Population dynamics and genetic changes of Picea abies in the South Carpathians revealed by pollen and ancient DNA analyses

Background: Studies on allele length polymorphism designate several glacial refugia for Norway spruce (Picea abies) in the South Carpathian Mountains, but infer only limited expansion from these refugia after the last glaciation. To better understand the genetic dynamics of a South Carpathian spruce lineage, we compared ancient DNA from 10,700 and 11,000-year-old spruce pollen and macrofossils retrieved from Holocene lake sediment in the Retezat Mountains with DNA extracted from extant material from the same site. We used eight primer pairs that amplified short and variable regions of the spruce cpDNA. In addition, from the same lake sediment we obtained a 15,000-years-long pollen accumulation rate (PAR) record for spruce that helped us to infer changes in population size at this site.Results: We obtained successful amplifications for Norway spruce from 17 out of 462 pollen grains tested, while the macrofossil material provided 22 DNA sequences. Two fossil sequences were found to be unique to the ancient material. Population genetic statistics showed higher genetic diversity in the ancient individuals compared to the extant ones. Similarly, statistically significant Ks and Kst values showed a considerable level of differentiation between extant and ancient populations at the same loci.Lateglacial and Holocene PAR values suggested that population size of the ancient population was small, in the range of 1/10 or 1/5 of the extant population. PAR analysis also detected two periods of rapid population growths (from ca. 11,100 and 3900 calibrated years before present (cal yr BP)) and three bottlenecks (around 9180, 7200 and 2200 cal yr BP), likely triggered by climatic change and human impact.Conclusion: Our results suggest that the paternal lineages observed today in the Retezat Mountains persisted at this site at least since the early Holocene. Combination of the results from the genetic and the PAR analyses furthermore suggests that the higher level of genetic variation found in the ancient populations and the loss of ancient allele types detected in the extant individuals were likely due to the repeated bottlenecks during the Holocene; however our limited sample size did not allow us to exclude sampling effect.This study demonstrates how past population size changes inferred from PAR records can be efficiently used in combination with ancient DNA studies. The joint application of palaeoecological and population genetics analyses proved to be a powerful tool to understand the influence of past population demographic changes on the haplotype diversity and genetic composition of forest tree species.