Michael Knapp

Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments

Significance Outside of permafrost, no contiguous DNA sequences have been generated from material older than ∼120,000 y. By improving our ability to sequence very short DNA fragments, we have recovered the mitochondrial genome sequence of a >300,000-y-old cave bear from Sima de los Huesos, a Spanish cave site that is famous for its rich collection of Middle Pleistocene human fossils. This finding demonstrates that DNA can survive for hundreds of thousands of years outside of permafrost and opens the prospect of making more samples from this time period accessible to genetic studies. Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.

Environmental DNA for wildlife biology and biodiversity monitoring

Extraction and identification of DNA from an environmental sample has proven noteworthy recently in detecting and monitoring not only common species, but also those that are endangered, invasive, or elusive. Particular attributes of so-called environmental DNA (eDNA) analysis render it a potent tool for elucidating mechanistic insights in ecological and evolutionary processes. Foremost among these is an improved ability to explore ecosystem-level processes, the generation of quantitative indices for analyses of species, community diversity, and dynamics, and novel opportunities through the use of time-serial samples and unprecedented sensitivity for detecting rare or difficult-to-sample taxa. Although technical challenges remain, here we examine the current frontiers of eDNA, outline key aspects requiring improvement, and suggest future developments and innovations for research.

Direct multiplex sequencing (DMPS): A novel method for targeted high-throughput sequencing of ancient and highly degraded DNA

Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA. Here, we present a novel and highly sensitive method for targeted sequencing of ancient and degraded DNA, which couples multiplex PCR directly with sample barcoding and high-throughput sequencing. Using this approach, we obtained a 96% complete mitochondrial genome data set from 31 cave bear (Ursus spelaeus) samples using only two 454 Life Sciences (Roche) GS FLX runs. In contrast to previous studies relying only on short sequence fragments, the overlapping portion of our data comprises almost 10 kb of replicated mitochondrial genome sequence, allowing for the unambiguous differentiation of three major cave bear clades. Our method opens up the opportunity to simultaneously generate many kilobases of overlapping sequence data from large sets of difficult samples, such as museum specimens, medical collections, or forensic samples. Embedded in our approach, we present a new protocol for the construction of barcoded sequencing libraries, which is compatible with all current high-throughput technologies and can be performed entirely in plate setup.

Next Generation Sequencing of Ancient DNA: Requirements, Strategies and Perspectives

The invention of next-generation-sequencing has revolutionized almost all fields of genetics, but few have profited from it as much as the field of ancient DNA research. From its beginnings as an interesting but rather marginal discipline, ancient DNA research is now on its way into the centre of evolutionary biology. In less than a year from its invention next-generation-sequencing had increased the amount of DNA sequence data available from extinct organisms by several orders of magnitude. Ancient DNA research is now not only adding a temporal aspect to evolutionary studies and allowing for the observation of evolution in real time, it also provides important data to help understand the origins of our own species. Here we review progress that has been made in next-generation-sequencing of ancient DNA over the past five years and evaluate sequencing strategies and future directions.

Withering Away—25,000 Years of Genetic Decline Preceded Cave Bear Extinction

The causes of the late Pleistocene megafaunal extinctions are still enigmatic. Although the fossil record can provide approximations for when a species went extinct, the timing of its disappearance alone cannot resolve the causes and mode of the decline preceding its extinction. However, ancient DNA analyses can reveal population size changes over time and narrow down potential causes of extinction. Here, we present an ancient DNA study comparing late Pleistocene population dynamics of two closely related species, cave and brown bears. We found that the decline of cave bears started approximately 25,000 years before their extinction, whereas brown bear population size remained constant. We conclude that neither the effects of climate change nor human hunting alone can be responsible for the decline of the cave bear and suggest that a complex of factors including human competition for cave sites lead to the cave bears extinction.

The Drowning of New Zealand and the Problem of Agathis

chondrial portraits of human populations using median networks. intraspecific network construction methods using simulated sequence data: Do existing algorithms outperform the global maximum parsimony approach? Syst. approach to the development of minimal phylogenetic trees. Development and application of three-tiered nuclear genetic markers for basal hexapods using single-stranded conformation polymorphism couple with targeted DNA sequencing.Max squeeze: Guaranteeing a minimal tree for population data. Mol. due to missing data in phylogenetic analyses including fossils: A critical review. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. population structure from population history: A cladistic analysis of the geographical distribution of mitochondrial DNA haplotypes in the tiger salamander, Ambystoma tigrinum. Genetics 140:767-782. This modified minimum spanning network (MSN) algorithm first constructs a network only from sequences that do not have missing data and then adds sequences with missing data: (1) calculate the distance between any two sequences and arrange these in increasing order; (2) put all distance comparisons that include sequences with missing data at variable sites aside to first consider only comparisons between sequences that do not have missing data. Let <5, be the smallest distance observed in the sample; (3) connect all haplotypes of different sub-networks that are at distance 5,; (4) repeat step (3) with second shortest distance (<

Setting the stage - building and working in an ancient DNA laboratory.

,+i) and so on until a network of all haplotypes is formed; (5) then consider comparisons with sequences that have missing data; (6) starting with the smallest distance (Sj), connect all haplotypes of different sub-networks that are at distance <

First DNA sequences from Asian cave bear fossils reveal deep divergences and complex phylogeographic patterns

/. When a sub-network (or haplotype) can be connected at more than one place on another sub-network at a given distance, connect it to all of them; (7) repeat step (6) with the next shortest distance (<5y+i) until all sequences with missing data are connected to the network. Eighty million years ago (Ma) the landmass that was to become New Zealand broke away from the Gondwanan supercontinent. During the subsequent Oligocene period (26 to 38 Ma), there was a significant reduction in the landmass of New Zealand (Cooper and Cooper, 1995, and references therein). However , whether or not New Zealand was completely submerged is a matter of controversy and recent debate

Identification of the remains of King Richard III

With the introduction of next generation high throughput sequencing in 2005 and the resulting revolution in genetics, ancient DNA research has rapidly developed from an interesting but marginal field within evolutionary biology into one that can contribute significantly to our understanding of evolution in general and the development of our own species in particular. While the amount of sequence data available from ancient human, other animal and plant remains has increased dramatically over the past five years, some key limitations of ancient DNA research remain. Most notably, reduction of contamination and the authentication of results are of utmost importance. A number of studies have addressed different aspects of sampling, DNA extraction and DNA manipulation in order to establish protocols that most efficiently generate reproducible and authentic results. As increasing numbers of researchers from different backgrounds become interested in using ancient DNA technology to address key questions, the need for practical guidelines on how to construct and use an ancient DNA facility arises. The aim of this article is therefore to provide practical tips for building a state-of-the-art ancient DNA facility. It is intended to help researchers new to the field of ancient DNA research generally, and those considering the application of next generation sequencing, in their planning process.

Discovery of lost diversity of paternal horse lineages using ancient DNA

Until recently, cave bears were believed to have only inhabited Europe. However, recent morphological evidence suggests that cave bears’ geographic range extended as far east as Transbaikalia, Eastern Siberia. These Asian cave bears were morphologically distinct from European cave bears. However, how they related to European lineages remains unclear, stressing the need to assess the phylogenetic and phylogeographic relationship between Asian cave bears and their European relatives. In this work, we address this issue using a 227 base‐pair fragment of the mitochondrial control region obtained from nine fossil bone samples from eight sites from the Urals, Caucasus, Altai Mountains, Ukraine and Yana River region in Eastern Siberia. Results of the phylogenetic analyses indicate that (i) the cave bear from the Yana River is most closely related to cave bears from the Caucasus region; (ii) the Caucasus/Yana group of bears is genetically very distinct from both European cave bears and brown bears, suggesting that these bears could represent an independent species; and (iii) the Western European cave bear lineage reached at least temporarily to the Altai Mountains, 7000 km east of their known centre of distribution. These results suggest that the diversity of cave bears was greater than previously believed, and that they could survive in a much wider range of ecological conditions than previously assumed. They also agree with recent studies on other extinct and extant species, such as wolves, hyenas and steppe bison, which have also revealed higher genetic and ecological diversity in Pleistocene populations than previously known.