Identification and characterization ofBotrylloides(Styelidae) species from Aotearoa New Zealand coasts

ABSTRACT Ascidians are marine filter feeder primitive chordates. Botrylloides ascidians possess diverse biological abilities like whole-body regeneration (WBR), hibernation/aestivation, blastogenesis, metamorphosis, and natural chimerism. However, the absence of distinctive morphological features often makes identification problematic. Botrylloides diegensis is an ascidian that has been misidentified in previous studies and is recorded in GenBank as Botrylloides leachii due to the high morphological similarity between the sister species. More available sequences and strategies around identification would help resolve some of the confusion currently surrounding its ambiguous nature. We collected several Botrylloides samples from 7 locations around New Zealand - Dunedin, Christchurch, Picton, Nelson, Whangateau, New Plymouth and Invercargill - and barcoded the species based on Cytochrome Oxidase I, Histone 3, 18S, and 28S ribosomal RNA markers. Network and Bayesian trees confirmed the presence of three Botrylloides species: B. diegensis, B. jacksonianum , and B. aff. anceps . Additionally, morphotypes of three species were investigated regarding zooid size, area, tentacle numbers and colonial arrangement.