Metabarcoding approaches use total and typically degraded DNA from environmental samples to analyse biotic assemblages and can potentially be carried out for any kinds of organisms in an ecosystem. These analyses rely on specific markers, here called metabarcodes, which should be optimized for taxonomic resolution, minimal bias in amplification of the target organism group and short sequence length. Using bioinformatic tools, we developed metabarcodes for several groups of organisms: fungi, bryophytes, enchytraeids, beetles and birds. The ability of these metabarcodes to amplify the target groups was systematically evaluated by (1) in silico PCRs using all standard sequences in the EMBL public database as templates, (2) in vitro PCRs of DNA extracts from surface soil samples from a site in Varanger, northern Norway, and (3) in vitro PCRs of DNA extracts from permanently frozen sediment samples of late-Pleistocene age (~ 16 000–50 000 yr BP) from two Siberian sites, Duvanny Yar and Main River. Comparison of the results from the in silico PCR with those obtained in vitro showed that the in silico approach offered a reliable estimate of the suitability of a marker. All target groups were detected in the environmental DNA, but we found large variation in the level of detection among the groups and between modern and ancient samples. Success rates for the Pleistocene samples were highest for fungal DNA, whereas bryophyte, beetle and bird sequences could also be retrieved, but to a much lesser degree. The metabarcoding approach has considerable potential for biodiversity screening of modern samples and also as a paleoecological tool.

Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results due to competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA we first optimised the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (C. antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analysis, the PCR can be biased towards amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified.

Although ancient DNA from sediments (sedaDNA) has been used to investigate past ecosystems, the approach has never been directly compared to the traditional methods of pollen and macrofossil analysis. We conducted a comparative survey of 18 ancient permafrost samples spanning the Late Pleistocene (46–12.5 thousand years ago), from the Taymyr Peninsula in northern Siberia. The results show that pollen, macrofossils and sedaDNA are complementary rather than overlapping, and in combination reveal more detailed information on plant palaeocommunities than can be achieved by each individual approach. SedaDNA and macrofossils share greater overlap in plant identifications than with pollen, suggesting that sedaDNA is local in origin. These two proxies also permit identification to lower taxonomic levels than pollen, enabling investigation of temporal changes in species composition and the determination of indicator species to describe environmental changes. Combining data from all three proxies, reveals an area continually dominated by a mosaic vegetation of tundra-steppe, pioneer and wet-indicator plants. Such vegetational stability is unexpected, given the severe climate changes taking place in the northern hemisphere during this time, with changes in average annual temperatures of > 22ºC. This may explain the abundance of ice-age mammals such as horse and bison in Taymyr Peninsula during the Pleistocene, and why it acted as a refugium for the last mainland woolly mammoth. Our finding reveals the benefits of combining sedaDNA, pollen and macrofossil for palaeovegetational reconstruction and add to the increasing evidence suggesting large areas of the northern hemisphere remained ecologically stable during the Late Pleistocene.

Nunataks are isolated bedrocks protruding through ice sheets. They vary in age, but represent island environments in “oceans” of ice through which organism dispersals and replacements can be studied over time. The J.A.D. Jensen’s Nunataks at the southern Greenland ice sheet are the most isolated nunataks on the northern hemisphere - some 30 km from the nearest biological source. They constitute around 2 km2 of ice-free land that was established in the early Holocene. We have investigated the changes in plant composition at these nunataks using both the results of surveys of the flora over the last 130 years, and through reconstruction of the vegetation from the end of the Holocene Thermal Maximum (5528±75 cal yr BP) using meta-barcoding of plant DNA recovered from the nunatak sediments (sedaDNA). Our results show that several of the plant species detected with sedaDNA are described from earlier vegetation surveys on the nunataks (in 1878, 1967 and 2009). In 1967, a much higher biodiversity was detected than from any other of the studied periods. While this may be related to differences in sampling efforts for the oldest period, it is not the case when comparing the 1967 and 2009 levels where the botanical survey was exhaustive. As no animals and humans are found on the nunataks, this change in diversity over a period of just 42 years must relate to environmental changes likely being climate-driven. This suggests that even the flora of fairly small and isolated ice-free areas reacts quickly to a changing climate.