Phylogenetic analysis and sex identification of captive African grey parrot (Psittacus erithacus) in Uganda and Japan

Abstract

The African grey parrot (Psittacus erithacus) is endangered due to habitat loss, poaching, and illegal trade. In 2022, Chubu University and the Uganda Wildlife Education Centre–Conservation Area (UWEC-CA) have initiated an ex-situ conservation project for rehabilitation and future reintroduction. Noninvasively genetic sampling was conducted using feces from four parrots at UWEC-CA and naturally shed feathers from 22 parrots at 11 Japanese zoos. Non-invasive genetic sampling enabled reliable sex identification and mitochondrial sequencing of parrots in Uganda and Japan. Phylogenetic analysis revealed two major clades, including a Gabon-associated lineage within which all UWEC-CA parrots clustered, providing baseline genetic context relevant to ex-situ conservation management and evidence-based reintroduction.

The African grey parrot (Psittacus erithacus) is a large parrot species distributed across forested regions of Central and Eastern Africa (Fig. 1). In the wild, the species primarily inhabit lowland tropical rain forests, forest edges, and forest savanna mosaics across West and Central Africa, where it forms cohesive social flocks and relies on large tree cavities for nesting. Due to its high cognitive ability and exceptional capacity for vocal mimicry, it has long been highly valued in the international pet trade. Intensive harvesting for trade, together with ongoing habitat loss, has caused severe population declines, leading to its current classification as Endangered on the IUCN Red List and its inclusion in Appendix I of Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) [3]. Despite these regulations, illegal trade continues, and many parrots are confiscated and relocated to zoos or wildlife management facilities across the world without proper documentation of their geographic origin and breeding history.

Fig. 1.

Distribution of the Grey Parrot (Psittacus erithacus). The map was redrawn based on the IUCN Red List (2023) at https://www.iucnredlist.org/ja/search/map?taxonomies=22673063&searchType=species (accessed 27 May 2026).

For effective ex-situ conservation, captive breeding, and reintroduction programs, the availability of fundamental genetic information—such as; sex, population of origin, and genetic diversity—is essential. This need has become increasingly urgent as wild populations continue to decline and as confiscated individuals are frequently transferred to conservation facilities without reliable background records or breeding history. This requirement is particularly critical for African grey parrots, which exhibit no obvious sexual dimorphism and frequently arrive at conservation facilities without documentation of their provenance.

Conventional genetic analyses using blood and tissue samples are invasive and undesirable for rescued or unaccustomed individuals like parrots kept at Uganda Wildlife Education Centre-Conservation Area (UWEC-CA) because of animal welfare concerns. Such methodology is also unpractical for the wild parrots with the same reason. Therefore, the development and validation of noninvasive genetic methods using naturally shed feathers and fecal samples represent an important methodological advance [2, 5]

Since 2022, Chubu University and the UWEC-CA have collaborated on African grey parrot conservation project supported by the Japan International Cooperation Agency (JICA), with the main aim of establishing practical ex-situ conservation protocols. Our study demonstrates that DNA extracted from feces and naturally shed feathers can be used for reliable sex determination using the chromo-helicase DNA-binding (CHD) gene, and mitochondrial DNA–based phylogenetic analysis. This non-invasive approach provides baseline genetic data for African grey parrots housed at UWEC-CA and at Japanese zoos with minimal stress to animals. Our results highlight the utility of noninvasive genetic sampling as a powerful tool for the genetic management of confiscated, captive, and endangered avian species in general.

In this study, our ultimate goal was to support evidence-based management and the potential reintroduction of confiscated African grey parrots by characterizing the geographic patterns of mitochondrial lineages in African grey parrots. To achieve this while minimizing stress to parrots, we also aimed to evaluate whether non-invasive samples such as feces and naturally shed feathers can provide sufficiently reliable genetic information for sex identification and phylogenetic analysis.

Four fresh fecal samples were collected noninvasively from the floors of rescue cages at UWEC-CA, where twelve parrots were housed. Individual assignment of samples at the time of collection was not possible because the parrots were not restrained during sampling. These parrots had varied histories prior to admission, with some confiscated from illegal traders at border towns, potentially from neighboring countries. Naturally shed feathers were collected from six parrots housed at Toyama Municipal Family Park Zoo. After optimizing the protocol, naturally shed feathers were collected from 16 parrots kept at 10 Japanese zoos. Some parrots in Japanese zoos had records of breeders or importers (Supplementary Table 1), but many parrots imported into Japan before 2017, when CITES regulations on African grey parrots were strengthened had no records. Following process were independently conducted in Uganda and in Japan respectively for Ugandan samples and for Japanese samples.

Genomic DNA was extracted from fecal samples using the QIAamp Fast DNA Stool Mini Kit according to the manufacturer’s instructions (QIAGEN N.V., Venlo, Netherlands). DNA was extracted from a single 2–3 mm tip of the shed feather shaft using the NucleoSpin Tissue Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), with minor modifications: incubation in Buffer T1 at 56°C was extended to 48 hr, incubation in Buffer B3 at 70°C was extended to 45 min, and DNA was eluted with 80 μL of Buffer BE after incubation at 70°C for 20 min [12].

Extracted DNA was subjected to PCR amplification for sex determination and mitochondrial DNA (mtDNA) phylogenetic analysis. Sex determination was performed using primers targeting the chromodomain helicase DNA-binding (CHD) gene: 2550F (5′-GTTACTGATTCGTCTACGAGA-3′) and 2718R (5′-ATTGAAATGATCCAGTGCTTG-3′) previously reported by Vucicevic et al. [11]. Approximately 1,200 bp of the mtDNA control region [7] was amplified using primers Pe-mt-F-2 (5′-CCCACATATCAAACCAGAGT-3′) and Pe-mt-R-2 (5′-GGAGTTGGTGATGGAGATTA-3′). Because African grey parrots possess two control regions due to duplication [4], these primers were designed to amplify control region 1 (CR1) based on the African grey parrot genome sequence (GCA_009867235.2) using the Primer3Plus website, with Pe-mt-F-2 located in the cyt b gene and the reverse primer (Pe-mt-R-2) in the tRNA^Thr gene. Positive control DNA samples for sex determination were also independently prepared in Uganda and in Japan. The individuals used as controls had been previously sexed using DNA extracted from shed feathers collected during routine ringing procedures in Uganda, and the results were later supported by behavioral observations such as breeding behaviors. In Japan, shed feathers were collected from captive individuals whose sex had already been identified and recorded.

PCR was performed using Ex Taq DNA polymerase (Takara Bio Inc., Kusatsu, Japan). Each 25 μL reaction contained 2.5 μL of 10× Ex Taq buffer, 2.0 μL of 2.5 mM dNTP mixture, 0.5 μL each of 10 μM forward and reverse primers, 0.125 μL of Ex Taq polymerase, 1.0 μL of template DNA, and 18.375 μL of nuclease-free water. Thermal cycling for sex determination consisted of an initial denaturation at 95°C for 4 min, followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 45 sec, with a final extension at 72°C for 4 min. PCR products were visualized by gel electrophoresis on 2% agarose gels stained with Midori Green Advance (NIPPON Genetics Co., Ltd., Tokyo, Japan). Males exhibited a single CHD band, whereas females exhibited two bands (Supplementary Figs. 1 and 2).

For mtDNA control region amplification, thermal cycling conditions consisted of an initial denaturation at 94°C for 10 min, followed by 35 cycles of 94°C for 10 sec, 57°C for 30 sec, and 72°C for 1 min, with a final extension at 72°C for 10 min. Amplicons were purified by isopropanol precipitation and dissolved in nuclease-free water. Sequencing was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit and a 3730xl DNA Analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA) in Japan (Hokkaido Bio System Co., Ltd., Sapporo, Japan), and a SeqStudio Genetic Analyzer (Thermo Fisher Scientific Inc.) in Uganda (Department of Immunology and Molecular Biology, Makerere University). Sequencing primers included Pe-mt-F-2, Pe-mt-R-2, Pe-mt-seqF (5′-ATGTACTCCTTGCCTCGTAA-3′), and Pe-mt-seqR (5′-TTACGAGGCAAGGAGTACAT-3′). Determined sequences were deposited in GenBank under accession numbers from LC912693 to LC912707.

Phylogenetic analyses were conducted using mtDNA sequences obtained from 26 individuals, together with previously reported haplotypes from captive individuals of wild origin [7]. Phylogenetic trees were constructed using the neighbor-joining method [8] based on Kimura’s two-parameter model [6], with Poicephalus robustus (GenBank accession AY082083) as an outgroup, using MEGA version 12.1 [10]. Bootstrap support values were calculated from 1,000 random re-samplings. A median-joining network of evolutionary relationships among available haplotypes was constructed using NETWORK version 10.2 [1].

Sex identification was successfully performed using both fecal samples and single shed feather tips (Supplementary Figs. 1 and 2). Among the four fecal samples from UWEC-CA, one male and three females were identified. Among the parrots housed in Japanese zoos, thirteen were males and seven were females. Sex determination was unsuccessful on two feather samples possibly due to the poor quality of the collected feathers or poor storage conditions of collected feather [9]. Nevertheless, the results demonstrate that fecal samples can be used as an alternative to blood sampling for sex determination. As sampling was entirely noninvasive and required no physical restraint, this protocol offers clear advantages over blood sampling. Furthermore, this approach may be applicable to free-ranging parrots in the wild, as fecal samples can be collected beneath trees where parrots are observed feeding or resting.

The mitochondrial DNA (mtDNA) control region was successfully amplified and sequenced from all 26 parrots housed at UWEC-CA and in Japanese zoos using either fecal or naturally shed feather samples, indicating that noninvasively collected samples are suitable for phylogenetic analysis. We identified 20 haplotypes (Hap1–Hap20), including four samples from UWEC-CA and sixteen from Japanese zoos (Table 1). Of these 20 haplotypes, five (Hap1–Hap5) were identical to haplotypes reported previously [7], and the remaining 15 newly determined sequences were deposited in GenBank under accession numbers (LC912693-LC912707). Although fecal samples from UWEC-CA were collected without individual identification, they exhibited distinct haplotypes, indicating that these samples can be treated as originating from different individuals. High haplotype diversity was observed in both Ugandan and Japanese settings, suggesting that these populations include individuals originating from a range of geographic regions or breeding facilities.

Table 1. Haplotype of African grey parrots in UWEC-CA and Japanese Zoos.

	UWEC-CA	Japanese Zoo	Accession No.	Clade
Hap1	-	3	AY082093	Gabon
Hap2	-	2	AY082087	Gabon
Hap3	1	1	AY082090	Gabon
Hap4	-	1	AY082088	Gabon
Hap5	-	1	AY082086	Gabon
Hap6	-	1	LC912693	Gabon
Hap7	-	1	LC912694	Gabon
Hap8	-	1	LC912695	Gabon
Hap9	-	1	LC912696	Gabon
Hap10	-	1	LC912697	Gabon
Hap11	-	1	LC912698	Gabon
Hap12	-	1	LC912699	Gabon
Hap13	-	1	LC912700	Gabon
Hap14	1	-	LC912701	Gabon
Hap15	1	-	LC912702	Gabon
Hap16	1	-	LC912703	Gabon
Hap17	-	2	LC912704	Unknown origin
Hap18	-	2	LC912705	Unknown origin
Hap19	-	1	LC912706	Unknown origin
Hap20	-	1	LC912707	Unknown origin
Number of individuals	4	22
Number of haplotypes	4	17
Haplotype diversity	1.000	0.974
Nucleotide diversity	0.0057	0.0077

Phylogenetic analysis of the 20 haplotypes together with reference sequences revealed that the haplotypes revealed two major clades (Fig. 2). One clade contained nine previously reported sequences (AY082085–AY082093), which likely represent Gabonese lineages, as these sequences were obtained from individuals captured in Gabon [7]. The other clade contained four sequences (AY082094–AY082097), whose geographic origin remains uncertain, as the corresponding individuals were captive birds sampled in São Tomé and Príncipe and may have been captured on the African mainland. All four haplotypes obtained from parrots housed at UWEC-CA clustered within the Gabon-associated clade. Among parrots housed in Japanese zoos, 16 individuals carried mtDNA haplotypes that clustered also within the Gabon-associated clade, whereas six carried haplotypes belonging to the clade of unknown origin (Fig. 2; Table 1).

Fig. 2.

Phylogenetic tree among grey parrot mtDNA haplotypes based on neighbor-joining method.

Importantly, Hap3, which is identical to AY082090 reported from Gabon [7], was shared by parrots sampled from Ugandan and Japanese settings (Table 1). This finding indicates that Hap3 is a commonly observed mtDNA haplotype within the Gabon-associated clade. Its occurrence in parrots from different facilities may simply reflect shared maternal lineages, although the precise geographic origins of these individuals remain unknown. Its central position in the haplotype network is also consistent with this interpretation (Supplementary Fig. 3).

Overall, these results suggest a close phylogenetic relationship between parrots housed at UWEC-CA and mtDNA lineages associated with Gabon-associated clade (Fig. 2 and Supplementary Fig. 3). Uganda represents the eastern limit of the distribution of African grey parrots and is geographically distant from Gabon. Nevertheless, the close genetic relationship observed between parrots housed in Uganda and Gabon-associated lineages may indicate limited phylogeographic structuring across parts of the species’ range. However, this interpretation remains tentative and requires further validation through broader sampling, particularly in central African countries such as the Democratic Republic of Congo.

Although birth origins were known for only five individuals in Japanese zoos, three parrots originating from a breeding facility in the Philippines—whose haplotypes were Hap1, Hap2, and Hap11—clustered within the same clade. This observation suggests that mtDNA lineages may reflect maternal geographic structure, but the current dataset is insufficient to draw firm conclusions. Accumulation of comparable genetic data from confiscated, captive, and wild populations will be essential for improving inference of potential geographic origins and for supporting evidence-based reintroduction and conservation management.

Ethics statement: All procedures conducted in Uganda were approved by the Uganda National Council for Science and Technology (UNCST; NS744ES), the Institutional Animal Care and Use Committee of Makerere University College of Veterinary Medicine, Animal Resources and Biosecurity (SVAR_IACUC/257/2025), the Uganda Wildlife Conservation Education Centre (UWEC/ED/27), and the Uganda Wildlife Authority (UWA; COD/96/05). In Japan, shed feather sampling at zoos was conducted in accordance with the Animal Welfare Standards of the Japanese Association of Zoos and Aquariums (JAZA).

CONFLICTS OF INTEREST

The authors declare no potential conflict of interests.

Funding Statement

This study was conducted with financial support from the JICA Grassroot Project, Grant-in-Aid for the Fund for the Promotion of Joint International Research (24KK0200) from the Japan Society for the Promotion of Science (JSPS), and the Suntory World Bird Fund.