Special Issue "Novel and Neglected Areas of Ancient DNA Research"

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Population and Evolutionary Genetics and Genomics".

Deadline for manuscript submissions: closed (31 May 2017)

Special Issue Editor

Guest Editor
Prof. Dr. Michael Hofreiter

Institute for Biochemistry and Biology, Department of Mathematics and Natural Sciences, University of Potsdam, 14476 Potsdam, Golm, Germany
Website | E-Mail
Phone: +49 0331 977 6321
Fax: +49 0331 977 6389
Interests: adaptation, ancient DNA, bears, DNA extraction, DNA sequencing, genetic diversity, hybridization, phylogenetics, Pleistocene mammals, population genetics, proboscideans, reticulate evolution, speciation

Special Issue Information

Dear Colleagues,

Research involving DNA from historical or ancient samples has flourished during the last 10 years. However, the majority of studies have focused either on hominids, or on a relatively limited number of large bodied and charismatic mammalian taxa. Such biases limit the scope of unique insights that ancient DNA can contribute to the study of evolution and ecology. Furthermore, ongoing technological developments in ancient DNA research carry huge potential for other, seemingly unrelated fields of basic and applied scientific research. Achieving this potential will require exploration into subject areas not typically investigated by the ancient DNA community. In this Special Issue of Genes, we present a set of review and research articles focused on “Novel and Neglected Areas of Ancient DNA Research”. This collection of works documents the current state of knowledge on ancient DNA research of neglected taxa, documents new and promising technical developments enabling future applications of ancient DNA, and also presents new research findings, addressing diverse and underexplored research areas. We hope that this special issue will inspire and motivate the next generation of ancient DNA researchers to broaden and diversify the scope of future ancient DNA research, fulfilling the potential of this most valuable tool for the study of evolution and ecology.

Prof. Dr. Michael Hofreiter
Guest Editor

Manuscript Submission Information

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Published Papers (14 papers)

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Research

Jump to: Review

Open AccessArticle Metagenomic Composition Analysis of an Ancient Sequenced Polar Bear Jawbone from Svalbard
Received: 29 June 2018 / Revised: 3 September 2018 / Accepted: 3 September 2018 / Published: 6 September 2018
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Abstract
The sequencing of ancient DNA samples provides a novel way to find, characterize, and distinguish exogenous genomes of endogenous targets. After sequencing, computational composition analysis enables filtering of undesired sources in the focal organism, with the purpose of improving the quality of assemblies
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The sequencing of ancient DNA samples provides a novel way to find, characterize, and distinguish exogenous genomes of endogenous targets. After sequencing, computational composition analysis enables filtering of undesired sources in the focal organism, with the purpose of improving the quality of assemblies and subsequent data analysis. More importantly, such analysis allows extinct and extant species to be identified without requiring a specific or new sequencing run. However, the identification of exogenous organisms is a complex task, given the nature and degradation of the samples, and the evident necessity of using efficient computational tools, which rely on algorithms that are both fast and highly sensitive. In this work, we relied on a fast and highly sensitive tool, FALCON-meta, which measures similarity against whole-genome reference databases, to analyse the metagenomic composition of an ancient polar bear (Ursus maritimus) jawbone fossil. The fossil was collected in Svalbard, Norway, and has an estimated age of 110,000 to 130,000 years. The FASTQ samples contained 349 GB of nonamplified shotgun sequencing data. We identified and localized, relative to the FASTQ samples, the genomes with significant similarities to reference microbial genomes, including those of viruses, bacteria, and archaea, and to fungal, mitochondrial, and plastidial sequences. Among other striking features, we found significant similarities between modern-human, some bacterial and viral sequences (contamination) and the organelle sequences of wild carrot and tomato relative to the whole samples. For each exogenous candidate, we ran a damage pattern analysis, which in addition to revealing shallow levels of damage in the plant candidates, identified the source as contamination. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessArticle Ancient DNA from Giant Panda (Ailuropoda melanoleuca) of South-Western China Reveals Genetic Diversity Loss during the Holocene
Received: 24 February 2018 / Revised: 26 March 2018 / Accepted: 27 March 2018 / Published: 6 April 2018
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Abstract
The giant panda was widely distributed in China and south-eastern Asia during the middle to late Pleistocene, prior to its habitat becoming rapidly reduced in the Holocene. While conservation reserves have been established and population numbers of the giant panda have recently increased,
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The giant panda was widely distributed in China and south-eastern Asia during the middle to late Pleistocene, prior to its habitat becoming rapidly reduced in the Holocene. While conservation reserves have been established and population numbers of the giant panda have recently increased, the interpretation of its genetic diversity remains controversial. Previous analyses, surprisingly, have indicated relatively high levels of genetic diversity raising issues concerning the efficiency and usefulness of reintroducing individuals from captive populations. However, due to a lack of DNA data from fossil specimens, it is unknown whether genetic diversity was even higher prior to the most recent population decline. We amplified complete cytb and 12s rRNA, partial 16s rRNA and ND1, and control region sequences from the mitochondrial genomes of two Holocene panda specimens. We estimated genetic diversity and population demography by analyzing the ancient mitochondrial DNA sequences alongside those from modern giant pandas, as well as from other members of the bear family (Ursidae). Phylogenetic analyses show that one of the ancient haplotypes is sister to all sampled modern pandas and the second ancient individual is nested among the modern haplotypes, suggesting that genetic diversity may indeed have been higher earlier during the Holocene. Bayesian skyline plot analysis supports this view and indicates a slight decline in female effective population size starting around 6000 years B.P., followed by a recovery around 2000 years ago. Therefore, while the genetic diversity of the giant panda has been affected by recent habitat contraction, it still harbors substantial genetic diversity. Moreover, while its still low population numbers require continued conservation efforts, there seem to be no immediate threats from the perspective of genetic evolutionary potential. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessArticle Testing of Alignment Parameters for Ancient Samples: Evaluating and Optimizing Mapping Parameters for Ancient Samples Using the TAPAS Tool
Received: 20 December 2017 / Revised: 6 March 2018 / Accepted: 8 March 2018 / Published: 13 March 2018
Cited by 1 | PDF Full-text (1850 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
High-throughput sequence data retrieved from ancient or other degraded samples has led to unprecedented insights into the evolutionary history of many species, but the analysis of such sequences also poses specific computational challenges. The most commonly used approach involves mapping sequence reads to
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High-throughput sequence data retrieved from ancient or other degraded samples has led to unprecedented insights into the evolutionary history of many species, but the analysis of such sequences also poses specific computational challenges. The most commonly used approach involves mapping sequence reads to a reference genome. However, this process becomes increasingly challenging with an elevated genetic distance between target and reference or with the presence of contaminant sequences with high sequence similarity to the target species. The evaluation and testing of mapping efficiency and stringency are thus paramount for the reliable identification and analysis of ancient sequences. In this paper, we present ‘TAPAS’, (Testing of Alignment Parameters for Ancient Samples), a computational tool that enables the systematic testing of mapping tools for ancient data by simulating sequence data reflecting the properties of an ancient dataset and performing test runs using the mapping software and parameter settings of interest. We showcase TAPAS by using it to assess and improve mapping strategy for a degraded sample from a banded linsang (Prionodon linsang), for which no closely related reference is currently available. This enables a 1.8-fold increase of the number of mapped reads without sacrificing mapping specificity. The increase of mapped reads effectively reduces the need for additional sequencing, thus making more economical use of time, resources, and sample material. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessArticle Ancient DNA Resolves the History of Tetragnatha (Araneae, Tetragnathidae) Spiders on Rapa Nui
Genes 2017, 8(12), 403; https://doi.org/10.3390/genes8120403
Received: 11 October 2017 / Revised: 2 December 2017 / Accepted: 13 December 2017 / Published: 20 December 2017
Cited by 2 | PDF Full-text (9285 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Rapa Nui is one of the most remote islands in the world. As a young island, its biota is a consequence of both natural dispersals over the last ~1 million years and recent human introductions. It therefore provides an opportunity to study a
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Rapa Nui is one of the most remote islands in the world. As a young island, its biota is a consequence of both natural dispersals over the last ~1 million years and recent human introductions. It therefore provides an opportunity to study a unique community assemblage. Here, we extract DNA from museum-preserved and newly field-collected spiders from the genus Tetragnatha to explore their history on Rapa Nui. Using an optimized protocol to recover ancient DNA from museum-preserved spiders, we sequence and assemble partial mitochondrial genomes from nine Tetragnatha species, two of which were found on Rapa Nui, and estimate the evolutionary relationships between these and other Tetragnatha species. Our phylogeny shows that the two Rapa Nui species are not closely related. One, the possibly extinct, T. paschae, is nested within a circumtropical species complex (T. nitens), and the other (Tetragnatha sp. Rapa Nui) appears to be a recent human introduction. Our results highlight the power of ancient DNA approaches in identifying cryptic and rare species, which can contribute to our understanding of the global distribution of biodiversity in all taxonomic lineages. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessArticle The History of Tree and Shrub Taxa on Bol'shoy Lyakhovsky Island (New Siberian Archipelago) since the Last Interglacial Uncovered by Sedimentary Ancient DNA and Pollen Data
Genes 2017, 8(10), 273; https://doi.org/10.3390/genes8100273
Received: 27 July 2017 / Revised: 27 September 2017 / Accepted: 4 October 2017 / Published: 13 October 2017
Cited by 3 | PDF Full-text (5173 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Ecosystem boundaries, such as the Arctic-Boreal treeline, are strongly coupled with climate and were spatially highly dynamic during past glacial-interglacial cycles. Only a few studies cover vegetation changes since the last interglacial, as most of the former landscapes are inundated and difficult to
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Ecosystem boundaries, such as the Arctic-Boreal treeline, are strongly coupled with climate and were spatially highly dynamic during past glacial-interglacial cycles. Only a few studies cover vegetation changes since the last interglacial, as most of the former landscapes are inundated and difficult to access. Using pollen analysis and sedimentary ancient DNA (sedaDNA) metabarcoding, we reveal vegetation changes on Bol’shoy Lyakhovsky Island since the last interglacial from permafrost sediments. Last interglacial samples depict high levels of floral diversity with the presence of trees (Larix, Picea, Populus) and shrubs (Alnus, Betula, Ribes, Cornus, Saliceae) on the currently treeless island. After the Last Glacial Maximum, Larix re-colonised the island but disappeared along with most shrub taxa. This was probably caused by Holocene sea-level rise, which led to increased oceanic conditions on the island. Additionally, we applied two newly developed larch-specific chloroplast markers to evaluate their potential for tracking past population dynamics from environmental samples. The novel markers were successfully re-sequenced and exhibited two variants of each marker in last interglacial samples. SedaDNA can track vegetation changes as well as genetic changes across geographic space through time and can improve our understanding of past processes that shape modern patterns. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessArticle Complete Mitochondrial Genome Sequencing of a Burial from a Romano–Christian Cemetery in the Dakhleh Oasis, Egypt: Preliminary Indications
Genes 2017, 8(10), 262; https://doi.org/10.3390/genes8100262
Received: 16 June 2017 / Revised: 15 September 2017 / Accepted: 26 September 2017 / Published: 6 October 2017
Cited by 3 | PDF Full-text (914 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The curse of ancient Egyptian DNA was lifted by a recent study which sequenced the mitochondrial genomes (mtGenome) of 90 ancient Egyptians from the archaeological site of Abusir el-Meleq. Surprisingly, these ancient inhabitants were more closely related to those from the Near East
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The curse of ancient Egyptian DNA was lifted by a recent study which sequenced the mitochondrial genomes (mtGenome) of 90 ancient Egyptians from the archaeological site of Abusir el-Meleq. Surprisingly, these ancient inhabitants were more closely related to those from the Near East than to contemporary Egyptians. It has been accepted that the timeless highway of the Nile River seeded Egypt with African genetic influence, well before pre-Dynastic times. Here we report on the successful recovery and analysis of the complete mtGenome from a burial recovered from a remote Romano–Christian cemetery, Kellis 2 (K2). K2 serviced the ancient municipality of Kellis, a village located in the Dakhleh Oasis in the southwest desert in Egypt. The data were obtained by high throughput sequencing (HTS) performed independently at two ancient DNA facilities (Armed Forces DNA Identification Laboratory, Dover, DE, USA and Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA). These efforts produced concordant haplotypes representing a U1a1a haplogroup lineage. This result indicates that Near Eastern maternal influence previously identified at Abusir el-Meleq was also present further south, in ancient Kellis during the Romano–Christian period. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessArticle Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin
Genes 2017, 8(10), 237; https://doi.org/10.3390/genes8100237
Received: 11 August 2017 / Revised: 6 September 2017 / Accepted: 18 September 2017 / Published: 21 September 2017
Cited by 9 | PDF Full-text (613 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established
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The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for final implementation in forensic genetic laboratories. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessArticle Detection of G1138A Mutation of the FGFR3 Gene in Tooth Material from a 180-Year-Old Museological Achondroplastic Skeleton
Received: 24 July 2017 / Revised: 18 August 2017 / Accepted: 22 August 2017 / Published: 29 August 2017
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Abstract
Throughout the last four centuries, many anatomical museums across the world have collected teratological specimens that became precious objects. These can be regarded as spirits of the past which have captured the morphology of diseases through time. These valuable and irreplaceable specimens can
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Throughout the last four centuries, many anatomical museums across the world have collected teratological specimens that became precious objects. These can be regarded as spirits of the past which have captured the morphology of diseases through time. These valuable and irreplaceable specimens can be perfectly used in contemporary dysmorphological or genetic research. Unfortunately, due to the historical nature of these specimens and the regularly used aggressive preservation fluids, DNA degradation is often present. Furthermore, the use of material for DNA extraction is restricted to preserve the appearance of these valuable museological specimens. Thus, the most challenging part in this perspective is to harvest sufficient DNA of good quality for further testing without damaging the specimens. Besides fixated specimens, most teratological collections contain dried skeletal and teeth materials which are an excellent source to extract DNA. We here present a DNA-based method that enables genetic identification of the G1138A mutation of the FGFR3 gene in a 180-year-old achondroplastic skeleton, confirming the previously morphologically determined disease. Nuclear DNA was extracted from a premolar tooth and the mutation was found using Sanger sequencing of a small region of the FGFR3 gene. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessArticle Identifying Bird Remains Using Ancient DNA Barcoding
Received: 29 May 2017 / Revised: 11 June 2017 / Accepted: 15 June 2017 / Published: 21 June 2017
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Abstract
Bird remains that are difficult to identify taxonomically using morphological methods, are common in the palaeontological record. Other types of challenging avian material include artefacts and food items from endangered taxa, as well as remains from aircraft strikes. We here present a DNA-based
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Bird remains that are difficult to identify taxonomically using morphological methods, are common in the palaeontological record. Other types of challenging avian material include artefacts and food items from endangered taxa, as well as remains from aircraft strikes. We here present a DNA-based method that enables taxonomic identification of bird remains, even from material where the DNA is heavily degraded. The method is based on the amplification and sequencing of two short variable parts of the 16S region in the mitochondrial genome. To demonstrate the applicability of this approach, we evaluated the method on a set of Holocene and Late Pleistocene postcranial bird bones from several palaeontological and archaeological sites in Europe with good success. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
Open AccessArticle Endogenous Retroviral Insertions Indicate a Secondary Introduction of Domestic Sheep Lineages to the Caucasus and Central Asia between the Bronze and Iron Age
Received: 12 May 2017 / Revised: 29 May 2017 / Accepted: 13 June 2017 / Published: 20 June 2017
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Abstract
Sheep were one of the first livestock species domesticated by humans. After initial domestication in the Middle East they were spread across Eurasia. The modern distribution of endogenous Jaagsiekte sheep retrovirus insertions in domestic sheep breeds suggests that over the course of millennia,
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Sheep were one of the first livestock species domesticated by humans. After initial domestication in the Middle East they were spread across Eurasia. The modern distribution of endogenous Jaagsiekte sheep retrovirus insertions in domestic sheep breeds suggests that over the course of millennia, successive introductions of improved lineages and selection for wool quality occurred in the Mediterranean region and most of Asia. Here we present a novel ancient DNA approach using data of endogenous retroviral insertions in Bronze and Iron Age domestic sheep from the Caucasus and Pamir mountain areas. Our findings support a secondary introduction of wool sheep from the Middle East between the Late Bronze Age and Iron Age into most areas of Eurasia. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessArticle An ‛Aukward’ Tale: A Genetic Approach to Discover the Whereabouts of the Last Great Auks
Received: 30 May 2017 / Accepted: 9 June 2017 / Published: 15 June 2017
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Abstract
One hundred and seventy-three years ago, the last two Great Auks, Pinguinus impennis, ever reliably seen were killed. Their internal organs can be found in the collections of the Natural History Museum of Denmark, but the location of their skins has remained
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One hundred and seventy-three years ago, the last two Great Auks, Pinguinus impennis, ever reliably seen were killed. Their internal organs can be found in the collections of the Natural History Museum of Denmark, but the location of their skins has remained a mystery. In 1999, Great Auk expert Errol Fuller proposed a list of five potential candidate skins in museums around the world. Here we take a palaeogenomic approach to test which—if any—of Fuller’s candidate skins likely belong to either of the two birds. Using mitochondrial genomes from the five candidate birds (housed in museums in Bremen, Brussels, Kiel, Los Angeles, and Oldenburg) and the organs of the last two known individuals, we partially solve the mystery that has been on Great Auk scholars’ minds for generations and make new suggestions as to the whereabouts of the still-missing skin from these two birds. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Review

Jump to: Research

Open AccessReview The Small and the Dead: A Review of Ancient DNA Studies Analysing Micromammal Species
Genes 2017, 8(11), 312; https://doi.org/10.3390/genes8110312
Received: 23 August 2017 / Revised: 13 October 2017 / Accepted: 13 October 2017 / Published: 8 November 2017
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Abstract
The field of ancient DNA (aDNA) has recently been in a state of exponential growth, largely driven by the uptake of Next Generation Sequencing (NGS) techniques. Much of this work has focused on the mammalian megafauna and ancient humans, with comparatively less studies
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The field of ancient DNA (aDNA) has recently been in a state of exponential growth, largely driven by the uptake of Next Generation Sequencing (NGS) techniques. Much of this work has focused on the mammalian megafauna and ancient humans, with comparatively less studies looking at micromammal fauna, despite the potential of these species in testing evolutionary, environmental and taxonomic theories. Several factors make micromammal fauna ideally suited for aDNA extraction and sequencing. Micromammal subfossil assemblages often include the large number of individuals appropriate for population level analyses, and, furthermore, the assemblages are frequently found in cave sites where the constant temperature and sheltered environment provide favourable conditions for DNA preservation. This review looks at studies that include the use of aDNA in molecular analysis of micromammal fauna, in order to examine the wide array of questions that can be answered in the study of small mammals using new palaeogenetic techniques. This study highlights the bias in current aDNA studies and assesses the future use of aDNA as a tool for the study of micromammal fauna. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessReview Time to Spread Your Wings: A Review of the Avian Ancient DNA Field
Received: 31 May 2017 / Revised: 27 June 2017 / Accepted: 10 July 2017 / Published: 18 July 2017
Cited by 1 | PDF Full-text (3563 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Ancient DNA (aDNA) has the ability to inform the evolutionary history of both extant and extinct taxa; however, the use of aDNA in the study of avian evolution is lacking in comparison to other vertebrates, despite birds being one of the most species-rich
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Ancient DNA (aDNA) has the ability to inform the evolutionary history of both extant and extinct taxa; however, the use of aDNA in the study of avian evolution is lacking in comparison to other vertebrates, despite birds being one of the most species-rich vertebrate classes. Here, we review the field of “avian ancient DNA” by summarising the past three decades of literature on this topic. Most studies over this time have used avian aDNA to reconstruct phylogenetic relationships and clarify taxonomy based on the sequencing of a few mitochondrial loci, but recent studies are moving toward using a comparative genomics approach to address developmental and functional questions. Applying aDNA analysis with more practical outcomes in mind (such as managing conservation) is another increasingly popular trend among studies that utilise avian aDNA, but the majority of these have yet to influence management policy. We find that while there have been advances in extracting aDNA from a variety of avian substrates including eggshell, feathers, and coprolites, there is a bias in the temporal focus; the majority of the ca. 150 studies reviewed here obtained aDNA from late Holocene (100–1000 yBP) material, with few studies investigating Pleistocene-aged material. In addition, we identify and discuss several other issues within the field that require future attention. With more than one quarter of Holocene bird extinctions occurring in the last several hundred years, it is more important than ever to understand the mechanisms driving the evolution and extinction of bird species through the use of aDNA. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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Open AccessReview Novel Substrates as Sources of Ancient DNA: Prospects and Hurdles
Received: 30 May 2017 / Revised: 22 June 2017 / Accepted: 10 July 2017 / Published: 13 July 2017
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Abstract
Following the discovery in the late 1980s that hard tissues such as bones and teeth preserve genetic information, the field of ancient DNA analysis has typically concentrated upon these substrates. The onset of high-throughput sequencing, combined with optimized DNA recovery methods, has enabled
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Following the discovery in the late 1980s that hard tissues such as bones and teeth preserve genetic information, the field of ancient DNA analysis has typically concentrated upon these substrates. The onset of high-throughput sequencing, combined with optimized DNA recovery methods, has enabled the analysis of a myriad of ancient species and specimens worldwide, dating back to the Middle Pleistocene. Despite the growing sophistication of analytical techniques, the genetic analysis of substrates other than bone and dentine remain comparatively “novel”. Here, we review analyses of other biological substrates which offer great potential for elucidating phylogenetic relationships, paleoenvironments, and microbial ecosystems including (1) archaeological artifacts and ecofacts; (2) calcified and/or mineralized biological deposits; and (3) biological and cultural archives. We conclude that there is a pressing need for more refined models of DNA preservation and bespoke tools for DNA extraction and analysis to authenticate and maximize the utility of the data obtained. With such tools in place the potential for neglected or underexploited substrates to provide a unique insight into phylogenetics, microbial evolution and evolutionary processes will be realized. Full article
(This article belongs to the Special Issue Novel and Neglected Areas of Ancient DNA Research)
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