Ancient DNA from lake sediments: bridging the gap between paleoecology and genetics

Abstract

Background: Quaternary plant ecology in much of the world has historically relied on morphological identification of macro- and microfossils from sediments of small freshwater lakes. Here, we report new protocols that reliably yield DNA sequence data from Holocene plant macrofossils and bulk lake sediment used to infer ecological change. This will allow changes in census populations, estimated from fossils and associated sediment, to be directly associated with population genetic changes.

Results: We successfully sequenced DNA from 64 samples (out of 126) comprised of bulk sediment and seeds, leaf fragments, budscales, and samaras extracted from Holocene lake sediments in the western Great Lakes region of North America. Overall, DNA yields were low. However, we were able to reliably amplify samples with as few as 10 copies of a short cpDNA fragment with little detectable PCR inhibition. Our success rate was highest for sediments < 2000 years old, but we were able to successfully amplify DNA from samples up to 4600 years old. DNA sequences matched the taxonomic identity of the macrofossil from which they were extracted 79% of the time. Exceptions suggest that DNA molecules from surrounding nearby sediments may permeate or adhere to macrofossils in sediments.

Conclusions: An ability to extract ancient DNA from Holocene sediments potentially allows exciting new insights into the genetic consequences of long-term environmental change. The low DNA copy numbers we found in fossil material and the discovery of multiple sequence variants from single macrofossil extractions highlight the need for careful experimental and laboratory protocols. Further application of these protocols should lead to better understanding of the ecological and evolutionary consequences of environmental change.

Figure 1

Sample origins. North American map indicating the location of sampled lakes (numbers). The modern range of beech is shaded in gray. The contemporary ranges of all other sampled species are throughout the sampled range. Bar graphs depict the number of macrofossils and bulk sediments taken from each lake's sediments through time.

Figure 2

Inhibition quantitation assay. Graph depicts fluorescence values against cycle of appearance along a 45 cycle quantitative PCR reaction. Gray lines are 10 samples from our ancient DNA extracts and 10 1:10 dilutions of those samples, all spiked with a 250 copy standard. Black lines are the dilution series of mammoth DNA from 31,250 copies to 0.4 copies, with the 250 copy standard highlighted in bold. Solid red lines are negative controls and dashed red lines are our "weak" negative controls, used here to illustrate the risks associated with not separating pre- and post-PCR processing.

Figure 3

Probability of PCR success. Results of the binomial general additive model (black curve) comparing PCR success (1) to failure (0) relative to sample age. Data are slightly 'jittered' to reduce overlap of points. Gray curves are simulations in which data were thinned to an equal sampling effort.

Figure 4

DNA yield. DNA quantity extracted from modern live material (live), senescent material (sen), and macrofossils and bulk sediments (ancient).

Figure 5

Sequence variation. Cladogram representing the relationship between haplotypes. Each Symbol represents a different haplotype. Each mutational step is represented by a hash mark. Cladogram symbols and identifications correlate with Table 2. Cladogram was constructed using TCS [46].

Figure 6

Shifts in genetic composition. Sediments less than 1200 years old (upper grey band) are genetically distinct from sediments over 1700 years old (lower grey band). This trend holds in comparisons limited to Ackerman and Tower Lakes, with samples spanning both time periods.