LUSITA MEILANA 1 *, Yusli Wardiatno 1, Nurlisa A Butet 1, Majariana Krisanti 1, Hefni Effendi 2

Transcription

1 Assessment of The Phylogenetic Relationship among Indonesian Horseshoe Crabs Collected from Java Island Using mtdna COI Gene in Combination with Morphological Characters LUSITA MEILANA 1 *, Yusli Wardiatno 1, Nurlisa A Butet 1, Majariana Krisanti 1, Hefni Effendi 2 1 Department of Aquatic Resources Management, Faculty of Fisheries and Marine Science, Bogor Agricultural University, Kampus IPB Darmaga, Bogor 16680, Indonesia 2 Center for Environmental Research of Bogor Agricultural University, Jl. Dramaga Bogor, Indonesia *Corresponding author: lusitameilana@yahoo.co.id

2 Horseshoe Crabs (Mimi/ Belangkas) Background

3 Limulus polyphemus Tachypleus tridentatus Tachypleus gigas Carcinoscorpius rotundicauda

4 Limulus polyphemus Tachypleus tridentatus Tachypleus gigas Carcinoscorpius rotundicauda

5 Background Horseshoe Crabs (Mimi/ Belangkas) utilization

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7 Rp ( $ )

8 Rp ( $ 2-3 )

9 Background Horseshoe Crabs (Mimi/ Belangkas) utilization life cycle

10 building houses on the beach Mangrove Degradation High activities Land Degradation

11 17 artificial islands in the North Coast Java Island Environmental Impact Assessment (EIA)

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14 Background Horseshoe Crabs (Mimi/ Belangkas) utilization related studies (Indonesian Horseshoe Crabs genetics - few) life cycle

15 Authors Tittle Years Fachrul MF Biological Aspects of Horseshoe Crabs (Xilphosura) 1989 Eidman M, Samosir AM, Aktani U Purnimo YA Suparta Biological Studies of Horseshoe Crabs (Subclass Xiphosura) in the Development and Utilization Context of Marine Biological Resources for the Pharmaceutical Industry Needs in Indonesia Reproductive Biology of Horseshoe Crabs Carcinoscorpius rotundicauda (Latreille) female were caught in the waters of Rembang, Central Java morphometric diversity of Horseshoe Crabs, Tachypleus gigas (MULLER) and Carcinoscorpius rotundicouda (Latreille) in Pandeglang Coastal Waters, West Java and Rembang Beach, Central Java Santoso AR Spawning and development of embryo Tachypleus gigas (Muller) 1992 Pratiwi R Horseshoe Crabs Spreaders Death Protected 1993 Balisani SL Ismurwanti C Rahmalia E Vauziyah C Eidman M, Mayunar, Redjeki S. Muslihah Development Studies of Carcinoscorpius rotundicauda Embryo (Latreille), the Results of Semi Natural and Artificial spawning Preliminary studies of the diazinon-60 EC concentration effect against embryonic development and hatching eggs Carcinoscorpius rotundicauda (Latreille). Artificial fertilization and early studies of Saponin Concentration and The Influence On The Embryos And Larvae Tachypleus Gigas (Muller) Development Tachypleus gigas embryonic development (Muller) of Banten Gulf waters at various salinity media Gonadal maturation Carcinoscorpius rotundicauda, (Latreille) and Tachypleus gigas (Muller) with different types of feed Some Aspects of Tachypleus gigas Reproductive Biology in Mayangan waters, Subang Regency, West Java Rubuyanto E Population studies (Xiphosura) in the Kuala Tungkal waters, Tanjabbar Districtr Jambi Mulya MB Preservation and Utilization of Genetic Resources Carcinosscorpius rotundicauda, L and Tachypleus gigas, M 2014

16 Objectives of the study 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

17 Locations

18 sampling periods and methods Sampling periods : All samples were taken during October 2014 March 2015 Sampling methods:. Random sampling Fisherman s boat Bare hand Laboratory Analysis: Molecular Biology Laboratory Aquatic Resources Management (MSP) and Integrated Laboratory Fisheries and Marine Faculty, Bogor Agricultural University (FPIK IPB)

19 Morphological analysis 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)).

20 Molecular analysis DNA Isolation and extraction DNA quality test COI gene amplification by PCR PCR quality test Nucleotide Sequencing

21 Morphological Results = T. gigas = T. tridentatus = C. rotundicauda

22 Sampling locations T. gigas T. tridentatus C. rotundicauda male female male female male female Total Banten Subang Semarang Demak Rembang Gresik Surabaya

23 Prosoma Maximum Width (cm) Body length (cm) 250 C. rotundicauda T. tridentatus T. gigas 250 C. rotundicauda T. tridentatus T. gigas 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

24 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.

25 C. rotundicauda

26 55,61 Similarity (%) 70,40 85,20 100,00 Semarang Demak Rembang Gresik Location Surabaya Subang 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.

27 Multinomial logistic Observed Predicted Semarang Demak Rembang Gresik Surabaya Subang Percent Correct Semarang % Demak % Rembang % Gresik % Surabaya % Subang % Overall Percentage 0.9% 5.2% 18.3% 24.3% 33.0% 18.3% 100.0% Based on the Pseudo R-Square table was obtained R 2 = 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

28 T. gigas

29 20,01 Similarity 46,67 73,34 100,00 Semarang Demak Subang Location Banten Surabaya T, gigas Surabaya form separate groups (based on morphological characters) : in this area has the body and limbs size, larger.

30 Predicted Observed Semarang Demak Surabaya Subang Banten Percent Correct Semarang % Demak % Surabaya % Subang % Banten % Overall Percentage Multinomial logistic 3.5% 36.8% 19.3% 31.6% 8.8% 100.0% Based on the Pseudo R-Square table was obtained R 2 = 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

31 T. tridentatus

32 20,01 Similarity (%) 46,67 73,34 100,00 Semarang Banten Location Gresik Subang 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.

33 Observed Predicted Semarang Gresik Subang Banten Percent Correct Semarang % Gresik % Subang % Banten % Overall Percentage Multinomial logistic 6.3% 9.4% 53.1% 31.3% 100.0% Based on the Pseudo R-Square table was obtained R 2 = 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

34 Molecular Results 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 DNA electrophoresis, pre-test results of PCR products on 1% agarose gel, Left to right column: 1kb marker; sample 1; sample 2 etc

35 C._rotundicauda_Demak CC DMK C._rotundicauda_Rembang CC RMG C._rotundicauda_Subang CC SBG T._gigas_Banten TG BTN T._gigas_Demak TG DMK T._gigas_Subang TG SBG T._gigas_Semarang TG SMG T._gigas_Surabaya AF370828_C._rotundicauda HQ588752_T._tridentatus TG SRB CC TT TG KJ825849_T._gigas

36 Construction of phylogeny tree based on COI gene

37 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

38 Thank you

39 T gigas1 Semarang T tridentatus1 Semarang T tridentatus4 Subang T gigas1 Demak JF T gigas KJ T gigas KJ T gigas JQ T tridentatus JN T tridentatus FJ T tridentatus JX C rotundicauda JQ C rotundicauda AF C rotundicauda JF C rotundicauda C.rotundicauda1 Demak C.rotundicauda2 Subang C.rotundicauda4 Rembang HQ Limulus polyphemus HQ Limulus polyphemus HQ Limulus polyphemus AF Limulus polyphemus HM Opilio parietinus 10

40 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)