Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus.

Recent genomic data have revealed multiple interactions between Neanderthals and modern humans, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)-the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution.

Ancient gene flow from early modern humans into Eastern Neanderthals.

It has been shown that Neanderthals contributed genetically to modern humans outside Africa 47,000-65,000 years ago. Here we analyse the genomes of a Neanderthal and a Denisovan from the Altai Mountains in Siberia together with the sequences of chromosome 21 of two Neanderthals from Spain and Croatia. We find that a population that diverged early from other modern humans in Africa contributed genetically to the ancestors of Neanderthals from the Altai Mountains roughly 100,000 years ago. By contrast, we do not detect such a genetic contribution in the Denisovan or the two European Neanderthals. We conclude that in addition to later interbreeding events, the ancestors of Neanderthals from the Altai Mountains and early modern humans met and interbred, possibly in the Near East, many thousands of years earlier than previously thought.

Comparative anatomy and 3D geometric morphometrics of the El Sidrón atlases (C1).

The first cervical vertebra (atlas, C1) is an important element of the vertebral column because it connects the cranial base with the cervical column, thus helping to maintain head posture and contributing to neck mobility. However, few atlases are preserved in the fossil record because of the fragility of this vertebra. Consequently, only eight well-preserved atlases from adult Neandertals have been recovered and described. Here, we present nine new atlas remains from the El Sidrón Neandertal site (Asturias, Spain), two of which (SD-1643 and SD-1605/1595) are sufficiently well preserved to allow for a detailed comparative and three-dimensional geometric morphometric analysis. We compared standard linear measurements of SD-1643 and SD-1605/1595 with those of other Neandertal atlases and carried out three-dimensional geometric morphometric analyses to compare size and shape of SD-1643 and SD-1605/1595 with those of 28 Pan (Pan troglodytes and Pan paniscus), a broad comparative sample of 55 anatomically modern humans from African and European populations, and other fossil hominins (Neandertals, Homo antecessor, Paranthropus boisei). The El Sidrón atlas fossils show typical features of the Neandertal atlas morphology, such as caudal projection of the anterior tubercle, gracility of both the posterior tubercle and the tuberosity for the insertion of the transverse ligament, and an anteroposteriorly elongated neural canal. Furthermore, when compared with atlases from the other taxa, Neandertals exhibit species-specific features of atlas morphology including a relatively lower lateral mass height, relatively narrower transverse foramina, and flatter and more horizontally oriented articular facets. Some of these features fit with previous suggestions of shorter overall length of the cervical spine and potential differences in craniocervical posture and mobility. Our results may support a different spinopelvic alignment in this species, as the atlas morphology suggests reduced cervical lordosis.

The study of the lower limb entheses in the Neanderthal sample from El Sidrón (Asturias, Spain): How much musculoskeletal variability did Neanderthals accumulate?

Entheses have rarely been systematically studied in the field of human evolution. However, the investigation of their morphological variability (e.g., robusticity) could provide new insight into their evolutionary significance in the European Neanderthal populations. The aim of this work is to study the entheses and joint features of the lower limbs of El Sidrón Neanderthals (Spain; 49 ka), using standardized scoring methods developed on modern samples. Paleobiology, growth, and development of both juveniles and adults from El Sidrón are studied and compared with those of Krapina Neanderthals (Croatia, 130 ka) and extant humans. The morphological patterns of the gluteus maximus and vastus intermedius entheses in El Sidrón, Krapina, and modern humans differ from one another. Both Neanderthal groups show a definite enthesis design for the gluteus maximus, with little intrapopulation variability with respect to modern humans, who are characterized by a wider range of morphological variability. The gluteus maximus enthesis in the El Sidrón sample shows the osseous features of fibrous entheses, as in modern humans, whereas the Krapina sample shows the aspects of fibrocartilaginous ones. The morphology and anatomical pattern of this enthesis has already been established during growth in all three human groups. One of two and three of five adult femurs from El Sidrón and from Krapina, respectively, show the imprint of the vastus intermedius, which is absent among juveniles from those Neanderthal samples and in modern samples. The scant intrapopulation and the high interpopulation variability in the two Neanderthal samples is likely due to a long-term history of small, isolated populations with high levels of inbreeding, who also lived in different ecological conditions. The comparison of different anatomical entheseal patterns (fibrous vs. fibrocartilaginous) in the Neanderthals and modern humans provides additional elements in the discussion of their functional and genetic origin.

Analyses of the neandertal patellae from El Sidrón (Asturias, Spain) with implications for the evolution of body form in Homo.

The evolution of the body form in Homo and its potential morphological connection to the arrangement of different skeletal systems is of major interest in human evolution. Patella morphology as part of the knee is potentially influenced by body form. Here, we describe for the first time the patellae remains recovered at El Sidrón Neandertal site and analyze them in a comparative evolutionary framework. We aim to clarify whether morphometric features frequently observed in Neandertal and modern human patellae are retained from a primitive anatomical arrangement or whether they represent derived features (apomorphies). For this purpose, we combine analyses of discrete features, classic anthropological measurements, and 3D geometric morphometrics based on generalized Procrustes analysis, mean size and shape comparisons, and principal components analysis. We found a size increment of the patella in hominin evolution, with large species showing a larger patella. Modern humans and Neandertals exhibit overall larger patellae, with maximum values observed in the latter, likely as a consequence of their broader body shape. Also, some Neandertals display a thicker patella, which has been linked to larger quadriceps muscles. However, Neandertals retain a primitive morphology in their patellar articular surfaces, with similar-sized lateral and medial articular facets, leading to a more symmetrical internal face. This feature is inherited from a primitive Homo ancestor and suggests a different configuration of the knee in Neandertals. Conversely, Homo sapiens exhibits an autoapomorphic patellar anatomy with expanded lateral articular facets. We propose that these distinct configurations of the patella within Homo may be a consequence of different body forms rather than specific functional adaptations of the knee. Thus, the slender body form of modern humans may entail a medial reorientation of the tibial tuberosity (patellar ligament), allowing lateral surface expansion. These anatomical evolutionary variations may involve subtle secondary differences in bipedalism within Homo.

Relationship between vertical facial pattern and brain structure and shape.

OBJECTIVES: Dolichofacial (long-faced) and brachyfacial (short-faced) individuals show specific and well-differentiated craniofacial morphology. Here, we hypothesise that differences in the basicranial orientation and topology between dolicho- and brachyfacial subjects could be associated with differences in the supporting brain tissues. MATERIAL AND METHODS: Brain volumes (total intracranial, grey matter, and white matter volume), cortical thickness, and the volumes and shapes of fifteen subcortical nuclei were assessed on the basis of magnetic resonance imaging in 185 subjects. Global, voxel-wise and shape analyses, as well as multiple regression models, were generated to evaluate the association between vertical facial variations (dolicho- and brachyfacial spectrum) and brain morphology. RESULTS: Several differences in brain anatomy between dolicho- and brachyfacial subjects, along with relevant associations between vertical facial indices and brain structure and shape, were found. The most relevant finding of this study is related to the strong association of vertical facial indices with the volumes and shapes of subcortical nuclei, as the dolichofacial pattern increased, the bilateral hippocampus and brain stem expanded, while the left caudate, right pallidus, right amygdala, and right accumbens decreased in volume. CONCLUSIONS: Long- and short-faced human subjects present differences in brain structure and shape. CLINICAL SIGNIFICANT: The results of our study increase the clinician's knowledge about brain structure in dolicho- and brachyfacial patients. The findings could be of interest since the affected brain areas are involved in higher cognitive functions in humans, including language, memory, and attention.

Primary visual cortex in neandertals as revealed from the occipital remains from the El Sidrón site, with emphasis on the new SD-2300 specimen.

The comparative analysis of the endocranial surface of the El Sidrón new occipital fragment SD-2300 shows meaningful differences in the configuration of the occipital pole region between neandertals and anatomically modern humans (AMH). The particular asymmetries found in neandertals in the venous sinus drainage and the petalial patterns are recognizable in this new specimen as well. In addition, the supra- and infracalcarine fossae of the occipital pole region appear to deviate obliquely from the mid-line when compared with sapiens. Due to the excellent preservation conditions of SD-2300, the main sulci and gyri of the occipital pole area have been identified, this degree of detail being uncommon in a fossil specimen; in general, the gyrification pattern is similar to AMH, but with some notable differences. Particularly interesting is the description of the lunate and the calcarine sulci. The lunate sulcus is located close to the occipital pole, in a similar posterior position to in other Homo species. Regarding the calcarine sulcus, there are significant differences in the primary visual cortex, with the V1 area, or Brodmann area 17, being larger in Homo neanderthalensis than in Homo sapiens. This may lead to greater visual acuity in neandertals than in sapiens.

Over 100 years of Krapina: New insights into the Neanderthal thorax from the study of rib cross-sectional morphology.

The Krapina costal sample was studied by Gorjanovic-Kramberger in the early twentieth century. He pointed out unique features in the sample such as the rounder rib cross-section, which was recently confirmed in other Neanderthal specimens. Round rib cross-sections are characteristic of Homo ergaster, suggesting this may be plesiomorphic for Pleistocene Homo, but it is unknown whether Homo antecessor also had this rib shape. Furthermore, the influence of allometry on the cross-sectional shape of ribs is still unknown. The large costal sample from Krapina allows us to address these issues. We quantified cross-section morphology at the midshaft throughout a closed curve of one landmark and nine sliding semilandmarks in the Krapina costal remains (n = 7), as well as in other Neanderthals (n = 50), H. antecessor (n = 3) and modern humans, both fossil (n = 12) and recent (n = 160). We used principal components analysis and mean comparisons to explore interspecific differences, regression analysis to investigate allometry, and partial least squares analysis to examine covariation of cross-section shape and overall rib morphology. Neanderthal cross-sections tended to be larger than those of recent humans except for the Krapina and Tabun remains. Regarding shape, inter-group differences were found only in the diaphragmatic thorax, where Neanderthal and H. antecessor ribs were statistically significantly rounder than those of modern humans. Allometry accounted for covariation of size on shape, but the Neandertal and modern human trajectories had different slopes. While our results based on the Krapina costal sample are similar to previous findings, we also make several new insights: 1) the cross-section morphology observed in Neanderthals was probably present in H. antecessor, albeit less marked; 2) the distinct roundness of Neanderthal cross-sections is not related to size; 3) rounder cross-sections are correlated with ribs presenting less curvature in cranial view and a low degree of torsion in recent humans. These results are important for the interpretation of fragmentary Neanderthal costal remains, and the fact that the differences are marked only in the diaphragmatic thorax could have implications for breathing kinematics.

New Neandertal wrist bones from El Sidrón, Spain (1994-2009).

Twenty-nine carpal bones of Homo neanderthalensis have been recovered from the site of El Sidrón (Asturias, Spain) during excavations between 1994 and 2009, alongside ∼2500 other Neandertal skeletal elements dated to ∼49,000 years ago. All bones of the wrist are represented, including adult scaphoids (n = 6), lunates (n = 2), triquetra (n = 4), pisiforms (n = 2), trapezia (n = 2), trapezoids (n = 5), capitates (n = 5), and hamates (n = 2), as well as one fragmentary and possibly juvenile scaphoid. Several of these carpals appear to belong to the complete right wrist of a single individual. Here we provide qualitative and quantitative morphological descriptions of these carpals, within a comparative context of other European and Near Eastern Neandertals, early and recent Homo sapiens, and other fossil hominins, including Homo antecessor, Homo naledi, and australopiths. Overall, the El Sidrón carpals show characteristics that typically distinguish Neandertals from H. sapiens, such as a relatively flat first metacarpal facet on the trapezium and a more laterally oriented second metacarpal facet on the capitate. However, there are some distinctive features of the El Sidrón carpals compared with most other Neandertals. For example, the tubercle of the trapezium is small with limited projection, while the scaphoid tubercle and hamate hamulus are among the largest seen in other Neandertals. Furthermore, three of the six adult scaphoids show a distinctive os-centrale portion, while another is a bipartite scaphoid with a truncated tubercle. The high frequency of rare carpal morphologies supports other evidence of a close genetic relationship among the Neandertals found at El Sidrón.

Evolutionary anatomy of the Neandertal ulna and radius in the light of the new El Sidrón sample.

This paper aims to improve our understanding of the phylogenetic trait polarity related to hominin forearm evolution, in particular those traits traditionally defined as "Neandertal features." To this aim, twelve adult and adolescent fragmented forelimb elements (including ulnae and radii) of Homo neanderthalensis recovered from the site of El Sidrón (Asturias, Spain) were examined comparatively using three-dimensional geometric and traditional morphometrics. Mean centroid size and shape comparisons, principal components analysis, and phylogenetic signal analysis were undertaken. Our investigations revealed that the proximal region of the ulna discriminated best between Neandertals and modern humans, with fewer taxonomically-informative features in the distal ulna and radius. Compared to modern humans, the divergent features in the Neandertal ulna are an increase in olecranon breadth (a derived trait), lower coronoid length (primitive), and anterior orientation of the trochlear notch (primitive). In the Neandertal radius, we observe a larger neck length (primitive), medial orientation of the radial tubercle (secondarily primitive), and a curved diaphysis (secondarily primitive). Anatomically, we identified three units of evolutionary change: 1) the olecranon and its fossa, 2) the coronoid-radius neck complex, and 3) the tubercle and radial diaphysis. Based on our data, forearm evolution followed a mosaic pattern in which some features were inherited from a pre-Homo ancestor, others originated in some post-ergaster and pre-antecessor populations, and other characters emerged in the specific Homo sapiens and H. neanderthalensis lineages, sometimes appearing as secondarily primitive. Future investigations might consider the diverse phylogenetic origin of apomorphies while at the same time seeking to elucidate their functional meaning.

Dietary reconstruction of the El Sidrón Neandertal familial group (Spain) in the context of other Neandertal and modern hunter-gatherer groups. A molar microwear texture analysis.

Here, we present the analysis of occlusal molar microwear textures of eight individuals from the El Sidrón Neandertal group (Spain). The aims of the study were: 1) to document potential age-, sex-, and maternal lineage-related differences in diet within a Neandertal familial group, and 2) to place the diet of El Sidrón individuals in the context of those of other Neandertal groups. This study also offers an interpretation of the diet of the El Sidrón Neandertals by comparing their microwear signatures to those of recent hunter-gatherer populations with diverse but known diets. The intra-group examination of the microwear signatures are consistent with the females of the El Sidrón group having had more abrasive diets or having used their teeth in more para-masticatory activities than did the males. Aside from the potential sex-related differences in diet, no additional intra-group dietary separation, such as by age group or maternal lineage, was observed. In comparison to other Neandertals, El Sidrón individuals, as a group, have microwear signatures most similar to those of other Neandertals from wooded habitats and different from those that lived in more open habitats. This result is expected based on the available paleoenvironmental reconstructions from El Sidrón Cave. The diet of the El Sidrón Neandertals, just like their Neandertal counterparts from similar wooded habitats, is interpreted as having been mixed, consisting of both meat and vegetable foods.

The costal remains of the El Sidrón Neanderthal site (Asturias, northern Spain) and their importance for understanding Neanderthal thorax morphology.

The study of the Neanderthal thorax has attracted the attention of the scientific community for more than a century. It is agreed that Neanderthals have a more capacious thorax than modern humans, but whether this was caused by a medio-lateral or an antero-posterior expansion of the thorax is still debated, and is key to understanding breathing biomechanics and body shape in Neanderthals. The fragile nature of ribs, the metameric structure of the thorax and difficulties in quantifying thorax morphology all contribute to uncertainty regarding precise aspects of Neanderthal thoracic shape. The El Sidrón site has yielded costal remains from the upper to the lower thorax, as well as several proximal rib ends (frequently missing in the Neanderthal record), which help to shed light on Neanderthal thorax shape. We compared the El Sidrón costal elements with ribs from recent modern humans as well as with fossil modern humans and other Neanderthals through traditional morphometric methods and 3D geometric morphometrics, combined with missing data estimation and virtual reconstruction (at the 1st, 5th and 11th costal levels). Our results show that Neanderthals have larger rib heads and articular tubercles than their modern human counterparts. Neanderthal 1st ribs are smaller than in modern humans, whereas 5th and 11th ribs are considerably larger. When we articulated mean ribs (size and shape) with their corresponding vertebral elements, we observed that compared to modern humans the Neanderthal thorax is medio-laterally expanded at every level, especially at T5 and T11. Therefore, in the light of evidence from the El Sidrón costal remains, we hypothesize that the volumetric expansion of the Neanderthal thorax proposed by previous authors would mainly be produced by a medio-lateral expansion of the thorax.

Three-dimensional morphometrics of thoracic vertebrae in Neandertals and the fossil evidence from El Sidrón (Asturias, Northern Spain).

Well preserved thoracic vertebrae of Neandertals are rare. However, such fossils are important as their three-dimensional (3D) spatial configuration can contribute to the understanding of the size and shape of the thoracic spine and the entire thorax. This is because the vertebral body and transverse processes provide the articulation and attachment sites for the ribs. Dorsal orientation of the transverse processes relative to the vertebral body also rotates the attached ribs in a way that could affect thorax width. Previous research indicates possible evidence for greater dorsal orientation of the transverse processes and small vertebral body heights in Neandertals, but their 3D vertebral structure has not yet been addressed. Here we present 15 new vertebral remains from the El Sidrón Neandertals (Asturias, Northern Spain) and used 3D geometric morphometrics to address the above issues by comparing two particularly well preserved El Sidrón remains (SD-1619, SD-1641) with thoracic vertebrae from other Neandertals and a sample of anatomically modern humans. Centroid sizes of El Sidrón vertebrae are within the human range. Neandertals have larger T1 and probably also T2. The El Sidrón vertebrae are similar in 3D shape to those of other Neandertals, which differ from Homo sapiens particularly in central-lower regions (T6-T10) of the thoracic spine. Differences include more dorsally and cranially oriented transverse processes, less caudally oriented spinous processes, and vertebral bodies that are anteroposteriorly and craniocaudally short. The results fit with current reconstructions of Neandertal thorax morphology.

Right-handed fossil humans.

Fossil hominids often processed material held between their upper and lower teeth. Pulling with one hand and cutting with the other, they occasionally left impact cut marks on the lip (labial) surface of their incisors and canines. From these actions, it possible to determine the dominant hand used. The frequency of these oblique striations in an array of fossil hominins documents the typically modern pattern of 9 right- to 1 left-hander. This ratio among living Homo sapiens differs from that among chimpanzees and bonobos and more distant primate relatives. Together, all studies of living people affirm that dominant right-handedness is a uniquely modern human trait. The same pattern extends deep into our past. Thus far, the majority of inferred right-handed fossils come from Europe, but a single maxilla from a Homo habilis, OH-65, shows a predominance of right oblique scratches, thus extending right-handedness into the early Pleistocene of Africa. Other studies show right-handedness in more recent African, Chinese, and Levantine fossils, but the sample compiled for non-European fossil specimens remains small. Fossil specimens from Sima del los Huesos and a variety of European Neandertal sites are predominately right-handed. We argue the 9:1 handedness ratio in Neandertals and the earlier inhabitants of Europe constitutes evidence for a modern pattern of handedness well before the appearance of modern Homo sapiens.

Evidence of toothpick groove formation in Neandertal anterior and posterior teeth.

OBJECTIVES: During the microscopic examination of the Neandertal dentitions from El Sidrón (Spain) and Hortus (France), we found unusual fine parallel microstriations on the mesial and distal sides of all tooth types, near the cervix. As its appearance was similar to toothpick grooves described in other Homo species, it could correspond to early stages on its formation. To test this hypothesis we developed an experimental replication of a groove using grass stalks. MATERIALS AND METHODS: Comparisons between 204 isolated Neandertal teeth and the two experimental dental specimens corroborate that the marks correspond to initial stages of toothpick groove formation, and we propose a five-grade recording scale that summarized the groove formation process. RESULTS: Using this new recording procedure, we found that Hortus individuals have higher incidence of this trait (eight individuals out of nine) than the El Sidrón individuals (nine out of 11). Toothpick grooves from El Sidrón show the earliest stages of development, whereas the grooves found on Hortus Neandertals were well-developed. Toothpick grooves were also found in 21 incisors and canines. CONCLUSIONS: These differences could be due to the more advanced occlusal dental wear in Hortus individuals, maybe age-related and with a more meat-based diet maybe favoring the inclusion of food debris and thus probing as the cleaning methodology. Our results allow the identification and characterization of incipient toothpick grooves on the human fossil record and contribute to increase our knowledge on Neandertals behavioral and oral care habits.

Neandertal talus bones from El Sidrón site (Asturias, Spain): A 3D geometric morphometrics analysis.

OBJECTIVES: The El Sidrón tali sample is assessed in an evolutionary framework. We aim to explore the relationship between Neandertal talus morphology and body size/shape. We test the hypothesis 1: talar Neandertal traits are influenced by body size, and the hypothesis 2: shape variables independent of body size correspond to inherited primitive features. MATERIALS AND METHODS: We quantify 35 landmarks through 3D geometric morphometrics techniques to describe H. neanderthalensis-H. sapiens shape variation, by Mean Shape Comparisons, Principal Component, Phenetic Clusters, Minimum spanning tree analyses and partial least square and regression of talus shape on body variables. Shape variation correlated to body size is compared to Neandertals-Modern Humans (MH) evolutionary shape variation. The Neandertal sample is compared to early hominins. RESULTS: Neandertal talus presents trochlear hypertrophy, a larger equality of trochlear rims, a shorter neck, a more expanded head, curvature and an anterior location of the medial malleolar facet, an expanded and projected lateral malleolar facet and laterally expanded posterior calcaneal facet compared to MH. DISCUSSION: The Neandertal talocrural joint morphology is influenced by body size. The other Neandertal talus traits do not co-vary with it or not follow the same co-variation pattern as MH. Besides, the trochlear hypertrophy, the trochlear rims equality and the short neck could be inherited primitive features; the medial malleolar facet morphology could be an inherited primitive feature or a secondarily primitive trait; and the calcaneal posterior facet would be an autapomorphic feature of the Neandertal lineage.

Extreme population differences in the human zinc transporter ZIP4 (SLC39A4) are explained by positive selection in Sub-Saharan Africa.

Extreme differences in allele frequency between West Africans and Eurasians were observed for a leucine-to-valine substitution (Leu372Val) in the human intestinal zinc uptake transporter, ZIP4, yet no further evidence was found for a selective sweep around the ZIP4 gene (SLC39A4). By interrogating allele frequencies in more than 100 diverse human populations and resequencing Neanderthal DNA, we confirmed the ancestral state of this locus and found a strong geographical gradient for the derived allele (Val372), with near fixation in West Africa. In extensive coalescent simulations, we show that the extreme differences in allele frequency, yet absence of a classical sweep signature, can be explained by the effect of a local recombination hotspot, together with directional selection favoring the Val372 allele in Sub-Saharan Africans. The possible functional effect of the Leu372Val substitution, together with two pathological mutations at the same codon (Leu372Pro and Leu372Arg) that cause acrodermatitis enteropathica (a disease phenotype characterized by extreme zinc deficiency), was investigated by transient overexpression of human ZIP4 protein in HeLa cells. Both acrodermatitis mutations cause absence of the ZIP4 transporter cell surface expression and nearly absent zinc uptake, while the Val372 variant displayed significantly reduced surface protein expression, reduced basal levels of intracellular zinc, and reduced zinc uptake in comparison with the Leu372 variant. We speculate that reduced zinc uptake by the ZIP4-derived Val372 isoform may act by starving certain pathogens of zinc, and hence may have been advantageous in Sub-Saharan Africa. Moreover, these functional results may indicate differences in zinc homeostasis among modern human populations with possible relevance for disease risk.

Patterns of coding variation in the complete exomes of three Neandertals.

We present the DNA sequence of 17,367 protein-coding genes in two Neandertals from Spain and Croatia and analyze them together with the genome sequence recently determined from a Neandertal from southern Siberia. Comparisons with present-day humans from Africa, Europe, and Asia reveal that genetic diversity among Neandertals was remarkably low, and that they carried a higher proportion of amino acid-changing (nonsynonymous) alleles inferred to alter protein structure or function than present-day humans. Thus, Neandertals across Eurasia had a smaller long-term effective population than present-day humans. We also identify amino acid substitutions in Neandertals and present-day humans that may underlie phenotypic differences between the two groups. We find that genes involved in skeletal morphology have changed more in the lineage leading to Neandertals than in the ancestral lineage common to archaic and modern humans, whereas genes involved in behavior and pigmentation have changed more on the modern human lineage.