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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]

Horseshoe Crabs (Mimi/ Belangkas)

Background

Limulus polyphemus Tachypleus tridentatus

Tachypleus gigas Carcinoscorpius rotundicauda

utilization

Background


Rp 3000-10000

( $ 0.2 - 0.7)

Rp 30000-50000

( $ 2-3 )

life cycle

Background

utilizationHorseshoe Crabs (Mimi/ Belangkas)

building houses on the beach

Land Degradation

Mangrove Degradation

High activities

17 artificial islands in the North Coast Java Island

Environmental Impact Assessment (EIA)

http://suarajakarta.co/news/

related studies

(Indonesian Horseshoe Crabs genetics - few)

Background

utilization


life cycle

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

•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

Locations

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

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

Molecular analysis

DNA Isolation and extraction

DNA quality test

COI gene amplification by PCR

PCR quality test

Nucleotide Sequencing

= T. gigas = T. tridentatus = C. rotundicauda

Morphological Results

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

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

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.

C. rotundicauda

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.

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

T. gigas

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.

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% .



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



T. tridentatus

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.

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% .



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



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

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

Construction of phylogeny tree based on COI gene

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

Thank you

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



AF216203 Limulus polyphemus

HM367070 Opilio parietinus

10

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)

assessment of the phylogenetic relationship among ... · demak, rembang, pantai kenjeran surabaya,...

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