Ayta Negritos: Highest Levels of Denisovan DNA

FIG. 2.



—Results of ADMIXTURE assuming k = 2 to k = 7. Each vertical line represents an individual and the different colors represent various ancestry components. Negrito group labels are indicated in red text.

Phylogenetic Relationships
To investigate the relationships among Negritos, we constructed a Neighbor-Joining (NJ) tree (fig. 3A). This tree shows that following the split from Europeans, the Papuans/Melanesians, Andamanese, Malaysian, and Philippine Negritos all appear basal to other Southeast and East Asian populations, in that branching order. The Neighbor-Net network in supplementary figure S7 in the Supplementary Material online shows a relatively long split (in blue color) that groups the Andamanese and Malaysian Negritos while a short split (in red color) groups the Andamanese and Philippine Negritos together. There is another split that separates all three groups of Negritos (except Batak) from the remaining populations. These splits suggest some shared genetic components that may not be immediately evident from other methods such as phylogenetic trees or PCA.

FIG. 3.



(A) Neighbor-joining tree constructed from Nei’s standard genetic distance. (B) Maximum-likelihood tree generated using Treemix, assuming five geneflow events, with only three shown for clarity. Numbers in red and blue text represent average migration weights and bootstrap supports for branches out of 1,000 bootstrap replicates, respectively. Only bootstrap values >50% are shown.

The treemix result assuming five gene flow events is shown in figure 3B. For visual clarity, only three gene flow events are shown. The gene flow from Denisovan to Papuan was observed 811 times out of 1,000 bootstrap replicates, and is consistent with previous reports (Reich et al. 2011; Meyer et al. 2012; Malaspinas et al. 2016). Interestingly, a separate gene flow event from Denisovan to some Philippine Negritos (Agta and Aeta) was also inferred. Out of 1,000 bootstrap replicates, this gene flow event was observed 733 times. Assuming the migration weight is analogous to gene flow proportion, the estimated gene flow from Denisovan to Papuan and from Denisovan to Philippine Negritos were 4.6% and 1.4%, respectively. The gene flow event with the highest migration weight was from the Malaysian non-Negritos to the Malaysian Negritos, at 47%. However, it was only observed 69 times out of 1,000 bootstrap replicates. In the remaining bootstrap replicates, the gene flow directions were from various positions along the internal branches of the Southeast/East Asian cluster towards Malaysian Negritos. Other inferred events involved gene flows from the French to Cambodians and from Malaysian Negritos to non-Negrito Southeast Asians and Han Chinese (supplementary fig. S8, Supplementary Material online). The topology of the treemix output was different from the NJ tree which assumed no gene flow (fig. 3A). The low bootstrap probabilities suggest that the treemix tree topology may not be reliable.

Discussion
This study presents an encompassing view of early human migrations into Southeast Asia (SEA) by analyzing genomic data of Negrito groups from three geographical regions. Although most of these Negrito groups currently reside in rural and sometimes inaccessible areas, they are far from being genetic isolates, as shown by PCA, ADMIXTURE, and D-statistics analyses. A clear difference was observed between the Mamanwa individuals from this sampling and the ones collected fairly recently (<10 years ago) on the Neighbor-Net network (supplementary fig. S11, Supplementary Material online). Recently sampled Mamanwa were closer to the Manobo, a non-Negrito group. This suggests admixture has been and probably still is occurring between the Mamanwa and Manobo. Alternatively it may imply a bias in sampling of individuals that represent a population. The consequence of this admixture is also reflected in mtDNA and Y-chromosomal diversity of the Philippine Negritos (Delfin et al. 2011, 2014).

An early study in the 1980s based on classic genetic markers hypothesized a dual origin of the Philippine Negrito populations. Genetic distance and phylogenetic analyses indicated that the Mamanwa were distinct from the Aeta and other populations. Together with the fact that the mean stature of Mamanwas was taller than the Aeta, it was postulated that while the Aeta originated from Sundaland, the Mamanwa had roots in Wallacea, south of Mindanao (Omoto 1984). In this study, however, this hypothesis was not substantiated by the genome-wide phylogenetic analyses.

Divergence time estimates using an LD based method yielded similar results even when using different SNP data sets. However, our divergence time estimates might possibly be underestimated. In equations Ne = 1/(4c) * [(1/r2)−2] and T = 2Ne*Fst (24), where Ne is effective population size, c is recombination distance between loci, r2 is the measure of LD, and T is divergence time in generations, the estimate of T decreases with increasing LD (r2). However, large blocks of SNPs in high LD in populations that have experienced extreme genetic drift from severe bottlenecks and/or recent admixture, may lead to an underestimation of T. Indeed our results suggest that Malaysian and Philippine Negritos did experience admixture with their neighboring non-Negrito populations (figs. 1 and 2; supplementary fig. 6, Supplementary Material online). We tried to mitigate the effects of recent admixture by filtering out admixed individuals based on PCA and ADMIXTURE results.

Both our NJ (fig. 3A) and UPGMA (supplementary fig. S10, Supplementary Material online) trees show that after divergence from Europeans, the ancestral Asians subsequently split into Papuans, Negritos and East Asians, implying a one-wave colonization of Asia. This is in agreement with a previous survey using a less dense SNP microarray (HUGO Pan-Asian SNP Consortium 2009) and another study using genome sequences of Andamanese individuals (Mondal et al. 2016). This is in contrast to the study based on whole genome sequences that suggested Australian Aboriginal/Papuan first split from European/East Asians 60 ka, and later Europeans and East Asians diverged 40 ka (Malaspinas et al. 2016). This implies a two-wave migration into Asia (Reich et al. 2011), and other studies using uniparental loci and HLA genes also seem to support that idea (Oppenheimer 2012; Di et al. 2015). Although our results appear to support the single-wave model, care should be taken in interpreting the NJ and UPGMA trees, because they do not assume gene flow after population differentiation. It may be possible that East Asians and Negritos appear close in the phylogenetic tree as a result of a long-term admixture. The method applied in the treemix software (Pickrell and Pritchard 2012) may be suitable for inferring gene flow events within a phylogenetic tree, but the tree topology assuming gene flow has very low bootstrap support (fig. 3B).

We did not observe any direct links between the different Negrito groups and the African Pygmies (Biaka) (fig. 3B and supplementary fig. S12, Supplementary Material online). This is in agreement with previous results (Omoto et al. 1978, 1981; Basu et al. 2015) and suggests that observed morphological similarities among the Negritos and African pygmies are more likely due to convergent evolution. The Neighbor-Net network (supplementary fig. S7, Supplementary Material online) suggests possible common links among all three Negrito groups (except highly admixed Batak in the Palawan Island), which are not obvious in methods like PCA and ADMIXTURE (figs. 1 and 2). Previous SNP analyses suggested a link between the Andamanese and Malaysian Negritos (Chaubey and Endicott 2013; Aghakhanian et al. 2015), but studies that have shown links among all the three Negrito groups are few (Reich et al. 2011). We identified 4,313 SNPs that could probably represent genomic regions that are shared among the common ancestors of the three Negrito groups. Some of these regions may be related to the common phenotypes—such as skin pigmentation, height, and facial morphology—apparent in all Negritos (Stokowski et al. 2007; Wood et al. 2014; Adhikari et al. 2016). These SNPs are also found in IL4 and CDH13 genes which are related to malarial resistance and were candidates for positive selection in the Malaysian Negritos (Liu et al. 2015). These observed patterns may have resulted from natural selection or alternatively via genetic drift, and further analyses are required to confirm or exclude either case.

Previous studies have reported that Papuans, Melanesians, and Australian Aboriginal retain high proportions of Denisovan ancestry, ranging from 3% to 6% (Reich et al. 2011; Meyer et al. 2012; Malaspinas et al. 2016). Here we report that the Aeta have the highest proportion of Denisovan ancestry among the four Philippine Negrito groups but Andamanese and Malaysian Negritos show very low signals (<1%) of Denisovan introgression. To explain these observations, we propose four possible scenarios of Denisovan introgression (supplementary fig. S13, Supplementary Material online). Two scenarios (differing by invoking either a single wave or two waves of migration to Asia) involve a single introgression event occurred in the common ancestor of Papuans and Negritos, followed by parallel losses in Andamanese, Malaysian Negritos and East Asians (supplementary fig. S13A and B, Supplementary Material online). The differences in the distribution of Denisovan blocks between Papuans and Philippine Negritos (fig. 5) may be attributed to genetic drift or subsequent gene flow from populations that initially lacked Denisovan ancestry, such as non-Negrito Austronesian-speaking groups (supplementary fig. S6, Supplementary Material online).

Alternatively, independent episodes of Denisovan admixture may have occurred in Papuan and Philippine Negrito lineages (supplementary fig. S13C and D, Supplementary Material online). If the Denisovan admixture took place in the common ancestor of Papuans and Aeta, it must have occurred after 50 ka, which is our estimate of Papuan-European divergence. Indeed, Malaspinas et al. (2016) estimated that the Denisovan admixture happened 44 ka in the Papuan/Australian Aborigine common ancestor. Although multiple episodes of Denisovan introgression appears to be the most parsimonious model, parallel loss of Denisovan ancestry in multiple lineages could also occur via genetic drift or purifying selection if the Denisovan genetic components are deleterious, as has been argued for Neanderthal introgression (Sankararaman et al. 2014).

In summary, we demonstrated that the Negritos of Andaman Islands, Malay Peninsula, and Philippine Islands represent one of the earliest branches of anatomically modern humans to have reached SEA, befitting the term the “First Sundaland People” instead of “Negritos.” The interactions they had with the environment, the pre-existing archaic humans in the region, and much later with agriculturalist migrants from the Asian mainland have all shaped their current genetic and cultural diversity.