Abstract

A fully sequenced high-quality genome has revealed in 2010 the existence of a human population in Asia, the Denisovans, related to and contemporaneous with Neanderthals. Only five skeletal remains are known from Denisovans, mostly molars; the proximal fragment of a fifth finger phalanx used to generate the genome, however, was too incomplete to yield useful morphological information. Here, we demonstrate through ancient DNA analysis that a distal fragment of a fifth finger phalanx from the Denisova Cave is the larger, missing part of this phalanx. Our morphometric analysis shows that its dimensions and shape are within the variability of Homo sapiens and distinct from the Neanderthal fifth finger phalanges. Thus, unlike Denisovan molars, which display archaic characteristics not found in modern humans, the only morphologically informative Denisovan postcranial bone identified to date is suggested here to be plesiomorphic and shared between Denisovans and modern humans.

INTRODUCTION

In 2010, a small fragment of a finger phalanx recovered from the Denisova Cave (Denisova 3) in southern Siberia yielded a mitochondrial and a draft genomic sequence that changed our view of the evolution of the Late Pleistocene hominin lineages in Eurasia (1, 2), revealing a previously unknown archaic human population. The phylogenetic analysis of the Denisova 3 mitogenome yielded a divergence date from the ancestors of Homo sapiens and Neanderthals of around 1 million years (Ma) ago (1.3 to 0.7 Ma ago) (1, 3, 4), i.e., much earlier than the mitogenomes of the Neanderthals from the Late Pleistocene that diverged about 500 thousand years (ka) ago [690 to 350 ka ago; (3)] (Fig. 1). The nuclear genome, however, suggests a much more recent common ancestor between European Neanderthals (Vindija) and Denisovans dating to around 400 ka ago [440 to 390 ka ago; (5)], characterizing Denisovans as a sister group to Neanderthals (3, 58) (Fig. 1). Later, traces of an even more archaic human have been identified in the Denisova 3 nuclear genome (7), and a mitochondrial sequence related to that of Denisova 3 has been found in a ca. 400,000-year-old specimen from Sima de los Huesos (Spain), the nuclear genome of which is more closely related to Neanderthals than to Denisovans (3, 9). Together, these data suggest that the Denisovan mitogenome was either replaced with that of a more archaic human following an admixture event or represents the mitogenome of the common ancestors of Neanderthals and Denisovans before its replacement in the lineage of the Late Pleistocene Neanderthals (Fig. 1) (24, 910). The mitogenome of the late Neanderthals either could result from an introgression (i.e., replacement of the mitogenome following admixture) from early anatomically modern humans (AMHs) early after the separation of the AMH and Neanderthal populations, as proposed in one study (4), or could be due to incomplete lineage sorting given the uncertainties in the methods to estimate the dates and the wide confidence intervals of the dates proposed (Fig. 1). Furthermore, the comparison of the Denisova 3 nuclear genome with the genomic sequence of a roughly 100,000-year-old Neanderthal from the Denisova Cave revealed that Denisovans had also experienced gene flow from a Neanderthal population (Fig. 1) (7). Recently, a bone fragment, also from the Denisova Cave, has been found, through genomic analysis, to belong to a female individual that was the F1 hybrid of a Neanderthal mother and a Denisovan father (11). Her maternal Neanderthal contribution is more closely related to the genome of the 40,000-year-old European Neanderthal from Vindija (5) than to that of the ~100,000-year-old Neanderthal from the Denisova Cave. Furthermore, the paternal Denisovan genome of the hybrid appears to bear traces of an ancient Neanderthal admixture (11). These data indicate that gene flow between Neanderthals and Denisovans was not a rare occurrence.

Fig. 1 Model of the phylogeny of Neanderthal, Denisovan, and AMH populations over the past 1,400,000 years as deduced from both nuclear (blue envelope) and mitochondrial genomes (red lines).

The vertical axis represents time in thousands of years (ka) ago. Population divergence dates estimated from genomic data and mitochondrial genome bifurcation date estimations originate from Prüfer et al. and Meyer et al., respectively (3, 5). Markers on the left indicate the means of the estimates for dates, and error bars indicate 95% confidence intervals. Gene flow events inferred from genome sequences are represented as dotted blue arrows (see text).

” data-hide-link-title=”0″ data-icon-position=”” href=”https://advances.sciencemag.org/content/advances/5/9/eaaw3950/F1.large.jpg?width=800&height=600&carousel=1″ rel=”gallery-fragment-images-2094537582″ title=”Model of the phylogeny of Neanderthal, Denisovan, and AMH populations over the past 1,400,000 years as deduced from both nuclear (blue envelope) and mitochondrial genomes (red lines). The vertical axis represents time in thousands of years (ka) ago. Population divergence dates estimated from genomic data and mitochondrial genome bifurcation date estimations originate from Prüfer et al. and Meyer et al., respectively (3, 5). Markers on the left indicate the means of the estimates for dates, and error bars indicate 95% confidence intervals. Gene flow events inferred from genome sequences are represented as dotted blue arrows (see text).”>

Fig. 1 Model of the phylogeny of Neanderthal, Denisovan, and AMH populations over the past 1,400,000 years as deduced from both nuclear (blue envelope) and mitochondrial genomes (red lines).

The vertical axis represents time in thousands of years (ka) ago. Population divergence dates estimated from genomic data and mitochondrial genome bifurcation date estimations originate from Prüfer et al. and Meyer et al., respectively (3, 5). Markers on the left indicate the means of the estimates for dates, and error bars indicate 95% confidence intervals. Gene flow events inferred from genome sequences are represented as dotted blue arrows (see text).

Molecular dating methods based on mitochondrial sequences indicate that Denisovans must have inhabited the Altai region for over tens of thousands of years (12, 13). Despite the fact that all Denisovan mitochondrial sequences come from the same archeological site, Denisova Cave, the mitochondrial diversity of Denisovans is higher than that of Neanderthals spanning from Spain to the Caucasus (12). As inferred from the high-coverage Denisova 3 genome, the Altai population of Denisovans is characterized by low nuclear genome diversity, consistent with a prolonged small population size (10). Neanderthal populations also appeared to have been small, as assessed through the analysis of both the Altai and the Vindija genomes (5, 7). On the basis of the modeling, it has been proposed that despite reduced nuclear diversity of the individual local populations, the overall nuclear diversity of the Neanderthal metapopulation was higher (8), although this point remains under discussion as it varies with the modeling methods (6, 14). The extent of the Denisovan metapopulation diversity is still awaiting genomic characterization of remains originating from beyond the Denisova Cave, but the presence of Denisovan ancestry in modern human genomes suggests that there were at least two distinct Denisovan populations (15). Indeed, the comparison of the genomic sequence of Denisova 3 with the genomes of present-day humans has revealed interbreeding between Denisovans and early AMHs ancestral to present-day human populations not only in Southeast Asia, above all in Melanesians, but also in mainland East Asia (e.g., 1518). The Denisovan ancestry in Melanesians appears to originate from a Denisovan population distantly related to that of the Denisova 3 specimen, and a similar ancestry can also be found in East Asia, particularly in Chinese and Japanese (15). In East Asians, a second Denisovan introgression from a Denisovan population more closely related to the Denisova 3 specimen was also detected (15). In some cases, the introgression proved to be adaptive, for example, in Tibetans (19) and Inuits (20).

The distribution and diversity of Denisovan DNA in present-day human populations suggest that Denisovans were once widely distributed throughout Asia (15, 18). This evidence stands in contrast to the scarcity of unambiguously identified remains and of associated characteristic morphological features. What little morphological information that is available comes from a mandible from Xiahe on the Tibetan Plateau and three teeth from the Denisova Cave (2, 12, 13, 21). Denisovan mitochondrial genome sequences and low amounts of nuclear DNA have been recovered from a deciduous molar (Denisova 2) and two large-sized permanent molars (Denisova 4 and 8) (1, 12, 13), while the mandible has been identified as Denisovan based on proteomic information (21). The morphology of the Xiahe mandible is similar to that of the Middle Pleistocene specimens, such as the Chinese Lantian and Zhoukoudian, with features of the dental arcade shape that separate it from Homo erectus (21). It harbors some traits reminiscent of Neanderthals, while other Neanderthal-specific features are lacking (21). Thus, the rare Denisovan human remains identified to date show affinity to Middle Pleistocene hominins (2, 12, 13), particularly to those from China (21) and, to a lesser extent, to the Neanderthal lineage (12). The permanent molars from the Denisova Cave show complex occlusal morphology (1, 12, 13). Whether these peculiar characteristics of the molars are the consequence of introgression from a more archaic Eurasian population remains to be seen but cannot be excluded since such a low-level introgression has been identified in the Denisova 3 genome (7).

Despite the importance of the Denisovan population for the study of human evolution, identification of Denisovan postcranial remains relies presently only on genomic data, since these remains of Denisovans exhibiting diagnostic features have yet to be reported. Progress in the identification of Denisovan skeletal remains would be instrumental for our understanding of this human lineage, for the identification of Denisovan remains, and for our ability to better characterize Denisovan population genomic diversity. Here, we report the morphometric analysis of a phalanx fragment that we show through its mitochondrial sequence to be the larger distal part of the original Denisova 3 phalanx, the genome of which had been published in 2010 and 2012 (1, 2, 10). In 2009, the phalanx was cut into two parts. The pictures of the phalanx taken by the Russian scientific team prior to its cutting, however, have been lost. The smaller proximal part of the bone was sent to the Max Planck Institute (MPI) for Evolutionary Anthropology in Leipzig, Germany, and sampling for paleogenomic analysis was performed. The larger distal part was sent to the University of Berkeley, CA, USA, and, in 2010, from there to the “Institut Jacques Monod” (IJM) in Paris, France, where it was measured and photographed and analyzed genetically. It was then returned to the University of Berkeley in 2011.

The present analysis of both phalanx parts represents the first morphological study of nondental remains of this mysterious population that has inhabited Asia for hundreds of thousands of years, has interbred sometimes with Neanderthals and possibly with more archaic Eurasian humans, and continues to endure in the genomes of some present-day human populations.

RESULTS AND DISCUSSION

Complete mitogenome sequence of the Denisova 3 fifth distal manual phalanx allows unambiguous matching of the two parts of the Denisovan phalanx

The Denisova 3 phalanx (2008 Д-2/ 91) was identified in 2008 in layer 11.2 of the East Gallery, square D2, of the Denisova Cave, the date of which is assumed to be more than 50 ka ago (1, 2). To unambiguously match this distal phalanx fragment to the previously described proximal fragment, we retrieved its complete mitogenome [mitochondrial DNA (mtDNA)] sequence using DNA extraction and sequence capture procedures previously described (2224). In total, 5838 unique reads were recovered, yielding a 26.7-fold coverage of the mitochondrial genome, of which each base was covered a minimum of twice (42 bases at twofold coverage) and a maximum of 70 times. The resulting consensus was identical to the previously published sequence (1) and represents the first replication of the Denisova mtDNA sequence outside of the MPI for Evolutionary Anthropology. This analysis also identified the previously proposed variable length in vivo of the polycytosine run at rCRS (revised Cambridge Reference Sequence) position 5889 (1), here found to be between 9 and 14 residues in length. This sequence identity indicates that the two phalanx fragments belong to the same individual.

The exceptional preservation of endogenous DNA in the Denisovan phalanx is evident not only in its high endogenous DNA content but also in the length of the DNA fragments recovered. While an endogenous DNA content of 70% was recovered from the proximal fragment using the more sensitive single-stranded DNA library preparation method (10, 22), shotgun sequencing of the distal fragment analyzed in this study prepared using a double-stranded library method contained 11.3% endogenous content. This is in line with the ~6-fold increase in endogenous content reported between these two methods (10, 22). Previous analyses of the distribution of the endogenous DNA fragment lengths were performed using only merged reads, which prohibits the identification of endogenous molecules longer than 134 nucleotides. The paired-end mapping strategy used in this study reveals a more complete endogenous fragment length distribution. We show all Denisova-mapping DNA fragments to have a mean distribution of 86.7 base pairs (bp), with a median of 81.3 (Fig. 2). The mean value is similar to that first reported [mean, 85.3 bp; (1)], although these two means are skewed toward larger fragments since the library construction method used for these two studies did not recover the greater part of shorter fragments found in libraries prepared with the single-stranded library method (10). The longest fragment containing diagnostic Denisovan nucleotide sites recovered in this study was 236 bp.

Fig. 2 Distribution of DNA fragment lengths mapping to the Denisova mitochondrial sequence.

Fragment lengths given in 10-bp bins are shown. Median is indicated by the dotted orange line. Read pairs that did not overlap sufficiently to be merged (i.e., longer than 134 nucleotides) but that could be mapped as paired-end reads were analyzed individually, and fragments carrying Denisovan single nucleotide polymorphisms (SNPs) were kept.

” data-hide-link-title=”0″ data-icon-position=”” href=”https://advances.sciencemag.org/content/advances/5/9/eaaw3950/F2.large.jpg?width=800&height=600&carousel=1″ rel=”gallery-fragment-images-2094537582″ title=”Distribution of DNA fragment lengths mapping to the Denisova mitochondrial sequence. Fragment lengths given in 10-bp bins are shown. Median is indicated by the dotted orange line. Read pairs that did not overlap sufficiently to be merged (i.e., longer than 134 nucleotides) but that could be mapped as paired-end reads were analyzed individually, and fragments carrying Denisovan single nucleotide polymorphisms (SNPs) were kept.”>

Fig. 2 Distribution of DNA fragment lengths mapping to the Denisova mitochondrial sequence.

Fragment lengths given in 10-bp bins are shown. Median is indicated by the dotted orange line. Read pairs that did not overlap sufficiently to be merged (i.e., longer than 134 nucleotides) but that could be mapped as paired-end reads were analyzed individually, and fragments carrying Denisovan single nucleotide polymorphisms (SNPs) were kept.

In contrast to the analyses of the proximal half of the phalanx, which reported low levels of modern human DNA contamination (0.35%) (1), the distal half showed the presence of a much higher modern human mitochondrial contaminant (12.1% detected by a similar method). Further investigation revealed that this contaminant could be attributed to a single haplogroup, J1b1a1. Since no member of the IJM laboratory in Paris where the genetic analysis was performed carried mitochondrial haplogroup J, we suspect this contamination to have occurred at some point during the previous handling of the sample, prior to its preparation for genetic analysis.

Anatomical description

When the mitogenome analysis of the proximal epiphysis from the metaphyseal surface indicated that the specimen was not a recent modern human, it was digitized through microcomputed tomography (μCT) at the Department of Human Evolution at the MPI for Evolutionary Anthropology, Leipzig (courtesy of H. Temming and J.-J. Hublin). The reconstructed image based on these scans is shown in Fig. 3. Further sampling for nuclear DNA analysis was performed on this specimen (2), and two small holes on the articular surface witness the drilling procedure. The proximal fragment of the phalanx is now composed of two pieces (the proximal epiphysis and the remains of the dorsal part of the diaphysis) (Fig. 3).