The temporal and spatial coarseness of megafaunal fossil records complicates attempts to to disentangle the relative impacts of climate change, ecosystem restructuring, and human activities associated with the Late Quaternary extinctions. Advances in the extraction and identification of ancient DNA that was shed into the environment and preserved for millennia in sediment now provides a way to augment discontinuous palaeontological assemblages. Here, we present a 30,000-year sedimentary ancient DNA (sedaDNA) record derived from loessal permafrost silts in the Klondike region of Yukon, Canada. We observe a substantial turnover in ecosystem composition between 13,500 and 10,000 calendar years ago with the rise of woody shrubs and the disappearance of the mammoth-steppe (steppe-tundra) ecosystem. We also identify a lingering signal of Equus sp. (North American horse) and Mammuthus primigenius (woolly mammoth) at multiple sites persisting thousands of years after their supposed extinction from the fossil record.

The earliest eukaryotes recorded in continental environments are non-pollen palynomorphs (NPP) in Mesoproterozoic strata, and NPP provide our best insights into lacustrine ecosystems through the Paleogene. They have been underexploited in studies of younger lake sediments, either ignored or only qualitatively observed, because many NPP are destroyed by standard processing techniques for pollen and embryophyte spores. The palaeoenvironmental potential of palynomorphs, with representatives from all eukaryotic kingdoms as well as cyanobacteria and from all trophic levels in various lacustrine environments, has been recognized by a few Quaternary palynologists in the past few decades. NPP have proven particularly valuable in archaeological and environmental monitoring studies of human impact on freshwater ecosystems, with spores of some fungi and eggs/ egg cases of some flatworms and roundworms associated with feces of humans and livestock, and the acid-resistant remains of various life stages of cyanobacteria, algae, and their aquatic consumers responding to increased turbidity and nutrient influx associated with permanent human settlements, particularly those associated with agricultural activity. Descriptions of NPP commonly encountered in Quaternary lake sediments and case studies illustrating applications to various research questions should encourage more palynologists that ‘Quaternary non-pollen palynomorphs' deserve our attention!’, to quote Prof. Bas van Geel, undisputed Father of NPP Research.Supplementary material at https://doi.org/10.6084/m9.figshare.c.5244661

To reconstruct a mastodon diet and provide a ‘snapshot’ view of environmental conditions in eastern Canada prior to the onset of the Wisconsinan glaciation, we analysed the faunal and floral components of dung associated with juvenile mastodon remains from East Milford, Nova Scotia, dated to 74.9 ± 5.0 ka cal BP. The diverse assemblage of pollen, non-pollen palynomorphs, plant macrofossils and macroinvertebrate remains in the dung suggests that the mastodons lived in a spruce-dominated mixed coniferous-deciduous forest with a strong boreal aspect interspersed with wetlands rich in charophytes, sedges, cattails, bulrushes and bryophytes. The abundance of spruce needles and birch samaras in the dung sample is consistent with an inferred browsing behaviour, having been reported for other mammutid species previously. The limited diversity and near-absence of coprophilous fungi, such as <i>Sporormiella</i>, in the dung could have an impact on understanding the influence of feeding strategies on the presence of coprophilous taxa in sedimentary records, and thus interpretations of megafaunal abundance. The dung also yielded the earliest known Canadian remains of the bark beetle <i>Polygraphus </i>cf. <i>rufipennis</i>, gemmulae of the freshwater sponge <i>Eunapius </i>cf. <i>fragilis </i>and loricae of the rotifer <i>Keratella cochlearis</i>.

&lt;p&gt;The multitude of factors alleged to have contributed to the late Quaternary mass extinction of some two-thirds of Earth&amp;#8217;s megafauna is complicated by the coarse record of buried macro-fossils. In response, micro-methods such as ancient DNA have been increasingly able to augment discontinuous palaeontological records to investigate the relative timings of vegetation turnover versus megafaunal extirpations&amp;#8212;all in the absence of biological tissues. Here, we present sedimentary ancient DNA data retrieved using the PalaeoChip Arctic-1.0 bait-set diachronically identifying fauna and flora from permafrost cores recovered from the Klondike region of central Yukon, Canada dating between 30,000&amp;#8211;6000 calendar years BP. We observe a substantial turnover in ecosystem composition between 13,000&amp;#8211;10,000 BP with the rise of woody shrubs and the disappearance of mammoth-steppe vegetation. We also identify a lingering signal of &lt;em&gt;Equus&lt;/em&gt; sp. (North American horse) and &lt;em&gt;Mammuthus primigenius&lt;/em&gt; (woolly mammoth) from multiple samples thousands of years after their last dated macro-fossils, possibly as late as the mid-Holocene.&lt;/p&gt;