Investigating demic versus cultural diffusion and sex bias in the spread of Austronesian languages in Vietnam.

Austronesian (AN) is the second-largest language family in the world, particularly widespread in Island Southeast Asia (ISEA) and Oceania. In Mainland Southeast Asia (MSEA), groups speaking these languages are concentrated in the highlands of Vietnam. However, our knowledge of the spread of AN-speaking populations in MSEA remains limited; in particular, it is not clear if AN languages were spread by demic or cultural diffusion. In this study, we present and analyze new data consisting of complete mitogenomes from 369 individuals and 847 Y-chromosomal single nucleotide polymorphisms (SNPs) from 170 individuals from all five Vietnamese Austronesian groups (VN-AN) and five neighboring Vietnamese Austroasiatic groups (VN-AA). We found genetic signals consistent with matrilocality in some, but not all, of the VN-AN groups. Population affinity analyses indicated connections between the AN-speaking Giarai and certain Taiwanese AN groups (Rukai, Paiwan, and Bunun). However, overall, there were closer genetic affinities between VN-AN groups and neighboring VN-AA groups, suggesting language shifts. Our study provides insights into the genetic structure of AN-speaking communities in MSEA, characterized by some contact with Taiwan and language shift in neighboring groups, indicating that the expansion of AN speakers in MSEA was a combination of cultural and demic diffusion.

Metagenomic and paleopathological analyses of a historic documented collection explore ancient dental calculus as a diagnostic tool.

Dental calculus is a microbial biofilm that contains biomolecules from oral commensals and pathogens, including those potentially related to cause of death (CoD). To assess the utility of calculus as a diagnostically informative substrate, in conjunction with paleopathological analysis, calculus samples from 39 individuals in the Smithsonian Institution's Robert J. Terry Collection with CoDs of either syphilis or tuberculosis were assessed via shotgun metagenomic sequencing for the presence of Treponema pallidum subsp. pallidum and Mycobacterium tuberculosis complex (MTBC) DNA. Paleopathological analysis revealed that frequencies of skeletal lesions associated with these diseases were partially inconsistent with diagnostic criteria. Although recovery of T. p. pallidum DNA from individuals with a syphilis CoD was elusive, MTBC DNA was identified in at least one individual with a tuberculosis CoD. The authenticity of MTBC DNA was confirmed using targeted quantitative PCR assays, MTBC genome enrichment, and in silico bioinformatic analyses; however, the lineage of the MTBC strain present could not be determined. Overall, our study highlights the utility of dental calculus for molecular detection of tuberculosis in the archaeological record and underscores the effect of museum preparation techniques and extensive handling on pathogen DNA preservation in skeletal collections.

Combining machine learning and optimization for the operational patient-bed assignment problem.

Assigning inpatients to hospital beds impacts patient satisfaction and the workload of nurses and doctors. The assignment is subject to unknown inpatient arrivals, in particular for emergency patients. Hospitals, therefore, need to deal with uncertainty on actual bed requirements and potential shortage situations as bed capacities are limited. This paper develops a model and solution approach for solving the patient bed-assignment problem that is based on a machine learning (ML) approach to forecasting emergency patients. First, it contributes by improving the anticipation of emergency patients using ML approaches, incorporating weather data, time and dates, important local and regional events, as well as current and historical occupancy levels. Drawing on real-life data from a large case hospital, we were able to improve forecasting accuracy for emergency inpatient arrivals. We achieved up to 17% better root mean square error (RMSE) when using ML methods compared to a baseline approach relying on averages for historical arrival rates. We further show that the ML methods outperform time series forecasts. Second, we develop a new hyper-heuristic for solving real-life problem instances based on the pilot method and a specialized greedy look-ahead (GLA) heuristic. When applying the hyper-heuristic in test sets we were able to increase the objective function by up to 5.3% in comparison to the benchmark approach in [40]. A benchmark with a Genetic Algorithm shows also the superiority of the hyper-heuristic. Third, the combination of ML for emergency patient admission forecasting with advanced optimization through the hyper-heuristic allowed us to obtain an improvement of up to 3.3% on a real-life problem.

Natural products from reconstructed bacterial genomes of the Middle and Upper Paleolithic.

Major advances over the past decade in the field of ancient DNA are providing access to past paleogenomic diversity, but the diverse functions and biosynthetic capabilities of this growing paleome remain largely elusive. We investigated the dental calculus of 12 Neanderthals and 52 anatomically modern humans ranging from 100,000 years ago to the present and reconstructed 459 bacterial metagenome-assembled genomes. We identified a biosynthetic gene cluster shared by seven Middle and Upper Paleolithic individuals that allows for the heterologous production of a class of previously unknown metabolites that we name "paleofurans." This paleobiotechnological approach demonstrates that viable biosynthetic machinery can be produced from the preserved genetic material of ancient organisms, allowing access to natural products from the Pleistocene and providing a promising area for natural product exploration.

Ancient genomes from the last three millennia support multiple human dispersals into Wallacea.

Previous research indicates that human genetic diversity in Wallacea-islands in present-day Eastern Indonesia and Timor-Leste that were never part of the Sunda or Sahul continental shelves-has been shaped by complex interactions between migrating Austronesian farmers and indigenous hunter-gatherer communities. Yet, inferences based on present-day groups proved insufficient to disentangle this region's demographic movements and admixture timings. Here, we investigate the spatio-temporal patterns of variation in Wallacea based on genome-wide data from 16 ancient individuals (2600-250 years BP) from the North Moluccas, Sulawesi and East Nusa Tenggara. While ancestry in the northern islands primarily reflects contact between Austronesian- and Papuan-related groups, ancestry in the southern islands reveals additional contributions from Mainland Southeast Asia that seem to predate the arrival of Austronesians. Admixture time estimates further support multiple and/or continuous admixture involving Papuan- and Asian-related groups throughout Wallacea. Our results clarify previously debated times of admixture and suggest that the Neolithic dispersals into Island Southeast Asia are associated with the spread of multiple genetic ancestries.

Stone Age Yersinia pestis genomes shed light on the early evolution, diversity, and ecology of plague.

The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague's formative years in terms of its early evolution and ecology.

Ancient Metagenomic Studies: Considerations for the Wider Scientific Community.

Like modern metagenomics, ancient metagenomics is a highly data-rich discipline, with the added challenge that the DNA of interest is degraded and, depending on the sample type, in low abundance. This requires the application of specialized measures during molecular experiments and computational analyses. Furthermore, researchers often work with finite sample sizes, which impedes optimal experimental design and control of confounding factors, and with ethically sensitive samples necessitating the consideration of additional guidelines. In September 2020, early career researchers in the field of ancient metagenomics met (Standards, Precautions & Advances in Ancient Metagenomics 2 [SPAAM2] community meeting) to discuss the state of the field and how to address current challenges. Here, in an effort to bridge the gap between ancient and modern metagenomics, we highlight and reflect upon some common misconceptions, provide a brief overview of the challenges in our field, and point toward useful resources for potential reviewers and newcomers to the field.

PyDamage: automated ancient damage identification and estimation for contigs in ancient DNA de novo assembly.

DNA de novo assembly can be used to reconstruct longer stretches of DNA (contigs), including genes and even genomes, from short DNA sequencing reads. Applying this technique to metagenomic data derived from archaeological remains, such as paleofeces and dental calculus, we can investigate past microbiome functional diversity that may be absent or underrepresented in the modern microbiome gene catalogue. However, compared to modern samples, ancient samples are often burdened with environmental contamination, resulting in metagenomic datasets that represent mixtures of ancient and modern DNA. The ability to rapidly and reliably establish the authenticity and integrity of ancient samples is essential for ancient DNA studies, and the ability to distinguish between ancient and modern sequences is particularly important for ancient microbiome studies. Characteristic patterns of ancient DNA damage, namely DNA fragmentation and cytosine deamination (observed as C-to-T transitions) are typically used to authenticate ancient samples and sequences, but existing tools for inspecting and filtering aDNA damage either compute it at the read level, which leads to high data loss and lower quality when used in combination with de novo assembly, or require manual inspection, which is impractical for ancient assemblies that typically contain tens to hundreds of thousands of contigs. To address these challenges, we designed PyDamage, a robust, automated approach for aDNA damage estimation and authentication of de novo assembled aDNA. PyDamage uses a likelihood ratio based approach to discriminate between truly ancient contigs and contigs originating from modern contamination. We test PyDamage on both on simulated aDNA data and archaeological paleofeces, and we demonstrate its ability to reliably and automatically identify contigs bearing DNA damage characteristic of aDNA. Coupled with aDNA de novo assembly, Pydamage opens up new doors to explore functional diversity in ancient metagenomic datasets.

Reconstruction of ancient microbial genomes from the human gut.

Loss of gut microbial diversity1-6 in industrial populations is associated with chronic diseases7, underscoring the importance of studying our ancestral gut microbiome. However, relatively little is known about the composition of pre-industrial gut microbiomes. Here we performed a large-scale de novo assembly of microbial genomes from palaeofaeces. From eight authenticated human palaeofaeces samples (1,000-2,000 years old) with well-preserved DNA from southwestern USA and Mexico, we reconstructed 498 medium- and high-quality microbial genomes. Among the 181 genomes with the strongest evidence of being ancient and of human gut origin, 39% represent previously undescribed species-level genome bins. Tip dating suggests an approximate diversification timeline for the key human symbiont Methanobrevibacter smithii. In comparison to 789 present-day human gut microbiome samples from eight countries, the palaeofaeces samples are more similar to non-industrialized than industrialized human gut microbiomes. Functional profiling of the palaeofaeces samples reveals a markedly lower abundance of antibiotic-resistance and mucin-degrading genes, as well as enrichment of mobile genetic elements relative to industrial gut microbiomes. This study facilitates the discovery and characterization of previously undescribed gut microorganisms from ancient microbiomes and the investigation of the evolutionary history of the human gut microbiota through genome reconstruction from palaeofaeces.

Initial Upper Palaeolithic humans in Europe had recent Neanderthal ancestry.

Modern humans appeared in Europe by at least 45,000 years ago1-5, but the extent of their interactions with Neanderthals, who disappeared by about 40,000 years ago6, and their relationship to the broader expansion of modern humans outside Africa are poorly understood. Here we present genome-wide data from three individuals dated to between 45,930 and 42,580 years ago from Bacho Kiro Cave, Bulgaria1,2. They are the earliest Late Pleistocene modern humans known to have been recovered in Europe so far, and were found in association with an Initial Upper Palaeolithic artefact assemblage. Unlike two previously studied individuals of similar ages from Romania7 and Siberia8 who did not contribute detectably to later populations, these individuals are more closely related to present-day and ancient populations in East Asia and the Americas than to later west Eurasian populations. This indicates that they belonged to a modern human migration into Europe that was not previously known from the genetic record, and provides evidence that there was at least some continuity between the earliest modern humans in Europe and later people in Eurasia. Moreover, we find that all three individuals had Neanderthal ancestors a few generations back in their family history, confirming that the first European modern humans mixed with Neanderthals and suggesting that such mixing could have been common.

Community-curated and standardised metadata of published ancient metagenomic samples with AncientMetagenomeDir.

Ancient DNA and RNA are valuable data sources for a wide range of disciplines. Within the field of ancient metagenomics, the number of published genetic datasets has risen dramatically in recent years, and tracking this data for reuse is particularly important for large-scale ecological and evolutionary studies of individual taxa and communities of both microbes and eukaryotes. AncientMetagenomeDir (archived at https://doi.org/10.5281/zenodo.3980833 ) is a collection of annotated metagenomic sample lists derived from published studies that provide basic, standardised metadata and accession numbers to allow rapid data retrieval from online repositories. These tables are community-curated and span multiple sub-disciplines to ensure adequate breadth and consensus in metadata definitions, as well as longevity of the database. Internal guidelines and automated checks facilitate compatibility with established sequence-read archives and term-ontologies, and ensure consistency and interoperability for future meta-analyses. This collection will also assist in standardising metadata reporting for future ancient metagenomic studies.

Ancient DNA from Guam and the peopling of the Pacific.

Humans reached the Mariana Islands in the western Pacific by ∼3,500 y ago, contemporaneous with or even earlier than the initial peopling of Polynesia. They crossed more than 2,000 km of open ocean to get there, whereas voyages of similar length did not occur anywhere else until more than 2,000 y later. Yet, the settlement of Polynesia has received far more attention than the settlement of the Marianas. There is uncertainty over both the origin of the first colonizers of the Marianas (with different lines of evidence suggesting variously the Philippines, Indonesia, New Guinea, or the Bismarck Archipelago) as well as what, if any, relationship they might have had with the first colonizers of Polynesia. To address these questions, we obtained ancient DNA data from two skeletons from the Ritidian Beach Cave Site in northern Guam, dating to ∼2,200 y ago. Analyses of complete mitochondrial DNA genome sequences and genome-wide SNP data strongly support ancestry from the Philippines, in agreement with some interpretations of the linguistic and archaeological evidence, but in contradiction to results based on computer simulations of sea voyaging. We also find a close link between the ancient Guam skeletons and early Lapita individuals from Vanuatu and Tonga, suggesting that the Marianas and Polynesia were colonized from the same source population, and raising the possibility that the Marianas played a role in the eventual settlement of Polynesia.

Cultural variation impacts paternal and maternal genetic lineages of the Hmong-Mien and Sino-Tibetan groups from Thailand.

The Hmong-Mien (HM) and Sino-Tibetan (ST) speaking groups are known as hill tribes in Thailand; they were the subject of the first studies to show an impact of patrilocality vs. matrilocality on patterns of mitochondrial (mt) DNA vs. male-specific portion of the Y chromosome (MSY) variation. However, HM and ST groups have not been studied in as much detail as other Thai groups; here we report and analyze 234 partial MSY sequences (∼2.3 mB) and 416 complete mtDNA sequences from 14 populations that, when combined with our previous published data, provides the largest dataset yet for the hill tribes. We find a striking difference between Hmong and IuMien (Mien-speaking) groups: the Hmong are genetically different from both the IuMien and all other Thai groups, whereas the IuMien are genetically more similar to other linguistic groups than to the Hmong. In general, we find less of an impact of patrilocality vs. matrilocality on patterns of mtDNA vs. MSY variation than previous studies. However, there is a dramatic difference in the frequency of MSY and mtDNA lineages of Northeast Asian (NEA) origin vs. Southeast Asian (SEA) origin in HM vs. ST groups: HM groups have high frequencies of NEA MSY lineages but lower frequencies of NEA mtDNA lineages, while ST groups show the opposite. A potential explanation is that the ancestors of Thai HM groups were patrilocal, while the ancestors of Thai ST groups were matrilocal. Overall, these results attest to the impact of cultural practices on patterns of mtDNA vs. MSY variation.

CoproID predicts the source of coprolites and paleofeces using microbiome composition and host DNA content.

Shotgun metagenomics applied to archaeological feces (paleofeces) can bring new insights into the composition and functions of human and animal gut microbiota from the past. However, paleofeces often undergo physical distortions in archaeological sediments, making their source species difficult to identify on the basis of fecal morphology or microscopic features alone. Here we present a reproducible and scalable pipeline using both host and microbial DNA to infer the host source of fecal material. We apply this pipeline to newly sequenced archaeological specimens and show that we are able to distinguish morphologically similar human and canine paleofeces, as well as non-fecal sediments, from a range of archaeological contexts.

Comparison of extraction methods for recovering ancient microbial DNA from paleofeces.

OBJECTIVES: Paleofeces are valuable to archeologists and evolutionary biologists for their potential to yield health, dietary, and host information. As a rich source of preserved biomolecules from host-associated microorganisms, they can also provide insights into the recent evolution and changing ecology of the gut microbiome. However, there is currently no standard method for DNA extraction from paleofeces, which combine the dual challenges of complex biological composition and degraded DNA. Due to the scarcity and relatively poor preservation of paleofeces when compared with other archeological remains, it is important to use efficient methods that maximize ancient DNA (aDNA) recovery while also minimizing downstream taxonomic biases. METHODS: In this study, we use shotgun metagenomics to systematically compare the performance of five DNA extraction methods on a set of well-preserved human and dog paleofeces from Mexico (~1,300 BP). RESULTS: Our results show that all tested DNA extraction methods yield a consistent microbial taxonomic profile, but that methods optimized for ancient samples recover significantly more DNA. CONCLUSIONS: These results show promise for future studies that seek to explore the evolution of the human gut microbiome by comparing aDNA data with those generated in modern studies.

Sharing of heteroplasmies between human liver lobes varies across the mtDNA genome.

Mitochondrial DNA (mtDNA) heteroplasmy (intra-individual variation) varies among different human tissues and increases with age, suggesting that the majority of mtDNA heteroplasmies are acquired, rather than inherited. However, the extent to which heteroplasmic sites are shared across a tissue remains an open question. We therefore investigated heteroplasmy in two liver samples (one from each primary lobe) from 83 Europeans, sampled at autopsy. Minor allele frequencies (MAF) at heteroplasmic sites were significantly correlated between the two liver samples from an individual, with significantly more sharing of heteroplasmic sites in the control region than in the non-control region. We show that this increased sharing for the control region cannot be explained by recent mutations at just a few specific heteroplasmic sites or by the possible presence of 7S DNA. Moreover, we carried out simulations to show that there is significantly more sharing than would be predicted from random genetic drift from a common progenitor cell. We also observe a significant excess of non-synonymous vs. synonymous heteroplasmies in the protein-coding region, but significantly more sharing of synonymous heteroplasmies. These contrasting patterns for the control vs. the non-control region, and for non-synonymous vs. synonymous heteroplasmies, suggest that selection plays a role in heteroplasmy sharing.

Contrasting Paternal and Maternal Genetic Histories of Thai and Lao Populations.

The human demographic history of Mainland Southeast Asia (MSEA) has not been well studied; in particular, there have been very few sequence-based studies of variation in the male-specific portions of the Y chromosome (MSY). Here, we report new MSY sequences of ∼2.3 mB from 914 males and combine these with previous data for a total of 928 MSY sequences belonging to 59 populations from Thailand and Laos who speak languages belonging to three major Mainland Southeast Asia families: Austroasiatic, Tai-Kadai, and Sino-Tibetan. Among the 92 MSY haplogroups, two main MSY lineages (O1b1a1a* [O-M95*] and O2a* [O-M324*]) contribute substantially to the paternal genetic makeup of Thailand and Laos. We also analyze complete mitochondrial DNA genome sequences published previously from the same groups and find contrasting pattern of male and female genetic variation and demographic expansions, especially for the hill tribes, Mon, and some major Thai groups. In particular, we detect an effect of postmarital residence pattern on genetic diversity in patrilocal versus matrilocal groups. Additionally, both male and female demographic expansions were observed during the early Mesolithic (∼10 ka), with two later major male-specific expansions during the Neolithic period (∼4-5 ka) and the Bronze/Iron Age (∼2.0-2.5 ka). These two later expansions are characteristic of the modern Austroasiatic and Tai-Kadai groups, respectively, consistent with recent ancient DNA studies. We simulate MSY data based on three demographic models (continuous migration, demic diffusion, and cultural diffusion) of major Thai groups and find different results from mitochondrial DNA simulations, supporting contrasting male and female genetic histories.

Ancestral mitochondrial N lineage from the Neolithic 'green' Sahara.

Because Africa's climate hampers DNA preservation, knowledge of its genetic variability is mainly restricted to modern samples, even though population genetics dynamics and back-migrations from Eurasia may have modified haplotype frequencies, masking ancient genetic scenarios. Thanks to improved methodologies, ancient genetic data for the African continent are now increasingly available, starting to fill in the gap. Here we present newly obtained mitochondrial genomes from two ~7000-year-old individuals from Takarkori rockshelter, Libya, representing the earliest and first genetic data for the Sahara region. These individuals carry a novel mutation motif linked to the haplogroup N root. Our result demonstrates the presence of an ancestral lineage of the N haplogroup in the Holocene "Green Sahara", associated to a Middle Pastoral (Neolithic) context.

The role of matrilineality in shaping patterns of Y chromosome and mtDNA sequence variation in southwestern Angola.

Southwestern Angola is a region characterized by contact between indigenous foragers and incoming food-producers, involving genetic and cultural exchanges between peoples speaking Kx'a, Khoe-Kwadi, and Bantu languages. Although present-day Bantu speakers share a patrilocal residence pattern and matrilineal principle of clan and group membership, a highly stratified social setting divides dominant pastoralists from marginalized groups that subsist on alternative strategies and have previously been thought to have pre-Bantu origins. Here, we compare new high-resolution sequence data from 2.3 Mb of the male-specific region of the Y chromosome (MSY) from 170 individuals with previously reported mitochondrial DNA (mtDNA) genomes, to investigate the population history of seven representative southwestern Angolan groups (Himba, Kuvale, Kwisi, Kwepe, Twa, Tjimba, !Xun), and to study the causes and consequences of sex-biased processes in their genetic variation. We found no clear link between the formerly Kwadi-speaking Kwepe and pre-Bantu eastern African migrants, and no pre-Bantu MSY lineages among Bantu-speaking groups, except for small amounts of "Khoisan" introgression. We therefore propose that irrespective of their subsistence strategies, all Bantu-speaking groups of the area share a male Bantu origin. Additionally, we show that in Bantu-speaking groups, the levels of among-group and between-group variation are higher for mtDNA than for MSY. These results, together with our previous demonstration that the matriclanic systems of southwestern Angolan Bantu groups are genealogically consistent, suggest that matrilineality strongly enhances both female population sizes and interpopulation mtDNA variation.