English: Phylogenetic tree of mtDNA haplogroup N1b. Time scale (ka) based on ML estimations for mitogenome sequences. The fourth major Ashkenazi founder mtDNA falls within haplogroup N1b. The distribution of N1b is much more focused on the Near East than that of haplogroup K, and the distinctive Ashkenazi N1b2 subclade has accordingly being assigned to a Levantine source. N1b2 has until now been found exclusively in Ashkenazim, and although it dates to only ~2.3 ka, it diverged from other N1b lineages ~20 ka. N1b2 can be recognized in the HVS-I database by the variant 16176A, but Behar et al. tested 14 Near Eastern samples (and some east Europeans) with this motif and identified it as a parallel mutation. Therefore, despite the long branch leading to N1b2, no Near Eastern samples are known to belong to it. In our unpublished database of 6991 HVS-I sequences, however, we identified two Italian samples with the 16176A marker, which we completely sequenced. We confirmed that they belong to N1b2 but diverge before the Ashkenazi lineages ~5 ka, nesting the Ashkenazi cluster. This striking result suggests that the Italian lineages may be relicts of a dispersal from the Near East into Europe before 5 ka, and that N1b2 was assimilated into the ancestral Ashkenazi population on the north Mediterranean ~2 ka. Although we found only two samples suggesting an Italian ancestry for N1b2, the control-region database available for inspection is very large (28,418 HVS-I sequences from Europe, the Near East and the Caucasus, of which 278, or ~1%, were N1b). Moreover, the conclusion is supported by our previous founder analysis of N1b HVS-I sequences, which dated the dispersal into Europe to the late Pleistocene/early Holocene.