DNA metabarcoding is an increasingly popular method to characterize and quantify biodiversity in environmental samples. Metabarcoding approaches simultaneously amplify a short, variable genomic region, or "barcode," from a broad taxonomic group via the polymerase chain reaction (PCR), using universal primers that anneal to flanking conserved regions. Results of these experiments are reported as occurrence data, which provide a list of taxa amplified from the sample, or relative abundance data, which measure the relative contribution of each taxon to the overall composition of amplified product. The accuracy of both occurrence and relative abundance estimates can be affected by a variety of biological and technical biases. For example, taxa with larger biomass may be better represented in environmental samples than those with smaller biomass. Here, we explore how polymerase choice, a potential source of technical bias, might influence results in metabarcoding experiments. We compared potential biases of six commercially available polymerases using a combination of mixtures of amplifiable synthetic sequences and real sedimentary DNA extracts. We find that polymerase choice can affect both occurrence and relative abundance estimates and that the main source of this bias appears to be polymerase preference for sequences with specific GC contents. We further recommend an experimental approach for metabarcoding based on results of our synthetic experiments.

Relict woolly mammoth (Mammuthus primigenius) populations survived on several small Beringian islands for thousands of years after mainland populations went extinct. Here we present multiproxy paleoenvironmental records to investigate the timing, causes, and consequences of mammoth disappearance from St. Paul Island, Alaska. Five independent indicators of extinction show that mammoths survived on St. Paul until 5,600 ± 100 y ago. Vegetation composition remained stable during the extinction window, and there is no evidence of human presence on the island before 1787 CE, suggesting that these factors were not extinction drivers. Instead, the extinction coincided with declining freshwater resources and drier climates between 7,850 and 5,600 y ago, as inferred from sedimentary magnetic susceptibility, oxygen isotopes, and diatom and cladoceran assemblages in a sediment core from a freshwater lake on the island, and stable nitrogen isotopes from mammoth remains. Contrary to other extinction models for the St. Paul mammoth population, this evidence indicates that this mammoth population died out because of the synergistic effects of shrinking island area and freshwater scarcity caused by rising sea levels and regional climate change. Degradation of water quality by intensified mammoth activity around the lake likely exacerbated the situation. The St. Paul mammoth demise is now one of the best-dated prehistoric extinctions, highlighting freshwater limitation as an overlooked extinction driver and underscoring the vulnerability of small island populations to environmental change, even in the absence of human influence. mammoth | extinction | Holocene | St. Paul Island | ancient DNA

Holocene relative sea-level (RSL) curves for the U.S. Gulf Coast are in mutual confl ict, with some characterized by a smooth RSL rise akin to widely accepted eustatic sea-level curves versus others, including several recent ones, that are characterized by a conspicuous "stair-step" pattern with prolonged (millennium-scale) RSL stillstands alternating with rapid (meterscale) rises. In addition, recent work in Texas and Alabama has revitalized the notion of a middle Holocene RSL highstand, estimated at 2 m above present mean sea level. An extensive sampling program in the Mississippi Delta (Louisiana) focused on the collection of basal peats that accumulated during the initial transgression of the pre-existing, consolidated Pleistocene basement. We used stable carbon isotope ratios to demonstrate that many of these samples accumulated in environments affected by frequent saltwater intrusion in the <30 cm zone between mean spring high water and mean sea level, and we selected plant macrofossils that were subjected to AMS 14 C dating. Nearly 30 sea-level index points from a ~20 km 2 study area on the eastern margin of the delta suggest that RSL rise followed a relatively smooth trend for the time interval 8000-3000 cal yr B.P., thus questioning the occurrence of major RSL stillstands alternating with abrupt rises. Given the narrow error envelope defi ned by our data set, any sea-level fl uctuations, if present, would have amplitudes of <1 m. Although a true middle Holocene highstand never occurred in the Mississippi Delta, the high level of detail of our time series enables a rigorous test of this hypothesis. Correction of our data set for a hypothetical tectonic subsidence rate of 1.1 mm yr-1 (assuming a constant subsidence rate compared to the tectonically relatively stable adjacent coast of Texas) leads to sea levels near 2 m above present during the time interval 6000-4000 cal yr B.P. However, this model also implies a RSL position near-2 m around 8000 cal yr B.P., which is inconsistent both with data of this age from Texas, as well as with widely accepted sea-level data from elsewhere. We therefore conclude that a middle Holocene highstand for the U.S. Gulf Coast is highly unlikely, and that the entire area is still responding glacio-isostatically, by means of forebulge collapse, to the melting of the Laurentide Ice Sheet.

The genus Cucurbita (squashes, pumpkins, gourds) contains numerous domesticated lineages with ancient New World origins. It was broadly distributed in the past but has declined to the point that several of the crops' progenitor species are scarce or unknown in the wild. We hypothesize that Holocene ecological shifts and megafaunal extinctions severely impacted wild Cucurbita, whereas their domestic counterparts adapted to changing conditions via symbiosis with human cultivators. First, we used high-throughput sequencing to analyze complete plastid genomes of 91 total Cucurbita samples, comprising ancient (n = 19), modern wild (n = 30), and modern domestic (n = 42) taxa. This analysis demonstrates independent domestication in eastern North America, evidence of a previously unknown pathway to domestication in northeastern Mexico, and broad archaeological distributions of taxa currently unknown in the wild. Further, sequence similarity between distant wild populations suggests recent fragmentation. Collectively, these results point to wild-type declines coinciding with widespread domestication. Second, we hypothesize that the disappearance of large herbivores struck a critical ecological blow against wild Cucurbita, and we take initial steps to consider this hypothesis through crossmammal analyses of bitter taste receptor gene repertoires. Directly, megafauna consumed Cucurbita fruits and dispersed their seeds; wild Cucurbita were likely left without mutualistic dispersal partners in the Holocene because they are unpalatable to smaller surviving mammals with more bitter taste receptor genes. Indirectly, megafauna maintained mosaic-like landscapes ideal for Cucurbita, and vegetative changes following the megafaunal extinctions likely crowded out their disturbed-ground niche. Thus, anthropogenic landscapes provided favorable growth habitats and willing dispersal partners in the wake of ecological upheaval.evolutionary ecology | sensory ecology | TAS2R genes | ancient DNA | archaeogenomics

Bottle gourd (Lagenaria siceraria) was one of the first domesticated plants, and the only one with a global distribution during pre-Columbian times. Although native to Africa, bottle gourd was in use by humans in east Asia, possibly as early as 11,000 y ago (BP) and in the Americas by 10,000 BP. Despite its utilitarian importance to diverse human populations, it remains unresolved how the bottle gourd came to be so widely distributed, and in particular how and when it arrived in the New World. A previous study using ancient DNA concluded that Paleoindians transported already domesticated gourds to the Americas from Asia when colonizing the New World [Erickson et al. (2005) Proc Natl Acad Sci USA 102 (51):18315-18320]. However, this scenario requires the propagation of tropical-adapted bottle gourds across the Arctic. Here, we isolate 86,000 base pairs of plastid DNA from a geographically broad sample of archaeological and living bottle gourds. In contrast to the earlier results, we find that all pre-Columbian bottle gourds are most closely related to African gourds, not Asian gourds.Ocean-current drift modeling shows that wild African gourds could have simply floated across the Atlantic during the Late Pleistocene. Once they arrived in the New World, naturalized gourd populations likely became established in the Neotropics via dispersal by megafaunal mammals. These wild populations were domesticated in several distinct New World locales, most likely near established centers of food crop domestication.long-distance dispersal | New World domestication | archaeogenomics