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Home , Denisova Cave, Krapina, Mezmaiskaya cave, Neanderthal, Neanderthal anatomy, Neanderthal behavior

WHAT’S IN A : A COMPARATIVE ANALYSIS

Taylorlyn Stephan Oberlin College Dept. of Advised by Prof. Amy Margaris

TABLE OF CONTENTS

I. Abstract – pg. 3 II. Introduction – pg. 3-4 III. Historical Background – pg. 4-5 a. Fig. 1 – pg. 5 IV. Methods – pg. 5-8 a. Figs. 2 and 3 – pg. 6 V. Genomic Definitions – pg. 8-9 VI. Site Introduction – pg. 9-10 a. Fig 4 – pg. 10 VII. El Sidron – pg. 10-14 a. Table – pg. 10-12 b. Figs. 5-7 – pg. 12 c. Figs. 8 and 9 – pg. 13 VIII. Mezmaiskaya – pg. 14-18 a. Table – pg. 14-16 b. Figs. 10 and 11 – pg. 16 IX. Shanidar – pg. 18-22 a. Table – pg. 19-20 b. Figs. 12 and 13 – pg.21 X. Vindija – pg. 22-28 a. Table – pg. 23-25 b. Fig. 14 – pg. 25 c. Figs. 15-18 – pg. 26 XI. The Neanderthal Project – pg. 28-32 a. Table – pg. 29 b. Fig. 19 – pg. 29 c. Figs. 20 and 21 – pg. 30 XII. Discussion – pg. 32- 36 XIII. Conclusion – pg. 36-38 XIV. Bibliography – pg. 38-42

2 ABSTRACT In this analysis, I seek to understand how three separate lines of evidence – skeletal , , and genomics – are used separately and in tandem to produce taxonomic classifications in Neanderthal and paleoanthropological research more generally. To do so, I have selected four sites as case studies: El Sidrón , , , and . El Sidrón, Mezmaiskaya, and Vindija all have detailed archaeological records and have yielded Neanderthal DNA. Shanidar is one of the oldest and most -documented Neanderthal sites. Alongside the four sites listed above, the findings of the full-coverage Neanderthal genome will be used as a “site” of sorts to understand how can inform and supplement morphological and archaeological data. Ultimately, the data presented here is more useful to contextualize the meta- interactions between paleoanthropological subdivisions rather than to answer, “what is a Neanderthal?”.

INTRODUCTION In order to explore the limits of Neanderthal morphology, behavior, and genetics, we must first acknowledge traditional definitions of . The defines them as “members of neanderthalensis; our closest extinct relative”. The European Archive, which hosts all of the genome sequences derived from Neanderthals, classifies the organism of origin as Homo sapiens neanderthalensis. But among anthropologists, the reigning definition of Neanderthals tends to omit a taxonomic name in favor of labeling them as: “Late human populations living in and parts of Western Asia until approximately 30,000 ago…this group was morphologically different from its contemporaries and from later human populations”.30 Traditional morphology guidelines paint Neanderthals as short and squat people, possessing large and wide noses, thick and pronounced brow ridges, and sometimes even larger than modern .6, 24, 26, 28, 34 Despite these common descriptive approximations of Neanderthal morphology, there is no consensus in regarding what exactly defines a Neanderthal. No current numerical standards decisively defining the limits of Neanderthal morphology yet exist, and it is even difficult to find clearly photographed Neanderthal skeletal and cultural remains. In recent years, our understanding of has changed rapidly. Historically, Neanderthals were associated with the lithic () and were believed to deliberately bury their dead.3, 40, 70, 71, 73, 82 Recent evidence shows that they produced art and jewelry.48, 81 But irrefutable evidence of language and more sophisticated tool building currently remain elusive

All citations in this document will follow the format dictated by the journal . The only differences are that journal 3 titles are not abbreviated and the bibliography is in alphabetical order. Science published the draft sequence of the Neanderthal genome and since then has published many significant findings on Neanderthal and biology. for most extinct hominins classified as Neanderthals; capabilities that have sometimes been used to define all modern humans excluding their Neanderthal contemporaries. But as I will discuss, archaeological evidence is often used to support a previous designation rather than explicitly define one in the way that morphological and anatomical data are used. The latest form of evidence in paleoanthropology, ancient DNA, promises to objectively find the genetic basis of human evolutionary history and link anatomical observations. Yet, as we will see, genomic data only complicate and contradict previous morphological and archaeological-based classification of Neanderthals. Additionally, is also subject to computational and statistical biases, comparable to the consequences of solely using morphological and behavioral landmarks. In this analysis, I seek to understand how three separate lines of evidence – skeletal morphology, archaeology, and genomics – are used separately and in tandem to produce taxonomic classifications in Neanderthal and paleoanthropological research more generally. To do so, I have selected four sites as case studies: El Sidrón Cave, Mezmaiskaya Cave, Shanidar Cave, and Vindija Cave. El Sidrón, Mezmaiskaya, and Vindija all have detailed archaeological records and have yielded Neanderthal DNA. Shanidar is one of the oldest and most well-documented Neanderthal sites. Alongside the four sites listed above, the findings of the full-coverage Neanderthal genome will be used as a “site” of sorts to understand how genetics can inform and supplement morphological and archaeological data. Ultimately, the data presented here is more useful to contextualize the meta- interactions between paleoanthropological subdivisions rather than to answer, “what is a Neanderthal?”.

HISTORICAL BACKGROUND Neanderthal were the first set of fossilized hominins to ever be found and identified as belonging to an ancient extinct human lineage. The Neanderthal was uncovered in 1856 by German miners in Neander Valley. Among the fragments were: a case, , arm bones, , and scapulae.76 Type are the original remnants of a species uncovered and are used as the standard for all other species identifications. In retrospect, scholars realized that fossils from and (1829) were also Neanderthals rather than long-dead arthritic humans.76 To date, Neanderthal remains have been exclusively found in Europe and Asia unlike their contemporaries, anatomically modern Homo sapiens (AMH), of which are found across the world. To explain this unequal population distribution, two models have emerged: the regional continuity and replacement models. Put forth by the physical anthropologist Wolpoff, the regional continuity model states that

4 later members of Homo evolved from independently from regional hominin groups.14, 30 This model supports clinal variation and seen in smaller populations. The replacement model, championed by paleontologists including Stringer and Andrews, is less popular than the regional continuity hypothesis. The latter suggests that all modern Homo sapiens are descended from archaic Homo sapiens that recently migrated out of ; overtaking Neanderthal and other Homo populations residing in .11, 74 Further complicating ideas about Neanderthal evolutionary origins are paleoanthropologists (like Harvati et al) who believe that Neanderthals are an exception to the regional continuity model based on their extreme morphology.11, 29

Fig. 1: Neanderthal geographic distribution Credit: Open Access

METHODS To address the questions posed in the abstract, I will utilize morphological landmarks, archaeology, and genomics in this analysis. The following methodology can be used as a guideline to explain how I will interpret the published data.

Morphological Landmarks Skeletal morphology is used to ascertain phenotypes and variation within a population. Biological anthropologists use morphology to set species limits based on accepted parameters of variation. To best understand how changes in skeletal form influence hominin species definitions,

5 cranial landmarks of interest based on qualitative and quantitative analyses will used to explore the limits of Neanderthal species classifications. There are eight general categories of craniofacial landmarks used to evaluate Neanderthal morphology.28 The first is the shape and orientation of the infra-orbital region – which is typically classified as “inflated” in Neanderthal specimens. Second is the degree of antero-inferior glabella projection relative to the and is used in differentiating pre- and early Neanderthals. Third, the position and sagittal orientation of the face is used; generally relatively forward. A strong juxtamastoid eminence is expected, which results in the crest of the digastric fossa border. Occipital plane convexity, or occipital “bunning” is a fifth classical Neanderthal trait. Reduced mastoid processes, high mid-facial prognathism, and large piriform (nasal) apertures are the final groups of Neanderthal skeletal classifications.28 Despite these categories, morphological-based speciation presents a number of difficulties. Landmark descriptions are subject to human bias, fossil availability and preservation conditions, what kind of tools and measurement system was utilized, and small sample sizes. There is no way to circumvent these constraints, so in this study, all data obtained from morphological analyses will strictly be used in context of its site and comparable landmark data. have been selected over other skeletal remains because they have the greatest number of landmarks and vary the most in diagnostic features. Additionally, cranial remains are more likely to survive in archaeological context than other parts of the skeleton due to their robusticity.

Fig. 2 and 3: Skeletal Landmarks Credit: Leon Chaitow (2005) and Pearson Education (2000)

Archaeology As one of the oldest disciplines dedicated to studying the past, archaeology is an essential tool in paleoanthropology. Key components of archaeological analysis include: site dating, inventory of

6 faunal and lithic remains, and traces of art and other artifacts that serve as behavioral indicators. Among the multiple techniques available to date archaeological sites, the most common used in paleoanthropology is via accelerator mass spectrometry – a chronometric dating tool. Radiocarbon dating compares the amount of radioactive 14C to its stable isotope, 12C. The first component, faunal analysis, is typically carried out by zooarchaeologists and the findings are included in supplemental research papers. In a faunal analysis, one would expect an inventory of all of the identifiable species found at the archaeological site as well as their comparative abundance and minimum number of individuals (MNI).54 data can be used to make inferences about diet, climate, and other aspects of Neanderthal behavior.31, 54 Secondly, lithics, or stone tools, are among the most reliable and abundant artifact remnants from Neanderthal sites. Almost all Neanderthal tools belong to the Mousterian Industry, named for a site in with plentiful lithic remains. Mousterian tools are produced by a flintknapping reduction technique called the Levallois: flakes are removed from prepared cores and typically gain a tortoise shell pattern.40 The requires pre-planning and allows the maker to closely control the tool size and shape.40 A second lithic industry, the Châtelperronian, arose from the Mousterian and is the earliest transitional lithic industry found in the Upper .32 Châtelperronian tools were originally found in France as well, but evidence of this has been found as far east as . Hallmarks of the Châtelperronian include denticulate tools and flint knifes with only one cutting edge.32 has been used to approximate cognitive capabilities – based on the complexity and design of the tools – as well as hunting and object production behavior. Other compelling insight to Neanderthal sociocultural abilities comes from the presence of art. Evidence of art is comes from cave drawings, traces of , and modified objects: beads, shells, , and stone figurines. The most famous traces of Neanderthal art primarily come from in France and Western Europe, but – a site in close proximity to Vindija in Croatia – boasts a large trove of modified eagle talons likely used as jewelry.48, 81 Supplementary behavioral evidence is inferred from traces of fire, deliberate burying of the dead, and the presence of healed fractures and other pathological skeletal morphology.31, 48 Although my original intent was to study how three specializations including archaeology use their respective evidence to assert species definitions, I have found that archaeological evidence is most often used to support and augment morphological data. This is especially true in light of the fact that both Neanderthals and early modern humans used tools; complicating our understanding of inter-group interaction and social behaviors. Nonetheless, I have included all of the

7 archaeological evidence I have found in my analysis to provide a more complete understanding of each of the sites’ available data.

Genomics Genomics is a rapidly emerging field that studies entire and their structure, , and functional information. Under the right conditions, DNA can exist within partially fossilized bones for thousands of years.27, 52 Complete fossilization of bones removes all of the original tissue and organic matter and replaces or permineralizes it with other elements. DNA will be completely degraded approximately 100,000 years after death, but if skeletal remains are only exteriorly fossilized, rapidly buried, and unexposed to pH, light, chemical, and other changes, then ancient DNA can be sequenced.52, 53 In 1997, the first Neanderthal mitochondrial sequence was derived from an individual found in Vindija Cave.47 Since the creation of the Neanderthal in 2006, five full mitochondrial genomes and a complete nuclear genome have been sequenced.9, 10, 20, 21, 22, 47, 52, 62 To successfully sequence Neanderthal genomes, researchers needed to learn how extract ancient DNA. First, a large enough sample of bone is required.53 Next, the sample is drilled to collect the bone dust and soaked overnight in an extraction buffer that releases DNA into solution. Silica is added to attach to the DNA and the resultant sample is centrifuged and undergoes a secondary wash solution. Primers are added to the DNA extract pellet and polymerase chain reaction (PCR) gels run to replicate the original DNA in a sufficient quantity for analysis.53 After this process was repeated countless times, all sequenced Neanderthal genomes became available open access on the European Nucleotide Archive (ENA). Through the UCSC genome browser, anyone can view Neanderthal genomic sequences with comparative human and genome alignments. The UCSC Neanderthal Genome Analysis Consortium also provides several supplementary sequences and their preliminary analysis for download.

GENOMIC DEFINITIONS To grasp the genomic data available, one must first understand the basics of genetics. Genes and alleles are the basic units for measuring .45 Genes are heredity units transferred from parent to offspring and comprise a distinct sequence of assembled as part of a chromosome. The sequence order within genes determines what nucleic acids and polypeptides can synthesize, essentially building the organism. Alleles are alternative forms of genes that arise from .45 They are found at the same loci (location) on the chromosome, but form different products. For example, genes that encode follicles have many alleles that produce the various

8 hair colors we are accustomed to. Single nucleotide polymorphisms (SNPs) are variations of single base pairs within a DNA sequence.45 SNPs are the most common method for determining variation within populations. SNPs occur once on average for every 300 nucleotides, and there are approximately 10 million SNPs in the modern .45 SNPs are used to approximate genetic similarity and evolutionary history and are sometimes also called “haplotypes”. Transversions describe the substitution of purines (two-rings) to pyrimidines (one-rings) in DNA sequences.45 Transitions are point mutations that substitute purines for purines and pyrimidines for pyrimidines.45 Small insertions or deletions in genome sequences are called indels.45 Structural variation within genomes – (how many genes there are), gene deletion, gene insertion, and gene substitutions – is also useful in comparative analyses.45 For the purposes of this study, SNP variability, shared genes of interest, transversions, transitions, and indels between modern and Neanderthal populations will be the primary tools for analyzing genomic data.

SITE INTRODUCTION El Sidrón, Mezmaiskaya, Shanidar, and Vindija caves were all selected as case studies for this analysis. These caves collectively contribute a wealth of Neanderthal remains, each with well- preserved craniofacial fragments. Additionally, each site is well documented and has been analyzed in larger bodies of paleoanthropological literature. The primary fossils used in the Neanderthal Genome Sequencing project came from Vindija Cave, and smaller amounts of DNA from both El Sidrón and Mezmaiskaya have been sequenced as well. Shanidar has especially detailed faunal and lithic records. Together, these Neanderthal sites provide key archaeological, morphological, and genomic evidence for constructing an understanding of what a Neanderthal is, and they range geographically through Western Europe and Asia Minor. Below are descriptions of each selected Neanderthal site with accompanying tables summarizing the available morphometric, archaeological, and genomic data. By analyzing these sites individually and comparatively, I can dissect how site excavators and specialists interpreted the data available to support their own notions of Neanderthal .

Site MNI Date Location

El Sidrón Cave (ESC) 13 Mean calibrated age of 43,129,129 BP ,

Mezmaiskaya Cave (MC) 3 39,700 ± 1,100 BP Mountains,

Shanidar Cave (SC) 10 35,000-65,000 BP Bradost Mountains,

Vindija Cave (VC) 40 3 distinct strata of Neanderthal occupation, Croatia encompassing 28,000-29,000 BP and >42,000 BP

9 Fig. 4: All of the sites where DNA was retrieved for use in the full-coverage Neanderthal genome. Credit: Green et al 2008

EL SIDRÓN Introduction El Sidrón is part of an elevated terrain in Asturias, Spain.4, 63, 64, 67 The site is part of a larger cave network called “Galería de Río”. The first skeletal remains were accidentally uncovered approximately 200 meters from the entrance in 1994, spurring a series of excavations that began in 2000.4, 43 Geologically, El Sidrón is composed of Mesozoic/ sediment.6, 15, 64 All of the Neanderthal remains found were accumulated in a narrow deposit with maximum thickness of 227 cm. The state of preservation suggests that the bones had limited exposure to outside elements. Of the approximate 1,232 fragmentary human remains, there is little carnivore or other faunal modification and very few bones exhibit hydraulic abrasion.64 It is suggested that this site was an unintentional site based on the degree of skeletal articulation and association. Of the thirteen individuals, six adults, one , two juveniles, and three adolescents have been positively identified.4, 7, 64 Three specimens were dated using C14 accelerator mass spectrometry and calibrated with CalPal. The given date ranges from 43,129 ± 129 BP.15, 63 Secondary dating was done as well using amino acid racemization on gastropods. The samples dated between 39,000 ± 7,000 BP.15, 64

Table Morphometric Cranial Vault Cranial Base Teeth Facial Skeleton Mandible

Large occipital Higher N = 213 Low projection of High and thick vault petrosquamus the mastoid mandibular body with sinus Large and process no submental notch Marched nuchal shovel- torus Large foramen shaped Flattened glenoid Deep pterygoid fossa magnum fossa Open sutures High levels Developed mental Well-marked of dental Well-defined foramen Large supra- Waldeyer’s aplasia and superciliary fissure insular pit crest interproximal Inclined mylohyoid line Developed supra- High degree of (All data glabellar fossa Deep masseter pit and

10 asymmetry in the derived from Wide with a gonial eversion lingual contour Bastir et al crenate Round torus 2007 and Bastir enamel Short retromolar space et al 2010) (All data derived Shovel- from Bastir et al Flattened glenoid fossa (All data derived shaped 2010, Rosas et al from Bastir et al 2006, and Rosas et (All data derived from 2010 and Rosas et Labial al 2012) Rosas et al 2006) al 2014) complexity

Strong lingual tubercle

Accessory lingual cusp

Evidence of

(All data derived from Dean et al 2013, Radinin et al 2016, and Rosas et al 2006)

Archaeology Evidence of Lithics/Tools Art Faunal Behavioral Analysis Planned Burial?

No - fossils and A total of 415 No Non-hominin The significant amount archaeological lithic tools have skeletal remains of flakes found at the material were been found are pretty scarce site suggests that the likely transported area may have been to the cave 12.1% are tools Cervus elaphus, an used extensively for posthumously and or retouched unidentified large flintknapping had little artifacts, 85.8% bovid, gastropods movement post- knapping Evidence of human deposition remains, 3.1% (All data derived modification of human cores or core from Alonso et al bones - especially facial (All data derived fragments 2003 and Rosas et “defleshing” seen from Alonso et al al 2006) through cut marks, 2003) Minimum conchoidal scars, number of percussion pitting and flakes is 283 corresponding flakes samples Social stratification Utilized flint and quartzite (All data derived from (quartzite to a Rosas et al 2006 and lesser extent) Santamaria et al 2010)

47 tools: 11 Levallois, 11 sidescrapers,

11 natural-backed , 22 denticulates, notch, Tayac point, cordiform - in pieces

(All data derived from Santamaria et al 2010)

Genomics Sample Sequence Individual Genome Type Genes of Interest Accession #

SAMEA1033202 ERS007244 Sid1253 Mitochondrial and FOXP2 and TAS2R38 nuclear

Figures

Fig. 5: Mandibular scans. Note the retromolar space; mental foramen position; and pterygoid fossa. Credit: the Universidad de Oviedo

Fig. 6 and 7: Cranial base and posterior landmarks. Note the shape of the magnum foramen; petrosquamus sinus; and cranial curve. Credit: Bastir et al (2007)

12

Fig. 8: Pathological mandibles and teeth. Note the taurodontism and Fig. 9: Homo sapiens neanderthalensis aplasia (E) and cut marks (implied complete mitochondrial genome, isolate ; F). Credit: Rosas et al El Sidron 1253. Credit: European (2006) Nucleotide Archive

Discussion Morphologically, El Sidrón appears to meet the classical descriptors of Neanderthal craniofacial features. There are several instances of occipital bunning (a pronounced bulge at the back of the skull) within the sample and craniofacial asymmetry dominates. The multiple descriptions of deep fossae imply that the El Sidrón individuals were highly muscled, perhaps suggesting a difficult lifestyle or particular to heavy and inclement weather.41 The relatively broad width of the could be a consequence from a high cranial capacity or a well- developed nervous system. Dental analysis proposes that the El Sidrón population suffered from , with diets primarily composed of hard foods.16, 58 This population possibly had chronic pain related to high degrees of dental aplasia (defective development or absence of teeth) and taurodontism (tooth bodies are enlarged at expense of the roots).16, 58 While not a domestic site by any means, El Sidrón surprisingly appears to have had a thriving flintknapping industry. There is a significant amount of flakes on-site, which suggests a serious production operation.63, 67 Although there are plenty of tools, hominin-modified faunal remains are limited.67 Conversely, there are clear cut marks on Neanderthal remains and some show evidence of percussion pits. Combined with the presence of conchoidal scars, researchers confidently concluded

13 that cannibalism occurred at El Sidrón.58, 67 Assuming this is true, some degree of social stratification and concepts of punishment and/or ostracization likely existed in this Neanderthal cultural sphere. Cannibalism is a pretty severe act to commit, and in modern understandings of human behavior and ethics, it would seem like cannibalism is only considered as a last resort. These Neanderthals could have cannibalized their neighbors based on religious reasons (which simultaneously suggests a Neanderthal spiritual understanding), for food, or some other ritual purpose. In terms of genomics, the sequence derived from individual 1253 has been of particular interest because it contains an analog to the FOXP2 gene.41, 43 FOXP2 is required for language acquisition and development in humans. Another notable gene found in the El Sidrón sample is TAS2R38 which has a role in bitter taste perception in modern human populations.13 Individual 1253 had the heterozygous SNP that produces the “taster” phenotype. People with this allele are especially sensitive to foods with bitter undertones, an assumed evolutionary to prevent consuming toxic substances.13 It is unknown what selective and environmental pressures act on this gene to maintain taster and non-taster phenotypes in modern populations, but whatever they are, they acted on Neanderthals in the same way.13

MEZMAISKAYA CAVE Introduction Mezmaiskaya Cave is located in the northern of Russia, straddling what we now conceive as the boundary between Europe and Asia. Original site excavations were conducted by L.V. Golovanova during the 1987-97 field seasons.19 Two individuals were uncovered at Mezmaiskaya, including the first Neanderthal infant. AMS radiocarbon dating places the site firmly in the Middle-to-, between 39,700 ± 1,100 BP.19, 55 Geological surveys identified dolomite, clay, loam, and rubble deposits dating from the Holocene and . Some researchers have suggested that a Campanian ignimbrite volcanic eruption within the Caucasus mountain chain contributed to these Neanderthals’ demise.55

Table Morphometric Cranial Vault Cranial Base Teeth Facial Skeleton Mandible

Squama of the Only slightly 14 dental Zygomatic arch Not available frontal bone is concave crowns derived substantially denser from thicker than a Small, deep deciduous modern human is fossae teeth (approx. infant rectangular 7 month fetus Paramastoid or 2-month-old Frontal Very weak sulcus process baby) eminence less

14 “distinctly pronounced Intrajugular expressed” (All data process absent derived from (All data Very massive Golonova et al derived from (All data derived sphenoid bone 1999 and Gunz Golonova et al from Golonova et et al 2012) 1999, Gunz et al 1999, Gunz et al More oval al 2012, and 2012, and Ponce de foramen Ponce de Leon Leon et al 2008) magnum et al 2008)

(All data derived from Golonova et al 1999 and Gunz et al 2012)

Archaeology Evidence of Lithics/ Tools Art Faunal Behavioral Analysis Planned Burial?

Yes, there is no Re-touched No Lots of Approximately 5-7% of evidence of a lithics (end priscus, the bison + goat bones “burial pit” but scrapers, backed caucasica, Ovis appeared to have cut very strong blades, Gravette orientalis, marks evidence for the points) Cervus elaphus, infant to have been Ursus deningeri Due to high altitude, buried intentionally Bone awls, bone kudarensis, authors suggest that MC borers and bone Marmota seems to have been No evidence of borer-polishers paleocaucasica occupied during warmer or à Bovids, months to hunt other “ritual points Cervics, Equids, bison/sheep/goat behavior” and Ursids (regional usage) High proportion make up over (All data derived of bifacial 296 kg of the Possibly niche overlap from Golonova et implements (first faunal size class between Neanderthals + al 1999) evidence of present at other early members of bifacial Mezmaiskaya Homo instruments in this part of Evidence of cut (All data derived from Eastern Europe) marks and other Baryshnikov et al 1996) faunal Industry processing primarily categorized by Majority of high proportion bones appear to of blades, pre- have been dominance of broken in a sidescrapers, “relatively fresh scarcity of condition” bifacial tools → two different 3.2% of faunal occupation remains show industries evidence of Mezmaiskaya burning lithic industry has technical (All data features not derived from commonly found Baryshnikov et in other al 1996)

15 Neanderthal sites: core preparation that results in crested scars/tablets + detachment from striking platforms + flaking to produce bladelets for further tool production

(All data derived from Golonova et al 1999 and Baryshnikov et al 1996)

Genomics Sample Sequence Individual Genome Type Genes of Interest Accession#

SAMEA1706711 ERS007242 Mez1 and Mez2 mitochondrial Closest relative to the Denisova, another archaic hominin found in

Figures

Fig. 10: Virtual reconstruction of Fig. 11: Note how Mezmaiskaya Mezmaiskaya infant. Note overall and Vindija are more closely cranial shape and estimate size. related. Credit: Reich et al (2010) Credit: Ponce de Leon et al (2008)

Discussion The morphological data presented in the above table is primarily gleaned from the Mezmaiskaya infant . There are only three well-preserved examples of identified as Neanderthals: the one from Mezmaiskaya (estimated age range: seven-month fetus to two-month

16 newborn) and two from Dederiyeh Cave (Syria; estimated age range: 1.6-2 years).3, 19 In a virtual reconstruction study, Ponce de Leon et al found that the infant’s endocranial volume was approximately 399 cm3 at birth, which is comparable to AMH brain capacity (380-420 cm3).46, 55 This discovery prompted the conclusion that differences in Neanderthal and AMH cranial cases develop during life. Despite including either measurements or images of the Mezmaiskaya infant cranial fragments, the frontal bone squama has been described as “denser” and the frontal eminence “less pronounced” when compared with modern human infants. Other morphological descriptions of note include: a “substantially thicker” zygomatic process; an occipital bone with greater dimensions, thickness, density, and rectangular shape; a missing intrajugular process; “weakly expressed” sigmoid sinus sulcus; massive sphenoid bone; and an ovular, curved foramen magnum.5, 19, 23 Due to the high number of morphological variants discussed and the level of detail, it suggests that Neanderthal neonatal development varies considerably (or at this site, at least). To juxtapose, the Neanderthal infant at Dederiyeh Cave was described as having: a moderately developed mastoid process; a transverse torus on the occipital bone; wide and strongly protruding nasal aperture; “swept-back appearance of the zygomatic bone”; and receding symphysial profile or .3 Unfortunately, the authors’ studies on morphological variations of the Mezmaiskaya and Dederiyeh children do not overlap on any qualitative description nor provide comparative images or landmark measurements. Thus it is not possible to develop a quantitative profile on Neanderthal infant craniofacial morphologies, but it is clear that differences in the occipital bone and zygomatic process are important. Interestingly, these differences do not seem as significant in the mature Mezmaiskaya cranial material. The partial adult found in the same layer of Mezmaiskaya cave was classified as an “archaic human with clear affinities to the Neanderthals of Western and Central Europe”.19 The body exhibits a developed paramastoid process and oval foramen magnum, much like the infant.19 No further descriptions or images were provided, probably due to the high amount of fossil deformities that had accumulated over years of erosion. Archaeologically, Mezmaiskaya newborn shows evidence of intentional burial due to both the completeness and partial articulation of the body (the Dederiyeh infant was intentionally buried as well).3, 19 Soil analysis failed to reveal indications of ritual behavior associated with deliberate burial – such as grains from flowers or the presence of . Unlike the hominin remains, the faunal material found in association was largely fragmentary and dispersed. The lithic industry found in context with these Neanderthal remains is enormous and totals around 3,645 artifacts.5, 19 The assemblage has a high proportion of bifacial tools including sidescrapers, small handaxes,

17 endscrapers, and tools with converging edges. 5, 19 Most cores show parallel flaking patterns and rare instances of tool re-touching techniques. The faunal remains found in the matrix show stone tool cut marks on 7% of the bison bones and 5% of the goat, sheep, and red deer remains. 5, 19 The variation in placement and degree of force on the bone suggests that multiple steps of the butchering process were done on-site. This suggests that Mezmaiskaya Cave was a kill/processing site, and paleoecological research suggests that it was primarily occupied seasonally, with use mostly during the warmer months when were plentiful and able to be killed.5, 55 Since only two individuals were found in the cave, it is likely Mezmaiskaya was not typically used as a burial ground, and that the hominins found there likely died unexpectedly and at an inopportune time, so that they could not be relocated and buried elsewhere. In addition to the detailed anatomical and behavioral proceedings presented above, the Mezmaiskaya genome is of much interest for hominin phylogeny. Despite making up 2% of the full nuclear genome (compared to El Sidrón at .1%), there is very little data on genes of interest in the Mezmaiskaya population. Instead, most of the research utilizing the Mezmaiskaya mitochondrial genome has been used in comparative analyses against the Denisova: another archaic hominin from a Siberian cave.11, 39, 59, 62 To date, the only Denisova skeletal remains are a bone and two teeth.39, 53 The Neanderthals from Mezmaiskaya were more closely related to Denisova compared to other Neanderthal populations sequenced, although the Vindija Cave occupants were the second closest relatives (81 transversion substitutions versus 80).11, 39, 62 From this information, it is possible to conclude that there were likely interactions in eastern Eurasia between Neanderthals and Denisova individuals in addition to AMH – a relationship we would not have been able to discern without genome sequencing technology.

SHANIDAR CAVE Introduction Excavations at Shanidar Cave in Iraq ran from 1957 to 1960 under the direction of Ralph Solecki.70, 71, 72 The site is situated within the outer , is relatively well forested, and is only 2.5 kilometers from the Greater Zab River (a major tributary into the Tigris). Contemporary migrant Kurdish shepherds live in parts of Shanidar Cave during the winter months and it was undoubtedly a domestic site for Neanderthals as well.70 There are four major occupation layers at Shanidar, although layer D is primarily the Neanderthal occupation zone. Also called the “Mousterian Layer”, the strata with Neanderthal fossils dates to approximately 48,000 BP.70, 72 Solecki suggests that the Neanderthals at Shanidar Cave were “culturally stagnant” and estimates that over 2,000 generations of Neanderthals lived there during 60,000 years of accumulation.70 As a site, Shanidar is difficult to interpret because it has several phases of occupation and includes hominins clearly

18 attributed to Neanderthals and Homo sapiens, as well as indeterminate fossils. Stratigraphy integrity is always a concern, but the fear of bioturbation and artifact mixing is especially relevant to Shanidar’s archaeological record. Despite producing only ten Neanderthal-identified individuals, it is probably the best documented Neanderthal site. Preservation conditions have been generally good with many articulated skeletal features. Extensive pollen, faunal, and lithic studies show that the Shanidar Neanderthals were capable of complex behaviors.70, 77 Unfortunately for its occupants, rock falls appear to have been a common occurrence in the cave and likely killed most of the individuals there.72

Table Morphometric Cranial Vault Cranial Base Teeth Facial Skeleton Mandible

Unusually large Unusually small Retreating Frontal flatness Mandibular maxillary sinuses mastoid process molars condyles do not Relatively broad fully articulate with Large digastric fossa (All data No canine supratoral sulcus the sockets in derived from fossae temporal bones Small occipital Stewart et al Flat frontal arcs (evidence of condyles 1977) Heavily worn arthritis?) dentition Barely visible Zygomaxillary occipital torus Large tubercle suture more convex Larger dental present on each side arcade than High nasal bridge of the condyle Small ear openings comparable Neanderthal Retreating orbital Incipient chin (All data derived specimens margins lacking a gonial from Stewart et al angle 1977 and Trinkhaus Occlude fairly Prominent et al 1996) well glabella that Maxilla inflation anteriorly projects (All data Broad coronoid derived from Depressed nasal processes Stewart et al floor with no 1977 and evidence for a (All data derived Trinkhaus et al prenasal fossa from Stewart et al 1996) 1977 and Trinkhaus Zygomaxillary et al 1996) suture more convex

(All data derived from Stewart et al 1977 and Trinkhaus et al 1996)

Archaeology Evidence of Lithics/ Tools Art Faunal Behavioral Planned Burial? Analysis

Pollen grains found Unifacial flint No No fish remains Tools indicate that

19 near bodies - flakes vegetal foods (and evidence that Primary source of possibly acorns + mourners left Possibly used food was big grains) were part of flowers for the dead? for tipping game (rather than the Neanderthal diet and as fish or fowl) (All data derived -working + bitumen from Solecki 1963 instruments Ovis orientalis found - indicative of and Solecki 1975) gmelini, Capra trade? No Levallois hircus aegagrus, cores were Sus scofa Attila, (All data derived found but many Cervus elaphus from Solecki 1963) tools have maral, Dama “facetted butt” mesopotamica, Capreolus (All data capreolus, Canis derived from lupus palipes, Solecki 1963) Vulpes flavenses, Ursus arctossyriacus

Ovis orientalis, Capra hircus aegagrus, and Cervus elaphus comprise more than 90% of the faunal remains

43% of Ovis and 11% of Capra remains were immature animals, perhaps indicating practices

(All data derived from Perkins 1964)

20

Figures

Fig. 12 and 13: Note high nasal bridge; tubercles on condyles; and pronounced supra- orbital ridges. Credit: Trinkhaus et al (1996)

Discussion Morphologically, the ten Neanderthal individuals found at Shanidar are variable but ultimately express massive faces; large masto-occipital processes but large digastric fossae; receding and round gonial angles.70, 71, 72 Shanidar IV has extensive gonial ridging – a trait indicative of thickly muscled faces, despite the tendency for thinner Neanderthal mandibles.71 Shanidar I had only two large foramina on their lower jaw but all of the remaining Neanderthal samples exhibited multiple foramina.72 All individuals had small ear openings and large tubercles on each condyle – traits not discussed in other Neanderthal site reports. Unfortunately for this analysis, the archaeologists involved were primarily concerned with taking numerical measurements and attempting skeletal reconstruction.72, 77 For those reasons, it is difficult to qualitatively compare the traits presented in his site reports with other Neanderthal sites. Luckily, Shanidar’s archaeological material is much more abundant and useful.

21 While a variety of tools and artifacts have been found in Shanidar Cave, layer D is firmly Mousterian and includes “typical points, scrapers, and ” – all made with unifacial flakes.70 Over the entire Neanderthal occupation temporal zone, there were no major changes in tool type or function except for a brief variance in points. Using chemical trace analysis, researchers found that acorns and grains were processed using lithics found in the cave. In addition to vegetal material, big game comprised a large portion of the Shanidar Neanderthals’ diets.54 Despite being in close proximity to the Greater Zab River, fish and other aquatic remains are not common in the faunal assemblage.54 Pollen analysis on the plentiful grains found in association with some of the Neanderthal burial pits has led Solecki to suggest that they left flowers for their deceased – part of ritual behavior that could indicate a belief in the afterlife or the importance of remembering family members.71 Perhaps as a more non-traditional inference of social behavior, the skeletal pathology of Shanidar I and Shanidar V is of particular interest. Shanidar I shows evidence of arthritis and a semi- healed skull fracture. Shanidar V also had arthritis and metatarsals that appear to have recovered from a traumatic fracture.72, 77 Evidence of injury and long-term healing is a good sign that the Shanidar population took care of disabled and elderly members within their social groups. While it would undoubtedly rich the morphological and archaeological records at Shanidar, no genome sequences have been derived or attempted from these fossils.

VINDIJA CAVE Introduction Geographically, Vindija Cave (Croatia) is positioned between two other well-studied Neanderthal sites: Krapina, strongly associated with the Mousterian lithic industry; and Velika Pecina, a site with the earliest modern humans in Europe.69 Vindija Cave lies in a narrow gorge on the southwestern slope of Kriznjak Vrh in the Dinarid Mountains. The cave is about 50 meters deep and 28 meters wide, composed of sandstone and limestone. Many researchers have differing systems for measuring the site stratigraphy, but all concur on the interfaces what is known as the G level.35, 36, 39, 44, 80 The excavated material from Vindija Cave spans 25-45 ka: encompassing the emergence of modern humans, the Middle-Upper Paleolithic transition, and disappearance and extinction of Neanderthals.1 Geologist Vukovic found tools and faunal remains in 1928, and carried out excavations for 30 years in and outside of the cave Malez took over in July 1974 and fieldwork continued every season until 1986, when the fossilized human remains were discovered.1, 35 Within the G layer, approximately 40 Neanderthal individuals have been uncovered in a red-brown clay matrix. Two Neanderthal-identified

22 individuals in G1 dated to 29,080 ± 400 BP and 28,020 ± 360 BP (13C/15N) and the G3 Neanderthal is dated to >42,000 (14C).47, 69

Table Morphometric Cranial Vault Cranial Base Teeth Facial Skeleton Mandible

Broad sagittal Not available Strongly Most common Have symphyses more torus shovel- diagnostic vertical than “classic” shaped available is Neanderthal Vault broad supraorbital mandibles compared to Large region upper face and Have incipient high Anterior Supraorbital tori eminences, although teeth are are absolutely and not to the point of Medial antero- smaller but relatively thinner becoming a superior sinus not compared to recognizable “chin” walls very thin significantly Krapina and other so compared Neanderthal Dimensions of corpus Roof is very thick to populations + height of ramus Neanderthals indicate small General gracility found at the Nasal breadths specimen (lower of the temporal nearby site of are “markedly ramus height than fossae Krapina less” than any other Neanderthals in known the region) Lateral (All data Neanderthal supraorbital derived from Reduced maxillary element slightly Ahern et al More robust alveolar height = curved 2003, frontal crest nasal breadth Karavanic (Karavanic + Smith) Slightly higher 2000, Malez Frontal crest cranial vaults 1980, and torus-like and Medial pterygoid (Karavanic + Wolpoff et al moderately tubercle Smith) 1981) developed (All data derived from Well-developed Rounded Ahern et al 2003, Breschet’s sulcus forehead Karavanic 2000, Malez 1980, and (All data derived Sulcus Wolpoff et al 1981) from Ahern et al pronounced 2003, Ahern et al 2001, Karavanic Continuously 2000, Malez present 1980, and vermiculate bone Wolpoff et al (supraorbital) 1981) (All data derived from Ahern et al 2003, Ahern et al 2001, Karavanic 2000, and Malez 1980)

Archaeology Evidence of Lithics/Tools Art Faunal Behavioral Analysis Planned Burial?

23 No Level G1: 56 No Absence of Saiga No evidence for chipped stones, of tatarica and deliberate which 15 (26.8%) slight presence of fragmentation or are determined to be Rangifer trandus. modification of bodies tools. This includes: Additionally, found denticulated pieces, - thera side scrapers, a spelaea. Canis Evidence of bifacial tool, end lupus, and butchering in faunal scrapers, burins, Capreolus record (large quantity blade retouched on caprolus have of blades) both sides. One been found with (All data derived from visible flake damage Some faunal Ahern et al 2003, has been found. This material show Karavanic 1995, and author suggests that signs of burning Wolpoff et al 1981) the cultural material and defleshing in G1 should be marks attributed to the Abundance of industry. 51% of remains tools were produced highest in G of /tuff and compared to the 49% were made rest of the Vindija from and Cave stratigraphy other related material. The Ursus spelaeus presence of a split- () is base bone point in ubiquitous across G1 is the best the stratigraphic indication that sequence but is Upper Paleolithic or also the only Aurignacian large carnivore technology was in found in G use Gnaw marks are Level G3: 17% of rare and likely lithics transformed left by into tools. The rather than assemblage is hominins dominated by sidescrapers, (All data derived notched pieces, and from Ahern et al denticulates. There 2003, Jankovic et is some evidence of al 2006, and bifacial and blade Richards et al technology, but they 2000) very likely could be pseudo-tools or from crytoturbated Upper Paleolithic strata .62% of the tools were made from chert and tuff and 38% were made of quartz. Retouched flakes comprise ~32% of

24 the assemblage, followed by 28% notches/denticulates, 23% sidescrapers, 7% endscrapers, and 10% miscellaneous

(All data derived from Ahern et al 2003, Karavanic and Smith 2013, Karavanic 1995, and Jankovic et al 2006).

Genomics Sample Sequence Individual Genome Type Genes of Interest Accession #

SAMEA2061885 ERS234481 Vi33.15 Nuclear genome STAT2, HLA, MAPT, HACNS1

Figures

Fig. 14: Frontal facial fragments. Note thinner supraorbital tori. Credit: Ahern et al (2003)

25

Fig. 15-17: Anterior supraorbital; mandible; torus projection. Note shovel-shaped teeth and extreme torus curvature. Credit: Malez et al (1980)

Fig. 18: Chromosome 13: positions 31,674,044- 32,174,043. A close-up of the BRCA2 gene in the Vindija nuclear genome. Credit: Ensembl Genomes: Neanderthal Genome Browser

26 Discussion The Vindija sample is probably the largest collection of Neanderthals to significantly deviate from traditional understandings of Neanderthal morphology. Some researchers have postulated that during the Pleistocene, the Dinarid Mountain chain would have been covered in glaciers – thus separating Vindija Cave and other parts of Croatia from the rest of the Adriatic.1, 33 This geographical barrier would have blocked migration and mating; possibly explaining the “progressive” and extremely gracile morphology seen in this collection. Under classic , Neanderthals are assumed to have large noses as an adaptation to cold environments. But the Vindija sample has small nasal apertures, prompting the theory that these Neanderthals permanently and primarily occupied the cave and were sheltered from external environments.1, 35,36, 44 To explain peculiarities in Vindijan morphology, many researchers have asserted that they exhibit a mosaic of traits found in AMH populations and classical Neanderthal biology.1, 3, 44, 80Assuming this theory is correct, this implies a degree of regional evolutionary continuity, evidence of frequent interbreeding among the two populations, and potentially similar cultural attitudes and capabilities between these extinct hominins.1 In terms of behavioral evidence, Vindija has a rich faunal and lithic record. Many animal bones show signs of de-fleshing, burning, and cutting.1, 60 Gnaw marks from predators are rare, so almost all of the modification seen came from Neanderthals. The lithic assemblage suggests that Neanderthals used percussion and pressure techniques during the retouching phase of stone tool production.1 A re-analysis of the G3 lithic assemblage also concluded that Neanderthals were using raw materials similar to (from less local sources) like “Upper Paleolithic people” at the site.37, 38 The bone point found in the G3 layer has been a point of serious contention among archaeologists for its link to a later Upper Paleolithic tool style (the Aurignacian) rather than Châtelperronian.38 To refute this point, archaeologists Ahern et al argue that the bone point and other more sophisticated remains were truly excavated from areas without cryoturbation, and thus are an accurate example of the Vindija Neanderthal toolkit.1 In addition to traditional stone tools, bone tools have been found at Vindija. These are more delicate tools which could have been used in clothing production. This behavior could have been adopted from AMH populations living in the area, and analogous genes of interest shared between the Vindija and modern human genomes strongly support a physical relationship between both groups. Arising from genomic analyses solely of Vindija sequences, STAT2, HLA, HACNS1, and MAPT genes are of much interest.9, 20, 22 A haplotype at STAT2 is believed to have introgressed from Vindijan Neanderthals into modern humans, particularly in Melanesian populations. STAT2 is

27 involved in the innate and -mediated responses.49 Human leukocyte antigen (HLA) and MAPT are believed to have entered the modern human genome through both positive selection and interactions with archaic hominins.9, 20 HLA is a gene complex that encodes major histone complex (MHC) ; all of which are also involved in the immune response.20, 21 Some biologists have used this information to explain the frequency of autoimmune diseases in modern Eurasian populations – possibly individuals with high Neanderthal ancestry – as faulty Neanderthal gene interactions with exclusively human genes. The HACNS1 gene is an enhancer that supports tissue development and limb growth, including the development of opposable thumbs, which are particular to the human lineage.20 It makes sense that Neanderthals would have an analogous gene, considering conservation of limb skeletal morphology (save for relative limb proportions).57

THE NEANDERTHAL GENOME PROJECT Background The first Neanderthal “genome” was extracted from individuals at Vindija Cave and yielded a full mitochondrial sequence.22, 53 A few years after this feat, the Max Planck Institute of Evolutionary Anthropology announced the Neanderthal Genome Project whose goal was to continue sequencing mitochondrial Neanderthal DNA and eventually produce a complete nuclear genome. When first announced in 2006, the project received mixed reviews from archaeologists, paleontologists, geneticists, and evolutionary biologists alike. Critiques of the endeavor included what some deemed as too high a cost, a waste of resources, and a pointless endeavor when archaeological and morphological data was already available.11, 50, 51, 53, 75 The draft genome sequenced was published in Science in 2010 and the full-coverage Neanderthal nuclear genome sequence followed in 2013. To date, countless more research has been done using the finds from the above sequences to compare Neanderthals to modern human populations. To complete the full-coverage genome, researchers compiled sequences from the following fossils: Vindija Cave Vi33.16 (54.1%), Vi33.25 (46.6%), and Vi33.26 (45.2%); Neander Valley Feld1 (0.1%); El Sidrón Sid1253 (0.1%); Mezmaiskaya Mez1 (2%). The percentages in parentheses refer to what proportion each fossil contributed to the full coverage sequence. The fossil from Neander Valley is the type fossil, and its inclusion in the Neanderthal Genome Sequencing Project was vital for linking historical archaeological research and molecular analysis.

28 Table Genomics Study Secondary Individual Genome Type Genes of Interest Accession #

PRJEB2065 ERP000119 Vi33.16, Nuclear genome COX gene clusters; Vi33.25, Feld1, ABO genes; MC1R Sid1253, Mez1

Figures

Fig. 19: All of the sites to date where Neanderthal DNA has been found. Credit: Sanchez-Quinto and Lalueza-Fox (2015)

29

Fig. 20: Note how the three Vindija sequences Fig. 21: The evolutionary relationship between exhibit less variability than the three human modern humans, Neanderthals, and . Fig. 21: Krause et al (2010) sequences. Credit: Green et al (2010) Credit: Krause et al (2012) Discussion One of the first results from Neanderthal Genome Project was that Neanderthals and modern humans have a different : the last common ancestor for whom all modern humans can trace their mitochondrial lineage.10, 20, 53 The mitochondrial Eve for all modern humans existed between 100,000-200,000 years ago whereas the last mitochondrial common ancestor of humans and Neanderthals was much older: living approximately 500,000 years ago.21, 53 Creating a major challenge in detecting between Neanderthals and AMH, this was also a much earlier evolutionary divergence than paleoanthropologists could have assumed from the fossil record. It also seems contradictory when one estimates that the last common ancestor (not along the female line) for Neanderthals and anatomically modern humans existed 300,000 years ago. Other information obtained from the mitochondrial Neanderthal genomes was that they were outside of the accepted limits of human mitochondrial variation (derived from the Human Genome Project and its off- shoots); implying a distinct speciation event between Neanderthals and Homo sapiens.9, 22, 53 Biologists Briggs et al estimate that Neanderthal mitochondrial diversity only differs by 20.4 bases; ultimately making the Neanderthal mitochondrial genome a ⅓ less diverse compared to modern human groups.9 Moreover, this suggests that the Neanderthal population was small and breeding frequently occurred between closely related members. To further illustrate this example, biologists Green et al compared the draft Neanderthal, five modern human, and the complete chimpanzee genomes.20 They estimated that transversions were

30 responsible for restricted divergence events after finding 50 informative transversions between the Neanderthal and five modern human sequences. By using this method, they concluded that Neanderthals fell inside the range of variation of modern humans due to a greater overall divergence in the three Vindija genomes (~11%) versus modern humans (~8%).20 In addition, 13% of the 100 kb window sections that the Neanderthal genome was split into had sequence divergences approximately ~20% of the time.10, 20 The overall divergence in 100 kb chunks of all modern human genome is only 2.5-3.7%.20, 53 Lastly, they concurred that the vast majority of genetic variation, in the forms of substitutions and indels (~87%), occurred before Neanderthals separated from modern humans.20, 53 The draft genome was useful in determining basic deviations in each group’s sequence, but until the full-coverage genome became available in 2013, functional and more specialized research could not be done. Using the full-coverage sequence, researchers found 78 amino acid alterations in nucleotide positions where all humans are the same but differ from Neanderthals and .50, 53, 66 Mutations that change the amino acid product typically have high morphological and functional consequences. These kinds of mutations can likely explain the differences we see in both populations when comparing Neanderthals to modern humans. Preliminary research published since 2013 has thus centered on five important proteins involved in some combination of: 1) sperm motility positive selection, 2) synthesis of molecules that produce proteins, 3) skin cell attachment and healing processes, and 4) sweat gland and hair follicle morphology.53, 66 Additionally, skin-expressed genes comprise 3/6 of the genes with multiple fixed substitutions or start/stop codons in the Neanderthal genome from the human one; which suggests a selection for skin morphology and physiology changing in the hominin lineage.53, 66 Of the 94 Mb of segmental duplications in the nuclear Neanderthal genome, there are 111 possible Neanderthal-specific duplications; 81% of which also express excess sequence diversity.53 The are 5,615,438 SNPs in the Neanderthal genome compared to five modern human genomes, 10% of which Neanderthals have the derived and not ancestral alleles.53 Putative (segments with unknown proteins and functions but are believed to be genes) changes in these SNPs include a region on chromosome two involving the THADA gene (associated with type two diabetes in modern humans), which could have affected energy metabolism in Neanderthals.66, 68 A second putative SNP change is in RUNX2, the only gene in the human genome that causes cleidocranial dysplasia: a phenotype involving delayed sutures, hypoplastic/aplastic , a bell- shaped cage, and dental abnormalities.53 Several of these traits are actually quite similar to traditional Neanderthal skeletal morphology, demonstrating the relationship that genomic and morphologic data have.

31 Finally, the most compelling use of Neanderthal genomic research arose when researchers from several labs all separately arrived that the same conclusion: Neanderthals and humans successfully interbred in the past.20, 52 All contemporary humans who can trace their ancestry to some degree outside of Africa (i.e.: Europeans and Asians) have 1-4% of Neanderthal DNA in their genome.50, 53, 66, 68 Before the sequencing project, it was impossible to absolutely and conclusively argue that Neanderthals and modern humans interbred, despite mosaic morphology found in intermediate fossils. For this reason alone, the resources and efforts put into the sequencing project were vindicated, and a renewed interest in Neanderthal archaeology and morphology followed.

DISCUSSION Spatial Considerations Variation in morphology and behavior is inherent when considering populations distributed across wide geographic distances. Rosas et al’s study on comparative mandibular morphology between northern and southern Neanderthal populations suggests there is a gradient in mandibular breadth and discrete features – such as the gonion, which measures the angle of the mandible curvature.64 Additionally, geographic barriers including glacial events, mountain ranges, and bodies of water likely provided strong reproductive barriers in antiquity. These factors in mind, it is imperative to realize that in some regions, Neanderthal traits may align closer to nearby AMH than to Neanderthals outside of their immediate geographic range. Spatial distribution produces strong phenotypic variance, including but not limited to: hair color and texture, skin color, nasal aperture size and shape, and dentition.8, 75 Lastly, individual Neanderthal diets would have greatly impacted their skeletal appearance, and food availability is highly dependent on one’s immediate environment.60, 61 Neanderthals residing in areas with poor vegetation and skilled large predators would have less food security than Neanderthals residing in warmer climates with plentiful faunal prey. Altercations with larger animals could very well result in life-threatening injuries and disabilities, and thus appearing on one’s pathological anatomy.

Temporal Considerations Between and among the four sites selected as case studies, there is a sizeable temporal range of Neanderthal existence and occupation. While Shanidar and Vindija caves have specimens spanning 20,000 to 13,000 kya respectively, each site has been dated within the 40,000 BP range.15, 19, 33, 69, 70 For this reason, variation dependent on temporal factors should not be a factor when comparing each selected site in relation to the others. However, future research on Neanderthal taxonomy would be improved if sites with wider temporal ranges were considered (including early and late Neanderthals).

32 In conjunction with this direction, it would be prudent to speculate on the reasons behind the Neanderthals’ absolute demise. The fact that early Homo sapiens survived and flourished while the Neanderthal lineage went extinct could be the key for understanding our closest extinct human ancestors.

Morphology Skeletal morphology is a direct product of genetics and the environment. By studying morphology, we can understand how Neanderthals were able to manipulate their environment for food and shelter, as well as infer larger evolutionary patterns. Throughout the literature review, it quickly became apparent that the terms used to describe and compare Neanderthal anatomy were somewhat subjective and rarely considered regional phenotypic variation. For example, within the four sites: El Sidrón Neanderthals had broad and low faces; Shanidar individuals exhibited full-facial prognathism rather than mid-facial prognathism; the residents at Vindija were uniquely gracile with less pronounced tori projection and thickness; and the Mezmaiskaya fossils have especially dense frontal squama and massive sphenoid bones.1, 2, 4, 7, 19, 26, 72, 78 A more comparable instance of this phenomenon occurs when describing the cranial torus (each site save for Mezmaiskaya has one). The tori at El Sidrón are “round” and “marched”, the Shanidar torus is barely visible yet Vindija’s occipital torus is “broad”.1, 64, 77 All of these descriptors fail to produce a comprehensive understanding of how Neanderthal tori interrelate. Even if all researchers had included numerical values on torus size and shape, it is unlikely that it would provide much more insight to Neanderthal supraorbital anatomy. This is because while numbers are more readily relatable, taking discrete measurements on fragmentary remains is very difficult, dependent on the researcher taking the measurements, and the numerical system used. The clearest synchronization in craniofacial morphology across the sample is in the foramen magnums descriptions. At each location, authors attribute ovular shape and wide breadth to the spinal chord opening at the cranial base. However, “wide and ovular foramen magnum” is the prevailing description when also talking about early and contemporary modern humans, and thus is not a strong diagnostic tool for distinguishing hominin species. Furthermore, it is important to note that when talking about Neanderthal and other hominin morphology, in nearly every case researchers are directly comparing and contrasting the skeletal elements to modern humans. This further complicates our understanding of Neanderthal morphology as we assume that they must be substantially and significantly deviant from landmarks in Homo sapiens. In the case of Neanderthal remains at Sima de los Huesos – a site in the Atapuerca region of Spain – researchers originally classified the skeletal fragments as .11, 42 They

33 only realized their mistake after ancient DNA was obtained from bones found on site and compared to the Neanderthal draft genome. This example serves as a rigorous reminder of the subjectivity of morphology and the difficulties researchers have when trying to assign fossils species labels on the basis of skeletal anatomy alone.

Archaeology Unfortunately, the sites selected above have no direct evidence of art or objects used in ritual events. A reasonable future direction of this work would be to incorporate sites with a varied archaeological record – including irrefutable evidence of deliberate burial, remains of jewelry and toys, cave art, clothing or tools used to work leather for clothing purposes, and traces of pigment and dyes – to further analyze Neanderthal behavioral and cognitive capabilities. Likewise, it would be prudent to choose sites with full faunal analyses to elucidate: regional diets, reconstruct paleoclimates, and infer more cases of cannibalistic and butchering behavior. Good candidate sites include: Gorham’s Cave (Gibraltar), (France), Kolosovskaya (Crimea), Fumane (), Cueva de los Aviones (Spain), and Krapina (Croatia).25, 31, 81 Despite these shortcomings, each site provides rich lithic assemblages which can be used to understand Neanderthal toolkits and their tool-making capacities. Most researchers agree that Neanderthal lithics fall under the purview of the Mousterian industry and are categorized by unifacial flintknapping. Some sites, like Mezmaiskaya and El Sidrón, are particularly noted for retouching and repurposing stone tools after their original function is no longer required.5, 67 The high proportion of worked stone is also what allows archeologists to classify sites as burial, occupation, or work places: Shanidar and Vindija were likely heavily occupied by Neanderthals either annually or seasonally.33, 70 Mezmaiskaya has been proposed as a temporary occupancy location, particularly due to the high proportion of lithics and faunal remains corresponding to sheep, goat, and bison hunting.19 The deliberate burial at Mezmaiskaya further backs this hypothesis as it seems likely that Neanderthals would hesitate to burial individuals directly in their primary base. El Sidrón is an interesting archaeological case: it seems like the hominin remains were deposited in the cave posthumously, but there are over 400 lithics in the cave.4, 63 But the discrepancy can be explained by cryo- and bioturbation in the larger cave galleries connecting El Sidrón to the Galería de Río. Additionally, it is even possible that the site was used extensively for flintknapping and it was just accidental that Neanderthal remains ended up in there. In all archaeological excavations, it is of the utmost importance to consider biases in the record due to preservation conditions, excavation techniques, and preconceived notions about

34 Neanderthal culture that researchers duplicate in their analyses. Perhaps the better question when attempting to contextualize Neanderthal archaeology is what evidence is not there rather than what is. If we only used El Sidrón, Mezmaiskaya, Shanidar, and Vindija caves (or even Neander Valley!) as the blueprints for conceiving what constitutes a Neanderthal, we would be missing the wealth of information that contradicts and expands our understanding of extinct hominins.

Genomics Without archaeology backing the fossils used in the Neanderthal Genome Project, the genetic data would be entirely useless. The five bones in which all of the sequences were derived are from a very limited geography and temporality; three of the five even obtained from the same site in the same stratigraphic (or time) context. The incredibly small sample size of the genome would not hold up in many scientific research studies as it is likely not representative of the entire population. In contrast, the Human Genome Project used over 1,000 individuals to put together the full-coverage human genome. Even after acknowledging the inherent bias in only using five fossils, it is also imperative to recognize: site stratigraphy, chronometric dates, associated cultural artifacts, and have a larger understanding of archaic hominin interactions before beginning to analyze genomic sequences. Additionally, genomic research is completely dependent on the preservation of skeletal remains and ancient DNA. Even if DNA preserves, geneticists cannot access hominin fossils without the cooperation of archaeologists and paleontologists. Because of these constraints, genomic data is hopelessly skewed to sites with cooperative research technicians and local laws, as well as the preservation biases that haunt the archaeological record to begin with. All of the genetic data we have on Neanderthals comes almost entirely from three bones found in Vindija Cave: Vi33.16, Vi33.25, and Vi433.26. Mitochondrial and nuclear DNA from El Sidrón and Mezmaiskaya caves have both been sequenced as well, but neither gleaned a comparable amount of ancient DNA to the Vindija samples. Further excavations to find fossils with high DNA concentrations were actually carried out at El Sidrón in 2012, but researchers could not find any bones with the same high genetic yield.53 So in a field that relies so heavily on large sample sizes and “objective” data, the genomic information we have on Neanderthals is actually highly biased and dependent on one site at a very particular time and place. To put it more concisely, all of the biases in the Vindija archaeological and morphological record are amplified in the Neanderthal genome sequence. I believe that genomic data is still relevant in the field of paleoanthropology and has given us considerable leaps in understanding extinct members of our genus, but such conclusions should always be weighted against the other forms of evidence we have.

35

CONCLUSION Ultimately, I found it entirely impossible to answer the question, “what is a Neanderthal?” using data from only four sites and the full-coverage genome. I do not think it will ever be really possible to have a definitive answer to that query – even if one does an in-depth meta-analysis on every existing Neanderthal archaeological site and re-studies all of the available genomes. Instead, my research revealed much more about the relationship(s) between the various disciplines that have a stake in the question than hominin taxonomy. To elaborate on these relationships, I have provided anecdotes below. It seems as if “paleoanthropologists” are almost always trained physical anthropologists or paleontologists. The latter primarily study hominin remains and often attempt skeletal reconstruction, sacrificing in-depth geological, faunal, and artifact excavations – not to mention computational biology.77 Trinkhaus, one of the primary investigators at Shanidar Cave, has frequently spoken out against the Neanderthal Genome Project and the use of genetic sequencing technology on hominins; arguing that fossils alone are able to tell researchers about the evolutionary and genetic relationships in human lineages.53 Conversely, many involved in ancient DNA are biologists, biochemists, and geneticists with absolutely no training in archaeology or .42 Primary investigators involved in sequencing Neanderthal DNA do not seem to have the framework for understanding taphonomic processes and the importance of cultural artifacts. Most of the research currently being published on data derived from the Neanderthal Genome Project is written for biomedical audiences, which often serves to completely alienate anthropologists and archaeologists who have worked at the very sites from where the fossils are found.9, 13, 20, 21, 52, 66 To further the divide in traditional paleoanthropology and genomics is the over-reliance on computational evidence at the expense of morphology. Genomics can measure sequence divergence, a helpful tool no doubt in looking at functional differences between individuals, but sequence divergence does not always equal species divergence. This distinction has tangible implications for paleoanthropological research, including questions of interbreeding and social interactions. Additionally, identical DNA sequences can produce extremely dissimilar phenotypes (due to differential gene transcription) and even with sequence data available, behavioral interferences are still highly theoretical. Disputes about Neanderthal cognitive, culturally symbolic, and linguistic capabilities will not be solved with genomic data. Lastly, the very act of ancient DNA sequencing is a destructive process, one whose benefits must be weighted against the risks of damaging a bone's important morphological landmarks.50

36 But genomics is not the only destructive force in paleoanthropology – archaeological excavation is perhaps the most reliably damaging tool. If a site’s stratigraphy is not completely accurate, then all conclusions drawn from the excavated material are suspect. This is a particular accusation that Vindija Cave frequently receives based on the disbelief that Upper Paleolithic technology would be found in association with Neanderthal remains in G1.2, 37 In addition to poor excavation techniques, mixed artifact assemblages could also result from natural processes such as faunal and geologic activities. Due to the time period in which many Neanderthal sites were found – in the mid-to-late 1800s – we may have lost crucial behavioral evidence such as deliberate burial positions, art, and bone/wood tools, and decorative objects simply because the archaeologists did not know how to look for them or interpret them. Archaeology is typically seen as a soft science, even from biological anthropologists, and the lack of what some may call proper equipment to avoid contamination only compounds archaeology’s legitimacy in paleoanthropology. Many working on the Neanderthal genome were shocked when they say archaeologists and museum curators touching fossils without gloves and downright horrified when some licked the remains to prove they were not rocks or other organic materials.53 This actually led to a “sterile dig” attempt at El Sidrón Cave in 2012 in which excavators wore Hazmat suits and gloves when searching for fossils to sequence.53 Not surprisingly, this was an unsuccessful venture. To summarize, overall trends in morphology, archaeology, and genomics from El Sidrón, Mezmaiskaya, Shanidar, and Vindija show that Neanderthals do exhibit some range of diversity as a species designation. Despite internal squabbles among researchers at specific sites, the minimum classification of “Neanderthal” is compelling across the sample. Collectively, the morphology of these Neanderthals fall into classical phenotypes – robust supraorbital and zygomatic processes; the lack of incipient chins; high mid-to-full facial prognathism; and large, shovel-shaped, pathological dentition – but the Neanderthals at Vindija are extremely gracile compared to Shanidar. It is indisputable that Neanderthals buried their dead, had complex and pre-planned lithic assembly methodology, and participated in art production and other ritual behavior. But despite possessing these capabilities, they still never managed to produce a magnitude of cultural artifacts or modified their environment to the extent that Homo sapiens have done. Lastly, the genomic data available for El Sidrón, Mezmaiskaya, and Vindija does place them in separate clades from modern humans. To these degrees, the Neanderthal archetype presented in the introduction does seem to prevail. But this is probably a conclusion more reflective on the data available from El Sidrón, Mezmaiskaya, Shanidar and Vindija caves rather than Neanderthals as a whole. Regardless of whether we call them Homo neanderthalensis or Homo sapiens neanderthalensis, Neanderthals are our closest extinct

37 hominin relatives and researching their morphology, behavior, and genomes only serves to help us understand what makes us human. There are definite benefits to sorting many individuals into the same species (particularly for modern animal conservation efforts), but it seems trite to insist on firm designations in paleoanthropology. Save perhaps grant funding, there is no real advantage to assigning Neanderthals the labels Homo neanderthalensis or Homo sapiens neanderthalensis. The organisms we colloquially call Neanderthals did successfully and repeatedly interbreed with early humans, and they must have had comparable cognitive and behavioral capacities for these kinds of relationships to occur frequently. Biology is not as objective or clearly rational as many scientists would like to believe, and archaeological evidence from which we can infer behavior is of the same import when looking at extinct hominins. It is clear that there are repetitive traits that link Neanderthals as a group, but it has also become clear that these attributes were not so peculiar that Neanderthals and humans could not understand one another.

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