wi Astuti, Hidayat Ashari, and Siti N. Prijono

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Phylogenetic position of Psittacula parakeet bird from Enggano Island, Indonesia based on analyses of cytochrome b gene sequences. wi Astuti, Hidayat Ashari, and Siti N. Prijono Research Centre for Biology, Indonesian Insitute of Sciences, Gd, Widyasatwaloka, CSC, Jl. Jakarta-Bogor KM 46, Cibinong, West Jawa, Indonesia. 5th International Conference on Biodiversity. Madrid, Spain. 10th 12th March 2016

Phylogenetic position of Psittacula parakeet bird from Enggano Island, Indonesia based on analyses of cytochrome b gene sequences. CORRECTION in ABSTRACT 1. 2 rd sentence : ND2 (delete) ----- cyt b 2. 5 th sentence : ND2 and COI (delete) genes (delete) ---- gene 3. 9 th sentence : 3.70 % for (P.l. modesta vs P.l. defontainei), 2.50 % for (P.l. defontainei vs P.l. longicauda), and 1.60 % for (P.l. modesta vs P l. longicauda) ---- delete 2.16 % for (P.l. modesta vs P.l. defontainei), 1.51 % for (P.l. defontainei vs P.l. longicauda), and 2.37 % for (P.l. modesta vs P l. longicauda)

About Enggano Island of Indonesia MAP of Indonesia Indonesia hast housands islands; 5 are big : Sumatra is., Java is., Borneo/Kalimantan is., Celebes/Sulawesi is., and Papua is.

Enggano island Located in Indian Ocean, approx. 100 km SW offshore of mainland Sumatra. 5 17-5 31 S & 102 05-102 25 E. One of Indonesia s outermost island. Geologically unique = Oceanic Island. It had never been a part of Sundaland. Separated from mainland Sumatra by marine basin of 2000 m deep = A perfect oceanic island. Bird speciation proses? Biologically unique = high

ABOUT Psittacula Parakeets BIRD Psittaciformes Psittacidae Loriinae Cacatuinae Psittacinae Nestorini Micropsittini Psittaculini Strigopini Cacatuini Calopsittacini Psittrichasini Cyclopsittacini Psittacini Arini Platycercini Lophopsittaci ni extinct

Psittacula parakeets P. longicauda modesta (Enggano Is.) Psittacula cyanocephala (Linné 1766), India,Pakistan P. longicauda longicauda (Sumatra,Borneo, Nias, Bangka, Singapore) P. longicauda defontainei (Natuna is.) P.alexandri (Java is.)

In the world there are 15-16 species of Psittacula : P. eupatria, P. wardi, P. krameri, P. echo, P. exsul, P. (himalayana) himalayana, P. (himalayana) finschii, P. intermedia, P. cyanocephala, P. roseata, P. columboides, P. calthropae, P. derbiana, P. caniceps, P. alexandri, and P. longicauda In the world: 5 subspecies of P. longicauda : P. l. tytlery, P. l. nicobarica, and P. l. longicauda, P. l. defontainei, and P. l. modesta. in Indonesia: P. l. longicauda (Sumatra, Borneo, Riau Is.), P. l. defontainei (Natuna Is.), and P. l. modesta (Enggano Is.)

Distribution of Psittacula Parakeet forms (Groombridge J.J et al., 2004)

Distribution of Long-tailed Parakeet Psittacula longicauda (Boddaert 1783) Psittacula.l. longicauda Psittacula.l. modesta Psittacula.l. defontainei Psittacula.l. nicobarica Psittacula.l. tytleri (Source: http://home.online.nl/psittaculaworld/species/p-longicauda.htm)

MITOCHONDRIAL DNA The mitochondrial genome of vertebrates is a small, circular molecule of 15-20 kb, with a compact and conserved organization in most, but not all, of the studied species (Wolstenholme, 1992). In animals, it contains 37 genes: 13 protein-coding genes (3 units of cytochrome ocsidase (CO I-III), 7 subunit NADH-dehydrogenase (ND1-6 and ND 4L, 2 subunits ATPase (6 and 6L), cyt b), 22 transfer RNA genes (trnas) and 2 ribosomal RNA genes (12S and 16S rrnas), and the control region (D-loop). Because their structures were relatively simple and mutation rates were higher than nuclear DNA, the mtdna sequences were used frequently to estimate phylogenetic relationships among animal taxa, genetic diversity, population genetics and molecular evolution

About cytochromre b gene The cytochrome b gene (cyt b) from the mitochondrial genome contains speciesspecific information and has been used in phylogeny as well as in forensic investigations in previous studies. Thus, it has been confirmed the usefulness of cyt b analysis in identifying the biological origin of casework specimens (Parson, et al., 2000) Mitochondrial cyt b sequences have been used to understand the genetic diversity for better conservation management of threatened species animals Cytochrome-b gene (~1,143 bp) is reported as the most useful marker in recovering phylogenetic relationships among closely related taxa, at a variety of taxonomic levels

AIMS 1. How far the difergencies (sequence divergences) between Psittacula from Enggano island to others subspecies of Psittacula longicauda are 2. How far the difergencies (sequence divergences) between species of Psittacula are 3. Where phylogenetically the position of Psittacula longicauda modesta from Enggano island is 4. Genetically, whether Psittacula from Enggano is separated from others subspecies of Psittacula longicauda 5. Genetically, whether Psittacula from Enggano is as subspecies of P. longicauda or become a new species

METHODS Blood samples taken from each live bird preserved in 99% ethanol DNA, extracted from 5 20 mg of dry blood Cytochrome b gene Amplification by (PCR) DNA sequencing DNA sequences alignment, using MEGA5 softwarw Phylogenetic trees analyses (MEGA5 software)

Target fragment of cyochrome b gene in this studi amplified by PCR individu M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Target fragment of cytochrome b 1200-bp

Bases composition (%) in the 869-bp of cytochrome b gene of Psittacula Cytochrome b is protein coding --- amino acid --- codons Tumine (T) Citosine (C) Adenin (A) Guanine (G) Total No. Codon position % % % % base 1 1st codon 10.32 48.66 37.65 3.37 290 2 2nd codon 23.55 29.67 24.66 22.11 290 3 3rd codon 37.23 28.50 20.24 14.03 289 4 Total 23.68 35.62 27.52 13.17 869

RESULTS Genetic distances (bellow diagonal) and st. error (above diagonal) between species/subspecies of Psittacula N o. Species name 1 2 3 4 5 6 7 8 9 10 1 P. columboides 0,0122 0,0121 0,0095 0,0096 0,0108 0,0096 0,0096 0,0096 5 2 P. finschii 0,0926 0,0057 0,0078 0,0119 0,0106 0,0106 0,0099 0,0105 3 3 P. himalayana 0,0937 0,0380 0,0092 0,0111 0,0109 0,0123 0,0107 0,0116 6 4 P. cyanochepala 0,0749 0,0437 0,0651 0,0098 0,0105 0,0109 0,0107 0,0103 4 5 P. krameri 0,0595 0,0855 0,0764 0,0767 0,0121 0,0112 0,0105 0,0102 1 6 P. roseata 0,0808 0,0780 0,0851 0,0665 0,0825 0,0112 0,0106 0,0122 4 7 P. alexandri alexandri 0,0661 0,0900 0,1003 0,0888 0,0712 0,0825 0,0081 0,0047 3 8 P. longicaoda defontainei 0,0640 0,0866 0,0911 0,0878 0,0665 0,0712 0,0615 0,0080 5 9 P. longicaoda modesta 0,0652 0,0844 0,0896 0,0832 0,0667 0,0665 0,0547 0,0216 0,005 8 1 0 P. longicauda longicauda 0,0665 0,0886 0,0978 0,0874 0,0738 0,0852 0,0664 0,0151 0,0237

Species name VARIABLE SITES in the 869-bp of cytochrome b gene sequences Site 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 1 1 2 4 4 4 5 7 7 8 9 0 0 0 2 3 3 4 4 5 6 6 7 7 8 8 9 9 0 0 0 1 2 2 3 6 7 7 8 9 9 9 0 1 2 3 4 5 7 0 6 3 3 5 8 8 0 9 2 7 0 3 7 1 3 9 5 8 4 0 4 1 2 1 7 3 5 2 5 6 1 6 9 5 8 4 7 7 5 8 9 7 0 5 1 0 5 P. longicauda_longicauda_(cyt) C C C C C G C C A T T C A C T C T C C C C G C A G A A C T A T A C T T C G A T T C C A C C C G T T P.finschii.. T T T A T. G C.. C A C...... A T. A G. T.. C.. C C T A G C.. T G T T T A.. P. himalayana.. T T T A T. G C.. C A C.... T T A T T... T.. C.. C. T A. C.. T G T T T A. C P. cyanocephala.. T T T A. T. C.... C. C... T A.. A.. T.. C G T C C. A. C.. T... T. C C P. roseata T.... A... C C... C. C... T A..... T. C C. T.. T... C...... A. C P. longicauda_defontainei_1.......... C...................................... P.longicauda_defontainei_2.......... C...................................... P.longicauda_defontainei_3.......... C...................................... P. longicauda_defontainei_4.......... C...................................... P. alexandri_1. T.. T A. T. C C T.. C T. A.. T A.. A. G T C......... C C T.... T... P. alexandri_2. T.. T A. T. C C T.. C T. A.. T A.. A. G T C......... C C T.... T... P. alexandri_3. T.. T A. T. C C T.. C T. A.. T A.. A. G T C......... C C T.... T... P. alexandri_4. T.. T A. T. C C T.. C T. A.. T A.. A. G T C......... C C T.... T... P. longicauda_modesta_1....... T.......... T..... A........ C........... T... P. longicauda_modesta_2....... T.......... T..... A........ C........... T... P. longicauda_modesta_3....... T.......... T..... A........ C........... T... P. longicauda_modesta_4....... T.......... T..... A........ C........... T... 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 7 8 0 0 1 2 4 4 4 6 7 7 7 7 9 9 0 1 2 3 3 3 4 5 5 5 6 6 7 8 8 8 9 9 0 1 2 2 2 3 4 4 5 5 5 9 1 0 3 3 4 2 5 8 9 2 3 5 7 0 6 8 7 7 0 5 9 4 3 4 5 2 8 7 3 6 9 2 5 1 0 2 5 9 4 3 6 2 8 gicauda_longicauda_(cyt) C T C A T A C C T A A C A T C C C C C C C G G T C T C T C C C C G A A C C C C A C T T C A hii. C.. C G.. C. G. C C A. T A T T. A A C. C T..... A........ C C.. alayana. C.. C G.. C. G. C C A. T A T. T. A C. C T C.... A G G..... T. C T G nocephala.... C G. T C... C C A. T A... A A C. C T.. T. T A........ C C.. ata T C.. C G T. C G. T C C. T T G... A A C T C T C.. T. A. G A T. T G. C C.. gicauda_defontainei_1................................ A............ icauda_defontainei_2................................ A............ icauda_defontainei_3................................ A............ gicauda_defontainei_4................................ A............ andri_1. C. G C... C G... C....... A.... T C T... A. G.. T....... andri_2. C. G C... C G... C....... A.... T C T... A. G.. T....... andri_3. C T G C... C G... C....... A.... T C T... A. G.. T....... andri_4. C. G C... C G... C....... A.... T C T... A. G.. T....... gicauda_modesta_1........ C.................. C................. gicauda_modesta_2........ C.................. C................. gicauda_modesta_3........ C.................. C................. gicauda_modesta_4........ C.................. C.................

NJ tree of Psittacula based on 868 bp of cytochrome b gene sequences da247_psittacula_longicauda_defontainei_(cyt) 100 88 100 da248_psittacula_longicauda_defontainei_(cyt) da244_psittacula_longicauda_defontainei_(cyt) da246_psittacula_longicauda_defontainei_(cyt) Psittacula_longicauda_longicauda_(cyt) da253_psittacula_longicauda_modesta_(cyt) Natuna is. Sumatra/ Born 88 100 da254_psittacula_longicauda_modesta_(cyt) da255_psittacula_longicauda_modesta_(cyt) da256_psittacula_longicauda_modesta_(cyt) Enggano is. 59 99 100 68 26 da249_psittacula_alexandri_alexandri_(cyt) da250_psittacula_alexandri_alexandi_(cyt) da251_psittacula_alexandri_alexandri_(cyt) da252_psittacula_alexandri_alexandri_(cyt) Psittacula_columboides_(cyt) Java is. 97 74 94 92 96 Psittacula_krameri_krameri_(cyt) Psittacula_roseata_(cyt) Psittacula_cyanocephala_(cyt) Psittacula_finschii_(cyt) Psittacula_himalayana_(cyt) Cacatua_alba_(cyt) Non Indonesia 100 Cacatua_galerita_(cyt) Columba_livia_(cyt) Outgroup species Accipiter_bicolor_(cyt) 0.02

ML tree of Psittacula based on 868- bp of cytochrome b gene sequences Natuna is. Sumatra/ Borneo is. Enggano is. Java is. Non Indonesia Outgroup species

CONCLUSIONS 1. Sequence divergences within Psittacula longicauda : - P. l. modesta vs P. l. longicauca was 2.37 % 1. - P.l. modesta vs P.l. defontainei was 2.16 % - P. l. longicauda vs defontainei was 1. 51 % 2. Mean sequence divergences between species of Psittacula was 5.12 %, ranged from 3.80 % (P. finschii vs P. himalayana) to 10.03 % (P. himalayana vs P. alexandri 3. Genetically, Psittacula from Enggano is (P.l.modesta) was separated from others subspecies of Psittacula longicauda (P. l. longicauda + P. l. defontainei) 4. Phylogenetically, the position of Psittacula from Enggano island (Psittacula longicauda modesta) was sister group of others subspecies of Psittacula longicauda (P. l. longicauda + P. l. defontainei) 5. My opinion, genetically, Psittacula from Enggano (P. l. modesta) is still a subspecies of P. longicauda or was not a new species of Psittacula

Acknowledgements The recent study was supported by Enggano Expedition Project of Indonesian Institute of Sciences, Indonesia.

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