Multiplexing PCR allows the identification of within-species genetic diversity in ancient eDNA.

Sedimentary ancient DNA (sedaDNA) has rarely been used to obtain population-level data due to either a lack of taxonomic resolution for the molecular method used, limitations in the reference material or inefficient methods. Here, we present the potential of multiplexing different PCR primers to retrieve population-level genetic data from sedaDNA samples. Vaccinium uliginosum (Ericaceae) is a widespread species with a circumpolar distribution and three lineages in present-day populations. We searched 18 plastid genomes for intraspecific variable regions and developed 61 primer sets to target these. Initial multiplex PCR testing resulted in a final set of 38 primer sets. These primer sets were used to analyse 20 lake sedaDNA samples (11,200 cal. yr BP to present) from five different localities in northern Norway, the Alps and the Polar Urals. All known V. uliginosum lineages in these regions and all primer sets could be recovered from the sedaDNA data. For each sample on average 28.1 primer sets, representing 34.15 sequence variants, were recovered. All sediment samples were dominated by a single lineage, except three Alpine samples which had co-occurrence of two different lineages. Furthermore, lineage turnover was observed in the Alps and northern Norway, suggesting that present-day phylogeographical studies may overlook past genetic patterns. Multiplexing primer is a promising tool for generating population-level genetic information from sedaDNA. The relatively simple method, combined with high sensitivity, provides a scalable method which will allow researchers to track populations through time and space using environmental DNA.

Ancient DNA, lipid biomarkers and palaeoecological evidence reveals construction and life on early medieval lake settlements.

Direct evidence of ancient human occupation is typically established through archaeological excavation. Excavations are costly and destructive, and practically impossible in some lake and wetland environments. We present here an alternative approach, providing direct evidence from lake sediments using DNA metabarcoding, steroid lipid biomarkers (bile acids) and from traditional environmental analyses. Applied to an early Medieval Celtic settlement in Ireland (a crannog) this approach provides a site chronology and direct evidence of human occupation, crops, animal farming and on-site slaughtering. This is the first independently-dated, continuous molecular archive of human activity from an archeological site, demonstrating a link between animal husbandry, food resources, island use. These sites are under threat but are impossible to preserve in-situ so this approach can be used, with or without excavation, to produce a robust and full site chronology and provide direct evidence of occupation, the use of plants and animals, and activities such as butchery.

Persistence of arctic-alpine flora during 24,000 years of environmental change in the Polar Urals.

Plants adapted to extreme conditions can be at high risk from climate change; arctic-alpine plants, in particular, could "run out of space" as they are out-competed by expansion of woody vegetation. Mountain regions could potentially provide safe sites for arctic-alpine plants in a warmer climate, but empirical evidence is fragmentary. Here we present a 24,000-year record of species persistence based on sedimentary ancient DNA (sedaDNA) from Lake Bolshoye Shchuchye (Polar Urals). We provide robust evidence of long-term persistence of arctic-alpine plants through large-magnitude climate changes but document a decline in their diversity during a past expansion of woody vegetation. Nevertheless, most of the plants that were present during the last glacial interval, including all of the arctic-alpines, are still found in the region today. This underlines the conservation significance of mountain landscapes via their provision of a range of habitats that confer resilience to climate change, particularly for arctic-alpine taxa.

An AFLP clock for absolute dating of shallow-time evolutionary history--too good to be true?

A major drawback of Amplified Fragment Length Polymorphisms (AFLP) as genetic makers for phylogeographic studies is their lack of a temporal dimension. In a recent publication in Molecular Ecology, Kropf et al. (2009) proposed a molecular clock for AFLP. In this comment we evaluate the proposed approach both theoretically and empirically. A linear increase with time is a prerequisite to use a genetic distance as molecular clock. Testing the relationship between genetic distance and time in the data of Kropf et al. (2009) for linearity revealed that the relationship was in fact not linear for their pooled data, as well as for one of the three species analyzed. Also, the relationship was not linear in two new species, where divergence times could be inferred from macrofossils. When applying the proposed molecular clock to data from eight species, dates obtained were plausible in some cases, but very improbable in others. The suggested genetic distance was also influenced by intrapopulation genetic diversity, leading to a potential bias. In the future, investigations of AFLP mutation rates combined with phylogeographic modelling may contribute to adding a time scale to the understanding of AFLP data.

Nuclear vs. plastid data: complex Pleistocene history of a circumpolar key species.

To fully understand the contemporary genetic structure of plants, both nuclear and plastid markers are needed. Three chloroplast DNA (cpDNA) lineages, which probably diverged before the major Pleistocene glaciations, have been identified in the circumpolar/circumboreal Vaccinium uliginosum. Here we investigate its nuclear DNA variation using nuclear ribosomal internal transcribed spacer (ITS) sequences, DNA ploidy level measurements and amplified fragment length polymorphisms (AFLPs). We also extend the cpDNA dataset. Two ITS lineages, corresponding to diploids and tetraploids, respectively, were identified. However, both main sequence types apparently occurred in most individual plants but showed ploidy-biased homogenization and possibly reflect paralogy predating the origin of V. uliginosum. The ploidy levels were largely consistent with the cpDNA lineages, suggesting that the initial cpDNA divergence followed early polyploidizations. Five main AFLP groups were identified, consistent with recent glacial refugia in Beringia, western Siberia, the southern European mountains and areas south/east of the Scandinavian and Laurentide ice sheets. Except from the southern European mountains, there has been extensive expansion from all refugia, resulting in several contact zones. Surprisingly, the presumably older ploidy and cpDNA patterns were partly inconsistent with the main AFLP groups and more consistent with AFLP subgroups. A likely major driver causing the inconsistencies is recent nuclear gene flow via unreduced pollen from diploids to tetraploids. This may prevent cytoplasmic introgression and result in overlayed patterns formed by processes dominating at different time scales. The data also suggest more recent polyploidizations, as well as several chloroplast capture events, further complicating this scenario. This study highlights the importance of combining different marker systems to unravel intraspecific histories.

Impact of ice ages on circumpolar molecular diversity: insights from an ecological key species.

We address the impact of the ice age cycles on intraspecific cpDNA diversity, for the first time on the full circumboreal-circumarctic scale. The bird-dispersed bog bilberry (or arctic blueberry, Vaccinium uliginosum) is a key component of northern ecosystems and is here used to assess diversity in previously glaciated vs. unglaciated areas and the importance of Beringia as a refugium and source for interglacial expansion. Eighteen chloroplast DNA haplotypes were observed in and among 122 populations, grouping into three main lineages which probably diverged before, and thus were affected more or less independently by, all major glaciations. The boreal 'Amphi-Atlantic lineage' included one haplotype occurring throughout northern Europe and one occurring in eastern North America, suggesting expansion from at least two bottlenecked, glacial refugium populations. The boreal 'Beringian lineage' included seven haplotypes restricted to Beringia and the Pacific coast of USA. The 'Arctic-Alpine lineage' included nine haplotypes, one of them fully circumpolar. This lineage was unexpectedly diverse, also in previously glaciated areas, suggesting that it thrived on the vast tundras during the ice ages and recolonized deglaciated terrain over long distances. Its largest area of persistence during glaciations was probably situated in the north, stretching from Beringia and far into Eurasia, and it probably also survived the last glaciation in southern mountain ranges. Although Beringia apparently was important for the initial divergence and expansion of V. uliginosum as well as for continuous survival of both the Beringian and Arctic-Alpine lineages during all ice ages, this region played a minor role as a source for later interglacial expansions.