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Analyses of sedimentary DNA (sedDNA) have increased exponentially over the last decade and hold great potential to study the effects of anthropogenic stressors on lake biota over time. Herein, we synthesise the literature that has applied a sedDNA approach to track historical changes in lake biodiversity in response to anthropogenic impacts, with an emphasis on the past c. 200 years. We identified the following research themes that are of particular relevance: (1) eutrophication and climate change as key drivers of limnetic communities; (2) increasing homogenisation of limnetic communities across large spatial scales; and (3) the dynamics and effects of invasive species as traced in lake sediment archives. Altogether, this review highlights the potential of sedDNA to draw a more comprehensive picture of the response of lake biota to anthropogenic stressors, opening up new avenues in the field of paleoecology by unrevealing a hidden historical biodiversity, building new paleo-indicators, and reflecting either taxonomic or functional attributes. Broadly, sedDNA analyses provide new perspectives that can inform ecosystem management, conservation, and restoration by offering an approach to measure ecological integrity and vulnerability, as well as ecosystem functioning. © 2022 The Authors. Freshwater Biology published by John Wiley & Sons Ltd.

Siberian larch (Larix Mill.) forests dominate vast areas of northern Russia and contribute important ecosystem services to the world. It is important to understand the past dynamics of larches in order to predict their likely response to a changing climate in the future. Sedimentary ancient DNA extracted from lake sediment cores can serve as archives to study past vegetation. However, the traditional method of studying sedimentary ancient DNA - metabarcoding - focuses on small fragments which cannot resolve Larix to species level nor allow a detailed study of population dynamics. Here we use shotgun sequencing and hybridization capture with long-range PCR-generated baits covering the complete Larix chloroplast genome to study Larix populations from a sediment core reaching back to 6700 years from the Taymyr region in northern Siberia. In comparison to shotgun sequencing, hybridization capture results in an increase of taxonomically classified reads by several orders of magnitude and the recovery of complete chloroplast genomes of Larix. Variation in the chloroplast reads corroborate an invasion of Larix gmelinii into the range of Larix sibirica before 6700 years ago. Since then, both species have been present at the site, although larch populations have decreased with only a few trees remaining in what was once a forested area. This study demonstrates for the first time that hybridization capture applied directly to ancient DNA of plants extracted from lake sediments can provide genome-scale information and is a viable tool for studying past genomic changes in populations of single species, irrespective of a preservation as macrofossil. published

Climate warming alters plant composition and population dynamics of arctic ecosystems. In particular, an increase in relative abundance and cover of deciduous shrub species (shrubification) has been recorded. We inferred genetic variation of common shrub species (Alnus alnobetula, Betula nana, Salix sp.) through time. Chloroplast genomes were assembled from modern plants (n = 15) from the Siberian forest-tundra ecotone. Sedimentary ancient DNA (sedaDNA; n = 4) was retrieved from a lake on the southern Taymyr Peninsula and analyzed by metagenomics shotgun sequencing and a hybridization capture approach. For A. alnobetula, analyses of modern DNA showed low intraspecies genetic variability and a clear geographical structure in haplotype distribution. In contrast, B. nana showed high intraspecies genetic diversity and weak geographical structure. Analyses of sedaDNA revealed a decreasing relative abundance of Alnus since 5,400 cal yr BP, whereas Betula and Salix increased. A comparison between genetic variations identified in modern DNA and sedaDNA showed that Alnus variants were maintained over the last 6,700 years in the Taymyr region. In accordance with modern individuals, the variants retrieved from Betula and Salix sedaDNA showed higher genetic diversity. The success of the hybridization capture in retrieving diverged sequences demonstrates the high potential for future studies of plant biodiversity as well as specific genetic variation on ancient DNA from lake sediments. Overall, our results suggest that shrubification has species-specific trajectories. The low genetic diversity in A. alnobetula suggests a local population recruitment and growth response of the already present communities, whereas the higher genetic variability and lack of geographical structure in B. nana may indicate a recruitment from different populations due to more efficient seed dispersal, increasing the genetic connectivity over long distances.

The modern, over 250-m-deep basin of Lake Constance represents the underfilled northern part of an over 400-m-deep, glacially overdeepened trough, which reaches well into the Alps at its southern end. The overdeepening was formed by repeated glacial advance-retreat cycles of the Rhine Glacier throughout the Middle to Late Pleistocene. A seismic survey of Lake Constance revealed a Quaternary sediment fill of more than 150 m thickness representing at least the last glacial cycle. The stratified sedimentary fill consists at the base of ice-contact deposits on top of the molasse bedrock, overlain by glaciolacustrine to lacustrine sediments. During the successful field test of a newly developed, mid-size coring system ("HIPERCORIG"), the longest core (HIBO19) ever taken in Lake Constance was retrieved with an overall length of 24 m. The sediments recovered consist of a nearly continuous succession of lacustrine silts and sands including more than 12 m of Late Glacial sediment at the base. 14 lithotypes were identified through petrophysical and geochemical analyses. In combination with a 14C- and OSL-based age-depth model, the core was divided into three main chronostratigraphic units. The basal age of ~13.7 ka BP dates the base of the succession back to the Bølling-Allerød interstadial, with overlying strata representing a complete and thick Younger-Dryas to Holocene succession. The sediments offer a high-resolution insight into the evolution of paleo-Lake Constance from a cold, postglacial to a more productive, warm Holocene lake. The Late Glacial succession is dominated by massive, m-thick sand beds reflecting episodic sedimentation pulses. They are most likely linked to a subaquatic channel system originating in the river Seefelder Aach, which is, despite the Holocene drape, still apparent in today's lake bathymetry. The overlying Holocene succession reveals a prominent, several cm-thick, double-turbiditic event layer representing the most distal impact of the Flimser Bergsturz, the largest known rockslide of the Alps that occurred over 100 km upstream the river Rhine at ~9.5 ka BP. Furthermore, lithologic variations in the Holocene succession document the varying sediment loads of the river Rhine and the endogenic production representing a multitude of environmental changes.

Climate change has a major impact on arctic and boreal terrestrial ecosystems as warming leads to northward treeline shifts, inducing consequences for heterotrophic organisms associated with the plant taxa. To unravel ecological dependencies, we address how long-term climatic changes have shaped the palaeo-ecosystems at selected sites in Siberia. We investigated sedimentary ancient DNA from five lakes spanning the last 47,000 years, using the ITS1 marker for fungi and the chloroplast P6 loop marker for vegetation metabarcoding. After bioinformatic processing with the OBItools pipeline, we obtained 706 unique fungal operational taxonomic units (OTUs) and 243 amplicon sequence variants (ASVs) for the plants. We show higher OTU numbers in dry forest tundra as well as boreal forests compared to wet southern tundra. The most abundant fungal taxa in our dataset are Pseudeurotiaceae, Mortierella, Sordariomyceta, Exophiala, Oidiodendron, Protoventuria, Candida vartiovaarae, Pseudeurotium, Gryganskiella fimbricystis, and Trichosporiella cerebriformis. The overall fungal composition is explained by the plant composition as revealed by redundancy analysis. The fungal functional groups show antagonistic relationships in their climate susceptibility. The advance of woody taxa in response to past warming led to an increase in the abundance of mycorrhizae, lichens, and parasites, while yeast and saprotroph distribution declined. We also show co-occurrences between Salicaceae, Larix, and Alnus and their associated pathogens and detect higher mycorrhizal fungus diversity with the presence of Pinaceae. Under future warming, we can expect feedback between fungus compositional and plant diversity changes which will affect forest advance and stability in arctic regions.

SummaryFungi are crucial organisms in most ecosystems as they exert ecological key functions and are closely associated with land plants. Fungal community changes may therefore help reveal biodiversity changes in past ecosystems. Lake sediments contain DNA of organisms in the catchment area, which allows reconstructing past biodiversity by using metabarcoding of ancient sedimentary DNA. We developed a novel PCR primer combination for fungal metabarcoding targeting a short amplicon to account for length bias of amplification due to ancient DNA degradation. In-silico PCRs showed higher diversity using this primer combination than using previously established fungal metabarcoding primers. We analyzed existing data from sediment cores from four artic and one boreal lake in Siberia. These cores had been stored for 2–22 years and examined degradation effects of ancient DNA and storage time-related bias in fungal communities. Amplicon size differed between fungal divisions, however, we observed no significant effect of sample age on amplicon length and GC content, suggesting robust results. We also found no indication of post-coring fungal growth during storage distorting ancient fungal communities. Terrestrial soil fungi, including mycorrhizal fungi and saprotrophs, were predominant in all lakes, which supports the use of lake sedimentary ancient DNA for reconstructing terrestrial communities.

Climate change has a major impact on arctic and boreal terrestrial ecosystems as warming leads to northward treeline shifts, inducing consequences for heterotrophic organisms associated with the plant taxa. To unravel ecological dependencies, we address how long-term climatic changes have shaped the co-occurrence of plants and fungi across selected sites in Siberia.We investigated sedimentary ancient DNA from five lakes spanning the last 47,000 years, using the ITS1 marker for fungi and the chloroplast P6 loop marker for vegetation metabarcoding. We obtained 706 unique fungal operational taxonomic units (OTUs) and 243 taxa for the plants. We show higher OTU numbers in dry forest tundra as well as boreal forests compared to wet southern tundra. The most abundant fungal taxa in our dataset are Pseudeurotiaceae, Mortierella, Sordariomyceta, Exophiala, Oidiodendron, Protoventuria, Candida vartiovaarae, Pseudeurotium, Gryganskiella fimbricystis, and Trichosporiella cerebriformis. The overall fungal composition is explained by the plant composition as revealed by redundancy analysis. The fungal functional groups show antagonistic relationships in their climate susceptibility. The advance of woody taxa in response to past warming led to an increase in the abundance of mycorrhizae, lichens, and parasites, while yeast and saprotroph distribution declined. We also show co-occurrences between Salicaceae, Larix, and Alnus and their associated pathogens and detect higher mycorrhizal fungus diversity with the presence of Pinaceae. Under future warming, we can expect feedbacks between fungus composition and plant diversity changes which will affect forest advance, species diversity, and ecosystem stability in arctic regions. published

Due to similarities in morphological features together with strong dispersal abilities, it was thought that some groups of zooplankton (e.g. rotifers, copepods, and cladocerans) have cosmopolitan distributions. In the particular case of cladocerans, recent molecular studies using DNA barcode regions have indicated a different picture, including the existence of multiple regional endemic species and geographical phylogroups; even at very small geographical scales. This has demostrated that cladocera species are less widely distributed than assumed. Morphological identifications of these animals require expertise and high taxonomic specialization. Even so, species identifications are hampered by the small size of the organisms (especially from the littoral zone) and by the sampling cost for obtaining rare species and both parthenogenetic and gamogenetic specimens. The use of molecular techniques can provide new tools to identify cryptic diversity, and by being added to taxonomical approaches, provide more precise data of biodiversity. However, the accuracy of species assignment in metabarcoding relies on the availability of a DNA reference library, which is challenging in areas with high endemicity rates such as the Iberian Peninsula. A preliminary compilation of the available molecular data for the cytochrome (COI) in public repositories (Barcode of Life Data Systems and NCBI GenBank) shows that the available sequences only cover ~60% of the Iberian freshwater cladocerans. The family Daphniidae is very well represented, while the family Chydoridae, which contained most of the Iberian endemism, is underrepresented. We have identified the gaps, and are now focusing on collecting the target organisms to fill the missing taxa of the Iberian library. A compendium of the sampling points, species recovered so far, pitfalls, and future strategy is presented here. The effective completion of a DNA database for cladocerans will have applications not only in biomonitoring programs but to develop DNA-based methods in paleolimnology.

Freshwater crayfish are among the most threatened animal taxa in Central Europe. Effective conservation measures for endangered native and management of invasive alien crayfishes requires knowledge about distribution, monitoring of existing and early detection of newly established populations. eDNA has recently emerged as a promsing, highly sensitive, and non-invasive detection tool in this regard. To evaluate eDNA as detection tool for freshwater crayfish, we developed a novel set of specific eDNA-assays for all native (Austropotamobius torrentium, Austropotamobius pallipes, Astacus astacus) and the most relevant invasive crayfish species (Pacifastacus leniusculus, Faxonius limosus, Faxonius immunis) in Central Europe. To ensure specificity each primer pair was tested in silico, in vitro, and in situ, including a total of 13 lotic and lentic waterbodies (Fig. 1). Moreover, we assessed the influence of spatio-temporal variables (distance to upstream population, season, and stream size) on eDNA detection in seven streams using two different detection methods (qualitative endpoint PCR and quantitative droplet digital PCR, ddPCR). The newly developed eDNA assays successfully detected all crayfish species across different lotic and lentic habitats. Multiple linear mixed-effects analysis revealed a significant effect of distance and season on eDNA detection rate (endpoint PCR) and eDNA-concentration (ddPCR). Year-round detection was successful up to 7 km downstream of the source population, although detectability was lowest in winter. eDNA detection rate further decreased with increasing stream size. Finally, eDNA-concentration correlated positively with estimated upstream population size. Overall, our study provides easily applicable eDNA assays for six crayfish species, enabling year-round detection, which represents a clear benefit over conventional methods. Due to its high sensitivity, eDNA detection is also suitable for the targeted search of as-yet unrecorded or newly emerging populations. Using quantitative ddPCR might further allow for a rough estimation of population size, provided that the identified spatio-temporal factors are accounted for. We therefore recommend implementing eDNA-detection as a complementary survey tool, particularly for a large-scale screening of data-deficient catchments or a year-round monitoring (Chucholl et al. 2021).

Climate change has a great impact on boreal ecosystems including Siberian larch forests. As a consequence of warming, larch grow is possible in areas where climate used to be too cold, leading to a shift of the tree line into more arctic regions. Most plants co-exist in symbiosis with heterotrophic organisms surrounding their root system. In arctic ecosystems, mycorrhizal fungi are a prerequisite for plant establishment and survival because they support nutrient uptake from nutrient-poor soils and maintain the water supply. Until now, however, knowledge about the co-variation of vegetation and fungi is poor. Certainly, the understanding of dynamic changes in biotic interactions is important to understand adaptation mechanisms of ecosystems to climate change.We investigated sedimentary ancient DNA from Lake Levinson Lessing, Taymyr Peninsula (Arctic Siberia, tundra), Lake Lama, Lake Kyutyunda (both northern Siberia, tundra-taiga transition zone) and Lake Bolshoe Toko (southern Siberia, forest area) covering the last about 45.000 years using ITS primers for fungi along with the chloroplast P6 loop marker for vegetation metabarcoding. We found changes in the fungal communities that are in broad agreement with vegetation turnover. To our knowledge, this is the first broad ecological study on lake sediment cores to analyze fungal biodiversity in relation to vegetation change on millennial time scales.

The modern basin of trinational Lake Constance, between Switzerland, Germany, and Austria, represents the underfilled northern part of a glacially overdeepened trough. It is over 400 m deep and reaches well into the Alps at its southern end. The overdeepening was formed by the numerous glacial advance-retreat cycles of the Rhine Glacier throughout the Middle to Late Quaternary. A seismic survey of Lake Constance revealed a Quaternary sediment fill of over 150 m thickness under the modern lake floor in a maximal water depth of >250 m. This sedimentary sequence represents at least the last glacial cycle with ice-contact deposits at the base on top of the Molasse bedrock overlain by glaciolacustrine to lacustrine sediments. During the successful field test of a newly developed mid-size coring system ("HIPERCORIG"), the longest core ever taken in Lake Constance was recovered with an overall length of 24 m. The drill core, taken in a water depth of 200 m, consists of a nearly continuous succession of lacustrine sediments including over 12 m of pre-Holocene sediment at the base. The entire core was petrophysically and geochemically analyzed, sedimentologically described, and 14 lithotypes were identified. In combination with a 14C- and OSL-based age-depth model, the core was divided into three main chronostratigraphic units. The basal age of ~13.7 ka BP places the base of the section back into the Bølling-Allerød interstadial whereas the overlying strata represent a complete Younger-Dryas and Holocene section.The sediments offer a high-resolution insight into the evolution of Paleolake Constance from a cold postglacial to a more productive warm Holocene lake. The Late Glacial sections are dominated by massive, m-thick sand beds reflecting episodic sedimentation pulses. They are most likely linked with a subaquatic channel system that is still apparent in today's lake bathymetry despite the Holocene drape. This channel system was fed from a Late Glacial river from the north; provenance analysis of the initially unexpected sands together with hydrologic considerations will document whether this inflowing high-discharge river represented a local catchment (i.e. northern lake shore) or an Alpine signal (i.e. from the south) provided by the Rhine glacier. Tentative pore water hydrogeochemical and isotope analyses indicate a still active flow system at depth. The overlying Holocene section reveals a prominent, several cm-thick double-turbiditic event layer representing the most distal impact of the "Flimser Bergsturz", the largest known rock slide of the Alps that occurred over 100 km upstream the Rhine River at ~9.5 ka BP. Furthermore, lithologic variations in the Holocene section document the varying sediment load of the Rhine and of the endogenic production representing a multitude of environmental changes.

The advent of pastoralism in Eastern Africa is one of the most significant cultural transformations in the continent’s history. Traditionally, herding origins and its spreading routes have been studied in the lowlands and described as a complex and lengthy process that began before 4 ka BP and lasted until 1.3 ka BP. This cultural transition has long been argued to have been a process involving both environmental change and population movements. Given the current patchy archaeological data, most studies studies conclude that no single factor can be identified as a driver of the onset of herding in Eastern Africa, but almost all evidence is from lowland areas. The higher elevations of the Eastern African mountains are sensitive to climate and environmental change, so may be ideal for testing hypotheses of human-environmental relationships. However, the history of pastoralism in the African highlands, especially its connection with regional herding migrations and Holocene climate change, has thus far been poorly explored with few available records. In this contribution, we provide evidence of early pastoral activities at high altitude in the Bale Mountains of southwest Ethiopia. We present a 4000-year multiproxy palaeoecological lacustrine sequence from Garba Guracha, a cirque lake at 3950 m asl, combining palaeoclimatic and palaeoenvironmental proxies. Our record indicates the distinctive presence of faecal fungal spores (Sporormiella, Cercospora, Podospora) and the expansion of pollen and sedaDNA from ruderal plants as early as 3.5 ka. To our knowledge, this is the highest altitude record of early animal husbandry traces on the continent. Coeval with the expansion of pastoralism indicators in Garba Guracha, we find important changes in the lake’s diatom community, as well as climate fluctuations reconstructed from biomarkers; these may be critical for understanding human occupation at high altitudes. However, archaeological studies conducted in the Garba Guracha basin have proved unfruitful in finding permanent settlements of herders, suggesting hypotheses of seasonal resource use. We discuss different scenarios of pastoral expansion on the Eastern African highlands under changing local climates, as well as the general context of pastoralist migration across Eastern Africa.

Lake sedimentary ancient DNA has become a recognized source of information on past biodiversity change, but our understanding of its distribution and taphonomy is still limited. Here we collected 40 surface sediment samples from Lake Constance in southern Germany and characterized sedimentary DNA (sedDNA) heterogeneity through four metabarcoding PCRs on general eukaryotes, vascular plants, cyanobacteria and copepods. We observed the variability of overall sedDNA being strongly correlated with sediment sample geographical locations, although this pattern varies within taxonomic groups. Specifically, geographical coordination and water depth explain 10.7% variability of terrestrial vascular plant sedDNA distribution, but the distribution of copepods is patchy and not correlated with sampling locations. Out study indicates that sedDNA is not distributed uniformly across the lake but rather linked to the movement of lake water and the local presence of the organisms.

International audience; The use of lake sedimentary DNA to track the long-term changes in both terrestrial and aquatic biota is a rapidly advancing field in paleoecological research. Although largely applied nowadays, knowledge gaps remain in this field and there is therefore still research to be conducted to ensure the reliability of the sedimentary DNA signal. Building on the most recent literature and seven original case studies, we synthesize the state-of-the-art analytical procedures for effective sampling, extraction, amplification, quantification and/or generation of DNA inventories from sedimentary ancient DNA (sedaDNA) via high-throughput sequencing technologies. We provide recommendations based on current knowledge and best practises.

Studies along elevational gradients worldwide usually find the highest plant taxa richness in mid-elevation forest belts. Hence, an increase in upper elevation diversity is expected in the course of warming-related treeline rise. Here, we use a time-series approach to infer past taxa richness from sedimentary ancient DNA from the south-eastern Tibetan Plateau over the last ~18,000 years. We find the highest total plant taxa richness during the cool phase after glacier retreat when the area contained extensive and diverse alpine habitats (14–10 ka); followed by a decline when forests expanded during the warm early- to mid-Holocene (10–3.6 ka). Livestock grazing since 3.6 ka promoted plant taxa richness only weakly. Based on these inferred dependencies, our simulation yields a substantive decrease in plant taxa richness in response to warming-related alpine habitat loss over the next centuries. Accordingly, efforts of Tibetan biodiversity conservation should include conclusions from palaeoecological evidence., Long-term sedimentary ancient DNA (sedaDNA) records can help inform how biodiversity will likely respond to future climate change. Here, Liu et al. reconstruct plant diversity at the margin of the Tibetan Plateau over the last ~18,000 years using sedaDNA and use this record to predict future diversity change.

During the 20th century, many lakes in the Northern Hemisphere were affected by increasing human population and urbanisation along their shorelines and catchment, resulting in aquatic eutrophication. Ecosystem monitoring commenced only after the changes became apparent, precluding any examination of timing and dynamics of initial community change in the past and comparison of pre- and post-impact communities. Peri-Alpine Lake Constance (Germany) underwent a mid-century period of eutrophication followed by re-oligotrophication since the 1980s, and is now experiencing warm temperatures. We extended the period for which monitoring data of indicator organisms exists by analysing historical environmental DNA (eDNA) from a sediment core dating back some 110 years. Using three metabarcoding markers - for microbial eukaryotes, diatoms and cyanobacteria - we revealed two major breakpoints of community change, in the 1930s and the mid-1990s. In our core, the latest response was exhibited by diatoms, which are classically used as paleo-bioindicators for the trophic state of lakes. Following re-oligotrophication, overall diversity values reverted to similar ones of the early 20th century, but multivariate analysis indicated that the present community is substantially dissimilar. Community changes of all three groups were strongly correlated to phosphorus concentration changes, whereas significant relationships to temperature were only observed when we did not account for temporal autocorrelation. Our results indicate that each microbial group analysed exhibited a unique response, highlighting the particular strength of multimarker analysis of eDNA, which is not limited to organisms with visible remains and can therefore discover yet unknown responses and abiotic-biotic relationships. published

Sedimentary ancient DNA has by now become a recognized source of information on past biodiversity change, but our understanding of its dynamics and taphonomy is still limited. While for environmental DNA in water, dedicated investigations on its provenance and degradation are being increasingly carried out, we know very little about sedimentary DNA, in particular with respect to aquatic organisms. We are therefore conducting investigations on the distribution of DNA in surface sediments and a short sediment core, with a focus on aquatic communities in the large and heterogeneous Lake Constance. Targeted organisms range from phyto- and zooplankton to fish and waterbirds. Initial results and comparison with sightings of rare species indicate that the DNA is not distributed uniformly or widely across the lake, especially for multicellular animals, but rather linked to the local presence of the organisms. This has implications for our understanding of how DNA enters the sediment and for paleoecological inferences derived from these records.

Siberian larch (Larix Mill.) forests dominate vast areas of northern Russia and contribute important ecosystem services to the earth. To be able to predict future responses of these forests to a changing climate, it is important to understand also past dynamics of larch populations. One well-preserved archive to study vegetation changes of the past is sedimentary ancient DNA (sedaDNA) extracted from lake sediment cores. We studied a lake sediment core covering 6700 calibrated years BP, from the Taymyr region in northern Siberia. To enrich the sedaDNA for DNA of our focal species Larix, we combine shotgun sequencing and hybridization capture with long-range PCR-generated baits covering the complete Larix chloroplast genome. In comparison to shotgun sequencing, hybridization capture results in an increase of taxonomically classified reads by several orders of magnitude and the recovery of near-complete chloroplast genomes of Larix. Variation in the chloroplast reads confirm an invasion of Larix gmelinii into the range of Larix sibirica before 6700 years ago. In this time span, both species can be detected at the site, although larch populations have decreased from a forested area to a single-tree tundra at present. This study demonstrates for the first time that hybridization capture applied to ancient DNA from lake sediments can provide genome-scale information and is a viable tool for studying past changes of a specific taxon.

In recent decades Arctic and Alpine terrestrial ecosystems experienced an increase in aquatic plant biomass due to global warming, which motivates the investigation of aquatic plant diversity in High Arctic and Alpine regions, whereof so far only sparse data exist. Aquatic plants are important primary producers, food resource and supply habitat structure and thus have been widely used to infer the ecological status of modern lakes. Identification of past aquatic plants using macrofossil records only partly reflects the past community structure due to differences in spatial distribution, preservation and seed abundance of taxa. Thus, in our study we applied sedimentary DNA analyses to detect aquatic plant diversity in modern surface samples of over 200 lakes from various localities across Northern, Eastern and Central Siberia and the Tibetan plateau and selected lake core samples (covering Holocene timescales) from these regions. We applied metabarcoding of the trnL marker and used Illumina technology for NGS amplicon sequencing of PCR products and performed OBITools pipeline for bioinformatic analyses and taxonomic assignment. Firstly, our study aims to evaluate if the trnL marker typically used for detecting terrestrial plant diversity can deliver valuable information on the composition of aquatic plants. Secondly, we will use ordination analyses to test which environmental variables (e.g. lake water depth, pH and conductivity) shape the diversity of genetically detected aquatic plants. Thirdly, we will analyze past genetic aquatic plant diversity from Holocene lake cores and compare it with the modern genetic data set to reconstruct putative drivers of past diversity changes. So far, we identified free-floating (Nymphoides, Ceratophyllum), submerged (Potamogeton sp.), wetland taxa (Caltha, Carex, Juncus) and bryophytes (Sphagnum) in modern and past genetic data sets. Further statistical analyses are pending and will be finalized and presented at EGU.

Due to methodological challenges there are only a few studies that focus on macrophyte dynamics in large lakes despite their notable role in a lake’s ecosystem functioning. This study investigates composition and productivity changes of the submerged vegetation of Lake Karakul, Pamir Mountains (Tajikistan), using sedimentary ancient DNA metabarcoding and elemental (C/N) and isotopic (δ13C, δ15N) measurements of Stuckenia cf. pamirica (Baagøe) Z. Kaplan (Potamogetonaceae) leaf remains. No Stuckenia cf. pamirica leaf remains were found for 28.7–26.1 cal ka BP, when both Potamogetonaceae and Chara (L.) DNA sequences were recorded, suggesting sparse submerged vegetation at the coring site. This agrees with the inference of a deep lake reached using geochemical proxies. From 26.1 to 17.5 cal ka BP a few macrophyte remains and high numbers of Potamogetonaceae sequences were recovered: lake level was probably low, as suggested by other studies on the lake. Another phase of increased numbers of Chara sequences and the absence of Stuckenia cf. pamirica leaf remains was found between 17.5 and 12.2 cal ka BP, which coincides with a lake-level transgression at Lake Karakul as indicated by paleo-shoreline investigations. Analyses of macrophyte remains reveal intermediate paleo-productivity from 6.9 cal ka BP and high paleo-productivity from 2.2 cal ka BP onwards. From comparisons with other studies, we suggest that lake-level changes are the main driver for the submerged vegetation composition and productivity at the coring site in Lake Karakul and underline our conclusions by depicting the present-day distribution of Stuckenia cf. pamirica and Chara within the lake.

Reliable information on past and present vegetation is important to project future changes, especially for rapidly transitioning areas such as the boreal treeline. To study past vegetation, pollen analysis is common, while current vegetation is usually assessed by field surveys. Application of detailed sedimentary DNA (sedDNA) records has the potential to enhance our understanding of vegetation changes, but studies systematically investigating the power of this proxy are rare to date. This study compares sedDNA metabarcoding and pollen records from surface sediments of 31 lakes along a north-south gradient of increasing forest cover in northern Siberia (Taymyr peninsula) with data from field surveys in the surroundings of the lakes. SedDNA metabarcoding recorded 114 plant taxa, about half of them to species level, while pollen analyses identified 43 taxa; both exceeding the 31 taxa found by vegetation field surveys. Increasing Larix percentages from north to south were consistently recorded by all three methods and principal component analyses based on percentage data of vegetation surveys and DNA sequences separated tundra from forested sites. Comparisons of the ordinations using Procrustes and PROTEST analyses show a significant fit among all compared pairs of records. Despite similarities of sedDNA and pollen records, certain idiosyncrasies, such as high percentages of Alnus and Betula in all pollen and high percentages of Salix in all sedDNA spectra are observable. Our results from the tundra to single-tree tundra transition zone show that sedDNA analyses perform better than pollen in recording site-specific richness (i.e. presence/absence of taxa in the vicinity of the lake) and perform as well as pollen in tracing vegetation composition. This article is protected by copyright. All rights reserved.

AimThis study investigates taxonomic and phylogenetic diversity in diatom genera to evaluate assembly rules for eukaryotic microbes across the Siberian tree line. We first analysed how phylogenetic distance relates to taxonomic richness and turnover. Second, we used relatedness indices to evaluate if environmental filtering or competition influences the assemblies in space and through time. Third, we used distance‐based ordination to test which environmental variables shape diatom turnover.LocationYakutia and Taymyria, Russia: we sampled 78 surface sediments and a sediment core, extending to 7,000 years before present, to capture the forest–tundra transition in space and time respectively.TaxonArctic freshwater diatoms.MethodsWe applied metabarcoding to retrieve diatom diversity from surface and core sedimentary DNA. The taxonomic assignment binned sequence types (lineages) into genera and created taxonomic (abundance of lineages within different genera) and phylogenetic datasets (phylogenetic distances of lineages within different genera).ResultsContrary to our expectations, we find a unimodal relationship between phylogenetic distance and richness in diatom genera. We discern a positive relationship between phylogenetic distance and taxonomic turnover in spatially and temporally distributed diatom genera. Furthermore, we reveal positive relatedness indices in diatom genera across the spatial environmental gradient and predominantly in time slices at a single location, with very few exceptions assuming effects of competition. Distance‐based ordination of taxonomic and phylogenetic turnover indicates that lake environment variables, like HCO3− and water depth, largely explain diatom turnover.Main conclusionPhylogenetic and abiotic assembly rules are important in understanding the regional assembly of diatom genera across lakes in the Siberian tree line ecotone. Using a space—time approach we are able to exclude the influence of geography and elucidate that lake environmental variables primarily shape the assemblies. We conclude that some diatom genera have greater capabilities to adapt to environmental changes, whereas others will be putatively replaced or lost due to the displacement of the Arctic tundra biome under recent global warming. published

The past centuries have seen tremendous turnovers in species distributions and biodiversity due to anthropogenic impacts on a global scale. The processes are ongoing and mostly not well documented. Long‐term records of biotic change can be recovered from sedimentary deposits, but traditional analyses were restricted to organisms that leave behind visible traces and molecular genetic tools were mostly employed on samples that promised good DNA preservation. In this issue of Molecular Ecology, Shaw, Weyrich, Hallegraeff and Cooper (2019) and Gomez Cabrera et al. (2019) present two studies on marine sedimentary records from warm environments, in which they successfully analyze ancient environmental DNA (aeDNA) on a decadal and centennial scale. Notably, the studies were conducted on novel samples with nonoptimal preservation conditions for ancient DNA ‐ historical collections of ship ballast tank sediments from Australia and two coral reef cores spanning up to 750 years (Figure 1) ‐ but yielded a high diversity of taxa. This highlights that aeDNA is a promising tool to globally study biodiversity history on scales of decades to centuries ‐ the timeframe most relevant to human society in the context of both current climate change and direct anthropogenic modifications of the environment. published

Environmental DNA preserved in sediments is rapidly gaining importance as a tool in paleoecology. Sampling procedures for sedimentary ancient DNA (sedaDNA) have to be well planned to ensure clean subsampling of the inside of sediment cores and avoid introducing contamination. Additionally, ancient DNA extraction protocols may need to be optimized for the recovery of DNA from sediments, which may contain inhibitors. Here we describe procedures for subsampling both nonfrozen and frozen sediment cores, and we describe an efficient method for ancient DNA extraction from such samples.

Environmental DNA preserved in sediments is rapidly gaining importance as a tool in paleoecology. Sampling procedures for sedimentary ancient DNA (sedaDNA) have to be well planned to ensure clean subsampling of the inside of sediment cores and avoid introducing contamination. Additionally, ancient DNA extraction protocols may need to be optimized for the recovery of DNA from sediments, which may contain inhibitors. Here we describe procedures for subsampling both nonfrozen and frozen sediment cores, and we describe an efficient method for ancient DNA extraction from such samples.

Giresse et al. (1) criticize both our paleoclimatic reconstruction and our inferred anthropogenic origin of the late Holocene rainforest crisis (LHRC) (2). However, their argumentation, which is combined with alleged evidence for a climatic change during the LHRC, lacks strong support. Citing studies describing both brief (weeklong) and limited periods of leaf wax production in deciduous trees, Giresse et al. (1) conclude that leaf waxes cannot record the environmental variability of a full season. However, this argument is flawed, as evergreen and subtropical deciduous trees produce leaf waxes over much longer timescales—their δD (δDwax) values have been shown to capture environmental variability on even seasonal timescales (3, 4 … [↵][1]1To whom correspondence should be addressed. Email: yannickgarcin{at}yahoo.fr. [1]: #xref-corresp-1-1

A strong temperature increase in the Arctic is expected to lead to latitudinal treeline shift. This tundra-taiga turnover would cause a positive vegetation-climate feedback due to albedo decrease. However, reliable estimates of tree migration rates are currently lacking due to the complex processes involved in forest establishment, which depend strongly on seed dispersal. We aim to fill this gap using LAVESI, an individual-based and spatially explicit Larix vegetation simulator. LAVESI was designed to simulate plots within homogeneous forests. Here, we improve the implementation of the seed dispersal function via field-based investigations. We inferred the effective seed dispersal distances of a typical open forest stand on the southern Taymyr Peninsula (north-central Siberia) from genetic parentage analysis using eight highly polymorphic nuclear microsatellite loci. The parentage analysis gives effective seed dispersal distances (median ~ 10 m) close to the seed parents. A comparison between simulated and observed effective seed dispersal distances reveals an overestimation of recruits close to the releasing tree and a shorter dispersal distance generally. We thus adapted our model and used it to simulate south-to-north transects: a slow-moving treeline front was revealed. The colonisation of the tundra areas was assisted by occasional long-distance seed dispersal events beyond the treeline area. The treeline (~ 1 tree ha−1) advanced by ~ 1.6 m yr−1, whereas the forest line (~ 100 trees ha−1) advanced by only ~ 0.6 m yr−1. We conclude that the treeline in north-central Siberia currently lags behind the current strong warming and will continue to lag in the near future.

Clist et al. (1) challenge our conclusions (2), criticizing our archaeological synthesis to maintain that the late Holocene rainforest crisis (LHRC) in Western Central Africa (WCA) was not triggered by human activity. Clist et al. (1) claim that the archaeological 14C dates we used were not critically evaluated, as we were more concerned with the quantity of dates rather than their quality. However, a careful reading of our article (2) and its associated SI Appendix unambiguously documents that we cautiously weighted the radiometric dates by applying a binning in space and time to correct for investigator bias and oversampling at different sites, following refs. 3⇓–5; this … [↵][1]1To whom correspondence should be addressed. Email: yannickgarcin{at}yahoo.fr. [1]: #xref-corresp-1-1

Arctic treelines are facing a strong temperature increase as a result of recent global warming, causing possible changes in forest extent, which will alter vegetation-climate feedbacks. However, the mode and strength of the response is rather unclear, as potential changes are happening in areas that are very remote and difficult to access, and empirical data are still largely lacking. Here, we assessed the current population structure and genetic differentiation of Larix Mill. tree stands within the northernmost latitudinal treeline reaching ~ 72° N in the southern lowlands of the Taymyr Peninsula (~ 100° E). We sampled 743 individuals belonging to different height classes (seedlings, saplings, trees) at 11 locations along a gradient from ‘single tree’ tundra over ‘forest line’ to ‘dense forest’ stands and conducted investigations applying eight highly polymorphic nuclear microsatellites. Results suggest a high diversity within sub-populations (HE = 0.826–0.893), coupled, however, with heterozygote deficits in all sub-populations, but pronounced in ‘forest line’ stands. Overall, genetic differentiation of sub-populations is low (FST = 0.005), indicating a region-wide high gene flow, although ‘forest line’ stands harbour few rare and private alleles, likely indicating greater local reproduction. ‘Single tree’ stands, located beyond the northern forest line, are currently not involved in treeline expansion, but show signs of a long-term refuge, namely asexual reproduction and change of growth-form from erect to creeping growth, possibly having persisted for thousands of years. The lack of differentiation between the sub-populations points to a sufficiently high dispersal potential, and thus a rapid northward migration of the Siberian arctic treeline under recent global warming seems potentially unconstrained, but observations show it to be unexpectedly slow.

High Arctic environments are particularly sensitive to climate changes, but retrieval of paleoecological data is challenging due to low productivity and biomass. At the same time, Arctic soils and sediments have proven exceptional for long-term DNA presentation due to their constantly low temperatures. Lake sediments contain DNA paleorecords of the surrounding ecosystems and can be used to retrieve a variety of organismal groups from a single sample. In this study, we analyzed vascular plant, bryophyte, algal (in particular diatom) and copepod DNA retrieved from a sediment core spanning the Holocene, taken from Bliss Lake on the northernmost coast of Greenland. A previous multi-proxy study including microscopic diatom analyses showed that this lake experienced changes between marine and lacustrine conditions. We inferred the same environmental changes from algal DNA preserved in the sediment core. Our DNA record was stratigraphically coherent, with no indication of leaching between layers, and our cross-taxon comparisons were in accordance with previously inferred local ecosystem changes. Authentic ancient plant DNA was retrieved from nearly all layers, both from the marine and the limnic phases, and distinct temporal changes in plant presence were recovered. The plant DNA was mostly in agreement with expected vegetation history, but very early occurrences of vascular plants, including the woody Empetrum nigrum, document terrestrial vegetation very shortly after glacial retreat. Our study shows that multi-taxon metabarcoding of sedimentary ancient DNA from lake cores is a valuable tool both for terrestrial and aquatic paleoecology, even in low-productivity ecosystems such as the High Arctic. (C) 2015 Elsevier Ltd. All rights reserved.

(IF 9.58; Q1); International audience; A potential human footprint on Western Central African rainforests before the Common Era has become the focus of an ongoing controversy. Between 3,000 y ago and 2,000 y ago, regional pollen sequences indicate a replacement of mature rainforests by a forest–savannah mosaic including pioneer trees. Although some studies suggested an anthropogenic influence on this forest fragmentation, current interpretations based on pollen data attribute the ‘‘rainforest crisis’’ to climate change toward a drier, more seasonal climate. A rigorous test of this hypothesis, however, requires climate proxies independent of vegetation changes. Here we resolve this controversy through a continuous 10,500-y record of both vegetation and hydrological changes from Lake Barombi in Southwest Cameroon based on changes in carbon and hydrogen isotope compositions of plant waxes. δ13C-inferred vegetation changes confirm a prominent and abrupt appearance of C4 plants in the Lake Barombi catchment, at 2,600 calendar years before AD 1950 (cal y BP), followed by an equally sudden return to rainforest vegetation at 2,020 cal y BP. δD values from the same plant wax compounds, however, show no simultaneous hydrological change. Based on the combination of these data with a comprehensive regional archaeological database we provide evidence that humans triggered the rainforest fragmentation 2,600 y ago. Our findings suggest that technological developments, including agricultural practices and iron metallurgy, possibly related to the large-scale Bantu expansion, significantly impacted the ecosystems before the Common Era.

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. published

Diatom diversity in lakes of northwest Yakutia (Siberia) was investigated by microscopic and genetic analysis of surface and cored lake sediments, to evaluate the use of sedimentary DNA for paleolimnological diatom studies and to identify obscure genetic diversity that cannot be detected by microscopic methods. Two short (76 and 73 bp) and one longer (577 bp) fragments of the ribulose 1,5- bisphosphate carboxylase/oxygenase (rbcL) gene, encoding the large subunit of the rbcL, were used as genetic markers. Diverse morphological assemblages of diatoms, dominated by small benthic fragilarioid taxa, were retrieved from the sediments of each lake. These minute fragilarioid taxa were examined by scanning electron microscopy, revealing diverse morphotypes in Staurosira and Staurosirella from the different lakes. Genetic analyses indicated a dominance of haplotypes that were assigned to fragilarioid taxa and less genetic diversity in other diatom taxa. The long rbcL_577 amplicon identified considerable diversification among haplotypes clustering within the Staurosira/Staurosirella genera, revealing 19 different haplotypes whose spatial distribution appears to be primarily related to the latitude of the lakes, which corresponds to a vegetation and climate gradient. Our rbcL markers are valuable tools for tracking differences between diatom lineages that are not visible in their morphologies. These markers revealed putatively high genetic diversity within the Staurosira/Staurosirella species complex, at a finer scale than is possible to resolve by microscopic determination. The rbcL markers may provide additional reliable information on the diversity of barely distinguishable minute benthic fragilarioids. Environmental sequencing may thus allow the tracking of spatial and temporal diversification in Siberian lakes, especially in the context of diatom responses to recent environmental changes, which remains a matter of controversy.

International audience; 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.

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.

Arctic and alpine treelines worldwide differ in their reactions to climate change. A northward advance of or densification within the treeline ecotone will likely influence climate-vegetation feedback mechanisms. In our study, which was conducted in the Taimyr Depression in the North Siberian Lowlands, w present a combined field-and model-based approach helping us to better understand the population processes involved in the responses of the whole treeline ecotone, spanning from closed forest to single-tree tundra, to climate warming. Using information on stand structure, tree age, and seed quality and quantity from seven sites, we investigate effects of intra-specific competition and seed availability on the specific impact of recent climate warming on larch stands. Field data show that tree density is highest in the forest-tundra, and average tree size decreases from closed forest to single-tree tundra. Age-structure analyses indicate that the trees in the closed forest and forest-tundra have been present for at least similar to 240 yr. At all sites except the most southerly ones, past establishment is positively correlated with regional temperature increase. In the single-tree tundra, however, a change in growth form from krummholz to erect trees, beginning similar to 130 yr ago, rather than establishment date has been recorded. Seed mass decreases from south to north, while seed quantity increases. Simulations with LAVESI (Larix Vegetation Simulator) further suggest that relative density changes strongly in response to a warming signal in the forest-tundra while intra-specific competition limits densification in the closed forest and seed limitation hinders densification in the single-tree tundra. We find striking differences in strength and timing of responses to recent climate warming. While forest-tundra stands recently densified, recruitment is almost non-existent at the southern and northern end of the ecotone due to autecological processes. Palaeo-treelines may therefore be inappropriate to infer past temperature changes at a fine scale. Moreover, a lagged treeline response to past warming will, via feedback mechanisms, influence climate change in the future.

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.

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.

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-1,5bisphosphate 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.

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.