Neanderthals and humans interbred in Europe for 5,400 years

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Neanderthals and humans interbred in Europe for 5,400 years May 18, 2023 19:51:35 GMT

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Post by Admin on May 18, 2023 19:51:35 GMT

Material and methods
Selection criteria of the probands
To ensure genotype calling rate, consistency across individuals and phylogenetic positioning in relation to anatomically modern humans, we did not consider contaminated, admixed, low-depth and archaic genomes with abundant uncalled positions in the loci understudy. We hence retained only high-quality genomes from one Denisovan (Denisova 3) and three Neanderthal individuals i.e., Altaï Neanderthal (Denisova 5), Vindija 33.19, and Chagyrskaya 8 [13–16] (S1 Table). These four probands are representative of the two archaic human-related species that spanned over 50,000 years of the Late Pleistocene and across approximately 5,000 km of Eurasia.

Presentation of the blood groups under study
We studied 7 blood group systems covering 11 genes: ABO including H system and Secretor status (ISBT 001 and 018, ABO, FUT1 and FUT2 genes), Rh (ISBT 004, RHD and RHCE genes), Kell and the covalently linked Kx protein (ISBT 006, KEL and XK genes), Duffy (ISBT 008, ACKR1 gene), Kidd (ISBT 009, SLC14A1 gene), MNS (ISBT 002, GYPB gene), and Diego and its Band 3-Memphis variant (ISBT 010, SLC4A1 gene) (S2 Table).

Exploration procedure for blood group alleles
For the probands and blood groups under study, we downloaded the already published [13–16] and curated *.vcf and *.bam(.bai) chromosome files available at the Genome Projects website of the Max Planck Department of Evolutionary Genetics (https://www.eva.mpg.de/genetics/genome-projects.html, S1 Table). For genotype calling filters, see the Supplementary Information of [13–16] and the readme files at cdna.eva.mpg.de/neandertal/. Briefly, the filters included a coverage filter stratified by GC content, minimum coverage of 10, Heng Li’s Mappability 35, Mapping Quality (MQ) of 25, no tandem repeats and no indels.

Then, we briefly proceeded in a two-step screening of the blood group loci. First, we first gathered the genotypes at the key functional changes with depth, allele counts, quality and Phred scores probability using vcftools [17] (S2 Table, S1 File). Second, we browsed all exomes regions within the coding bounds (i.e. from the initiation ATG to the stop codons), in search for additional variation from hg19 (S3 Table, S1 File). While doing so, we paid specific attention to the following five points.

Consideration of the reference sequence.
We aligned with the reference blood group gene sequences used by the ISBT against the GRCh37 (hg19) with nucleotide labelling according to the sense (5’-3’) strand (S2 Table). We noticed that for six loci we studied (ABO, KEL, GYPB, RHCE, SCL4A1 and FUT1) the hg19 reference sequence opens by default onto the antisense strand (3’ → 5’) in the NCBI graphic window and the ancient genome browser, although their cDNA is conventionally the sense strand (5’ → 3’). This has two consequences: the chromosomal coordinates are decreasing as we progress throughout the coding strand (5’ → 3’) of these genes (from exon ’n’ to exon ’n+1’) and overall, nucleotides should be reversed-complemented.

ABO genotype calls.
We inferred the ABO alleles according to the functional approach developed by [18, 19] for pure and chimeric A-B transferase cDNAs. In conformity with this approach, we identified the ABO alleles by 4 letters corresponding to the 4 main amino acid changes in the catalytic site of the glycosyltransferase of pure A or B allele positions, preceded by the presence or not of the G in position c.261 (rs8176719) (i.e. G-AAAA meaning 4 SNPs of A allele generating A phenotype) and the deletion or not of the C at position c.1061 (rs56392308) to differentiate the A1 and A2 alleles. We achieved the ABO allele identification with the screening of all exons and collected the genotypes at 39 additional loci previously identified as responsible for various ABO alleles [20] (S2 Table; Fig A in S1 File). Special attention has been taken to FUT2 whose amino acid numbering in NBCI and hg19 should be rectified to get the correct amino-acid changes as mentioned by the ISBT. This is due to the fact that the initiation codon is the third ATG at the beginning of exon 2 (19: 49,206,247) [21].

RHD and RHCE genotype calls.
For RHD and RCE, while browsing the exons in search for variation with hg19, we gathered the genotypes of the key changes of the RH*Ce, *CE, *ce, and *cE alleles. Any variation with hg19 was consolidated with The Human Rhesusbase.com [22], Erythrogene.com [11] and screenshots of the bam sequences (Figs B-D in S1 File).

For any identification of a variant, we searched for it in all four archaic genomes. In addition, for any call at two key variants of our findings, namely c.733G>C (RHD) and c.712A>G (RHCE), we searched for the other polymorphisms that usually constitute the haplotypes made with them, respectively RHD*DBU, *DLX, *DV, *DVI, *DBS, *DBT, *DUC2 and *ceAR, *ceEK, *ceBI, *ce*SM, and *ceHAR. For this, we browsed both vcf and bam alignment by varying the MQ threshold (S2 Table; Fig D in S1 File).

Identification of indels.
Because indels could have been filtered out in the making the vcf files, all ABO, RHD and RHCE, notably the ABO c.261delG, c.1061delC, and RHCE 209bp insert have been double-checked from the specific indels vcf files (http://ftp.eva.mpg.de/neandertal/Vindija/VCF/indels/) and bam alignments using Integrative Genomics Viewer (IGV [23]) (S2 Table and Fig A in S1 File).

Low-mapped variants.
The screenshots of the bam alignments in simultaneously the four archaic individuals have highlighted a difference in depth and MQ between reference and alternate alleles. This is especially true for variants with very low frequency in modern humans reference panel such as rs17418085 (RHD), rs150073306 (RHD), rs1132763 (RHCE), and rs1132764 (RHCE) in the 3 Neandertals (alternate allele) in comparison with Denisova 3, homozygous for the reference alleles (Figs C and D in S1 File). Hence, these loci may suffer from reference bias, which is known to strongly reduce the depth and mapping of the reads with the alternate alleles [24, 25], and consequently, the genotype accuracy indexes at these loci. Hence, in cases where variants have been called in the released VCFs of some probands but filtered out in the others, we screenshotted the indexed alignments with hg19 using IGV [23] to manually call genotypes with allocated reads count and MQ cut-off (i.e. value above which the reads are not visualized) (S1 File).

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Neanderthals and humans interbred in Europe for 5,400 years May 19, 2023 18:55:06 GMT

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Post by Admin on May 19, 2023 18:55:06 GMT

Results
Detailed information for the blood group systems, genotypes and phenotypes as well as for other polymorphisms identified in these archaic hominins is presented in Tables 1 and 2 and the principal information is shown in Figs 1 and 2.

Fig 1. ABO system.

A, Nomenclature of the archaic ABO alleles [18]. B, Different observed genotypes and inferred phenotypes in Neandertal and Denisova. Red: polymorphisms characteristic of the ABO*A allele, blue: polymorphisms characteristic of the ABO*B allele, brown: deletion characteristic of the ABO*O01 allele.

Fig 2. Representation of the different RHD and RHCE genotypes in Neandertal and Denisova.

Black font, unknown allees in modern humans; blue font, known alleles in modern humans; gray shadow, allele background found in modern humans.

The results of the analysis for each blood group system can be resumed as follows:

ABO system. We found the most common phenotypes present in modern human populations: A1, B and O resulting from the combination of 3 different alleles (Fig 1). Two samples are heterozygous: Chagyrskaya 8 with group A1 (delG-AAAA/G-BAAA without C deletion at position 1061) and Vindija 33.19 with group B (delG-AAAA/G-BABB) and two are homozygous: Altaï with group A1 (G-BAAA/G-BAAA without C deletion at position 1061) and Denisova-3 with group O (delG-AAAA/delG-AAAA).

H/Se system. All the samples present a common FUT1 allele. Concerning FUT2 locus, 3 alleles are found: the conventional functional FUT2*01 allele and two non-functional alleles: FUT2*01N.04 and FUT2*01N: c.714A, p.238stop. Although FUT2*01N.04 (Neanderthal Vindija and Chagyrskaya) is presently found in South East Asian and Oceanian populations [26–28], the latter allele, present in a single dose in Denisova-3, has never been described in modern humans [10, 11].

RH system. All of the backgrounds are based on the Dce (R0) haplotype as described in Africa [8] and we found only one complete Dce (R0) haplotype corresponding to the wild type described in modern human populations [8]. This complete R0 haplotype is present, at a single dose, in the Denisova-3 sample. The other haplotypes are variants encoding partial antigens D, c and e, which are missing some epitopes. All of these haplotypes, whether wild or variant, are related, phylogenetically speaking, to the African RHD*DIVa cluster [8]. From the three Neanderthal genomes, we have identified one potential novel RHD variant sharing typical SNP combinations assigned to this cluster (Fig 2). This variant exhibited the three SNPs that characterize the partial RHD*DIII type4 (or RHD*03.04 defined by c.186T, c.410T and c.455C), combined with two additional SNPs, c.602C>G and c.733G>C. This latter SNP defining the partial RHD*DUC2 allele. This new combination has been reported for the first time in only one modern Australian Aborigine [29] and occurred at a homozygous level in Altaï, and at least at a heterozygous level in Vindija and Chagyrskaya, with a normal RHD Exon-9 sequence (c.1170T, c.1193A) for all of them. This new RHD variants segregated with RHCE*ce allele variants although the RHD*DIII type4 has been reported to segregate with RHCE*Ce in modern humans [30].

Furthermore, we found the presence of at least three RHCE*ceEK variant alleles (c.48G>C, c.712A>G, c.787A>G and possibly c.800T>A) in Altai Neandertal, Vindija and Chagyrskaya, and two wild-type RHCE*ce in Denisova-3. Each of the Neanderthal archaic genomes has one other African RHCE*ce variant in cis. The RHCE*ceEK allele is mostly encountered in people of African descent [31]. When present in double dose, it encodes a rare phenotype defined by the absence of public antigen RH:-18. The anti-RH18 antibody has been reported to be responsible for haemolytic transfusion reactions and haemolytic disease of the foetus and new-born (HDFN) [32].

MNS, KEL, Duffy, Kidd and Diego systems. All four archaic samples present the deduced phenotype S-s+, K negative, Fy(a-b+), Jk (a+b-) and Di (a-b+) due to double-dose of MNS*04, KEL*02, FY*02, JK*01, DI*02 respectively. The three Neanderthals present the deduced phenotype Js(a+b-) of the Kell system due to double-dose of the ancestral KEL*06 allele, which is exclusive to African populations with 1% of Js(a+b-) phenotype [11]. On the contrary, Denisova 3 presents the deduced Js(a-b+) phenotype resulting from a double-dose of the antithetical KEL*07 allele. Lastly, the three Neanderthals are carriers of the Band 3-Memphis variant (according to rs5036).

Last Edit: May 19, 2023 18:55:47 GMT by Admin

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Neanderthals and humans interbred in Europe for 5,400 years May 20, 2023 20:52:42 GMT

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Post by Admin on May 20, 2023 20:52:42 GMT

Discussion
Neanderthals are a human hunter-gatherer fossil population that lived in Eurasia between 250 kya and 38 kya before being totally replaced throughout their territory by Homo sapiens. Morphological features progressively evolved from their African ancestors (Homo heidelbergensis) and adapted to the cooler climate of Europe [33]. Their arrival in Europe marks a major cultural change with the importation of a new tool, well known in Africa since at least 1.5 mya and in the Levant, the handaxe [34]. The Denisovans are also an extinct human population but bone record is too fragmentary. Their "discovery" and phylogeny relies only on genetic studies [35, 36].

Neanderthal populations were never large, structured in small interconnected groups (about 20 individuals) that never outnumbered 70,000 individuals at the time of their "golden age" (the "emian" interglacial at OIS 5, around 120 kya) [37]. The small size of the population has to be correlated with the partial geographic isolation of Neanderthals caused by European climatic fluctuations during Pleistocene. It was at this time that the Neanderthals, well identified by singular morphological features, spread eastwards carrying the same lithic assemblages and technology as far east as the Altai mountains [38] where they encountered the Denisovans. Genetic data has also pointed out the low genetic diversity of Neanderthals, with a demographic depression peak in the Altai where a very consanguineous individual was found [14], and genetic continuity across Europe from 120 kya until the disappearance of the population around 40 kya [39]. This low variability is also visible in the morphology of the Neanderthals, which remained the same during the last 100 kya of their existence throughout their territory from the Atlantic to the Altai [40]. In this view, our results provide four main points relevant for the origin, vulnerabilities and dispersion of Neanderthal and Denisova (Figs 3 and 4).

Fig 3. Erythroid blood group distribution from Denisova and Neanderthal archaic genomes.

Branching matches nuclear DNA tree topology [43]. Blue, Neanderthal lineage; red, Denisovan lineage. Made with Natural Earth. Free vector and raster map data @ naturalearthdata.com.

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Neanderthals and humans interbred in Europe for 5,400 years May 21, 2023 21:18:20 GMT

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Post by Admin on May 21, 2023 21:18:20 GMT

Fig 4. Introgression scan of modern humans in the RHD region.

Introgression tracts were called at confidence > = 0.2 and <0.5 (15.7 Kb, light purple) and confidence > = 0.5 and <0.9 (4.6 Kb, dark purple) in 5 haplotypes from 2 HGDP-CEPH populations (1: Papuans Sepik; 2: Brahuis). Alleles carried by these haplotypes at 5 key SNPs for the RHD*DIII type 4 with c.602G and DUC2 are shown in green or orange, whether they are the ancestral or derived allele in the human lineage. The bottom panel shows the position of these SNPs on the RHD map.

Consolidation of an African origin for Neanderthal and Denisova
First, the analysis of archaic blood groups anchors the lineage of Eurasian Neanderthals and Denisovans to Africa via: the absence of the antigen combination RhC, RhE, K, Fya, Jkb and S (14% of sub-Saharan African populations, 0.06% in non-African populations [10]), the presence of a double-dose of ancestral forms of the RH, Kell, Duffy, Kidd, MNS and Diego blood groups (respectively the haplotype Dce/Dce, KEL*02/*02, KEL*06/*06, FY*02/*02, JK*01/*01, MNS*04/*04 and Memphis form of Band 3 system [8, 41, 42], and RHD, RHCE variants phylogenetically linked or presently exclusive to African clusters and populations [31]. These features are in accordance with a Neanderthal and Denisovan gene pool pre-dating the exit of Homo sapiens from Africa [43].

A (mysterious) genetic link between Neanderthals and Australia
The new partial RHD allele, RHD*DIII type4 with c.602G and c.733C, reported in the three Neanderthal individuals, and not in Denisova 3, was unknown in modern humans until 2019 and its description as a new variant in an individual from the First Nation of Australia with a RHD Exon-9 deletion or rearrangement with RHCE Exon-9 [29]. Thus, this polymorphism is not a new variant in the historical sense of the term, as it was already present around 100 kya in Neanderthals. This result fuels the discussion about admixture events between the different lineages and also about the early dispersal of Homo sapiens via the Arabian Peninsula [44] towards Australia [45] and Oceania [46]. In order to assess whether the Neanderthal RHD*DIII type4 with c.602G and c.733C arose in modern humans from shared ancestry or introgression, we have modelled high-coverage modern chromosomes from Asia and Oceania (HGDP-CEPH collection, publicly available, [47]) as a mosaic of modern and archaic states using a hidden-Markov model (see S2 File for detailed methods). We found a probable introgressed tract of 15.7 Kb spanning over 4 exons of RHD in 5 individuals: 2 HGDP-Brahuis and 3 HGDP-Sepik Papuans (Fig 4). The 3 Sepik Papuans are heterozygous carriers of the derived allele at rs150073306 which defines the DUC2 allele (raising thus the MAF of the derived allele nearly to 19% in that population—see S2 File), for which Altai is homozygous. When phased, there is an almost identical haplotype to Altai and the Australian Aborigine in Papuan HGDP00546. A parsimonious hypothesis to explain the relic of a RHD haplotype common amongst all Neanderthals in 2 modern Oceanians only, would suggest that the RHD*DIII type4 with c.602G and c.733C profile have been carried by Levantine Neanderthals and passed to modern humans before 65 kya and the route towards SouthEast Asia [48, 49]. This assumption remains however speculative as the archaic tract was called at a low confidence level (>20%). A shorter tract (4.6Kb) was still called at a confidence level of >50% but not at higher confidence thresholds (>90%). Further analyses would be required to validate our assumption.

The signature of environmental pressure
Our analysis of the ABO and FUT2 genes supports the fact that pathogens have played a major role in the survival and selection of genetic variants in modern and archaic hominins [50]. The molecular bases of the archaic ABO phenotypes, particularly for A and B groups, are identical to those found in non-human primates, which confirms an ancient balanced selection shared by Primate species [51]. Noteworthy is the presence of two non-secretor FUT2 polymorphisms in Neanderthal and Denisovan individuals. To date, the non-secretor phenotype has not been found in non-human primates [51] and shown to confer more resistance to some viruses with a notable resistance to Norovirus (Norwalk-like, Caliciviridae family) infection [52–54], which is a major cause of acute gastroenteritis worldwide. Given the short existence of the Norwalk-like virus in humans (post-Neolithic [55]), two scenarios would explain the occurrence of the non-secretor trait in archaic humans. FUT2 could have evolved neutrally in hominins before the late occurrence of selective pressure, in the same way as for glucose transporters (GLUT 2 and 12) and G6PD2 with malaria [56]. Or, given that hominids likely hosted some viruses [57], a Norwalk-like virus (or a sister group) could have already existed 90,000 years ago across Eurasia, causing enough mortality in young children to select two convergent FUT2 alleles. Note that the occurrence of the Neanderthal non-secretor allele in Oceanians [26–28] remains open.

Demographic ‘fragility’
Lastly, our study highlights unfavorable characteristics that can lead to "demographic fragility". This fragility can be evoked on the basis of two elements: a low genetic diversity and the possible presence of HDFN. Indeed, the large number of shared alleles by the four archaic genomes despite their geographical and temporal distribution may be related to the deduced inbreeding situation in Neanderthals [14–16, 58], known to be a source of low adaptability. Meanwhile, the Neanderthal RH allele variants encode for partial RhD, Rhc and Rhe antigens, only Denisova 3 presents a complete form in terms of epitopes, such as they are described in their "wild" forms in modern humans. Partial RhD, Rhc and Rhe antigens lacking epitopes may induce an immune response when exposed to complete antigens [59, 60]. Moreover, when the RHCE*ceEK allele is present in a double dose (a situation which may turn out to be frequent in view of its presence in the 3 Neanderthals), in addition to the presence of partial Rhc and Rhe antigens, it encodes a phenotype defined by the absence of an Rh antigen named RH18. Today, this antigen is considered to be a high frequency antigen in the modern human population. Thus, a Neanderthal mother with partial RhD, Rhc, and Rhe phenotypes and sometimes RH:-18, carrying a Denisovan foetus expressing complete forms of RhD, Rhc and Rhe antigens and expressing the RH18 antigen, would have been prone to be immune to missing epitopes and synthesize anti-RhD, anti-Rhc, anti-Rhe and even anti-RH18 antibodies. These antibodies are known to have an important clinical significance in terms of HDFN [32]. These elements could have contributed to weakening the descendants to the point of leading to their demise especially combined with the competition with Homo sapiens for the same ecological niche [61].

Conclusions
Analyses of blood group systems of Neanderthals and Denisovans contributed to a better understanding of their origin, expansion and encounters with Homo sapiens. Blood group profiles revealed polymorphism at the ABO locus, ancestral and African-origin alleles, and a RH haplotype presently secluded in Oceania, plausible relic of introgression events into modern humans prior their expansion towards Southeast Asia. An additional contribution is the reduced variability of many alleles and the possible presence of haemolytic disease of the foetus and new-born, which reinforces the notion of high inbreeding, weak demography and endangered reproductive success of the late Neanderthals, giving to our species the great opportunity to spread throughout the world.

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Neanderthals and humans interbred in Europe for 5,400 years Jun 9, 2023 5:30:22 GMT

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Post by Admin on Jun 9, 2023 5:30:22 GMT

Recent scientific discoveries have shown that Neanderthal genes comprise some 1 to 4% of the genome of present-day humans whose ancestors migrated out of Africa, but the question remained open on how much those genes are still actively influencing human traits—until now.

A multi-institution research team including Cornell has developed a new suite of computational genetic tools to address the genetic effects of interbreeding between humans of non-African ancestry and Neanderthals that took place some 50,000 years ago. (The study applies only to descendants of those who migrated from Africa before Neanderthals died out, and in particular, those of European ancestry.)

In a study published in eLife, the researchers reported that some Neanderthal genes are responsible for certain traits in modern humans, including several with a significant influence on the immune system. Overall, however, the study shows that modern human genes are winning out over successive generations.

"Interestingly, we found that several of the identified genes involved in modern human immune, metabolic and developmental systems might have influenced human evolution after the ancestors' migration out of Africa," said study co-lead author April (Xinzhu) Wei, an assistant professor of computational biology in the College of Arts and Sciences. "We have made our custom software available for free download and use by anyone interested in further research."

Using a vast dataset from the UK Biobank consisting of genetic and trait information of nearly 300,000 Brits of non-African ancestry, the researchers analyzed more than 235,000 genetic variants likely to have originated from Neanderthals. They found that 4,303 of those differences in DNA are playing a substantial role in modern humans and influencing 47 distinct genetic traits, such as how fast someone can burn calories or a person's natural immune resistance to certain diseases.

Unlike previous studies that could not fully exclude genes from modern human variants, the new study leveraged more precise statistical methods to focus on the variants attributable to Neanderthal genes.

While the study used a dataset of almost exclusively white individuals living in the United Kingdom, the new computational methods developed by the team could offer a path forward in gleaning evolutionary insights from other large databases to delve deeper into archaic humans' genetic influences on modern humans.

"For scientists studying human evolution interested in understanding how interbreeding with archaic humans tens of thousands of years ago still shapes the biology of many present-day humans, this study can fill in some of those blanks," said senior investigator Sriram Sankararaman, an associate professor at the University of California, Los Angeles. "More broadly, our findings can also provide new insights for evolutionary biologists looking at how the echoes of these types of events may have both beneficial and detrimental consequences."

More information: Xinzhu Wei et al, The lingering effects of Neanderthal introgression on human complex traits, eLife (2023). DOI: 10.7554/eLife.80757

Journal information: eLife

Provided by Cornell University

elifesciences.org/articles/80757