Mitochondrial DNA reveals complex genetic structuring in a stenoendemic conifer Picea omorika [(Pan.) Purk.] caused by its long persistence within the refugial Balkan region

Picea omorika (Pan.) Purk. is a relict from the Arcto-Tertiary flora with its entire current natural range confined to an area of only 10,000 km(2) within the Balkans, a region well known as a Quaternary refugium. We have amplified the second intron of the mitochondrial NADH dehydrogenase subunit1 gene in 200 trees originating from ten natural populations to assess the phylogeographic structure and history of this conifer. Five haplotypes harbouring different numbers of 34-bp minisatellites were detected, revealing haplotypic richness of 3.007 and gene diversities H (S) = 0.075 and H (T) = 0.225. More interestingly, despite the very small distribution range of P. omorika and its dispersal by wind, non-random distribution of haplotypes was observed, resulting in an unexpectedly high estimate of population differentiation (G (ST) = 0.668), and 56.8% of molecular variation assigned to variation among populations. Those findings suggest substantial isolation of populations and their partitioning into two gene pools characterized by different history and levels of genetic diversity, and very limited seed flow in this species (Nm = 0.25). They support the hypothesised early arrival of P. omorika in the Balkan region, and residence within this refugium during several ice ages at least. We demonstrate that the assessment of genetic diversity and structuring are not straightforward in species confined to refugial regions, and that past microvicariance might bias formal phylogeographic (G (ST) = N (ST) = 0.668) and isolation-by-distance analysis (r = 0.028, P gt 0.05)