Diversity of The Ornate Lorikeet ( Trichoglossus ornatus ) Birds Based on Mitochondrial DNA Protein Coding Gene

Ornate lorikeet (Trichoglossus ornatus) is an endemic bird in Sulawesi. Endemism is one of the factors in declining bird’s population. In the case of the birds conservation programme, information about gene diversity is important for basic strategy. Mitochondrial DNA of animals consists of protein coding genes including ND2 gene. This study informs diversity of the Ornate Lorikeet (Trichoglossus ornatus) birds based on DNA sequences of ND2 gene. DNA total was extracted from blood samples of 21 birds. PCR (Polymerase Chain Reaction) was performed and successfully amplified a single DNA fragment of ND2 gene for all birds. DNA fragments were sequenced and totally 997 base pairs were analyzed. NJ tree was constructed using MEGA5. All DNA sequence data showed that between the birds there were 20 polymorphic (segregating) sites with mean genetic distance was 0.004 ± 0.002 (ranged from 0,000 – 0,008), and had 17 sequence haplotypes (HTor1HTo17). Haplotype diversity (Hd) was 0.967 ± 0.30387 and nucleotide diversity (Pi) was 0.00439 ± 0.0012. Genetic diversity information could be potential relevance to the breeding management for conservation of the birds.

ND2 gene has been widely used as a genetic marker for uncovering genetic diversity of birds and for the study of phylogeny in vertebrates including birds (Townsend et al., 2007;Verónica et al. 2009, Mu et al., 2012).This paper takes into account the diversity of Ornate lorikeet (T.ornatus) birds which are endemic in Sulawesi Island based on DNA sequences of the mitochondrial protein coding ND2 gene.This genetic diversity information of T. ornatus can be used as basic information for its breeding strategies and conservation action plan either in-situ or ex-situ to keep the population in order to be sustainable.

METHODS
This study was conducted on 2015 and used blood samples from 21 Ornate lorikeets (T.ornatus) an endemic bird of Sulawesi.The samples were genetic material collection of Genetic Laboratory of Zoology Division, Research Center for Biology -LIPI.Each blood sample was preserved in absolute ethanol (96%) and kept in refrigerator at 4 ° C. The DNA total was extracted from about 10 mg of blood using a QIAGEN Mini Kit and follows the procedures provided by manufacture.
The quality and quantity of DNA was checked in agarose gel through the electrophoresis process then visualized using photos under UV light.The extracted DNA solution was then diluted to a certain concentration for further use in PCR analysis (Polymerase Chain Reaction).DNA fragments of the ND2 gene from each bird were amplified through the PCR process using a pair of DNA nucleotide primer L5216 and H6313 under PCR conditions following Sorenson et al. (1999).Each PCR product then sequenced.DNA sequencing process is done by using the services of First Base company.
All DNA sequence data from all birds studied, then aligned together in a conventional alignment process using eyes on MEGA5 (software).Nucleotide composition, nucleotide variation including number and their position, nucleotide sites, and parsimony information sites were calculated using MEGA5.The number of sequence haplotypes (H), haplotype diversity (Hd) and nucleotide diversity (Pi) were calculated using the DnSP software.A neighbor-joining (NJ) analysis was then performed to create a NJ tree and to know the groupings of the individual birds.The grouping confidence value (bootstrap value) is obtained by 1000 repetitions on MEGA 5. Trichoglossus haematodus , Psittaculirostris edwardsii, and P. desmarestii were ougroup species.

INTRODUCTION
Ornate lorikeet (Trichoglossus ornatus) bird is one of parrot birds and belongs to the family Psittacidae (Forshaw andCooper, 1989 Juniper andParr, 1998).The bird is endemic in the Sulawesi island of Indonesia.They spread in Sulawesi and surrounding islands including the islands of Togian, Peleng, Banggai and Tukang Besi (Juniper and Parr, 1998;Forshaw, 2010).
Its endemic presence is in an island and its capture is for trade and as a pet, as well as habitat destruction and fragmented habitats may be the cause of the decline of its population in nature.Currently, this bird population is declining (BirdLife International, 2016) and the conservation status by the IUCN Red Least is Least Concern ver 3.1 (IUCN, 2017) and included in CITES Appendix II.
The population decline of these birds can lead to a decrease in its genetic diversity (Rivers et al., 2014).Genetic diversity is one of the three levels of biodiversity (genetics, species, and ecosystems) required in organism conservation (Sharma and Sharma, 2013) and as a basic or essential for the survival and adaptability of organisms to environmental change (Boettcher et al., 2010;Pauls et al., 2013).The effects of declining of genetic diversity or low genetic variation in wildlife and livestock can affect to a decrease in the ability of birds to adapt to their environment (Barrett and Schluter, 2008) and to the disease attacks (Toro et al., 2011), so that its ability to survive becomes decreasing.The inability to adapt to changing conditions greatly increases the risk of extinction (Riversa et al., 2014).Genetic variation and diversity within a population explain the presence of different alleles in the population for specific genes.The presence of genetic variation indicates that individuals of a population have varied alleles, meaning that individuals can differ in genotypes (Toro and Caballero, 2005), so that in conservation or breeding selection, variations of individuals is more concerned (Toro et al., 2011).Perhaps the most important is how to make local breeds more able to breed through conservation measures and how to prioritize conservation.In both cases, the assessment and management of genetic diversity are key aspects (Hiemstra et al., 2010;Soini et al., 2012;Boettcher et al., 2015).
The use of genetic markers is necessary and important in estimating genetic diversity, either between individuals or within species (Avise, 2004).Mitochondrial DNA is one of the genetic markers (Arif and Khan, 2009;Barker et al., 2012) in which there is a ND2 protein coding gene.The

RESULTS AND DISSCUSION
The PCR process successfully amplified the single DNA fragment of the ND2 gene which the length was 1040 base pairs of all 21 samples of Trichoglossus ornatus.DNA sequence data obtained from each sample varied in length, therefore only 997 base pairs were used for genetic diversity analysis.There were no insertion, deletion, and stop codon in all sequence data analyzed.The absence of a stop codon indicates that the DNA sequences are from the protein coding gene (Li and Gaur, 1991).DNA is extracted from blood samples, the consequence is that DNA sequence data can be contaminated by nuclear pseudogene (Sorenson and Fleicher, 1996).The absence of insertion and deletion indicated that there was no pseudogene contamination in all T. ornatus sequence data analyzed in this study.This means that the amplified DNA fragment is the true target fragment of the ND2 gene.
DNA sequence contained AT-rich; where the composition of A + T (53.90%) was higher than C + G (46.10%) (Figure 1) as found in birds and other vertebrates (Udin et al., 2015).The composition of the nucleotide base (A, C, G, T) in the total codon positions of the DNA sequence of the ND2 in T. ornatus studied were higest in cytosine (T), followed by adenine (A), thymine (T), and guanine (G) respectively (Figure 1).The DNA sequences produced the lowest of guanine (G) as found in other vertebrates including bird, fish, and mammal.Cytosine (C) was highest as it occurs in others aves and fish (Udin et al., 2015).These results are also consistent with those of the Javan parakeets (Psittacula alexandri alexandri) (Astuti, 2017) and Litlle spiderhunter (Arachnothera longirostra) of passerine birds (Moyle et al., 2011;Prijono et al., 2017).The number of nucleotide variable and parsimony sites were most widely at the third codon position, followed by the first codon position and second codon position, respectively (Table 1).Most variable in the third codon position because the substitution of the base nucleotide is synonymous and has the same amino acids (Li and Gaur, 1991).
The number of synonymous polymorphic sites is 15 sites, more abundant than non synonymous polymorphic (5 sites) (Table 5), since substitution of nucleotide bases is easier to occur than non-synonymous one.In synonymous polymorphic, although nucleotide base substitution and nucleotide base component of amino acids are different but the type or name of amino acids is the same.The substitution of nucleotide base is more common in the position of the third codon than in the first codon position or in the second codon (Li and Gaur, 1991).2).Transitional substitution was more abundant than transversional substitution.This fenomena is quite possible occured because transitional substitution is nucleotide substitutions of purine bases to other purine bases or from pyrimidine bases to other pyrimidine bases.
Transversional substitution occurs from the purine bases to the pyrimidine or from pyrimidine to purin bases.Therefore, transitional substitutions are more frequent or easier to occur than trasversional substitutions (Li and Gaur, 1991), and the nucleotide variations were mainly related to transitions (Mu et al., 2012).Transitional substution occurring in T. ornatus birds in recent study of 4, ie 2 substitutions from C to T and the otherwise C to T. While, the transversional substitution was only 1 that is from G to C.
Table 3. Variations of nucleotide bases and haplotypes in 997 bp of ND2 gene at Ornate lorikeets (T.ornatus) The average number of nucleotide differences within 21 ND2 gene sequence data was 4 and the value of Tajima test (D) was 0.79609 with P> 0.01 not significant (Table 5).
Haplotype diversity of T. ornatus was high (0.967 ± 0.30387) and nucleotide diversity was 0.00439 ± 0.00125 (Table 5).The percentage of haplotype diversity was relatively high and nucleotide diversity was relatively low.This condition was also shown in the endemic populations of European starling (Sturnus vulgaris granti) birds which had high haplotype diversities ranged from 0.767 to 0.900 (Veronica et al., 2009), and was also in the mitochondrial genes in other bird species such as Cacatua alba and C. moluccensis (Astuti, 2011), Pterodroma magentae (Taiko) (Lowrence et al., 2008), and other vertebrate species such as Lontra felina (Valqui et al., 2010).High haploty- The genetic distance (genetic divergence) between individuals (intraspecific distance) based on ND2 on T. ornatus birds studied ranged from 0.001% to 0.008% (Table 5).This value indicates that the individual birds are still in the same bird species.In other bird species of passerine birds the divergence of ND2 sequence between populations in the different islands was 0.7% (Verónica et al., 2009) lower than divergence in one island (1.2%) % (Townsend et al., 2007).The genetic distance of ND2 between individuals (intraspecific) in Arachnothera longirostra birds population were ranged from 0.25% to 0.76 (Java) and 0.10% (Sumatra) (Priyono et al., 2017), in Hirundo rustica (European swallow) was 0.25 % -1.6% (Dor et al., 2010), and 0.24% -0.58 % were in Psittacula alexandri alexandri (Javan parakeets) (Astuti, 2017).NJ phylogenetic tree presents the groupings of T.ornatus birds.Individual bird's number 7, 13, 15, and 17 that shared the HTo7ND2 haplotype (haplotype 7) was clustered together with 0% genetic distance.Individual 12 and 14 who have HTo12ND2 haplotype (haplotype 12) clustered together.All twenty one (21) T. ornatus birds grouped together and separated from T. haematodus by a genetic distance (divergence) of 3.1 ± 0.05 % and supported by 100% bootstrap value.The value of interspecies divergence in birds varies depending on the bird species and its speciation process.For example, Hirundo birds interspecies divergence ranged from 0.7% to 11.6% (Dor et al. 2010), and 8.3% ~ 26.7% between different fish species (Mu et al, 2012).T.ornatus and T. haematodus are separated from outgroup species (Psitacula edwarsii and P. desmarestii) with 100% bootstrap value.
The results of this study reveal the genetic diversity of birds T. ornatus.This is expected to be useful as basic information for breeding programs and strategies and action plans in its conservation programs both of in-situ and ex-situ.The genetic diversity of T. ornatus birds in this study is relatively high.It provides promising hopes and prospects in its conservation so that it can be used sustainably.

CONCLUSION
The haplotype diversity among the 21 birds studied were relatively high indicating that genetic diversity among individuals of the Trichoglossus ornatus bird was high as well, even though this bird is an endemic bird.High genetic diversity gives a good prospect for conservation of this bird.

Figure 1 .
Figure 1 .Graph of nucleotide base composition of 977 bp (total codon position) of the ND2 gene in Ornate lorikeet (Trichoglossus ornatus).

Figure 2 .
Figure 2. Graph of nucleotide base composition (%) at each codon position in the 997 bp of ND2 gene sequence of the Ornate lorikeet (Trichoglossus ornatus)

Table 1 .
The number of variable sites and the parsimony informative sites (PI) in each codon position of the ND2 gene of Ornate lorikeet (Tricho-= 5, singleton =4) 323 332 Note: PI = Parsimony Informative The total number of substitutions (varia-tions) of nucleotide occurred at 20 sites; consisting of 15 transitional sites and 5 transversional sites (Table

Table 2 .
(Ran et al., 2008 )n and transversion substitutions and its ratio at each codon positionWu and Fang, 2005)and apparently low of nucleotide diversity values in T. ornatus studied may be due to the birds having low population histories, such as those occurring in Muntiacus crinifrons (Black muntjac)(Ran et al., 2008 ).