Geometric morphometrics and paleoproteomics enlighten the paleodiversity of Pongo.

Pleistocene Pongo teeth show substantial variation in size and morphology, fueling taxonomic debates about the paleodiversity of the genus. We investigated prominent features of the enamel-dentine-junction junction (EDJ)-phylogenetically informative internal structures-of 71 fossil Pongo lower molars from various sites by applying geometric morphometrics and conducted paleoproteomic analyses from enamel proteins to attempt to identify extinct orangutan species. Forty-three orangutan lower molars representing Pongo pygmaeus and Pongo abelii were included for comparison. The shape of the EDJ was analyzed by placing five landmarks on the tip of the main dentine horns, and 142 semilandmarks along the marginal ridges connecting the dentine horns. Paleoproteomic analyses were conducted on 15 teeth of Late Pleistocene Pongo using high-resolution tandem mass spectrometry. The geometric morphometric results show variations in EDJ shape regarding aspects of the height and position of the dentine horns and connecting ridges. Despite the issue of molar position and sample size, modern molars are distinguished from fossil counterparts by their elongated tooth outline and narrowly positioned dentine horns. Proteomic results show that neither a distinction of P. pygmaeus and P. abelii, nor a consistent allocation of fossil specimens to extant species is feasible. Based on the EDJ shape, the (late) Middle to Late Pleistocene Pongo samples from Vietnam share the same morphospace, supporting the previous allocation to P. devosi, although substantial overlap with Chinese fossils could also indicate close affinities with P. weidenreichi. The hypothesis that both species represent one chronospecies cannot be ruled out. Two fossil specimens, one from Tam Hay Marklot (Laos, Late Pleistocene), and another from Sangiran (Java, Early to Middle Pleistocene), along with some specimens within the Punung sample (Java), exhibit affinities with Pongo abelii. The Punung fossils might represent a mix of early Late Pleistocene and later specimens (terminal Pleistocene to Holocene) related to modern Pongo. The taxonomy and phylogeny of the complete Punung sample needs to be further investigated.

Palaeoenvironments and hominin evolutionary dynamics in southeast Asia.

Secure environmental contexts are crucial for hominin interpretation and comparison. The discovery of a Denisovan individual and associated fauna at Tam Ngu Hao 2 (Cobra) Cave, Laos, dating back to 164-131 ka, allows for environmental comparisons between this (sub)tropical site and the Palearctic Denisovan sites of Denisova Cave (Russia) and Baishiya Karst Cave (China). Denisovans from northern latitudes foraged in a mix of forested and open landscapes, including tundra and steppe. Using stable isotope values from the Cobra Cave assemblage, we demonstrate that, despite the presence of nearby canopy forests, the Denisovan individual from Cobra Cave primarily consumed plants and/or animals from open forests and savannah. Using faunal evidence and proxy indicators of climates, results herein highlight a local expansion of rainforest at ~ 130 ka, raising questions about how Denisovans responded to this local climate change. Comparing the diet and habitat of the archaic hominin from Cobra Cave with those of early Homo sapiens from Tam Pà Ling Cave (46-43 ka), Laos, it appears that only our species was able to exploit rainforest resources.

Early presence of Homo sapiens in Southeast Asia by 86-68 kyr at Tam Pà Ling, Northern Laos.

The timing of the first arrival of Homo sapiens in East Asia from Africa and the degree to which they interbred with or replaced local archaic populations is controversial. Previous discoveries from Tam Pà Ling cave (Laos) identified H. sapiens in Southeast Asia by at least 46 kyr. We report on a recently discovered frontal bone (TPL 6) and tibial fragment (TPL 7) found in the deepest layers of TPL. Bayesian modeling of luminescence dating of sediments and U-series and combined U-series-ESR dating of mammalian teeth reveals a depositional sequence spanning ~86 kyr. TPL 6 confirms the presence of H. sapiens by 70 ± 3 kyr, and TPL 7 extends this range to 77 ± 9 kyr, supporting an early dispersal of H. sapiens into Southeast Asia. Geometric morphometric analyses of TPL 6 suggest descent from a gracile immigrant population rather than evolution from or admixture with local archaic populations.

Tooth enamel nitrogen isotope composition records trophic position: a tool for reconstructing food webs.

Nitrogen isotopes are widely used to study the trophic position of animals in modern food webs; however, their application in the fossil record is severely limited by degradation of organic material during fossilization. In this study, we show that the nitrogen isotope composition of organic matter preserved in mammalian tooth enamel (δ15Nenamel) records diet and trophic position. The δ15Nenamel of modern African mammals shows a 3.7‰ increase between herbivores and carnivores as expected from trophic enrichment, and there is a strong positive correlation between δ15Nenamel and δ15Nbone-collagen values from the same individuals. Additionally, δ15Nenamel values of Late Pleistocene fossil teeth preserve diet and trophic level information, despite complete diagenetic loss of collagen in the same specimens. We demonstrate that δ15Nenamel represents a powerful geochemical proxy for diet that is applicable to fossils and can help delineate major dietary transitions in ancient vertebrate lineages.

Dietary strategies of Pleistocene Pongo sp. and Homo erectus on Java (Indonesia).

During the Early to Middle Pleistocene, Java was inhabited by hominid taxa of great diversity. However, their seasonal dietary strategies have never been explored. We undertook geochemical analyses of orangutan (Pongo sp.), Homo erectus and other mammalian Pleistocene teeth from Sangiran. We reconstructed past dietary strategies at subweekly resolution and inferred seasonal ecological patterns. Histologically controlled spatially resolved elemental analyses by laser-based plasma mass spectrometry confirmed the preservation of authentic biogenic signals despite the effect of spatially restricted diagenetic overprint. The Sr/Ca record of faunal remains is in line with expected trophic positions, contextualizing fossil hominid diet. Pongo sp. displays marked seasonal cycles with ~3 month-long strongly elevated Sr/Ca peaks, reflecting contrasting plant food consumption presumably during the monsoon season, while lower Sr/Ca ratios suggest different food availability during the dry season. In contrast, omnivorous H. erectus shows low and less accentuated intra-annual Sr/Ca variability compared to Pongo sp., with δ13C data of one individual indicating a dietary shift from C4 to a mix of C3 and C4 plants. Our data suggest that H. erectus on Java was maximizing the resources available in more open mosaic habitats and was less dependent on variations in seasonal resource availability. While still influenced by seasonal food availability, we infer that H. erectus was affected to a lesser degree than Pongo sp., which inhabited monsoonal rain forests on Java. We suggest that H. erectus maintained a greater degree of nutritional independence by exploiting the regional diversity of food resources across the seasons.

A Middle Pleistocene Denisovan molar from the Annamite Chain of northern Laos.

The Pleistocene presence of the genus Homo in continental Southeast Asia is primarily evidenced by a sparse stone tool record and rare human remains. Here we report a Middle Pleistocene hominin specimen from Laos, with the discovery of a molar from the Tam Ngu Hao 2 (Cobra Cave) limestone cave in the Annamite Mountains. The age of the fossil-bearing breccia ranges between 164-131 kyr, based on the Bayesian modelling of luminescence dating of the sedimentary matrix from which it was recovered, U-series dating of an overlying flowstone, and U-series-ESR dating of associated faunal teeth. Analyses of the internal structure of the molar in tandem with palaeoproteomic analyses of the enamel indicate that the tooth derives from a young, likely female, Homo individual. The close morphological affinities with the Xiahe specimen from China indicate that they belong to the same taxon and that Tam Ngu Hao 2 most likely represents a Denisovan.

Trophic ecology of a Late Pleistocene early modern human from tropical Southeast Asia inferred from zinc isotopes.

Tam Pà Ling, a cave site in northeastern Laos, has yielded the earliest skeletal evidence of Homo sapiens in mainland Southeast Asia. The reliance of Pleistocene humans in rainforest settings on plant or animal resources is still largely unstudied, mainly due to poor collagen preservation in fossils from tropical environments precluding stable nitrogen isotope analysis, the classical trophic level proxy. However, isotopic ratios of zinc (Zn) in bioapatite constitute a promising proxy to infer trophic and dietary information from fossil vertebrates, even under adverse tropical taphonomic conditions. Here, we analyzed the zinc isotope composition (66Zn/64Zn expressed as δ66Zn value) in the enamel of two teeth of the Late Pleistocene (63-46 ka) H. sapiens individual (TPL1) from Tam Pà Ling, as well as 76 mammal teeth from the same site and the nearby Nam Lot cave. The human individual exhibits relatively low enamel δ66Zn values (+0.24‰) consistent with an omnivorous diet, suggesting a dietary reliance on both plant and animal matter. These findings offer direct evidence of the broad utilization of resources from tropical rainforests by one of the earliest known anatomically modern humans in Southeast Asia.

A multi-proxy approach to exploring Homo sapiens' arrival, environments and adaptations in Southeast Asia.

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.

Zinc isotopes in Late Pleistocene fossil teeth from a Southeast Asian cave setting preserve paleodietary information.

Stable carbon and nitrogen isotope ratios of collagen from bone and dentin have frequently been used for dietary reconstruction, but this method is limited by protein preservation. Isotopes of the trace element zinc (Zn) in bioapatite constitute a promising proxy to infer dietary information from extant and extinct vertebrates. The 66Zn/64Zn ratio (expressed as δ66Zn value) shows an enrichment of the heavy isotope in mammals along each trophic step. However, preservation of diet-related δ66Zn values in fossil teeth has not been assessed yet. Here, we analyzed enamel of fossil teeth from the Late Pleistocene (38.4-13.5 ka) mammalian assemblage of the Tam Hay Marklot (THM) cave in northeastern Laos, to reconstruct the food web and assess the preservation of original δ66Zn values. Distinct enamel δ66Zn values of the fossil taxa (δ66Zncarnivore < δ66Znomnivore < δ66Znherbivore) according to their expected feeding habits were observed, with a trophic carnivore-herbivore spacing of +0.60‰ and omnivores having intermediate values. Zn and trace element concentration profiles similar to those of modern teeth also indicate minimal impact of diagenesis on the enamel. While further work is needed to explore preservation for settings with different taphonomic conditions, the diet-related δ66Zn values in fossil enamel from THM cave suggest an excellent long-term preservation potential, even under tropical conditions that are well known to be adverse for collagen preservation. Zinc isotopes could thus provide a new tool to assess the diet of fossil hominins and associated fauna, as well as trophic relationships in past food webs.

Enamel proteome shows that Gigantopithecus was an early diverging pongine.

Gigantopithecus blacki was a giant hominid that inhabited densely forested environments of Southeast Asia during the Pleistocene epoch1. Its evolutionary relationships to other great ape species, and the divergence of these species during the Middle and Late Miocene epoch (16-5.3 million years ago), remain unclear2,3. Hypotheses regarding the relationships between Gigantopithecus and extinct and extant hominids are wide ranging but difficult to substantiate because of its highly derived dentognathic morphology, the absence of cranial and post-cranial remains1,3-6, and the lack of independent molecular validation. We retrieved dental enamel proteome sequences from a 1.9-million-year-old G. blacki molar found in Chuifeng Cave, China7,8. The thermal age of these protein sequences is approximately five times greater than that of any previously published mammalian proteome or genome. We demonstrate that Gigantopithecus is a sister clade to orangutans (genus Pongo) with a common ancestor about 12-10 million years ago, implying that the divergence of Gigantopithecus from Pongo forms part of the Miocene radiation of great apes. In addition, we hypothesize that the expression of alpha-2-HS-glycoprotein, which has not been previously observed in enamel proteomes, had a role in the biomineralization of the thick enamel crowns that characterize the large molars in Gigantopithecus9,10. The survival of an Early Pleistocene dental enamel proteome in the subtropics further expands the scope of palaeoproteomic analysis into geographical areas and time periods previously considered incompatible with the preservation of substantial amounts of genetic information.

A distinguishing feature of Pongo upper molars and its implications for the taxonomic identification of isolated hominid teeth from the Pleistocene of Asia.

OBJECTIVES: The taxonomic status of isolated hominoid teeth from the Asian Pleistocene has long been controversial due to difficulties distinguishing between pongine and hominin molars given their high degree of morphometrical variation and overlap. Here, we combine nonmetric and geometric morphometric data to document a dental pattern that appears to be taxonomically diagnostic among Pongo. We focus on the protoconule, a cuspule of well-documented evolutionary history, as well as on shape differences of the mesial fovea of the upper molars. MATERIALS AND METHODS: We examined 469 upper molars of eight hominid genera (Australopithecus, Paranthropus, Homo, Meganthropus, Sivapithecus, Pan, Gorilla, and Pongo), including representatives of Homo erectus and extinct and recent Pongo. Analyses were conducted at the enamel-dentine junction to overcome the limitations introduced by dental wear. RESULTS: We found that a moderate or large protoconule is present in ~80% of Pleistocene and extant Pongo. Conversely, a moderate to pronounced protoconule in hominins, Meganthropus, and African great apes occurs in low frequencies (~0-20%). Canonical variate analyses for the mesial fovea show that Pleistocene and extant Pongo cluster together and are clearly differentiated from all other groups, except for Sivapithecus. DISCUSSION: This study suggests that the protoconule and the shape of the mesial fovea in upper molars are useful features for the taxonomic identification of isolated hominid teeth. By identifying these new features, our results can contribute to the better understanding of hominoid evolutionary history and biogeography during the Asian Pleistocene. However, we emphasize that the reported features should be used in combination with other diagnostic variables for the most accurate taxonomic assessments.

Evidence for increased hominid diversity in the Early to Middle Pleistocene of Indonesia.

Since the first discovery of Pithecanthropus (Homo) erectus by E. Dubois at Trinil in 1891, over 200 hominid dentognathic remains have been collected from the Early to Middle Pleistocene deposits of Java, Indonesia, forming the largest palaeoanthropological collection in South East Asia. Most of these fossils are currently attributed to H. erectus. However, because of the substantial morphological and metric variation in the Indonesian assemblage, some robust specimens, such as the partial mandibles Sangiran 5 and Sangiran 6a, were formerly variably allocated to other taxa (Meganthropus palaeojavanicus, Pithecanthropus dubius, Pongo sp.). To resolve the taxonomic uncertainty surrounding these and other contentious Indonesian hominid specimens, we used occlusal fingerprint analysis (OFA) to reconstruct their chewing kinematics; we also used various morphometric approaches based on microtomography to examine the internal dental structures. Our results confirm the presence of Meganthropus as a Pleistocene Indonesian hominid distinct from Pongo, Gigantopithecus and Homo, and further reveal that Dubois's H. erectus paratype molars from 1891 are not hominin (human lineage), but instead are more likely to belong to Meganthropus.

The prehistoric peopling of Southeast Asia.

The human occupation history of Southeast Asia (SEA) remains heavily debated. Current evidence suggests that SEA was occupied by Hòabìnhian hunter-gatherers until ~4000 years ago, when farming economies developed and expanded, restricting foraging groups to remote habitats. Some argue that agricultural development was indigenous; others favor the "two-layer" hypothesis that posits a southward expansion of farmers giving rise to present-day Southeast Asian genetic diversity. By sequencing 26 ancient human genomes (25 from SEA, 1 Japanese Jomon), we show that neither interpretation fits the complexity of Southeast Asian history: Both Hòabìnhian hunter-gatherers and East Asian farmers contributed to current Southeast Asian diversity, with further migrations affecting island SEA and Vietnam. Our results help resolve one of the long-standing controversies in Southeast Asian prehistory.

Dental phenotypic shape variation supports a multiple dispersal model for anatomically modern humans in Southeast Asia.

The population history of anatomically modern humans (AMH) in Southeast Asia (SEA) is a highly debated topic. The impact of sea level variations related to the Last Glacial Maximum (LGM) and the Neolithic diffusion on past population dispersals are two key issues. We have investigated competing AMH dispersal hypotheses in SEA through the analysis of dental phenotype shape variation on the basis of very large archaeological samples employing two complementary approaches. We first explored the structure of between- and within-group shape variation of permanent human molar crowns. Second, we undertook a direct test of competing hypotheses through a modeling approach. Our results identify a significant LGM-mediated AMH expansion and a strong biological impact of the spread of Neolithic farmers into SEA during the Holocene. The present work thus favors a "multiple AMH dispersal" hypothesis for the population history of SEA, reconciling phenotypic and recent genomic data.

Early modern humans and morphological variation in Southeast Asia: fossil evidence from Tam Pa Ling, Laos.

Little is known about the timing of modern human emergence and occupation in Eastern Eurasia. However a rapid migration out of Africa into Southeast Asia by at least 60 ka is supported by archaeological, paleogenetic and paleoanthropological data. Recent discoveries in Laos, a modern human cranium (TPL1) from Tam Pa Ling's cave, provided the first evidence for the presence of early modern humans in mainland Southeast Asia by 63-46 ka. In the current study, a complete human mandible representing a second individual, TPL 2, is described using discrete traits and geometric morphometrics with an emphasis on determining its population affinity. The TPL2 mandible has a chin and other discrete traits consistent with early modern humans, but it retains a robust lateral corpus and internal corporal morphology typical of archaic humans across the Old World. The mosaic morphology of TPL2 and the fully modern human morphology of TPL1 suggest that a large range of morphological variation was present in early modern human populations residing in the eastern Eurasia by MIS 3.

Anatomically modern human in Southeast Asia (Laos) by 46 ka.

Uncertainties surround the timing of modern human emergence and occupation in East and Southeast Asia. Although genetic and archeological data indicate a rapid migration out of Africa and into Southeast Asia by at least 60 ka, mainland Southeast Asia is notable for its absence of fossil evidence for early modern human occupation. Here we report on a modern human cranium from Tam Pa Ling, Laos, which was recovered from a secure stratigraphic context. Radiocarbon and luminescence dating of the surrounding sediments provide a minimum age of 51-46 ka, and direct U-dating of the bone indicates a maximum age of ~63 ka. The cranium has a derived modern human morphology in features of the frontal, occipital, maxillae, and dentition. It is also differentiated from western Eurasian archaic humans in aspects of its temporal, occipital, and dental morphology. In the context of an increasingly documented archaic-modern morphological mosaic among the earliest modern humans in western Eurasia, Tam Pa Ling establishes a definitively modern population in Southeast Asia at ~50 ka cal BP. As such, it provides the earliest skeletal evidence for fully modern humans in mainland Southeast Asia.

Discovery of a second human molar and cranium fragment in the late Middle to Late Pleistocene cave of Ma U'Oi (Northern Vietnam).

In November 2002, during the second season of work by a Vietnamese-French-Japanese team, we discovered a human molar and a fragment of an occipital bone in the late Middle to Late Pleistocene cave of Ma U'Oi (Bacon et al., Geobios. 37 (2004) 305). The layer from which this material comes is the same as that in which a human lower molar was found in 2001. Both molars can be attributed to archaic Homo, and both exhibit archaic and modern traits.