Montiel Et Al 2007 Believers vs Skeptics

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Recent Advances in Molecular Biology and Evolution: Applications to Biological Anthropology, 2007: ISBN: 978-81-308-0198-8 Editors: Cristina Santos and Manuela Lima

Ancient DNA and Biological Anthropology: Believers vs. skeptics

Montiel, R.1,3, Francalacci, P.2 and Malgosa, A.31Departamento de Biologia – CIRN, Universidade dos Açores, Portugal 2Dipartimento di Zoologia e Genetica Evoluzionistica, Università di Sassari Italy; 3Departament de Biologia Animal, Biologia Vegetal i Ecologia Universitat Autònoma de Barcelona, Spain

Abstract The first-ever published paper on ancient DNA (aDNA) analysis (published in China) reported on DNA extraction from an ancient human mummy. A few years later, the first paper on human remains published in the West, also reported on the extraction and analysis of DNA from a human mummy. Since then, the potential of ancient DNA analysis in anthropological studies has been widely acknowledged. However, the contamination/authenticity problem, which

Correspondence/Reprint request: Dr. Rafael Montiel, Departamento de Biologia-CIRN, Universidade dos Açores, Rua da Mãe de Deus, Apartado 1422, 9501-801 Ponta Delgada (Açores) Codex, Portugal E-mail: [email protected]

Montiel, R. et al. 2

affects the entire aDNA field, is particularly acute in human studies, and hampers the full development of this discipline. For some researchers, this problem makes the study of ancient human populations unfeasible and/or impractical. On the other hand, aDNA studies of human populations are published every year, reflecting the deep interest that anthropologists and archaeologists have in the unique genetic information that can be directly retrieved by studying them. Thus, the general application of aDNA techniques to the study of human populations is a contentious issue, and the question remains open as to what extent following the stringent criteria suggested by Cooper and Poinar is useful in the authentication of ancient human DNA sequences. In practice, the complete set of suggested controls is rarely followed in human studies, and even when applied carefully these criteria may not be sufficient to guarantee authenticity. Furthermore, some authors are proposing flexibility and the intelligent use of these authenticity criteria in order to avoid the “checklist” approach, which can result in the publication of non-authentic results and in rejection of some interesting and probably authentic ones. On the other hand, although there have been several papers recommending appropriate experimental designs for ancient DNA studies, there have been few attempts at statistical analysis to establish the confidence of the results obtained. In our view, ancient DNA recovery and analysis from human populations requires a specific frame and set of controls that in a cost-effective way allows anthropologically relevant information which may be considered authentic within certain confidence limits to be retrieved from ancient human populations.

I. Introduction I.1. An overview of aDNA and the contamination problem Ancient DNA (aDNA) is defined as DNA recovered from naturally or artificially preserved biological tissues. Two papers published in the middle 80’s in Nature are considered the starting point for this research field [1, 2] and a few years later, the kind of tissues analyzed and their antiquity had increased exponentially [3]. Some works could be considered as hallmarks in the aDNA field as they have contributed to its growth and consolidation. In this sense, the possibility of applying PCR to DNA analysis of ancient tissues [4, 5] marks the real beginning of the explosion in this field. Subsequently, the criticism and recommendations published by Lindahl [6, 7] represented an inflexion point. Lindahl’s most relevant recommendations were later put into practice by several important laboratories [e.g., 8, 9]. A great advance was represented by the publication of authenticated results from >40,000-year-old Mammoth samples [8-10]. These achievements were followed by the tour de force represented by the recovery of mtDNA sequences from the Neanderthal type specimen [11]. This work was followed by a series of Neanderthal studies

Ancient DNA and Biological Anthropology 3

[12-20], that led to the establishment of the field of Neanderthal genetics [21-23]. The next big step in the ancient DNA field was made recently, when Noonan et al. [24] and Poinar et al. [25] showed how new approaches, like metagenomics and the application of new sequencing technologies may raise aDNA to the genomic level. These new methodologies are already being used for the study of Neanderthal genomics [26, 27], a dream that has come true. In the past, the most spectacular reports presented sequences recovered from samples of millions years old, including insects entombed in amber [28, 29] and dinosaur bones [30]. Soon afterwards, however, it was shown that the recovered sequences were more likely the product of DNA contamination ([31, 32] for amber studies; and [33-36] for dinosaur bones). These studies, now considered a fiasco [37], raised several doubts about the entire field. The criticism was supported by the work of Lindahl [38, 39], who, while studying the stability of DNA in aqueous solutions, concluded that informative segments of DNA could not last for more than 100,000 years under any conditions [6, 7]. Although the stability of DNA in time is a controversial issue, it now seems clear that if conditions are optimal, DNA may last for 500,000 or even a million years [25, 40-42]. Nevertheless, the risk of amplifying exogenous (contaminant) DNA in ancient DNA studies has become clearer than ever. The severity of this problem may be understood by considering that aDNA is strongly damaged, and, therefore, any trace amount of fresh contaminant DNA may outcompete it in downstream analyses. The problem is exacerbated in ancient human studies, since the researchers belong to the same species as the object of study and the potential sources of contamination increase with each step of the analysis. Despite the acknowledged potential of aDNA studies in Anthropology, the ever-present problem of contamination has hindered the application of this technique to the study of ancient human populations. The advice of one of the leading groups in this field is, with some exceptions, not to work with ancient human samples [43]. On the other hand, studies on ancient human populations are still published every year [e.g., 44–48] and the question remains if the standard authenticity criteria [49] are adequate for ancient human remains and ancient populational studies. We know that contamination is an inherent problem in the field, and as it will be ever-present, we need to learn how to deal with it in order to achieve authentic results. Only if we face the problem squarely may we come to understand it in depth and learn how to handle it. I.2. Why study ancient human populations The focus of aDNA studies, why do them and what should be studied, can be a source of controversy among scholars in this field. For example, Pääbo et al. [43] suggest that human aDNA analysis is only feasible in distinct and isolated populations but not in historic populations that are too similar to recent


populations. According to them, the proposal to study cemeteries from less than 1,000 years ago makes no sense because no useful molecular evolutionary information could be acquired from this short a time span. Setting aside technical problems, in our view, this way of thinking suffers from at least two weaknesses. In fact, even more recent series (<300 BP, for instance) could be useful for studying mitochondrial (and perhaps nuclear STR) molecular evolution, because it could be possible to follow family lineages and to calculate the number of mutations generated, thus extending genealogical studies of mtDNA mutation. Certainly, the analysis of descendents could run into problems of ethics and data protection, but this is a different matter to deal with. On the other hand, if evolution is the main focus of our research, we should consider that mutation is not the only mechanism fueling evolution. Migration, natural selection and genetic drift also play fundamental roles in evolution. The effects of migration and genetic drift may be assessed by analyzing populations of any antiquity.

The second weakness in the argument of Pääbo et al. [43] is related to the focus of the research: aDNA analysis on protohistoric and historical populations could be interesting beyond questions of molecular evolution. The study of ancient populations is the foundation of knowledge about our history. This knowledge comes especially from Biological Anthropology, which offers the opportunity to learn directly about people who lived before us, providing information on populational and family relationships, intragrupal variability, and the inception and diffusion of diseases, and helping us to ascertain what they were like, what they looked like, where they came from, how they lived, what their lifestyles were, and so on. One of the questions whose answer is most sought after is: “Where do we come from?” Sometimes it is only rhetorical, but often it is a real question about our biological origins. The answer to this question can be found in the study of human remains and, now more than ever, in paleogenetics. I.2.1. Very ancient populations The farther one goes back in time, the more difficult it is to talk about populations in the strict sense of the word, because fossil and archaeological records are very scarce. Often, the earliest remains are represented by isolated bones, or individuals in the best of cases. Usually, fossils from Australopithecus or from the first Homo, for example, are represented by a single tooth, or a partial skeleton in cases of excellent preservation. There are still, however, noteworthy and important exceptions, such as the large sample of Homo heidelbergensis from La Sima de los Huesos in Atapuerca, Spain [50], or those of Homo neanderthalensis from the Krapina site in Croatia [51]. Note, however, that the exceptions correspond to a more modern species of hominid lineage. Therefore, data about our more ancient forefathers are subject


to uncertainty and great doubts. Also, it is difficult to precisely specify limits on studies of human origins: How far do we want to (or can we) go back? Do we focus on genus, species, or local population? Besides the final objective of the study, the methodology that we endeavor to apply almost always imposes its own limits. For aDNA studies, the objective cannot be focused too far back. An important question to consider is that the final aim of Biological Anthropology is to achieve understanding about populations, transcending knowledge about individuals. In this sense, Biological Anthropology explores human variation using biological principles and methods. It deals with the place of humans in nature, the pattern of our evolution as a species, and the way in which individuals and populations interact with their environment. Populations in the strict sense can be studied since the Neolithic, or over the last 8,000 years BP. This time frame can be feasibly covered by aDNA techniques in many regions. The kind of problems that can be solved by aDNA analysis are usually related to population movements. The spread of the Neolithic in Europe is one of the most interesting areas for anthropological analysis. Cultural or biological diffusion or both together, could explain the spread of the Neolithic around the Mediterranean Arc [52-55] and aDNA analysis could offer conclusive data in this regard. In Asia and America the study of ancient human populations may help to solve questions about population diffusion in both continents, in addition to those pertaining to the peopling of the Americas. Nevertheless, the study of the peopling of the Americas poses a major sampling problem. The earliest settlers are unknown, as is the moment when the peopling took place. The latest information shows that the most ancient remains hardly go beyond the Holocene, and remains from North and South America are very similar in antiquity. The geographical situation of the Americas at that time (glaciers covered almost all the North American territory) made interments difficult and also complicate the possibility of recovering ancient human remains. Therefore, even the smallest Amerindian specimens which could provide some genetic data have an enormous interest. On the other hand, the study of American populations of between 2,000 and 9,000 years old could provide relevant information on other problems related to the settlement of the Americas. After different hypotheses about the origin of Amerindian populations (tripartite wave from linguistics, morphology and genetics [56], two-wave [57], or one single migration based on binary polymorphisms and NRY-STRs [58]) another problem arises about the peopling of the New World. How did humans colonize the two Americas in such a short time? The study of regional and temporal genetic continuity might provide important data to test several hypotheses. In general, population density in ancient times was very low, and this, along with the movement of peoples, created isolates in which founder and bottleneck effects might be


relevant. Paleogenetics can help both to track migratory routes and to characterize isolated populations. I.2.2. Relatively recent populations Problems in more recent populations are similar to those in ancient ones, but higher sampling rates enable us to pose new questions. For instance, in the European context, Roman or Medieval necropolises offer an exceptional possibility not only for understanding human variability, but also for interpreting and reconstructing European history. In America, the study of populations before and after contact with Europeans provides a unique context for understanding many aspects of population interaction and their effects on the genetic pool and genetic function. In reference to Europe, below are two cases that exemplify the kind of questions that may be solved by analyzing ancient populations of different ages.

1. On the island of Majorca, there are three major necropolises located on the Bay of Alcudia in the north. Two of them, the Necropolis of Son Real and the Necropolis of S’Illot des Porros, correspond to a Talayotic autochthonous culture [59] with parallels to other Mediterranean islands, such as Sardinia. The first question to try to solve is if the Talayotic culture was the result of demic or exclusively cultural expansion. Carbon-14 and archaeological dating (VI-II B.C.) overlap the two cemeteries, Son Real being probably slightly more ancient. Son Real is on the coast and S’Illot des Porros is located on a rocky island just in front of Son Real. This geographical, temporal and archaeological situation poses various queries: (i) Do both necropolises correspond to the same population? Morphological data [60] shows very similar characteristics, but some traits indicate more slender individuals in S’Illot des Porros; (ii) if both necropolises are from a single and contemporary population, was an elite buried on the rocky island? In the necropolis of S’Illot des Porros two funerary rites are observed, inhumation and cremation, and dating is not conclusive enough to totally separate both; (iii) are rites only a cultural change, or do they correspond to replacing populations? The third necropolis, Can Reiners, is over the ruins of the roman city of Pollentia in the modern town of Alcudia. The site is from the Early Middle Ages, almost 1,000 years later than Talayotic ones. In this lapse of time, the Romans conquered Majorca (123 B.C.) some centuries after their empire declined; (iv) do the people buried in Can Reiners belong to an autochthonous population or were they different from Majorcans?; and (v) can influences coming from the Roman Empire and other European and Mediterranean populations be identified and evaluated?

2. In Medieval times, Muslims occupied the Iberian Peninsula for eight centuries, the Al-Andalus area being the longest occupied. In some regions,


continuity between previous communities existed. The genetic study of Muslims cemeteries [61], easy to identify by funerary rituals, can determine the genetic composition of the medieval population in order to evaluate the impact of North African migrations and to assess the evolutionary changes of the population up to the present.

There are many other studies showing the interesting questions that can be addressed by analyzing aDNA in American, European and Asian populations. Some of these are reviewed in Section II.2 below. New questions are concerned with Paleomicrobiology applied to knowledge of infectious human diseases [62]. Problems such as the place of origin of venereal syphilis, whether Europe or America, and its subsequent diffusion, or the correct differential diagnosis of periostitis in pathological bones are some of the passionate controversies in which aDNA can offer precious and sometimes conclusive information. II. The believers: From single individuals to population studies II.1. First studies on ancient humans II.1.1. The early Chinese experience The first-ever published aDNA paper (Fig. 1), appeared in 1981 [63], reported on the extraction of DNA from two ancient Chinese mummies. Motivated by previous studies indicating good preservation at the organs and tissues levels, Wang and Lu decided to extract DNA from the liver of a 2,000-year-old mummy from the Han Dynasty to verify if the preservation was also good at the molecular level. As controls, the livers of a mummy from the Ming Dynasty (the Ming Dynasty lasted from 1268 to 1644 AD, so the sample was at least 500 years old) and of a non-cancer patient were studied. The samples were subjected to several treatments with proteinases (pepsin and trypsin), followed by phenol extraction and chromatography through a DEAE-Cellulose column. From 0.28g of the Han Dynasty liver, 13.9µg of DNA were obtained, which presented a molecular weight similar to that of the tRNAAla (i.e., highly degraded). The authors concluded that their results offered a scientific basis for the preservation of ancient bodies at the molecular level [63]. Today, assessing the authenticity of Wang and Lu’s results is not relevant. It is highly probable that some small fraction of the DNA extracted belonged to the mummy but, in our opinion, the relevance of their work lies in the author’s determination in searching for scientific proof for the possibility of DNA preservation in ancient tissues. This indicates an early recognition of the potential that such studies might have. The fact that the sample came from human remains is also very significant. Since this paper, the Chinese scientific


Figure 1. Title, authors, and institution (in parentheses) of the first-ever published article reporting aDNA analysis [63]. community has been active in research on aDNA. A quick hierarchical search of CNKI database (Chinese National Knowledge Infrastructure; www.cnki.net) revealed that, at the time of writing, more than 500 papers have been published in Chinese journals. The search was performed on abstracts in the Biology and Medicine catalogs by using the following keywords: Human, Ancient, and DNA, in that order. II.1.2. Pääbo’s work Perhaps with the same motivations as Wang and Lu, Svante Pääbo, conducted systematic studies on ancient Egyptian mummies. As artificial mummification in ancient Egypt was practiced for thousands of years, and as climatic conditions have also promoted natural mummification, Pääbo considered that Egyptian mummies represented a unique source of ancient human remains that could be useful for molecular genetic studies. His first studies, published in 1985 [2, 64], comprised a histological analysis of the different tissues of each of 23 mummies. By staining with ethidium bromide Pääbo was able to detect DNA in the cellular nucleus of the cartilaginous tissue of the external ear of one mummy and in the epidermis and subcutaneous tissues from the face of another. However, the extracted DNA from the latter was impossible to clone due to modifications in its pyrimidines. Nevertheless, Pääbo was able to clone DNA extracted from the skin of a boy who died at one year old which presented good histological conditions and, by using an Alu probe, he identified one clone containing a 3.4kb insert. The probe hybridized with two segments of the insert, and one of them was sequenced. Comparison of the sequence obtained (900 bp) with an Alu consensus sequence showed that there were no significant modifications in the mummy’s DNA after death. In Pääbo’s opinion, these results established the viability of successfully cloning substantial fragments of nuclear DNA from biological remains of considerable antiquity, although he remarked that most of the analyzed samples lacked nucleic acids. Analyzing these results in the light of current knowledge, it is hard to sustain that the cloned fragment indeed came from the mummy’s DNA (i.e.,


that it was authentic), as noted by several researchers [e.g., 42, 65]. Although, technically speaking, the intrinsic value of these results could be reduced to the detection of nucleic acids by ethidium bromide staining, these papers, along with the work of Higuchi et al. [1], have the unique scientific merit of having launched aDNA research, motivating hundreds of researchers to engage in the recovery of DNA from ancient (human) samples. Furthermore, Pääbo’s contributions to the aDNA field since then have been continual and extremely relevant, so it is more than fair to consider him as the founder of the aDNA field (and, of course, of its applications in anthropology). II.1.3. Other studies on ancient humans Other early research involved the analysis of single (or a few) individuals. Doran et al. [66], published a study in which human DNA was detected in brain tissue from ~8,000-year-old individuals that had been preserved in a small swampy pond in central Florida, but it was not possible to clone it. Later Pääbo et al. [67] published an mtDNA sequence of a 7,000-year-old brain from an individual recovered from Little Salt Spring, Florida. This study was pioneering in several respects: besides the use of PCR in human samples, in this paper early authenticity criteria were formulated for aDNA in general, and for ancient human DNA in particular. In 1989, the possibility of recovering DNA from ancient bone was investigated. These works were specifically addressed to recovering ancient human DNA. Rocio Vargas, a researcher from the Instituto de Investigaciones Antropológicas (Mexico), published the lesser known of these studies in a Mexican scientific journal [68], reporting the recovery of genetic material from human bones dated between 650 and 750 years old. At the same time, Japanese researchers led by S. Horai published in a Japanese journal a study on the recovery of DNA from bones ranging from 60 to 6,000 years old [69]. However, the work that has had the greatest impact in the scientific community was the one published in Nature by Hagelberg et al. [70], who extracted DNA from bones of between 300 and 5,500 years old. In showing the feasibility of recovering DNA from hard tissues, these studies widened the potential sources of aDNA, increasing the expectations for these techniques to address relevant anthropological questions. Hänni et al. [71] advanced a step further, when they showed that ancient human teeth were also good, if not better, sources of aDNA. Lawlor et al. [72] cloned and characterized HLA genes from 7,500-year-old archaeological remains. Handt et al. [73] published another important study on an ancient human, an approximately 5,000-year-old mummified human body found in the Tyrolean Alps. One DNA sequence of a hypervariable segment of the mitochondrial control region was determined independently in two different laboratories from internal samples from the body, providing evidence of authenticity [73]. This work showed the potential of aDNA studies


when single individuals are analyzed, as interesting conclusions relevant for disciplines other than genetics were obtained. Di Benedetto et al. [74] examined five human remains from the Eastern Italian Alps dated between 14,000 and 3,000 years ago. This work represents an early demonstration of the authenticity framework recently described by Cooper and Poinar [49]. Mitochondrial DNA sequences from three of the five individuals analyzed were obtained by amplifying and cloning five overlapping fragments after assessing biochemical preservation by amino acid racemization studies and DNA quantification. The cloning procedures, along with the multiple controls and extractions performed and the independent analysis carried out in another laboratory, represented such a great amount of effort that the authors concluded that sample sizes for human studies would remain small [74]. Nevertheless, other works have shown how difficult it is to authenticate aDNA when single or very few individuals are analyzed. Adcock et al. [75] extracted DNA from a morphologically gracile individual, Lake Mungo 3, dated to 60,000 years BP. The amplified mtDNA sequence allowed relevant conclusions about the origin of anatomically modern humans to be drawn. However, serious doubts about the authenticity of these results have been raised [76, 77]. Caramelli et al. [78] typed the mtDNA hypervariable region I (HVRI) of two anatomically modern Homo sapiens individuals of the Cro-Magnon type dated to about 23 and 25 thousand years ago. In this study, all the criteria suggested by Cooper and Poinar [49] were followed, although the reliability of the results has been questioned (see [79]; and [80] for a reply) and signs of cross-contamination have been detected [81] (see Section III.2.1). II.2. Human aDNA for population studies II.2.1. Early population studies The next move in human aDNA studies was to develop population-level studies. This is where aDNA may reach its full application in Biological Anthropology, which is concerned with “variation in time and space.” In fact, after a decade of aDNA research, “for the real anthropological potential of ancient DNA to be realized, more studies analyzing the sorts of populations and addressing the sorts of questions that anthropologists are interested in were needed” [82]. Also, as Pääbo et al. [67] asserted, the study of numerous individuals from ancient populations may provide proof of authenticity. Actually, they considered that this approach would provide “indisputable proof”, however, in light of the following discussion of the transcendence of the contamination problem, this affirmation now seems to have been hasty. Among the first population-level studies, the works of Horai et al. [83], Kurosaki et al. [84] and Stone and Stoneking [85] stand out. Horai et al. analyzed 11 ancient individuals from two different sites, and compared their results with modern data in order to address the question of the origin of the


Japanese [83]. Although the work of Kurosaki et al. was not a populational study as such, their work was the first to analyze putatively related individuals, attempting to achieve individual identification and assess kinship by analyzing short tandem repeats (STR) [84]. Works from the following years were almost all concerned with the study of ancient Native American samples. Stone and Stoneking [85] characterized mtDNA from 50 pre-Columbian individuals from a Prehistoric Oneota cemetery at Norris Farms, Illinois, that dates to A.D. 1300. This work represents the first contribution of the aDNA field to the problem of the peopling of the Americas (for a review of this subject, see the chapter by Solorzano et al. in this book). One important question in the study of ancient populations is to what extent individuals from different dates found at the same site may be considered a single population. Hauswirth et al. [86] addressed this question at the Windover site (7,000-8,000 BP). By analyzing DNA from multiple individuals spanning nearly the full range of estimated burial dates, they confirmed the hypothesis that there is a persistence of both nuclear and mitochondrial haplotypes at Windover throughout its entire period of use. Thus, according to their results, Windover can be considered a single population. Hauswirth et al. [86] also used their data to estimate the mutation rate of human mtDNA, obtaining rates within the range of other independently calculated values. Merriwether et al. [87] studied ancient Native American DNA and showed that all four lineages were present before European contact in North America, and that at least two were present in South America, lending support to the single-wave hypothesis for migration into the New World. In this period, aDNA population data from Pacific islanders were also published [88, 89]. An interesting study was published by Béraud-Colomb et al. [90], in which 10 individuals, ranging in age from 620 to 12,000 years old, from Morocco, the Sahara, Sudan, Ethiopia, Italy, Sardinia, and France, were analyzed. Five of them were successfully characterized for polymorphisms of the β-globin gene. According to the authors, this study showed the feasibility of systematically studying nuclear DNA polymorphisms of ancient populations. About this work, Stoneking [82] remarked that the exhaustive laboratory procedures appeared to meet all of the informal guidelines suggested by the ancient DNA community for avoiding contamination, in addition to being “presented in such painstaking detail.” However, some controversy arose about whether enough criteria were used to consider these results as authentic [91, 92]. II.2.2. Advanced population studies and their implications “Second generation” population studies were published beginning in 1995. In these studies, well-defined populations were analyzed and the results were more relevant than simply showing that the retrieval of ancient human samples was possible. Stone et al. [93] analyzed 20 more individuals from Norris


Farms, Illinois. The sex of 19 individuals was accurately determined using molecular genetic techniques as compared with standard osteological methods. This high match rate supports the authenticity of the results. In fact, it was proposed that matching in sex determination could be used as an authenticity criterion [94]. It is worth noting that sex determination relies on an analysis of nuclear DNA (from sexual chromosomes). Lalueza et al. [95] extracted DNA from the bones and teeth of 60 individuals from four extinct human populations from Tierra del Fuego, Patagonia. Their study, almost entirely based on Restriction Enzyme (RFLP) analysis, revealed the complete absence of two of the four primary mitochondrial haplogroups present in contemporary Amerindians, namely A and B. Stone and Stoneking [96, 97] complemented their study of the prehistoric Oneota cemetery at Norris Farms, obtaining mtDNA results from 108 of the 152 individuals analyzed. Their data gave support to the single-wave hypothesis for the colonization of the Americas. Samples from China have been analyzed by some of the authors of the Kurosaki’s paper [84]. In these studies 2,000-year-old and 2,500-year-old individuals from Shandong Province were analyzed [98, 99], and the results were used to investigate temporal changes in population genetic structure. In Europe, Izagirre and de la Rúa [100] studied 121 dental samples from four prehistoric Basque sites by RFLP analysis; their results led to discussion about the origin of the V haplogroup in Europe. Montiel et al. [101] published the first mtDNA sequences from a European series. Forty-four individuals from a 16th-century necropolis were analyzed, from which 28 control region sequences were obtained. This study was concerned with the authentication process of results from European populations, however, the data allowed population comparison and conclusions about the mtDNA mutation rate to be made [65]. To gain insights into the origin of the Guanches, the first known inhabitants of the Canary Islands, Maca-Meyer et al. [102] analyzed 131 teeth corresponding to 129 different individuals, from 15 archaeological sites sampled from four of the seven Canary Islands and dated to around 1,000 years old. Informative mtDNA sequences were obtained from a total of 71 individuals, allowing conclusions to be drawn about the origin of the Guanches and modern mtDNA composition in the Canary Islands [102]. This study was complemented by a study of mtDNA diversity in 17th–18th century remains from Tenerife, also conducted by Maca-Meyer et al. [103]. More recently, Haak et al. [104] successfully extracted and sequenced mtDNA fragments from 24 out of 57 Neolithic skeletons from various locations in Germany, Austria, and Hungary, concluding that these first Neolithic farmers did not have a strong genetic influence on modern European female lineages and supporting a Paleolithic ancestry for modern Europeans. Currently, many ancient population studies are being published, which can be said to belong to “third generation” studies, in which an effort has been


made to meet, as far as possible, the authenticity criteria proposed by Cooper and Poinar [49]. Some representative examples have been chosen from among these and the percentages of efficiency, of samples cloned, and of samples independently replicated can be seen in Table 1. Lalueza-Fox et al. [105] analyzed 47 pre-Columbian Ciboneys from Cuba, obtaining sequences from 17 of them (36.2%). One PCR product of 3 different samples was cloned, and one sample was sent to an independent laboratory for replication. Vernesi et al. [106] analyzed 80 Etruscan samples, and after discarding 32 samples that presented bad preservation conditions, two extracts were prepared from the remaining 48 (60%) and three overlapping fragments were amplified and cloned for each of them. Each of the three fragments was sequenced at least eight times (two extracts × two PCRs × two clones or more). The entire procedure was repeated in an independent laboratory for three bone specimens and identical sequences were obtained [106]. Lalueza-Fox et al. [107] analyzed 36 teeth retrieved from Kazakh archaeological sites, recovering sequences from 29 of them (80.6%). Eight samples were cloned and a subsample of six teeth was sent for independent replication, which was accomplished for four of them. Sampietro et al. [44] analyzed 20 teeth from 17 pre-Roman Iberians, cloned 8 samples and sent 2 for independent replication. Alzualde et al. [45, 108] studied 123 teeth belonging to 65 individuals recovered from the late ancient (6th to 7th Century A.D.) cemetery of Aldaieta (in the Basque Country). Amplification was successful in 56 individuals (86.2%); 10 were cloned and 9 Table 1. Studies in which an effort has been made to meet the “standard criteria” of Cooper and Poinar [49].

Individual specimens

Reference Analyzed Successful Duplicated b aa c Quantified Cloned Replicated d

Lalueza-Fox et al. [105] 47 17 (36.2%) 1 --- --- 3 1

Vernessi et al. [106] a 80 48 (60%) 48 80 77 48 3

Lalueza-Fox et al. [107] 36 29 (80%) 5 --- --- 8 4

Sampietro et al. [44] 17 17 (100%) 1 2 5 8 2

Alzualde et al. [108] 76 67 (88.2%) 25 --- 9 10 9

Alzualde et al. [45] 65 56 (86.2%) 55 --- 9 10 9

Töpf et al. [47] 156 48 (30.8%) 48 --- ? 2 4 a samples discarded by amino acid analysis (n=3) and quantification (n=29) were not used for amplification. b at least two independent extracts were analyzed (intra-laboratory). c amino acid analysis. d analysis repeated in a second or third laboratory.


independently replicated. Töpf et al. [47] analyzed 156 individuals from Britain (4th to 11th Century A.D.). A total of 48 (30.8%) DNA sequences could be authenticated and were included in the population analyses; cloning was carried out for two samples, and three independent laboratories replicated four samples. Quantification of aDNA present was estimated by the number of cycles needed to obtain amplification. Other studies have been published that do not follow the standard criteria but provide data supporting authenticity, like duplication, phylogenetic sense, molecular sexing, etc. Keyser-Tracqui et al. [109] analyzed 99 skeletons exhumed from the Egyin Gol necropolis (Mongolia) dating from >2,000 years ago and obtained partial genealogical reconstruction using biparental, paternal, and maternal genetic systems in 62 of them (62.6%). During excavation and curation, samples were handled with gloves by a reduced number of anthropologists wearing face masks. DNA extracted from all people handling the material or working in the laboratory was genetically typed and then compared with the results of all the ancient samples. Mooder et al. [46] analyzed 82 Siberian individuals excavated from the Lokomotiv and Ust’-Ida cemeteries, successfully sequencing 70 of them (85.4%). All samples were extracted in duplicate. Casas et al. [61] analyzed bone and teeth samples belonging to 71 individuals collected from the medieval Moorish town of Madinat Baguh (Priego de Cordoba, Spain). Characterization of mtDNA was achieved for 61 of them (85.9%). Ten samples were replicated in independent laboratories and the mtDNA HVRI of the operators working with the medieval samples was sequenced in order to be compared with the ancient sequences obtained. Changchun et al. [110] extracted DNA from thighbone samples from 18 ancient Xianbei remains (4th and 5th centuries A.D.), obtaining mtDNA sequences from 16 of them (88.9%). Two independent extractions were performed and the sequences were compared to those of the handlers. Shinoda et al. [111] examined 57 ancient Peruvian Highlanders, from which 35 (61.4%) were successfully analyzed. No work on human mtDNA had previously been conducted in their laboratory. Burger et al. [48] analyzed 51 bone samples from early Holocene sites in central and eastern Europe. Using a range of authentication criteria, the authors obtained high confidence genotypes from eight early Neolithic and one Mesolithic skeletons from central, northeast, and southeast Europe. We may consider this paper as one of the first of the “fourth generation” of papers, in which a flexible approach for authenticity is used as advocated by Gilbert et al. [112] (see Section IV.3 below). II.2.3. Other studies Amplification of nuclear DNA is regarded as much more difficult than mtDNA. Therefore, of particular note is the work of S. Hummel, B. Herrmann and co-workers who, since at least 1991 [113], have published results in peer-


reviewed journals reporting analysis of nuclear STRs [114, 115] including Y-chromosomal STRs [116] and megaplex [117] and multiplex [118] methodologies, as well as other nuclear genetic markers, like the ABO blood group [119], the ∆F508 mutation [120], and the CCR5-∆32 HIV resistance gene [121], all with high efficiency in remains of up to 3,000 years old. Other studies characterizing nuclear DNA include those of Ricaut and co-workers in Sytho-Siberian individuals dating back 2,500 years [122], in a Neolithic Siberian skeleton dated to 3,600 years B.P. [123], and in skeletons from the south of France dated to 400-1,000 A.D. [124]. Other issues in the field are related to molecular damage of aDNA, which was investigated at the beginning [64] and has been the subject of subsequent research since [e.g., 40, 125]. The work of Gilbert et al. has been specifically concerned with characterizing postmortem damage in human mtDNA [126, 127]. Their work has provided a frame for assessing the preservation conditions and authenticity of ancient human remains. Although the works on Neanderthal aDNA [11-20, 26, 27] are extremely relevant for Biological Anthropology, this issue is not reviewed or discussed here because the problems related with authenticity in Neanderthal studies are different to some extent from the problems presented by more recent Homo sapiens populational studies. III. The skeptics: The criticism III.1 Detection of contaminating human DNA The severity of human DNA contamination was realized early on by researchers who amplified human specific mitochondrial DNA (mtDNA) from animal bone extracts [94, 128]. Recent work has underscored this problem by detecting human contamination in paleontological tooth samples from animals [41] and the extensive human contamination that affects museum animal specimens [129, 130]. Wandeler et al. [129] analyzed the teeth of 279 red fox individuals and detected human DNA in 110 samples (40.0%) by microsatellite typing (HLABC-CA2). Malmström et al. [130] analyzed bones and teeth from 29 museum dogs and found human mtDNA contamination in all of them, likely produced before the arrival of the samples to the laboratory [130]. In other cases, human contamination has been detected when modern European sequences have been determined from Amerindian samples [131]. Similarly, contamination with modern human DNA (i.e., from Homo sapiens) has been detected in Neanderthal bones [e.g., 11, 16]. In general, the reasoning goes that if human DNA is contaminating non-human remains it is likely that it may also be present in ancient human samples, but in the latter case, detecting the contaminant sequences is highly problematic, if not totally impossible [112]. The problem becomes more


serious if the researchers and the samples share the same phylogeographic context (i.e., belong to the same population or even the same continent) because in this case researchers and subjects will present similar or identical genetic markers. The ubiquity of human DNA contamination leads some researchers to conclude that many published studies that report ancient human DNA sequences are unreliable [41]. However, we must consider that museum specimens, some paleontological samples, and many Neanderthal bones and teeth have been subjected to intensive analyses and scrutiny by numerous researchers. On the other hand, many human series, except those used for reference and, therefore, openly available for study, are maintained at University laboratories, where they are usually handled by a few specialists. III.2. Criticism III.2.1. Direct criticism Based on the dramatic results showing the ubiquity of human DNA contamination, several researchers have criticized some published works analyzing ancient human samples. In a comment on the work of Béraud-Colomb et al. [90], Stoneking stated that the ability to reproducibly amplify a different DNA sequence from each of several bones is an indication that the sequences are not due to contamination by a single contemporary DNA source [82]. In response, Cooper [91] argued that when a group of bones originates from differing archaeological sites or museums and is consequently handled (and contaminated) by different individuals, as in the work of Béraud-Colomb et al. [90], different reproducible sequences could be retrieved from the different individuals analyzed. Therefore, in Cooper’s opinion [91], there appears to be little reason to believe that Béraud-Colomb’s results are authentic. Nevertheless, Cooper’s reasoning is not applicable to human series from a single site that have been handled by a reduced group of researchers nor, of course, to series recently excavated in which a single specialist, whose DNA has been extracted to type the relevant markers, conducted the sampling for aDNA using the proper material and equipment. In a review published in 2001, Hofreiter et al. [41] stated that the first short nuclear DNA that is likely to be genuine was determined “only recently”, citing the work of Greenwood et al. [132], implicitly regarding all previous reports on nuclear DNA as non-authentic, and even mentioning the work of Lawlor et al. [72]. In the same review, Hofreiter et al. [41] published the interesting results (in their Box 2) of an experiment designed to show the problem of human contamination and to support their criticism of some published works. They started by describing how two independent extracts were obtained from two well preserved ~30,000-year-old teeth, which were previously subjected to analysis by aspartic acid racemization (founding values of 0.10-0.11, lower than that observed for the Neanderthal-type specimen of


0.12). A segment of the mtDNA control region was amplified twice from one of the teeth and direct sequencing revealed a reproducible human 93 bp sequence. When the experiment was repeated, the same sequence was obtained. The authors remarked that, at this point, most studies of human remains would be published making the claim that the DNA sequences stemmed from the human teeth and gave as an example the works of Adcock et al. [75]; Oota et al. [98]; Wang et al. [99]; Izagirre and de la Rúa [100]; Schultes et al. [116]; and Monsalve et al. [133]. However, when the two control region amplification products were cloned and 20 clones from each were sequenced, a total of 20 different human sequences were found among the clones, indicating exogenous contamination had occurred. Finally, to make their point, Hofreiter et al. [41] revealed that the teeth analyzed actually came from cave bears that were found alongside remains of Late Pleistocene humans in Upper Cave, Zhokouchien, China. So, all human sequences obtained, either from cloning or by direct sequencing, came from contamination events. In conclusion, the authors stated that failure to adhere to all the criteria they presented (in their Box 1) would undoubtedly result in the publication of false results. Although this is a remarkably useful experiment to illustrate the human contamination problem, some of its features may be potentially misleading, especially when assessing the authenticity of population-level human studies. Indeed, some relevant questions remain open. To start with, it is important to note that Hofreiter et al. [41] found 20 different sequences in 40 clones. This means that some of these 20 sequences may actually correspond to singletons produced during the cloning process (i.e., artifacts). Another important point to note is that the cloning results were from a single tooth, so the real extent of human contamination in Upper Cave, or in their laboratory, was not assessed. Finally, Hofreiter et al. [41] did not discuss possible sources of the contamination detected, and this is extremely relevant since it leaves open the possibility that some of the human sequences they found are actually ancient. Some authors have proposed that contamination may have been produced before the remains were buried (in this case, if the ancient humans had eaten bear or vice versa) or, more probably, after they were deposited and before they were retrieved, especially when the remains of several individuals (and/or species) lay together due to infiltrations during decomposition or during diagenetic processes [134, 135]. These questions may be addressed by analyzing several teeth from both humans and bears from the same cave and characterizing, if possible, the mtDNA of archaeologists and other participants in the study, as has been done in several human population studies [e.g., 61, 109, 110, 136]. Furthermore, if DNA from the archeologists is not available, the analysis of more cave teeth and their comparison with sequences from human teeth may highlight the contaminant sequences and help in the authentication of other different sequences found in human series. In the


meantime, a direct comparison of single-tooth results with population-level studies, like some of those cited by Hofreiter et al. [41], is undoubtedly biased. In a more recent review, Pääbo et al. [43] cited the works of Pääbo [2]; Adcock et al. [75]; Caramelli et al. [78]; Oota et al. [98]; Hänni et al. [137]; Oota et al. [138]; Vernesi et al. [139]; and Yao et al. [140] as examples in which it is impossible to establish authenticity because a DNA sequence identical or similar to contemporary humans was determined, although further discussion was not provided. Along different lines, Bandelt [81, 141] has analyzed and criticized specific published “ancient” mtDNA sequences. Based on a worldwide mtDNA phylogenetic tree, as well as on a precise (mathematical) knowledge of its variation, Bandelt and co-workers [see 142] have engaged in a crusade to show that, very often, bad quality mtDNA sequence data are being used to obtain flawed conclusions, with relevant implications for the study of the association of mtDNA with complex diseases [e.g., 143], for forensic genetics [e.g., 144], and for ancient DNA studies [81, 141]. In 2004, Bandelt [141] criticized the work of Vernesi et al. [106] highlighting some mutations that were out of phylogenetic and mutation rate context (although see the reply by Barbujani et al. [145] for a different assessment). In 2005, in a paper whose purpose was to propose an authentication strategy to be applied a posteriori, Bandelt [81] criticized the data of García-Bour et al. [146] because they contained some haplotypes with clear phylogeographic inconsistencies. He also took issue with the data of Caramelli et al. [78], and Vernesi et al. [106, 139] because some of the sequences presented seemed to arise from recombination among cross-contaminants, and further criticized the data of Vernesi et al. [106] for presenting an abnormal mutational spectrum. It is worthy of note that Bandelt’s criticism was used to exemplify how a detailed analysis of data in the light of an edited worldwide database may be useful in authenticating ancient human sequences; it was not intended as a generalization to establish a position against the analysis of aDNA in humans. III.2.2. General conclusions about human studies For Pääbo et al. [43], it is impossible to establish authenticity in cases where a DNA sequence identical or similar to contemporary humans is determined, even with a rigorous application of the established criteria. According to them, the only possible exceptions are unusual instances in which relatively rare variants are expected that are not present in the researchers. These might include Native American remains, isolated populations such as the Andaman Islanders, or extremely well-preserved remains retaining large amounts of DNA. However, they remark that the study of mtDNA variants in individuals from a 1,000-year-old graveyard would provide little information of value. This is because they consider that very few, if any, mutations could


be expected to have appeared in that time span. However, this opinion does not take into consideration works showing that with a proper experimental design, three generations are enough to detect new mtDNA mutations [see 147] and, more relevantly, that the study of past human populations could provide interesting clues in areas other than molecular evolution, as discussed in Section I.2 of this chapter. Willerslev and Cooper [42] discuss the problem of ancient human studies, highlighting the ubiquity of human contamination in bone and teeth and stating that such contamination is impossible to clean despite extensive treatment with UV irradiation and bleach. Their arguments are based on unpublished results by M.T.P. Gilbert, although published systematic studies have shown the contrary [e.g., 148]. For this reason, Willerslev and Cooper [42] recall the urgent need for disposable gloves and face-masks during excavation and handling of archaeological specimens. Also, Willerslev and Cooper [42] criticize the use of control region sequences to determine mtDNA haplogroups since the PCR may introduce misleading errors, and they indicate that a far more reliable approach is to characterize multiple variable positions around the mitochondrial genome in order to define a haplotype, citing the work of Maca-Meyer et al. [102] on ancient samples from the Canary Islands. In fact, this approach has been used as an explicit authenticity criterion in previous studies [96, 101, 131]. Furthermore, we developed a formal framework to assess the probability of regarding a haplogroup as authentic due to independently produced PCR errors that occurred in “diagnostic” sites [65], which will be discussed below. As has been seen, these researchers consider that aDNA should only be studied in a few ancient human populations that present very particular characteristics. Furthermore, Gilbert et al. [112] warn that ancient population studies may be flawed in their interpretations due to a theoretical vacuum, similar to the “osteological paradox” [149], and that researchers may be seduced to fill with histories the interpretation of complex series of historical processes from the “meager variation seen in small-sized burial samples.” However, this should not preclude ancient populations studies since Science is progressive, and only with such data can an adequate theoretical framework be developed. On the other hand, recent studies showing the feasibility of decontaminating ancient human samples [148, 150, 151], are initiating a new wave of optimism in which the retrieval of authentic ancient human DNA is seen as possible. IV. Criteria of authenticity IV.1. Early criteria As soon as the contamination problem was realized, a need was recognized for establishing authenticity. Pääbo et al. [67] proposed that a


failure in PCR amplification of large fragments from ancient DNA extracts could be an additional criterion of authenticity. This early criterion was later developed into the “appropriate molecular behavior” criterion (see below). The necessity of a stringent protocol for authentication is particularly felt whenever the possibility of using the phylogenetic criterion is unavailable. The first published criteria of authenticity [5] were limited to three points: (i) testing of control extracts to detect contamination introduced during the extraction procedure, (ii) more than one extract should be prepared from each sample and both should yield identical results, and (iii) there should be an inverse correlation between amplification efficiency and the size of the amplification product. In addition, Pääbo et al. [152] pointed out that the sequences could be ancient (namely, not derived from modern contaminations) but not authentic, both because of post mortem changes that resulted in random substitutions regardless of the nucleotide position within the codon, and the jumping polymerase effect, which originates mosaic sequences [153]. The major source of contamination of modern DNA was soon recognized in the amplicons (target DNA fragments produced by previous PCR reactions), which can spread through the aerosol produced by pipetting and other manipulations of the lab ware. Their ubiquity in the laboratory environment is a nightmare for any researcher involved in the ancient DNA field, and they are ultimately responsible for a very insidious cause of contamination, the “carrier effect” (contaminating molecules adhering to the surface of laboratory plasticware and released by the addition of the ancient extract) [128, 154]. In fact, the spread of amplicons affects all works using PCR, not only in the aDNA field, so standards for the proper conduct of PCR experiments which included separation of working areas were published very early [155]. In order to circumvent the carryover of amplicons, Glenn and Braun [156] proposed the use of uracyle in the nucleotide mix of every PCR reaction and the addition of Uracil-N-DNA-glycosylase in subsequent reactions in order to selectively eliminate the contaminating U-amplicons. However, this protocol has been only sporadically applied in ancient DNA studies, possibly because the amplified fragments containing uracyle are not suitable for downstream analyses, including RFLP. IV.2. The first integrated criteria The initial period of enthusiasm about ancient DNA studies led to flawed results on specimens of paleontological interest millions of years old [28-30, 157], which raised serious criticism about the feasibility and repeatability of the analysis. Lindahl [7] argued that the chemical instability of DNA, subject to hydrolysis, oxidation and nonenzymatic methylation, will introduce diagenetic changes over time that will eventually limit the possibility of recovering genetic information from organic material to a short period of time.


It was in response to Lindahl’s criticism that authenticity criteria became the subject of formal proposals. Handt et al. [128] published the first set of integrated criteria which needed to be fulfilled before a sequence could be claimed as ancient. It comprised the following six points: (i) the strict physical separation of the laboratory areas where the ancient samples are processed; (ii) specially dedicated laboratory clothing in order to avoid contamination carried by the researchers and accurate cleaning of the work areas with 5% sodium hypoclorite and UV irradiation; (iii) routine monitoring of contamination – two extraction controls and one PCR control; (iv) at least two extractions per sample performed at different moments and preferably from different parts of the sample, with the obligation to report incongruent results, if any, in the publication; (v) consistency with the phylogenetic criterion; and (vi) an inverse relationship between amplification efficiency and the molecular length of the amplified fragment. In a more recent paper, Béraud-Colomb et al. [90] strengthened the abovementioned points i, ii, and iii, and added more precautions, including the sterilization of all buffers by both autoclave and filtration, the use of dedicated pipettes sterilized by UV irradiation, and the use of aerosol-resistant pipette tips. This paper generated a discussion about the need to perform independent replication [82, 91], which was later established as another criterion of authenticity, although some discussion is still going on about when independent replication should be requested [42, 43].

IV.3. Second generation criteria (Do it right or not at all) Concerns about the rigor of research on ancient DNA and the fact that high-profile journals continued to publish studies that did not meet the necessary requirements, prompted Cooper and Poinar [49] to publish the most comprehensive and stringent list of criteria of authenticity, further proposing that aDNA studies must comply with them in order to be publishable or funded. The nine key criteria proposed, which will be discussed below, were: A physically isolated work area, Control amplifications, Appropriate molecular behavior, Reproducibility (intra-laboratory), Cloning, Independent replication (inter-laboratory), Biochemical preservation (especially assessment of amino acid changes), Quantification, and Associated remains (e.g., the study of faunal remains as negative controls for human PCR amplifications). Although recognizing that adherence to these criteria as part of routine good practice is both expensive and time-consuming, Cooper and Poinar stated that “failure to do so can only lead to an increasing number of dubious claims, which will bring the entire field into further disrepute,” in allusion to the amber and dinosaur fiascos [49]. The fulfillment of all these “standard criteria” was encouraged by Willerslev and Cooper [42], who further proposed additional criteria, like a time-dependent pattern of damage and diversity (which provides strong


support for antiquity in microbial studies); decontamination of reagents and specimens (particularly important in ancient human and microbial studies); and uracil-N-glycosylase (UNG) treatment to eliminate some post-mortem damage (here the treatment is applied to samples, not to U-amplicons as referred to above). In the view of Willerslev and Cooper [42], “appropriate laboratory facilities and controls, independent replication and cloning of amplification products are essential and should not be compromised.”

IV.4 Third generation criteria (The tenth commandment) A third generation of criteria were recently published which relaxed to a certain extent the stringency of the criteria according to the nature and characteristics of the samples and gave more weight to scientific judgment of the reliability of results. Furthermore, it was recognized that all sources of errors do not occur in all studies so it was not strictly necessary to adhere to each and every criterion in every case [43; 112]. As an example of flexibility, Pääbo et al. [43] consider that biochemical analyses of preservation may be superfluous when specimens are obviously well preserved. The advance in Neanderthal studies represents another example: the first two Neanderthal DNA sequences were replicated in a second laboratory, but as subsequent Neanderthal DNA sequences have been found to be similar to the first ones determined, repetition in another laboratory is, in the opinion of Pääbo et al. [43], “extravagant.” This view is clearly opposed to that of Willerslev and Cooper [42], for whom “the routine independent replication of results could maintain the highest standards of experimental practice in participating laboratories, and this psychological factor should not be undervalued in such contamination-prone research.” In fact, the only sign of relaxation in the arguments of Willerslev and Cooper [42] is their recommendation to replicate and clone just a subset of the samples analyzed. In the opinion of Gilbert et al. [112], the criteria were intended to assist in determining the authenticity of a study, but they cannot replace a crucial consideration of the problem. Thus, the criteria should not be used as a finite checklist that would guarantee authenticity, nor should some of them be used and others avoided without justification. Therefore, Gilbert et al. [112] advocate that ancient DNA researchers should take a more cognitive approach to assessing the reliability and conclusions of their data. Instead of planning or assessing studies by using criteria as checklists, consideration should be given on a case-by-case basis as to whether the evidence presented is strong enough to satisfy authenticity criteria, given the specific problems of each case. Consequently, Gilbert et al. [112] place the responsibility on authors to self-assess their work in light of the problems inherent to the field, and they end up by adding a “tenth commandment” to the original nine points: “Thou shalt interpret the veracity of the data by a critical consideration of all available information.”


V. The standard criteria and human population studies V.1. A critical review of the criteria Here we review the standard criteria and comment about their application to human population-level studies. Physically isolated work areas. If the study is conducted in a laboratory in which the different work areas have been separated for the different steps of the analysis, the results will have a higher probability of being authentic. DNA extraction must be separated from PCR setup and post-PCR procedures. Logistic separation, in which each area has all the material and equipment, is essential to avoid transferring contaminating molecules between areas. Temporal separation, in which the different procedures are conducted at different times, may increase the efficiency of physical separation [101]. Also, restricting the direction in which persons move between areas (always from the low to the high DNA concentration rooms) may be as efficient as positive pressure chambers [42]. It should be noted, however, that while these procedures are intended to decrease the contamination risk in laboratory analyses, they are not effective when contamination has happened before the arrival of the samples at the laboratory. Negative controls. Both for DNA extraction and amplification blank controls must be performed in which negative results are expected unless the reagents or material are contaminated, or contamination is introduced during the procedure. The lower the sample-to-control ratio, the more reliable the procedure will be. However, it should be borne in mind that clean controls do not imply an absence of contamination, due to phenomena like the carrier effect [154], and that neither do contaminated controls imply contaminated samples [e.g., 158]. The latter could be the result of a single-tube contamination phenomenon [65] so that different samples in the same batch, including blank controls, may become contaminated independently. Therefore, negative controls are only useful in an integrated authentication framework [65, 101, 159]. Positive controls should be avoided as they represent an increased risk of cross-contamination. Two procedures may be used to optimize conditions for PCR when new primers are going to be tested. One involves collaboration between two laboratories in such a way that all the optimization procedures are carried out in one laboratory while the other is devoted exclusively to aDNA work [131], and the other consists in optimizing conditions directly on aDNA samples [160]. Appropriate molecular behavior. As aDNA is highly fragmented, more PCR product for smaller fragments is expected to be obtained, as well as more mtDNA than nuclear DNA, since there are many more copies of mitochondrial than nuclear DNA, increasing the probability of preservation for the former. However, results from some researchers have shown that the interaction of DNA molecules is far more complex. In some instances it was easier to


amplify aDNA that was larger than contaminant DNA fragments [131], weakening the universality of this criterion. Furthermore, some primers may preferentially amplify nuclear mitochondrial insertions (numts) instead of actual mtDNA [11]. Reproducibility. At least two extracts from each specimen must be analyzed and the results must be concordant. In case of disagreement, a third or a fourth sample from the same individual must be characterized. Discordant results may show that contamination has occurred, although concordant results do not guarantee authenticity, because if the sample was extensively contaminated, several extracts may contain the same exogenous sequence [41]. This is especially true for samples that do not contain endogenous DNA or where it is highly damaged. On the other hand, in many cases there is not enough material (teeth or bones) to replicate every individual analyzed. In population-level studies, problematic samples could be discarded. Cloning. PCR products must be cloned and multiple clones must be sequenced to assess the integrity of the amplified DNA. This procedure may reveal different sequences amplified at different ratios. Ideally, overlapping fragments must be amplified and cloned. For some authors, this is an absolute requirement for proof of authenticity; although it may render population-level studies impractical. According to Bower et al. [161], at least 20 clones must be sequenced in order to be >95% confident of identifying the most abundant sequence present at 70% in an ancient sample. So, for example, if we must analyze two extracts per individual and amplify (at least) two overlapping fragments, we should sequence at least 80 clones per individual. A 25-individual study implies sequencing at least 2,000 clones for the cloning experiments to be meaningful. What is more, systematic cloning has the drawback that the number of DNA copies of the fragment under study is increased several times, which also increases the crossover contamination risk. For example, in the study of Vernesi et al. [106], each fragment was cloned and sequenced at least eight times, so whatever the initial cross-contamination risk (without cloning) was, it was increased by a factor of eight. Moreover, additional procedures must be devised to ensure the destruction of all specific DNA inserts harbored in bacteria (note that this will not be accomplished by just autoclaving residual cultures and petri dishes). It is worth noting that when applying the same methodology used to analyze the data of Vernesi et al. [106], Bandelt [81] was unable to detect signs of cross-contamination in the data of Keyser-Tracqui et al. [109], which were produced without cloning any of the amplified fragments. On the other hand, if the initial template is abundant (>1,000 copies), PCR products will be more homogeneous and the cloning procedure will not add new information [43]. For example, when analyzing an exceptionally well-preserved Neanderthal sample, Ovchinnikov et al. [13] obtained the same sequence by either sequencing amplicons directly


or by cloning and then sequencing them. Therefore, the cloning criteria may, and should be, relaxed if quantification indicates good preservation. Quantification. When there are few DNA molecules available for PCR, different amplifications from one single extract may be discordant because the first PCR cycles are critical, and different minority sequences (damaged or contaminating) may be differentially amplified in different PCR experiments. However, when the number of starting molecules is good (>1,000 [43]), independent amplifications will likely give the same result. Since quantification implies additional sample handling, thus increasing the contamination risk, in some cases it would be better to analyze results from independent extracts from the same individual, looking for discrepancies before proceeding to quantify. This alternative may save many hours of work and at the same time avoid increased contamination risks in population studies, where many samples must be analyzed and where the result of the whole series may be more indicative of sample preservation and authenticity of results than an exhaustive sample-by-sample analysis. Furthermore, quantification does not reveal whether some or all of the starting molecules are contaminants [42, 131]. Independent replication. This is an extension of replication in one lab, but must be performed by a second (and a third) laboratory. Ideally, samples must be sent directly from the museum or collector to avoid transfer contamination [41]. However, according to Stoneking [82], to require such independent analysis would cause more problems than it would solve, because of the destructive nature of the methodology and some logistic problems, like getting enough funding to do the same analyses in two independent laboratories. In fact, in many population studies, only a subset of samples is replicated (see Table 1). Biochemical preservation. The samples should present a good general level of preservation, and testing the racemization of amino acids is advised [40]. However, the relationship between amino acid racemization and DNA preservation is not well defined and may vary among different tissues and conditions [42]. Furthermore, as in the case of quantification, good biochemical preservation does not exclude contamination [131]. On the other hand, it is important to consider that an increase in the handling of samples implies an increase in the risk of contamination. In general, if the sample is well preserved it will contain a good number of endogenous molecules and, consequently, it will produce a homogeneous result. So, in some cases, additional handling of samples is not justified unless the result might be of great relevance. Also, in population-level studies, the percentage of samples that contain amplifiable DNA may be a better indicator of the general biochemical preservation at the site. Associated remains. It is advised to analyze fauna (or other associated remains) to assess human DNA contamination and the preservation conditions


of the site under study. However, results for these criteria must be taken with caution: even a well-preserved specimen may be contaminated. Moreover, preservation conditions may vary among samples from the same burial. Therefore, unsuccessful attempts to recover DNA from associated remains do not preclude preservation in other samples from the same site. On the other hand, detecting human contamination in associated remains does not mean that we cannot analyze the human series. On the contrary, this information may be integrated in the authentication process, because it may help us to characterize the contaminants introduced during excavation and handling. V.2. What is lacking Besides the problems in applying the standard criteria to ancient human population studies discussed above, there are some open questions that need to be addressed.

1. Many studies report replication of a subset of samples. However, it is not clear what the real utility of replicating one or two individuals in a population-level study is, and it seems that this is done just to satisfy in some way the standard criteria. If this is the case, then it is more dangerous to replicate one or two individuals than none at all, because sheltered behind this weak form of meeting a standard criterion, researchers may give more confidence to data that are not actually fully authenticated. According to Willerslev and Cooper [42], all ongoing work should feature the independent replication of a subset of perhaps 10% of samples, or of all key results. In our opinion, the number of samples submitted for independent replication, when conducted, could be based on the preservation conditions of the samples, as determined by the amplification efficiency observed for the specific population under analysis. Replicating one sample of 17 would be useless if a total of 47 were analyzed, because the efficiency would be extremely low (36.2%), and this might increase the risk of amplifying contaminant sequences. On the other hand, when the efficiency is good (>80%) a reduced subset will confirm that in general the work has been conducted properly, although authenticity must rely on some other criteria. Therefore, instead of requiring independent replication for the publishing of aDNA results, the reviewers could ask for some discussion about the number of samples replicated (or the absence of them) and their significance in the authentication process.

2. The same question remains for the number of samples to be cloned. Instead of systematically cloning all the amplified fragments of all the samples, which may also increase the risk of contamination, we believe that cloning must be conducted only on some selected samples. Those that have shown inconsistencies in direct sequencing might be selected,


although if the sample size were big and the efficiency good, it would be better to eliminate these samples from the analysis. The resources employed to clone a few problematic samples would be better used to extract and sequence additional samples. Alternatively, those samples with relevant results (for example a “new” interesting haplotype) would also be liable to be cloned or even submitted for independent replication. In any case, cloning results cannot replace other more relevant authenticity criteria, like the a posteriori analysis proposed by Bandelt [81].

3. Many researchers acknowledge that there is a high probability of contamination due to the transference (carryover) of amplicons from previous amplifications, and, consequently, laboratory areas are designed to avoid this. However, there is no real way of estimating this probability, which may differ among different laboratories according to the effectiveness of the physical and logistic separation of pre and post-PCR processes. One way to control and quantify the probability is to use an internal (modified) control to trace/determine contamination rates. This control may be amplified alongside aDNA samples, and then future amplifications of aDNA samples may be routinely tested for the presence of the modified control. The information on the frequency of carryover contamination provided by this control may be very useful when statistical approaches to authenticity are devised (see Section VI.2). The problem is that many researchers would be reluctant to accept that they detected the modified control in their pre-PCR laboratories. In presenting this idea at a recent international congress, one of us was confronted by an individual who said that if we detect such transferring of amplicons between areas in our laboratory, we should close it and never conduct aDNA studies again in that laboratory, because this kind of contamination should never happen. In our opinion, this attitude is clearly mistaken and could be extremely dangerous to the field, because the idea that contamination should never occur does not mean that it will never occur in fact. That is why we perform negative controls in the first place! Fortunately, in addition to ourselves [101], there are other researchers capable of acknowledging when contamination has happened and even reporting on the instances in their papers [e.g., 102, 105, 159]. In any case, closing the laboratory if contamination is detected seems totally uncalled for. For example, the DNA Commission of the International Society for Forensic Genetics recommends that “if either the extraction reagent blank or the PCR negative control yields a sequence that is the same as that of the evidence sample, the results from the evidence sample must be rejected and the analysis repeated” [162], but it does not suggest that the laboratory should be closed. Furthermore, the same Commission also states that “because of the sensitivity of detection of mtDNA analysis, low levels of


exogenous DNA contamination can be observed; yet low levels of contamination can be tolerated because reliable results can be obtained in the presence of contamination” [162]. This means that the forensic community has learned to live with contamination and to deal with it, even though forensic cases may involve life or death situations.

4. Another relevant issue is the characterization of the tempo and mode of human DNA contamination that occurred before the molecular analyses. Tracing back contaminant sequences by typing all personnel involved in the analysis of a given series may show at which step and in which amount contaminant templates are being introduced. To date, only one systematic study addressing this question has been performed and it showed that 17.13% of the cloned sequences could be unambiguously identified as contaminants, with those derived from the people involved in the retrieval and washing of the remains present in higher frequencies than those of the anthropologists and genetic researchers [136]. Studies like this are fundamental since they may reveal the phases of the analysis where contamination has more opportunities to occur and, consequently, where to apply supplementary measures to both avoid it and detect it. Indeed, the study by Sampietro et al. [136] is encouraging because it shows that if we were able to avoid contamination during handling (in series to be excavated) or to detect it (by typing the personnel involved), the authentication of ancient human DNA sequences would not be impossible. Bouwman et al. [163] analyzed the spatial distribution of contaminant DNA in ancient bones, proposing that it presents different distribution in relation to authentic aDNA, and this could also be used to authenticate ancient sequences.

5. As to the authentication process, it seems that training of field archaeologists for proper sample handling for DNA analysis is still lacking, especially for those that are not collaborating with any aDNA laboratory. However, this training could only be accomplished if we generalize the notion that the study of ancient human DNA is possible or, at least, that it will be possible in the future. By saying now that ancient human DNA is impossible to authenticate we are denying this possibility to the generations to come, those who will analyze the human series that are being retrieved at present.

6. The improvement of protocols, either for decontamination [148, 150, 151] or the search for new polymerases that are insensitive to molecular damage (e.g., Y-family polymerases [164]) is also relevant to the authentication process. Kemp and Smith [148] introduced contamination to ancient bones and determined which of several sodium hypochlorite treatments was best in eliminating surface contamination. The elimination of surface contamination from bone required immersion in at least 3.0% (w/v)


sodium hypochlorite for at least 15 minutes. Furthermore, according to Kemp and Smith [148], endogenous DNA proved to be quite stable even with extreme sodium hypochlorite treatments (6% for 21 h), probably because of its adsorption to hydroxyapatite in the bone. Salamon et al. [150] further demonstrated that relatively well preserved DNA is occluded within clusters of intergrown bone crystals that are resistant to disaggregation by NaOCl. They obtained reproducible authentic sequences from both modern and ancient animal bones, including human ones, from DNA extracts from crystal aggregates. The treatment with NaOCl also minimized modern DNA contamination [150]. More recently, Malström et al. [151] studied the efficiency of bleach incubation of bone powder and its relative detrimental effects on contaminant and authentic ancient DNA, noting that bleach treatment is significantly more detrimental to contaminant than to authentic ancient DNA in the bleached bone powder. So, if aDNA is being protected from these chemical treatments by its association with hydroxiapatite crystal then it would be interesting to investigate whether this association also provides protection to DNA from other treatments, like incubation with DNases. In fact, it is known that the binding of DNA to mineral surfaces may protect it against DNase I degradation [165-167]. Decontamination with DNase I could be far more effective than chemical or UV light decontamination, since damage to a template produced by the latter may render the template more prone to jumping-PCR [153]. On the other hand, if conditions are optimized, DNase I may completely reduce any DNA segment to free nucleotides if it is not protected in some way (in this case, by its association to hydroxyapatite).

VI. An integrative approach for human population studies VI.1. Flexibility and the intelligent use of authentication criteria It now seems clear that the “standard criteria” are not appropriate for human population studies, which require additional specific criteria which, ideally, will not represent an exponential increase in costs. Above all, it should be realized that the authentication process will be strengthened by increasing the sample size [101, 159] and, therefore, efficient authentication methods allowing the analysis of more samples should be preferred over those requiring more resources while limiting the number of samples to be analyzed. Furthermore, meta-analysis of published studies may provide the basis for the improvement of the authentication process. For example, in general, the efficiency in DNA recovery varies from 60 to 88% in most of the studies referred to in Section II.2.2 (Table 2). So, studies in which the efficiency is much


Table 2. Efficiency in the analysis of ancient human populations.

Individuals Reference Analyzed Successful Efficiency (%) Stone et al. [93] 20 19 95.0 Stone and Stoneking [96, 97] 152 108 71.1 Montiel et al. [101] 44 28 63.6 Keyser-Tracqui et al. [109] 99 62 62.6 Lalueza-Fox et al. [105] 47 17 36.2 Maca-Meyer et al. [102] 129 71 55.0 Vernessi et al. [106] a 80 48 60.0 Lalueza-Fox et al. [107] 36 29 80.6 Sampietro et al. [44] 17 17 100 Haak et al. [104] 57 24 42.1 Alzualde et al. [45] 65 56 86.2 Casas et al. [61] 71 61 85.9 Changchun et al. [110] 18 16 88.9 Mooder et al. [46] 82 70 85.4 Töpf et al. [47] 156 48 30.8 Shinoda et al. [111] 57 35 61.4 Burger et al. [48] 51 9 17.6

a samples discarded by aa analysis (n=3) and quantification (n=29) were not used for amplification.

lower than 60% should be submitted to more rigorous authentication criteria, because a lower efficiency indicates poor preservation conditions, which in turn will produce more artifacts and a higher risk of amplifying exogenous sequences. Correlations between amplification efficiency and preservation conditions like soil characteristics and age and temperature of the site could also be very useful in assessing authenticity in future studies. Another question is why at the same site some samples have aDNA while others do not. Correlations between amplification efficiency and soil microdifferentiation at the site would also be useful. The history of the process undergone by the series after recovery from the site should also be integrated in the authentication arguments [112]. Recently excavated series in which precautions were taken and DNA from the researchers was typed will be more likely to be authentic. Other series will require intensive decontamination treatments, and further information will need to be provided to support authenticity. In this framework, “it may be


possible to work with ancient human DNA, but the burden of proof lies heavily upon each researcher claiming to have such results” [130]. Gilbert et al. [112], suggested that authors should explicitly classify (with justification) their target aDNA studies into one of four risk categories which reflect how easy it is to generate erroneous data through contamination, as follows: Highest risk, High risk, Medium risk, and Low risk. In this classification, human studies would be in the “Highest risk” category [112]. However, within this broad category, different human population studies could be sub-classified, because well-preserved, well-excavated, and well-handled series would have a very different risk of being contaminated from the risk associated with, say, a museum collection, from which no control was taken at all. VI.2. The statistical approach By using statistical approaches, new strategies for authentication may emerge, and a new aDNA culture will be needed. This was envisioned in 1997 by Béraud-Colomb et al. [92], who stated that “the analysis of all these data by careful statistical and phylogenetical methods will eventually reveal inconsistencies and help in a progressive classification of sequences in ‘dubious’ or ‘probably real’ categories” [92]. Note that in this case the classification is based on statistical approaches and not on researchers’ considerations, as in the classification proposed by Gilbert et al. [112]. No human aDNA study could be considered as 100% authenticated, and we should start to use new statements for our results that, in this framework, would have a high, medium or low probability of being authentic. In 2001, we proposed that the relationship between efficiency and levels of contamination might be indicative of the probability of authenticity [101]. More recently, Spencer and Howe [159] developed a maximum-likelihood framework to estimate the probability of authenticity based on data of amplification efficiency and contamination detected in the controls. They developed their models taking into consideration two kinds of controls (extraction and amplification blanks), but these models could be extended to include other appropriate controls [159]. Nevertheless, for these approaches to be useful it is essential to record the number of controls used and the number of contamination events. In fact, a contaminated control should not be discarded, because it might provide useful information to trace back to contamination sources and also aid in the authentication process. Therefore, besides the Lindahl’s recommendation to publish negative results [7], in the early days of aDNA studies many researchers discussed in electronic bulletins the need to also publish contamination results, although the latter suggestion was more difficult to implement in the aDNA community. The number of controls used should also be reported, because contamination may occur even if it is not detected in the controls, and this probability might also be modeled


based on the number of controls used and the number of samples analyzed [159]. It is worth noting that, according to Spencer and Howe [159], with only a single sample we cannot confidently reject the possibility that a positive result is only contamination; on the contrary, our confidence in the data will increase by analyzing a large sample size and as the amplification efficiency increases. To improve the statistical approach, the probabilities of having undetected contamination and of introducing specific sequences are needed. The former may be estimated from the number of contamination events detected a posteriori. For example, Bandelt [81] detected some erroneous sequences in the studies he analyzed, but we should realize that other sequences in these same studies may also come from contamination and that we cannot distinguish them from the authentic ones since they do not have “strange” features. However, we can use the number of identified contamination events to estimate the probability of having other non-identified events. As far as the probability of introducing specific sequences is concerned, it may be estimated if we know the probable contaminants that could derive from people handling and analyzing the samples. Furthermore, with many studies like that of Sampietro et al. [136] we could obtain prior information on this probability [159] that would improve the models for general application. These priors may be fine-tuned considering the phylogenetic context of both the sample and the researchers. In this sense, ancient human sequences from an American sample excavated and analyzed by European researchers will have more probability of authenticity than sequences from European series analyzed by the same team. In any case, if decontamination methods prove to be reliable, the probability of authenticity of all human studies could increase substantially. Phylogenetic methods of authentication were used early on by Stone and Stoneking [96] for mtDNA data. However, taking into account the extent to which our knowledge of the world phylogeny of mtDNA has increased, more explicit and reliable approaches could be developed. Bandelt [81] showed how to use a worldwide database to look for phylogeographic inconsistencies, abnormal mutational spectra, and mosaic (chimeric) sequences. The latter might arise from in vitro recombination of different DNA molecules, mainly through jumping-PCR [153]. A mixture of different molecules may also be detected at the level of the whole mitochondrial genome by analyzing linked markers, which may be accomplished by taking advantage of the relationship between the markers that define the haplogroups [101]. We estimated that the probability of the Taq DNA polymerase producing two independent errors in two “diagnostic” sites of the haplogroup T (16294 and 15607) might be as low as 8.36 × 10-11 [65]. In this calculation, the error rate of the Taq reported by Eckert and Kunkel [168] was used, but it might actually be higher due to molecular damage. However, determination of haplogroups requires the screening of more than two sites in the mitochondrial genome and all of them


should ideally be screened in ancient populations. For example, the consistency of haplogroup assignment may be tested by analyzing at least nine positions in the case of European populations. Due to polymerase errors, to obtain the correct nucleotide in all nine positions it is necessary to have a confluence of nine independent misincorporations occurring in those specific sites. Screening many positions in a high number of individuals may seem tedious and expensive, but Endicott et al. [169] showed that multiplex SNP typing of mtDNA is a low-cost, high-throughput method for haplogroup determination in ancient samples. Furthermore, information from an edited worldwide database [e.g., 81] and the specific mutation rate at different positions observed in ancient mtDNA [e.g., 126] could be used in our model [65] to estimate more accurate error probabilities for the determination of each of the known mtDNA haplogroups. VII. Perspectives VII.1. Genomics and metagenomics in human population studies? The application of new techniques to the aDNA studies has showed us that we can obtain useful information from nuclear DNA after all, and although these techniques have their own contamination problems, we wonder if some day they will be used for the study of ancient human populations, especially considering that some of them are especially suited for aDNA analyses [26, 170]. In the study by Green et al. [26], a Neanderthal bone containing more Neanderthal DNA than contaminating Homo sapiens DNA was searched. This relationship was assessed estimating the proportion of mtDNA of each of both kinds. A similar search may be conducted in American populations (or other isolated populations), specifically determining the amount of Amerindian DNA versus DNA from Europe, assuming European teams conducted the excavation and analysis. However, the full potential of these techniques may only be achieved as decontamination methods are improved. One of the problems of a genomic approach involves the cost of the analysis, but it appears that this will change in the near future. The initial draft of the first human genome sequence, completed in 2001, cost an estimated US$300 million, while by the end of 2006 the cost of a full mammalian genome sequence was expected to be about US$100,000, a 3,000-fold cost reduction in just 6 years [171]. Furthermore, in September 2003, the J. Craig Venter Science Foundation offered US$500,000 to the first group capable of sequencing a human genome for US$1,000, and the competition became more heated in 2004, when the National Institutes of Health launched a US$70-million grant program to support researchers working to sequence a complete mammal-sized genome, initially for US$100,000 and ultimately for US$1,000 [171]. If expectations are achieved, in a few years we might start to think about


conducting ancient population genomic studies, provided we are able to put forward scientifically relevant hypotheses to test at this level and that we have learned to deal with the contamination problem. VIII. Conclusion Decontamination procedures, statistical and phylogenetic methods, information on the history of an analyzed series since its recovery from the burial site, DNA typing of the researchers involved, and inference of contaminant sequences from associated non-human material may increase the probability of the authenticity of ancient human DNA studies. Furthermore, when a series is excavated under controlled conditions, the results may also be considered as ranking below the “Highest risk” category proposed by Gilbert et al. [112] for human studies. These criteria, specifically addressed to authenticating ancient human population studies, may prove to be more effective than some of the “standard criteria” of Cooper and Poinar [49]. Particularly, the value of cloning all amplified fragments from all the individuals analyzed should be reconsidered, since it may be expensive and generate more problems than it solves due to the increased risk of cross-contamination. In this sense, the impressive work of Vernesi et al. [106] may be of great intrinsic value, since it can be used as a reference to compare data obtained by “full-cloning” procedures with those obtained by direct sequencing, provided Vernesi and co-workers or some other group is able to analyze the same series with the latter methodology. However, the researcher’s own criteria in the application of some of the standard criteria in specific situations and for some samples may help to solve more complicated problems. On the other hand, all the standard criteria should be rigorously followed, as far as possible, whenever the sample size is small. As the reader will have become aware while reading this chapter, we belong to the group of optimists as defined by Béraud-Colomb et al. [92], or “believers” in our terminology. However, everybody in this field is, in fact, part “believer” and part “skeptic”, so we hope that researchers in the future, when writing a historical review of the discipline, may say that “believers and skeptics together developed a strong and well-funded framework in which to conduct and authenticate aDNA studies in humans that has enabled us to obtain fundamental knowledge about molecular evolution, human migration, human history, paleopathology, evolutionary medicine, and [other integrative disciplines that may have emerged in the meantime].” IX. Acknowledgments The aDNA work at the UAB is supported by the Ministerio de Educación y Ciencia of Spain (Research grant CGL2005-02567). R.M. is a postdoctoral


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