DNA barcoding of recently diversified tree species: a case study on spruces based on 20 DNA fragments from three different genomes

Key message A tiered barcoding system comprising traditional cytoplasmic DNAs and multiple nuclear DNAs should be developed to barcode recently diversified tree species. Abstract In this study, we aimed to test whether certain nuclear genes are more powerful tools for barcoding spruce species than chloroplast and mitochondrial DNA fragments (DNAs). We sequenced 20 DNAs, including five chloroplast DNAs, two mitochondrial DNAs, and 13 nuclear genes, for a total of 123 individuals representing 37 spruce species and subspecies. We found that some nuclear genes did show higher discriminatory power than any of the chloroplast or mitochondrial DNA fragments we examined. However, no single DNA fragment could discriminate more than half of the spruce species, although a combination of all the nuclear genes greatly increased discrimination success (up to more than 89 % of the total number of species). These findings suggested that most spruce species are derived from recent diversification and still on their way to the final stage of speciation. A low level of accumulation of genetic variation along each species lineage renders it impossible to develop any single nuclear DNA fragment as a barcode. However, combining variations from multiple nuclear genes makes it possible to discriminate between most spruce species. A tiered barcoding system is therefore needed for spruces, and the second tier should comprise multiple nuclear DNAs developed specifically for this genus. These findings provide a case study on discriminating and barcoding recently diverged species, especially for those tree species with large effective population sizes and long life histories.