To reveal how a large gene family evolved in the highly organized genome, the present study sequenced 294 copies of 5S rRNA genes from six individuals of three populations of Ophiopogon xylorrhizus Wang et Dai, and 45 copies from its closest species, O. sylvicola Wang et Tang. Based on the sequences the gene-phylogeny was inferred through PAUP. The sequences were highly diversified. Of the 339 randomly cloned copies, only 13 pairs (3.8%) were identical, with length varied between 307 - 548 bp (average 438.6 bp). The sequence differentiation index (SDI) in O. xylorrhints was 0.078, in O. sylvicola was 0.032, and between these two species was 0.149. In the most parsimonious tree reconstructed, all of the 294 copies of O. xylorrhizus were grouped into one clade, and those of O. sylvicola were grouped into another clade with 100% bootstrap support. The result demonstrated that the copies of 5S rRNA genes originated from just one progenitor copy, 'founder copy', in both of the species respectively, followed by a series of proliferation, and little, if any, homogenization ever occurred in the copies. As the common ancestor of the two species must have home multiple copies of the genes, all the other copies were lost during or after the speciation. Additionally, the phylogeny clearly showed that the founder copy was proliferated continuously through time, not just once. O. xylorrhizus is an extremely endangered species in which high genetic diversity, selfing and depression on seedlings were observed previously. In our study the sequences from different populations or different individuals were mixed in the branches of the O. xylorrhizus clade, although they evolved independently. It is assumed that gene flow of the species happened much more frequently in the past, making the proliferated copies dispersed to different populations, and the species maintained an amount of genetic diversity. Therefore, the selfing and seedling depression would be the result of a recent event. We suggest that some changes in outer mechanis
