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Allorecognition, the ability of an organism to distinguish kin from non-kin, or self from non-self, has been studied extensively in a group of invertebrate chordates, the colonial ascidians called botryllids (Subphylum Tunicata, Class Ascidiacea, Family Styelidae). When two conspecific botryllid colonies come in contact, there are two potential outcomes to an allorecognition reaction: fusion or rejection. The rejection outcome of allorecognition varies by species, and has been classified by type (referred to as R-Type). R-Type is defined according to how far the fusion process progresses before the rejection begins, since the rejection reaction appears as an interference of the fusion process. Here, we map the evolution of R-Types onto an extended and robust phylogeny of the botryllids. In this study, we have reconstructed the largest phylogenomic tree of botryllids, including 97 samples and more than 40 different species, and mapped on it nine of the 13 species for which the R-Type is known. Based on the R-Type known in a single outgroup species (Symplegma reptans), we infer that at least R-Type B and E-like could be ancestral to the Botrylloides/Botryllus group. However, the application of ancestral character state reconstructions does not provide conclusive results since several clades show more than one equiparsimonious R-Type state. Notably, all R-Type A species are clustered together and certainly evolved later than other R-Types. Our phylogenomic tree has been built on 177 nuclear loci and nearly all clades are well supported. Moreover, our phylogenetic analyses also take into account the results of species delimitation analyses based on the mitochondrial COI gene and of careful morphological analyses of the samples. The implementation of this integrated taxonomic approach, combining morphological as well as nuclear and mitochondrial data, has allowed the description of six new species, and the identification of a number of putative unnamed taxa. Thus, our results also demonstrate the existence of an unexplored hidden diversity within botryllids. [ABSTRACT FROM AUTHOR]

Allorecognition, the ability of an organism to distinguish kin from non-kin, or self from non-self, has been studied extensively in a group of invertebrate chordates, the colonial ascidians called botryllids (Subphylum Tunicata, Class Ascidiacea, Family Styelidae). When two conspecific botryllid colonies come in contact, there are two potential outcomes to an allorecognition reaction: fusion or rejection. The rejection outcome of allorecognition varies by species, and has been classified by type (referred to as R-Type). R-Type is defined according to how far the fusion process progresses before the rejection begins, since the rejection reaction appears as an interference of the fusion process. Here, we map the evolution of R-Types onto an extended and robust phylogeny of the botryllids. In this study, we have reconstructed the largest phylogenomic tree of botryllids, including 97 samples and more than 40 different species, and mapped on it nine of the 13 species for which the R-Type is known. Based on the R-Type known in a single outgroup species (Symplegma reptans), we infer that at least R-Type B and E-like could be ancestral to the Botrylloides/Botryllus group. However, the application of ancestral character state reconstructions does not provide conclusive results since several clades show more than one equiparsimonious R-Type state. Notably, all R-Type A species are clustered together and certainly evolved later than other R-Types. Our phylogenomic tree has been built on 177 nuclear loci and nearly all clades are well supported. Moreover, our phylogenetic analyses also take into account the results of species delimitation analyses based on the mitochondrial COI gene and of careful morphological analyses of the samples. The implementation of this integrated taxonomic approach, combining morphological as well as nuclear and mitochondrial data, has allowed the description of six new species, and the identification of a number of putative unnamed taxa. Thus, our results also demonstrate the existence of an unexplored hidden diversity within botryllids.

Colonial ascidians are common occupants of hard surfaces in coastal marinas as fouling organisms on pier pilings and ship hulls and have significant financial impact. Methods to inhibit fouling often are time consuming and labor intensive, thus treatments that prevent settlement for long time periods are warranted. Trybutyltin (TBT) appeared to be an effective antifouling agent, but is highly toxic to non-target species and now globally prohibited. Medetomidine is the active ingredient in the anti-fouling paint, Selektope, that shows promise to prevent attachment of marine fouling species. Here, we assessed the potential of medetomidine on larval metamorphosis of the invasive ascidian Botrylloides violaceus in static bioassays. We exposed newly released tadpole larvae to eight concentrations (0.5 mg/L, 1 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, 40 mg/L, 50 mg/L) of medetomidine and recorded the number of larvae that were mobile, attached, metamorphosed or deceased after 1 h, 2 h, 4 h, 6 h, 24 h and 48 h. Larvae exposed to the highest concentrations of medetomidine quickly attached to the surface of Petri dishes, but failed to metamorphose. Larvae were mobile over the first 2 to 4 h of the experiment in the filtered sea water controls and low medetomidine concentrations. Larvae metamorphosed in all treatments except the two highest concentrations of medetomidine (40 and 50 mg/L), but ultimately all larvae exposed to medetomidine died. Medetomidine appears to have potential as a mechanism to control populations of B. violaceus , but we caution that further testing of non-target species is warranted. • The invasive ascidian Botrylloides violaceus can dominate benthic/fouling communities. • Medetomidine exposure enhances settlement of Botrylloides larvae. • Metamorphosis of larvae is inhibited by exposure to medetomidine. • Medetomidine has potential to be an effective antifouling treatment. [ABSTRACT FROM AUTHOR]

Tunicates are a diverse group of worldwide marine filter-feeders that are vertebrates' closest invertebrate relatives. Colonial tunicates are the only know chordates that have been shown to undergo whole-body regeneration (WBR). Botrylloides in particular can regenerate one fully functional adult from a minute fragment of their vascular system in as little as 10 days. This regenerative process relies on the proliferation of circulating stem cells, likely supported by the activity of some of the 11 identified types of hemocytes. To study and challenge WBR, it is thus important to have the capacity to isolate, analyze, and manipulate hemolymph in regenerating colonies. Here we present a microtransfusion technique that permits the collection of pure hemocytes, the quantification of their purity, their labeling, and reinjection into colonial tunicates. To exemplify our approach, we present in addition a protocol to analyze the isolated hemocytes using flow cytometry. Our approach is minimally invasive, does not induce lethality, and therefore allows repeated transfusion into exactly the same colony with minimal disruption to the process being studied., (© 2022. The Author(s).)