Speciation among sympatric lineages in the genus Palythoa (Cnidaria: Anthozoa: Zoantharia) revealed by morphological comparison, phylogenetic analyses and investigation of spawning period

Zoantharians are sessile marine invertebrates and colonial organisms possessing sexual and asexual reproductive ability. The zooxanthellate zoantharian genus Palythoa is widely distributed in coral reef ecosystems. In the Ryukyu Archipelago, Japan, sympatric Palythoa tuberculosa and P. mutuki are the dominant species of this genus in the intertidal zone. Previous phylogenetic analyses have shown that these two species are closely related, and additionally revealed a putative sympatric hybrid species (designated as Palythoa sp. yoron). In this study, we attempted to delineate Palythoa species boundaries and to clarify the relationships among these three groups plus another additional putative sympatric species (P. aff. mutuki) by multiple independent criteria. The morphology of these four lineages was clearly different; for example the number of tentacles was significantly different for each species group in all pairwise comparisons. From observations of gonadal development conducted in 2010 and 2011, P. sp. yoron and P. aff. mutuki appear to be reproductively isolated from P. tuberculosa. In the phylogenetic tree resulting from maximum likelihood analyses of the ITS-rDNA sequence alignment, P. tuberculosa and P. sp. yoron formed a very well supported monophyletic clade (NJ = 100%, ML = 95%, Bayes = 0.99). This study demonstrates that despite clear morphological and/or reproductive differences, P. tuberculosa and P. sp. yoron are phylogenetically entangled and closely related to each other, as are P. mutuki and P. aff. mutuki. Additionally, no single molecular marker was able to divide these four lineages into monophyletic clades by themselves, and a marker that has enough resolution to solve this molecular phylogenetic species complex is required. In summary, the morphological and reproductive results suggest these lineages are four separate species, and that incomplete genetic lineage sorting may prevent the accurate phylogenetic detection of distinct species with the DNA markers utilized in this study, demonstrating the value of morphological and reproductive data when examining closely related lineages.