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Post by Admin on May 13, 2021 21:01:29 GMT
Results Preservation of Oral Microbiota in Dental Calculus. Authenticating ancient DNA (aDNA) preservation is a necessary and essential step for all paleogenomic studies. However, these methods have been underdeveloped for ancient microbiomes. Here, we apply a multistep procedure of both conventional and new methods to evaluate and validate oral microbiome preservation in our dataset (SI Appendix, Fig. S2). First, we applied a reference-based metagenomic binning of reads to the National Center for Biotechnology Information (NCBI) nucleotide (nt) database (27) (Dataset S2) and then developed and applied a method to assess the decay of the cumulative percentage of known oral taxa in samples compared to a panel of oral and nonoral reference metagenomes (SI Appendix, Fig. S3A and section S3.4.1). This allowed us to remove samples that did not exhibit a taxonomic composition consistent with an oral origin. We then cross validated these results using SourceTracker (28) (SI Appendix, Fig. S3B) and inspection by principal coordinate analysis (PCoA, SI Appendix, Fig. S5 A–D). To samples exhibiting good oral microbiome preservation (Fig. 1B), we then applied the R package decontam (29) to detect and remove putative laboratory and environmental contaminants prior to downstream analysis (SI Appendix, section S3.6). Next, we examined each dataset and confirmed the presence of DNA damage characteristics of ancient samples, including short fragment lengths and elevated levels of cytosine to thymine deamination (Fig. 1C and SI Appendix, Fig. S4) (30). Finally, to reduce potentially spurious assignments for compositional analysis, we removed low-abundance taxa using thresholds optimized at different taxonomic levels (SI Appendix, Figs. S7 and S8 and sections S3.6 and S5.2). The resulting 89 well-preserved dental calculus datasets consist of samples ranging from the present day up to 100 ka. The Core African Hominid Oral Microbiome. We performed PCoA on our dataset of well-preserved samples and found considerable overlap in the microbial composition of African hominid dental calculus, as well as howler monkeys (Fig. 1B), suggesting the existence of a core microbiome that has been maintained for more than 8 My, based on fossil and molecular evidence of host divergence among African hominids (31, 32), and possibly since before the catarrhine–platyrrhine split ca. 40 Mya (33, 34). At the same time, small but significant differences were indicated by Permutational Multivariate Analysis of Variance (PERMANOVA) (35) at both the microbial genus and species levels between each hominid genus (100 bootstrap replicates, ɑ = 0.05; genus: F = 5.22 ± 1.42, df = 3, R2 = 0.27 ± 0.05, P = 0.001; species: F = 6.67 ± 2.52, df = 3, R2 = 0.32 ± 0.07, P = 0.001; SI Appendix, Fig. S5), and this pattern remained robust after controlling for unequal sample sizes (SI Appendix, section S4.2). Dental plaque biofilms in humans form by the microbial succession of early, bridging, and late colonizers (36), and in contrast to the gut, which has high interindividual variability at the microbial phylum level (37) and is sensitive to subsistence changes over short and long timescales (15, 38, 39), oral microbial communities have been found to be more stable and consistent, particularly at the genus level (40⇓–42), and even when challenged by antibiotics (43). Because of this, we sought to begin to define the African hominid core oral microbiome as a group and for each genus separately. For a microbial taxon to be considered “core” (44, 45), we required it to be present in at least two-thirds of the populations making up a given host genus, counting as present only those populations in which it is found in at least half of individuals to account for variation in preservation (SI Appendix, Fig. S9A and section S5.2). We then calculated the intersection of each core microbial genus (Fig. 2A) and species (Fig. 2B) across all host taxa (Dataset S3). Most “core” taxa are shared across all three African hominid genera (Gorilla, Pan, and Homo) and howler monkeys, whereas fewer are “core” only to African hominids (Gorilla, Pan, and Homo), Pan and Homo, or Homo (Fig. 2 and SI Appendix, Fig. S1). Despite smaller sample sizes than studies of present-day microbiomes, bootstrapping analysis to assess consistency of calculations supported most core microbiome assignments, with lower values possibly indicating taxa influenced by factors such as biofilm maturity (SI Appendix, section S5.3). This suggests a high degree of genus-level microbial taxonomic conservation during African hominid, and possibly broader primate, host evolution and speciation. Fig. 2. Core oral microbiome of African hominids shows a deep evolutionary conservation of biofilm structure. UpSet plots showing the number of microbial genera (A) and species (B) core to host groups and group combinations. (C) Core taxa of the human oral microbiome (inclusive of all African hominid and howler monkey ranks). Human biofilm spatial organization based on refs. 8 and 100. Taxa are colored by the broadest host group for which they are core. “Other” taxa are those that fall into paraphyletic host groupings (e.g., Alouatta:Homo). Dashed lines separate the biofilm into basal, intermediate, and peripheral regions (100). Taxa with unknown spatial location are marked with an asterisk (*); taxa core to Homo with any combination of other host genera at the species level but not at the genus level are marked with a dagger (†). Reference Dataset S3 for additional information. Core taxa at both the genus and species levels include well-known members of each stage of plaque biofilm formation (8, 36), including the early colonizers Streptococcus and Actinomyces, the bridging taxa Fusobacterium and Corynebacterium, and the late colonizers Porphyromonas and Treponema, although the latter two are “core” to only chimpanzees and Homo (Fig. 2C). Major periopathogens, bacteria associated with periodontal disease, are found among the different host core combinations, and, focusing on Porphyromonas and Tannerella specifically because of their clinical significance today, we find that their major virulence factors are shared across multiple primates and thus are not specific to modern humans (SI Appendix, Fig. S9 B and C). The presence of periopathogens within the core microbiome supports the hypothesis that they are not pathogens in a conventional sense but rather that their pathogenic character in present-day humans may be related to an imbalance between the biofilm and the host, as has been suggested by recent ecological studies (46). Although some of the African hominid “core” taxa are periopathogens or their close relatives, most core members are known today to play important structural and functional roles in the formation and maturation of plaque, implying deep coevolutionary relationships between these taxa and their hosts.
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Post by Admin on May 13, 2021 22:53:52 GMT
African Hominid Oral Microbiome Structure Shows a Weak Relationship with Host Phylogeny. Hierarchical clustering shows that calculus metagenomes tend to cluster by host genus, confirming intragroup similarity, but these relationships exhibit differences from host phylogeny (Fig. 3). We find, for example, that howler monkeys and gorillas fall together in a single clade and a subset of Homo clusters with chimpanzees. With respect to the latter, available metadata do not provide any clear associations with factors such as geography, time period, or disease to explain this pattern (SI Appendix, section S4.3). Overall, gorillas and howler monkeys are characterized by a wide diversity of aerobic and facultatively anaerobic taxa, while chimpanzees have higher levels of obligately anaerobic taxa, including many putative periopathogens (e.g., Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Filifactor alocis, and Fretibacterium fastidosum). Neanderthals consistently fall within the diversity of modern humans. Homo is notable for its high abundance of Streptococcus spp., while this genus is found at substantially lower levels in Pan. Fig. 3. African hominid dental calculus microbiomes cluster by host genus and other factors. Hierarchical clustering of howler monkeys, chimpanzees, gorillas, Neanderthals, and ancient and present-day modern humans based on species-level prokaryotic taxonomic assignments. Bacterial oxygen tolerance is associated with biofilm maturation stage in modern humans, and colored names indicate species corresponding to Socransky complexes (111) (reference SI Appendix section S5.1.1 for a summary). Microaerophilic is defined based on the BacDive database and is roughly synonymous to facultative anaerobe. The tree is schematic, and bifurcations are shown until all host genera are represented. Microbial species names are collapsed to genus level. Species and sample names can be located in SI Appendix, section S4.3. Many of the taxa identified in human and nonhuman primate dental calculus are poorly characterized, making further exploration difficult. Indeed, several species within the human core genera remain unnamed (Ottowia sp. oral taxon 894, Olsenella sp. oral taxon 807) or understudied (Pseudopropionibacterium propionicum, F. fastidiosum) and some even lack genus designations ([Eubacterium] minutum, TM7x, Anaerolinaceae bacterium oral taxon 439). Their absence from most discussions of the modern human oral microbiome points to a major gap in current oral microbiology research, and targeted investigation of these species is needed to identify their functional and structural roles within plaque biofilms (47⇓–49). Host genus patterns in community structure may be influenced by differences in salivary flow or composition (50), as well as differences in diet texture, quality, and nutrient content (51) (SI Appendix, sections S1, S5.1, and S5.6). We also investigated microbial community structure within modern humans, but in contrast to previous studies (13), we found no difference among broad dietary patterns or time periods (SI Appendix, Fig. S6 and section S4.5). These findings accord with the results of modern oral microbiome studies, which also show minimal, if any, broad and sustained compositional changes in response to diet (e.g., refs. 41, 52, and 53). The relative stability of the oral microbiome may be due in part to the extensive community interdependencies (54, 55) that have developed within these biofilms to metabolize complex host salivary glycoproteins, which are the major nutrient source for most members of the oral microbiota (56). This is in contrast to studies demonstrating strong associations between diet and taxonomic/functional composition in modern gut microbiomes (57, 58). Evolutionary Histories of Oral Microbial Species Reflect Homo Interactions. We next examined the phylogenies of individual microbial taxa to determine if host evolutionary relationships are reflected at the microbial genome level. To improve genome coverage and reduce potential noise from DNA damage, we selected a representative subset of well-preserved calculus samples across all host genera (n = 19; SI Appendix, Table S1 and Dataset S1) and constructed uracil-DNA glycosylase-treated (UDG) libraries to remove deaminated cytosines (59), which we then deeply sequenced and analyzed together with a subset of four of the present-day modern humans. Genome-level sequence reconstruction from diversity-rich ancient microbiomes is challenging due to both the highly fragmented nature of aDNA and the low relative abundance of each species, which makes strain separation difficult (SI Appendix, section S6). Furthermore, a lack of sufficient reference genomes for many commensal oral microbes increases mismapping and noise artifacts when identifying single-nucleotide polymorphisms (SNPs) (SI Appendix, Fig. S10). Nevertheless, despite these challenges and using representative genomes from core taxa, we were able to reconstruct phylogenetic trees with high bootstrap support on internal nodes for eight oral bacteria (Fig. 4 and SI Appendix, Fig. S11). Fig. 4. African hominid oral taxa cluster phylogenetically by host genus. Selected neighbor-joining SNP-based phylogenetic cladograms of representative core oral microbiome genomes from deep-sequenced calculus metagenomes (SI Appendix, section S6.6). Actinomyces and Tannerella trees are rooted on the branch leading to howler monkeys (Alouatta, blue), Fretibacterium tree is midpoint rooted. Positions refer to non-N nucleotide calls in the alignment. Node values represent node support out of 100 bootstrap replicates. Asterisk (*) represents the Upper Paleolithic individual from El Mirón (EMN001), which consistently falls near Neanderthal individuals. The remaining eight trees, with tip labels, are provided in SI Appendix, Fig. S11. As with compositional analysis, reconstructed genome-level sequences tend to cluster with those from the same host genus but do not closely reflect host phylogeny (Fig. 4 and SI Appendix, Figs. S11 and S12). Overall, genome-level sequences reconstructed from gorillas and chimpanzees fall closer to each other than do those of chimpanzees and Homo. Biases from the use of modern human-derived microbial reference genomes may in part contribute to this pattern, but microbial exchange due to overlapping territorial ranges of gorillas and chimpanzees throughout their evolution may also be a contributing factor. Within Homo, Neanderthals consistently group together, indicating shared within-species microbial diversity. However, we also note that the Upper Paleolithic individual from El Mirón in Iberia (18.6 ka) clusters in all trees with Neanderthals, rather than with other Pleistocene hunter-gatherers of the African Later Stone Age or more recent Holocene-era European or African populations. Recently published human genomic data including this individual has revealed that its associated genetic ancestry component was largely displaced across Europe after 14 ka (24, 60) during postglacial warming. Turning to our low-coverage metagenomic datasets, we assessed additional European Upper Paleolithic and Mesolithic groups (SI Appendix, section S6.6) and found that they show a similar pattern (albeit at lower resolution), with the oral taxa of individuals dated to before 14 ka mostly falling with Neanderthals and those after 14 ka mostly clustering with present-day modern humans (24, 60). This pattern suggests that the reconstructed oral bacterial genomes from El Mirón reflect a standing microbial diversity in Homo that was present in Europe during the Middle and Upper Paleolithic, but which was later replaced following subsequent migrations of modern human populations from elsewhere. Because oral microbiota are primarily inherited through caregivers (61, 62), additional sampling and ultradeep sequencing of Paleolithic European and Asian dental calculus may prove informative about the poorly understood interaction dynamics between archaic and modern humans.
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Post by Admin on May 14, 2021 20:03:07 GMT
Homo-Specific Shifts in Oral Biofilm Are Linked to Dietary Starch Availability. The metabolic potential of a microbial community, which is inferred from its total gene content, can offer insights into biofilm ecology and function that cannot be understood from taxonomy alone. To better characterize the metabolic and functional differences among hominid oral biofilms, we compared the gene content of dental calculus metagenomes from well-preserved samples of the larger sequencing dataset using two different methods of functional classification, HUMAnN2 (63) and AADDER (64), and found moderate concordance in overall results. Principal Components Analysis (PCA) of the protein-level functional assignments cluster host genera distinctly with a high degree of separation between hosts and functional content (Fig. 5A and SI Appendix, Figs. S13 and S14), whereas we observe only a moderate degree of separation in the taxonomic PCoA (Fig. 1B), suggesting that gene content of the taxa shared by hominids is more host-specific than taxonomic assignments, a pattern that has also been seen for other microbial systems (65). The genes that drive separation of Homo from nonhuman primates consistently relate to carbohydrate processing (SI Appendix, Fig. S14), are much more abundant in Homo, and largely derive from Streptococcus (SI Appendix, Fig. S13), something also observed in primate gut microbiomes (66). We therefore investigated the distribution of Streptococcus across our samples (Fig. 5B) using a classification system based on biochemical characteristics and genetic relatedness (67). We find that Streptococcus species belonging to the Mitis, Sanguinis, and Salivarius groups are dominant in Homo, while these same groups are effectively absent in chimpanzees, and nonhuman primates in general are characterized by much higher proportions of Streptococcus species in the Anginosus, Mutans, and Pyogenic groups (Fig. 5B). Fig. 5. Metabolic function and Streptococcus amylase-binding gene content is distinct between African hominid oral microbiomes. (A) PCA of microbial gene functions (SEED classification) clusters well-preserved samples by host genus (PERMANOVA R2 = 0.345). Homo is functionally distinct from nonhuman African hominids and howler monkeys, particularly with respect to carbohydrate metabolism (SI Appendix, Fig. S14). (B) Bar plot of proportion of alignments to different Streptococcus groups show differences between host genera. Color of squares below bars corresponds to legend in C. Amylase-binding activity has been observed among members of the Sanguinis, Mitis, and Salivarius groups (68). (C) Ratios of reads aligning to amylase-binding-protein annotated sequences versus a genus-wide Streptococcus “superreference” show higher values in Homo than nonhuman primates, based on a deep-sequenced subset of samples and four present-day modern humans. Note the ratio on the y-axes of abpA and abpB are scaled differently. The Mitis, Sanguinis, and Salivarius groups are notable for their ability to express amylase-binding proteins to capture salivary ɑ-amylase (68, 69), which they use for their own nutrient acquisition from dietary starch, as well as dental adhesion (70, 71). Amylase-binding protein genes (e.g., abpA and abpB) share no homology but rather confer a similar phenotype through convergent evolution, and they are found almost exclusively in oral Streptococcus species (68). Alpha-amylase is the most abundant enzyme in modern human saliva and modern humans express it at higher levels than any other hominid (50, 72). In contrast to most other nonhuman primates, modern humans exhibit high salivary ɑ-amylase (AMY1) copy number variation, with a reported range of up to 30 diploid copies (16, 73, 74). This copy number expansion is estimated to have occurred along the modern human lineage after the divergence from Neanderthals in the Middle Pleistocene (75, 76). It has been argued this increase relates to dietary shifts during the evolutionary history of modern humans and specifically to an increased reliance on starch-rich foods (20, 21). We next calculated the ratio of reads aligning to abpA and abpB sequences compared to all Streptococcus reads in the deep-sequenced dataset. We find that abpA and abpB reads are nearly absent in the nonhuman groups but are prevalent and significantly more abundant in Homo (Mann–Whitney U test Homo versus non-Homo: abpB, α = 0.05, U = 128, P = < 0.001, 95% CI = 0.686 to 0.851; abpA, α = 0.05, U = 112, P = < 0.001, 95% CI = 0.398 to 0.861). In particular, abpB is present in all deeply sequenced Homo individuals, and abpA is especially prevalent in modern humans (Fig. 5C). This suggests that oral streptococci evolved in association with changes in host diet and supports an early importance of starch-rich foods in Homo evolution.
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Post by Admin on May 14, 2021 22:40:17 GMT
Discussion Commensal microbes of the oral microbiome represent an underutilized and independent source of information about host evolutionary and ecological differences (15, 77). With generation times orders-of-magnitude shorter than their hosts and the ability to acquire new functions through horizontal gene transfer across distantly related groups, microbes are a particularly dynamic and temporally resolved system for understanding human evolution. After applying a rigorous strategy to identify, decontaminate, and authenticate well-preserved dental calculus specimens up to 100 ka, we identify a core group of 10 bacterial genera within the African hominid primate oral microbiome that are also shared with howler monkeys, suggesting that these microbial groups have played a key role in oral biofilms since before the catarrhine–platyrrhine split ca. 40 Mya (33, 34). Today, these core taxa are primarily involved in providing structural support within the dental plaque biofilm, and their study holds promise for understanding biofilm growth and maturation in the ancestral human microbiome (78, 79). Identifying the role of such taxa is critical for the successful long-term treatment, prevention, and control of dysbiotic biofilms, such as those found in dental and periodontal diseases (80).
Further, we identify 27 genus-level members of the Homo core oral microbiome, and these include many well-known and clinically relevant taxa, such as Streptococcus and the periopathogens P. gingivalis, T. forsythia, and T. denticola; however, nearly all of these are also core microbiome members of other African hominids. Only Veillonella parvula, a commensal species known to have a synergistic relationship with the cariopathogen Streptococcus mutans (81), is primarily found in humans. Surprisingly, not all members of the core Homo oral microbiome are well-known—three have no genus designation and several lack species names, revealing a major gap in oral microbiology research that in part relates to the difficulties in growing and propagating these microbes.
Focusing on oral microbiome evolution within Homo, we reconstruct authentic oral metagenomes of Neanderthals dating up to 100 ka and modern humans dating up to 30 ka, finding a high degree of similarity in microbial community structure, while also documenting indications of strain-level differences within core taxa. Interestingly, we find that Neanderthal-associated strain-level sequence variants are consistently present in Upper Paleolithic Europeans but not afterward, which accords with a described modern human genomic turnover around 14 ka (24, 60). Comparing human and nonhuman primates, we show that within Streptococcus, amylase-binding groups play a central role in the oral biofilms of Homo, likely aided by both their enhanced ability to colonize the dentition and their exclusive access to dietary starches. These Streptococcus groups and abpB are a general feature of Homo, suggesting that starch-rich foods, possibly modified by cooking (20) (SI Appendix, section S5.8), first became important early in Homo evolution prior to the split between Neanderthal and modern human lineages more than 600 ka (82, 83), a finding with potential implications for the energetics of Homo-associated encephalization (19⇓–21, 26). Subsequent copy number expansion of AMY1 in the modern human genome and the rise of abpA in oral streptococci may signal an even greater reliance on starch-rich foods by modern humans.
Further research on the evolution of abpA, abpB, and other amylase-binding proteins, including phylogenetic reconstruction and demographic modeling, promises to refine questions regarding biofilm formation and the nature and timing of dietary change in Homo. In addition, future research on non-African hominids (orangutans) and additional catarrhines, in particular cercopithecenes with high or unusual salivary amylase expression, such as gelada and hamadryas baboons (73, 84, 85), may yield further insights into the diverse evolutionary trajectories of primate oral microbiomes in response to habitat and dietary change. In addition, it is clear that more research on core genera is urgently needed, as many of the highly conserved and potentially key structural taxa in hominid oral biofilms are understudied and even lack formal names. Furthermore, future sequencing projects focusing on within-species genomic diversity will be critical to understanding microbiome evolution and coadaptation within the human lineage. This study demonstrates that integrating evolutionary studies of the modern human microbiome with wild primate and ancient Homo metagenomic data provides valuable insights into the ancestral states of the human oral microbiome, the nature of microbial–host relationships, and major events in the evolution of modern humans and Neanderthals.
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Post by Admin on Jun 18, 2021 2:54:32 GMT
More than 49,000 years ago, a family of Neanderthals set up camp in a cave high in Siberia’s Altai Mountains, overlooking a river valley where bison, red deer, and wild horses roamed. In the cave’s main gallery, a teenage girl lost a tooth, perhaps while gnawing on bison that her father or his kin had hunted in the sweeping grasslands.
Now, researchers have analyzed the genomes of this father and daughter and 12 of their relatives, many of whom sheltered in the same cave over less than 100 years. The new genomes almost double the number of Neanderthal genomes known and offer a glimpse of the Neanderthal population at the eastern end of their range, at a time when they were headed toward extinction.
The genomes also offer the first real clues to the social structure of a group of Neanderthals. In addition to identifying the first father-daughter pair, the genetic evidence suggests these males stayed in their family groups as adults, like men in many modern human societies, says geneticist Laurits Skov of the Max Planck Institute for Evolutionary Anthropology. He presented the work in a virtual talk at the ninth International Symposium on Biomolecular Archaeology earlier this month.
“It’s really remarkable that they managed to get genomes from seven males at one site,” says paleogeneticist Cosimo Posth at Tübingen University. “For this group in this cave, it is indeed suggestive that they lived in small groups of closely related males.”
Over the past decade, geneticists have sequenced the genomes of 19 Neanderthals. But that DNA mostly came from females who were distantly related and lived at sites across Europe and Asia anywhere between 400,000 and 50,000 years ago.
Computational biologist Benjamin Peter and paleogeneticist Svante Pääbo at Max Planck led the new study with a team including Skov, a postdoc. They extracted Neanderthal DNA from teeth, bone fragments, and a jawbone dug up during ongoing excavations at Chagyrskaya and Okladnikov caves by archaeologists at the Russian Academy of Sciences in Novosibirsk. Optically stimulated luminescence dates of the sediments around the teeth and bones suggest the Neanderthals lived between 49,000 and 59,000 years ago. Both caves are close—50 to 130 kilometers—to the famous Denisova Cave, which was inhabited by both Neanderthals and their close cousins, the Denisovans, off and on between 270,000 to 50,000 years ago.
The researchers analyzed DNA from more than 700,000 sites across the genomes from seven males and five females from Chagyrskaya, and from a male and a female from Okladnikov. They found family ties: The nuclear DNA from one Chagyrskaya bone fragment linked the father to the tooth shed by his teenage daughter. Some individuals shared two types of maternally inherited mitochondrial DNA (mtDNA). Those genomes hadn’t yet differentiated from each other, which happens in a few generations, so the individuals must have lived during the same century.
The DNA painted a bigger picture of Neanderthal society. Several Chagyrskaya males carried long chunks of identical nuclear DNA from the same recent ancestor. Their Y chromosomes were also similar and came from a modern human ancestor, like those of the only three other male Neanderthal genomes known. The nuclear DNA also showed they were more closely related to later Neanderthals in Spain than to earlier ones at neighboring Denisova, suggesting migration.
The similarities among the males suggest they belonged to a population of only hundreds of men who were fathering children—about the same number of breeding males as seen in endangered mountain gorillas today. “If you were to think of this Neanderthal population like [populations today], they would be an endangered population,” Skov says.
In contrast to the Y chromosome and nuclear DNA, the mtDNA of both males and females was relatively diverse, implying that more female ancestors contributed to the population than males. That could be a founder effect, in which the initial group included fewer fertile males than females. Or it could reflect the nature of Neanderthal society, says paleogeneticist Qiaomei Fu of the Chinese Academy of Sciences, who heard the talk. Either “fewer men than women contributed to the next generation, or women moved more frequently between groups,” she says.
To Skov, the evidence suggests the latter. He says modeling studies show it’s unlikely that a small group of migrants expanding from Europe into Siberia would include mostly females and few males. Instead, he thinks these Neanderthals lived in very small groups of 30 to 110 breeding adults, and that young females left their birth families to live with their mates’ families. Most modern human cultures are also patrilocal, underscoring another way that Neanderthals and modern humans were similar.
Posth cautions that 14 genomes can’t reveal the social lives of all Neanderthals. But he sees ominous signs in the males’ low diversity. The end was fast approaching for our closest cousins: In just 5000 to 10,000 years, they would be gone.
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