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.

International initiatives aimed at generating genomic resources, and particularly reference genomes, have flourished in recent years. Some focus on specific taxa, such as the Vertebrate Genomes Project, Bird Genome 10K Project, Bat1K Project, Global Invertebrate Genomics Alliance, 10 000 Plant Genomes Project, and 1000 Fungal Genomes project. Others focus on geographic regions, such as the California Conservation Genomics Project, Darwin Tree of Life for Britain and Ireland, Catalan Initiative for the Earth BioGenome Project in the Catalan territories, Endemixit in Italy, Norwegian Earth Biogenome Project, and SciLifeLab in Sweden, on applications such as the LOEWE Translational Biodiversity Genomics in Germany, or on ecological systems such as the Aquatic Symbiosis Genomics project. Collectively part of the Earth BioGenome Project (EBP), in Europe these initiatives are organized under the umbrella of the European Reference Genome Atlas (ERGA). A genome atlas of European biodiversityERGA is a pan-European scientific response to the current threats to biodiversity. Approximately one fifth of the ~200 000 eukaryotic species present in Europe can be inferred to be at risk of extinction according to the International Union for Conservation of Nature (IUCN) Red List classification (this estimate only considers the assessed species; https://www.iucn.org/regions/europe/our-work/biodiversity-conservation/european-red-list-threatened-species).ERGA aims to generate reference genomes of European eukaryotic species across the tree of life, including threatened, endemic, and keystone species, as well as pests and species important to agriculture, fisheries, and ecosystem function and stability. ERGA builds upon current genomic consortia in EU member states, EU Associated Countries, representatives of other countries within the European bioregion, and international collaborators. These reference genomes will address fundamental and applied questions in conservation, biology, and health. ERGA seeks to alert the EU about the potential of conservation genomics, and particularly the role of reference genomes, in biodiversity assessment, conservation strategies, and restoration efforts.

The extinct passenger pigeon was once the most abundant bird in North America, and possibly the world. While theory predicts that large populations will be more genetically diverse and respond more efficiently to selection, passenger pigeon genetic diversity was surprisingly low. To investigate this we analysed 41 mitochondrial and 4 nuclear genomes from passenger pigeons, and 2 genomes from band-tailed pigeons, passenger pigeons’ closest living relatives. We find that passenger pigeons’ large population size allowed for faster adaptive evolution and removal of harmful mutations, but that this drove a huge loss in neutral genetic diversity. These results demonstrate how great an impact selection can have on a vertebrate genome, and invalidate previous results that suggested population instability contributed to this species’ surprisingly rapid extinction.

9Palaeogenomics has greatly increased our knowledge of past evolutionary and ecological 10 change, but has been restricted to the study of species that preserve as fossils. Here we show 11 the potential of shotgun metagenomics to reveal population genomic information for a taxon 12 that does not preserve in the body fossil record, the algae Nannochloropsis. We shotgun 13 sequenced two lake sediment samples dated to the Last Glacial Maximum and identified N. 14 limnetica as the dominant taxon. We then reconstructed full chloroplast and mitochondrial 15 genomes to explore within-lake population genomic variation. This revealed at least two 16 major haplogroups for each organellar genome, which could be assigned to known varieties 17 of N. limnetica. The approach presented here demonstrates the utility of lake sedimentary 18 ancient DNA (sedaDNA) for population genomic analysis, thereby opening the door to 19 environmental palaeogenomics, which will unlock the full potential of sedaDNA. 20 Keywords 21 Sedimentary ancient DNA, palaeogenomics, shotgun metagenomics, haplotype diversity, ice 22 age, Nannochloropsis 23 24 29 2ancient DNA -the environment -has been almost entirely limited to inferring the presence or 30 absence of taxa through time [8][9][10][11][12][13][14] . However, a nuanced understanding of ecological and 31 evolutionary dynamics requires population genomic information. The direct recovery of this 32 information from cave sediment has recently been shown 12 , but -to our knowledge -has not 33 yet been demonstrated for lake sediments. 34Lake sediments provide an ideal source of sedimentary ancient DNA (sedaDNA) 35 that originates from both the catchment and the lake itself, as well as providing a stable 36 environment required for optimal aDNA preservation 15,16 . As a result, lake sedaDNA has 37 been used to infer the taxonomic composition of past communities 16,17 , regardless of whether 38 those taxa preserve in the body fossil record. The most commonly applied method is DNA 39 metabarcoding, which allows for the targeting of particular groups of organisms 18,19 . 40 However, the ability to confidently identify barcodes is constrained by the completeness of 41 appropriate reference databases, and the length and variability of the barcode targeted. Short 42 barcodes are necessarily targeted for fragmented aDNA 20 , which can therefore impede 43 species-level identification. An alternative approach is shotgun metagenomics, which is non-44 targeting and preserves aDNA damage patterns that, in contrast to metabarcoding, allows for 45 authentic aDNA to be distinguished from modern contamination 16,21-23 . For palaeogenomic 46 reconstruction however, either deep shotgun sequencing or target enrichment of sedaDNA is 47 required, which allows for robust species-level identification 12,14,24 , as well as the potential 48 exploration of population genomic variation. 49Andøya, an island located off the coast of northwest Norway, was partially 50 unglaciated during the Last Glacial Maximum (LGM, Figur...