Ancient DNA Sequences Research Articles

Geologically ancient DNA: fact or artefact?

Studies continue to report ancient DNA sequences and viable microbial cells that are many millions of years old. In this paper we evaluate some of the most extravagant claims of geologically ancient DNA. We conclude that although exciting, the reports suffer from inadequate experimental setup and insufficient authentication of results. Consequently, it remains doubtful whether amplifiable DNA sequences and viable bacteria can survive over geological timescales. To enhance the credibility of future studies and assist in discarding false-positive results, we propose a rigorous set of authentication criteria for work with geologically ancient DNA.

A canine X chromosome painting probe applied to four canid species: close relationship of a heterochromatic‐like sequence between the dog and the blue fox

Microdissection of chromosomes is an invaluable tool to physically isolate single chromosomes, chromosome-arms or chromosome-bands and, subsequently generate painting probes with which numerical or structural aberrations of chromosomes can be studied. In addition, such painting probes can be used to compare karyotypic relationships among mammalian species. For the present study a canine whole X chromosome painting probe was prepared by means of conventional microdissection and degenerate-oligonucleotide-primed PCR. The application of this paint to the chromosomes of the domestic dog, red fox, blue fox and Chinese raccoon dog revealed hybridization to the entire X chromosome and the pseudo-autosomal region of the Y chromosome in all four species analysed. In the blue fox the same paint revealed additional strong hybridization signals on the heterochromatic arms after low-stringent posthybridization washes. The present study indicates the existence of an ancient canid heterochromatic-like DNA sequence, which survived in the proximal part of the X chromosome of all species studied and, in addition, was involved in the formation of heterochromatic arms in the blue fox.

Geobiology and paleobiogeography: tracking the coevolution of the Earth and its biota

Paleobiogeographic research is an important area of geobiology that involves the study of the coevolution of the Earth and its biota by considering how tectonic and climatic changes have affected the evolution and distribution of organisms. The intellectual heritage of the discipline stretches back well before Darwin. Phylogenetic approaches to paleobiogeography have played an important part in the expansion of the field, and recent analyses have incorporated Geographic Information Systems (GIS). Each of these approaches, by enhancing the precision of paleobiogeography, helps make the discipline more relevant to geobiology. The interaction occurring between the geological and biological sciences in paleobiogeography is apparent in several areas. First, there is the emergence of new techniques such as the ability to analyze ancient DNA sequences. Also, paleobiogeographers and biogeographers have realized that geo-dispersal, a biogeographic process first identified through studies of the fossil record, can powerfully influence the evolution and distribution of biotas. Finally, biogeographers have recognized that paleontological incompleteness and extinction constrain our ability to reconstruct biogeographic patterns in the fossil record and the extant biota, respectively. Each of these developments suggests that further growth in paleobiogeography will involve important interactions between studies involving the fossil record and the extant biota, and this, along with the discipline's commitment to studying how tectonic and climatic changes have influenced evolution, reaffirms the validity of the synthetic field that is geobiology.

Genetic Analyses from Ancient DNA

About 20 years ago, DNA sequences were separately described from the quagga (a type of zebra) and an ancient Egyptian individual. What made these DNA sequences exceptional was that they were derived from 140- and 2400-year-old specimens. However, ancient DNA research, defined broadly as the retrieval of DNA sequences from museum specimens, archaeological finds, fossil remains, and other unusual sources of DNA, only really became feasible with the advent of techniques for the enzymatic amplification of specific DNA sequences. Today, reports of analyses of specimens hundreds, thousands, and even millions of years old are almost commonplace. But can all these results be believed? In this paper, we critically assess the state of ancient DNA research. In particular, we discuss the precautions and criteria necessary to ascertain to the greatest extent possible that results represent authentic ancient DNA sequences. We also highlight some significant results and areas of promising future research.

Using PEBBLE for the Evolutionary Analysis of Serially Sampled Molecular Sequences

The PEBBLE (Phylogenetics, Evolutionary Biology, and Bioinformatics in a moduLar Environment) application is a relative newcomer to the field of phylogenetic applications. Although designed as a customizable generalist application, PEBBLE was initially developed to implement procedures for the analysis of sequences associated with different sampling times, e.g., rapidly evolving viral genes sampled over the course of infection, or ancient DNA sequences. The basic protocol describes the use of PEBBLE to infer a phylogenetic tree using the sUPGMA algorithm, and the inference of substitution rate parameters using maximum likelihood. The alternate and support protocols describe the simulation capabilities of PEBBLE, and general use of the PEBBLE application, respectively.

Spiking of contemporary human template DNA with ancient DNA extracts induces mutations under PCR and generates nonauthentic mitochondrial sequences.

Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34 positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood.

Status and relationships of the extinct giant Canary Island lizard Gallotia goliath (Reptilia: Lacertidae), assessed using ancient mtDNA from its mummified remains

Ancient mitochondrial DNA sequences (378 base pairs of cytochrome b and 368 of 12S rRNA) extracted from a mummified extinct giant lizard, Gallotia goliath, from eastern Tenerife, Canary Islands, were used to assess the species status and relationship of this form within the genus. G. goliath is clearly a member of the G. simonyi group of the western Canary islands (Tenerife, La Gomera, El Hierro and La Palma) and is not closely related to the giant G. stehlini of Gran Canaria. Contrary to recent opinion, it is phylogenetically distinct, within the G. simonyi group, from the extant G. simonyi of El Hierro and also from the recently discovered live G. gomerana on La Gomera and from G. intermedia in north-western Tenerife. It may be the sister taxon of either all the other members of the G. simonyi group or of G. intermedia. The phylogenetic distinctness of G. goliath makes Tenerife unique among oceanic islands in having had one giant and two medium-sized lizard species that were probably substantially herbivorous, the others being G. intermedia and G. galloti. Gallotia shows great community differences on other islands in the Canaries, two having a single small species, one a single giant, and three a giant and a medium-sized form.

Non-random DNA damage resulting from heat treatment: implications for sequence analysis of ancient DNA

To mimic damage occurring during preservation of ancient DNA, we heated wheat DNA at 95 °C for 2–21 days. Samples were amplified by PCRs directed at a 181-bp segment of the mitochondrial atpA gene. PCR products were cloned and multiple sequences obtained. We anticipated that nucleotide changes indicating damage would be randomly distributed, but this was not the case. Of the 124 cloned sequences, 107 displayed the same adenine to guanine change, this change being seen in clones from all of the eight heated samples. Fifteen of the cloned sequences, from six samples, showed the same apparent deletion at position 92. If non-random sequence changes are a widespread feature of DNA degradation then current strategies for analysis of ancient DNA sequences must be revised, as these could lead to nucleotide variations that are artefacts resulting from DNA diagenesis being mistaken for genuine sequence features of the ancient DNA.

Extreme reversed sexual size dimorphism in the extinct New Zealand moa Dinornis.

The ratite moa (Aves; Dinornithiformes) were massive graviportal browsers weighing up to 250 kg (ref. 1) that dominated the New Zealand biota until their extinction approximately 500 yr ago. Despite an extensive Quaternary fossil record, moa taxonomy remains problematic and currently 11 species are recognized. Three Dinornis species were found throughout New Zealand and differed markedly in size (1-2 m height at back) and mass (from approximately 34 to 242 kg). Surprisingly, ancient mitochondrial DNA sequences show that the three species were genetically indistinguishable within each island, but formed separate North and South Island clades. Here we show, using the first sex-linked nuclear sequences from an extinct species, that on each island the three morphological forms actually represent just one species, whose size varied markedly according to sex and habitat. The largest females in this example of extreme reversed sexual size dimorphism were about 280% the weight and 150% the height of the largest males, which is unprecedented among birds and terrestrial mammals. The combination of molecular and palaeontological data highlights the difficulties of analysing extinct groups, even those with detailed fossil records.

Evidence for a genetic discontinuity between Neandertals and 24,000-year-old anatomically modern Europeans.

During the late Pleistocene, early anatomically modern humans coexisted in Europe with the anatomically archaic Neandertals for some thousand years. Under the recent variants of the multiregional model of human evolution, modern and archaic forms were different but related populations within a single evolving species, and both have contributed to the gene pool of current humans. Conversely, the Out-of-Africa model considers the transition between Neandertals and anatomically modern humans as the result of a demographic replacement, and hence it predicts a genetic discontinuity between them. Following the most stringent current standards for validation of ancient DNA sequences, we typed the mtDNA hypervariable region I of two anatomically modern Homo sapiens sapiens individuals of the Cro-Magnon type dated at about 23 and 25 thousand years ago. Here we show that the mtDNAs of these individuals fall well within the range of variation of today's humans, but differ sharply from the available sequences of the chronologically closer Neandertals. This discontinuity is difficult to reconcile with the hypothesis that both Neandertals and early anatomically modern humans contributed to the current European gene pool.

Diverse Plant and Animal Genetic Records from Holocene and Pleistocene Sediments

Genetic analyses of permafrost and temperate sediments reveal that plant and animal DNA may be preserved for long periods, even in the absence of obvious macrofossils. In Siberia, five permafrost cores ranging from 400,000 to 10,000 years old contained at least 19 different plant taxa, including the oldest authenticated ancient DNA sequences known, and megafaunal sequences including mammoth, bison, and horse. The genetic data record a number of dramatic changes in the taxonomic diversity and composition of Beringian vegetation and fauna. Temperate cave sediments in New Zealand also yielded DNA sequences of extinct biota, including two species of ratite moa, and 29 plant taxa characteristic of the prehuman environment. Therefore, many sedimentary deposits may contain unique, and widespread, genetic records of paleoenvironments.

A reanalysis of the ancient mitochondrial DNA sequences recovered from Neandertal bones.

Recent reports analyzing mitochondrial DNA sequences from Neandertal bones have claimed that Neandertals and modern humans are different species. The phylogenetic analyses carried out in these articles did not take into account the high substitution rate variation among sites observed in the human mitochondrial D-loop region and also lack an estimation of the parameters of the nucleotide substitution model. The separate phylogenetic position of Neandertals is not supported when these factors are considered. Our analysis shows that Neandertal-Human and Human-Human pairwise distance distributions overlap more than what previous studies suggested. We also show that the most ancient Neandertal HVI region is the most divergent when compared with modern human sequences. However, the opposite would be expected if the sequence had not been modified since the death of the specimen. Such incongruence is discussed in the light of diagenetic modifications in ancient Neandertal DNA sequences.

The Provenance of Type Specimens of Extinct Mascarene Island Giant Tortoises (Cylindraspis) Revealed by Ancient Mitochondrial DNA Sequences

thesis, University of Puerto Rico, Mayagiiez, Puerto Rico. MACLEAN, W. P. 1985. Water-loss rates of Sphaerodactylus parthenopion (Reptilia: Gekkonidae) the smallest amniote vertebrate. Comparative Biochemistry and Physiology 82A:759-761. MACLEAN, W. P., AND R. HOLT. 1979. Distribution patterns in St. Croix Sphaerodactylus lizards: the taxon cycle in action. Biotropica 11:189-195. MAUTZ, W. J. 1982. Patterns of evaporative water loss. In C. Gans and E H. Pough (eds.), Biology of the Reptilia. Vol. 12, pp. 443-481. Academic Press, New York. MEIER, A. J., AND R. E. NOBLE. 1990. Notes on the status and habits of the Desecheo gecko, Sphaerodactylus levinsi. Journal of Herpetology 24:426-428. MURPHY, R. W., E C. MCCOLLUM, G. C. GORMAN, AND R. THOMAS. 1984. Genetics of hybridizing populations of Puerto Rican Sphaerodactylus. Journal of Herpetology 18:93-105. RIVERO, J. A. 1998. The Amphibians and Reptiles of Puerto Rico. Universidad de Puerto Rico, Editorial Universitaria, San Juan, Puerto Rico. RODRIGUEZ-RAMIREZ, J., AND A. R. LEWIS. 1991. Reproduction in the Puerto Rican teiids Ameiva exsul and A. wetmorei. Herpetologica 47:395-403. SCHWARTZ, A., AND R. W. HENDERSON. 1991. Amphibians and Reptiles of the West Indies: Descriptions, Distributions, and Natural History. University of Florida Press, Gainesville. SHREVE, A. 1968. The notatus group of Sphaerodactylus (Sauria: Gekkonidae) in Hispaniola. Breviora 280: 1-24.

The Provenance of Type Specimens of Extinct Mascarene Island Giant Tortoises (Cylindraspis) Revealed by Ancient Mitochondrial DNA Sequences

The Provenance of Type Specimens of Extinct Mascarene Island Giant Tortoises (Cylindraspis) Revealed by Ancient Mitochondrial DNA Sequences

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In the present work, we report a molecular approach that contributes to determine the zoological origin of unidentified coprolites from Patagonia. Three coprolites with morphological characters attributable to human or carnivorous, from Patagonian archaeological sites, were analyzed. Molecular analysis was conducted following the Authenticity Criteria to Determine Ancient DNA (aDNA) sequences. Two replicates of 398 and 521 coprolites were amplified. Specific fragments of cytb and nadh5 genes were successfully sequenced. No PCR positives were reached to any aDNA replicates of coprolite 246. In the present work mammal coprolites aDNA between 2,700–5,100 years old were isolated. These results allowed us to establish coprolites identity as Puma concolor. This is the first genomic information produced in an Argentinian lab based on aDNA from Patagonian coprolites. In consequence, the present study would open the way to future paleogenomics studies in archaeological sites of the region.

Analysis of Five Ancient Equine Skeletons by Mitochondrial DNA Sequencing

Pompeii is a unique archaeological site because of the natural disaster occurred there makes it possible to perform many kinds of studies, including the biological and naturalistic. Morphological and morphometric analyses were conducted on five equine skeletons dated to 79 BC , found in a stable near the Casti Amanti house, to assess their taxonomic assignment. These studies classified four of the skeletons as donkeys and one as a mule. We report the molecular characterisation of the five equine skeletons based on their ancient DNA extraction and sequencing. Attention was focused on the D-loop region of mitochondrial DNA (mtDNA), compared to DNA from the living equine species Equus asinus and Equus caballus . The results of molecular analyses do not fit exactly with previous morphological and morphometric studies. Our data indicates that two of the skeletons could be horses or mules and three could be donkeys or hinnies. This result demonstrates the relevant contribution of modern molecular approaches t...

Ancient mitochondrial DNA and morphology elucidate an extinct island radiation of Indian Ocean giant tortoises (Cylindraspis).

Ancient mitochondrial DNA sequences were used for investigating the evolution of an entire clade of extinct vertebrates, the endemic tortoises (Cylindraspis) of the Mascarene Islands in the Indian Ocean. Mitochondrial DNA corroborates morphological evidence that there were five species of tortoise with the following relationships: Cylindraspis triserrata ((Cylindraspis vosmaeri and Cylindraspis peltastes) (Cylindraspis inepta and Cylindraspis indica)). Phylogeny indicates that the ancestor of the group first colonized Mauritius where speciation produced C. triserrata and the ancestor of the other species including a second sympatric Mauritian form, C. inepta. A propagule derived from this lineage colonized Rodrigues 590 km to the east, where a second within-island speciation took place producing the sympatric C. vosmaeri and C. peltastes. A recent colonization of Réunion 150 km to the southwest produced C. indica. In the virtual absence of predators, the defensive features of the shells of Mascarene tortoises were largely dismantled, apparently in two stages. 'Saddlebacked' shells with high fronts evolved independently on all three islands. This and other features, such as a derived jaw structure and small body size, may be associated with niche differentiation in sympatric species and may represent a striking example of parallel differentiation in a large terrestrial vertebrate. The history of Mascarene tortoises contrasts with that of the Galápagos, where only a single species is present and surviving populations are genetically much more similar. However, they too show some reduction in anti-predator mechanisms and multiple development of populations with saddlebacked shells.

Mitochondrial DNA from Myotragus balearicus, an extinct bovid from the Balearic Islands.

DNA was extracted from teeth and bones of Myotragus balearicus, a bovid that evolved in isolation on the Balearic Islands (Western Mediterranean) from the end of the Miocene, becoming extinct 4,000 years BP, after the arrival of humans in the islands. The numerous morphological apomorphies of Myotragus, most strikingly its dwarfism, frontal eyes, and ever-growing incisors, obscure its phylogenetic relationships with extant bovids. Therefore, the recovery of genetic information from Myotragus is of significant interest to help clarify the taxonomic position of this species. In this study we amplified and sequenced a 95 bp (base pair) fragment of the mtDNA cytochrome b gene from 6,000-year-old specimens of Myotragus. Several experimental controls, such as amino acid analysis, independent reproduction in two different laboratories, and cloning of the PCR product, support the authenticity of the ancient DNA sequence recovered. Phylogenetic comparison with orthologous sequences from supposedly related extant genera (serow, goral, mountain goat, chamois, takin, sheep, goat, Himalayan tahr, arctic musk ox, barbary sheep, blue sheep, and saiga) from the Caprinae subfamily suggests that Myotragus is related to some of these species. However, the real phylogenetic position of Myotragus is difficult to assess, due to the lack of resolution of the present molecular study, which can be partially attributed to the short length of the genetic fragment recovered.

Protein preservation and DNA retrieval from ancient tissues.

The retrieval of DNA from fossils remains controversial. To substantiate claims of DNA recovery, one needs additional information on the preservation of other molecules within the same sample. Flash pyrolysis with GC and MS was used to assess the quality of protein preservation in 11 archaeological and paleontological remains, some of which have yielded ancient DNA sequences authenticated via a number of criteria and some of which have consistently failed to yield any meaningful DNA. Several samples, including the Neanderthal-type specimen from which DNA sequences were recently reported, yielded abundant pyrolysis products assigned to 2,5-diketopiperazines of proline-containing dipeptides. The relative amounts of these products provide a good index of the amount of peptide hydrolysis and DNA preservation. Of these samples, four stem from arctic or subarctic regions, emphasizing the importance of cooler temperatures for the preservation of macromolecules. Flash pyrolysis with GC and MS offers a rapid and effective method for assessing fossils for the possibility of DNA preservation.

Ancient HTLV-I provirus DNA of Andean mummies

SP-28 In order to investigate the origin of HTLV-I prevailing among Andes Mongoloids, we isolated ancient HTLV-I provirus DNA from mummy bone marrow which were preserved in San Pedro de Atacama of North Chile. A total of 104 cases of mummy bone marrow DNA were rendered for PCR amplification of HTLV-I pX, β-globin and HLA-DQB1 genes. We successfully amplified an ancient HTLV-I pX DNA sequence from one of 104 mummies by prevention the cross-contamination from modern DNAs. The nucleotide sequence of the ancient HTLV-I provirus DNA showed very little variation in the 159 bp of HTLV-I pX gene as compared with contemporary HTLV-I provirus DNA sequence form Japanese and Chilean native HTLV-I carriers. We were also able to amplify 112 bp of β-globin gene and 267 bp of HLA DNA sequence relevant to HLA-DQB1*0502 from mummy DNAs. This is the first demonstration that ancient Andes mummy was infected with HTLV-I more than 1,500 years ago among aboriginal Andean Mongoloids migrated from the Asian Continent carrying with HTLV-I.