Significance Ancient DNA sequences from chickens provide an opportunity to study their human-mediated dispersal across the Pacific due to the significant genetic diversity and range of archaeological material available. We analyze ancient and modern material and reveal that previous studies have been impacted by contamination with modern chicken DNA and, that as a result, there is no evidence for Polynesian dispersal of chickens to pre-Columbian South America. We identify genetic markers of authentic ancient Polynesian chickens and use them to model early chicken dispersals across the Pacific. We find connections between chickens in the Micronesian and Bismarck Islands, but no evidence these were involved in dispersals further east. We also find clues about the origins of Polynesian chickens in the Philippines.

Abstract The human colonization of Remote Oceania remains one of the great feats of exploration in history, proceeding east from Asia across the vast expanse of the Pacific Ocean. Human commensal and domesticated species were widely transported as part of this diaspora, possibly as far as South America. We sequenced mitochondrial control region DNA from 122 modern and 22 ancient chicken specimens from Polynesia and Island Southeast Asia and used these together with Bayesian modeling methods to examine the human dispersal of chickens across this area. We show that specific techniques are essential to remove contaminating modern DNA from experiments, which appear to have impacted previous studies of Pacific chickens. In contrast to previous reports, we find that all ancient specimens and a high proportion of the modern chickens possess a group of unique, closely related haplotypes found only in the Pacific. This group of haplotypes appears to represent the authentic founding mitochondrial DNA chicken lineages transported across the Pacific, and allows the early dispersal of chickens across Micronesia and Polynesia to be modeled. Importantly, chickens carrying this genetic signature persist on several Pacific islands at high frequencies, suggesting that the original Polynesian chicken lineages may still survive. No early South American chicken samples have been detected with the diagnostic Polynesian mtDNA haplotypes, arguing against reports that chickens provide evidence of Polynesian contact with pre-European South America. Two modern specimens from the Philippines carry haplotypes similar to the ancient Pacific samples, providing clues about a potential homeland for the Polynesian chicken.

The colonization of the remote Pacific was one of the last great human migrations, but despite the recent nature of the events, the timing and routes remain an area of considerable debate. The first colonization of Western Polynesia occurred around 3,250–3,100 calendar years before present (cal B.P.) as part of the eastward migration of Lapita pottery-bearing peoples (1). This migration occurred only a few hundred years after the emergence of this distinctive pottery tradition in the Bismarck Archipelago around 3,470–3,250 cal B.P., although its antecedents can be traced to Island Southeast Asia (ISEA) (2⇓⇓–5). Following the initial movement into Western Polynesia, a prolonged 1,800-y hiatus, or “pause,” is apparent before further colonization (6), potentially relating to the need to develop sailing technology essential for crossing the vast ocean barrier to the east (between Samoa and the Society Islands, 2,400 km; Fig. 1). The huge navigational achievement of colonizing the remote East Polynesian triangle (an oceanic region roughly the size of North America) then occurred rapidly (<300 y) (6). Although the overall chronology of the eastern Pacific island colonization has recently been further resolved, the precise details of this intensive migratory episode remain unclear (6).

Fig. 1. Map showing samples and localities mentioned in this study. Samples from Vanuatu and Guam previously published in Dancause et al. (26) are underlined, with haplogroup frequencies of the chicken specimens indicated by pie charts (thick outlines indicate ancient samples). Colors refer to haplotype/haplogroup: D haplogroup in red, E haplogroup in blue, and all other haplotypes are in black. Gray arrows represent movements inferred from archaeological data (49), whereas black arrows represent routes tested in BayeSSC analysis (dashed arrows indicate movements tested in different scenarios, whereas solid arrows are constant across the different scenarios). 1, introduction of chickens within Near Oceania; 2, alternate hypothesis proposed by (38); 3, introduction of chickens from New Guinea into Micronesia; 4–7, spread of chickens from Western Polynesia into, and within, Eastern Polynesia. Dashed line indicates demarcation between Near and Remote Oceania, Western Polynesia is defined by a dashed circle, and Eastern Polynesia is indicated by a gray shaded triangle.

Human commensal and early domesticated species were widely, but not ubiquitously, dispersed as people colonized the Pacific. As a result, they provide an opportunity to study colonization events and subsequent movements for islands and regions where they were successfully introduced, especially through the use of biomolecular techniques, including ancient DNA. In the Asia–Pacific region, the complex histories of Pacific island colonizations have been investigated using the biological elements associated with these cultures, such as bottle gourds (7, 8), sweet potatoes (9), pigs (10, 11), dogs (12), Pacific rats (13), and chickens (14⇓⇓–17). However, studies of commensals and domesticates in the Pacific to date have provided limited resolution of dispersal routes, due to low amounts of genetic diversity in many groups and overwriting of genetic signals by subsequent introductions, especially for cotransported species like rats (10, 13, 18).

Ancient and modern DNA from chickens provide an opportunity to study human-mediated dispersal across the Pacific due to the extent of genetic and phenotypic diversity and the range of archaeological material available. Although recent studies of domestic chicken breeds have highlighted how the domestication process and subsequent breeding have resulted in a 70% loss of nuclear genetic diversity (19), substantial phylogeographic structure remains within the mitochondrial (mtDNA) sequences of chickens worldwide (20⇓⇓–23). Furthermore, an extensive reference dataset of >3,000 mtDNA control region (CR) sequences and >60 whole mtDNA genomes is available from across the natural range of wild and semiwild birds, as well as domestic breeds of chicken, permitting the reconstruction of phylogeographic patterns of domestic chickens and associated human cultures. Despite these intensive surveys, a resolved worldwide chicken mtDNA phylogeny has not been developed, and this is an essential prerequisite to interpreting short ancient DNA sequences. The current phylogenetic framework for chickens is based on Liu et al. (24), who identified nine highly divergent haplogroups (designated A–I) using mtDNA CR sequences, with an additional four recently described on the basis of whole mtDNA genomes (W–Z) (25). However, there is little information about the support for these topologies, and only neighbor-joining trees have been reported to date.

Phylogeographic studies have identified that one particular mtDNA lineage (CR haplogroup D) is largely limited to the Asia–Pacific region (24), whereas many of the other haplogroups are ubiquitous worldwide, potentially as a result of historical dispersal with European colonialists (e.g., haplogroups A, B, and E), and are therefore generally phylogeographically uninformative. Previous studies of modern and ancient chickens have identified both haplogroup D and E in the Pacific (14⇓⇓–17, 26), making interpretation of colonization history difficult due to potentially contrasting origins and dispersal histories (24). Indeed, the presence of haplogroup E in the Pacific has been used to infer a link between Polynesia and pre-Columbian South America, although both the phylogenetic signal and radiocarbon dating of the samples have been questioned (27⇓–29). This issue has recently taken on more significance as other studies of ancient genetic diversity in South America emphasize the importance of evidence for pre-European Polynesian contact (17, 30).

In this study, we first quantify the support for previously defined chicken mtDNA CR haplogroups using recently published whole mitochondrial genomes (WMGs) (25). We then use the resulting robust evolutionary framework to analyze the spatial and temporal patterns of mtDNA CR haplotypes in ancient and modern Pacific chickens to examine their origins in ISEA (31), the dispersal of chickens into Near Oceania and Western Polynesia, potential connections between the New Guinea region and Micronesia, and the claimed introduction of Polynesian chickens to South America (14).

Discussion Our results indicate that a small cluster of mtDNA haplogroup D sequences, defined by a diagnostic combination of four CR SNPs (which we term the “ancestral Polynesian motif”), represent the founding lineages of chickens transported as prehistoric domesticates across the Pacific and ultimately ending up in Polynesia (i.e., “Polynesian chickens”). We suggest that the most common haplotype in ancient samples (H239) represents the core mtDNA lineage of Polynesian chickens, and that the one- or two-step derivatives in ancient Pacific island specimens (Fig. 3) represent in situ evolution following colonization. This hypothesis is supported by the geographic distribution of the ancient daughter lineages, which are unique to each Pacific island group, and the elevated frequency of lineages with the four diagnostic SNPs in the eastern Pacific (SI Appendix, Fig. S10). Although mtDNA is maternally inherited as a single genetic locus, limiting the ability to recover complex colonization histories, our data establish clear hypotheses that can be tested with genomic data from both modern and ancient chickens, and other groups such as humans, commensals, and other domesticates. It is important to note that in situations like the Pacific, phylogeographic signals in domestic species are likely to represent processes of initial human dispersal and later trade patterns. Our findings contrast substantially with previous studies (14⇓–16), which we suggest stems from our strict adherence to contamination reduction measures—for example, the use of Shrimp DNase. By removing a key source of potential contamination with domestic chicken DNA (PCR reagents), the use of Shrimp DNase has allowed us to recharacterize the crucial ancient Rapa Nui sample from a prior study (PAQANA011) as haplogroup D and not, as previously reported, haplogroup E. Consequently, we cast doubt on the authenticity of other haplogroup E sequences reported from ancient Pacific chicken specimens, where such procedures were not used. Perhaps more importantly, we suggest it will be very difficult to categorically rule out contamination as the source of haplogroup E sequences in ancient samples, due to the sporadic presence of domesticate DNA in laboratory consumables (34) and the likelihood that any such contamination would result in haplogroup E sequences. Importantly, sequencing longer stretches of such contaminating templates (17) does not provide any additional support for authenticity. Our recharacterization of the Rapa Nui PAQANA011 specimen as haplogroup D has implications for the other E sequences reported by Storey et al. (14), including the putative ancient Chilean chicken sequence from El Arenal-1 used to propose a prehistoric link between Polynesia and South America. Coalescent simulations using “all ancient haplogroups” and the modern data found that a European–South America route was more likely than a direct link between haplogroup E chicken sequences in Polynesia and South America, due to the phylogeographic signals within the worldwide dataset showing more similarities between chickens from Europe and South America. Perhaps more generally, these findings highlight how haplogroup E sequences are uninformative in nature and lack phylogeographic signal worldwide. A clear understanding of the nature and extent of Polynesian contact with South America will require genomic analyses of both ancient and modern populations of humans, commensals, and domesticates. The distribution of the nine D haplotypes currently known to share the ancestral motif provides a unique genetic signature that can be used to trace the human dispersal of chickens through ISEA and the Pacific islands. Our reconstruction of the chicken colonization history of Micronesia highlights how simulations with CR data can provide sufficient phylogeographic signal to generate new hypotheses regarding trade and migration scenarios. Although it has been proposed that many commensals and domesticates are late arrivals to the Micronesian islands compared with humans (35), we have reconstructed a link between chickens from islands in the Bismarck Sea and Micronesia that dates to ∼3,850 B.P. Such an early date is broadly consistent with archaeological evidence for human settlement of Saipan at 3,300–3,500 B.P. (36) and Palau at almost 4,000 B.P. (35), however few comparably early zoo-archaeological remains have been found in Micronesia to date (10, 13, 37). The inferred link between chickens from the Bismarcks and Micronesia without subsequent eastward movement does not support a two-wave model of Polynesian origins (14, 15, 38) where an earlier Lapita migration wave (2,800–3,500 ya) was mixed with a second, later wave moving through Micronesia to Western Polynesia (1,500–2,000 ya). Our simulations suggest that there was little interaction between chickens from Micronesia and the islands further eastward. One caveat concerning the power of the simulation analysis is the small number of Micronesian samples [one ancient Fais (16) and five modern Guam (26) specimens] and the expected historical and recent turnover of chicken populations in the region. Reassuringly, the ancient Fais haplotype H260 is present in modern chickens from the Santa Cruz (n = 2) and Solomon Islands (n = 5), apparently surviving any later introgression. Our reconstruction of the colonization history of Micronesian chickens demonstrates the potential power of coalescent simulations to test hypothesized migration and trade routes in archaeology and anthropology. The only ISEA location where the ancestral SNP motif has been detected are Camiguin and Manila in the Philippines, and a link with this area is consistent with other lines of evidence about early Polynesian origins (3, 4, 31). The other Philippine chicken haplotypes are spread throughout the haplogroup D network (Fig. 3), reflecting relatively high genetic diversity (haplotype diversity = 0.89; SI Appendix, Table S9). Despite extensive European settlement in the Pacific region over the last few centuries, many native chicken populations appear to contain relatively high frequencies of founding mitochondrial lineages—for example, the Marquesas, Solomon Islands, Vanuatu (26), and the Santa Cruz Islands—suggesting a high level of genetic continuity on these islands since prehistoric times. In addition to the two ancient haplotypes detected in modern samples, many other D haplotypes are also present in modern Pacific chicken populations, from the Santa Cruz Islands, Solomon Islands, and Vanuatu (26). Therefore, Polynesian chickens may be one of the few examples where ancestral genetic patterns can still be observed in a domesticated species. Chickens on remote Pacific islands may also contain Polynesian nuclear genomic lineages, and if so, would represent one of the few surviving examples of precolonial domestic chickens.

Conclusion Although mtDNA lacks the power of genomic loci to reconstruct complex evolutionary histories, we show that an informative region of the chicken mitochondrial genome can be used to trace their human dispersal in the Pacific. The analysis of ancient and modern specimens reveals a unique Polynesian genetic signature, which can be traced back to ISEA, and promises to allow further resolution of migration and trading routes in the area. Importantly, we reveal that a previously reported connection between pre-European South America and Polynesian chickens most likely resulted from contamination with modern DNA, and that this issue is likely to confound ancient DNA studies involving haplogroup E chicken sequences. These observations reaffirm the potential of coalescent simulations of genetic data to evaluate new hypotheses regarding the dispersal of humans, commensals, and domesticates derived from archaeology. These hypotheses can be further grounded using genomic-scale studies in combination with direct dating and genetic investigation of new archaeological samples.

Acknowledgments We thank Jessica Metcalf, Peggy Macqueen, and other members of the Australian Centre for Ancient DNA for assistance; John Terrell for manuscript discussions; Richard Walter (Department of Anthropology, University of Otago) and Atholl Anderson (Australian National University) for providing access to the Niue samples; and Will Millard for collecting modern feather samples from New Guinea. G.L. and K.D. also thank Atholl Anderson, Hanneke Boon, James Wharram, Klaus Hymphendahl, Matt Fletcher, Ingo Isensee, and the Lapita Expedition for collecting feather samples from ISEA and Western Polynesia. This work was funded by the Australian Research Council and the University of Adelaide.