Gene ic S ock iden i ica ion o The A ican Hind
Gene ic S ock iden i ica ion o The A ican Hind Cephalopholis
Cephalopholis aeniops
aeniops
in he Cape Ve de a chipelago
in he Cape Ve de a chipelago
San os K
1
, Manen P
2
, Lopes E
1
, Quin ei o J
3
, Almeida C
1
, González N
2
The s ock concep is undamen al as a baseline in o ma ion in ishe ies managemen , whose applicable
de ini ion depends on he speci ic issues ha wan o be esol ed. One o he mos accep ed de ini ion is
e e ed as an in aspeci ic g oup o andomly ma ing indi iduals wi h spa ial and empo al in eg i y (Ihssen
e al. 1981). Ne e heless, he gene ic s ock concep is based on a ep oduc i ely isola ed uni , which is
gene ically di e en om o he s s ocks (O enden 1990). Those wo de ini ions sha e an impo an concep :
andom ma ing uni . The eby, popula ion gene ic analyses allow de ec ing he spa ial scale a which
e olu iona y p ocesses explain signi ican depa u es o admix u e o es biogeog aphic hypo hesis ha
elucida e he ex en o gene ic d i e ec ha enhance ep oduc i e isola ion. The A ican Hind
Cephalopholis aeniops is mainly dis ibu ed a he Eas e n A lan ic, om Ma oc o Angola, including Cape
Ve d, São Tomé and P incipe islands (Pas o 2002, ishbase 2014, bu see B i o e al. 2011 and Guide i e al.
2010). This species is one o he mos impo an comme cial deme sal species in he Cape Ve de a chipelago
and he consequences o popula ion educ ions due o an in ensi e ishing e o and he unknown
geog aphic dis ibu ion o he s ocks (Pas o 2002) a e he main conce ns owa ds ishe y’s sus ainabili y.
Hence, he aim o he p esen s udy was o es ima e island gene ic di e si y and o iden i y he gene ic s ocks
ac oss he a chipelago o delimi disc e e gene ic uni s o con ibu e o an e ec i e managemen in he
egion. A o al 62 samples o Cephalopholis aeniops we e collec ed a 5 islands o he Cape Ve d a chipelago (Figu e
1). A e collec ion, samples we e ixed in e hanol 96% and p ese ed a -20°C. DNA isola ion was pe o med
om a 30 mg o issue ollowing he E.Z.N.A. DNA Tissue ki (Omega Bio-Tek) p o ocol. The nucleo ide
sequences o he Cy och ome oxidase subuni I (COI) and he hype a iable Con ol Region (CR) o
mi ochond ial DNA we e ampli ied h ough PCR. The COI was ampli ied using he o wa d Fish F2 and he
e e se FishR2 p ime s (Wa d e al. 2005). The COI PCR p o ile was: 95°C o 5 min, 36 cycles o 95°C o 40 s,
50°C o 50 s and 72°C o 60s, and a inal ex ension o 72°C, 7 min. The CR was ampli ied using he
L15998/CSBDH p ime s (Quin ei o 2010), and he PCR p o ile was: 95°C o 3 min, 36 cycles o 95°C o 40 s,
59°C o 50 s and 72°C o 50s, and a inal ex ension o 72°C, 7 min. PCR p oduc s we e pu i ied using Exo-SAP-
I (A ime ix) p o ocol and luo escen ly labelled nucleo ide sequences we e ob ained using he BigDye 3.1
sequencing ki (Applied Biosys ems). Finally, he ex ension p oduc s we e pu i ied using Byg Dye XTe mina o
sequencing ki (Applied Biosys ems) and sequenced in o an ABI3500 Gene ic Analyze (Applied Biosys ems).
The nuclo ide sequences we e analyzed and aligned using BioEdi 7.0.0 and Clus alX . Nucleo ide (π) and
haplo ype (h
d
) di e si y we e es ima ed using DnaSP .5.10. In one hand, pai wise F
ST
alues we e es ima ed
and es ed, and in he o he a hie a chical pa i ioning o gene ic a ia ion among and wi hin islands was
es ima ed by an AMOVA using he A lequin 3.5 so wa e. Then, a NJ dis ance-based un oo ed ee was buil
using MEGA5. A Median-joining ne wo k o haplo ypes was cons uc ed using he Ne wo k .4.6 so wa e.
INTRODUCTION
METHODS
Figu e 4. Island haplo ype ne wo k and
neibou gh-joining ee o Cephalopholis
aeniops a he Cape Ve d islands, using he
con ol egion secuences. The a ea o he
ci cle is p opo ional o he equency o he
haplo ypes and b anch leng h is p opo ional
o he numbe o mu a ion posi ions.
Figu e 3. Island haplo ype ne wo k and neibou gh-joining ee o Cephalopholis
aeniops a he Cape Ve d islands, using he COI secuences. The a ea o he
ci cle is p opo ional o he equency o he haplo ypes and b anch leng h is
p opo ional o he numbe o mu a ion posi ions.
Sao Vicen e
Boa is a
S a Luzia
San iago
S o An ao
0.000.010.020.030.040.050.06
S o An ao
S Vicen e
S a Luzia
Boa is a
0.0000.0010.0020.0030.0040.0050.006
A o al o 9 haplo ype sequences o 654 bp o COI we e de ec ed. The maximum and he minimum
haplo ype di e si y alues we e egis e ed a San o An ão and Boa is a islands, espec i ely. The maximum
and minimum nucleo ide di e si y alues we e ob ained a San a Luzia and San o An ão and Boa is a
islands, espec i ely. No geog aphic pa e n o hd was de ec ed ac oss he sampled islands. (Table I, Fig. 2).
A o al o 34 haplo ype sequences o 420 bp o he con ol egion we e de ec ed. The maximum and he
minimum haplo ype di e si y alues we e egis e ed a São Vicen e and San a Luzia islands, espec i ely. The
maximum and minimum nucleo ide di e si y alues we e ob ained a San a Luzia and São Vicen e islands,
espec i ely. No geog aphic pa e n o hd was de ec ed ac oss he sampled islands (Table I, Fig. 2).
GENETIC DIVERSITY RESULTS
The haplo ype ne wo k o COI showed a s a pa e n in which he highes haplo ype equency was de ec ed
in all sampled islands. The haplo ypes wi h low equency a ose om his majo haplo ype h ough one single
mu a ion wi h he excep ion o he mos di e gen haplo ype which di e s in 3 single mu a ions in San a
Luzia. Fu he mo e, wo o hose one single mu a ion de i ed haplo ypes we e de ec ed a highe
equencies in all he islands (Fig 3). Un oo ed F
ST
dis ance-based NJ ee showed wo majo di e gen
clus e s o islands. The one con aining San o An ão and São Vicen e neighbou islands and he o he including
San a Luzia and Boa is a. The ypology o he ee showed a sligh geog aphic pa e n, almos among he
mos no hwes e n neighbou islands.
The haplo ype ne wo k o CR showed mo e complex ela ionships among haplo ypes due o he highe a e
o e olu ion o his DNAm egion. In his case, he highes equency haplo ype was no de ec ed Boa is a
Island and mo e han one single mu a ion cha ac e ized he de i ed haplo ypes. Un oo ed F
ST
dis ance-based
NJ ee showed wo majo di e gen clus e s composed by: São Vicen e, Boa is a and San a Luzia in one hand
and he o he by San iago and San o An ão (Fig. 4). As he leng h o he b anches was di ec ly ela ed o he
s eng h o gene ic di e gence among islands, he islands belonging o he i s clus e we e close ela ed
han hose ones o he second clus e . Finally, he ypology o he ee did no show a geog aphic pa e n.
MEDIAN-JOINING HAPLOTYPE NETWORK AND DISTANCE-BASED NEIGHBOUR-JOINING TREE RESULTS
DISCUSION & CONCLUSION
Boa is a San iago
Sao Vicen e San a Luzia
San o An ao
The highe gene ic di e si y pa ame e s o he DNAm CR e ealed he expec ed highe a e o e olu ion o
his hype a iable egion o he DNAm a bo h island and a chipelago scale. The low gene ic di e en ia ion
o CR DNAm among islands suppo ed he non de ec ion o nei he gene ic di e si y no NJ ee geog aphic
pa e n. Fu he mo e, he almos all p opo ion o gene ic a ia ion de ec ed wi hin islands and all o he
abo e elucida ed o CR DNAm ha gene low was an impo an homogenizing e olu iona y o ce which
lead bo h wi hin and among island e olu ion a he a chipelago. E enmo e, he ela ionship among
haplo ypes o bo h COI and RC median joinning ne wo ks suppo ed ha island isola ion was no enough o
lead o a haplo ype pa e n a he a chipelago. The la e could be explained as a esul o an expec ed
ounde e ec in oceanic islands, like Cape Ve d, which we e colonized by a small po ion o he gene pool o
he sou ce con inen al popula ions. Finally, despi e mo e sequences will be added o hose analyzed he e,
hese p elimina y esul s sugges ed wo complemen a y hypo hesis ha could elucida e he gene ic s ock/s
o Cephalopholis aeniops a he Cape Ve d. COI indica ed one gene ic s ock in he a ea and he
hype a iable bu he mo e esolu i e CR a he scale s udied he e poin ed ou ha unde a chipelago le el
analysis, mo e han one gene ic s ock migh be geog aphically delimi ed al hough i should be e alua ed a
ine scale and hence, mo e sequences need o be added o he analysis.
A)
Islands
n
h
n
h
San o An ão
17
7
0.846
±
0.064
0.00741
±
0.00164
20
5
0.742
±
0.073
0.00148
±
0.00025
São Vicen e
11
10
0.982
±
0.046
0.00658
±
0.00090
14
4
0.495
±
0.151
0.00084
±
0.00030
San a Luzia
19
15
0.959
±
0.036
0.01194
±
0.00225
12
4
0.561
±
0.154
0.00148
±
0.00063
Boa is a
8
7
0.964
±
0.077
0.00826
±
0.00169
20
5
0.442
±
0.133
0.00075
±
0.00026
San iago
7
5
0.905
±
0.103
0.00934
±
0.00199
0
0
Cape Ve d Islands
62
34
0.927
±
0.026
0.01088
±
0.00116
66
9
0.572
±
0.066
0.00113
±
0.00019
h
d
± SD
π ± SD
h
d
± SD
π ± SD
CONTROL REGION COI
Table I. Island gene ic di e si y pa ame e s o Cephalopholis aeniops in he Cape Ve d islands
Figu e 2. Island gene ic di e si y pa ame e s o Cephalopholis aeniops a he Cape Ve d a chipelago. Haplo ype di e si y (a) and nucleo ide di e si y (b) and i s
s anda d de ia ions o Region Con ol o DNAm we e es ima ed.
0.000
0.004
0.008
0.012
0.016
0.6
0.7
0.8
0.9
1.0
1.1
hd
Boa is a
San iago
Sao Vicen e
San a Luzia
San o An ao
B)
GENETIC DIFFERENTIATION AND AMOVA RESULTS
F
ST
alues in Table II showed Boa is a as he unique
island wi h non signi ican alues and slig h bu
signi ican gene ic di e en ia ion among hose islands
wi h bold F
ST
alues. The AMOVA showed slig h bu
signi ican di e en ia ion among island le el analysis
(F
ST
= 0.027, P < 0.05). Mo eo e , he 97% o he o al
a iance o he nucleo ide sequences da a se was
among he indi iduals wi hin islands (Table III).
Sou ce o
a ia ion
d. .
SS
Va iance
componen s
%
a ia ion
S a is ic P- alue
Among islands 4 2.425 0.0128 2.74 F
ST
= 0.027 0.041
Wi hin islands
57
25.849
0.45349
97.26
To al
61
28.274
0.46629
Table III. Hie a chical AMOVA o he DNAm RC sequences o Cephalopholis aeniops in he Cape Ve d islands.
S a. Luzia
S. Vicen e
Boa is a
San iago
S. Vicen e
0.0011
Boa is a
-0.0149
-0.0215
San iago
0.0648*
0.0887*
0.0648
S o. An ão
0.0247*
0.0149
0.0122
0.0982**
* p < 0.05; ** p < 0.01
Table II. Pai wise among islands F
ST
alues DNAm RC sequences o
Cephalopholis aeniops in he Cape Ve d a chipelago.
Figu e 1. S udy a ea and he loca ion o he sampling secuences o Cephalopholis aenipos aco ss he Cape Ve d
Islands. “n”: sample size ( ed: CR; whi e: COI)
S. ANTÃO
n= 17; n=20
S. VICENTE
n= 11; n=14
S. LÚZIA
n= 19; n= 12
BOAVISTA
n= 8; n=20
SANTIAGO
n=7 ; n= 0
B i o A, Clemen e S, He e a R. 2011. On he occu ence o he A ican hind, Cephalopholis aeniops, in he Cana y Islands (eas e n
sub opical A lan ic): in oduc ion o la ge-sized deme sal li o al ishes in ballas wa e o oil pla o ms? Biol In asions - In asion
No e. p 5.
Guide i P, Gia dina F, Azzu o E. 2010. A new eco d o Cephalopholis aeniops in he Medi e anean Sea, wi h conside a ions on
he Sicily channel as a biogeog aphical c oss oad o exo ic ish. Ma ine Biodi e si y Reco ds. Ma ine Biological Associa ion o he
Uni ed Kingdom; Vol. 3; e13; Published online: p 1-4.
Ihssen PE., Booke HE, Casselman JM, McGlade JM, Payne NR, U e FM. 1981. S ock iden i ica ion: Ma e ials and me hods. Can. J.
Fish. Aqua . Sci. 38: 1838-1855.
Pas o OT. 2002. Li e his o y and s ock assessmen o he a ican hind (Cephalopholis aeniops) (Valenciennes, 1828) in São
Vicen e - São Nicolau insula shel o he Cape Ve de a chipelago; Final P oyec 2002. The Uni ed Na ions Uni e si y (UNU) _
Fishe ies T aining P og amme, p 1- 45.
h p://www. ishbase.o g/Summa y/SpeciesCephalopholis&speciesname aeniops
REFERENCES
1. Dp o. de Engenha ias e Ciências do Ma , Uni e sidade de Cabo Ve de; 2. Dp o. Biología. Uni e sidad de Las Palmas de de G an Cana ia; 3. Dp o. Bioquímica e Bioloxia Molecula . Uni e sidade de San iago de Compos ela