Introduction

Humans have historically transported wildlife, such as the black francolin, from the Eastern Mediterranean and Middle East to the Indian subcontinent, impacting current biodiversity (1,2). The black francolin (Francolinus francolinus, Linnaeus, 1766), a terrestrial gamebird, which belongs to the Galliformes order of the family Phasianidae, is classified as Least Concern (3). Morphologically, the male is recognized by a black face and a dark rust-brown neck collar. At the same time, the brownish female has thick black-and-white patterns throughout the body. This bird inhabits a variety of habitats worldwide and eats grains, seeds, insects, and worms (4). This bird's threat in Asia, especially in Iraq, is due to increased predation, illegal hunting, trapping, habitat deterioration and fragmentation, continuous use of handguns for hunting, and chemical residues in agriculture. Additionally, human activities, including the expansion of urban areas, affect Iraq's biological diversity (5-7). Asian francolins are diverse in form, the environment, behavior, and their geographic distribution patterns (8). The bird challenge of morphological analysis (primarily of feather features) and of recognizing diverse developmental phases. Recently, the researchers began using DNA tools to conserve biological diversity and species taxonomy (9,10). Short fragments of DNA from a standard portion of the genome serve as a DNA barcode, the mitochondrial cytochrome c oxidase I (mtCOI) gene, to identify individuals at the species level, elucidate evolutionary relationships among closely related species, and recognize genetic diversity (11-13). DNA barcoding has gained wide acceptance in taxonomic research and in ecology and evolution for its ability to quickly and accurately identify species (14-16). Forcina et al. (17) conducted an evolutionary sequence analysis of the entire mitochondrial DNA control region in Asian francolin species. Furthermore, there are few studies on the mt COI gene in Iraqi birds. Balog et al. (18) assessed the genetic diversity of Iraqi Red and Raabi pigeons using the COI area.

The current study aimed to use a valuable mitochondrial DNA marker (the partial mtCOI gene) to identify native F. francolinus in Iraq and to determine genetic variation at the species level. Mitochondrial DNA is an effective marker, and the COI gene is used as a DNA barcode.

Materials and methods

Ethical approval

The experiment was approved by the local ethical committee under approval number 862 (8/10/2024) at the University of Baghdad, College of Science for Women, Department of Biology.

Sampling and DNA extraction

Fourteen F. francolinus (19) samples were gathered from various locations in Baghdad during the period from August to October 2024 (Figure 1). Genomic DNA was extracted from skin and feathers using the DNeasy Blood and Tissue Kit (Qiagen, Germany), as modified from (20,21). The DNA concentrations were measured using NanoDrop to verify the DNA quality of all samples at two wavelengths (260 and 280 nm).

Figure 1: Lateral view of the black francolin (Francolinus francolinus); A: Male, B: Female.

Amplification and sequencing:

The PCR amplification of the COI gene was done using specific primer pairs FrCO-FWD: 5′-ACAGCACTCAGCCTACTAAT-3′ and FrCO-REV: 5′-GGTGTCCGAAGAATCAGAAT-3′. Each PCR reaction had a final volume of 35 μl containing 3 μl of genomic DNA, 1.5 μl of each forward and reverse primer, and 17.5 μl of GoTaq® G2 Hot Start GreenMaster Mix 2X, and was completed by adding 11.5 μl of free nuclease-distilled water. The optimum conditions of COI amplification in the thermocycler were set as follows: one cycle of 95°C initial denaturation for 5 min, followed by thirty-three cycles of 95°C denaturation for 45 sec, annealing at 55°C/45 sec, 1 min for extension at 72°C, and one cycle for final extension at 72°C for 7 min. The reaction was held at 4°C for 15 min. The PCR products were electrophoresed on a 1.5% agarose gel, and the bands were visualized using a gel imaging system. The PCR outcomes of COI were sequenced using an automated DNA sequencer. The COI sequences were analyzed and aligned with reference sequences available at the National Center for Biotechnology Information (NCBI, USA) (https://blast.ncbi.nlm.nih.gov/Blast.).

Data analysis

The taxonomic rank of F. francolinus was assessed to align it with the most closely related species in NCBI. The Mega 11 program was used to estimate pairwise sequence distances for divergence analysis (22) and to construct a phylogenetic tree for this species. Some of the nucleotide sequences were unsuccessful. The phylogenetic tree was built using the Maximum Likelihood (ML) method and validated with 1000 bootstrap replicates (23) to determine the evolutionary history of F. francolinus.

Results

PCR amplification of the partial mtCOI gene was performed to identify F. francolinus. The PCR outcomes indicate that the 631 bp segment of the mt COI gene for the black francolin birds (Figure 2) was subsequently sent for sequencing. The sequence analysis confirms that the mt COI gene belongs to F. francolinus. The partial COI sequence of F. francolinus was registered in NCBI GenBank under accession numbers PV199482.1, PV199483.1, PV199484.1, PV199485.1, PV199486.1, PV199487.1, and PV199488.1, respectively. The intraspecific genetic distances ranged from 0.00% to 5.6%, which explains evolutionary divergences in the mtCOI sequence within F. francolinus (Table 1). The phylogenetic analysis revealed clear clustering patterns among F. francolinus individuals from different geographic regions. Iraqi samples formed a well-supported clade, exhibiting moderate bootstrap values, indicating a close genetic relationship and possible shared ancestry among these individuals. One Turkish sample (NLAVE321-11) was nested within this Iraqi clade, suggesting historical gene flow or a common ancestral lineage between populations in Iraq and adjacent regions of Turkey. The Turkish samples overall were placed in separate positions, reflecting genetic divergence within the population. Conversely, individuals from the USA and Cyprus formed clades, indicating significant genetic differentiation likely due to geographic isolation (Figure 3).

Figure 2: The PCR amplicons of the COI gene were electrophoresed on a 1.5% agarose gel at 70 V/60 min. The 631 bp product is indicated by a blue arrow. Lane DNA ladder (M), lanes (1-7) represent samples of PCR products.

Table 1: Pairwise distances for estimates of evolutionary divergence between COI sequences of F. francolinus

Figure 3: Phylogenetic tree analysis of the mtCOI sequence of F. francolinus by using Mega 11 (ML). This tree showed the geographical distribution in Iraq and in different countries (the sample from Iraq was marked with a blue circle).

Discussion

The global population growth, urbanization, deforestation, climate change, and illegal hunting have severely reduced wildlife habitats. Furthermore, species may adapt to their environment to survive or migrate, or their numbers may decline over time, which impacts bird diversity (6,24,25). Therefore, mitochondrial DNA is a powerful genomic tool for better understanding species and assessing the diversification of ecologically distinct species, thereby promoting biodiversity conservation (26,27). According to Khaliq et al. (28), the overhunt would threaten the survival of Francolinus pondicerianus in Pakistan. As a result, use mitochondrial DNA to better understand the genetic diversity of this bird and its conservation. Genetic diversity within the black francolin population was demonstrated through partial mitochondrial gene sequencing of individuals from various regions. Additionally, deforestation, flooding, and habitat destruction might result in further separation and smaller populations that may have decreased genetic diversity (29,30).

The black francolin is generally considered a non-migratory gamebird, typically remaining within the same area where it hatched. Nevertheless, it may undertake short-distance movements in response to environmental fluctuations, such as habitat degradation or seasonal changes in food availability, often in search of suitable breeding grounds or foraging habitats (31,32). Alternatively, the chicks or eggs of the black francolin in exposed nests may be susceptible to predation by raptors such as Elanus caeruleus (33). Due to its sensitivity to habitat disturbances, targeted conservation efforts and practical habitat management (e.g., maintaining forest sustainability) are vital to maintaining stable populations and preserving its ecological role in native ecosystems (32,34). The current study highlighted that the target COI genes were selected because of their widespread use in species taxonomy. Despite their partiality, these genes provide sufficient sequence divergence to meet the requirements of this study. Here, the COI genes were PCR-amplified using specifically designed primer pairs, which were demonstrated to be a stringent complement to the conserved region for species identification. These results are generally in agreement with other studies, but used universal primers to amplify conserved regions of the COI gene (15,35). DNA barcodes provide high-quality genetic information for species identification (36). Therefore, these findings will assist in developing the GenBank database and can be used for additional phylogenetic analysis on other avian species. The high intraspecific divergence (0.00%-5.6%) of the F. francolinus COI sequence among individuals from Iraq and different regions (e.g., USA, Cyprus, Turkey). This elevated divergence may reflect historical geographic isolation among populations, resulting in limited gene flow and the accumulation of substantial genetic differences over time. The phylogenetic analysis of the COI sequence revealed clear genetic structuring among F. francolinus populations across regions.

Iraqi samples form a monophyletic clade, suggesting localized evolution or limited gene flow with other populations. In contrast, the USA samples cluster separately with high bootstrap support, indicating significant genetic divergence from the Iraqi group. Turkish samples appeared in two separate branches within the tree. One Turkish sample was closely related to the Iraqi clade. In contrast, the others were more closely related to the USA and Cyprus samples. These findings may suggest population isolation due to geographic barriers in the Middle East and other regions. The phylogenetic structure observed supports the hypothesis of deep genetic divergence within species, potentially shaped by historical biogeographic events or habitat fragmentation (37). DNA sequence data in public repositories enable phylogenetic inference of avian species taxonomy (38). Therefore, a terrestrial gamebird is essential to combine ecological monitoring with molecular tools, such as genome-wide association studies, to further evaluate genetic diversity, population connectivity, and ecological adaptability of black francolins, thereby contributing to the development of conservation strategies (39,40).

Conclusion

This investigation demonstrates that genetic diversity is significant for assessing the viability of F. francolinus in Iraq. Seven novel COI haplotypes from Iraqi F. francolinus were deposited in GenBank (accessions PV199482.1-PV199488.1), representing the first such records from Mesopotamia. These genetic data suggest that Iraqi black francolins represent an evolutionarily significant unit meriting: (1) habitat corridors between remaining wetlands, and (2) stricter enforcement of hunting regulations.

Acknowledgment               

I want to express our appreciation and gratitude to the Department of Biology in the College of Science for Women at the University of Baghdad.

Conflict of interest

There is no conflict of interest.