Pristionchus pacificus Sommer, Carta, Kim, and Sternberg, 1996 is an important model organism in evolutionary biology that integrates developmental biology with ecology and population genetics. This species is part of a sub-complex of the genus Pristionchus that is considered to have originated in East Asia. Here, we describe two new species of Pristionchus, P. maxplancki and P. quartusdecimus, which were isolated from beetles in Japan, supporting the hypothesis that a region including Japan is the origin of diversification of the P. pacificus species complex. Phytogeny inferred from a partial small subunit rRNA gene and 25 ribosomal protein genes shows P. maxplancki to be the closest known outgroup to a triad of sibling species, including P. pacificus. Pristionchus quartusdecimus is a putative outgroup to the P. pacificus species complex, supporting a more ancient origin of Pristionchus species in the region. Species diagnoses are based on morphological and molecular characters, in addition to reproductive isolation for P. maxplancki. Members of the P. pacificus species complex as well as P. quartusdecimus are distinguished by stegostomatal structures, male genital papilla arrangement, and gubernaculum shape. The discovery of a new member of the P. pacificus species complex allows greater precision in polarizing and reconstructing ancestral states in the comparative model system centering on P. pacificus. Together with previous reports, these findings support an important biogeographic role of Japan in the evolution of the genus Pristionchus and the P. pacificus species complex, especially the associated phenotypic evolution of mouth morphology.
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