Humans have a comparatively higher rate of more polymorphisms in regulatory regions of the primate CCR5 gene, an immune system gene with both general and specific functions. This has been interpreted as allowing flexibility and diversity of gene expression in response to varying disease loads. A broad expression repertoire is useful to humans—the only globally distributed primate—due to our unique adaptive pattern that increased pathogen exposure and disease loads (e.g., sedentism, subsistence practices). The main objective of the study was to determine if the previously observed human pattern of increased variation extended to other members of our genus, Homo. The data for this study are mined from the published genomes of extinct hominins (four Neandertals and two Denisovans), an ancient human (Ust’-Ishim), and modern humans (1000 Genomes). An average of 15 polymorphisms per individual were found in human populations (with a total of 262 polymorphisms). There were 94 polymorphisms identified across extinct Homo (an average of 13 per individual) with 41 previously observed in modern humans and 53 novel polymorphisms (32 in Denisova and 21 in Neandertal). Neither the frequency nor distribution of polymorphisms across gene regions exhibit significant differences within the genus Homo. Thus, humans are not unique with regards to the increased frequency of regulatory polymorphisms and the evolution of variation patterns across CCR5 gene appears to have originated within the genus. A broader evolutionary perspective on regulatory flexibility may be that it provided an advantage during the transition to confrontational foraging (and later hunting) that altered human-environment interaction as well as during migration to Eurasia and encounters with novel pathogens.
Citation: Hoover KC (2018) Intragenus (Homo) variation in a chemokine receptor gene (CCR5). PLoS ONE 13(10): e0204989. https://doi.org/10.1371/journal.pone.0204989
Editor: Francesc Calafell, Universitat Pompeu Fabra, SPAIN
Received: September 27, 2017; Accepted: September 18, 2018; Published: October 2, 2018
Copyright: © 2018 Kara C. Hoover. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The author received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Chemokine receptors facilitate communication between cells and the environment [1, 2] and mediate the activity of chemokines, proteins secreted by the immune system genes to chemically recruit immune cells to infection sites via chemotaxis [2, 3]. The cell surface chemokine receptor CCR5 (a G protein-coupled receptor) is best known for its adaptive immune system role in binding the M-tropic human immunodeficiency virus (HIV) and creating a gateway to host cell infection [3–12]. In several mammals, CCR5 genes present high levels of gene conversion with the chromosomally adjacent CCR2 [13–18]. Primate CCR5 gene structure, open reading frame (ORF), and amino acid identity are evolutionary highly conserved [2, 19–21] and interspecific gene sequences are functionally similar . There is, however, common and significant variation across species outside conserved regions. Most of these polymorphisms are not deleterious to health and tolerated due to the redundancy of the chemokine family in ligand binding [2, 22]. New World Monkeys have a high number of functional polymorphisms due to lentivirus resistance . Further, humans have been found to have a significantly high number of cis-regulatory region polymorphisms in comparison to 36 non-human primate species of apes, Old World Monkeys, and New World Monkeys . Humans also have a specific a 32bp deletion in Exon 3, CCR5Δ32 [24, 25], that results in a non-functional protein [24–29]) associated with HIV-resistance and West Nile Virus susceptibility in northern European populations [30–42].
Located on Chromosome 3 (3p21), human CCR5 is 6,065 bases long with an ORF of 1,056 bases that codes for a protein with 352 residues. Two common transcripts (B with three exons and the more stable A with four) likely resulted from non-coding upstream polymorphisms in two separate gene promotors (the functionally weaker cis-acting promoter (PU) upstream of Exon 1 and the downstream promoter (PD) upstream of Exon 3 [19, 20]. These transcripts cause alternate splicing (differential inclusion or exclusion of exons) in messenger RNA that affects regulation of cell surface receptor expression levels [20–22].
The plasticity in regulation of gene expression via alternate transcripts and increased polymorphisms [20–22] makes human CCR5 particularly interesting from a broader evolutionary perspective. Homo has one of the broadest adaptive ranges of any species  and human CCR5’s ability to rapidly respond to new pathogens [19, 20] may have served an adaptive function during evolutionary migration and shifts in human-environment relationships with changes to subsistence. Our genomes carry vast evidence of past disease responses [44–46] that are shared across the genus and reflect a unique disease pattern for Homo. For example, there is strong evidence for increased disease risk via genetic load in extinct Homo and past human populations  and archaeological evidence for past disease treatment (ingestion of anti-biotic and anti-fungal non-food plants) in Neandertals [48–52]. CCR5 has been well studied due to its role in HIV infection (with a focus on natural selection acting on the 32bp deletion) but no work has explored variation within the genus Homo more broadly.
The plethora of research on the evolution CCR5 was conducted prior to the generation of deep coverage, high quality paleogenomes for extinct hominins, such as Neandertal species and the newer Denisova species. While paleogenomic sample sizes are not robust to make statements on selection or add to a discussion of other evolutionary forces acting on variation, they provide an evolutionary dimension to understanding the patterns of variation characterizing our genus and insights into possible adaptations to new environments, subsistence regimes, and pathogens . Plus, the sample of ancient genome is increasing every year. Just a few insights gained from a single paleogenomes include ground-breaking studies on evolution of skin color in humans and Neandertals  and the introgression of functionally adaptive polymorphisms into the human immune system genes from Altai Neandertal . Understanding the differences between derived and specific variation also enables potential differentiation of challenges we overcame as a genus such as obligate bipedalism  or high-altitude adaptation  versus challenges we overcame as a species such as the biocultural evolution of sickle-cell trait and malaria infection . Thus, the overall aim of this research is to place humans within the context Homo and examine if the pattern of humans having a significantly higher number of cis-regulatory region polymorphisms (compared to 36 non-human primate species of apes, Old World Monkeys, and New World Monkeys)  is specific or one that is shared by our genus.
Variation in CCR5 was examined in humans and extinct hominins to address the questions: are there shared patterns of variation across the genus for polymorphism frequency and is the distribution of polymorphisms across the gene suggestive of a common evolutionary trajectory? Based on previous studies on human-nonhuman primate gene structure and variation and the finding that human polymorphisms allow flexible CCR5 gene expression [19, 20], the expectation is that there is a shared pattern of variation that aided adaptation for members of the geographically and ecological dispersed genus Homo. Both expectations were met.
Modern human data is from the 1000 Genomes Project , which contains data for 2,504 individuals from 26 populations (Table 1). While coverage is low per individual, the data are robust enough to identify the majority of polymorphisms at a frequency of at least 1% in the populations studied, which is suitable for the current study. Extinct Homo data are: Denisova 3 , Denisova 2 , Vindija 33.19 Neandertal , Altai Neandertal , El Sidron Neandertal , Mezmaiskaya 1 Neandertal , and an ancient human that contributed no genes to modern populations, Ust’-Ishim —see Table 1 for accession numbers. These species are Pleistocene Eurasian hominins with Denisova representing an eastern Eurasian Pleistocene population and Neandertal a western one (with some overlap with Denisova in Siberia). All genomes have high coverage (excepting Mezmaiskaya and Sidron); contamination with modern human DNA is estimated to be less than 1% for the extinct hominins [61–63, 66, 67].
The human reference sequences for two common transcript variants for the CCR5 gene (NM_000579 and NM_001100168) were downloaded from the National Center for Biotechnology Information (NCBI). The modern human variation data (CCR5 and cis-acting elements) were downloaded from 1000 Genomes via ftp as variant call format (VCF) files (http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/). All data were downloaded to and analyzed using the University of Alaska Research Computing Systems. All files were aligned to the human genome GRCh37/hg19. VCF files for six extinct hominin species and one extinct human (Ust’-Ishim) were downloaded from the Max Planck Institute Leipzig. Ancient DNA often contains C-to-T deaminations at the end of reads . The lack of variation identified from paleogenomic sequence reads is unlikely to be a result of typical problems associated with ancient DNA sequence reads since chemical processes like deamination would increase SNPs (whether false or not). More significantly, the paleogenomes were generated using protocols that largely eliminates this error . Despite high levels of variation at this locus and evidence for balancing selection in humans at this locus, strong levels of introgression from inter-breeding with Neandertals in Eurasia have not been reported at this locus, as they have for other immune system loci in similar scenarios [27, 70–75]; introgression data from the Reich lab (https://reich.hms.harvard.edu/datasets)  confirm this is the case (S1 Table). Moreover, the African genetic variation is similar to European genetic variation which suggests that diversity was already present in modern humans prior to any admixture with archaic species in Europe.
Distribution of polymorphisms was guided by the structure provided by Mummidi et al.  and included promoter regions (PU and PD), ORF, and CCR5. The target area for PU was the most inclusive range (-1976 to +33) which avoided overlap with PD and because little difference was noted between putative PU regions studied by Mummidi et al . The target area for PD was the most productive range (+119 to +828). Significant difference in distribution of polymorphisms across gene structure for all samples was tested using Monte Carlo methods for the exact test.
Results and discussion
Previous research has examined gene structure , gene variation within primate species , and selection acting on the gene primarily in response to viral load [6, 10, 21, 22, 33, 36, 37, 40, 41, 77, 78]. The goal of this research was to establish if the pattern of human variation and distribution of polymorphisms in CCR5  is specific (i.e., unique in the human species) or genus-wide (i.e., a pattern shared by Homo).
Are there shared patterns of variation across the genus for polymorphism frequency? In the modern human sample, 262 known SNPs were observed (Tables 2 and S2 contains all 1000 Genomes variants). SNP frequency per individual (total SNPs in a population/total number of individuals) within the 26 populations ranged from 14 to 24, with East Asians exhibiting the highest variation and Africa and the Americas the least (Table 1). There were 94 polymorphisms identified across all extinct Homo samples (Altai, Denisova 3 and 2, Mezmaiskaya, Ust’-Ishim, Vindija, and El Sid), an average of 13 per individual included in analysis (Tables 1 and 2 and S1). No polymorphisms were found in the El Sidron specimen and, as a result, it is not included in the tables. Some polymorphisms in extinct Homo (n = 41) have been previously observed in modern humans (Table 2). There were 53 novel polymorphisms identified, 32 in Denisova (1 in Denisova 3, 31 in Denisova 2) and 21 in Mezmaiskaya. Table 2 summarizes extinct Homo polymorphisms.
Is the distribution of polymorphisms across the gene suggestive of a common evolutionary trajectory? The frequency of polymorphisms across gene structure are used rather than counts because the human sample is much larger and captures an exponentially greater number of polymorphisms as a result (see S3 Table for raw count summary). Both humans and extinct members of our genus exhibit more polymorphisms in gene regulatory regions (Table 3) suggesting a shared pattern of variation across Homo. When polymorphisms occur in both PU and PD, there is a greater frequency in the functionally stronger regulatory area, PD, but in four ancient samples (Altai, Denisova 3, Vindija, and Ust-Ishim), they only occur in PU (see S3 Table); only Denisova 2 and Mezmaiskaya had no polymorphisms in the ORF. The comparatively lower frequency across all samples reflects the conservation trend noted in primates [19, 20]. A structural analysis of the distribution of polymorphisms via an Exact Test indicated no significant statistical differences among all samples (results not shown). Given the expected frequency of polymorphism (based on the perception of CCR5 covered by an area of interest—see Table 3 footnotes), there is a significant pattern in the samples. First, modern humans and Denisova 2 have a greater than expected number of polymorphisms in the ORF (even if these are exceeded by polymorphisms in regulatory regions). All samples (except Denisova 2) have a greater than expected number of polymorphisms in the promoter regions.
Prior research found that humans have a potentially unique plasticity in gene expression due to the effect of alternate splicing . The distribution of polymorphisms across gene regions in Homo suggests plasticity in gene regulation and expression in response to viral loads, as noted in previous studies [19, 20]. The pattern of immune gene introgression, particularly regulatory haplotypes in the antiviral OAS gene cluster , has suggested that selective forces in our close relatives operated on expression, not protein variation—same as seen in non-human primate CCR5 variation [19, 20]—and those adaptations were also useful to humans. Thus, an increase in polymorphisms that allowed plasticity in regulation and expression in CCR5 makes sense even if it is not due to introgression. Without functional testing, the exact nature of the polymorphisms is not known other than by inference and comparative analysis, as done here. And, without more paleo-genomes to compare, we cannot know if the variation in these genomes represents true species variation but the data presented here indicate that the pattern is not human specific, rather one shared by recent members of Homo.
The expectation that extinct hominins and modern humans would share this pattern of increased variation in the regulatory areas of the gene is met in the current study. Our genus has several unique behavioral and genetic adaptations compared to nonhuman primates and these adaptations might hold some avenues for further research. For instance, a genus-wide shift in subsistence activities occurred during the Plio-Pleistocene (roughly 2 million years ago) from opportunistic non-confrontational scavenging to confrontational scavenging and, later, top predatory behaviors; this alteration to hominin-environment interaction brought hominins into greater and regular contact with animal carcasses [79–84]. Neandertals in Europe have also been shown to be active hunters and foragers  who experienced increased pathogen exposure and disease load as a result [48, 49, 51, 86]. Humans and European Neandertals would have shared similar ecological adaptive pressures in Europe—broad and varied—whereas Altai Neandertal (related to European Neandertals) and Denisova would have shared similar ecological adaptive pressures in Siberia with Ust’-Ishim—less varied. Key pathogens year-round in tropical to temperate zones are more likely to be viral (vector-borne) or bacterial (zoonotic) with transmission via interaction with the environment ; high latitude pathogens year-round are more likely parasitic due to the reliance on marine mammals  and the short season for viral vector reproductive cycles to transmit infection from insects to hominins . Evidence for gene introgression from extinct hominin species to modern humans is clustered (among other domains) in immune system genes [27, 72, 76, 89]; in particular, the OAS anti-viral gene cluster on Chromosome 12 shows signatures of positive selection [72, 76], which suggests that adaptation to Eurasian pathogens may have been partly facilitated by prior adaptative mutations to local viral loads. At a minimum, the environmental challenge faced by non-human members of Homo facilitated human adaptation to a new environment—a shared challenge with a similar solution.
While previous studies have examined variation in CCR5, particularly CCR5Δ32 which has a more recent origin [36–42], as a product of more recent human-disease interaction, the widespread pattern of increased variation in the gene across the genus Homo identified in this study suggests a potential evolutionary adaptation. A key event distinguishing members of the genus Homo from the last common ancestor with Australopithecus was the shift to confrontational scavenging and, later, hunting; this alteration to human-environment interaction added a new point of disease contact as evidenced by modern data showing hunting bushmeat (which ancient hominins did too ) alters disease exposure via introduction of retroviruses and other pathogens [90–94]. Given CCR5‘s role in both innate and adaptive immune system functioning, its plasticity may have provided an advantage to members of Homo across these varied disease ecologies and its potentially greater than normal interaction with the environment in foraging and hunting activities. As more ancient genomes become sequenced, we can have more robust data with which to work and invest resources into functional testing and experimentation of what function these polymorphisms might have had.
S1 Table. Altai neandertal introgression data.
- 1. Allen SJ, Crown SE, Handel TM. Chemokine: receptor structure, interactions, and antagonism. Annu Rev Immunol. 2007;25(1):787–820. pmid:17291188
- 2. Baggiolini M, Dewald B, Moser B. Interleukin-8 and related chemotactic cytokines—CXC and CC chemokines. Adv Immunol. 1994;55:97–179. pmid:8304236
- 3. Samson M, Labbe O, Mollereau C, Vassart G, Parmentier M. Molecular cloning and functional expression of a new human CC-chemokine receptor gene. Biochemistry. 1996;35(11):3362–7. pmid:8639485
- 4. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, et al. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell. 1996;85(7):1135–48. pmid:8674119
- 5. Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, et al. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature. 1996;381:667–73. pmid:8649512
- 6. Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM, et al. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science. 1996;272(5270):1955–8. pmid:8658171
- 7. Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, et al. A dual-tropic primary hiv-1 isolate that uses fusin and the β-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell. 1996;85(7):1149–58. https://doi.org/10.1016/S0092-8674(00)81314-8. pmid:8674120
- 8. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature. 1996;381:661–6. pmid:8649511
- 9. Farzan M, Choe H, Martin K, Marcon L, Hofmann W, Karlsson G, et al. Two orphan seven-transmembrane segment receptors which are expressed in CD4-positive cells support Simian Immunodeficiency Virus infection. J Exp Med. 1997;186(3):405. pmid:9236192
- 10. Liao F, Alkhatib G, Peden KWC, Sharma G, Berger EA, Farber JM. STRL33, A novel chemokine receptor–like protein, functions as a fusion cofactor for both macrophage-tropic and T Cell line–tropic HIV-1. J Exp Med. 1997;185(11):2015. pmid:9166430
- 11. Alkhatib G, Liao F, Berger EA, Farber JM, Peden KWC. A new SIV co-receptor, STRL33. Nature. 1997;388:238. pmid:9230431
- 12. Deng H, Unutmaz D, KewalRamani VN, Littman DR. Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature. 1997;388:296–300. pmid:9230441
- 13. Esteves PJ, Abrantes J, Van Der Loo W. Extensive gene conversion between CCR2 and CCR5 in domestic cat (Felis catus). Int J Immunogenet. 2007;34(5):321–4. pmid:17845301
- 14. Vàzquez-Salat N, Yuhki N, Beck T, O'Brien SJ, Murphy WJ. Gene conversion between mammalian CCR2 and CCR5 chemokine receptor genes: A potential mechanism for receptor dimerization. Genomics. 2007;90(2):213–24. https://doi.org/10.1016/j.ygeno.2007.04.009. pmid:17544254
- 15. Perelygin AA, Zharkikh AA, Astakhova NM, Lear TL, Brinton MA. Concerted Evolution of Vertebrate CCR2 and CCR5 Genes and the Origin of a Recombinant Equine CCR5/2 Gene. J Hered. 2008;99(5):500–11. pmid:18502735
- 16. Carmo CR, Esteves PJ, Ferrand N, van der Loo W. Genetic variation at chemokine receptor CCR5 in leporids: alteration at the 2nd extracellular domain by gene conversion with CCR2 in Oryctolagus, but not in Sylvilagus and Lepus species. Immunogenetics. 2006;58(5):494–501. pmid:16596402
- 17. Abrantes J, Carmo CR, Matthee CA, Yamada F, van der Loo W, Esteves PJ. A shared unusual genetic change at the chemokine receptor type 5 between Oryctolagus, Bunolagus and Pentalagus. Conservation Genetics. 2011;12(1):325–30.
- 18. Schroder KEE, Carey MP, Vanable PA. Methodological Challenges in Research on Sexual Risk Behavior: II. Accuracy of Self-Reports. Ann Behav Med. 2003;26(2):104–23. pmid:14534028
- 19. Mummidi S, Ahuja SS, McDaniel BL, Ahuja SK. The human CC chemokine receptor 5 (CCR5) gene. Multiple transcripts with 5'-end heterogeneity, dual promoter usage, and evidence for polymorphisms within the regulatory regions and noncoding exons. J Biol Chem. 1997;272(49):30662–71. pmid:9388201
- 20. Mummidi S, Bamshad M, Ahuja SS, Gonzalez E, Feuillet PM, Begum K, et al. Evolution of human and non-human primate CC chemokine receptor 5 gene and mRNA. Potential roles for haplotype and mRNA diversity, differential haplotype-specific transcriptional activity, and altered transcription factor binding to polymorphic nucleotides in the pathogenesis of HIV-1 and simian immunodeficiency virus. J Biol Chem. 2000;275(25):18946–61. pmid:10747879
- 21. Zhang YW, Ryder OA, Zhang YP. Sequence evolution of the CCR5 chemokine receptor gene in primates. Mol Biol Evol. 1999;16(9):1145–54. pmid:10486970
- 22. Zhang Y-W, Ryder OA, Zhang Y-P. Intra- and interspecific variation of the CCR5 gene in higher primates. Mol Biol Evol. 2003;20(10):1722–9. pmid:12949140
- 23. Ribeiro IP, Schrago CG, Soares EA, Pissinatti A, Seuanez HN, Russo CAM, et al. CCR5 chemokine receptor gene evolution in New World monkeys (Platyrrini, Primates): implication on resistance to lentiviruses. Infections, Genetics and Evolution. 2005;5:271–80.
- 24. Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, et al. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science. 1996;273(5283):1856–62. pmid:8791590
- 25. Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature. 1996;382(6593):722–5. pmid:8751444
- 26. Temme S, Zacharias M, Neumann J, Wohlfromm S, König A, Temme N, et al. A novel family of human leukocyte antigen class II receptors may have its origin in archaic human species. J Biol Chem. 2014;289(2):639–53. pmid:24214983
- 27. Abi-Rached L, Jobin MJ, Kulkarni S, McWhinnie A, Dalva K, Gragert L, et al. The shaping of modern human immune systems by multiregional admixture with archaic humans. Science. 2011;334(6052):89–94. pmid:21868630
- 28. Huang Y, Paxton WA, Wolinsky SM, Neumann AU, Zhang L, He T, et al. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat Med. 1996;2(11):1240–3. pmid:8898752
- 29. Zimmerman PA, Buckler-White A, Alkhatib G, Spalding T, Kubofcik J, Combadiere C, et al. Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk. Mol Med. 1997;3(1):23–36. pmid:9132277
- 30. Glass WG, McDermott DH, Lim JK, Lekhong S, Yu SF, Frank WA, et al. CCR5 deficiency increases risk of symptomatic West Nile Virus infection. J Exp Med. 2006;203(1):35–40. pmid:16418398
- 31. Lim JK, Louie CY, Glaser C, Jean C, Johnson B, Johnson H, et al. Genetic deficiency of chemokine receptor CCR5 is a strong risk factor for symptomatic West Nile virus infection: a meta-analysis of 4 cohorts in the US epidemic. J Infect Dis. 2008;197(2):262–5. pmid:18179388
- 32. Diamond MS, Klein RS. A genetic basis for human susceptibility of West Nile Virus. Trends Microbiol. 2006;14:287–89. pmid:16750369
- 33. Novembre J, Galvani AP, Slatkin M. The geographic spread of the CCR5 Delta32 HIV-resistance allele. PLoS Biol. 2005;3(11):e339. pmid:16216086
- 34. Novembre J, Johnson T, Bryc K, Kutalik Z, Boyko AR, Auton A, et al. Genes mirror geography within Europe. Nature. 2008;456(7218):98–101. pmid:18758442
- 35. Hoover KC, Barker CM. West Nile virus, climate change, and circumpolar vulnerability. Wiley Interdiscip Rev Clim Change. 2016;7(2):283–300.
- 36. Carrington M, Kissner T, Gerrard B, Ivanov S, O'Brien SJ, Dean M. Novel alleles of the chemokine-receptor gene CCR5. Am J Hum Genet. 1997;61(6):1261–7. pmid:9399903
- 37. Stephens JC, Reich DE, Goldstein DB, Shin HD, Smith MW, Carrington M, et al. Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes. Am J Hum Genet. 1998;62(6):1507–15. pmid:9585595
- 38. Galvani AP, Slatkin M. Evaluating plague and smallpox as historical selective pressures for the CCR5-Delta 32 HIV-resistance allele. Proc Natl Acad Sci U S A. 2003;100(25):15276–9. pmid:14645720
- 39. Limborska SA, Balanovsky OP, Balanovskaya EV, Slominsky PA, Schadrina MI, Livshits LA, et al. Analysis of CCR5Delta32 geographic distribution and its correlation with some climatic and geographic factors. Hum Hered. 2002;53(1):49–54. doi: 48605. pmid:11901272
- 40. Sabeti PC, Walsh E, Schaffner SF, Varilly P, Fry B, Hutcheson HB, et al. The case for selection at CCR5-Delta32. PLoS Biol. 2005;3(11):e378. pmid:16248677
- 41. Hummel S, Schmidt D, Kremeyer B, Herrmann B, Oppermann M. Detection of the CCR5-Delta32 HIV resistance gene in Bronze Age skeletons. Genes Immun. 2005;6(4):371–4. pmid:15815693
- 42. Mecsas J, Franklin G, Kuziel WA, Brubaker RR, Falkow S, Mosier DE. Evolutionary genetics: CCR5 mutation and plague protection. Nature. 2004;427:606. pmid:14961112
- 43. Winder IC, Devès MH, King GCP, Bailey GN, Inglis RH, Meredith-Williams M. Evolution and dispersal of the genus Homo: A landscape approach. J Hum Evol. 2015;87:48–65. https://doi.org/10.1016/j.jhevol.2015.07.002. pmid:26235482
- 44. Brinkworth JF. Infectious Disease and the Diversification of the Human Genome. Hum Biol. 2017;89(1). https://digitalcommons.wayne.edu/humbiol/vol89/iss1/4.
- 45. Quintana-Murci L. Genetic and epigenetic variation of human populations: An adaptive tale. Comptes Rendus Biologies. 2016;339(7):278–83. https://doi.org/10.1016/j.crvi.2016.04.005.
- 46. Karlsson EK, Kwiatkowski DP, Sabeti PC. Natural selection and infectious disease in human populations. Nature reviews Genetics. 2014;15(6):379–93. pmid:24776769
- 47. Berens AJ, Cooper TL, Lachance J. The genomic health of ancient hominins. Hum Biol Oceania. 2017;89(1). https://digitalcommons.wayne.edu/humbiol/vol89/iss1/2.
- 48. Hardy K, Buckley S, Collins MJ, Estalrrich A, Brothwell D, Copeland L, et al. Neanderthal medics? Evidence for food, cooking, and medicinal plants entrapped in dental calculus. Die Naturwissenschaften. 2012;99(8):617–26. pmid:22806252
- 49. Hardy K, Radini A, Buckley S, Blasco R, Copeland L, Burjachs F, et al. Diet and environment 1.2 million years ago revealed through analysis of dental calculus from Europe’s oldest hominin at Sima del Elefante, Spain. The Science of Nature. 2016;104(1):2. pmid:27981368
- 50. Weyrich LS, Duchene S, Soubrier J, Arriola L, Llamas B, Breen J, et al. Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus. Nature. 2017;544(7650):357–61. pmid:28273061
- 51. Hardy K, Buckley S, Huffman M. Doctors, chefs or hominin animals? Non-edible plants and Neanderthals. Antiquity. 2016;90(353):1373–9.
- 52. Essig A, Hofmann D, Munch D, Gayathri S, Kunzler M, Kallio PT, et al. Copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis. J Biol Chem. 2014;289(50):34953–64. pmid:25342741
- 53. Marciniak S, Perry GH. Harnessing ancient genomes to study the history of human adaptation. Nature Reviews Genetics. 2017;advance online publication. http://www.nature.com/nrg/journal/vaop/ncurrent/abs/nrg.2017.65.html. pmid:28890534
- 54. Olalde I, Allentoft ME, Sanchez-Quinto F, Santpere G, Chiang CWK, DeGiorgio M, et al. Derived immune and ancestral pigmentation alleles in a 7,000-year-old Mesolithic European. Nature. 2014;advance online publication. http://www.nature.com/nature/journal/vaop/ncurrent/abs/nature12960.html. pmid:24463515
- 55. Lalueza-Fox C, Römpler H, Caramelli D, Stäubert C, Catalano G, Hughes D, et al. A melanocortin 1 receptor allele suggests varying pigmentation among Neanderthals. Science. 2007;318(5855):1453–5. pmid:17962522
- 56. Racimo F, Sankararaman S, Nielsen R, Huerta-Sanchez E. Evidence for archaic adaptive introgression in humans. Nature Review Genetics. 2015;16(6):359–71. http://www.nature.com/nrg/journal/v16/n6/abs/nrg3936.html. pmid:25963373
- 57. Niemitz C. The evolution of the upright posture and gait—a review and a new synthesis. Die Naturwissenschaften. 2010;97(3):241–63. pmid:20127307
- 58. Huerta-Sanchez E, Jin X, Asan Bianba Z, Peter BM, Vinckenbosch N, et al. Altitude adaptation in Tibetans caused by introgression of Denisovan-like DNA. Nature. 2014;512:194–7. ]http://www.nature.com/nature/journal/v512/n7513/abs/nature13408.html. pmid:25043035
- 59. Livingstone FB. Anthropological implications of sickle cell gene distribution in West Africa. American Anthropologist. 1958;60(3):533–62.
- 60. Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, Handsaker RE, et al. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012;491(7422):56–65. pmid:23128226
- 61. Prufer K, Racimo F, Patterson N, Jay F, Sankararaman S, Sawyer S, et al. The complete genome sequence of a Neanderthal from the Altai Mountains. Nature. 2014;505(7481):43–9. pmid:24352235
- 62. Slon V, Viola B, Renaud G, Gansauge M-T, Benazzi S, Sawyer S, et al. A fourth Denisovan individual. Science Advances. 2017;3(7). pmid:28695206
- 63. Prüfer K, de Filippo C, Grote S, Mafessoni F, Korlević P, Hajdinjak M, et al. A high-coverage Neandertal genome from Vindija Cave in Croatia. Science. 2017; pmid:28982794
- 64. Castellano S, Parra G, Sánchez-Quinto FA, Racimo F, Kuhlwilm M, Kircher M, et al. Patterns of coding variation in the complete exomes of three Neandertals. Proceedings of the National Academy of Sciences. 2014;111(18):6666–71. pmid:24753607
- 65. Fu Q, Li H, Moorjani P, Jay F, Slepchenko SM, Bondarev AA, et al. Genome sequence of a 45,000-year-old modern human from western Siberia. Nature. 2014;514(7523):445–9. pmid:25341783
- 66. Reich D, Green RE, Kircher M, Krause J, Patterson N, Durand EY, et al. Genetic history of an archaic hominin group from Denisova Cave in Siberia. Nature. 2010;468(7327):1053–60. pmid:21179161
- 67. Meyer M, Kircher M, Gansauge M-T, Li H, Racimo F, Mallick S, et al. A High-Coverage Genome Sequence from an Archaic Denisovan Individual. Science. 2012;338(6104):222–6. pmid:22936568
- 68. Dabney J, Meyer M, Pääbo S. Ancient DNA damage. Cold Spring Harb Perspect Biol. 2013;5(7):a012567. pmid:23729639
- 69. Briggs AW, Stenzel U, Meyer M, Krause J, Kircher M, Pääbo S. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA. Nucleic Acids Res. 2010;38(6):e87–e. pmid:20028723
- 70. Gittelman Rachel M, Schraiber Joshua G, Vernot B, Mikacenic C, Wurfel Mark M, Akey Joshua M. Archaic hominin admixture facilitated adaptation to Out-of-Africa environments. Curr Biol. 2016;26(24):3375–82. pmid:27839976
- 71. Hu Y, Ding Q, Wang Y, Xu S, He Y, Wang M, et al. Investigating the evolutionary importance of Denisovan introgressions in Papua New Guineans and Australians. bioRxiv2015.
- 72. Mendez FL, Watkins JC, Hammer MF. Neandertal origin of genetic variation at the cluster of OAS immunity genes. Mol Biol Evol. 2013;30(4):798–801. pmid:23315957
- 73. Pimenoff VN, Mendes de Oliveira C, Bravo IG. Transmission between archaic and modern human ancestors during the evolution of the oncogenic human papillomavirus 16. Mol Biol Evol. 2017;34(1):4–19. pmid:28025273
- 74. Racimo F, Marnetto D, Huerta-Sánchez E. Signatures of archaic adaptive introgression in present-day human populations. Mol Biol Evol. 2017;34(2):296–317. pmid:27756828
- 75. Vernot B, Tucci S, Kelso J, Schraiber JG, Wolf AB, Gittelman RM, et al. Excavating Neandertal and Denisovan DNA from the genomes of Melanesian individuals. Science. 2016;352(6282):235–9. pmid:26989198
- 76. Sankararaman S, Mallick S, Dannemann M, Prufer K, Kelso J, Paabo S, et al. The genomic landscape of Neanderthal ancestry in present-day humans. Nature. 2014;507:354–7. pmid:24476815
- 77. Holmes EC. On the origin and evolution of the human immunodeficiency virus (HIV). Biol Rev Camb Philos Soc. 2001;76(2):239–54. pmid:11396848
- 78. Galvani AP, Slatkin M. Evaluating plague and smallpox as historical selective pressures for the CCR5-Δ32 HIV-resistance allele. Proc Natl Acad Sci U S A. 2003;100(25):15276–9. pmid:14645720
- 79. Brantingham PJ. Hominid–carnivore coevolution and invasion of the predatory guild. J Anthropol Archaeol. 1998;17(4):327–53. https://doi.org/10.1006/jaar.1998.0326.
- 80. de Heinzelin J, Clark JD, White T, Hart W, Renne P, WoldeGabriel G, et al. Environment and behavior of 2.5-million-year-old Bouri hominids. Science. 1999;284(5414):625–9. pmid:10213682
- 81. Domínguez-Rodrigo M. Hunting and scavenging by early humans: The state of the debate. J World Prehist. 2002;16(1):1–54.
- 82. Domínguez-Rodrigo M, Pickering TR. Early hominid hunting and scavenging: a zooarcheological review. Evol Anthr. 2003;12(6):275–82.
- 83. Shipman P, Bosler W, Davis KL, Behrensmeyer AK, Dunbar RIM, Groves CP, et al. Butchering of giant geladas at an Acheulian site Current Anthropology. 1981;22(3):257–68.
- 84. Moleón M, Sánchez-Zapata JA, Margalida A, Carrete M, Owen-Smith N, Donázar JA. Humans and Scavengers: The Evolution of Interactions and Ecosystem Services. Bioscience. 2014;64(5):394–403.
- 85. Richards MP, Pettitt PB, Trinkaus E, Smith FH, Paunović M, Karavanić I. Neanderthal diet at Vindija and Neanderthal predation: The evidence from stable isotopes. Proc Natl Acad Sci U S A. 2000;97(13):7663–6. pmid:10852955
- 86. Hardy BL. Climatic variability and plant food distribution in Pleistocene Europe: Implications for Neanderthal diet and subsistence. Quaternary Science Reviews. 2010;29(5–6):662–79. https://doi.org/10.1016/j.quascirev.2009.11.016.
- 87. Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL, et al. Global trends in emerging infectious diseases. Nature. 2008;451(7181):990–3. pmid:18288193
- 88. Dudley JP, Hoberg EP, Jenkins EJ, Parkinson AJ. Climate change in the North American Arctic: A one health perspective. EcoHealth. 2015;12(4):713–25. pmid:26070525
- 89. Mendez Fernando L, Watkins Joseph C, Hammer Michael F. A haplotype at STAT2 introgressed from Neanderthals and serves as a candidate of positive selection in Papua New Guinea. Am J Hum Genet. 2012;91(2):265–74. https://doi.org/10.1016/j.ajhg.2012.06.015. pmid:22883142
- 90. Steve A-M, Ahidjo A, Placide M-K, Caroline F, Mukulumanya M, Simon-Pierre N- K, et al. High prevalences and a wide genetic diversity of simian retroviruses in non-human primate bushmeat in rural areas of the Democratic Republic of Congo. EcoHealth. 2017;14(1):100–14. pmid:28050688
- 91. van Vliet N, Moreno J, Gomez J, Zhou W, Fa JE, Golden C, et al. Bushmeat and human health: assessing the evidence in tropical and sub-tropical forests. Ethnobiology and Conservation. 2017;6.
- 92. Kurpiers LA, Schulte-Herbrüggen B, Ejotre I, Reeder DM. Bushmeat and emerging infectious diseases: Lessons from Africa. In: Angelici FM, editor. Problematic Wildlife: A Cross-Disciplinary Approach; https://doi.org/10.1007/978-3-319-22246-2_24 Cham: Springer International Publishing; 2016. p. 507–51.
- 93. Mossoun A, Calvignac-Spencer S, Anoh AE, Pauly MS, Driscoll DA, Michel AO, et al. Bushmeat hunting and zoonotic transmission of simian T-Lymphotropic Virus 1 in tropical West and Central Africa. J Virol. 2017;91(10):e02479–16. pmid:28298599
- 94. Chen M, Reed RR, Lane AP. Acute inflammation regulates neuroregeneration through the NF-κB pathway in olfactory epithelium. Proc Natl Acad Sci U S A. 2017;114(30):8089–94. pmid:28696292