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Background  Hox gene clusters with at least 13 paralog group (PG) members are common in vertebrate genomes and in that of amphioxus. Ascidians, which belong to the subphylum Tunicata (Urochordata), are phylogenetically positioned between vertebrates and amphioxus, and traditionally divided into two groups: the Pleurogona and the Enterogona. An enterogonan ascidian,  Ciona intestinalis  ( Ci ), possesses nine Hox genes localized on two chromosomes; thus, the Hox gene cluster is disintegrated. We investigated the Hox gene cluster of a pleurogonan ascidian,  Halocynthia roretzi  ( Hr ) to investigate whether Hox gene cluster disintegration is common among ascidians, and if so, how such disintegration occurred during ascidian or tunicate evolution.    Results  Our phylogenetic analysis reveals that the  Hr  Hox gene complement comprises nine members, including one with a relatively divergent Hox homeodomain sequence. Eight of nine  Hr  Hox genes were orthologous to  Ci-Hox1 ,  2, 3, 4, 5, 10, 12  and  13.  Following the phylogenetic classification into 13 PGs, we designated  Hr  Hox genes as  Hox1, 2, 3, 4, 5, 10, 11/12/13.a ,  11/12/13.b  and  HoxX . To address the chromosomal arrangement of the nine Hox genes, we performed two-color chromosomal fluorescent in situ hybridization, which revealed that the nine Hox genes are localized on a single chromosome in  Hr , distinct from their arrangement in  Ci . We further examined the order of the nine Hox genes on the chromosome by chromosome/scaffold walking. This analysis suggested a gene order of  Hox1 ,  11/12/13.b, 11/12/13.a, 10, 5, X,  followed by either  Hox4, 3, 2  or  Hox2, 3, 4  on the chromosome. Based on the present results and those previously reported in  Ci , we discuss the establishment of the Hox gene complement and disintegration of Hox gene clusters during the course of ascidian or tunicate evolution.    Conclusions  The Hox gene cluster and the genome must have experienced extensive reorganization during the course of evolution from the ancestral tunicate to  Hr  and  Ci . Nevertheless, some features are shared in Hox gene components and gene arrangement on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to tunicates as well as later ascidian lineage-specific events.    Electronic supplementary material  The online version of this article (10.1186/s40851-017-0078-3) contains supplementary material, which is available to authorized users.

Hox gene cluster of the ascidian, Halocynthia roretzi, reveals multiple ancient steps of cluster disintegration during ascidian evolution