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.

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.

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.

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.

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.

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.

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

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.

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).

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

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.

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).

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.

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.

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%.

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.