Cyclostomes, comprising jawless vertebrates such as lampreys and hagfishes, are 
the sister group of living jawed vertebrates (gnathostomes) and hence an 
important group for understanding the origin and diversity of vertebrates. In 
vertebrates and other metazoans, Hox genes determine cell fate along the 
anteroposterior axis of embryos and are implicated in driving morphological 
diversity. Invertebrates contain a single Hox cluster (either intact or 
fragmented), whereas elephant shark, coelacanth, and tetrapods contain four Hox 
clusters owing to two rounds of whole-genome duplication ("1R" and "2R") during 
early vertebrate evolution. By contrast, most teleost fishes contain up to eight 
Hox clusters because of an additional "teleost-specific" genome duplication 
event. By sequencing bacterial artificial chromosome (BAC) clones and the whole 
genome, here we provide evidence for at least six Hox clusters in the Japanese 
lamprey (Lethenteron japonicum). This suggests that the lamprey lineage has 
experienced an additional genome duplication after 1R and 2R. The relative age 
of lamprey and human paralogs supports this hypothesis. Compared with 
gnathostome Hox clusters, lamprey Hox clusters are unusually large. Several 
conserved noncoding elements (CNEs) were predicted in the Hox clusters of 
lamprey, elephant shark, and human. Transgenic zebrafish assay indicated the 
potential of CNEs to function as enhancers. Interestingly, CNEs in individual 
lamprey Hox clusters are frequently conserved in multiple Hox clusters in 
elephant shark and human, implying a many-to-many orthology relationship between 
lamprey and gnathostome Hox clusters. Such a relationship suggests that the 
first two rounds of genome duplication may have occurred independently in the 
lamprey and gnathostome lineages.