Phylogeny and Genetic Diversity of Philippine Mallard Ducks Based on the Mitochondrial DNA D-loop Region

Abstract

As with other locally adapted species, Philippine mallard ducks risk population fragmentation and indiscriminate hybridization. To allow for targeted breed improvement, this study aimed to unravel the maternal genetic relationships, phylogeny, and levels of genetic diversity in Philippine mallard ducks. We sequenced the mitochondrial DNA D-loop region of 118 ducks sampled from the Southern Leyte, Samar, and Cebu Provinces. Sequence data analysis revealed nine transition base substitutions and eight distinct haplotypes. Overall haplotypic diversity (Hd = 0.666 ± 0.038) exceeded that reported for Javanese ducks, but was comparable to that of Thai, Chinese, and Indian domestic duck populations. The matrilineal phylogenetic tree positioned all Philippine mallard ducks within the Old-World Haplogroup A. Specifically, they clustered into subhaplogroup A3 (75/118), together with domestic ducks from China and Southeast Asia; putative ancestral subhaplogroups A0 (6/118) and A1a (5/118) in a basal position, showing close genetic affinities with Eurasian wild mallards and Eastern spot-billed ducks; and subhaplogroup A1b (6/118), showing genetic relatedness to the Indian Runner duck. Notably, 26 individuals formed a distinct cluster corresponding to subhaplogroup A2, which appeared to be unique to this population. This study provides the first mitochondrial DNA D-loop-based genetic characterization of Philippine mallard ducks, placing this population within the broader Haplogroup A lineage and highlighting how its genetics was shaped by island biogeography. Collectively, these results establish the basis for future research on animal genetic resources and phenomics, supporting the sustainable management of mallard duck populations in the Philippines.

Introduction

The Philippines relies heavily on livestock and poultry production, including ducks[1]. In rural communities, locally adapted Philippine mallard ducks and the improved egg-type ItikPINAS ducks are important sources of livelihood, and are the primary breeds for the production of balut (cooked embryonated eggs), salted eggs, and century eggs[2,3]. Ducks are spread throughout the country and are raised predominantly by smallholder farmers using traditional husbandry systems[3,4]. Besides their economic value, domestic ducks act as natural predators of insects and snails in rice fields and other wetland ecosystems[5,6].

Domestic ducks have become widely distributed across diverse geographical regions, with Asia accounting for approximately 89% of the global duck population[7]. Genomic data have revealed historical gene flow between Southeast Asian and southern Chinese duck populations[8], supporting the long-standing hypothesis that these regions represent primary centers of duck domestication dating back more than 2,000 years[9]. However, unlike their wild progenitors, domestic ducks have undergone extensive anthropogenic hybridization, selective breeding, and localized genetic introgression[8,10]. Although these processes have shaped key phenotypic traits, they may also contribute to population fragmentation and the potential loss of genetic integrity in locally adapted populations such as Philippine mallard ducks. Erosion of their distinct genetic makeup may compromise adaptive traits and ultimately affect their long-term viability and productivity.

Mitochondrial DNA (mtDNA) is used to reconstruct the evolutionary history and maternal lineage structure of domestic avian species. Ducks are classified into two major genetic lineages: Old-World (OW) Haplogroup A, predominant in Eurasia, and New-World (NW) Haplogroup B, originating in North America[11,12,13]. Phylogenetic relationships and genetic diversity underlying phenotypic variation have gained increasing attention in elucidating duck domestication in island landscapes. In the Philippines, studies on native pigs, wild boars[14,15] and native chickens[16,17,18] have demonstrated the value of phylogeographic approaches in characterizing genetic resources and documenting matrilineal inheritance patterns across the archipelago. Nevertheless, the genetics of Philippine mallard ducks remain poorly understood and there is no consensus regarding their origin or domestication history in Island Southeast Asia. Comprehensive population genetics and phylogeographic analyses could outline the migration patterns, diversity, and evolutionary processes that shape the genetic makeup of locally adapted Philippine mallard ducks.

Here, we generated 637-bp mtDNA D-loop sequences of Philippine mallard ducks, similar to those from previous phylogeographic studies[10,13], to compare publicly available datasets and reconstruct the matrilineal phylogeny of this breed. We aimed to provide the first detailed assessment of its genetic diversity within a global mtDNA framework.

Materials and Methods

Sampling and DNA Extraction

Blood samples were collected from a total of 118 Philippine mallard ducks across provinces in Cebu (N = 30 each in the northern and southern parts), Samar (N = 25), and Southern Leyte (N = 25) in the Visayas region (Table S1; Supplementary Fig. 1). To ensure that the samples were unrelated, birds from different known families and sites within each province were selected, in accordance with guidelines on the molecular characterization of animal genetic resources established by the Food and Agriculture Organization[19]. Blood samples were collected following ethical guidelines for the treatment and use of farm animals (Guide for the Care and Use of Agricultural Animals in Research and Teaching, Fourth Edition, 2020; www.asas.org/services/ag-guide). The owners provided personal consent to include their animals in this study. Genomic DNA was extracted from stored whole-blood samples using the phenol-chloroform method[20].

The final dataset was complemented with comparable mtDNA D-loop sequences of mallard ducks retrieved from GenBank (Table S2).

PCR Amplification and Sequencing

The target mtDNA control region was amplified following two steps. First, about 5.0-kb mtDNA D-loop fragments were amplified using a long-and-accurate PCR with 1.0 µL mallard duck DNA as template, 0.6 µL each of primers Cytb-Forward 5ʹ-TACACGAATCAGGCTCAAACAACCCCCTAGGCATC-3ʹ and 16S-Reverse 5ʹ-TGCACCATTAGGTTGTCCTGATCCAACATCGAGGT-3ʹ (10 pmol), 3.6 µL ddH₂O, 10.0 µL 2×PCR buffer, 4.0 µL 2 µM dNTPs, and 0.2 µL KOD-FX Neo Polymerase (TOYOBO, Osaka, Japan) in a 20-µL reaction.

The reaction was initiated by denaturation at 94 °C for 2 min, followed by 30 cycles of DNA denaturation at 98 °C for 10 s, annealing of primers at 66 °C for 30 s, and primer extension at 68 °C for 2 min and 30 s, using a GeneExplorer Thermal Cycler (BIOER Technology, Hangzhou, China). Next, 0.5 µL of the PCR product was used for segmental amplification of the target mtDNA D-loop region (718 bp) using 0.3 µL each of primers F-156 5′-GTTGCGGGGTTATTTGGTTA-3′ and R-H773 5′-CCATATACGCCAACCGTCTC-3′ (10 pmol) in a 20-μL reaction containing also 4.5 µL ddH₂O, 10.0 µL 2×PCR buffer, 4.0 µL 2 µM dNTPs, and 0.4 µL KOD-FX Neo Polymerase. PCR cycling included a preliminary denaturation at 94 °C for 5 min, followed by 35 cycles of DNA denaturation at 94 °C for 30 s, annealing of primers at 63 °C for 30 s, and primer extension at 72 °C for 1 min, and final extension at 72 °C for 7 min. The DNA fragments obtained by segmental amplification were cleaned and purified using exonuclease I to degrade the residual PCR primers and shrimp alkaline phosphatase to dephosphorylate the remaining dNTPs. Finally, the mtDNA D-loop fragments were directly sequenced using 3130/3130xl Genetic Analyzers (Applied Biosystems, Foster City, CA, USA).

Sequence Alignment and Genetic Diversity

A total of 118 partial mtDNA D-loop region sequences were generated in this study and were edited using the GeneStudio Pro tool (http://genestudio.com/). Ambiguous sites were trimmed and cleaned sequences (637 bp) were aligned in MEGA12[21] using ClustalW[22]. Aligned nucleotide sequences were viewed using BioEdit 7.2.5 software[23]. It should be noted that the analyzed samples did not represent the entire Philippine mallard duck population, as they were collected only from selected provinces in the Visayas region and were limited to sequences that passed quality control. Nonetheless, this dataset provides an adequate sample size for molecular characterization[19,24]. All newly generated sequences were deposited in the NCBI GenBank database under Accession No. PX505304-PX505422 (Table S1).

Genetic diversity indices, such as the number of haplotypes (Ht), haplotype diversity (Hd), and nucleotide diversity (π) were estimated using DnaSP v6.0 software[25].

Phylogenetic Reconstruction

To determine the genealogy of Philippine mallard ducks, reference sequences representing different mallard duck clades across Asia were included[12,13]. Phylogenetic reconstruction was performed using maximum likelihood in IQ-TREE[26] with the best-fit substitution model HKY+F+G4, based on the Bayesian Information Criterion determined by Modelfinder[27]. Statistical node support was calculated using ultrafast bootstrap support[28] and SH-aLRT[29] with 1,000 replicates.

A median-joining network was constructed using PopArt v1.7 software to infer evolutionary relationships between haplotypes[30]. The number and assignment of haplotypes were determined using DnaSP v6.0 software.

Population Genetic Structure

The population pairwise net genetic distance based on population pairwise F_ST (significant values were accepted at p < 0.05) was estimated using Arlequin v3.5.2.2 software (with 10,000 permutations)[31]. Genetic structures were estimated by analysis of molecular variance (AMOVA) implemented in Arlequin v3.5.2.2. The level of significance was evaluated based on 1,000 random permutations.

Results

Sequence Variation and Genetic Diversity

We analyzed the mtDNA D-loop region sequences of Philippine mallard ducks (N = 118) using Anas platyrhynchos Accession no. NC_009684 as reference sequence. Variations were aligned relative to the reference sequence in the hypervariable region, spanning positions 105–741 (637 bp) (Table 1). Nine nucleotide base transition substitutions were identified at the following base positions: 219 (T→C), 243 (A→G), 252 (C→T), 253 (T→C), 271 (T→C), 274 (C→T), 281 (G→A), 365 (A→), and 607 (C→T). These mutation sites reflect population diversification of Philippine mallard duck ecotypes in a localized landscape. The observed polymorphic sites and haplotype variations are summarized in Table S1.

Table 1. Sequence variation in the mtDNA D-loop region of Philippine mallard ducks.

	Nucleotide base transition mutations (nucleotide positions)
	219	243	252	253	271	274	281	365	607	GenBank Accession No.
Reference sequence	T	A	C	T	T	C	G	A	C	NC_009684
PMD D-loop sequence
SL2	C	.	T	C	C	.	.	.	T	PX505304
SL4	C	.	.	.	C	.	.	.	.	PX505305
SL5	C	.	.	.	C	.	.	.	.	PX505306
SL6	C	.	.	.	C	.	.	.	T	PX505307
SL17	C	.	.	.	C	.	.	.	T	PX505308
SL35	C	.	.	.	C	.	.	.	T	PX505309
SL36	C	.	.	.	C	.	.	.	T	PX505310
SL39	C	.	.	.	C	.	.	.	T	PX505311
SL40	C	.	.	.	C	.	.	.	T	PX505312
SL43	C	.	.	.	C	.	.	.	T	PX505313
SL44	C	.	.	.	C	.	.	.	T	PX505314
SL56	C	.	.	.	C	.	.	.	T	PX505315
SL59	C	.	.	.	C	.	.	.	T	PX505316
SL60	C	.	.	.	C	.	.	.	.	PX505317
SL66	C	.	.	.	C	.	.	.	.	PX505318
SL67	C	.	.	.	C	.	.	.	.	PX505319
SL82	C	.	.	.	C	.	.	.	T	PX505320
SL83	C	.	.	.	C	.	.	.	T	PX505321
SL84	C	.	.	.	C	.	.	.	T	PX505322
SL87	C	.	.	.	C	.	.	.	T	PX505323
SL88	C	.	.	.	C	.	.	.	T	PX505324
SL90	C	.	.	.	C	.	.	.	T	PX505325
SL94	C	.	.	.	C	.	.	.	T	PX505326
SL97	C	.	.	.	C	.	.	.	T	PX505327
SL99	C	.	.	.	C	.	.	.	T	PX505328
Leyte3	C	.	.	.	C	.	.	.	T	PX505329
Samar32	C	.	.	.	C	.	.	.	.	PX505330
Samar36	C	.	T	C	C	.	.	.	T	PX505331
Samar37	C	.	.	.	C	.	.	.	T	PX505332
Samar38	C	.	T	C	C	.	.	.	T	PX505333
Samar39	C	.	T	C	C	.	.	.	T	PX505334
Samar42	C	.	.	.	C	.	.	.	T	PX505335
Samar47	C	.	.	.	C	.	.	G	T	PX505336
Samar50	C	.	T	C	C	.	.	.	T	PX505337
Samar75	C	.	.	.	C	.	.	.	T	PX505338
Samar76	C	.	.	.	C	.	.	.	T	PX505339
Samar77	C	.	.	.	C	.	.	.	.	PX505340
Samar78	C	.	.	.	C	.	.	.	.	PX505341
Samar81	C	.	.	.	C	.	.	.	.	PX505342
Samar82	C	.	.	.	C	.	.	.	.	PX505343
Samar84	C	.	.	.	C	.	.	.	.	PX505344
Samar85	C	.	.	.	C	.	.	.	.	PX505345
Samar86	C	.	.	.	C	.	.	.	.	PX505346
Samar87	C	.	.	.	C	.	.	.	.	PX505347
Samar90	C	.	.	.	C	.	.	.	.	PX505348
Samar91	C	.	.	.	C	.	.	.	.	PX505349
Samar92	C	.	.	.	C	.	.	.	.	PX505350
Samar93	C	.	.	.	C	.	.	.	.	PX505351
Samar95	C	.	.	.	C	.	.	.	.	PX505352
Samar97	C	.	.	.	C	.	.	.	.	PX505353
Samar98	C	.	.	.	C	.	.	.	.	PX505354
AS43	C	.	T	.	C	T	.	.	T	PX505355
AS44	C	.	.	.	C	.	.	.	T	PX505356
AS45	C	.	.	.	C	.	.	.	T	PX505357
AS46	C	.	.	.	C	.	.	G	T	PX505358
AS49	C	.	T	.	C	T	.	.	T	PX505359
CA21	C	.	.	.	C	.	.	.	.	PX505360
CA24	C	.	.	.	C	.	.	.	T	PX505361
CA27	C	G	.	.	C	.	.	.	T	PX505362
CA28	C	.	.	.	C	.	A	.	T	PX505363
CA29	C	.	.	.	C	.	.	.	T	PX505364
CP1	C	G	.	.	C	.	.	.	T	PX505365
CP2	C	.	.	.	C	.	.	.	T	PX505366
CP5	C	G	.	.	C	.	.	.	T	PX505367
CP6	C	.	.	.	C	.	.	G	T	PX505368
SR13	C	.	.	.	C	.	.	.	T	PX505369
SR14	C	.	.	.	C	.	.	.	T	PX505370
SR15	C	G	.	.	C	.	.	.	T	PX505371
SR17	C	.	.	.	C	.	.	.	T	PX505372
SR20	C	.	.	.	C	.	.	.	.	PX505373
SR52	C	G	.	.	C	.	.	.	T	PX505374
SR53	C	.	.	.	C	.	.	.	T	PX505375
TB31	C	.	.	.	C	.	A	.	T	PX505376
TB32	C	.	.	.	C	.	.	.	T	PX505377
TB33	C	.	T	.	C	T	.	.	T	PX505378
TB34	C	.	.	.	C	.	.	.	T	PX505379
TB37	C	G	.	.	C	.	.	.	T	PX505380
TB39	C	.	.	.	C	.	.	.	T	PX505381
TB40	C	.	.	.	C	.	.	.	.	PX505382
TB59	C	G	T	.	C	T	.	.	T	PX505383
TB60	C	G	.	.	C	.	.	.	T	PX505384
AR1	C	.	.	.	C	.	.	.	T	PX505385
AR2	C	.	T	C	C	.	.	.	T	PX505386
AR3	C	.	.	.	C	.	.	.	T	PX505387
AR5	C	.	.	.	C	.	.	.	T	PX505388
AR6	C	.	.	.	C	.	.	.	.	PX505389
AR7	C	.	.	.	C	.	.	.	T	PX505390
AR8	C	.	.	.	C	.	.	.	T	PX505391
AR9	C	.	.	.	C	.	.	.	T	PX505392
BD20	C	.	.	.	C	.	.	.	T	PX505393
BD21	C	.	.	.	C	.	.	.	T	PX505394
BD22	C	.	.	.	C	.	.	.	T	PX505395
BD24	C	.	.	.	C	.	.	.	T	PX505396
BD25	C	G	.	.	C	.	.	.	T	PX505397
BD27	C	.	.	.	C	.	.	.	T	PX505398
BD28	C	.	.	.	C	.	.	.	T	PX505399
BD52	C	.	.	.	C	.	.	.	T	PX505400
CR12	C	.	.	.	C	.	.	.	T	PX505401
CR15	C	.	.	.	C	.	.	.	T	PX505402
CR16	C	.	.	.	C	.	.	.	T	PX505403
CR18	C	.	.	.	C	.	.	.	.	PX505404
CR19	C	.	.	.	C	.	.	.	T	PX505405
CR49	C	.	.	.	C	.	.	.	T	PX505406
GL40	C	.	.	.	C	.	.	G	T	PX505407
GL42	C	.	.	.	C	.	.	G	T	PX505408
GL44	C	.	.	.	C	.	.	.	T	PX505409
GL45	C	.	.	.	C	.	.	.	T	PX505410
GL46	C	G	.	.	C	.	.	.	T	PX505411
GL53	C	.	.	.	C	.	.	G	T	PX505412
PN31	C	.	.	.	C	.	.	.	T	PX505413
PN35	C	G	.	.	C	.	.	.	T	PX505414
PN37	C	.	.	.	C	.	.	.	T	PX505415
PN39	C	.	.	.	C	.	.	.	T	PX505416
PN55	C	.	.	.	C	.	.	.	T	PX505417
Itik-Pinas56	C	.	.	.	C	.	.	.	T	PX505418
Itik-Pinas57	C	.	.	.	C	.	.	.	T	PX505419
Itik-Pinas58	C	G	.	.	C	.	.	.	T	PX505420
Itik-Pinas60	C	.	.	.	C	.	.	.	T	PX505421
Bitik	C	.	T	.	C	T	.	.	T	PX505422

The genetic diversity indices of Philippine mallard ducks and other duck populations across Asia and Europe with comparable mtDNA D-loop sequences are reported in Table 2. Interestingly, Philippine mallard ducks revealed moderate haplotype diversity (Hd = 0.666) but consistently low nucleotide diversity (π = 0.0018), a pattern characteristic of recently derived or demographically expanding maternal lineages. This diversity level is higher than that observed in Javanese domestic ducks yet substantially lower than that of the more divergent European lineages. Philippine mallard ducks showed a similar haplotype richness as Korean native populations but markedly reduced nucleotide variation compared with Chinese domestic ducks, indicating a narrower maternal gene pool.

Table 2. Genetic diversity indices of Philippine mallard ducks estimated using mtDNA D-loop sequences (637 bp)

Region	Country	Molecular diversity indices				Reference
	(population)
		N	Ht	Hd	π
Southeast Asia	Philippines					This study
	Samar	25	4	0.560±0.097	0.0019±0.0004
	North Cebu	30	7	0.782±0.054	0.0021±0.0003
	South Cebu	38	6	0.486±0.094	0.0010±0.0002
	Southern Leyte	25	3	0.397±0.103	0.0009±0.0003
	Overall	118	8	0.666±0.038	0.0018±0.0002
	Indonesia
	Central Java	34	7	0.373±0.105	0.0012±0.0004	GenBank
	Thailand	10	3	0.511±0.165	0.0010±0.0004	GenBank
		10	-	0.756±0.130	0.0014±0.0004	Leekaew et al., 2008
East Asia	China					Li et al., 2010
	(domestic ducks)	238	-	0.645	0.115
	South Korea
	(white native)	21	-	0.633	0.0019	Sultana et al., 2016
	(black native)	20	-	0.689	0.0030
South Asia	India
	West Bengal	23	-	0.249±0.116	-	Gaur et al., 2017
	Odisha	26	-	0.351±0.117	-
	Tamil Nadu	33	-	0.770±0.054	-
	Bangladesh
	(deshi white)	20	-	0.337	0.0037	Sultana et al., 2016
	(deshi black)	36	-	0.829	0.0006
North-West Europe	Great Britain	21
	Netherlands	23	-	0.696±0.05	0.0034±0.0004	Kraus et al., 2011
	Norway	22
Central Europe	Austria	18
	Germany	24	-	0.929±0.018	0.0047±0.0003	Kraus et al., 2011
	Estonia	22

N, number of samples; Ht, number of haplotypes; Hd, Haplotypic diversity; π, nucleotide diversity

Matrilineal Phylogeny and Genetic Affinities of Philippine Mallard Ducks

Phylogenetic analysis revealed that all Philippine mallard ducks shared a common maternal origin within the OW Haplogroup A lineage, while also exhibiting locally differentiated maternal lineages. Such clustering patterns indicate close genetic affinities between duck populations from Southeast Asia, Eurasian domestic breeds, and wild mallards. Notably, none of the contemporary Philippine mallard duck sequences showed maternal ancestry with Anas luzonica (the endemic Philippine duck) and all were clearly distinct from the NW Haplogroup B lineages represented by North American accessions retrieved from GenBank. These results support the monophyly of Philippine mallard ducks within OW Haplogroup A, confirming their matrilineal structure and diversification within island Southeast Asia (Fig. 1; Supplementary Fig. 2).

Fig. 1.

(A) Phylogenetic tree of Philippine mallard ducks based on mtDNA D-loop nucleotide sequences (637 bp). The tree was constructed using comparable sequences of domestic mallard ducks and known breeds derived from NCBI GenBank (Table S2). Node dots correspond to bootstrap support values. Identified haplogroups are assigned with branch color highlights. Colored dots on branch tips correspond to putative subhaplogroup classifications (Red: subhaplogroup A3, Orange: subhaplogroup A2, Yellow-green: subhaplogroup A1b, Green: subhaplogroup A1a, Yellow: subhaplogroup A0). (B) Schematic classification tree for OW Haplogroup A. Nucleotide positions were scored relative to the reference sequence NC_009684. Mutational motifs (transition substitutions) are shown on the tips. The tree was visualized and edited in FigTree v1.4.4. (http://tree.bio.ed.ac.uk/software/figtree/).

The majority of Philippine mallard duck samples (63.56%) clustered within subhaplogroup A3, along with Southeast Asian and Chinese duck populations, suggesting a shared and widespread maternal lineage. In contrast, subhaplogroup A2 comprised 22.03% of the samples (26/118) and formed a distinct cluster unique to present-day Philippine mallard ducks. A smaller number of sequences occupied basal positions, including those assigned to the putative ancestral subhaplogroups A0 (6/118) and A1a (5/118), both of which exhibited close genetic relationships with Eurasian wild mallards and eastern spot-billed ducks. A minor cluster within subhaplogroup A1b (6/118) showed genetic affinity for the Indian Runner duck, suggesting a possible historical introgression or shared ancestry.

The median-joining network revealed consistent clustering of Philippine mallard ducks within OW haplogroup A, with haplotype 2 forming a widely shared central node across Asian mallard duck populations (Fig. 2). The branches radiating from haplotype 2 indicate a recent population expansion of mallard ducks in the region. A clear separation was observed between the OW-Haplogroup A mallard ducks across Asia and Haplogroup B populations that include Anas rubripes and Anas fulvigula.

Fig. 2.

Median-joining network of the mtDNA D-loop region depicting evolutionary relationships of Philippine mallard duck populations, together with comparable sequences of other domestic mallard duck populations and the endemic Anas luzonica. The area of each circle is proportional to the frequency of the corresponding haplotypes. The length of branches connecting to other haplotypes corresponds to mutational positions.

Analysis of molecular variance based on pairwise differences attributed 93.36% of the molecular variation between Philippine mallard ducks and the duck populations from Indonesia and Thailand to within-population genetic variation (Table 3). This pattern indicates weak genetic differentiation across mallard duck populations in Southeast Asia compared to Chinese and Indian ones. Similarly, no hierarchical genetic structure was detected between Philippine mallard ducks and duck populations in Southeast Asia, China, or India, as evidenced by the negative among-group variance, suggesting shared maternal ancestry. Consistent with the AMOVA results, population pairwise F_ST values supported a largely homogenous genetic structure within Southeast Asian and Chinese populations, ranging from F_ST = -0.01292 (between Chinese and Thai) to F_ST = 0.19202 (between Philippine mallard ducks and Chinese) (Table S3).

Table 3.  Population genetic structure estimated by AMOVA based on Philippine mallard duck mtDNA controlregion sequences.

Group	N	Source of variation (%)
		Among groups	Among populations within group	Within populations
PMD only	118		21.49**	78.51
A. (PMD vs. SEAa)	163	4.66	1.39	93.96**
B. (PMD vs SEA / CHIa)	189	-3.46	9.96**	93.50**
C. (PMD vs SEA / INDa)	176	-12.31	19.95**	92.35**
D. (PMD vs CHI / INDa)	158	-17.72	26.79**	90.93**

Significant fixation indices at * p < 0.05; ** p < 0.01; PMD – Philippine mallard duck; SEA – populations from Indonesia and Thailand; CHI, Chinese domestic ducks; IND, Indian mallard ducks. ^acomparable D-loop sequences were retrieved from GenBank.

Discussion

Understanding the matrilineal pool and genetic structure of Philippine mallard ducks is important not only for economic reasons, but also for the development of sustainable breeding strategies and the conservation of genetic resources. We identified sites in the mtDNA control region of Philippine mallard ducks that might reflect the diverse ecology of the archipelago, as well as the management and breeding practices of farmers that have shaped present-day ducks (Supplementary Fig. 3). The slightly more heterogeneous matrilineal gene pool of Philippine mallard ducks as opposed to other Southeast Asian mallard duck populations (e.g., Javanese ducks) may be attributed to variable transregional introgressions of ancestral lineages into and out of the archipelago, a phenomenon that drives also the unique genetic diversity of native Philippine pigs and native chickens[14,15,17,18].

The moderate-to-high intrapopulation genetic diversity detected in Philippine mallard ducks further suggests strong adaptability and reproductive success[32,33,34]. Notably, Philippine mallard ducks exhibited a higher haplotype diversity index (Hd = 0.666) than Javanese ducks (Hd = 0.373 ± 0.105)[35], deshi white ducks in Bangladesh (Hd = 0.337)[36], duck populations in West Bengal (Hd = 0.249 ± 0.116) and Odisha, India (Hd = 0.351±0.117)[37], as well as Vietnamese domestic ducks (Hd = 0.515)[38]; but comparable diversity as Chinese domestic ducks (Hd = 0.645)[39], Thai native ducks (Hd = 0.756 ± 0.130)[40], and the Tamil Nadu duck population in India (Hd = 0.770 ± 0.054)[37]. This genetic reservoir is crucial for selection and genetic improvement programs that target productivity, nutritional value, and enhanced stress and disease resistance, thereby contributing to food security under a changing climate[41,42,43,44]. The comparable or higher haplotype diversity of Philippine mallard ducks relative to most Southeast Asian datasets highlights links and shared maternal ancestry across the region. In contrast, Chinese and European domestic ducks exhibit substantially deeper maternal divergence and more diverse lineages than island populations such as Philippine mallard ducks.

The phylogenetic analyses presented here provide new insights into the matrilineal ancestry of the Philippine mallard ducks and clarify their placement within the global mtDNA landscape. Consistent with earlier population genetic studies, OW Haplogroup A—one of the two principal mallard macroclades—remains widely distributed across Eurasia[10,12,13]. Notably, despite extensive regional phylogeographic sampling[10,13,45], Philippine mallard ducks have not been explicitly represented within this Asian haplogroup. In the present study, their sequences formed a monophyletic assemblage within OW Haplogroup A, with the majority of individuals (63.56%) clustering in subhaplogroup A3, alongside Southeast Asian and Chinese domestic ducks and Eurasian wild mallards[10]. This pattern points to a shared maternal ancestry with widely distributed Asian domestic lineages and suggests potential signatures from wild mallard introgression. Such genetic relationships are consistent with ancient agricultural practices in water-rich environments, where ducks are valued for both meat and egg production[6].

An additional genetic structure was evident in subhaplogroup A1, where a subset of Philippine mallard duck sequences clustered with Eurasian wild mallards and Anas zonorhyncha (eastern spot-billed duck). The inclusion of A. zonorhyncha in this clade raises the possibility of maternal introgression into Philippine mallard duck populations[46]. Although the number of Philippine mallard duck samples in this cluster was limited, the pattern aligned with previous hypotheses of introgressive hybridization between mallards and eastern spot-billed ducks[47,48]. However, these results do not support the proposition that A. zonorhyncha is the primary maternal origin of Eurasian mallards[49]. Rather, the close morphological, behavioral, and genetic affinities between these species[50] likely facilitate occasional interspecific hybridization, a phenomenon well documented among Anas species[51].

The presence of subhaplogroup A2 in the present study suggests a geographic structure unique to the island landscape, indicating that this lineage may have followed a distinct evolutionary trajectory. The emergence of matrilineal lineages is consistent with geographic isolation, founder effects, and early colonization events, which often shape genetic diversity in island systems[14,17]. Comparable patterns of island-specific lineages have been reported in other avian taxa, including Hawaiian ducks[52], Pacific black ducks[53], and Philippine domestic chickens[17], highlighting the capacity of insular environments to generate and maintain genetic distinctiveness.

In contrast, no Philippine mallard duck sequences exhibited affinity with A. luzonica or NW Haplogroup B, indicating that their maternal ancestry of PMD was firmly rooted within the OW Haplogroup A lineage. Taken together, these findings reveal a complex evolutionary history characterized by a shared Asian domestic ancestry, episodic interspecific introgression, and localized lineage diversification shaped by island biogeography.

Population pairwise F_ST values further supported the weak genetic structure observed between Southeast Asian, Chinese and Indian mallard populations[12]. This pattern was consistent with the relatively high gene flow facilitated by extensive intracontinental migration across Asia[54,55,56]. Mallards possess exceptional flight capabilities and rely on widespread stopover sites that often serve as temporary breeding grounds[54], thereby reducing opportunities for strong regional genetic differentiation. This dynamic was reflected in the high within-population variance components observed in Southeast Asian mallard ducks. Besides natural dispersal, historical trade routes that connected Southeast Asia with China and extended westward to India[57] likely facilitated maternal gene flow among mallard populations.

In conclusion, the present study provides the first mtDNA D-loop-based genetic characterization of the Philippine mallard ducks, offering new insights into their matrilineal phylogeny and genetic diversity. Starting from a previously unsampled island population, we provide compelling evidence that Philippine mallard ducks belong exclusively to OW Haplogroup A, exhibiting high intrapopulation diversity and signatures of gene flow with neighboring Asian mallard populations. These findings not only refine the phylogeographic context of Philippine mallard ducks within the broader Eurasian mallard lineage, but also underscore the genetic distinctiveness associated with island biogeography and historical connections. Collectively, this work provides the basis for future animal genetic resources and phenomics research, and supports the development of targeted breed improvement and conservation strategies aimed at promoting sustainable mallard duck production in the Philippines.

Acknowledgments

We wish to thank the Department of Agriculture–Regional Field Office VIII, the Local Government Units of the provinces of Southern Leyte, Samar, and Cebu, and the Office of the Vice President for Research, Extension, and Innovation, Visayas State University, for their generous support of this research. We are grateful to the farmers for their assistance during sample collection. The team wishes to thank Sweet Charish Godinez for creating the base maps.

Author Contributions

Cyrill John P. Godinez and Masahide Nishibori conceived the study and designed the experiments; Cyrill John P. Godinez, Erik John S. Putan, Carla M. Cabardo, Leonard Andrew C. Ceballos, and Cloi D. Adolfo collected the samples; Cyrill John P. Godinez, Justine Bennette H. Millado, and Masahide Nishibori provided laboratory resources; Cyrill John P. Godinez, Erik John S. Putan, Carla M. Cabardo, Justine Bennette H. Millado, and Masae Hirose performed the experiments and genetic analyses. Cyrill John P. Godinez wrote the manuscript. All authors have reviewed and approved the final manuscript.

Funding

This work was funded by the Japan–ASEAN Science, Technology, and Innovation Platform (JASTIP), Kyoto University, under a program led by Dr. Yasuyuki Kono and Dr. Hiroshi Kamitakahara.

Conflict of Interest

The authors declare no conflict of interests.

Supplementary Materials

The online version contains supplementary material available at

https://doi.org/10.2141/jpsa.2026012

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