Laura Saskia Epp

Fifty thousand years of Arctic vegetation and megafaunal diet

Although it is generally agreed that the Arctic flora is among the youngest and least diverse on Earth, the processes that shaped it are poorly understood. Here we present 50 thousand years (kyr) of Arctic vegetation history, derived from the first large-scale ancient DNA metabarcoding study of circumpolar plant diversity. For this interval we also explore nematode diversity as a proxy for modelling vegetation cover and soil quality, and diets of herbivorous megafaunal mammals, many of which became extinct around 10 kyr bp (before present). For much of the period investigated, Arctic vegetation consisted of dry steppe-tundra dominated by forbs (non-graminoid herbaceous vascular plants). During the Last Glacial Maximum (25–15 kyr bp), diversity declined markedly, although forbs remained dominant. Much changed after 10 kyr bp, with the appearance of moist tundra dominated by woody plants and graminoids. Our analyses indicate that both graminoids and forbs would have featured in megafaunal diets. As such, our findings question the predominance of a Late Quaternary graminoid-dominated Arctic mammoth steppe.

New environmental metabarcodes for analysing soil DNA: potential for studying past and present ecosystems

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 (i) in silico PCRs using all standard sequences in the EMBL public database as templates, (ii) in vitro PCRs of DNA extracts from surface soil samples from a site in Varanger, northern Norway and (iii) in vitro PCRs of DNA extracts from permanently frozen sediment samples of late‐Pleistocene age (∼16 000–50 000 years 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 palaeoecological tool.

Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

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 because of 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 optimized 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 (Coelodonta 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 analyses, the PCR can be biased towards the 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.

Fungal palaeodiversity revealed using high-throughput metabarcoding of ancient DNA from arctic permafrost.

The taxonomic and ecological diversity of ancient fungal communities was assessed by combining next generation sequencing and metabarcoding of DNA preserved in permafrost. Twenty-six sediment samples dated 16 000-32 000 radiocarbon years old from two localities in Siberia were analysed for fungal ITS. We detected 75 fungal OTUs from 21 orders representing three phyla, although rarefaction analyses suggested that the full diversity was not recovered despite generating an average of 6677 ± 3811 (mean ± SD) sequences per sample and that preservation bias likely has considerable effect on the recovered DNA. Most OTUs (75.4%) represented ascomycetes. Due to insufficient sequencing depth, DNA degradation and putative preservation biases in our samples, the recovered taxa probably do not represent the complete historic fungal community, and it is difficult to determine whether the fungal communities varied geographically or experienced a composition shift within the period of 16 000-32 000 bp. However, annotation of OTUs to functional ecological groups provided a wealth of information on the historic communities. About one-third of the OTUs are presumed plant-associates (pathogens, saprotrophs and endophytes) typical of graminoid- and forb-rich habitats. We also detected putative insect pathogens, coprophiles and keratinophiles likely associated with ancient insect and herbivore faunas. The detection of putative insect pathogens, mycoparasites, aquatic fungi and endophytes broadens our previous knowledge of the diversity of fungi present in Beringian palaeoecosystems. A large group of putatively psychrophilic/psychrotolerant fungi was also detected, most likely representing a modern, metabolically active fungal community.

Use of ancient sedimentary DNA as a novel conservation tool for high-altitude tropical biodiversity.

Conservation of biodiversity may in the future increasingly depend upon the availability of scientific information to set suitable restoration targets. In traditional paleoecology, sediment-based pollen provides a means to define preanthropogenic impact conditions, but problems in establishing the exact provenance and ecologically meaningful levels of taxonomic resolution of the evidence are limiting. We explored the extent to which the use of sedimentary ancient DNA (sedaDNA) may complement pollen data in reconstructing past alpine environments in the tropics. We constructed a record of afro-alpine plants retrieved from DNA preserved in sediment cores from 2 volcanic crater sites in the Albertine Rift, eastern Africa. The record extended well beyond the onset of substantial anthropogenic effects on tropical mountains. To ensure high-quality taxonomic inference from the sedaDNA sequences, we built an extensive DNA reference library covering the majority of the afro-alpine flora, by sequencing DNA from taxonomically verified specimens. Comparisons with pollen records from the same sediment cores showed that plant diversity recovered with sedaDNA improved vegetation reconstructions based on pollen records by revealing both additional taxa and providing increased taxonomic resolution. Furthermore, combining the 2 measures assisted in distinguishing vegetation change at different geographic scales; sedaDNA almost exclusively reflects local vegetation, whereas pollen can potentially originate from a wide area that in highlands in particular can span several ecozones. Our results suggest that sedaDNA may provide information on restoration targets and the nature and magnitude of human-induced environmental changes, including in high conservation priority, biodiversity hotspots, where understanding of preanthropogenic impact (or reference) conditions is highly limited.

Hidden diversity in diatoms of Kenyan Lake Naivasha: a genetic approach detects temporal variation

This study provides insights into the morphological and genetic diversity in diatoms occurring in core sediments from tropical lakes in Kenya. We developed a genetic survey technique specific for diatoms utilizing a short region (76–67 bp) of the ribulose‐1,5‐bisphosphate carboxylase/oxygenase large subunit (rbcL) gene as genetic barcode. Our analyses (i) validated the use of rbcL as a barcoding marker for diatoms, applied to sediment samples, (ii) showed a significant correlation between the results obtained by morphological and molecular data and (iii) indicated temporal variation in diatom assemblages on the inter‐ and intra‐specific level. Diatom assemblages from a short core from Lake Naivasha show a drastic shift over the last 200 years, as littoral species (e.g. Navicula) are replaced by more planktonic ones (e.g. Aulacoseira). Within that same period, we detected periodic changes in the respective frequencies of distinct haplotype groups of Navicula, which coincide with wet and dry periods of Lake Naivasha between 1820 and 1938 AD. Our genetic analyses on historical lake sediments revealed inter‐ and intra‐specific variation in diatoms, which is partially hidden behind single morphotypes. The occurrence of particular genetic lineages is probably correlated with environmental factors.

Highly overlapping winter diet in two sympatric lemming species revealed by DNA metabarcoding

Sympatric species are expected to minimize competition by partitioning resources, especially when these are limited. Herbivores inhabiting the High Arctic in winter are a prime example of a situation where food availability is anticipated to be low, and thus reduced diet overlap is expected. We present here the first assessment of diet overlap of high arctic lemmings during winter based on DNA metabarcoding of feces. In contrast to previous analyses based on microhistology, we found that the diets of both collared (Dicrostonyx groenlandicus) and brown lemmings (Lemmus trimucronatus) on Bylot Island were dominated by Salix while mosses, which were significantly consumed only by the brown lemming, were a relatively minor food item. The most abundant plant taxon, Cassiope tetragona, which alone composes more than 50% of the available plant biomass, was not detected in feces and can thus be considered to be non-food. Most plant taxa that were identified as food items were consumed in proportion to their availability and none were clearly selected for. The resulting high diet overlap, together with a lack of habitat segregation, indicates a high potential for resource competition between the two lemming species. However, Salix is abundant in the winter habitats of lemmings on Bylot Island and the non-Salix portion of the diets differed between the two species. Also, lemming grazing impact on vegetation during winter in the study area is negligible. Hence, it seems likely that the high potential for resource competition predicted between these two species did not translate into actual competition. This illustrates that even in environments with low primary productivity food resources do not necessarily generate strong competition among herbivores.

Shedding new light on the diet of Norwegian lemmings: DNA metabarcoding of stomach content

Lemmings are key herbivores in many arctic food webs, and their population dynamics have major impacts on the functioning of tundra systems. However, current knowledge of lemming diet is limited, hampering evaluation of lemming–vegetation interactions. This lack of knowledge is mainly due to methodological challenges, as previously used microhistological methods result in large proportions of poorly resolved plant taxa. We analyzed diets of Norwegian lemmings (Lemmus lemmus) in three different habitats using a new method, DNA metabarcoding of stomach contents. To achieve detailed information on ingested vascular plants, bryophytes, and fungi, we amplified short fragments of chloroplast DNA (for plants; P6 loop of the trnL intron) and nuclear ribosomal DNA (for fungi; ITS1-region). Our results revealed that lemming diets were dominated by grasses, mainly Avenella flexuosa, and mosses, mainly Dicranum spp., but that a variety of other food items were also eaten. Vascular plant composition of the diets differed between heath, meadow, and wetland habitats, whereas bryophyte composition did not. Also, a variety of fungal taxa were retrieved, but as most of the identified taxa belong to micromycetes, they were unlikely to be consumed as food. The role of fungi in the diet of lemmings remains to be investigated. We suggest that there may be substantial variation between habitats and regions in lemming diet.

Molecular profiling of diatom assemblages in tropical lake sediments using taxon‐specific PCR and Denaturing High‐Performance Liquid Chromatography (PCR‐DHPLC)

Here we present a protocol to genetically detect diatoms in sediments of the Kenyan tropical Lake Naivasha, based on taxon‐specific PCR amplification of short fragments (approximately 100 bp) of the small subunit ribosomal (SSU) gene and subsequent separation of species‐specific PCR products by PCR‐based denaturing high‐performance liquid chromatography (DHPLC). An evaluation of amplicons differing in primer specificity to diatoms and length of the fragments amplified demonstrated that the number of different diatom sequence types detected after cloning of the PCR products critically depended on the specificity of the primers to diatoms and the length of the amplified fragments whereby shorter fragments yielded more species of diatoms. The DHPLC was able to discriminate between very short amplicons based on the sequence difference, even if the fragments were of identical length and if the amplicons differed only in a small number of nucleotides. Generally, the method identified the dominant sequence types from mixed amplifications. A comparison with microscopic analysis of the sediment samples revealed that the sequence types identified in the molecular assessment corresponded well with the most dominant species. In summary, the PCR‐based DHPLC protocol offers a fast, reliable and cost‐efficient possibility to study DNA from sediments and other environmental samples with unknown organismic content, even for very short DNA fragments.

Genetic data from algae sedimentary DNA reflect the influence of environment over geography

Genetic investigations on eukaryotic plankton confirmed the existence of modern biogeographic patterns, but analyses of palaeoecological data exploring the temporal variability of these patterns have rarely been presented. Ancient sedimentary DNA proved suitable for investigations of past assemblage turnover in the course of environmental change, but genetic relatedness of the identified lineages has not yet been undertaken. Here, we investigate the relatedness of diatom lineages in Siberian lakes along environmental gradients (i.e. across treeline transects), over geographic distance and through time (i.e. the last 7000 years) using modern and ancient sedimentary DNA. Our results indicate that closely-related Staurosira lineages occur in similar environments and less-related lineages in dissimilar environments, in our case different vegetation and co-varying climatic and limnic variables across treeline transects. Thus our study reveals that environmental conditions rather than geographic distance is reflected by diatom-relatedness patterns in space and time. We tentatively speculate that the detected relatedness pattern in Staurosira across the treeline could be a result of adaptation to diverse environmental conditions across the arctic boreal treeline, however, a geographically-driven divergence and subsequent repopulation of ecologically different habitats might also be a potential explanation for the observed pattern.