Adaptive response of prokaryotic communities to extreme pollution flooding in a Paleolithic rock art cave (Pindal Cave, northern Spain).

A flood event affecting Pindal Cave, a UNESCO World Heritage site, introduced a substantial amount of external sediments and waste into the cave. This event led to the burial of preexisting sediments, altering the biogeochemical characteristics of the cave ecosystem by introducing heightened levels of organic matter, nitrogen compounds, phosphorus, and heavy metals. The sediments included particulate matter and waste from a cattle farm located within the water catchment area of the cavity, along with diverse microorganisms, reshaping the cave microbial community. This study addresses the ongoing influence of a cattle farm on the cave ecosystem and aims to understand the adaptive responses of the underground microbial community to the sudden influx of waste allochthonous material. Here, we show that the flood event had an immediate and profound effect on the cave microbial community, marked by a significant increase in methanogenic archaea, denitrifying bacteria, and other microorganisms commonly associated with mammalian intestinal tracts. Furthermore, our findings reveal that one year after the flood, microorganisms related to the flood decreased, while the increase in inorganic forms of ammonium and nitrate suggests potential nitrification, aligning with increased abundances of corresponding functional genes involved in nitrogen cycling. The results reveal that the impact of pollution was neither recent nor isolated, and it was decisive in stopping livestock activity near the cave. The influence of the cattle farm has persisted since its establishment over the impluvium area, and this influence endures even a year after the flood. Our study emphasizes the dynamic interplay between natural events, anthropogenic activities, and microbial communities, offering insights into the resilience of cave ecosystems. Understanding microbial adaptation in response to environmental disturbances, as demonstrated in this cave ecosystem, has implications for broader ecological studies and underscores the importance of considering temporal dynamics in conservation efforts.

The evolutionary history of Neanderthal and Denisovan Y chromosomes.

Ancient DNA has provided new insights into many aspects of human history. However, we lack comprehensive studies of the Y chromosomes of Denisovans and Neanderthals because the majority of specimens that have been sequenced to sufficient coverage are female. Sequencing Y chromosomes from two Denisovans and three Neanderthals shows that the Y chromosomes of Denisovans split around 700 thousand years ago from a lineage shared by Neanderthals and modern human Y chromosomes, which diverged from each other around 370 thousand years ago. The phylogenetic relationships of archaic and modern human Y chromosomes differ from the population relationships inferred from the autosomal genomes and mirror mitochondrial DNA phylogenies, indicating replacement of both the mitochondrial and Y chromosomal gene pools in late Neanderthals. This replacement is plausible if the low effective population size of Neanderthals resulted in an increased genetic load in Neanderthals relative to modern humans.

Correction: Chronological reassessment of the Middle to Upper Paleolithic transition and Early Upper Paleolithic cultures in Cantabrian Spain.

[This corrects the article DOI: 10.1371/journal.pone.0194708.].

Chronological reassessment of the Middle to Upper Paleolithic transition and Early Upper Paleolithic cultures in Cantabrian Spain.

Methodological advances in dating the Middle to Upper Paleolithic transition provide a better understanding of the replacement of local Neanderthal populations by Anatomically Modern Humans. Today we know that this replacement was not a single, pan-European event, but rather it took place at different times in different regions. Thus, local conditions could have played a role. Iberia represents a significant macro-region to study this process. Northern Atlantic Spain contains evidence of both Mousterian and Early Upper Paleolithic occupations, although most of them are not properly dated, thus hindering the chances of an adequate interpretation. Here we present 46 new radiocarbon dates conducted using ultrafiltration pre-treatment method of anthropogenically manipulated bones from 13 sites in the Cantabrian region containing Mousterian, Aurignacian and Gravettian levels, of which 30 are considered relevant. These dates, alongside previously reported ones, were integrated into a Bayesian age model to reconstruct an absolute timescale for the transitional period. According to it, the Mousterian disappeared in the region by 47.9-45.1ka cal BP, while the Châtelperronian lasted between 42.6k and 41.5ka cal BP. The Mousterian and Châtelperronian did not overlap, indicating that the latter might be either intrusive or an offshoot of the Mousterian. The new chronology also suggests that the Aurignacian appears between 43.3-40.5ka cal BP overlapping with the Châtelperronian, and ended around 34.6-33.1ka cal BP, after the Gravettian had already been established in the region. This evidence indicates that Neanderthals and AMH co-existed <1,000 years, with the caveat that no diagnostic human remains have been found with the latest Mousterian, Châtelperronian or earliest Aurignacian in Cantabrian Spain.

Response to Comment on "The growth pattern of Neandertals, reconstructed from a juvenile skeleton from El Sidrón (Spain)".

The comment by DeSilva challenges our suggestion that brain growth of the El Sidrón J1 Neandertal was still incomplete at 7.7 years of age. Evidence suggests that endocranial volume is likely to represent less than 90% adult size at El Sidrón as well as Neandertal male plus Krapina samples, in line with further evidence from endocranial surface histology and dural sinus groove size.

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.

The growth pattern of Neandertals, reconstructed from a juvenile skeleton from El Sidrón (Spain).

Ontogenetic studies help us understand the processes of evolutionary change. Previous studies on Neandertals have focused mainly on dental development and inferred an accelerated pace of general growth. We report on a juvenile partial skeleton (El Sidrón J1) preserving cranio-dental and postcranial remains. We used dental histology to estimate the age at death to be 7.7 years. Maturation of most elements fell within the expected range of modern humans at this age. The exceptions were the atlas and mid-thoracic vertebrae, which remained at the 5- to 6-year stage of development. Furthermore, endocranial features suggest that brain growth was not yet completed. The vertebral maturation pattern and extended brain growth most likely reflect Neandertal physiology and ontogenetic energy constraints rather than any fundamental difference in the overall pace of growth in this extinct human.

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.

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.

Neandertal and Denisovan DNA from Pleistocene sediments.

Although a rich record of Pleistocene human-associated archaeological assemblages exists, the scarcity of hominin fossils often impedes the understanding of which hominins occupied a site. Using targeted enrichment of mitochondrial DNA, we show that cave sediments represent a rich source of ancient mammalian DNA that often includes traces of hominin DNA, even at sites and in layers where no hominin remains have been discovered. By automation-assisted screening of numerous sediment samples, we detected Neandertal DNA in eight archaeological layers from four caves in Eurasia. In Denisova Cave, we retrieved Denisovan DNA in a Middle Pleistocene layer near the bottom of the stratigraphy. Our work opens the possibility of detecting the presence of hominin groups at sites and in areas where no skeletal remains are found.

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.

Adult Neandertal clavicles from the El Sidrón site (Asturias, Spain) in the context of Homo pectoral girdle evolution.

We undertook a three-dimensional geometric morphometric (3DGM) analysis on 12 new Neandertal clavicle specimens from the El Sidrón site (Spain), dated to 49,000 years ago. The 3DGM methods were applied in a comparative framework in order to improve our understanding of trait polarity in features related to Homo pectoral girdle evolution, using other Neandertals, Homo sapiens, Pan, ATD6-50 (Homo antecessor), and KNM-WT 15000 (Homo ergaster/erectus) in the reference collection. Twenty-nine homologous landmarks were measured for each clavicle. Variation and morphological similarities were assessed through principal component analysis, conducted separately for the complete clavicle and the diaphysis. On average, Neandertal clavicles had significantly larger muscular entheses, double dorsal curvature, clavicle torsion, and cranial orientation of the acromial end than non-Neandertal clavicles; the El Sidrón clavicles fit this pattern. Variation within the samples was large, with extensive overlap between Homo species; only chimpanzee specimens clearly differed from the other specimens in morphometric terms. Taken together, our morphometric analyses are consistent with the following phylogenetic sequence. The primitive condition of the clavicle is manifest in the cranial orientation of both the acromial and sternal ends. The derived condition expressed in the H. sapiens + Neandertal clade is defined by caudal rotation of both the sternal and acromial ends, but with variation in the number of acromia remaining in a certain cranial orientation. Finally, the autapomorphic Neandertal condition is defined by secondarily acquired primitive cranial re-orientation of the acromial end, which varies from individual to individual. These results suggest that the pace of phylogenetic change in the pectoral girdle does not seem to follow that of other postcranial skeletal features.

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.

Possible Further Evidence of Low Genetic Diversity in the El Sidrón (Asturias, Spain) Neandertal Group: Congenital Clefts of the Atlas.

We present here the first cases in Neandertals of congenital clefts of the arch of the atlas. Two atlases from El Sidrón, northern Spain, present respectively a defect of the posterior (frequency in extant modern human populations ranging from 0.73% to 3.84%), and anterior (frequency in extant modern human populations ranging from 0.087% to 0.1%) arch, a condition in most cases not associated with any clinical manifestation. The fact that two out of three observable atlases present a low frequency congenital condition, together with previously reported evidence of retained deciduous mandibular canine in two out of ten dentitions from El Sidrón, supports the previous observation based on genetic evidence that these Neandertals constituted a group with close genetic relations. Some have proposed for humans and other species that the presence of skeletal congenital conditions, although without clinical significance, could be used as a signal of endogamy or inbreeding. In the present case this interpretation would fit the general scenario of high incidence of rare conditions among Pleistocene humans and the specific scenariothat emerges from Neandertal paleogenetics, which points to long-term small and decreasing population size with reduced and isolated groups. Adverse environmental factors affecting early pregnancies would constitute an alternative, non-exclusive, explanation for a high incidence of congenital conditions. Further support or rejection of these interpretations will come from new genetic and skeletal evidence from Neandertal remains.

A geometric morphometrics comparative analysis of Neandertal humeri (epiphyses-fused) from the El Sidrón cave site (Asturias, Spain).

A new collection of 49,000 year old Neandertal fossil humeri from the El Sidrón cave site (Asturias, Spain) is presented. A total of 49 humeral remains were recovered, representing 10 left and 8 right humeri from adults, adolescents, and a juvenile (not included in the analyses). 3D geometric morphometric (GM) methods as well as classic anthropological variables were employed to conduct a broad comparative analysis by means of mean centroid size and shape comparisons, principal components analysis, and cluster studies. Due to the fragmentary nature of the fossils, comparisons were organized in independent analyses according to different humeral portions: distal epiphysis, diaphysis, proximal epiphysis, and the complete humerus. From a multivariate viewpoint, 3D-GM analyses revealed major differences among taxonomic groups, supporting the value of the humerus in systematic classification. Notably, the Australopithecus anamensis (KP-271) and Homo ergaster Nariokotome (KNM-WT 15000) distal humerus consistently clusters close to those of modern humans, which may imply a primitive condition for Homo sapiens morphology. Australopithecus specimens show a high degree of dispersion in the morphospace. The El Sidrón sample perfectly fits into the classic Neandertal pattern, previously described as having a relatively wide olecranon fossa, as well as thin lateral and medial distodorsal pillars. These characteristics were also typical of the Sima de los Huesos (Atapuerca) sample, African mid-Pleistocene Bodo specimen, and Lower Pleistocene TD6-Atapuerca remains and may be considered as a derived state. Finally, we hypothesize that most of the features thought to be different between Neandertals and modern humans might be associated with structural differences in the pectoral girdle and shoulder joint.

The relevance of the first ribs of the El Sidrón site (Asturias, Spain) for the understanding of the Neandertal thorax.

Reconstructing the morphology of the Neanderthal rib cage not only provides information about the general evolution of human body shape but also aids understanding of functional anatomy and energetics. Despite this paleobiological importance there is still debate about the nature and extent of variations in the size and shape of the Neandertal thorax. The El Sidrón Neandertals can be used to contribute to this debate, providing new costal remains ranging from fully preserved and undistorted ribs to highly fragmented elements. Six first ribs are particularly well preserved and offer the opportunity to analyze thorax morphology in Neandertals. The aims of this paper are to present this new material, to compare the ontogenetic trajectories of the first ribs between Neandertals and modern humans, and, using geometric morphometrics, to test the hypothesis of morphological integration between the first rib and overall thorax morphology. The first ribs of the El Sidrón adult Neandertals are smaller in centroid size and tend to be less curved when compared with those of modern humans, but are similar to Kebara 2. Our results further show that the straightening of the first ribs is significantly correlated with a straightening of the ribs of the upper thorax (R = 0.66; p < 0.0001) in modern humans, suggesting modularity in the upper and lower thorax units as reported in other hominins. It also supports the hypothesis that the upper thorax of Neandertals differs in shape from modern humans with more anteriorly projecting upper ribs during inspiration. These differences could have biomechanical consequences and account for stronger muscle attachments in Neandertals. Different upper thorax shape would also imply a different spatial arrangement of the shoulder girdle and articulation with the humerus (torsion) and its connection to the upper thorax. Future research should address these inferences in the context of Neandertal overall body morphology.

The timing and spatiotemporal patterning of Neanderthal disappearance.

The timing of Neanderthal disappearance and the extent to which they overlapped with the earliest incoming anatomically modern humans (AMHs) in Eurasia are key questions in palaeoanthropology. Determining the spatiotemporal relationship between the two populations is crucial if we are to understand the processes, timing and reasons leading to the disappearance of Neanderthals and the likelihood of cultural and genetic exchange. Serious technical challenges, however, have hindered reliable dating of the period, as the radiocarbon method reaches its limit at ∼50,000 years ago. Here we apply improved accelerator mass spectrometry (14)C techniques to construct robust chronologies from 40 key Mousterian and Neanderthal archaeological sites, ranging from Russia to Spain. Bayesian age modelling was used to generate probability distribution functions to determine the latest appearance date. We show that the Mousterian ended by 41,030-39,260 calibrated years bp (at 95.4% probability) across Europe. We also demonstrate that succeeding 'transitional' archaeological industries, one of which has been linked with Neanderthals (Châtelperronian), end at a similar time. Our data indicate that the disappearance of Neanderthals occurred at different times in different regions. Comparing the data with results obtained from the earliest dated AMH sites in Europe, associated with the Uluzzian technocomplex, allows us to quantify the temporal overlap between the two human groups. The results reveal a significant overlap of 2,600-5,400 years (at 95.4% probability). This has important implications for models seeking to explain the cultural, technological and biological elements involved in the replacement of Neanderthals by AMHs. A mosaic of populations in Europe during the Middle to Upper Palaeolithic transition suggests that there was ample time for the transmission of cultural and symbolic behaviours, as well as possible genetic exchanges, between the two groups.

Temporal lobe sulcal pattern and the bony impressions in the middle cranial fossa: the case of the el Sidrón (Spain) neandertal sample.

Correspondence between temporal lobe sulcal pattern and bony impressions on the middle cranial fossae (MCF) was analyzed. MCF bone remains (SD-359, SD-315, and SD-1219) from the El Sidrón (Spain) neandertal site are analyzed in this context. Direct comparison of the soft and hard tissues from the same individual was studied by means of: 1) dissection of two human heads; 2) optic (white light) surface scans; 3) computed tomography and magnetic resonance of the same head. The inferior temporal sulcus and gyrus are the features most strongly influencing MCF bone surface. The Superior temporal sulcus and middle temporal and fusiform gyri also leave imprints. Temporal lobe form differs between Homo sapiens and neandertals. A wider and larger post-arcuate fossa (posterior limit of Brodmann area 20 and the anterior portion of area 37) is present in modern humans as compared to neandertals. However other traits of the MCF surface are similar in these two large-brained human groups. A conspicuous variation is appreciated in the more vertical location of the inferior temporal gyrus in H. sapiens. In parallel, structures of the lower surface of the temporal lobe are more sagittally orientated. Grooves accommodating the fusiform and the lower temporal sulci become grossly parallel to the temporal squama. These differences can be understood within the context of a supero-lateral deployment of the lobe in H. sapiens, a pattern previously identified (Bastir et al., Nat Commun 2 (2011) 588-595). Regarding dural sinus pattern, a higher incidence of petrosquamous sinus is detected in neandertal samples.

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.

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.