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Research on ancient and forensic DNA is related in many ways, and the two fields must deal with similar obstacles. Therefore, communication between these two communities has the potential to improve results in both research fields. Here, we present the insights gained in the ancient DNA community with regard to analyzing DNA from aged skeletal material and the potential use of the developed protocols in forensic work. We discuss the various steps, from choosing samples for DNA extraction to deciding between classical PCR amplification and massively parallel sequencing approaches. Based on the progress made in ancient DNA analyses combined with the requirements of forensic work, we suggest that there is substantial potential for incorporating ancient DNA approaches into forensic protocols, a process that has already begun to a considerable extent. However, taking full advantage of the experiences gained from ancient DNA work will require comparative studies by the forensic DNA community to tailor the methods developed for ancient samples to the specific needs of forensic studies and case work. If successful, in our view, the benefits for both communities would be considerable., (Copyright © 2021 Elsevier B.V. All rights reserved.)

By combining high-throughput sequencing with target enrichment ('hybridization capture'), researchers are able to obtain molecular data from genomic regions of interest for projects that are otherwise constrained by sample quality (e.g. degraded and contamination-rich samples) or a lack of a priori sequence information (e.g. studies on nonmodel species). Despite the use of hybridization capture in various fields of research for many years, the impact of enrichment conditions on capture success is not yet thoroughly understood. We evaluated the impact of a key parameter--hybridization temperature--on the capture success of mitochondrial genomes across the carnivoran family Felidae. Capture was carried out for a range of sample types (fresh, archival, ancient) with varying levels of sequence divergence between bait and target (i.e. across a range of species) using pools of individually indexed libraries on Agilent SureSelect(™) arrays. Our results suggest that hybridization capture protocols require specific optimization for the sample type that is being investigated. Hybridization temperature affected the proportion of on-target sequences following capture: for degraded samples, we obtained the best results with a hybridization temperature of 65 °C, while a touchdown approach (65 °C down to 50 °C) yielded the best results for fresh samples. Evaluation of capture performance at a regional scale (sliding window approach) revealed no significant improvement in the recovery of DNA fragments with high sequence divergence from the bait at any of the tested hybridization temperatures, suggesting that hybridization temperature may not be the critical parameter for the enrichment of divergent fragments., (© 2015 John Wiley & Sons Ltd.)

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.

Technological innovations such as next generation sequencing and DNA hybridisation enrichment have resulted in multi-fold increases in both the quantity of ancient DNA sequence data and the time depth for DNA retrieval. To date, over 30 ancient genomes have been sequenced, moving from 0.7× coverage (mammoth) in 2008 to more than 50× coverage (Neanderthal) in 2014. Studies of rapid evolutionary changes, such as the evolution and spread of pathogens and the genetic responses of hosts, or the genetics of domestication and climatic adaptation, are developing swiftly and the importance of palaeogenomics for investigating evolutionary processes during the last million years is likely to increase considerably. However, these new datasets require new methods of data processing and analysis, as well as conceptual changes in interpreting the results. In this review we highlight important areas of future technical and conceptual progress and discuss research topics in the rapidly growing field of palaeogenomics., (© 2015 WILEY Periodicals, Inc.)

The great majority of phenotypic characteristics are complex traits, complicating the identification of the genes underlying their expression. However, both methodological and theoretical progress in genome-wide association studies have resulted in a much better understanding of the underlying genetics of many phenotypic traits, including externally visible characteristics (EVCs) such as eye and hair color. Consequently, it has become possible to predict EVCs from human samples lacking phenotypic information. Predicting EVCs from genetic evidence is clearly appealing for forensic applications involving the personal identification of human remains. Now, a recent paper has reported the genetic determination of eye and hair color in samples up to 800 years old. The ability to predict EVCs from ancient human remains opens up promising perspectives for ancient DNA research, as this could allow studies to directly address archaeological and evolutionary questions related to the temporal and geographical origins of the genetic variants underlying phenotypes., (© 2013 WILEY Periodicals, Inc.)

Marine mammals have long generation times and broad, difficult to sample distributions, which makes inferring evolutionary and demographic changes using field studies of extant populations challenging. However, molecular analyses from sub-fossil or historical materials of marine mammals such as bone, tooth, baleen, skin, fur, whiskers and scrimshaw using ancient DNA (aDNA) approaches provide an opportunity for investigating such changes over evolutionary and ecological timescales. Here, we review the application of aDNA techniques to the study of marine mammals. Most of the studies have focused on detecting changes in genetic diversity following periods of exploitation and environmental change. To date, these studies have shown that even small sample sizes can provide useful information on historical genetic diversity. Ancient DNA has also been used in investigations of changes in distribution and range of marine mammal species; we review these studies and discuss the limitations of such 'presence only' studies. Combining aDNA data with stable isotopes can provide further insights into changes in ecology and we review past studies and suggest future potential applications. We also discuss studies reconstructing inter- and intra-specific phylogenies from aDNA sequences and discuss how aDNA sequences could be used to estimate mutation rates. Finally, we highlight some of the problems of aDNA studies on marine mammals, such as obtaining sufficient sample sizes and calibrating for the marine reservoir effect when radiocarbon-dating such wide-ranging species., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

We extracted DNA from 39 Danish aurochs specimens and successfully amplified and sequenced a 252 base pair long fragment of the multivariable region I of the mitochondrial control region from 11 specimens. The sequences from these specimens dated back to 9830-2865 14Cyr BP and represent the first study of genetic variation of Danish aurochs. In addition, for all specimens we address correlations between the ability to obtain DNA sequences and various parameters such as the age of the sample, the collagen content, the museum storage period, Danish geography and whether the specimens were found in an archeological or geological context. We find that aurochs from southern Scandinavia display a star-shaped population genetic structure, that is indicative of a local and relatively recent diversification from a few ancestral haplotypes that may have originated in the ancestral Western European population before migration northwards during the retreat of the glaciers. Scenarios suggesting several invasions of genetically distinct aurochs are not supported by these analyses. Rather, our results suggest that a single continuous migration northward occurred. Our findings also suggest, although with only limited support, that aurochs in Northwestern Europe underwent a population expansion beginning shortly after the retreat of the glacial ice from Denmark and had a stable population size until the population decline that must have occurred prior to extinction. The absence of haplotypes similar to modern domestic cattle in our aurochs suggests that introgression between these species must have been limited, if it occurred at all. We found that the successful recovery of genetic material for PCR amplification correlates with sample age and local geographic conditions. However, contrary to other studies, we found no significant correlation between length of time in museum storage or the type of the locality in which a specimen was discovered (archeological or geological) and amplification success. Finally, we found large variances in our estimates of collagen content preventing an evaluation of this as an indicator of preservation quality., (Copyright © 2011. Published by Elsevier GmbH.)

Natural history museums around the world hold millions of animal and plant specimens that are potentially amenable to genetic analyses. With more and more populations and species becoming extinct, the importance of these specimens for phylogenetic and phylogeographic analyses is rapidly increasing. However, as most DNA extraction methods damage the specimens, nondestructive extraction methods are useful to balance the demands of molecular biologists, morphologists, and museum curators. Here, I describe a method for nondestructive DNA extraction from bony specimens (i.e., bones and teeth). In this method, the specimens are soaked in extraction buffer, and DNA is then purified from the soaking solution using adsorption to silica. The method reliably yields mitochondrial and often also nuclear DNA. The method has been adapted to DNA extraction from other types of specimens such as arthropods.

Recent studies using ancient DNA have been instrumental in advancing understanding of the impact of Holocene climate change on biodiversity. Ancient DNA has been used to track demography, migration and diversity, and is providing new insights into the long-term dynamics of species and population distributions. The Holocene is key to understanding how the past has impacted on the present, as it bridges the gap between contemporary phylogeographic studies and those with inference on Pleistocene patterns, based on ancient DNA studies. Here, we examine the major patterns of Holocene faunal population dynamics and connectivity; highlighting the dynamic nature of species and population responses to Holocene climatic change, thereby providing an 'analogue' for understanding potential impacts of future change., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Global temperature increased by approximately half a degree (Celsius) within the last 150 years. Even this moderate warming had major impacts on Earth's ecological and biological systems, especially in the Arctic where the magnitude of abiotic changes even exceeds those in temperate and tropical biomes. Therefore, understanding the biological consequences of climate change on high latitudes is of critical importance for future conservation of the species living in this habitat. The past 25,000 years can be used as a model for such changes, as they were marked by prominent climatic changes that influenced geographical distribution, demographic history and pattern of genetic variation of many extant species. We sequenced ancient and modern DNA of the collared lemming (Dicrostonyx torquatus), which is a key species of the arctic biota, from a single site (Pymva Shor, Northern Pre Urals, Russia) to see if climate warming events after the Last Glacial Maximum had detectable effects on the genetic variation of this arctic rodent species, which is strongly associated with a cold and dry climate., Results: Using three dimensional network reconstructions we found a dramatic decline in genetic diversity following the LGM. Model-based approaches such as Approximate Bayesian Computation and Markov Chain Monte Carlo based Bayesian inference show that there is evidence for a population decline in the collared lemming following the LGM, with the population size dropping to a minimum during the Greenland Interstadial 1 (Bølling/Allerød) warming phase at 14.5 kyrs BP., Conclusion: Our results show that previous climate warming events had a strong influence on genetic diversity and population size of collared lemmings. Due to its already severely compromised genetic diversity a similar population reduction as a result of the predicted future climate change could completely abolish the remaining genetic diversity in this population. Local population extinctions of collared lemmings would have severe effects on the arctic ecosystem, as collared lemmings are a key species in the trophic interactions and ecosystem processes in the Arctic.

Background: Deciphering the behavioral repertoire of great apes is a challenge for several reasons. First, due to their elusive behavior in dense forest environments, great ape populations are often difficult to observe. Second, members of the genus Pan are known to display a great variety in their behavioral repertoire; thus, observations from one population are not necessarily representative for other populations. For example, bonobos (Pan paniscus) are generally believed to consume almost no vertebrate prey. However, recent observations show that at least some bonobo populations may consume vertebrate prey more commonly than previously believed. We investigated the extent of their meat consumption using PCR amplification of vertebrate mitochondrial DNA (mtDNA) segments from DNA extracted from bonobo feces. As a control we also attempted PCR amplifications from gorilla feces, a species assumed to be strictly herbivorous., Principal Findings: We found evidence for consumption of a variety of mammalian species in about 16% of the samples investigated. Moreover, 40% of the positive DNA amplifications originated from arboreal monkeys. However, we also found duiker and monkey mtDNA in the gorilla feces, albeit in somewhat lower percentages. Notably, the DNA sequences isolated from the two ape species fit best to the species living in the respective regions. This result suggests that the sequences are of regional origin and do not represent laboratory contaminants., Conclusions: Our results allow at least three possible and mutually not exclusive conclusions. First, all results may represent contamination of the feces by vertebrate DNA from the local environment. Thus, studies investigating a species' diet from feces DNA may be unreliable due to the low copy number of DNA originating from diet items. Second, there is some inherent difference between the bonobo and gorilla feces, with only the later ones being contaminated. Third, similar to bonobos, for which the consumption of monkeys has only recently been documented, the gorilla population investigated (for which very little observational data are as yet available) may occasionally consume small vertebrates. Although the last explanation is speculative, it should not be discarded a-priori given that observational studies continue to unravel new behaviors in great ape species.

High-throughput sequencing technologies have opened up a new avenue for studying extinct organisms. Here we identify and quantify biases introduced by particular characteristics of ancient DNA samples. These analyses demonstrate the importance of closely related genomic sequence for correctly identifying and classifying bona fide endogenous DNA fragments. We show that more accurate genome divergence estimates from ancient DNA sequence can be attained using at least two outgroup genomes and appropriate filtering.

Many rare and valuable ancient specimens now carry the scars of ancient DNA research, as questions of population genetics and phylogeography require larger sample sets. This fuels the demand for reliable techniques to screen for DNA preservation prior to destructive sampling. Only one such technique has been widely adopted: the extent of aspartic acid racemization (AAR). The kinetics of AAR are believed to be similar to the rate of DNA depurination and therefore a good measure of the likelihood of DNA survival. Moreover, AAR analysis is only minimally destructive. We report the first comprehensive test of AAR using 91 bone and teeth samples from temperate and high-latitude sites that were analysed for DNA. While the AAR range of all specimens was low (0.02-0.17), no correlation was found between the extent of AAR and DNA amplification success. Additional heating experiments and surveys of the literature indicated that d/l Asx is low in bones until almost all the collagen is lost. This is because aspartic acid is retained in the bone within the constrained environment of the collagen triple helix, where it cannot racemize for steric reasons. Only if the helix denatures to soluble gelatin can Asx racemize readily, but this soluble gelatine is readily lost in most burial environments. We conclude that Asx d/l is not a useful screening technique for ancient DNA from bone.

In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.

It is difficult to obtain fossil data from the 10% of Earth's terrestrial surface that is covered by thick glaciers and ice sheets, and hence, knowledge of the paleoenvironments of these regions has remained limited. We show that DNA and amino acids from buried organisms can be recovered from the basal sections of deep ice cores, enabling reconstructions of past flora and fauna. We show that high-altitude southern Greenland, currently lying below more than 2 kilometers of ice, was inhabited by a diverse array of conifer trees and insects within the past million years. The results provide direct evidence in support of a forested southern Greenland and suggest that many deep ice cores may contain genetic records of paleoenvironments in their basal sections.

Ancient DNA analyses rely on the extraction of the tiny amounts of DNA remaining in samples that are hundreds to tens of thousands of years old. Despite the critical role extraction efficiency plays in this field of research, no study has comprehensively compared ancient DNA extraction techniques to date. There are a wide range of methods currently in use, which rely on such disparate principles as spin columns, alcohol precipitation, or binding to silica. We have compared a number of these methods using quantitative PCR and then optimized each step of the most promising method. We found that most chemicals routinely added to ancient DNA extraction buffers do not increase, and sometimes even decrease, DNA yields. Consequently, our optimized method uses a buffer consisting solely of EDTA and proteinase K for bone digestion and binding DNA to silica via guanidinium thiocyanate for DNA purification. In a comparison with published methods, this minimalist approach, on average, outperforms all other methods in terms of DNA yields as measured using quantitative PCR. We also found that the addition of bovine serum albumin (BSA) to the PCR helps to overcome inhibitors in ancient DNA extracts. Finally, we observed a marked difference in the performance between different types of DNA polymerases, as measured by amplification success.

This method is designed to maximize recovery of PCR-amplifiable DNA from ancient bone and teeth specimens and at the same time to minimize co-extraction of substances that inhibit PCR. This is achieved by a combination of DNA extraction from bone powder using a buffer consisting solely of EDTA and proteinase K, and purification of the DNA by binding to silica in the presence of high concentrations of guanidinium thiocyanate. All steps are performed at room temperature (20-23 degrees C), thereby reducing further degradation of the already damaged and fragile ancient DNA and providing an optimal trade-off between DNA release and degradation. Furthermore, the purification step removes most of the various types of PCR inhibitors present in ancient bone samples, thereby optimizing the amount of ancient DNA available for subsequent enzymatic manipulation, such as PCR amplification. The protocol presented here allows DNA extraction from ancient bone and teeth with a minimum of working steps and equipment and yields DNA extracts within 2 working days.

Due in part to scarcity of material, no published study has yet cladistically addressed the systematics of living and fossil Tenrecidae (Mammalia, Afrotheria). Using a noninvasive technique for sampling nuclear DNA from museum specimens, we investigate the evolution of the Tenrecidae and assess the extent to which tenrecids fit patterns of relationships proposed for other terrestrial mammals on Madagascar. Application of several tree-reconstruction techniques on sequences of the nuclear growth hormone receptor gene and morphological data for all recognized tenrecid genera supports monophyly of Malagasy tenrecids to the exclusion of the two living African genera. However, both parsimony and Bayesian methods favor a close relationship between fossil African tenrecs and the Malagasy Geogale, supporting the hypothesis of island paraphyly, but not polyphyly. More generally, the noninvasive extraction technique can be applied with minimal risk to rare/unique specimens and, by better utilizing museum collections for genetic work, can greatly mitigate field expenses and disturbance of natural populations.

This method is designed to assemble long, continuous DNA sequences using minimal amounts of fragmented ancient DNA as template. This is achieved by a two-step approach. In the first step, multiple fragments are simultaneously amplified in a single multiplex reaction. Subsequently, each of the generated fragments is amplified individually using a single primer pair, in a standard simplex (monoplex) PCR. The ability to amplify multiple fragments simultaneously in the first step allows the generation of large amounts of sequence from rare template DNA, whereas the second nested step increases specificity and decreases amplification of contaminating DNA. In contrast to current protocols using many template-consuming simplex PCRs, the method described allows amplification of several kilobases of sequence in just one reaction. It thus combines optimal template usage with a high specificity and can be performed within a day.

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.

Sengis (order Macroscelidea) are small mammals endemic to Africa. The taxonomy and phylogeny of sengis has been difficult to resolve due to a lack of clear morphological apomorphies. Molecular phylogenies have already significantly revised sengi systematics, but until now no molecular phylogeny has included all 20 extant species. In addition, the age of origin of the sengi crown clade and the divergence age of its two extant families remain unclear. Two recently published studies based on different datasets and age-calibration parameters (DNA type, outgroup selection, fossil calibration points) proposed highly different divergent age estimates and evolutionary scenarios. We obtained nuclear and mitochondrial DNA from mainly museum specimens using target enrichment of single-stranded DNA libraries to generate the first phylogeny of all extant macroscelidean species. We then explored the effects of different parameters (type of DNA, ratio of ingroup to outgroup sampling, number and type of fossil calibration points) and their resulting impacts on age estimates for the origin and initial diversification of Macroscelidea. We show that, even after correcting for substitution saturation, both using mitochondrial DNA in conjunction with nuclear DNA or alone results in much older ages and different branch lengths than when using nuclear DNA alone. We further show that the former effect can be attributed to insufficient amounts of nuclear data. If multiple calibration points are included, the age of the sengi crown group fossil prior has minimal impact on the estimated time frame of sengi evolution. In contrast, the inclusion or exclusion of outgroup fossil priors has a major effect on the resulting node ages. We also find that a reduced sampling of ingroup species does not significantly affect overall age estimates and that terminal specific substitution rates can serve as a means to evaluate the biological likeliness of the produced temporal estimates. Our study demonstrates how commonly varied parameters in temporal calibration of phylogenies affect age estimates. Dated phylogenies should therefore always be seen in the context of the dataset which was used to produce them.

The grey wolf (Canis lupus) was the first species to give rise to a domestic population, and they remained widespread throughout the last Ice Age when many other large mammal species went extinct. Little is known, however, about the history and possible extinction of past wolf populations or when and where the wolf progenitors of the present-day dog lineage (Canis familiaris) lived1-8. Here we analysed 72 ancient wolf genomes spanning the last 100,000 years from Europe, Siberia and North America. We found that wolf populations were highly connected throughout the Late Pleistocene, with levels of differentiation an order of magnitude lower than they are today. This population connectivity allowed us to detect natural selection across the time series, including rapid fixation of mutations in the gene IFT88 40,000-30,000 years ago. We show that dogs are overall more closely related to ancient wolves from eastern Eurasia than to those from western Eurasia, suggesting a domestication process in the east. However, we also found that dogs in the Near East and Africa derive up to half of their ancestry from a distinct population related to modern southwest Eurasian wolves, reflecting either an independent domestication process or admixture from local wolves. None of the analysed ancient wolf genomes is a direct match for either of these dog ancestries, meaning that the exact progenitor populations remain to be located.

Temporal genomic data hold great potential for studying evolutionary processes such as speciation. However, sampling across speciation events would, in many cases, require genomic time series that stretch well back into the Early Pleistocene subepoch. Although theoretical models suggest that DNA should survive on this timescale1, the oldest genomic data recovered so far are from a horse specimen dated to 780-560 thousand years ago2. Here we report the recovery of genome-wide data from three mammoth specimens dating to the Early and Middle Pleistocene subepochs, two of which are more than one million years old. We find that two distinct mammoth lineages were present in eastern Siberia during the Early Pleistocene. One of these lineages gave rise to the woolly mammoth and the other represents a previously unrecognized lineage that was ancestral to the first mammoths to colonize North America. Our analyses reveal that the Columbian mammoth of North America traces its ancestry to a Middle Pleistocene hybridization between these two lineages, with roughly equal admixture proportions. Finally, we show that the majority of protein-coding changes associated with cold adaptation in woolly mammoths were already present one million years ago. These findings highlight the potential of deep-time palaeogenomics to expand our understanding of speciation and long-term adaptive evolution.

The great auk was once abundant and distributed across the North Atlantic. It is now extinct, having been heavily exploited for its eggs, meat, and feathers. We investigated the impact of human hunting on its demise by integrating genetic data, GPS-based ocean current data, and analyses of population viability. We sequenced complete mitochondrial genomes of 41 individuals from across the species' geographic range and reconstructed population structure and population dynamics throughout the Holocene. Taken together, our data do not provide any evidence that great auks were at risk of extinction prior to the onset of intensive human hunting in the early 16th century. In addition, our population viability analyses reveal that even if the great auk had not been under threat by environmental change, human hunting alone could have been sufficient to cause its extinction. Our results emphasise the vulnerability of even abundant and widespread species to intense and localised exploitation.

The straight-tusked elephants Palaeoloxodon spp. were widespread across Eurasia during the Pleistocene. Phylogenetic reconstructions using morphological traits have grouped them with Asian elephants (Elephas maximus), and many paleontologists place Palaeoloxodon within Elephas. Here, we report the recovery of full mitochondrial genomes from four and partial nuclear genomes from two P. antiquus fossils. These fossils were collected at two sites in Germany, Neumark-Nord and Weimar-Ehringsdorf, and likely date to interglacial periods ~120 and ~244 thousand years ago, respectively. Unexpectedly, nuclear and mitochondrial DNA analyses suggest that P. antiquus was a close relative of extant African forest elephants (Loxodonta cyclotis). Species previously referred to Palaeoloxodon are thus most parsimoniously explained as having diverged from the lineage of Loxodonta, indicating that Loxodonta has not been constrained to Africa. Our results demonstrate that the current picture of elephant evolution is in need of substantial revision.

Near the end of the Pleistocene epoch, populations of the woolly mammoth (Mammuthus primigenius) were distributed across parts of three continents, from western Europe and northern Asia through Beringia to the Atlantic seaboard of North America. Nonetheless, questions about the connectivity and temporal continuity of mammoth populations and species remain unanswered. We use a combination of targeted enrichment and high-throughput sequencing to assemble and interpret a data set of 143 mammoth mitochondrial genomes, sampled from fossils recovered from across their Holarctic range. Our dataset includes 54 previously unpublished mitochondrial genomes and significantly increases the coverage of the Eurasian range of the species. The resulting global phylogeny confirms that the Late Pleistocene mammoth population comprised three distinct mitochondrial lineages that began to diverge ~1.0-2.0 million years ago (Ma). We also find that mammoth mitochondrial lineages were strongly geographically partitioned throughout the Pleistocene. In combination, our genetic results and the pattern of morphological variation in time and space suggest that male-mediated gene flow, rather than large-scale dispersals, was important in the Pleistocene evolutionary history of mammoths.

Retracing complex population processes that precede extreme bottlenecks may be impossible using data from living individuals. The wisent (Bison bonasus), Europe's largest terrestrial mammal, exemplifies such a population history, having gone extinct in the wild but subsequently restored by captive breeding efforts. Using low coverage genomic data from modern and historical individuals, we investigate population processes occurring before and after this extinction. Analysis of aligned genomes supports the division of wisent into two previously recognized subspecies, but almost half of the genomic alignment contradicts this population history as a result of incomplete lineage sorting and admixture. Admixture between subspecies populations occurred prior to extinction and subsequently during the captive breeding program. Admixture with the Bos cattle lineage is also widespread but results from ancient events rather than recent hybridization with domestics. Our study demonstrates the huge potential of historical genomes for both studying evolutionary histories and for guiding conservation strategies.

Leopard complex spotting is inherited by the incompletely dominant locus, LP, which also causes congenital stationary night blindness in homozygous horses. We investigated an associated single nucleotide polymorphism in the TRPM1 gene in 96 archaeological bones from 31 localities from Late Pleistocene (approx. 17 000 YBP) to medieval times. The first genetic evidence of LP spotting in Europe dates back to the Pleistocene. We tested for temporal changes in the LP associated allele frequency and estimated coefficients of selection by means of approximate Bayesian computation analyses. Our results show that at least some of the observed frequency changes are congruent with shifts in artificial selection pressure for the leopard complex spotting phenotype. In early domestic horses from Kirklareli-Kanligecit (Turkey) dating to 2700-2200 BC, a remarkably high number of leopard spotted horses (six of 10 individuals) was detected including one adult homozygote. However, LP seems to have largely disappeared during the late Bronze Age, suggesting selection against this phenotype in early domestic horses. During the Iron Age, LP reappeared, probably by reintroduction into the domestic gene pool from wild animals. This picture of alternating selective regimes might explain how genetic diversity was maintained in domestic animals despite selection for specific traits at different times.

After centuries of human hunting, the Eurasian beaver Castor fiber had disappeared from most of its original range by the end of the 19th century. The surviving relict populations are characterized by both low genetic diversity and strong phylogeographical structure. However, it remains unclear whether these attributes are the result of a human-induced, late Holocene bottleneck or already existed prior to this reduction in range. To investigate genetic diversity in Eurasian beaver populations during the Holocene, we obtained mitochondrial control region DNA sequences from 48 ancient beaver samples and added 152 modern sequences from GenBank. Phylogeographical analyses of the data indicate a differentiation of European beaver populations into three mitochondrial clades. The two main clades occur in western and eastern Europe, respectively, with an early Holocene contact zone in eastern Europe near a present-day contact zone. A divergent and previously unknown clade of beavers from the Danube Basin survived until at least 6000 years ago, but went extinct during the transition to modern times. Finally, we identify a recent decline in effective population size of Eurasian beavers, with a stronger bottleneck signal in the western than in the eastern clade. Our results suggest that the low genetic diversity and the strong phylogeographical structure in recent beavers are artefacts of human hunting-associated population reductions. While beaver populations have been growing rapidly since the late 19th century, genetic diversity within modern beaver populations remains considerably reduced compared to what was present prior to the period of human hunting and habitat reduction.

The Torrener Bärenhöhle (Cave of Torren) is an alpine bear cave in the limestone Alps near Salzburg (Austria). The entrance of this cave is located in the riverbed of a periodically flowing stream that floods the caverns during snowmelt or after heavy rainfall. Due to these flooding events, the fossiliferous layers were repeatedly destroyed and the fossil remains – mainly cave bear bones – were distributed over the cave's entrance hall. The fossil collections have taken place since 1924. During the last decades, numerous bones scattered over the cave floor have been collected again and again. In this study, we conduct metric and morphological analyses of this fossil material as well as DNA analyses in an attempt to clarify the taxonomic position of the cave bear remains. The chronological status of the bear remains has not yet been clarified, because the few samples that have been analysed so far were beyond the range of 14C dating, indicating they are older than 49,000 cal yr BP. The following taxa are represented in small numbers: Brown bear (Ursus arctos L.), wolf (Canis lupus L.), cave lion (Panthera spelaeus Goldfuss, 1810), European bison (Bison bonasus L.) and beaver (Castor fiber L.). [ABSTRACT FROM AUTHOR]

Millions of scientific specimens are housed in museum collections, a large part of which are fluid preserved. The use of formaldehyde as fixative and subsequent storage in ethanol is especially common in ichthyology and herpetology. This type of preservation damages DNA and reduces the chance of successful retrieval of genetic data. We applied ancient DNA extraction and single stranded library construction protocols to a variety of vertebrate samples obtained from wet collections and of different ages. Our results show that almost all samples tested yielded endogenous DNA. Archival DNA extraction was successful across different tissue types as well as using small amounts of tissue. Conversion of archival DNA fragments into single‐stranded libraries resulted in usable data even for samples with initially undetectable DNA amounts. Subsequent target capture approaches for mitochondrial DNA using homemade baits on a subset of 30 samples resulted in almost complete mitochondrial genome sequences in several instances. Thus, application of ancient DNA methodology makes wet collection specimens, including type material as well as rare, old or extinct species, accessible for genetic and genomic analyses. Our results, accompanied by detailed step‐by‐step protocols, are a large step forward to open the DNA archive of museum wet collections for scientific studies. [ABSTRACT FROM AUTHOR]

Targeted capture coupled with high‐throughput sequencing can be used to gain information about nuclear sequence variation at hundreds to thousands of loci. Divergent reference capture makes use of molecular data of one species to enrich target loci in other (related) species. This is particularly valuable for nonmodel organisms, for which often no a priori knowledge exists regarding these loci. Here, we have used targeted capture to obtain data for 809 nuclear coding DNA sequences (CDS) in a nonmodel organism, the Eurasian lynx Lynx lynx, using baits designed with the help of the published genome of a related model organism (the domestic cat Felis catus). Using this approach, we were able to survey intraspecific variation at hundreds of nuclear loci in L. lynx across the species' European range. A large set of biallelic candidate SNPs was then evaluated using a high‐throughput SNP genotyping platform (Fluidigm), which we then reduced to a final 96 SNP‐panel based on assay performance and reliability; validation was carried out with 100 additional Eurasian lynx samples not included in the SNP discovery phase. The 96 SNP‐panel developed from CDS performed very successfully in the identification of individuals and in population genetic structure inference (including the assignment of individuals to their source population). In keeping with recent studies, our results show that genic SNPs can be valuable for genetic monitoring of wildlife species. [ABSTRACT FROM AUTHOR]