Retroposon insertions provide insights into deep lagomorph evolution.

The homogenous mammalian order Lagomorpha comprises about 80 species in two families, Ochotonidae (pikas) and Leporidae (rabbits and hares). However, the phylogenetic relationships among leporids are controversial. Molecular data, particularly from mitochondrial sequences, give highly homoplasious signals. To resolve the controversy between mitochondrial and nuclear data, we analyzed genomic orthologous retroposon insertion sites, a virtually homoplasy-free marker system. From a differential screen of rabbit genomic data for intronic retroposon insertions of CSINE elements, we polymerase chain reaction-amplified and sequenced 11 retroposons in eight representative lagomorphs. We found three retroposons shared among all lagomorphs but absent in outgroups, four confirmed the monophyly of leporids, and three significantly supported Pronolagus as the sister group to all other leporids. One retroposon supported the monophyly of Lepus. The position of Pronolagus outside of the remaining leporids supports the sequence-based signals of nuclear genes and clearly refutes the misleading signals of mitochondrial genes.