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Assessment of The Phylogenetic Relationship among
Indonesian Horseshoe Crabs Collected from Java
Island Using mtDNA COI Gene in Combination with
Morphological Characters
LUSITA MEILANA1*, Yusli Wardiatno 1, Nurlisa A Butet1, Majariana Krisanti1,
Hefni Effendi2
1Department of Aquatic Resources Management, Faculty of Fisheries and Marine
Science, Bogor Agricultural University, Kampus IPB Darmaga, Bogor 16680,
Indonesia2 Center for Environmental Research of Bogor Agricultural University, Jl. Dramaga
Bogor, Indonesia
*Corresponding author: [email protected]
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Horseshoe Crabs (Mimi/ Belangkas)
Background
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Limulus polyphemus Tachypleus tridentatus
Tachypleus gigas Carcinoscorpius rotundicauda
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Limulus polyphemus Tachypleus tridentatus
Tachypleus gigas Carcinoscorpius rotundicauda
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utilization
Background
Horseshoe Crabs (Mimi/ Belangkas)
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Rp 3000-10000
( $ 0.2 - 0.7)
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Rp 30000-50000
( $ 2-3 )
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life cycle
Background
utilizationHorseshoe Crabs (Mimi/ Belangkas)
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building houses on the beach
Land Degradation
Mangrove Degradation
High activities
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17 artificial islands in the North Coast Java Island
Environmental Impact Assessment (EIA)
http://suarajakarta.co/news/
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related studies
(Indonesian Horseshoe Crabs genetics - few)
Background
utilization
Horseshoe Crabs (Mimi/ Belangkas)
life cycle
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Authors Tittle Years
Fachrul MF Biological Aspects of Horseshoe Crabs (Xilphosura) 1989
Eidman M, Samosir AM,
Aktani U
Biological Studies of Horseshoe Crabs (Subclass Xiphosura) in the Development and
Utilization Context of Marine Biological Resources for the Pharmaceutical Industry Needs in
Indonesia
1992
Purnimo YA Reproductive Biology of Horseshoe Crabs Carcinoscorpius rotundicauda (Latreille) female
were caught in the waters of Rembang, Central Java
1992
Suparta morphometric diversity of Horseshoe Crabs, Tachypleus gigas (MULLER) and Carcinoscorpius
rotundicouda (Latreille) in Pandeglang Coastal Waters, West Java and Rembang Beach, Central
Java
1992
Santoso AR Spawning and development of embryo Tachypleus gigas (Muller) 1992
Pratiwi R “Horseshoe Crabs” Spreaders Death Protected 1993
Balisani SL Development Studies of Carcinoscorpius rotundicauda Embryo (Latreille), the Results of Semi
Natural and Artificial spawning
1994
Ismurwanti C Preliminary studies of the diazinon-60 EC concentration effect against embryonic development
and hatching eggs Carcinoscorpius rotundicauda (Latreille).
1994
Rahmalia E Artificial fertilization and early studies of Saponin Concentration and The Influence On The
Embryos And Larvae Tachypleus Gigas (Muller) Development
1995
Vauziyah C Tachypleus gigas embryonic development (Muller) of Banten Gulf waters at various salinity
media
1995
Eidman M, Mayunar,
Redjeki S.
Gonadal maturation Carcinoscorpius rotundicauda, (Latreille) and Tachypleus gigas (Muller)
with different types of feed
1997
Muslihah Some Aspects of Tachypleus gigas Reproductive Biology in Mayangan waters, Subang
Regency, West Java
2004
Rubuyanto E Population studies (Xiphosura) in the Kuala Tungkal waters , Tanjabbar Districtr Jambi 2012
Mulya MB Preservation and Utilization of Genetic Resources Carcinosscorpius rotundicauda, L and
Tachypleus gigas, M
2014
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•to assess the horseshoe crabs morphological characters
in Segara Menyan Subang, Pelabuan Banten, Semarang,
Demak, Rembang, Pantai Kenjeran Surabaya, and
Campur Rejo Gresik
•to establish phylogenetic relationship among Indonesian
horseshoe crabs (T. gigas, T. tridentatus, C.
rotundicauda) of coastal areas of Java Island based on
COI gene molecular marker
Objectives of the study
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Sampling methods:.
sampling periods and methods
Sampling periods :All samples were taken during October 2014 – March 2015
Laboratory Analysis: Molecular Biology Laboratory Aquatic Resources Management (MSP) and Integrated
Laboratory Fisheries and Marine Faculty, Bogor Agricultural University (FPIK IPB)
Bare handRandom sampling Fisherman’s boat
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2
Caption: XX1: (1. total length (X1), 2. telson length (X2), 3. body length (X3), 4. prosoma length (X4), 5. median
ridge Length (X5), 6. front occeli length (X6), 7. ophistoma Length (X7), 8. ventral messel thick (X8), 9. maximum
width of prosoma (X9), 10. distance between the two compound eyes (X10), 11. distance between auriculata spine
(X11), 12. distance between the marginal process (X12), 13. distance between posterior angle (X13), 14. distance
between anal angle (X14), 15. mid-telson high (X15), 16. mid-telson width) (X16) , XX2: (17. marginal spine 1
length (X17), 18. spine marginal II length (X18), 19. spine marginal III length (x19), 20. spine marginal IV length
(X20), 21. marginal spine V length ( X21), 22. marginal spine VI length (X22), 23. diameter claw of chelicera (X23),
24. claw diameter of pedipalpi (X24), 25. claw diameter of foot path I (X25), 26. claw diameter of foot II ( X26), 27.
claw diameter of foot path III (X27), 28. claw diameter of foot IV (X28)).
Morphological analysis
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Molecular analysis
DNA Isolation and extraction
DNA quality test
COI gene amplification by PCR
PCR quality test
Nucleotide Sequencing
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= T. gigas = T. tridentatus = C. rotundicauda
Morphological Results
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Sampling
locationsT. gigas T. tridentatus C. rotundicauda
Total
male female male female male female
Banten 6 11 17
Subang 19 17 4 18 58
Semarang 1 2 2 2 7
Demak 10 13 2 4 29
Rembang 18 3 21
Gresik 3 9 20 32
Surabaya 7 4 37 4 52
216
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Based on the maximum width and body length of prosoma three species, the overall obtained size
large to small in sequence :
T. gigas > T. tridentatus > C. rotundicauda
0
50
100
150
200
250
Pro
so
ma M
axim
um
Wid
th (
cm
)
C. rotundicauda T. tridentatus T. gigas
0
50
100
150
200
250
Bo
dy l
en
gth
(cm
)
C. rotundicauda T. tridentatus T. gigas
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Dendogram formed based on similarities and differences in the 14 Horseshoe crabs
morphological characteristics obtained information that T. gigas and T. tridentatus
have similar characteristics, but C. rotundicauda has its own character.
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SubangSurabayaGresikRembangDemakSemarang
55,61
70,40
85,20
100,00
Location
Sim
ilari
ty (
%)
C. rotundicauda Subang form separate groups (based on morphological characters) :
in this area has the body and limbs size, smaller.
Rembang has a larger maximum width of prosoma.
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ObservedPredicted
Semarang Demak Rembang Gresik Surabaya Subang Percent Correct
Semarang 1 100.0%
Demak 0 6 100.0%
Rembang 0 0 21 100.0%
Gresik 0 0 0 28 100.0%
Surabaya 0 0 0 0 38 100.0%
Subang 0 0 0 0 0 21 100.0%
Overall
Percentage0.9% 5.2% 18.3% 24.3% 33.0% 18.3% 100.0%
Based on the Pseudo R-Square table was obtained R2 = 95.3%.
Based on the classification table, the model has the ability to distinguish between
population groups by 100% (individual was classified as an original individual from
Semarang (n = 1), Demak (n = 6), Rembang (n = 21), Gresik (n = 28 ), Surabaya (n =
38), and Subang (21)).
Percent correct can reach up to 100% because of the individuals number that
allegedly found a bit of each region
Multinomial logistic
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SurabayaBantenSubangDemakSemarang
20,01
46,67
73,34
100,00
Location
Sim
ilari
ty
T, gigas Surabaya form separate groups (based on morphological characters) : in
this area has the body and limbs size, larger.
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ObservedPredicted
Semarang Demak Surabaya Subang Banten Percent Correct
Semarang 2 100.0%
Demak 0 21 100.0%
Surabaya 0 0 11 100.0%
Subang 0 0 0 18 100.0%
Banten 0 0 0 0 5 100.0%
Overall
Percentage3.5% 36.8% 19.3% 31.6% 8.8% 100.0%
Based on the Pseudo R-Square table was obtained R2 = 93.8% .
Based on the classification table, the model has the ability to distinguish between
population groups by 100% (individual was classified as an original individual from
Semarang (n=2), Demak (n=21), Surabaya (n=11), Subang (18), dan Banten (n=5)).
Percent correct can reach up to 100% because of the number of individuals who
allegedly found a bit of each region
Multinomial logistic
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SubangGresikBantenSemarang
20,01
46,67
73,34
100,00
Location
Sim
ilari
ty (
%)
T, tridentatus Subang form separate groups (based on morphological characters) :
in this area has the body and limbs size, smaller. Gresik relatively large body size
and limb than Semarang and Banten.
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Observed Predicted
Semarang Gresik Subang Banten Percent Correct
Semarang 2 100.0%
Gresik 0 3 100.0%
Subang 0 0 17 100.0%
Banten 0 0 0 10 100.0%
Overall
Percentage6.3% 9.4% 53.1% 31.3% 100.0%
Based on the Pseudo R-Square table was obtained R2 = 88.8% .
Based on the classification table, the model has the ability to distinguish between
population groups by 100% (individual was classified as an original individual from
Semarang (n=2), Gresik (n=3), Subang (n=17), dan Banten (n=10)).
Percent correct can reach up to 100% because of the number of individuals who
allegedly found a bit of each region
Multinomial logistic
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Molecular Results
DNA electrophoresis, pre-test results of PCR products on 1% agarose gel,
Left to right column: 1kb marker; sample 1; sample 2 etc
1 : TT SBG
2 : CC SBG
3 : TG SMG
4 : TT SBG
5 : TG DMK
6 : CC DMK
7 : CC RMG
8 : TG SBG
9 : TG SBG
10 : TG BTN
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CC
DMK
CC
RMG
CC
SBG
TG
BTN
TG
DMK
TG
SBG
TG
SMG
TG
SRB CC TT TG
C._rotundicauda_Demak
C._rotundicauda_Rembang 0.002
C._rotundicauda_Subang 0.002 0.000
T._gigas_Banten 0.140 0.138 0.138
T._gigas_Demak 0.140 0.138 0.138 0.000
T._gigas_Subang 0.140 0.138 0.138 0.007 0.007
T._gigas_Semarang 0.140 0.138 0.138 0.007 0.007 0.000
T._gigas_Surabaya 0.143 0.140 0.140 0.009 0.009 0.002 0.002
AF370828_C._rotundicauda 0.132 0.129 0.129 0.107 0.107 0.112 0.112 0.114
HQ588752_T._tridentatus 0.129 0.127 0.127 0.088 0.088 0.088 0.088 0.090 0.114
KJ825849_T._gigas 0.136 0.134 0.134 0.015 0.015 0.015 0.015 0.018 0.107 0.086
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Construction of phylogeny tree based on COI gene
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Conclusions
• morphological and molecular inconsistency, individual was
classified as an original individual from each locations according to
morphological analysis
•Indonesia has different nucleotides character compared to other
countries (distinct), C. rotundicauda allegedly is a subspecies but not
yet convinced.
• Need conservation for all locations
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T gigas1 Semarang
T tridentatus1 Semarang
T tridentatus4 Subang
T gigas1 Demak
JF896114 T gigas
KJ825849 T gigas
KJ825848 T gigas
JQ739210 T tridentatus
JN018216 T tridentatus
FJ860267 T tridentatus
JX437074 C rotundicauda
JQ178358 C rotundicauda
AF370828 C rotundicauda
JF896106 C rotundicauda
C.rotundicauda1 Demak
C.rotundicauda2 Subang
C.rotundicauda4 Rembang
HQ588747 Limulus polyphemus
HQ588751 Limulus polyphemus
HQ588753 Limulus polyphemus
AF216203 Limulus polyphemus
HM367070 Opilio parietinus
10
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conservation
SK Menteri kehutanan No. 12/Kpts-II/1987
(Minister of Forestry Decree)
Peraturan Pemerintah RI No. 7/1999)
(Mulya 2004; Rubiyanto 2012 (Indonesia
Government Regulation)