A multi-proxy approach to exploring Homo sapiens’ arrival, environments and adaptations in Southeast Asia
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Published: 26 October 2021
A multi-proxy approach to exploring Homo sapiens’ arrival, environments and adaptations in Southeast Asia
Anne-Marie Bacon, Nicolas Bourgon, Frido Welker, Enrico Cappellini, Denis Fiorillo, Olivier Tombret, Nguyen Thi Mai Huong, Nguyen Anh Tuan, Thongsa Sayavonkhamdy, Viengkeo Souksavatdy, Phonephanh Sichanthongtip, Pierre-Olivier Antoine, Philippe Duringer,
Jean-Luc Ponche, Kira Westaway, Renaud Joannes-Boyau, Quentin Boesch, Eric Suzzoni, Sébastien Frangeul, Elise Patole-Edoumba, Alexandra Zachwieja, Laura Shackelford, Fabrice Demeter, Jean-Jacques Hublin & Élise Dufour
Scientific Reports volume 11, Article number: 21080 (2021) Cite this article

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Abstract
The capability of Pleistocene hominins to successfully adapt to different types of tropical forested environments has long been debated. In order to investigate environmental changes in Southeast Asia during a critical period for the turnover of hominin
species, we analysed palaeoenvironmental proxies from five late Middle to Late Pleistocene faunas. Human teeth discoveries have been reported at Duoi U’Oi, Vietnam (70–60 ka) and Nam Lot, Laos (86–72 ka). However, the use of palaeoproteomics
allowed us to discard the latter, and, to date, no human remains older than ~ 70 ka are documented in the area. Our findings indicate that tropical rainforests were highly sensitive to climatic changes over that period, with significant fluctuations
of the canopy forests. Locally, large-bodied faunas were resilient to these fluctuations until the cooling period of the Marine Isotope Stage 4 (MIS 4; 74–59 ka) that transformed the overall biotope. Then, under strong selective pressures, populations
with new phenotypic characteristics emerged while some other species disappeared. We argue that this climate-driven shift offered new foraging opportunities for hominins in a novel rainforest environment and was most likely a key factor in the settlement
and dispersal of our species during MIS 4 in SE Asia.

Introduction
Although the earliest forms of Homo occupied diverse C3-C4 environmental niches in Africa1, the genus is generally seen as primarily being adapted to open environments2,3. In Asia, early Homo erectus likely inhabited areas devoid of forests along river
valleys in north China and Java and a niche partitioning between archaic humans and other large primates living in heavily forested habitats has been proposed4,5,6,7,8.

During the Late Pleistocene, the Far East witnessed a major turnover of hominins with the extinction of the last H. erectus in Indonesia8, the likely presence of the last Denisovans in several parts of the continent9 and eventually the replacement of all
archaic groups following the arrival of Homo sapiens10. On a continental scale, it has been suggested that the shift from open habitats (mixed savannah and woodland) to rainforest habitats at the transition between the Middle Pleistocene and the Late
Pleistocene triggered the decline of archaic hominins, unable to adapt to these new environments11.

Determining the palaeoenvironmental context facing different hominin species in Southeast (SE) Asia thus has the potential to feed into the debates relating to the uniqueness of our species. However, in the Pleistocene tropical Indochinese subregion,
rare dental remains tentatively assigned to hominins have often been reinterpreted as remains of great apes (mainly orangutans of the Pongo genus)5,12,13,14, making it difficult to firmly associate hominins with records of past vegetation in many cases.

The dispersal route of H. sapiens towards southern China likely crossed Indochina15, but the timing of this event, its process —one or several waves possibly since ~ 100 thousand years ago (ka)16,17—and how H. sapiens adapted to rainforest
environments remain unresolved. Certainly, the paucity of detailed chronology for several SE Asian sites contributes to obscuring our understanding of the period. To date, the earliest indisputable archaeological evidence of hominin adaptation to Asian
tropical rainforests is actually quite recent and dated to ~ 73–63 ka in Lida Ajer, Sumatra18.

Here, we seek to try and address some of these crucial issues for the understanding of the evolution of our species by analysing five mammalian faunas from Vietnam and Laos, whose age ranges fall within different Marine Isotope Stages (MIS): Coc Muoi (
148–117 ka, MIS 6–5), Tam Hang South (94–60 ka, MIS 5–4), Nam Lot (86–72 ka, MIS 5), Duoi U’Oi (70–60 ka, MIS 4) and Tam Hay Marklot (38.4–13.5 ka, MIS 3–2)19,20,21 (Fig. 1). From a palaeoecological point of view, the crown dimensions
and stable isotopic measurements of identified taxa from these faunas are proxies for environmental reconstruction7,11,20,22,23,24 and a primary source of information on biotas occupied by hominins. In the area studied, the earliest occurrence of H.
sapiens is documented by skeletal remains of several individuals from ~ 70 ka at Tam Pà Ling (~ 70–46 ka25,26) and by two teeth at Duoi U’Oi (70–60 ka22). However, an older putative hominin specimen associated with the Nam Lot assemblage (86�
��72 ka22) opens the possibility of an even earlier arrival27. We thus used palaeoproteomics28,29,30 to resolve the specific assignment of this specimen based on its dental enamel proteome, with the goal of better contextualizing the arrival of modern
humans locally.

Figure 1
figure1
(A) Location of sites in northern Laos (Tam Hang South, Nam Lot, and Tam Hay Marklot) and northern Vietnam (Coc Muoi and Duoi U’Oi). (B) Sanbao, Dongee, and Hulu Chinese caves δ18O records showing millennial-scale climate shifts related to changes in
East Asian summer monsoon intensity for the last 224 ka (modified after87). The decreases in δ18O values (‰, VPDB, left ordinate axis) correspond to increases in precipitation, i.e., the amount effect77. The right ordinate axis corresponds to the
Northern Hemisphere summer insolation (65°N, W m−2). The age intervals of the faunas have been placed below the curve of δ18O records, from the oldest (right) to the youngest (left).

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During the late Middle to the Late Pleistocene, local faunas were composed of a large proportion of modern taxa associated with a few archaic taxa23,31,32,33,34,35. Overall, the faunas were similar to those of other continents at the time as they were
dominated by megaherbivores (> 1000 kg up to 5000 kg36), including elephant, stegodon, giant tapir, and several species of rhinoceroses and large bovids37. This similarity also extends to the local association of species with different ecologies and to
their discrepancy with present-day spatial distributions (e.g., orangutans with pandas or tigers with hyenas). Most palaeontologists now consider that this unexpected association of species, named non-analogue faunas (38,39,40,41,42, but see Ref.43),
results from the different responses of species to environmental changes that is, in an “individualistic manner”— according to their life-histories44,45,46,47.

Using classical zooarchaeological approaches, the analysis of SE Asian mammalian faunas for palaeoecological reconstructions has failed to detect major functional changes in mammalian communities37,48,49, with the palaeontological record appearing to be
somewhat uniform. Indeed, in this tropical area, the species display broad ecological ranges, both latitudinal and altitudinal. Still, the question remains as to how species survived climatic shifts during the Pleistocene and adapted to non-analogue
climates, exhibiting different sets of environmental variables (structure of the vegetation, rates of insolation, seasonality, amount of rainfall, etc.) than those of today50,51. Furthermore, the evolution of various lineages at the infraspecific level
is generally unknown because movements of populations are seldom traceable in fossil records. An additional limitation of the studies of species dynamics in tropical Asia is the absence of preserved DNA in the fossil remains, which prevents the
reconstruction of the genetic history of mammalian lineages. In Eurasia, most molecular analyses of ancient DNA (aDNA) focused on Beringian faunas, emphasising that the Late Pleistocene was a dynamic period for cold-adapted mammals influenced by climate
changes52,53,54,55,56,57,58,59,60,61,62,63,64. These studies demonstrate various processes (i.e., contraction of populations, local extinction, migration, replacement by new populations, or interspecies competition) resulting in the success of new and
better-adapted populations over time47,65,66,67. In the meantime, the influence of climate cooling on warm-adapted populations remains largely unknown.

Using morphometric and particularly isotopic proxies from teeth, it is however possible to address various environmental issues and specifically to assess the effects of large climate oscillations on rainforest ecosystems at a regional scale and their
impacts on the mammalian communities and associated hominins, as indicated by the growing body of research20,22,24,68,69,70,71,72,73.

Our dataset contains several hundreds of isolated teeth of mammals belonging to six mammalian Orders, i.e., Artiodactyla, Perissodactyla, Proboscidea, Carnivora, Primates, and large Rodentia (Methods, Supplementary Materials and Methods, Supplementary
Figs. S1–S3, Supplementary Tables S3–S4). All sites are located in a narrow latitudinal belt between 23° and 20° running through the northern regions of Laos and Vietnam (Fig. 1A). The location of the sites minimizes the variations in species body
size due to abiotic parameters related to latitudinal distribution, i.e., cline effect (temperature, distance from the coast, rain seasonality, amount of rainfall). However, the five sites are located at various altitudes, ranging from lowland sites at
the level of the alluvial plain (Duoi U’Oi) to medium mountain sites (Nam Lot and Tam Hang South).

First, we compared carbon (δ13Capatite) and oxygen (δ18O) isotope measurements from dental enamel from a corpus of 335 specimens belonging to a large spectrum of taxa, using new data from Coc Muoi, Duoi U’Oi, and Tam Hang South, and those already
published of Nam Lot and Tam Hay Marklot20,22. We estimated the δ13Ccarbon source values in the diet of animals to specifically analyse the changes in proportions of C3-plants (trees, bushes, shrubs, and grasses) versus C4-plants (grasses, sedges) over
the studied period74. The δ13C of bioapatite allows the reconstruction of palaeoenvironments based on these isotopically-distinct carbon sources. We also used the δ13Ccarbon source to differentiate the C3 canopy forests from other C3 forested habitats
to reveal local fluctuations of the tropical rainforests in relation to climatic changes75,76. δ18O values were used to provide additional palaeoecological information related to variation in abiotic conditions (latitude, climate, temperature, moisture
content, amount, and isotopic composition of precipitation)77,78,79,80,81,82,83,84 (“Methods”).

Additionally, we used original morphometric data -the dental crown area of 213 specimens belonging to five taxa among herbivores and omnivores, the sambar deer (Rusa unicolor), the muntjac (Muntiacus sp.), the serow (Capricornis sumatraensis), the boar (
Sus scrofa), and the macaque (Macaca sp.)- to detect significant phenotypic changes through time within their lineages. Combining the proxies based on stable isotope data with these morphometric data enabled us to identify which climate shifts had the
most substantial impact on the mammalian communities in relation to rainforest dynamics.

Our fourth proxy takes into account the type of digestive physiology, using the ratio of ruminants versus hindgut fermenting herbivores by body mass category42, as an indicator of the expansion of open landscapes (primarily through the occurrence of
exclusive grazing taxa) and therefore the contraction of rainforests.

Finally, we discuss how the climate changes that occurred during the Late Pleistocene might have influenced the adaptation of the first H. sapiens locally, and more widely in the SE Asian region. For that purpose, we used available climatic records, e.g.,
pollen data85,86, and Chinese caves δ18O data from speleothems87, as other relevant sources of information.

Results
Rejection of an early Homo species presence at Nam Lot
MS/MS spectra unambiguously assign the Nam Lot incisor (NL 433) to the genus Pongo (orangutans) with no unique and high-confidence matches to the genus Homo29,30. For those positions where we have proteomic data for the Nam Lot specimen, no sequence
differences exist between Pongo abelii and P. pygmaeus in our reference sequences. As a result, we assign the specimen to the genus Pongo without further species specification (Supplementary Methods and Results).

Stable isotope data
The δ13Csource and δ18Oapatite values of specimens belonging to all taxonomic groups are shown in Figs. 2 and 3 and Supplementary Tables S5–S7. The values (δ13Capatite, δ13Ccarbon source, and δ18Oapatite) for all specimens and reference standards
are presented in Supplementary Annexes S1–S2. With regards to the new data measured here, the δ13Ccarbon source values for Coc Muoi, Duoi U’Oi and Tam Hang South range from − 33.8 to − 18.1 ‰ (average δ13Ccarbon source = − 28.0 ± 2.
4 ‰ (1 σ), n = 84), − 34.3 to − 15.1 ‰ (average δ13Ccarbon source = − 28.4 ± 3.0 ‰ (1 σ), n = 60) and − 30.0 to − 12.0 ‰ (average δ13Ccarbon source = − 25.0 ± 3.6 ‰ (1 σ), n = 62), respectively (
Fig. 2).

Figure 2
figure2
Histogram distribution of the relative frequency (%) in δ13Ccarbon source values for all taxa in the five SE Asian faunas, following a chronological sequence from the oldest (left) to the youngest (right). Each bin represents a spacing of 1‰. Shaded
areas represent δ13Ccarbon source values associated with closed-canopy forests (δ13Ccarbon source < −27.2‰); intermediate rainforests and woodland biomes (δ13Ccarbon source >  − 27.2 ‰ and < − 21.3 ‰; and savannah-like
environments (δ13Ccarbon source > − 15.3‰). The white area (δ13Ccarbon source > − 21.3 ‰ and < − 15.3 ‰) consists of values resulting from the combined consumption of both C3 and C4 resources, and does not correspond to any
specific ecological environment. The dashed red line represents the mean δ13Ccarbon source value in each site.

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Figure 3
figure3
Distribution in δ18O values for all taxa in the five SE Asian faunas, following a chronological sequence from the oldest (left) to the youngest (right): Coc Muoi (), Tam Hang South (), Duoi U’Oi () and previously published data from Nam Lot () and Tam
Hay Marklot (). The outline of the violin plots represents kernel probability density, where the width shows the proportion of the data found there. The boxes from the box and whisker plots inside the violin plots represent the 25th–75th percentiles,
with the median as a bold horizontal line.

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The new δ18Oapatite values obtained for the three sites range from − 9.8 to − 3.9 ‰ (average δ18Oapatite = − 6.1 ± 1.2 ‰ (1 σ), n = 84), − 9.7 to − 2.5 ‰ (average δ18Oapatite = − 6.6 ± 1.3 ‰ (1 σ), n = 
60) and − 9.4 to − 2.8 ‰ (average δ18Oapatite = − 6.7 ± 1.4 ‰ (1 σ), n = 62), respectively for Coc Muoi, Duoi U’Oi and Tam Hang South (Fig. 3).

Statistically significant differences between sites, from both the novel (Coc Muoi, Tam Hang South, and Duoi U’Oi) and published data (Nam Lot and Tam Hay Marklot), were determined through Kruskal–Wallis one-way analysis of variance for δ13Ccarbon
source (H(4) = 83.3, p-value < 2.2e−16) and for δ18Oapatite (H(4) = 25.5, p-value = 4.019e−05). Post-hoc Dunn’s test pair-wise comparisons draw out the sites from Vietnam as distinct from the sites in Laos regarding their δ
13Ccarbon source values. Tam Hay Marklot and Nam Lot also appear to be significantly different from each other. Finally, δ18Oapatite values from Tam Hang South are identified as significantly different from those of all other sites except Duoi U’Oi,
while Duoi U’Oi itself is being drawn out as significantly different to Nam Lot and Tam Hay Marklot (Supplementary Tables S8, S9).

Broadly, the ranges and medians of δ18O values fluctuated (Fig. 3), in accordance with δ13Ccarbon source values (Fig. 2). However, the distribution of the δ13Ccarbon source values highlights that the C3 forested environments (canopy forests,
intermediate rainforests and woodlands) remained predominant over the period studied. Furthermore, when we look at the percentages of specimens according to the distribution of δ13Ccarbon source values associated to the different biomes in Table 1, the
data demonstrate that environmental conditions changed significantly through the Coc Muoi—Tam Hay Marklot temporal series. Tropical forests were thus apparently sensitive to climate change. Our results particularly illustrate the dynamics of the canopy
forests (δ13Ccarbon source <  − 27.2 ‰), and show their potential for contraction across space and time: Coc Muoi (65.4%), Tam Hang South (27.4%), Nam Lot (42.1%), Duoi U’Oi (73.3%), and Tam Hay Marklot (26.3%).

Table 1 Percentage (%) and number of specimens (n/N) in the five faunas according to the distribution of δ13Ccarbon source values (‰ VPDB) from the oldest (top) to the youngest (bottom).
Full size table
Distribution of herbivore species by body mass and digestive strategy
The sequence of the faunas by body mass and digestive strategy is presented in Fig. 4 and Supplementary Table S10. In the three oldest faunas, hindgut fermenting taxa, i.e., non-ruminant taxa, including seven large herbivores (> 350 kg) and
megaherbivores (> 1000 kg) belonging to the following genera, Megatapirus, Tapirus, Stegodon, Elephas, Rhinoceros, and Dicerorhinus (vs. only one ruminant Bos species), dominated the biomass. Duoi U’Oi with a ratio “ruminant vs. non-ruminant taxa”
of 4:7 shows a change in the composition of megaherbivores with the absence of Megatapirus (> 350 kg) and Stegodon (> 1000 kg). However, hindgut fermenting herbivores remain predominant since the loss in the diversity of large-bodied archaic taxa is
not compensated by an increase in ruminants. Tam Hay Marklot marks a shift that represents small- to medium-sized ruminants (18 to 350 kg) (among which Rucervus eldii, Axis porcinus and Naemorhedus caudatus) becoming predominant (ratio ruminant vs. non-
ruminant taxa of 8:6). This trend apparently continued to the present, as seen in the increase of grazing species in current faunas at these latitudes (ratio 9:4) (Fig. 4).

Figure 4
figure4
Number of species by body mass category and digestive strategy in the five faunas, following a chronological sequence from the oldest (left) to the youngest (right). The ratio refers to the number of ruminants versus non-ruminant taxa. See Supplementary
Table S10 for the list of taxa within each body mass category.

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Crown area dimensions as a phenotypic signal
The dimensional ranges of crown areas of p3 (Rusa unicolor and Sus scrofa) and m3 (Macaca sp., Muntiacus sp., and Capricornis sumatraensis) differ between sites (Fig. 5A). However, statistical analyses were limited by unbalanced sample size for some
sites, and only R. unicolor (n = 65) and S. scrofa (n = 61) were analysed with the Kruskal–Wallis test (H(4) = 21.09, p-value = 0.0003 and H(4) = 14.25, p-value = 0.007, respectively). Post-hoc Dunn’s test pair-wise
comparisons draw out R. unicolor from Coc Muoi as significantly differing from those of Nam Lot (p-value < 0.005) and Duoi U’Oi (p-value < 0.05); and R. unicolor of Nam Lot differing from that of Tam Hay Marklot (p < 0.05). S. scrofa
samples also show significant differences between populations (p-value < 0.05): Coc Muoi vs. Nam Lot; Tam Hang South vs. Duoi U’Oi; Nam Lot vs. Duoi U’Oi and Tam Hay Marklot (Supplementary Tables S11 and S12).

Figure 5
figure5
Distribution of crown area dimensions (A) and δ13Ccarbon source values (B) in five taxa (the boxes represent the 25th–75th percentiles, median and whisker plots), following a chronological sequence from the oldest (top) to the youngest (bottom). CM
Coc Muoi, THS Tam Hang South, NL Nam Lot, DU Duoi U’Oi, THM Tam Hay Marklot. See Supplementary Table S13 for the number of specimens.

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Overall, there is an appearance of cumulative effects from Coc Muoi to Nam Lot, whereby populations follow a directional evolution towards either a greater (R. unicolor, S. scrofa, and Macaca sp.) or a smaller (Muntiacus sp. and C. sumatraensis) crown
area surface according to the taxon. In the overall faunal sequence, Duoi U’Oi marks a shift with a change in this directional evolution. This shift is particularly notable in S. scrofa, but the five taxa studied seemingly appear affected by this
reversal of dimensions in lineages (Fig. 5A). This reversal is used here as a signal indicating that populations with new phenotypic characteristics emerged, either due to adaptation or replacement of populations (through extinction or assimilation), in
the face of high selective pressures. Therefore, in the Coc Muoi—Tam Hay Marklot temporal series, Duoi U’Oi seems to mark some kind of turnover in populations.

Discussion
From the available record discussed here, no clear human presence in the area prior to ~ 70 ka can be demonstrated. However, the ability to obtain protein sequence information from tropical areas30 and to distinguish between Pongo and Homo, as shown
by the results of our palaeoproteomic analysis of the Nam Lot incisor (86–72 ka), opens up the possibility to directly address the question of early H. sapiens presence in SE Asia in the future.

The relative similarity in δ13Ccarbon source and δ18O values between Coc Muoi (148–117 ka) and Duoi U’Oi (70–60 ka) suggests that climatic conditions induced a C3-dominated ecosystem in two distinct periods. As shown in the curves of the Sanbao/
Hulu δ18O Chinese caves records87 in Fig. 1B, and considering the age ranges of the faunas, the predominance of these forested ecosystems could be associated with two high-amplitude drops in monsoon intensity, during MIS 6 for Coc Muoi (MIS 6.288,89)
and during MIS 4 for Duoi U’Oi (Fig. 2 in87). In Coc Muoi and Duoi U’Oi, closed rainforests contained most of the mammalian biomass composed primarily of browsers weighing up to ~ 5000 kg (Fig. 4 and Supplementary Table S10). However, on closer
inspection, the two sites reveal marked differences in the species relying on canopy forests for their diet (Fig. 6). Firstly, Duoi U’Oi marks a decline in the diversity of megaherbivores with the absence of two archaic taxa: the giant tapir
Megatapirus augustus and the proboscidean Stegodon orientalis. Both sites are situated in the same vegetation zone < 400 m above sea level (asl), and other sources of variability are reduced, supporting the hypothesis of a predominant climatic effect
on mammalian communities.

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