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.

Detection of DNA in lake sediments holds promise as a tool to study processes like extinction, colonization, adaptation and evolutionary divergence. However, low concentrations make sediment DNA difficult to detect, leading to high false negative rates. Additionally, contamination could potentially lead to high false positive rates. Careful laboratory procedures can reduce false positive and negative rates, but should not be assumed to completely eliminate them. Therefore, methods are needed that identify potential false positive and negative results, and use this information to judge the plausibility of different interpretations of DNA data from natural archives. We developed a Bayesian algorithm to infer the colonization history of a species using records of DNA from lake‐sediment cores, explicitly labelling some observations as false positive or false negative. We illustrate the method by analysing DNA of whitefish (Coregonus lavaretus L.) from sediment cores covering the past 10,000 years from two central Swedish lakes. We provide the algorithm as an R‐script, and the data from this study as example input files. In one lake, Stora Lögdasjön, where connectivity with the proto‐Baltic Sea and the degree of whitefish ecotype differentiation suggested colonization immediately after deglaciation, DNA was indeed successfully recovered and amplified throughout the post‐glacial sediment. For this lake, we found no loss of detection probability over time, but a high false negative rate. In the other lake, Hotagen, where connectivity and ecotype differentiation suggested colonization long after deglaciation, DNA was amplified only in the upper part of the sediment, and colonization was estimated at 2,200 bp based on the assumption that successful amplicons represent whitefish presence. Here the earliest amplification represents a false positive with a posterior probability of 41%, which increases the uncertainty in the estimated time of colonization. Complementing careful laboratory procedures aimed at preventing contamination, our method estimates contamination rates from the data. By combining these results with estimates of false negative rates, our models facilitate unbiased interpretation of data from natural DNA archives.

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Measures of environmental DNA (eDNA) concentrations in water samples have the potential to be both a cost‐efficient and a nondestructive method to estimate fish population abundance. However, the inherent temporal and spatial variability in abiotic and biotic conditions in aquatic systems have been suggested to be a major obstacle to determine relationships between fish eDNA concentrations and fish population abundance. Moreover, once water samples are collected, methodological biases are common, which introduces additional sources of variation to potential relationships between eDNA concentrations and fish population abundance. Here, we evaluate the performance of applying the droplet digital PCR (ddPCR) method to estimate fish population abundance in experimental enclosures. Using large‐scale enclosure ecosystems that contain populations of nine‐spined stickleback (Pungitius pungitius), we compared the concentrations of fish eDNA (COI mitochondrial region, 134 bp) obtained with the ddPCR method with high precision estimates of fish population abundance (i.e., number of individuals) and biomass. To evaluate the effects of contrasted concentrations of humic substances (potential PCR inhibitors) on the performance of ddPCR assays, we manipulated natural dissolved organic carbon (DOC) concentrations (range 4–11 mg/L) in the enclosures. Additionally, water temperature (+2°C) was manipulated in half of the enclosures. Results showed positive relationships between eDNA concentration and fish abundance and biomass estimates although unexplained variation remained. Still and importantly, fish eDNA estimates from high DOC enclosures were not lowered by potential inhibitory effects with our procedure. Finally, water temperature (although only 2°C difference) was neither detected as a significant factor influencing fish eDNA estimates. Altogether, our work highlights that ddPCR‐based eDNA is a promising method for future quantification of fish population abundance in natural systems.

1. Loss of habitat and changes in the spatial configuration of habitats are major drivers of species extinctions, but the responses to these drivers differ between organisms. To advance theory on how extinction risk from different types of habitat alteration relates to species-specific traits, there is a need for studies of the long-term extinction dynamic of individual species.2. The goal of this study was to quantify how habitat area and the spatial configuration of habitats affect extinction rate of an aquatic top predator, the northern pike Esox lucius L.3. We recorded the presence/absence of northern pike in 398 isolated habitat fragments, each one consisting of a number of interconnected lakes. Time since isolation of the habitat fragments, caused by cut-off from the main dispersal source in the Baltic Sea, varied between 0 and 10,000 years. Using survival regression, we analysed how pike population survival was affected by time since isolation, habitat size and habitat subdivision. The approach builds on the assumptions that pike colonized all fragments before isolation and that current absences result from extinctions. We verified these assumptions by testing (a) if pike was present in the region throughout the entire time period when the lakes formed and (b) if pike typically colonize lakes that are formed today. We also addressed the likelihood that unrecorded anthropogenic introductions could bias our estimates of extinction rate. 4. Our results supported the interpretation that current patterns of presence/absence in our study system are shaped by extinctions. Further, we found that time since isolation and fragment area had strong effects on pike population survival.In contrast, spatial habitat subdivision (i.e. if a fragment contained few large lakes or many small lakes) and other environmental covariates describing climate and productivity were unrelated to pike survival. Over all, extinction rate was high in young fragments and decreased sharply with increasing fragment age. 5.Our study demonstrates how the link between extinction rate and habitat size and spatial structure can be quantified. More similar studies may help us find generalizations that can guide management of habitat size and connectivity. K E Y W O R D Saquatic ecosystems, connectivity, extinction, fragmentation, habitat age, habitat area | 1203Journal of Animal Ecology ENGLUND Et aL.

Purpose Intact lake sediments reflect the development of terrestrial ecosystems. This development can be understood by decoding mineral and geochemical information of sedimentary archives. Therefore, we characterized a Holocene lake sediment core and revealed bulk to micro-scale variations via a combination of geochemical techniques and statistical methods. Methods A 2.3 m sediment core was collected from Hotagen, a lake in west-central Sweden; a sediment sample was collected every 5 cm. A part of each sediment sample was kept untreated (named bulk) and another part was size-fractionated into < 4, 4–16, 16–64, and > 64 µm subsamples. Characterization was then made with respect to grain size distribution (GSD), physico-chemical parameters, geochemical properties, organic composition, and mineralogy. The sediments were investigated at bulk, micro-, and elemental scales using powder X-ray diffraction (XRD), diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), and scanning electron microscopy coupled to energy-dispersive X-ray spectroscopy (SEM–EDX). Results The deepest sediment was identified as glacial till dating back to the Late Pleistocene. The bulk sediments showed a clear distinction between 0–195 cm (unit 1, U1) and 200–225 cm (unit 2, U2) depths. Quartz and feldspar minerals decreased and organic matter and clay minerals increased from the till towards the lower limit of U1. The development in the sedimentary properties marked the transformation of the terrestrial ecosystem from glacier-covered land to vegetated areas. This development was also well reflected by the appearance of X-ray amorphous materials and the formation of distinct organo-mineral aggregates; chlorite was the predominant clay mineral in these aggregates. The geochemical variation between U2 and U1 sediments was further established by resolving the DRIFT spectral components through multivariate curve resolution alternating least square (MCR-ALS). The U1 sediments settled over a period of ~ 7500 years and showed comparable mineral, geochemical, and organic composition. However, the size-fractionated sediments, mainly < 4 µm, showed diverse mineral and geochemical composition. Indeed, these sediments were distinct by containing relatively higher amounts of X-ray amorphous materials and clay minerals, the latter had variable Na, Mg, and K contents. Conclusion The combined use of geochemical and statistical approaches used in this study followed the mineral and geochemical development of sediments that had settled during the Late Pleistocene and Early Holocene Epochs. Finally, the U2 sediments marked the terrestrial ecosystem development that occurred during the late glaciation, deglaciation, and post-glaciation periods. Graphical abstract

Sedimentary environmental DNA (sed-eDNA) coupled with metabarcoding is increasingly exploited for ecological studies, but application of the method to resolve fish dynamics in lakes still needs better validation. This study (1) evaluated the sed-eDNA yields from the commonly used DNeasy PowerSoil DNA Kit from mineral-rich and organic-rich sediments and (2) examined the viability of fish sed-eDNA recovery and detection in surface sediment samples from 13 Swedish mountain lakes, with organic contents of 18–52%, by using conventional PCR and droplet digital PCR. Based on concurrent fish-population surveys these lakes contain arctic char and brown trout. We show that, compared to other specifically designed lysis buffers, the DNeasy PowerSoil DNA Kit is less effective to recover DNA from organic-rich sediments and almost 50% of the extracted DNA was lost during purification steps. The amplification of fish sed-eDNA using conventional PCR with teleo primers failed to detect positive signals; whereas ddPCR assays enabled quantification of amplifiable DNA in all the extracts. However, further molecular cloning of the positive ddPCR droplets from one sediment sample revealed amplified sequences of unidentified origin that cannot be aligned well to fish. Thus the performance of the teleo primers for quantification of fish sed-eDNA detection requires further examination. For detection of fish sed-eDNA for ecological studies, we suggest that DNA extraction methods and primers should be carefully selected and the performance of ddPCR to detect DNA at low quantities needs to be further scrutinized to circumvent the pitfalls of false positives.