Tunicates are marine filter-feeding invertebrates that can be found worldwide and which are the closest phylogenetic group to the vertebrates (Craniata). Of particular interest, colonial tunicates are the only known chordates that can undergo Whole-Body Regeneration (WBR) via vascular budding. In Botrylloides anceps, a fully functional adult regenerates from a fragment of the vascular system in around 2 weeks after amputation. In this chapter, we present protocols to collect B. anceps colonies, confirm their species, breed them in the lab, monitor WBR and perform histological staining on cryosections.

Botrylloides niger is an invasive marine filter-feeding invertebrate that is believed to originate from the West Atlantic region. This species of colonial tunicate has been observed on several locations along the coasts of Israel and around the Suez Canal but it has not yet been reported on the coasts of the northeastern Mediterranean Sea (NEMS), suggesting an ongoing Lessepsian migration. However, the extent of this invasion might be concealed by reports of other potentially misidentified species of Botrylloides, given that the strong morphological similarities within this genus renders taxonomical identification particularly challenging. In this study, we performed a phylogeographic and morphological analysis of B. niger in the NEMS. We collected 241 samples from 8 sampling stations covering 824 km of coastlines of NEMS. We reported 14 different morphotypes, of which the orange-brown, orange and brown-striped morphs were the most abundant. Using the mitochondrial cytochrome C oxidase I (COI), one of the four most commonly used DNA barcoding marker, we identified 4 haplotypes with the Konacık (H4) and the Mezitli (H3) ones being the most diverged. The COI haplotypes clustered with the reference B. niger sequences from GenBank and separated from sister Botrylloides species with high confidence. We confirmed our identification using the three additional barcoding markers (Histone 3, 18S rRNA and 28S rRNA), which all matched with over 99% similarity to the reference sequences. In addition, we monitored the Kızkalesi station for a year and applied temporal analysis to the colonies collected. The colonies regressed during winter while resettled and expanded during summer. We performed gene flow analysis on our spatial data that identified a possible population subdivision at the sampling site of Side, which might be caused by a local freshwater input. Overall, we here present the first report on the presence of Botrylloides niger in the NEMS, we show that this species is commonly present throughout this region and with a particularly high morphological as well as genomic diversity.

Botrylloides niger (class Ascidiacea) is an invasive marine filter-feeding invertebrate that is believed to originate from the West Atlantic region. This species of colonial tunicate has been observed in several locations along the coasts of Israel and around the Suez Canal, but it has not yet been reported on the coasts of the Northeastern Mediterranean Sea (NEMS), suggesting an ongoing Lessepsian migration. However, the extent of this invasion might be concealed by reports of other potentially misidentified species of Botrylloides, given that the strong morphological similarities within this genus renders taxonomical identification particularly challenging. In this study, we performed a phylogeographic and morphological analysis of B. niger in the NEMS. We collected 238 samples from 8 sampling stations covering 824 km of the coastlines of NEMS. We reported 14 different morphotypes, of which the orange-brown, orange, and brown-striped morphs were the most abundant. Using the mitochondrial cytochrome C oxidase I (COI) as a DNA barcode marker, we identified 4 haplotypes. The COI haplotypes clustered with the reference B. niger sequences from GenBank and differed significantly from the sister Botrylloides species. We confirmed our identification using three additional barcoding markers (Histone 3, 18S rRNA, and 28S rRNA), which all matched with over 99% similarity to reference sequences. In addition, we monitored a station for a year and conducted a temporal analysis of the collected colonies. The colonies were absent during the winter and spring, while new colonies were established in the summer and expanded during autumn. We performed demographic population analysis on our spatial data that identified a possible population subdivision at a sampling site, which might have been caused by local freshwater input. Herein, we present the first report on the presence of Botrylloides niger in the NEMS. This study represents a key step toward understanding the diversity and the propagation of this highly invasive species of colonial ascidians, both within the Mediterranean basin as well as globally.