers&A,&Harpending&C.&1992.&Population&growth&makes&waves&in&the&distribution&of&
pairwise&genetic&differences.&Mol&Biol&Evol&9:&552<569.&
&
Rozas&J,&Sánchez<DelBarrio&JC,&Messeguer&X,&et&al.&2003.&DnaSP,&DNA&polymorphism&
analyses&by&the&coalescent&and&other&methods.&Bioinformatics&19:&2496<2497.&
&
Sá,& R.& M.,& Ferreira& da& Silva,& M.;& Sousa,& F.;& and& Minhos,& T.& 2012.& The& trade& and&
ethnobiological& use& of& chimpanzee& body& parts& in& Guinea<Bissau:& Implications& for&
Conservation.&Traffic&Bulletin&24&(1):&31<34.&
&
Shimada&M,&Hayakawa&S,&et&al.&2004.&Mitochondrial&DNA&Genealogy&of&Chimpanzees&
in&the&Nimba&Mountains&and&Bossou,&West&Africa.&American&Journal&of&Primatology&
64:&261<&275.&
&
Stöver& B,& Müller& K.& 2010.& TreeGraph& 2:& Combining& and& visualizing& evidence& from&
different&phylogenetic&analyses.&BMC&Informatics&11:7.&
&
Sousa,&F.M.&(2009).&&Pan%troglodytes%verus%densities&and&the&use&of&natural&resources&in&Gadamael&área,&Guinea<Bissau.&MSc&thesis.&Lisbon&University.&Lisbon,&Portugal.&
&
Sousa,&C.,&Gippoliti,&S.,&and&Akhlas,&M.& (2005).&Republic&of&Guinea<Bissau.& In:&World%Atlas%of%Great%Apes%and%their%Conservation,&Caldecott& J,&Miles&L& (Eds).&University&of&
California&Press,&London.&pp.362–365.&&
&
Sousa,& J.,&Barata,&A.V.,& Sousa,&C.,&Casanova,&C.,& and&Vicente,& L.& (2011).&Chimpanzee&
oil<palm&use&in&southern&Cantanhez&National&Park,&Guinea<Bissau.&American%Journal%of%Primatology&73:1–13.&&
Taberlet& P,&Griffin& S,&Goossens&B,&Questiau& S,&Manceau&V,& Escaravage&N,&Waits& LP,&
Bouvet&J.&1996.&Reliable&genotyping&of&samples&with&very& low&DNA&quantities&using&
PCR.&Nucleic&Acids&Research&24:&3189<3194.&
&
Telles&MPC,& Diniz<Filho& JAF.& 2005.&Multiple&mantel& tests& and& isolation<by<distance,&
taking&into&account&long<term&historical&divergence.&Genet&Mol&Res&4,&742–748.&&
&
! 61!
Valière& N.& 2002.& GIMLET:& a& computer& program& for& analysing& genetic& individual&
identification&data.&Molecular&Ecology&Notes%2:&377–379.&&
van& Oosterhout& C,& Hutchinson& WF,& Wills& DP,& Shipley& P.& 2004.& Micro<checker:&
software& for& identifying& and& correcting& genotyping& errors& in& microsatellite& data.&
Molecular&Ecology&Notes&4:&535<538.&
&
Yoshikawa,&M.,& H.& Ogawa,& T.& Sakamaki,& and& G.& Idani.& 2008.& Population& density& of&
chimpanzees&in&Tanzania.&Pan&Africa&News&15(2):&17<20.&
& &
! 62!
'
'
'
'
'
PART'B'!!
FINANCIAL'STATEMENT'!
! 63!
! 64!
APPENDIX'!Publications%&1.& Sá,& R.M.& 2013.& Phylogeography,& conservation& genetics& and& parasitology& of&chimpanzees& (Pan% troglodytes% verus)& in& Guinea<Bissau,& West& Africa.& PhD& Thesis.&Universidade&Nova&de&Lisboa.&&2.& Sá,& R.M.,& Ferreira& da& Silva,& M.,& Sousa,& F.M.,& Minhós,& T.,& 2012.& The& trade& andðnobiological& use& of& chimpanzee& body& parts& in& Guinea<Bissau:& implications& for&conservation.&Traffic&Bulletin.&24,&31–34.&&3.&Sá,&R.M.;&Marra,&A.;&Koops,&K.;&Minhos,&T.;&Ferreira&da&Silva,&M.;&Van&Schijndel,&J.;&Sousa,& C.;& && Bruford,&M.W.& 2013Inferring& the& Evolutionary& History& of& a& peripheral&and&endangered&ape&population:&the&chimpanzees&from&Guinea<Bissau.&PLOS&ONE&[&in%prep.].&&
International%Meetings%%1.&Sá,&R.&M.&2013.&Perspectives&of&population&genetics&for&primate&survival&in&Guinea<Bissau:& progress& and& prospects.& I& Bioanthropological& Meeting.& Coimbra.& Coimbra&University,&Portugal.&&2.& Sá,& R.& M.& 2013.& Genética& de& Conservação& e& Parasitologia& dos& chimpanzés& na&Guiné<Bissau.& Seminário& no& Instituto& da& Biodiversidade& e& Áreas& Protegidas.& Bissau,&Guiné<Bissau.&&3.& Sá,& R.& M.& 2012.& Genetic& Characterization& of& chimpanzees& in& Guinea<Bissau.& 10&years&of&the&Chimbo&Foundation.&World&Wildlife&Fund.&Zeist.&Netherlands.&&4.& Sá,& R.& 2011.&mtDNA& variation& and& patterns& of& parasitism& in& the& chimpanzees& of&Guinea<Bissau.& Implications& for& their& conservation.& Leverhulme& Centre& for& Human&Evolutionary&Research,&Cambridge&University,&Cambridge,&UK&25th&February.&&5.&Sá,&R.,&Sousa,&C.&&&Bruford,&M.&Mitochondrial&genetic&variation&of&chimpanzees&in&Cantanhez& Woodland& National& Park,& Guinea<Bissau.& VI& Congress& of& the& European&Federation&for&Primatology;& IIIrd& Iberian&Primatological&Congress,&Almada,&Portugal,&14<17&September&2011.&
