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 for present-day
populations. We searched 18 plastid genomes for intraspecific variable
regions and developed 61 primers to target these. Initial multiplex PCR
testing resulted in a final set of 38 primers. These primers 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 primers
could be recovered from the sedaDNA data, where for each sample
28.1 primers containing 34.15 variant sequences were obtained on average.
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 primers is a promising tool for
generating population-level genetic information from sedaDNA. The
relatively simple method, combined with high sensitivity, provides a
scalable method that will allow researchers to track populations through
time and space using environmental DNA.