Characterizing DNA preservation in degraded specimens of Amara alpina (Carabidae: Coleoptera)

DNA preserved in degraded beetle (Coleoptera) specimens, including those derived from dry‐stored museum and ancient permafrost‐preserved environments, could provide a valuable resource for researchers interested in species and population histories over timescales from decades to millenia. However, the potential of these samples as genetic resources is currently unassessed. Here, using Sanger and Illumina shotgun sequence data, we explored DNA preservation in specimens of the ground beetle Amara alpina, from both museum and ancient environments. Nearly all museum specimens had amplifiable DNA, with the maximum amplifiable fragment length decreasing with age. Amplification of DNA was only possible in 45% of ancient specimens. Preserved mitochondrial DNA fragments were significantly longer than those of nuclear DNA in both museum and ancient specimens. Metagenomic characterization of extracted DNA demonstrated that parasite‐derived sequences, including Wolbachia and Spiroplasma, are recoverable from museum beetle specimens. Ancient DNA extracts contained beetle DNA in amounts comparable to museum specimens. Overall, our data demonstrate that there is great potential for both museum and ancient specimens of beetles in future genetic studies, and we see no reason why this would not be the case for other orders of insect.     

A Statistical Approach to Identify Ancient Template DNA

One of the key problems in the study of ancient DNA is that of authenticating sequences obtained from PCR amplifications of highly degraded samples. Contamination of ancient samples and postmortem damage to endogenous DNA templates are the major obstacles facing researchers in this task. In particular, the authentication of sequences obtained from ancient human remains is thought by many to be rather challenging. We propose a novel approach, based on the c statistic, that can be employed to help identify the sequence motif of an endogenous template, based on a sample of sequences that reflect the nucleotide composition of individual template molecules obtained from ancient tissues (such as cloned products from a PCR amplification). The c statistic exploits as information the most common form of postmortem damage observed among clone sequences in ancient DNA studies, namely, lesion-induced substitutions caused by cytosine deamination events. Analyses of simulated sets of templates with miscoding lesions and real sets of clone sequences from the literature indicate that the c-based approach is highly effective in identifying endogenous sequence motifs, even when they are not present among the sampled clones. The proposed approach is likely to be of general use to researchers working with DNA from ancient tissues, particularly from human remains, where authentication of results has been most challenging. [Reviewing Editor: Dr. Magnus Nordborg]     

Proboscidean DNA from Museum and Fossil Specimens: An Assessment of Ancient DNA Extraction and Amplification Techniques

Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.     

The never‐ending story of geologically ancient DNA: was the model plant Arabidopsis the source of Miocene Dominican amber?

Studies characterizing geologically ancient DNA in plants are rare, and all have reportedly obtained plastid DNA sequences from Miocene fossils in a remarkable state of preservation. Recently, a group made the extraordinary claim of having amplified a geologically ancient Miocene plastid DNA fragment (the rbcL gene) from Dominican amber nuggets, and the organismal source of this DNA was identified as Hymenaea protera (Fabaceae), the plant that produced the fossilized Dominican amber. Assuming that the Miocene sequence is error‐free, reanalysis of the sequence indicates it is probably a technical artifact or an rbcL pseudogene. Furthermore, BLAST similarity searches and phylogenetic analyses strongly suggest that the putative Miocene sequence retrieved from fossilized amber is in fact a modern contaminant from one of the most widely used model plants, Arabidopsis thaliana. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 234–240.     

Ancient hyaenas highlight the old problem of estimating evolutionary rates

Phylogenetic analyses of ancient DNA data can provide a timeline for evolutionary change even in the absence of fossils. The power to infer the evolutionary rate is, however, highly dependent on the number and age of samples, the information content of the sequence data and the demographic history of the sampled population. In this issue of Molecular Ecology, Sheng et al. ( 2014 ) analysed mitochondrial DNA sequences isolated from a combination of ancient and present‐day hyaenas, including three Pleistocene samples from China. Using an evolutionary rate inferred from the ages of the ancient sequences, they recalibrated the timing of hyaena diversification and suggest a much more recent evolutionary history than was believed previously. Their results highlight the importance of accurately estimating the evolutionary rate when inferring timescales of geographical and evolutionary diversification.     

Difference in the location inDasycladaceae of a DNA sequence homologous to theDrosophila per locus

The period (per) locus ofDrosophila melanogaster has a fundamental role on the expression of biological rhythms. A DNA sequence, which is homologous to a short region of theDrosophila per locus, has been found at different locations in various species of Dasycladaceae. InBatophora oerstedii, one of the phylogenetically oldest member of Dasycladaceae, a DNA sequence homologous to theDrosophila per locus was detected only in the chloroplast genome but not in the nuclear genome. In contrast, inAcetabularia cliftonii which in phylogeny branched off Batophora 350 million years ago, like in higher plants, theper locus homologous sequence is located in the nuclear rather than the chloroplast genome. The difference in the location of this sequence in phylogenetically separated species of the ancient unicellular and uninucleate green algae suggests gene translocation between the chloroplast genome and the nuclear genome during evolution.Abbreviations nDNA nuclear DNA - ctDNA chloroplast DNA     

Histone Synthesis inTrypanosoma cruzi

Trypanosoma cruziis an ancient, parasitic eukaryote which does not undergo chromatin condensation during cell division. This behavior may be explained if one considers the strong amino acid sequence divergence ofTrypanosomahistones compared to higher eukaryotes. In the latter organisms histone synthesis is coupled to DNA replication. Considering the nonconserved amino acid sequence ofT. cruzihistones, as well as the absence of chromatin condensation in this organism, we have studied histone synthesis in relation to DNA replication in this parasite. We have found that core histones and a fraction of histone H1 are synthesized concomitantly to DNA replication. However, another fraction of histone H1 is constitutively synthesized.     

Characterization of a family of tandemly repeated DNA sequences in Triticeae

The recombinant plasmid dpTa1 has an insert of relic wheat DNA that represents a family of tandemly organized DNA sequences with a monomeric length of approximately 340 bp. This insert was used to investigate the structural organization of this element in the genomes of 58 species within the tribe Triticeae and in 7 species representing other tribes of the Poaceae. The main characteristic of the genomic organization of dpTa1 is a classical ladder-type pattern which is typical for tandemly organized sequences. The dpTa1 sequence is present in all of the genomes of the Triticeae species examined and in 1 species from a closely related tribe (Bromus inermis, Bromeae). DNA from Hordelymus europaeus (Triticeae) did not hybridize under the standard conditions used in this study. Prolonged exposure was necessary to obtain a weak signal. Our data suggest that the dpTa1 family is quite old in evolutionary terms, probably more ancient than the tribe Triticeae. The dpTa1 sequence is more abundant in the D-genome of wheat than in other genomes in Triticeae. DNA from several species also have bands in addition to the tandem repeats. The dpTa1 sequence contains short direct and inverted subrepeats and is homologous to a tandemly repeated DNA sequence from Hordeum chilense.     

The value of DNA sequence data for studying landscape genetics

In a recent Opinion article in Molecular Ecology, Wang (2010) emphasizes the fact that current patterns of genetic differentiation among populations reflect processes that have acted over temporal scales ranging from contemporary to ancient. He draws a sharp distinction between the fields of phylogeography (as the study of historical processes) and landscape genetics (which he restricts to very recent processes). Wang characterizes DNA sequence data as being inappropriate for the study of contemporary population processes and further states that studies which only include mitochondrial DNA or chloroplast DNA data cannot be considered part of landscape genetics. In this response, we clarify the generally accepted view that DNA sequence data can be analysed with methods that separate contemporary and historical processes. To illustrate this point, we summarize the study of Vandergast et al. (2007), which Wang mischaracterizes as being confused in terms of temporal scale. Although additional focus should be placed on the important issue of correct data interpretation, we disagree strongly with the implication that contemporary and historic processes cannot be separated in the analyses of DNA sequence data.     

Non-repetitive DNA sequence divergence in phylogenetically diploid and tetraploid teleostean species of the family cyprinidae and the order isospondyli

Non-repetitive DNA of anciently tetraploid teleostean species was analysed for the presence of duplicated sequences. Closely related diploid species were investigated in comparison. From the reassociation kinetics of total nuclear DNA, rate constants and fraction sizes of classes of repetitive and non-repetitive sequences were determined. DNA fractions enriched in the slowest renaturing sequence class were prepared and subjected to reassociation. The rate constants of these reactions were compared with the values expected for single-copy DNA from analytical genome size determinations. From reassociated DNA enriched in non-repetitive sequences also the melting temperatures were determined as a measure of internal base sequence heterogeneity. It has been shown that the two ancient tetraploids Cyprinus carpio and Thymallus thymallus are, with regard to the thermal stability of reassociated non-repetitive DNA, and with regard to the correspondence of reaction rates with the values expected for single copy DNA, indistinguishable from diploid controls (Rutilus rutilus, Clupea harengus and Sprattus sprattus). The tetraploid species Salmo irideus, Salvelinus fontinalis and Coregonus lavaretus appear as very recent tetraploids with regard to these criteria. The significance of the results for estimating the time of occurence of polyploidisation events in these taxa is discussed.     

Very old DNA

The verification of DNA sequences obtained from very old tissue sources as indeed ancient is a major point of discussion in the ancient DNA field. Proper controls and the use of the phylogenetic approach are the general methods employed for verification of the ancient DNA. Most studies have reported the recovery of extremely small amounts of nucleic acids which are sheared into rather small fragments. In addition, problems such as ‘PCR jumping’ can produce spurious sequence information. These observations suggest that random amplification techniques and the development of primers for highly informative but short target regions are essential for the further development of the ancient DNA field.     

Intragenomic DNA sequence homologies in the chicken and other members of the classAves: DNA re-association under reduced stringency conditions

Summary We have investigated the intragenomic DNA sequence homologies of twelve species of birds representing five orders, and emphasizing Galliformes. This study differs in two important ways from the classical approaches taken in constructing and evaluating phylogenies based on DNA sequence similarities. Comparisons are made on the basis of sequence homologieswithin genomes of related birds, rather than between genomes. DNA is reassociated at 50°C in 0.5M phosphate buffer; these conditions allow formation and detection of duplexes containing more mismatch than would normally be permitted using more stringent conditions, affording an opportunity to observe more ancient sequence homologies. Thermal stability profiles of DNA duplexes formed under these conditions are the basis of comparison; three general patterns were observed. This approach emphasizes differences in sequence composition between genomes while the more traditional method of intergenomic tracer DNA hybridization at higher stringency emphasizes sequence similarities.No correlation was found between taxonomic position and intragenomic sequence composition, either within or between lineages. The thermal stability profiles of DNA duplexes formed within avian genomes did not reflect the biological similarities inferred from morphology, karyotype, and studies of interspecific hybridization. While all of the differences observed could have occurred over geological time, it was surprising that the genomes of the domestic chicken and the Red Jungle Fowl (Gallus gallus) differ in their sequence compositions. It appears that amplification/reduction events and/or positional changes occur rather often during evolution of a lineage.Abbreviations SDS sodium dodecyl sulphate - PB equimolar sodium phosphate buffer pH 6.8 - Cot concentration of DNA in moles of nucleotide per liter times the incubation time in seconds - Equiv. or Equivalent Cot Cot corrected for the monovalent cation concentration effect on re-association rate - HAP hydroxylapatite - Te_1/2 temperature at which one-half the DNA has eluted from HAP - SSC 0.15M sodium chloride-0.015M sodium citrate     

DNA sequences repaired at pachytene exhibit strong homology among distantly related higher plants

Moderately repetitive DNA sequences in Lilium (cv Enchantment) which undergo a meiotic-specific repair synthesis during pachytene (P-DNA) were previously shown to exist as families of very low internal sequence divergence. The present study concerns P-DNA sequence preservation among higher plants. The relative abundance of these sequences in a variety of plant species and their divergence relative to Enchantment P-DNA was determined through C₀t analysis and thermal denaturation of hybrid duplexes. Nearly all of the P-DNA sequence families of Enchantment were found to be present in the genomes of a number of monocot species and the dicot Vicia faba. Sequence content is highly conserved, with less than 6% divergence between Lilium and distantly related species such as Zea mays and Secale cereale. However, the number of repeats per P-DNA family varies considerably in different species, being particularly low among the Poales. P-DNA differs from most high thermal stability (HTS) sequence families of Enchantment which, although exhibiting a high degree of internal homology, are not present as repetitive DNA in the genomes of the other species examined. For most HTS families, the lack of internal divergence probably reflects their fairly recent introduction into the moderately repetitive DNA class, while P-DNA sequences represent evolutionarily ancient families which are the products of strong selective pressure for an indispensable meiotic function.     

Repeated DNA sequences inTriticum (Poaceae): Chromosomal mapping and its bearing on the evolution of B and G genomes

The somatic chromosomes ofTriticum turgicum var.durum cv. Langdon andT. dicoccoides (AABB tetraploids),T. timopheevii, andT. araraticum (AAGG tetraploids) were assayed for distribution patterns of a highly repeated 120bp DNA sequence by in situ hybridization. The repeated sequence appears to be an ancient sequence shared withSecale andAegilops. The distribution patterns of the chromosomes were compared to the patterns of the A and B genome chromosomes ofT. aestivum cv. Chinese Spring (AABBDD hexaploid).T. turgidum andT. dicoccoides were observed to have identical in situ hybridization patterns. In both species, nine chromosomes with a total of 21 sites of hybridization were observed. The pattern, with few exceptions, was identical to that of Chinese Spring.T. araraticum andT. timopheevii were observed to have different patterns. InT. araraticum, six chromosomes with 21 total hybridization sites are present while inT. timopheevii nine chromosomes with 19 total sites exist. Major differences in hybridization patterns were observed between the B and G genomes. The divergence of the tetraploid wheats in this study appears to have resulted in changes in location, not in amount, of the ancient repeated sequence.     

Unusual and Strongly Structured Sequence Variation in a Complex Satellite DNA Family from the Nematode Meloidogyne chitwoodi

An AluI satellite DNA family has been isolated in the genome of the root-knot nematode Meloidogyne chitwoodi. This repeated sequence was shown to be present at approximately 11,400 copies per haploid genome, and represents about 3.5% of the total genomic DNA. Nineteen monomers were cloned and sequenced. Their length ranged from 142 to 180 bp, and their A + T content was high (from 65.7 to 79.1%), with frequent runs of As and Ts. An unexpected heterogeneity in primary structure was observed between monomers, and multiple alignment analysis showed that the 19 repeats could be unambiguously clustered in six subfamilies. A consensus sequence has been deduced for each subfamily, within which the number of positions conserved is very high, ranging from 86.7% to 98.6%. Even though blocks of conserved regions could be observed, multiple alignment of the six consensus sequences did not enable the establishment of a general unambiguous consensus sequence. Screening of the six consensus sequences for evidence of internal repeated subunits revealed a 6-bp motif (AAATTT), present in both direct and inverted orientation. This motif was found up to nine times in the consensus sequences, also with the occurrence of degenerated subrepeats. Along with the meiotic parthenogenetic mode of reproduction of this nematode, such structural features may argue for the evolution of this satellite DNA family either (1) from a common ancestral sequence by amplification followed by mechanisms of sequence divergence, or (2) through independent mutations of the ancestral sequence in isolated amphimictic nematode populations and subsequent hybridization events. Overall, our results suggest the ancient origin of this satellite DNA family, and may reflect for M. chitwoodi a phylogenetic position close to the ancestral amphimictic forms of root-knot nematodes. Received: 23 April 1997 / Accepted: 9 July 1997     

Co‐amplification of cytochrome b and D‐loop mtDNA fragments for the identification of degraded DNA samples

We propose a simple and effective approach to simultaneously co‐amplify both cytochrome b and D‐loop fragments to evaluate DNA preservation and to monitor possible contaminations in the analysis of degraded animal DNA samples. We have applied this approach to over 200 ancient salmon samples and 25 ancient whale DNA samples, clearly demonstrating its multiple benefits for analysis of degraded DNA samples, and the ease in which co‐amplification can be optimized for different taxa. This simple, cost‐efficient and genomic DNA‐saving approach can be used routinely in the analysis of minute and degraded DNA samples in wildlife forensics, food inspection, conservation biology and ancient faunal remains.     

古代DNA序列的分析与甄别──兼评恐龙DNA研究

从古代生物中获取的古代ＤＮＡ序列为进一步从分子水平上认识古代生命及其演化提供了新的证据。分析和甄别古代ＤＮＡ序列是古代ＤＮＡ研究的关键步骤之一。本文着重讨论甄别古代ＤＮＡ序列的主要标准如：野外取样与实验室控制,ＤＮＡ“行为”分析、系统发育验证和实验结果的可重要性,并在此基础上评述近期发表的有关恐龙ＤＮＡ的研究。     

Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies

PCR analyses of ancient and degraded DNA suffer from their extreme sensitivity to contamination by modern DNA originating, in particular, from carryover contamination with previously amplified or cloned material. Any strategy for limiting carryover contamination would also have to be compatible with the particular requirements of ancient DNA analyses. These include the need (i) to amplify short PCR products due to template fragmentation; (ii) to clone PCR products in order to track possible base misincorporation resulting from damaged templates; and (iii) to avoid incomplete decontamination causing artifactual sequence transformation. Here we show that the enzymatic decontamination procedures based upon dUTP- and uracil-N-glycosylase (UNG) can be adapted to meet the specific requirements of ancient DNA research. Thus, efficiency can be improved to vastly reduce the amplification of fragments < or = 100 bp. Secondly, the use of an Escherichia coli strain deficient in both UNG and dUTPase allows for the cloning of uracil-containing PCR products and offers protection from plasmid DNA contamination, and, lastly, PCR products amplified from UNG-degraded material are free of misleading sequence modifications.     

An Alphoid-Like Satellite DNA Sequence Is Present in the Genome of a Lacertid Lizard

A PstI DNA family was isolated from the genome of a lacertid, Lacerta graeca. The 185-bp monomeric unit (pGPS) was cloned and hybridized to DNAs and chromosomes of several lacertid species. The data showed that pGPS hybridizes to the (1) centromeric or pericentromeric heterochromatin of almost all the chromosomes of L. graeca and (2) genomic DNA of species phylogenetically related and unrelated to L. graeca. The presence of pGPS even in species immunologically apart more than 30 million years suggests that this repeated family might be either very ancient or have been conserved during evolution due to its functional role. The latter hypothesis might be supported by the results of sequence analysis which showed some homology with both several alphoid sequences of primates and the CDEIII centromeric sequence of yeast. Segments of the satellite sequence are similar to the mammalian CENP-B box. These observations suggest that pGPS might have a role in determining the centromeric function in lacertid lizards. Received: 6 February 1997 / Accepted: 14 May 1997     

Using next-generation sequencing for molecular reconstruction of past Arctic vegetation and climate

Palaeoenvironments and former climates are typically inferred from pollen and macrofossil records. This approach is time-consuming and suffers from low taxonomic resolution and biased taxon sampling. Here, we test an alternative DNA-based approach utilizing the P6 loop in the chloroplast trnL (UAA) intron; a short (13–158 bp) and variable region with highly conserved flanking sequences. For taxonomic reference, a whole trnL intron sequence database was constructed from recently collected material of 842 species, representing all widespread and/or ecologically important taxa of the species-poor arctic flora. The P6 loop alone allowed identification of all families, most genera (>75%) and one-third of the species, thus providing much higher taxonomic resolution than pollen records. The suitability of the P6 loop for analysis of samples containing degraded ancient DNA from a mixture of species is demonstrated by high-throughput parallel pyrosequencing of permafrost-preserved DNA and reconstruction of two plant communities from the last glacial period. Our approach opens new possibilities for DNA-based assessment of ancient as well as modern biodiversity of many groups of organisms using environmental samples.