Genetic diversity and phylogeography of Polynesian Sandalwood (Santalum insulare) by chloroplastic and nuclear microsatellite markers: definition of genetical provenances in Pacific Islands and Asia

1. REGIONAL WORKSHOP ON SANDALWOOD RESEARCH, DEVELOPMENT AANNDD EEXXTTEENNSSIIOONN
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Genetic diversity and phylogeography of
Polynesian Sandalwood (Santalum insulare) by
chloroplastic and nuclear microsatellite
markers: definition of genetical provenances
Jean-François Butaud a,c , Jean-Marc Bouvet b, Emeline
Lhuillier b, Fanny Rives b & Daniel Verhaegen b
• a Forest department, Rural development service (SDR), Tahiti
• b Genetic Laboratory, CIRAD-forêt, Montpellier, France
• c Natural Products Laboratory (LCSN), University of French
Polynesia (UPF), Tahiti

2. Introduction and main parts of this
presentation
• Molecular studies on chloroplast and nuclear
DNA with microsatellite markers
• Leaf samples from 10 islands (9 in FP and
Mitiaro from Cook Islands)
• Aims : genetic diversity and structure,
phylogeography of S. insulare in Eastern
Polynesia

3. Distribution
of
Polynesian
sandalwood
in Eastern
Polynesia

4. Chloroplast DNA study
• Chloroplast DNA haploid and maternally inherited :
transmission by seed and not by pollen
• No recombination linked with sexual reproduction :
low rythm of mutations
• Informations on the phylogeography of S.
• Estimation of the neutral diversity (not submited to
natural selection)
• This study used 4 markers and defined the chlorotype
by a combination of 4 alleles

5. Chloroplast DNA study – Genetic diversity
• Marquesas show the highest diversity
• Relatively to its size Australs-Cook is diverse
Archipelago Island n na ne He
Society
Tahiti 65 4 1.21 0.19
Moorea 78 3 1.96 0.49
Raiatea 14 2 1.32 0.25
Total 157 5 1.66 0.4
Marquesas
Nuku Hiva 56 4 1.45 0.31
Ua Pou 7 1 1 0
Hiva Oa 37 3 1.1 0.06
Tahuata 23 5 3.17 0.71
Fatu Hiva 1 1 1 0
Total 124 9 3.02 0.67
Australs-Cook
Raivavae 48 2 1.65 0.40
Rapa 14 1 1 0
Mitiaro 2 1 1 0
Total 64 3 2.42 0.59
Eastern Polynesia 11 islands 345 17 5.47 0.82

6. Chloroplast DNA study – Genetic structure
• 36% of variation
between
archipelago, 31%
between islands
and 33 within
islands
• 69% within the
Australs, 64%
Marquesas, 21%
Society
100
Nuku Hiva
Fatuiva
674
Australs-Cook islands
Rapa
Moorea
Tahiti
Ua-Pou
327
Hiva Oa Tahuata
417
691
557
Raiatea
950
900
Raivavae
Cook
561
Society islands
Marquesas islands
Australs-Cook islands
Marquesas islands

7. Chloroplast DNA study – Geographical distribution
• Geographical distribution of chlorotypes
• Homogeneous Society, Marquesas 2 groups, Rapa isolated in Australs-Cook
• Mitiaro and Raivavae have the same chlorotype
Minimum spanning network of
the chlorotypes :
2 groups on the tips, 1 in the
middle
Correspondance with geography
K
J
M
C
B
P
A
F G
N
L H
D
I Q
E
O

8. Chloroplast DNA study – Discussion
• Genetic diversity of S. insulare as high or higher than diversity continental of
species : wide range of habitats and numerous islands
• High differentiation between archipelagoes and islands : low migrant number
due to oceanic distances
• Australs-Cook :
- high differentiation of Rapa due to geological history
- similarity between Mitiaro and Raivavae : exchange in the same geological
archipelago
• Complex structure in Marquesas
• Recent genetic exchange in the more homogeneous Society
• Phylogeography : chlorotypes endemic to each archipelago and Australs
between Society and Marquesas
– One successful colonisation on each archipelago
– Colonisation of Eastern Polynesia possibly by Australs-Cook archipelago (geological
age, geological story of Tuamotu…)
• Pattern of pseudo-vicariance / Complex of metapopulations in each archipelago
with no recent relations

9. Distribution
of
Polynesian
sandalwood
in Eastern
Polynesia

10. Nuclear DNA study
• Nuclea DNA di- or polyploid, pater- and
maternally inherited
• Recombination linked with sexual reproduction :
high rythm of mutations
• Informations on the phylogeography of S. but
mainly on the intra-population dynamism
• Estimation of the neutral diversity (not submited
to natural selection)
• This study used 8 markers and defined genotype
by a combination of 2x8=16 alleles

11. Nuclear DNA study – Clonality
• 45% of clones depending of the islands
• Linked with man induced vegetative multiplication
Populations Samples Genotype number % of clones
Marquesas 144 62 57
Fatu Hiva 3 2 33
Hiva Oa 36 6 83
Tahuata 22 9 59
Nuku Hiva 76 40 47
Ua Pou 7 5 29
Society 164 58 65
Tahiti 77 40 48
Moorea 72 16 78
Raiatea 15 2 87
Australs 76 36 53
Raivavae 62 34 42
Rapa 14 2 86
Total 384 156 45

12. Nuclear DNA study – Genetic diversity
• Society and Marquesas diversity are similar
• Australs is less diverse
Populations N NA R Ho He
MARQUESAS 64 6.13 5.72 0.41 0.55
FATU HIVA 2 1.75 1.44 0.44 0.44
HIVA OA 8 3.00 1.44 0.48 0.44
TAHUATA 9 3.00 1.45 0.40 0.45
NUKU HIVA 40 4.63 1.49 0.47 0.49
UA POU 5 2.63 1.44 0.50 0.44
SOCIETY 60 6.50 5.69 0.37 0.51
TAHITI 41 5.00 1.42 0.37 0.42
MOOREA 17 3.13 1.37 0.36 0.37
RAIATEA 2 2.13 1.56 0.44 0.56
AUSTRALS 38 3.25 3.23 0.29 0.33
RAPA 2 1.50 1.27 0.31 0.27
RAIVAVAE 36 2.50 1.28 0.29 0.28
TOTAL 162 8.75 - 0.39 0.69

13. Nuclear DNA study – Genetic structure
• 27% of the
variation between
archipelago, 24%
between islands
and 50 within
islands
• Between islands
Fst=0,5 (0,68
between Rapa and
Raivavae ; 0,07
between HO and
Tahuata)
• Between
archipelago
Fst>0,3

14. Nuclear DNA study – Multilocus genotypes
• Nuku Hiva
splited in high
and low
elevation
populations
• South Marquesas
homogeneous
• Ua Pou in
unexpected place
Marquesas
Nuku Hiva
"Haut"
Ua
Pou
UP-C
Hohoi
Teepo
UP-D
Poutetainui
F-A
Omoa
Tahioae Toovii
Vaioa
NH-V NH-U
NH-AE NH-AB
Haut TD
HO-A
Hanaava
o HO-B
Vaikooi
Vaikooi HO-C
HO-E
Mokoa
u
HO-F
Taaoa
NH-H Haut TD
TU-B
Côte Nord
Est
TU-G TU-E
Côte
Sud
TU-D
Côte
Sud
TU-H
Hanami
ai
Vaitahu Faanui
TU-I
NH-B
NH-E
Haut
TD
NH-F
NH-G
NH-I
NH-K NH-J
NH-L
Maauu NH-M
NH-N
Vaiteheii
NH-O
NH-Q
NH-S
Tahioae
Vaioa
NH-W
NH-X
NH-Y
NH-Z
NH-AA
NH-AF
NH-AG
NH-AJ
NH-AL
Vaiteheii NH-AM
NH-AN
UP-A
Haakuti
Vaihaoa
UP-B
Hakaheta
u
UP-E
60
91
86
63
59
69
83
60
Fatu
Hiva
Haut
de
Terre
Désert
e
Haut TD
Toovii crêtes
Toovii crêtes
Toovii crêtes
Toovii
Haut TDToovii
Tahioae
Vaioa
Toovii
Motuhee
Nuku
Hiva
"Bas"
Hiva Oa
0 0.2 Tahuata

15. Nuclear DNA study – Multilocus genotypes
• Group with
Raiatea and
Moorea
• Segregation of
some very low
elevation
populations of
Tahiti
Society
Orohena
crêtes
Aorai
sommet
Orohena crêtes
T-Y
T-Z
0 0.2
Moorea
M-B
T-AB
M-I M-K
Fairurani
M-C
Fairurani
M-E
M-D
M-F
M-G
M-H
M-L
M-M M-O M-P
T-D
T-E
T-F
T-G
T-H
T-I
T-J
T-L
T-M
T-O
T-P
T-Q
T-R
T-S
T-T
T-U
T-V
Côte Ouest
T-AA
T-AD
T-AE
T-AF
T-AG
T-AJ
Pic Vert
T-AM
T-AN
R-A
Fetuna
50
92
62 90
78
62
58
55
73
75
52
Fairurani
Tohiea
Rotui Rotui
Rotui
Raiatea
Pic Vert
basses pentes
Aorai
sommet
Aorai sommet
Pic
Vert
Orohena
crêtes
Pic Vert
T-AK
Aorai
sommet
Orohena crêtes
Aorai
sommet
Pic Vert
Aorai
sommet Tahiti
var. alticola
+ Pic Vert
Tahiti
var. insulare
- Pic Vert

16. Nuclear DNA study – Multilocus genotypes
• High differentiation
between Rapa and
Raivavae
• No structuration
inside Raivavae
between high island
or motu populations
Australs
RV-P
0 0.
2
RP-B
RP-A
RV-A
RV-B
RV-Y
RV-Z
RV-C
RV-D
Motu
Est
Raivavae île principale
Motu
Vaiamanu
RV-E
RV-F
et Raivavae motu
RV-H
RV-I
RV-J
RV-K
RV-L
RV-M
RV-N
RV-O
RV-Q
RV-R
RV-T
RV-V
RV-W
RV-X
RV-AA
RV-AC
RV-AD
RV-AE
RV-AF
100
65
52
52
Rapa
Karapoo rahi
Anatakuri Nako
Vaianaua
Petits
Motu
Petits Motu
Motu Sud Ouest
Motu
Est
Anatonu
Est
Anatonu
Ouest
Motu
Est Motu
Vaiamanu
Motu
Est
Anatonu
Est
Anatonu
Ouest
Anatonu
Ouest

17. Nuclear DNA study – Discussion
• Clonality by vegetative multiplication as a natural adaptation
strategy in hard ecological conditions and advantageous today in
a man-degraded environment
• Genetic diversity low compared to continental species :
fondation effect, no regeneration by seed, overexploitation
• High differentiation between archipelagoes and between
islands : metapopulations isolated by ocean
• Possible colonisation of Eastern Polynesia by Australs due to
geological history (need of confirmation by phylogeny)
• Intra-island differentiation with the example of Nuku Hiva :
fragmentation due to overexploitation or natural differentiation
in different ecological areas

18. Conclusion and definition of genetic provenances / origins
• Concordance between 2 studies
• Australs Sandalwood is the least diverse and the most endangered
• Phylogeographical hypothesis : Hawaii (age, botany), Australs-Cook (age and
genetic) and by Australs : Marquesas (most diverse) and Society (least diverse)
• Botanical varieties : it doesn’t match with Tahiti and Marquesas
• Provenances for conservation and management units
Archipelago Provenance Variety
Austral Islands Rapa margaretae
Raivavae raivavense
Cook Islands Mitiaro mitiaro
Pitcairn Islands Henderson hendersonense
Society Islands Tahiti alticola et insulare
Moorea and Raiatea raiateense
Marquesas Islands
Nuku Hiva high elevation marchionense
Nuku Hiva low elevation marchionense
Ua Pou marchionense
Hiva Oa and Tahuata marchionense
Fatu Hiva marchionense

19. Distribution
of
Polynesian
sandalwood
in Eastern
Polynesia

20. Acknowledgments
• Lex Thomson and CI foresters for sending leaves
from Mitiaro sandalwood
• Alexandre Vaillant and Mireille Poitel from CIRAD
for molecular analysis
• Workers of SDR in all the islands visited
• Funds for research from the Ministry of
Environment and the Ministry of Overseas Territory
• Research programme on Biodiversity in the Austral
Islands from the Research delegation of French
Polynesia