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Post by Admin on Feb 6, 2023 21:26:26 GMT
Extinct lineages clustered with each other and closest to East Asian living humans Amino acid sequences for the sampled ORs were the basis of a cladogram, which visualizes relationships across samples (Figure S2). Extinct lineages formed a clade with Vindija Neanderthal being the most distinct, which was unexpected because genome-wide studies have indicated that Vindija is most genetically similar to Chagyrskaya Neanderthal.28 The clade of extinct lineages (Neanderthal and Denisova) was closest to the ancient human Ust’-Ishim. Ancient samples were closest to living East Asians and then living South Asians—both groups harbor genetic signatures of introgression with Neanderthals and Denisovans.16 Testing for differences in protein function due to SNPs indicates a shared functional repertoire with differences in sensitivity more than specificity.  Figure 2 Regression results for dose responses (A) Comparison of human (xaxis) and Neanderthal (yaxis) OR responses to odorants in vitro, (B) Comparison of human (xaxis) and Denisovan (yaxis) OR responses to odorants in vitro. (C) Boxplots of in vitro OR responses for all samples showing median, box boundaries (first and third quartiles), and two whiskers (upper whisker extends to the largest value no further than 1.5 inter-quartile range from the third quartile; lower whisker extends to the smallest value at most 1.5 inter-quartile range of first quartile, outliers identified with red asterisk), (D) OR response by lineage and gene. Each OR is represented by a different color and each point represents the natural log of the response to an odorant. Dotted lines correspond to the linear regression for the entire set of ORs responses for a given lineage. The corresponding equation and R2 values are shown on the regression plot. Raw and analyzed data are in DataS1. Of the 29 new genes examined and additional genomes examined for 7D4, we identified 11 genes containing 14 novel variants that were subjected to functional testing. Because gene function in not reliably predictable for ORs from sequence data,29,30 we directly measured the functional responses of ORs containing novel variants. Each OR protein, expressed in a cell line, was screened against seven odorants previously identified in the literature as evoking responses: OR1A1,29,31 OR1C1,30 OR2C1,29 OR10J5,29 OR5P3,29 and OR10G3.30 Dose response assays for the top screening responses included seven concentrations of the odors delivered separately. To compare dose responses, we computed an Activity Index based on the potency and efficacy of an OR/odorant pair (Figure 4). We distinguish between genes and proteins by prefixing proteins with OR and eliminating the prefix when referring to genes (e.g., 1A1 for the gene, OR1A1 for the protein). Functional testing included 784 OR/odorant pairs with at least 1 concentration in total—42 odorants on 2 ORs (living and extinct) for a total of 84 OR/odorant pairs (Figure S3), 2 ORs against 350 odorants for a total of 700 OR/odorant pairs (Figure S4). There were only three Neanderthal genes containing novel variants across all three Neanderthal samples. Their dose responses were not correlated with those of contemporary humans (Figures 2A, 2C and 2D ). Only OR1C1 had a detectable response, but it was significantly lower than that of living humans (Figures 3A , 3C, 3D, 4, S2, and S3). Despite the higher number of novel OR variants in Denisovans and higher dose responses compared to contemporary humans (Figures 2C and 2D), the OR responses for six genes and those for human reference were significantly correlated (R2 = 0.87) (Figure 2B). Sampled Denisovan ORs were less sensitive to odors that contemporary humans perceive as floral but much more sensitive to odors perceived as spicy, balsamic, or unpleasant (e.g., sulfur 4x greater and balsamic 3x greater than in contemporary humans) (Figure 4). Higher dose responses in Denisovan ORs appear to be driven by two amino acid variations in two ORs containing novel variants (Figures 3 and S3). |
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Post by Admin on Feb 9, 2023 18:11:51 GMT
 Figure 3 Homology model of the human consensus odorant receptor The transmembrane domains are represented in tubes and numbered from 1 to 7. The location of each of the variants is pointed on the human consensus OR homology model by a colored ring. For each variant, dotted lines connect the variant location to a panel showing the ORs dose-response to odorants that were significantly activating ORs in screening. The xaxis of panels represents the logarithmic transformation of the odorant concentration (M) and the yaxis the normalized luminescence (Luciferase (L) normalized by Renilla (R) luminescence, see STAR Methods) generated by the OR activation in luciferase assay. The empty vector pCI is added as a negative control. Error bars are SEM (standard error of the mean) of n = 4 replicates. The Homo lineage origin of the OR variant is color coded in the OR homology model and the dose-responses as modern human = orange, Denisova = blue, Neanderthal Vindija = light purple, Neanderthal Chagyrskaya = dark purple. Statistical significance of OR response between of the human and Homo lineage versions is assessed by Extra sum-of-square F test on the dose-response and shown as ∗∗∗p<0.001, ∗∗0.001<p<0.01, ∗0.01<p<0.05, ns non-significant. Raw and analyzed data are in DataS1.  Figure 4 Comparison of OR activity index for human and extinct lineages Color coding is from low (purple) to high (mustard). Odor qualities are from Good Scents Company.32 |
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Post by Admin on Feb 12, 2023 17:38:06 GMT
Despite using the same DNA sequence as previous studies of OR2B1130 and OR6P1,25 neither responded to any of the ∼350 odors (100 μM) against which they were tested (Figure S4)—neither did the extinct lineages. In previous studies, OR2B1130 and OR6P125 responded strongly to coumarin and Anisaldehyde, respectively, at concentrations higher than 100 μM. We found that such concentrations often cause cell toxicity or OR non-specific cell responses.
OR1A1. All but 2-heptanone induced a response in the screening assay (Figure S3A) and the highest responses were for allyl phenylacetate, a honey-like odor (particularly for Denisovan) (Figures 4 and 3). Neither of the two Denisovan variants (V461I1.54, T257 M6.54) was in an amino acid region critical for mammalian OR function nor were they involved in the odorant binding cavity—perhaps explaining their minimal functional impact.
OR1C1. The only significant response was from Chagyrskaya Neanderthal to androstadienone (Figure S3B) and it was weak (Figure 4 and 3). Chagyrskaya Neanderthal OR1C1 variant Y120H3.48 is part of the highly conserved MAY3.48DRY motif involved in the activation of mammalian ORs (Figure 3 and S3B), which might explain why this variant alters function.23,33,34
OR2C1. Screening assay responses were strong but not statistically significant (Figure S3A). The dose response assay for octanethiol produced a statistically significant response in the Denisovan version of this OR (Figures 4 and 3). The shared C149 W4.50 corresponds to the conserved W4.50. The W residue is highly conserved in GPCRs but less so in ORs (58%). The location of the novel Denisovan I214 T5.54 in TM5 is below residues involved in canonical ligand binding cavity and it points into the receptor rather than the surface. In addition, prior functional tests for C149 found a similar response.25 The C149W allele may stop protein function and may have produced octanethiol-specific anosmia.
OR5P3. In the screening assay, Vindija Neanderthal did not have a significant response but the human reference responded to five of the seven odorants (Figure S3B). Vindija dose responses, which included higher concentrations of the top three responses for contemporary humans (coumarin and both enantiomers of carvone), did not exceed control (Figure 3). The cell surface expression for Vindija indicated that the OR protein was present at the cell surface, albeit at a slightly lower level than that in contemporary humans. Vindija F159 L4.58 is in the extra-cellular part of TM4 (Figure 3), near 4.53, which is involved in mouse OR trafficking.35 We observed slightly lower trafficking of the Vindija protein. A similar mutation (S155 A4.56) in human OR1A2 decreases in vitro responses to (S)-(−)-citronellal.31 If OR5P3 F159 L4.58 is involved in odorant binding, the mutation of this position from phenylalanine to leucine might prevent the π-π stacking interaction between the aromatic residue and coumarin. We conclude that the Vindija protein is not functional and this might be attributable to many potential reasons (a few examples are lack of odorant binding, or fail in activation mechanism, or fail to bind the G protein).
OR10G3. The screening assay revealed significant responses for all seven odors in the Denisovan OR and in the human reference (Figure S3A). Denisovan variants had significantly stronger dose responses to vanillin and ethyl vanillin compared to contemporary humans (Figure 4 and 3). Neither of the Denisovan variants (S73G2.52, E197 K5.36) was located in conserved amino acid regions (Figure 3). TM2 is not involved in odorant binding or receptor function, implying that S73G2.52 probably did not change the receptor response. TM5 (E197 K5.36) forms part of the binding cavity but position 5.36 is located at the very limit of ECL2. K at this position is a rare residue in contemporary human ORs (3.6%), suggesting a functional, adaptive reason for this change. The location of the variant suggests it may be involved in ligand entry.
OR10J5. There were three significant screening responses to lyral, helional and eugenol (Figure S3A). The Denisovan response to top odor lyral was lower than that of the human reference (Figures 4 and 3). The G21RNter variant of OR10J5 found in the Denisovan is located at the very end of the N terminal end, just before the start of TM1. The role of this region in OR function is undetermined.
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Post by Admin on Feb 15, 2023 18:50:26 GMT
Discussion
We tested 784 OR/odorant pairs on ten novel missense variants, which were located in 8 genes out of 30 (29 new genes and OR7D4 for the new Neanderthal samples). Five were functionally different than contemporary humans (OR1C1, OR2C1, OR5P3, OR10G3, OR10J5), one the same (OR1A1) and two without identifiable ligands (OR2B11, OR6P1). Given the small percentage of genes with variants altering OR function, members of the genus Homo likely shared an olfactory repertoire based on our sample, with Neanderthals and Denisovans smelling the same range of odors we do but having different dose responses to those odors. When OR function was altered by a novel OR, the difference was in sensitivity rather than specificity. Novel Denisovan OR variants (OR1A1, OR2C1, OR10G3, OR10J5) were twice as responsive as human equivalents to odors contemporary humans perceive as spicy, balsamic, and unpleasant (Figure 4), but not to odors perceived as floral. Given the human specialization of olfactory sensory neurons to food-based odors,4 the variation in sensitivity to food odors in an extinct lineage in our sample is indirect evidence of a similar emphasis. Novel Neanderthal variants were three times less responsive than human ORs, including reduced responses to odors perceived as green, floral, and spicy (Figure 4). There is some correlation in Neanderthal skull morphology that suggests their olfactory bulbs were smaller than contemporary humans,13 but the link between bulb size and olfactory acuity is unclear.36,37
Given that most variants are shared by the genus Homo, the small number of novel variants in extinct lineages may reflect adaptations following dispersals from Africa and after the split with humans. For example, the Denisovan OR strong response to honey-like odors (allyl phenyl acetate) may reflect the evolutionary olfactory ecology of the lineage. Honey is the most energy-dense natural food and is a prized component of extant hunter-gatherer diets (except where bees are rare or absent)—even great apes have a ‘honey tooth’.38 The hunt for sugar is as old as the earliest stem primates 54 mya who notably had a high prevalence of caries after a shift to a diet high in fruit39—such an emphasis on sweet is allied with the increased sensitivity to vanilla odors in our study. Denisovan’s increased sensitivity to sweet odors suggests an evolutionary tuning to sources of natural sugar. Energy-dense foods like sugars are sought by larger-brained primates40 and, based on oral microbiome data, Neanderthals and contemporary humans share functional adaptations in nutrient metabolism, including starch digestion, that are not found in our closest ape relatives.41 The high response to vanilla odors further suggests a tuning to sweet—an odor-taste pairing common in contemporary humans.42
Although local ecological adaptive pressures may have acted on ORs in extinct lineages to produce the few novel variants observed, extinct lineages were less variable in OR genes and proteins compared to 1000 Genomes. Differences in sample sizes might account for some of the striking differences, but the reduced variation is probably because of genetic drift effect or gene conservation.28 Purifying selection has been observed in chimpanzee ORs compared to a mix of relaxed and positive selection in human ORs43 and extinct lineages may also have been subject to this as well (as suggested by the presence of fewer variants, most of which code for synonymous proteins). The mean of Fst values across genes comparing 1000 Genomes to extinct lineages (11%) is higher than those for 1000 Genomes population comparisons (4%), which suggests that there are structural differences between them and us that reflect both explanations—drift and conservation. Denisovans have been previously noted to exhibit geographical and temporal population structure.16,17 Based on our data the last common ancestor shared by Homo (contemporary humans, Neanderthals, Denisova, and others) and Pan (chimpanzees, bonobos) had a conserved set of ORs. Contemporary humans derived away from the pattern of conservation more recently, with evolutionary pressure toward increased missense variation.
Despite sharing a genetic and functional olfactory repertoire with Neanderthals and Denisovans (shared variants suggest a shared olfactory world), contemporary humans have greater genetic variability and diversity (across populations) than extinct lineages. Such variation and diversity indicates a broad olfactory repertoire44 and may reflect cultural adaptations following our migrations from Africa. Relaxed selection on OR genes for groups no longer engaging in traditional lifestyles is possible.45 In our sample, 1000 Genomes groups outside Africa were less variable in most OR genes than those in Africa in our sample and other research has found that greater OR gene enrichment in African hunter-gatherer groups (Hadza and Pygmies) but not African agricultural (Yoruba) and pastoral (Maasai) groups.46 Tanzanian Sandawe hunter-gatherers show no OR allelic enrichment, however, which undermines the case for relaxed selection.46 High allelic diversity and OR generalization on a broad scale may have functional implications, such as increasing the effective size of the olfactory repertoire.44
Our data provide insights into how the dispersal of human lineages outside of Africa (Denisova, Neanderthal, ancient human) may have affected olfactory gene repertoire and function. Understanding the evolutionary genetics and functional significance of observed OR variability in and among human populations and extinct relatives sheds light on the role of olfaction in key aspects of human culture, and perhaps our current success as a global species. The applications of these methods to a large dataset addressing the function of genetic variation in extinct hominins is an advance in the study of human evolution and allows future work with odorant receptors (and other proteins that can be created in the lab) and their adaptive function in human evolutionary timescales. Understanding our unique OR allelic diversity and its evolution is an important challenge to olfactory science.
Limitations of the study
The primary limitation of the study is outside our control: the small sample sizes available for the extinct human lineages that met our inclusion criteria. Although there are an increasing number of ancient genomes available, each team uses different methods for handling damaged DNA and variant calls may be true genetic variation or the result of different methods. We limited our sample to the largest available sample across lineages of interest that was generated by one team using the same methods for damaged genomes. Another limitation is that we are unable to conduct sophisticated population genetics analysis with sample sizes as low as 1 (Denisova and Ust’-Ishim, respectively) and as high as 3 (if Altai, Chagyrskaya, and Vindija Neanderthals are grouped together despite having different ecological challenges). Although the data generated are robust for examining how odorant receptors function to bind odors, we cannot gain insights into how these odors may have been perceived by the extinct lineages. A final limitation is our current knowledge of odorant-OR and genotype-phenotype associations. Deorphanizing ORs with potential agonists is outside the scope of this paper. Many recent deorphanized ORs47,48,49 have yet to undergo extensive testing against a vast array of agonists. Our approach was to focus on the well-established ORs supported by multiple lab testing, given our focus on applying established OR-odorant pairs to the question of human olfactory evolution.
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Post by Admin on Mar 12, 2023 18:40:13 GMT
Bow-and-arrow, technology of the first modern humans in Europe 54,000 years ago at Mandrin, France Abstract Consensus in archaeology has posited that mechanically propelled weapons, such as bow-and-arrow or spear-thrower-and-dart combinations, appeared abruptly in the Eurasian record with the arrival of anatomically and behaviorally modern humans and the Upper Paleolithic (UP) after 45,000 to 42,000 years (ka) ago, while evidence for weapon use during the preceding Middle Paleolithic (MP) in Eurasia remains sparse. The ballistic features of MP points suggest that they were used on hand-cast spears, whereas UP lithic weapons are focused on microlithic technologies commonly interpreted as mechanically propelled projectiles, a crucial innovation distinguishing UP societies from preceding ones. Here, we present the earliest evidence for mechanically propelled projectile technology in Eurasia from Layer E of Grotte Mandrin 54 ka ago in Mediterranean France, demonstrated via use-wear and impact damage analyses. These technologies, associated with the oldest modern human remains currently known from Europe, represent the technical background of these populations during their first incursion into the continent.  Fig. 1. Mandrin E lithic artifacts. (1 to 13) Distal of nanopoints, micropoints, and points. (14 to 17) Distal of Soyons points. (18 to 22) Mesial of bladelets, micropoints, and points. (23 to 35) Proximal of nanopoints, micropoints, and points. (36 to 47) Sub-full and full nanopoints, micropoints, and points. INTRODUCTION Consensus in the archaeology of human origins has posited that mechanically propelled weapons, such as bow-and-arrow or spear-thrower-and-dart combinations, appeared abruptly in the Eurasian archaeological record with the arrival of anatomically and behaviorally modern humans and the Upper Paleolithic (UP) after 45 to 42 thousand years (ka) ago (note S1) (1–3). Here, we present the earliest evidence for bow-and-arrow technology in Eurasia from Layer E of Grotte Mandrin in Mediterranean France. These projectile technologies represent the technical background of expanding modern humans during their first incursion into Europe ~54 ka ago (4). The production of lithic artifacts in Mandrin’s Layer E was focused on standardized tiny points, some clustering around only 1 cm in length (Figs. 1 and 2), thus far unseen in archaeological assemblages of this age and representing a main structural difference between Neanderthal and modern human social and material organization. These technologies may have given modern humans a competitive advantage over local Neanderthal societies.  Fig. 2. Mandrin E. Lithic points. (A) Large point (1) versus nanopoint (2). (B) Neronian micropoints and nanopoints; (1 to 3) elongated nanopoints, (4) pointed nanopoint, (5 and 6) nanopoints, and (7 and 8) micropoints. Graphic scale is 1 Euro cent (diameter, 16.25 mm). Grotte Mandrin is a vaulted rock shelter directly overlooking the middle valley of the Rhône River. Mandrin records a reference archaeological succession, for it contains all of the phases currently known for the last Neanderthal societies, right up to the emergence of the UP (5–7). Each archaeological layer has yielded a rich lithic industry and paleontological remains (4). Layer E yielded 2267 lithic elements attributed to the Neronian, a “culture” entirely oriented toward the production of standardized Levallois points, technologically obtained after laminar phases (5–7). Quantitatively, blades, bladelets, and a variety of points represent 75.1% of all blanks. The production of these points was technically highly controlled and focused on two categories: larger points from 30 to 60 mm in maximum length and microlithic points below 30-mm maximum length and sometimes as small as 10 mm, termed here “nanopoints.” The distinction between these two categories is technological, not based on size. The larger points were produced on the basis of laminar technology, initiated by a crested blade extraction and followed by unipolar blade production that configured the core geometry to extract technologically well-defined points. The micro/nanopoints were not produced simply as a smaller version of that process on very reduced cores but rather via “core-on-flake” knapping of blanks produced while making the larger points (Figs. 1 and 2, fig. S1, and note S2) (5). www.science.org/doi/10.1126/sciadv.add4675 |
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