GENETIC CHARACTERISTICS OF LONG-TAILED PARAKEETS (Psittacula longicauda modesta Fraser, 1845) FROM ENGGANO ISLAND

ARTICLE HIGLIGHTS
- Genetic variation found in long-tailed parakeets native to Enggano Island
- Genetic information shows population differences among island parakeets
- Enggano Island population displays unique genetic characteristics
- Results support conservation efforts for long-tailed parakeets on Enggano Island

ABSTRACT
The Enggano Long-tailed Parakeet is considered as an agricultural pest by farmers on Enggano Island, leading to a continuing decline in its wild population. This study investigated the genetic characteristics, nucleotide composition, single-nucleotide polymorphisms (SNPs), species-specific barcodes, genetic distances, and phylogenetic relationships of this subspecies using the mitochondrial cytochrome oxidase subunit I (COI) gene. Fourteen blood samples were collected from community-owned captive birds. Total genomic DNA was extracted using the DNeasy® Blood and Tissue Kit following the Qiagen Spin-Column Protocol. DNA amplification was performed by Polymerase Chain Reaction (PCR) using specific primers at the Zoology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Bengkulu. The amplified DNA was separated on a 2.4% agarose gel and visualized under ultraviolet light. Samples displaying clear DNA bands were subsequently sequenced by Apical Scientific. Sequence data were processed and aligned using MEGA version 11.0, resulting in a 665 bp COI fragment suitable for analysis. A total of 19 SNPs and 12 species-specific barcode sites were identified, indicating a distinct genetic signature in the Enggano population. Conserved sites were highly dominant (97.14%), while variable sites accounted for only 2.86%. Nucleotide composition analysis revealed a higher GC content (50.50%) than AT (49.47%). Genetic distance analysis showed 6 – 11.3% divergence between Psittacula longicauda modesta and other Psittacula species. Phylogenetic reconstruction placed all Enggano individuals in a well-supported monophyletic cluster (bootstrap value = 100), confirming their genetic separation from other parakeet species. These findings indicate low genetic diversity within the Enggano population, likely resulting from geographic isolation and small population size. The identified barcode sites underscore the strong diagnostic potential of the COI gene for molecular identification and taxonomic differentiation. Overall, this study establishes the first genetic baseline for Psittacula longicauda modesta, expands molecular resources available for the genus Psittacula, and provides valuable insights for conservation planning and taxonomic assessment of this endemic subspecies.

INTRODUCTION

The Long-tailed Parakeet (Psittacula longicauda) belongs to family Psittacidae (H.B.W., 2022). This species measures approximately 40 – 42 cm in body length and exhibits yellowish-green plumage on the upper and lower parts of the body, with additional coloration in specific regions, including bluish-purple (tail), pink (sides of the head), black (neck), and dark green (around the eyes). It inhabits mangrove forests, primary and secondary forests, forest edges, open woodlands, and oil palm plantations. Its diet consists mainly of various fruits, including berries, figs, and papayas (N.E.S.T., 2023).

In Indonesia, P. longicauda is protected under the Ministrial Regulation of the (Ministry of Environment and Forestry of the Republic of Indonesia, 2018) and is listed as Vulnerable by the IUCN (MyBIS, 2024). Despite its conservation status, the species is permitted for export under the Ministrial Regulation of the (Indonesia, 2018) and is included in CITES Appendix II (Indonesia, 2018). A 2015 expedition to Enggano Island confirmed the presence of the Long-tailed Parakeet (P. longicauda). The expedition identified the Long-tailed Parakeet as one of 14 endemic bird species on Enggano Island (Maryanto et al., n.d.).

The Enggano Long-tailed Parakeet is frequently hunted by local residents of Enggano Island and subsequently sold to other inhabitants or to fishermen from Java. These fishermen transport the birds directly to Java, such as Muara Angke (Jakarta) and Merak (Banten), making illegal capture and trade difficult for authorities to detect (Iqbal et al., 2020). Such activities have significantly contributed to the species population decline. Reduced population size increases the risk of extinction, beginning with the loss of genetic diversity (Willoughby et al., 2015). Therefore, conserving the Long-tailed Parakeet on Enggano Island is essential for maintaining ecological stability. Preliminary genetic studies are needed to assess its genetic diversity as an indicator of population health and persistence.

The COI gene is a widely used molecular marker due to its advantages, including maternal inheritance, absence of recombination, high copy number, and suitability for DNA barcoding(Imtiaz et al., 2017). The COI nucleotide sequence obtained in this study represents a novel genetic contribution, as no genetic data for the Long-tailed Parakeet (P. longicauda) are currently available in GenBank. This research aimed to characterize the species nucleotide characters, composition, codon usage, single nucleotide polymorphisms (SNPs), species-specific barcodes, genetic distances, and phylogenetic relationships.

MATERIALS AND METHODS

Sample Collection

Blood samples from 14 Enggano Long- tailed Parakeets (Psittacula longicauda modesta) were collected from four villages on Enggano Island: Kahyapu, Banjarsari, Kaana, and Meok. Sampling was conducted on 6 June 2023 and 12 June 2023. Blood was drawn from the pectoral vein at the base of the wing using a syringe, with approximately 0.5 – 1 mL collected from each individual and transferred into tubes containing EDTA (Jarulis & RH, 2021). After blood collection, the puncture site was cleaned with 76% alcohol to prevent infection and then covered with a sterile dressing. The 14 parakeets were subsequently given sugar water using a dropper. All blood samples were stored in a freezer at -20 °C (Lecce I et al., 2022). A total of 14 individuals were successfully sampled in this study.

The sample size obtained was influenced by ethical considerations, as Psittacula longicauda modesta is an endemic subspecies on Enggano Island, and excessive handling may increase stress and pose risks to the individuals. Therefore, only birds that could be safely accessed through local owners at the time of sampling were included. Additionally, a sample size of 10 – 20 individuals is commonly considered adequate for preliminary mitochondrial DNA analyses in Psittacidae. Thus, the 14 individuals sampled here are regarded as sufficiently representative for establishing a baseline assessment of the genetic characteristics of this subspecies. After blood collection, all birds were returned to their owners.

This research was conducted under ethical approval from the Research Ethics Committee, Institute for Research and Community Service, University of Bengkulu (Approval No. 107/ KER-LPPM/EC/2025). All sampling procedures followed institutional guidelines for the ethical treatment of animals and were designed to minimize stress to the birds. Molecular analyses were performed at the Zoology and Biotechnology Laboratory, Department of Biology, University of Bengkulu.

Isolation

Total DNA isolation was performed using the Dneasy® Blood Kit and Tissue Kit (catalog number 69504, 50 samples) following a modified Qiagen Spin-column Protocol. The quality and quantity of the isolated DNA were assessed using electrophoresis on a 1.2% agarose gel. The DNA samples were then stored at -20 °C in a freezer before amplification.

Amplification and Sequencing

The amplification of the COI gene was performed using the Polymerase Chain Reaction (PCR) procedure. Two primers, Forward and Reverse, were designed using the Primer 3 software, available online (accessible at http://bio-info.ut.ee/primer3-0.4.0/primer3). One pair of primers was named PLCOIF ( 5’- TATTCTCAACCAACCACAAAGACAT- 3’) and PLCOIR (5’-CAAAGAATCAGAAGAGGTGTTGGTA-3’). This study utilized the COI gene sequence from the mitochondrial genome of Psittacula alexandri (GenBank ID: NC_045378), a taxonomically related species to Psittacula longicauda, as the reference for primer design. The COI sequence used was 1548 bp in length, with a product size of 696 bp. Amplification was conducted using a SimpliAmp™ Thermal Cycler (Applied Biosystems).

The PCR reaction mixture (total volume of 25 μL) consisted of 12.5 μL of GoTaq® Green Master Mix 2X, 0.75 μL of forward primer, 0.75 μL of reverse primer, 1.5 μL of DNA template, and 9.5 μL of Nuclease-Free Water (ddH2O). The PCR cycling conditions for COI gene amplification were as follows: 95 °C for pre-denaturation (2 minutes), 95 °C for denaturation (1 minute), 58 °C for annealing (45 seconds), 72 °C for elongation (1 minute), 72 °C for post-elongation (5 minutes), and 4 °C for cooling. The denaturation-elongation cycle was repeated 30 times. PCR products (3 μL) were visualized using 2.4% agarose gel electrophoresis, and the results were documented using an ultraviolet transilluminator (λ=320 nm). High-quality DNA bands were sent to Apical

Scientific for sequencing.

Data Analysis

The nucleotide sequences obtained from sequencing were edited and aligned using ClustalW in the Molecular Evolutionary Genetics Analysis software (MEGA) version 11 (Tamura et al., 2021). Analyses of genetic characteristics, nucleotide composition, single nucleotide polymorphisms (SNPs), and species barcoding were also performed using MEGA version 11. The COI gene sequences were edited using BIOEDIT software version 7.0.9. All samples were successfully aligned, yielding a final sequence length of 665 bp.

The COI sequences from each individual were compared with sequences available in GenBank using the Basic Local Alignment Search Tool for nucleotides (BLASTn) to assess sequence similarity. Genetic distances among individuals were calculated using the Kimura 2-Parameter (K2P) model. Phylogenetic reconstruction was conducted using the Neighbor-Joining algorithm with the K2P model, and the robustness of the resulting tree was evaluated with 1000 bootstrap replications (Tamura et al., 2021).

Eight COI gene sequences from the genus Psittacula were retrieved from GenBank and used as reference sequences (P. cyanocephala NC_054153; P. cyanocephala MT433093; P. roseata NC_045379; P. roseata MK986661; P. echo OR240864; P. echo OR240830; P. alexandri NC_045378; P. alexandri MK986660). One COI gene sequence from another species in the family Psittacidae, Polytelis alexandrae (JQ175945), was included as the outgroup.

RESULTS AND DISCUSSION

COI Gene Band of the Enggano Long- Tailed Parakeets

This study utilized one of the mitochondrial genes, the COI gene. PCR visualization produced clear and distinct DNA bands Figure 1, indicating that the samples were suitable for sequencing to obtain the nucleotide composition of the COI gene.

Figure 1.Study location in mangroves ecosystem in Sekotong, West Lombok, West Nusa Tenggara, Indonesia

Based on the sequencing results, the lengths of the obtained COI sequences varied, with the forward sequences ranging from 670 to 673 base pairs (bp) and the reverse sequences ranging from 667 to 696 bp. The sequences were subsequently edited using BioEdit and MEGA 11, resulting in a final COI sequence length of 665 bp.

The agarose gel electrophoresis results were analyzed based on the position of the DNA bands after migration during electrophoresis. A DNA ladder was used as a reference to determine the fragment size corresponding to the expected target. The DNA bands displayed in the gel image confirm that the specific primers successfully annealed to the target mitochondrial DNA region. By comparing the band positions with the molecular marker, the length of the amplified fragments was verified.

The COI gene is located between nucleotide positions 5365 and 6912 of the avian mitochondrial genome (Li & Duan, 2019). Based on this information, the 665 bp COI sequence obtained in the present study represents 36.18% of the full COI gene length. This sequence corresponds to the early portion of the COI gene, specifically between nucleotide positions 20 and 715.

Nucleotide Character, Codon Position and Nucleotide Composition

The COI gene length of the Long-tailed Parakeets from Enggano was 665 bp Table 1.

Table 1.Nucleotide character and codon position of Psittacula longicauda modesta COI gene at 665 bp

Character	n	Total Base	Number (%)
Conservative site (%)	14	665	646 (97.14)
Parsimony site (%)	14	665	10 (1.50)
Singleton site (%)	14	665	9 (1.35)
Variable site (%)	14	665	19 (2.86)
Codon position for thymine	14	665
First codon	14	665	12.6
Second codon	14	665	17.3
Third codon	14	665	42
Codon position for cytosine	14	665
First codon	14	665	46
Second codon	14	665	27.4
Third codon	14	665	28.7
Codon position for adenine	14	665
First codon	14	665	36.7
Second codon	14	665	24.9
Third codon	14	665	14.9
Codon position for guanine	14	665
First codon	14	665	4.7
Second codon	14	665	30.3
Third codon	14	665	14.4

This sequence comprised 646 conserved sites (97.14%), 10 parsimony-informative sites (1.50%), and 9 singleton sites (1.35%). Thymine was the most common nucleotide in the third codon position (42%), while cytosine and adenine were predominant in the second codon position (46% and 36.7%, respectively). Guanine also showed a notable frequency in the second codon position (30.3%).

Compared with other species within the order Psittaciformes, this COI gene length is shorter than that of the green parakeet (Psittacara brevipes) from Socorro, which is 701 bp (Martínez-Gómez et al., 2017), but longer than the 625 bp reported for five Accipitridae species (Zein, 2018) and 32 Ardeidae species (Huang et al., 2016). The length obtained in this study falls within the typical range used for species identification, making it a valuable reference for Psittacula longicauda modesta.

Nucleotide composition analysis revealed that certain nucleotides dominate specific codon positions, such as thymine in the third position, cytosine in the first, and guanine in the second. These patterns may reflect mutation pressure and natural selection influencing codon usage. Previous studies of mitochondrial COI gene composition in various taxa have reported a predominance of cytosine and adenine exceeding 35% (Uddin et al., 2018). In this study, cytosine exhibited the highest proportion at 34.10%, while guanine showed the lowest at 16.50%; the GC content was 50.50%, and the AT content was 49.47% Table 2.

Table 2.Nucleotide composition of Psittacula longicauda modesta COI gene at 665 bp

Sample code	Thymine (T)	Cytosine (C)	Adenine (A)	Guanine (G)	GC content (%)	AT content (%)	Total base
PLCOI1	23.90	34.00	25.70	16.40	50.40	49.60	665
PLCOI2	23.90	33.80	25.70	16.50	50.30	49.60	665
PLCOI3	24.10	34.00	25.40	16.50	50.50	49.50	665
PLCOI4	23.80	34.10	25.60	16.50	50.60	49.40	665
PLCOI5	23.90	34.10	25.60	16.40	50.50	49.50	665
PLCOI6	23.90	34.10	25.40	16.50	50.60	49.30	665
PLCOI7	23.90	34.10	25.40	16.50	50.60	49.30	665
PLCOI8	24.10	34.00	25.60	16.40	50.40	49.70	665
PLCOI9	24.10	34.10	25.40	16.40	50.50	49.50	665
PLCOI10	24.10	33.80	25.70	16.40	50.20	49.80	665
PLCOI11	23.90	34.30	25.40	16.40	50.70	49.30	665
PLCOI12	24.10	34.00	25.40	16.50	50.50	49.50	665
PLCOI13	23.90	34.10	25.40	16.50	50.60	49.30	665
PLCOI14	23.90	34.10	25.40	16.50	50.60	49.30	665
Average	24.00	34.10	25.50	16.50	50.50	49.47	665

This composition differs from several species within the same family (Psittacidae), where AT content commonly exceeds GC content. Examples include the nanday parakeet (Aratinga nenday) (AT: 54.03%, GC: 45.97%), Fischer’s lovebird (Agapornis fischeri) (AT: 51.1%, GC: 48.9%), and Lilian’s lovebird (Agapornis lilianae) (AT: 51.16%, GC: 48.84%) (Liu et al., 2019);(Liu et al., 2019); (Chen et al., 2019). Similarly, the Sandpiper family (Scolopacidae) exhibits a lower GC content (47.32%) relative to AT content (52.68%) (Huang & Tu, 2016).

Identification of Species Based on BLASTn (Nucleotide BLAST)

The identified species from the BLASTn results represents sequences used as references for comparison with the tested samples, originating from China Table 3.

Table 3.Species identification result based on BLASTn System database

Species and Sample Code	Identified Species (Top 3)	BLASTnAccess code	Percent Identity (%)	Query cover (%)	Country of origin
P. longicauda 1 (PLCOI1)	Psittacula alexandri	NC_045378.1	94.84	100	Dehong Wildlife Rescue Centre, China
P. longicauda 2 (PLCOI2)	Psittacula alexandri	NC_045378.1	94.20	98	Dehong Wildlife Rescue Centre, China
P. longicauda 3 (PLCOI3)	Psittacula alexandri	NC_045378.1	94.69	100	Dehong Wildlife Rescue Centre, China
P. longicauda 4 (PLCOI4)	Psittacula alexandri	NC_045378.1	94.26	100	Dehong Wildlife Rescue Centre, China
P. longicauda 5 (PLCOI5)	Psittacula alexandri	NC_045378.1	94.27	100	Dehong Wildlife Rescue Centre, China
P. longicauda 6 (PLCOI6)	Psittacula alexandri	NC_045378.1	94.56	100	Dehong Wildlife Rescue Centre, China
P. longicauda 7 (PLCOI7)	Psittacula alexandri	NC_045378.1	94.43	100	Dehong Wildlife Rescue Centre, China
P. longicauda 8 (PLCOI8)	Psittacula alexandri	NC_045378.1	94.42	100	Dehong Wildlife Rescue Centre, China
P. longicauda 9 (PLCOI9)	Psittacula alexandri	NC_045378.1	94.70	100	Dehong Wildlife Rescue Centre, China
P. longicauda 10 (PLCOI10)	Psittacula alexandri	NC_045378.1	94.70	100	Dehong Wildlife Rescue Centre, China
P. longicauda 11 (PLCOI11)	Psittacula alexandri	NC_045378.1	94.13	100	Dehong Wildlife Rescue Centre, China
P. longicauda 12 (PLCOI12)	Psittacula alexandri	NC_045378.1	94.44	100	Dehong Wildlife Rescue Centre, China
P. longicauda 13 (PLCOI13)	Psittacula alexandri	NC_045378.1	94.20	98	Dehong Wildlife Rescue Centre, China
P. longicauda 14 (PLCOI14)	Psittacula alexandri	NC_045378.1	94.29	100	Dehong Wildlife Rescue Centre, China

P. longicauda and P. alexandri, suggesting potential co-evolutionary patterns within neighboring Asian regions (Juniper & Parr, 1998). The BLASTn data further indicates that the reference specimens used for comparison are sourced from China, reflecting the international scope of the database, which comprises genetic information from diverse regions (Altschul et al., 1990).

It is important to emphasize that although the identified closest matches originate from these countries, this does not imply that the original specimens of P. longicauda are from those regions. Instead, they represent the available reference sequences within global databases. This identification highlights significant phylogenetic affinities between P. longicauda and P. alexandri, contributing to a deeper understanding of potential evolutionary relationships among Asian parakeet species.

Moreover, the geographic distribution of reference sequences underscores the importance of using globally curated databases to obtain accurate and meaningful results. Notably, no COI gene sequences of P. longicauda are currently available in GenBank, making the present sequence the first of its kind to be reported globally. This confirms that the analyzed sequence corresponds to P. longicauda, while sharing close genetic similarity with P. alexandri.

Single Nucleotide Polymorphism

A total of 19 single nucleotide polymorphisms (SNPs) were identified in the COI gene sequences of Long-tailed Parakeets from Enggano, occurring at nucleotide sites 5 – 12, 92, 331, and 654 – 663 Table 4 & 5. (Cendron et al., 2020);(Kyrkjeeide et al., 2020).

Table 4.Intrapopulation single nucleotide polymorphisms (SNPs) of Psittacula longicauda modesta based on the 665 bp COI gene sequence (nucleotide sites: 5 – 331)

Species	Sample code	Nucleotide site
		5	6	7	8	10	11	12	92	331
Psittacula longicauda modesta	PLCOI1K	T	A	T	A	G	A	A	C	A
P. l modesta	PLCOI2K	G	T	.	C	.	.	.	A	.
P. l modesta	PLCOI3K	.	.	.	T	.	.	.	.	G
P. l modesta	PLCOI4K	.	.	.	C	.	.	.	.	G
P. l modesta	PLCOI5K	.	.	.	C	C	T	G	A	.
P. l modesta	PLCOI6K	.	.	.	C	.	.	.	.	G
P. l modesta	PLCOI7K	.	.	.	C	.	.	.	.	G
P. l modesta	PLCOI8K	.	T	.	C	.	.	.	A	.
P. l modesta	PLCOI9K	.	T	.	C	.	.	.	.	.
P. l modesta	PLCOI10K	.	T	.	.	.	.	.	A	.
P. l modesta	PLCOI11KN	.	T	.	C	.	.	.	.	.
P. l modesta	PLCOI12KN	.	T	.	C	.	.	.	A	.
P. l modesta	PLCOI13M	.	.	.	T	.	.	.	.	G
P. l modesta	PLCOI14M	.	.	.	C	.	.	.	.	G

Table 5.Intrapopulation single nucleotide polymorphisms (SNPs) of Psittacula longicauda modesta based on the 665 bp COI gene sequence (nucleotide sites: 654 – 663)

Species	Sample code	Nucleotide site
		654	655	656	657	658	659	660	661	662	663
Psittacula longicauda modesta	PLCOI2K	T	C	T	G	A	A	T	T	T	T
P. l modesta	PLCOI2K	C	T	G	A	.	.	.	.	T	.
P. l modesta	PLCOI3K	.	.	.	.	.	.	.	.	T	C
P. l modesta	PLCOI4K	.	.	.	.	.	.	A	A	T	C
P. l modesta	PLCOI5K	.	.	.	.	.	.	.	.	.	.
P. l modesta	PLCOI6K	.	.	.	.	.	.	.	.	T	C
P. l modesta	PLCOI7K	.	.	.	.	.	.	.	.	T	C
P. l modesta	PLCOI8K	.	.	.	.	.	.	.	.	.	.
P. l modesta	PLCOI9K	.	.	.	.	.	.	.	.	.	.
P. l modesta	PLCOI10K	.	.	.	.	.	.	.	.	.	.
P. l modesta	PLCOI11KN	.	.	G	A	.	T	.	C	.	.
P. l modesta	PLCOI12KN	.	.	.	.	G	.	.	.	.	.
P. l modesta	PLCOI13M	.	T	G	A	.	T	.	.	.	C
P. l modesta	PLCOI14M	.	.	.	.	.	.	.	.		C

Several nucleotide sites were conserved across all samples, indicated by dot symbols (.), suggesting functional or evolutionary constraints at these positions. SNP mapping, therefore, provides important insights into genetic variation and evolutionary processes within populations (JDRDS et al., 2017). The concentration of SNPs in the terminal regions of the COI gene suggests that these regions may be less conserved than the central portion of the gene. Reduced conservation allows the accumulation of mutations, particularly synonymous substitutions, without affecting the function of the encoded protein. As noted These SNPs were predominantly located toward the terminal regions of the COI gene. SNPs are important markers for monitoring genetic diversity, as low levels of variation may increase population vulnerability to threats such as disease outbreaks or environmental change. Consequently, SNP characterization in species such as Psittacula longicauda is valuable for assessing conservation status and understanding population structure (Xia et al., 2019). However, SNP markers generally exhibit lower polymorphism compared to simple sequence repeat (SSR) markers due to their bi- or triallelic nature by (Castle, 2011), mutations tend to accumulate in regions of the genome that are under weaker functional constraints, whereas highly conserved regions generally exhibit lower levels of genetic variation.

Species Barcode

The COI gene has been widely applied as a DNA barcode for species discrimination. In the present study, analysis of Long-tailed Parakeet (Psittacula longicauda) samples against various GenBank reference sequences identified twelve barcoding sites at positions 94, 97, 136, 226, 316, 451, 487, 502, 547, 556, 559, and 665 Table 6.

Table 6.Species barcode of Psittacula longicauda modesta COI gene at 665 bp

Species	Nucleotide Sequence
	94	97	136	226	316	451	487	502	547	556	559	665
Psittacula longicauda 1 (PLCOI1_Enggano_ Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 2 (PLCOI2_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 3 (PLCOI3_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 4 (PLCOI4_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 5 (PLCOI5_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 6 (PLCOI6_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 7 (PLCOI7_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 8 (PLCOI8_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 9 (PLCOI9_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 10 (PLCOI10_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 11 (PLCOI11_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 12 (PLCOI12_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 13 (PLCOI13_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula longicauda 14 (PLCOI14_Enggano_Indonesia)	G	G	T	G	G	T	T	T	C	G	C	T
Psittacula alexandri NC_045378 China	A	A	C	A	A	C	C	C	A	A	T	G
Psittacula alexandri MK986660 China	A	A	C	A	A	C	C	C	A	A	T	G
Psittacula echo OR240864 India	A	A	C	A	A	C	C	C	A	A	T	G
Psittacula echo OR240830 India	A	A	C	A	A	C	C	C	A	A	T	G
Psittacula cyanocephala NC_054153 India	A	A	C	A	A	C	C	C	A	A	T	G
Psittacula cyanocephala MT433093 India	A	A	C	A	A	C	C	C	A	A	T	G
Psittacula roseata NC_045379 China	A	A	C	A	A	C	C	C	A	A	T	G
Psittacula roseata MK986661 China	A	A	C	A	A	C	C	C	A	A	T	G

These sites function as diagnostic markers that distinguish P. longicauda from Enggano from other Psittacula species worldwide, with guanine and thymine being the predominant nucleotides.

Notably, nucleotide substitutions at positions 94, 136, 226, and 316 differentiated P. longicauda from Indonesia from P. alexandri, P. echo, P. cyanocephala, and P. roseata originating from China and India. These nucleotide variations facilitated accurate species identification and were consistent with conserved mitochondrial regions commonly employed in DNA barcoding studies. Furthermore, DNA barcoding supports not only reliable species identification (Dimitriou et al., 2017) but also plays a crucial role in monitoring illegal wildlife trade and conserving endangered species (Gonçalves et al., 2015);(Tizard et al., 2019).

The Enggano population of P. longicauda exhibits unique genetic characteristics and may require targeted conservation efforts, as DNA barcoding can reveal genetic variations indicative of declining or threatened species (Li et al., 2014);(Li & Duan, 2019).

Based on the COI gene, the intrapopulation genetic distance of Psittacula longicauda modesta from Enggano (n = 14) showed a minimum value of 0.0%, a maximum of 1.8%, and an average of 0.9% Table 7. The minimum value of 0.0% indicates the presence of individuals with identical COI nucleotide sequences. Meanwhile, the maximum value of 1.8% is considered very low, suggesting minimal nucleotide variation within the population. These results indicate that the Enggano population is genetically homogeneous.

In contrast, the intragenus genetic distance ranged from 6.0% to 11.3%, with an average of 8.3%. Furthermore, the intrafamily genetic distance showed a minimum value of 10.4%, a maximum of 11.3%, and an average of 10.6%. Similar genetic distance patterns have been reported in previous studies, including those on the order Anseriformes (Sun et al., 2017). Based on interspecific genetic distance values, the species within the genus Psittacula most closely related to P. longicauda modesta is P. alexandri, while the most distantly related taxa are P. echo and Polytelis alexandrae. The observed interspecific genetic distances in this study meet commonly accepted criteria for species delineation Table 7.

The average genetic distance between Long- tailed Parakeet individuals (ingroup) and the pink- necked parakeet (Polytelis alexandrae), which belongs to the same family (Psittacidae), was 0.106 (10.6%) Table 7. This value is consistent with the findings of Hebert et al. (2004), who reported an average interspecific genetic distance of 7.93% and an average intraspecific genetic distance of 0.43%.

Table 7.Percentages of genetic distance intrapopulation (Enggano), intragenus (Psittacula spp.) and intrafamily (outgroup)

Genetic distance	Maximum (%)	Minimum (%)	Average (%)
Intrapopulation (Enggano)	1.8	0.0	0.9
Intragenus (Psittacula spp)	11.3	6.0	8.3
Intrafamily (outgroup)	11.3	10.4	10.7
Genetic distance between P.l and P.a	6.9	6	6.3
Genetic distance between P.l and P.e	11.3	10.1	10.5
Genetic distance between P.l and P.c	9.4	8.4	8.7
Genetic distance between P.l and P.r	8.5	7.5	7.8
Genetic distance between P.l and P.al	11.3	10.4	10.6

Genetic Distance

The intrapopulation and intragenus genetic distances of the Long-tailed Parakeet are presented in Table 7.

Phylogenetic

Phylogenetic reconstruction was performed using the Neighbor-Joining algorithm with the Kimura 2-parameter model and 1,000 bootstrap replicates, revealing a clear separation between the Long-tailed Parakeet and other Psittacula species Figure 2.

Figure 2.

The outgroup species, Polytelis alexandrae from Australia, did not cluster within any of the three groups (groups 1, 2, or 3) of the genus Psittacula. The phylogenetic tree further showed that the Enggano Long-tailed Parakeet population is divided into three nodes with bootstrap support values of 70, 32, and 38, respectively. Bootstrap values are commonly used to evaluate the reliability of clades in phylogenetic analyses (Horiike, 2016).

In addition, Psittacula alexandri appeared to be the species most closely related to the Enggano Long-tailed Parakeet population and was clearly separated from other Psittacula species within group three. This finding is consistent with (Braun et al., 2019), who reported that P. longicauda is the sister taxon to P. alexandri and P. derbiana. The COI-based phylogenetic tree constructed in this study provided insights into intraspecific differentiation among parakeet populations. Phylogenetic inference using COI gene sequences is considered reliable for resolving evolutionary relationships at the species level. The branches of the tree represent evolutionary relationships by tracing lineages back to their most recent common ancestors (Patwardhan et al., 2014); (Chen & Field, 2020);(Olsson & Alström, 2020).

This study successfully revealed previously unavailable genetic information on the Long- tailed Parakeet population from Enggano. The COI gene sequence identified in this study exhibited 12 nucleotide differences between the Enggano population and four other species within the genus Psittacula, representing a novel finding. Therefore, COI gene sequence may serve as an effective molecular marker for species identification. Moreover, this genetic information has practical applications for monitoring the trade of Long-tailed Parakeets, particularly for tracing the geographic origin of confiscated or captive individuals.

Low Genetic Diversity and Conservation Implications

The low genetic diversity observed in Psittacula longicauda modesta is reflected by the very small intrapopulation genetic distance (0.0 – 1.8%), the high proportion of conserved nucleotide sites (97.14%), and the limited number of SNPs detected in the COI gene. Such patterns are commonly reported in populations isolated on small islands, where restricted habitat size, dispersal barriers, and limited gene flow from external populations reduce the introduction of new haplotypes (Willoughby et al., 2015); (Vijay et al., 2017).

Similar conditions have been documented in other island-endemic bird species, such as the Norfolk parakeet Cyanoramphus cookii, which has experienced multiple population bottlenecks resulting in reduced genetic diversity and increased inbreeding due to small population size and long-term isolation (Genetic diversity and inbreeding in an endangered island-dwelling parrot population following repeated population bottlenecks, 2024). These findings suggest that island isolation and restricted gene flow are general drivers of declining genetic diversity in insular bird populations.

Several biological, ecological, and anthropogenic factors may contribute to the low genetic diversity observed in P. l. modesta. Enggano Island has a limited habitat area, which inherently constrains population size. In addition, pressures such as illegal capture, habitat modification, and human disturbance may further reduce effective population size and potentially induce genetic bottlenecks. Reduced genetic diversity associated with such factors has also been reported in other island-endemic birds, including the Seychelles Magpie-Robin, which exhibits extremely low mitochondrial variation due to a combination of island isolation and historical population declines (Cavill et al., 2022), and the Grenada Dove Leptotila wellsi, which shows low mtDNA diversity based on non-invasive sampling approaches (Peters et al., 2024). The high proportion of conserved sites in the COI sequences of P. l. modesta may indicate long-term population stability without the introduction of new genetic variation, or the influence of purifying selection acting to maintain functional mitochondrial regions.

From a conservation perspective, low genetic diversity has several important implications. Populations with limited genetic variation are more susceptible to inbreeding depression, which can negatively affect fitness, reproductive success, and resilience to environmental stress. Studies on C. cookii and L. wellsi demonstrate that island- endemic birds with low genetic diversity often exhibit reduced adaptive potential and heightened vulnerability to environmental change or emerging diseases. For island-endemic taxa such as P. l. modesta, these factors may collectively increase the long-term risk of population decline or eventual extinction if left unmanaged.

Taken together, these findings highlight the need for comprehensive conservation strategies, including habitat protection, strict enforcement against illegal capture and trade, and long-term monitoring of wild populations. Evidence from other island bird species emphasizes the importance of incorporating population genetic considerations into conservation planning, including the potential need to enhance gene flow or implement genetic management strategies to mitigate inbreeding.

CONCLUSION

This study provided the first genetic baseline for Psittacula longicauda modesta from Enggano Island through the characterization of a 665 bp COI gene fragment. The nucleotide character, nucleotide composition, nucleotide variation, and identified SNPs indicate that the Enggano population possesses COI gene characteristics unique to this island, while exhibiting low genetic diversity. Such reduced variability may reflect evolutionary pressures associated with geographic isolation, small population size, and habitat disturbance linked to human activities. These factors warrant attention in conservation planning, particularly as the species is increasingly regarded as an agricultural pest and its wild population continues to decline.

The identification of twelve species-specific barcode sites distinguishing P. l. modesta from other Psittacula species demonstrated the strong diagnostic utility of the COI gene and supported the application of COI gene as an effective marker for molecular identification, taxonomic refinement, and wildlife trade monitoring. Genetic distance analyses were consistent with taxonomic boundaries, together with the distinct clustering observed in the phylogenetic reconstruction, which further confirm that the Enggano population represents a discrete evolutionary lineage, likely shaped by long-term island isolation.

Overall, the findings of this study expand the available genetic resources for the genus Psittacula and provide a valuable foundation for future work on island evolution, population genetics, and evidence-based conservation strategies. The COI dataset generated in this study can contribute to clarifying the taxonomic status of P. l. modesta and informing targeted management efforts for this endemic Enggano population.

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