Botryllus schlosseri (Pallas, 1766)

Botryllus schlosseri (Pallas, 1766) View in CoL , a colonial ascidian of subfamily Botryllinae , has in the last decades

*Corresponding author. E-mail: carmela.gissi@uniba.it

$ These authors contributed equally to this work

† Current address: Centro Piattaforme Tecnologiche, Università di Verona, Piazzale A. Scuro 10, 37134 Verona, Italy

[Version of record, published online 31 March 2020; http:// zoobank.org/ urn:lsid:zoobank.org:pub:DB18D5A7-0E96-458E-B3C0-5E93327359AC ]

become a reference organism, not only in fields such as evolutionary developmental biology and immunobiology, but also for studying biological processes such as stem cell migration, apoptosis, regeneration and ageing ( Sabbadin, 1982; Manni et al., 2007, 2019; Rosengarten & Nicotra, 2011; Voskoboynik & Weissman, 2015). Botryllus schlosseri colonies propagate asexually by cyclical waves of blastogenesis (i.e. palleal budding) and during colony growth processes such as apoptosis and stem cell migration occur synchronously and massively ( Rinkevich et al., 2013; Rosner et al., 2013; Franchi et al., 2016; Manni et al., 2019). In addition to its role as a model organism, Botryllus schlosseri is a cosmopolitan fouling species and one of the most widespread marine invaders ( Berrill, 1950; Carlton, 2005; Ben-Shlomo et al., 2010; Bock et al., 2012; Lord, 2017), and is potentially able to damage the industry of shellfish aquaculture ( Carman et al., 2010) and other marine industrial activities ( Carver et al., 2006).

In the context of the above-mentioned importance of this species, its taxonomy needs to be unequivocally and fully resolved, and the identification of the relative specimens has to occur beyond doubt based on clear diagnostic characters. However, in the last decade molecular data has shown that Botryllus schlosseri is a “species complex” consisting of five genetically highly divergent clades, named from A to E, each corresponding to a cryptic species ( Lopez-Legentil et al., 2006; Bock et al., 2012). Clade A is globally distributed and clade E is present only in European waters, whereas the other clades are geographically restricted to a few localities ( Stach & Turbeville, 2002; Lopez-Legentil et al., 2006; Perez-Portela et al., 2009; Bock et al., 2012; Yund et al., 2015; Nydam et al., 2017; Reem et al., 2017). The five clades have been identified for the first time by Lopez-Legentil et al. (2006) based on a short fragment of the mitochondrial (mt) gene cox1, but they were not described as cryptic species but just as clades characterized by high nucleotide diversity, low haplotype diversity and restricted gene flow. Using both cox1 and nuclear microsatellites, Bock et al. (2012) have put forward the hypothesis that these clades correspond to cryptic species. Indeed, Bock et al. observed a high interclade (10.8–16.5%) and a comparatively low intraclade (0.8–3.8%) cox1 divergence and, at microsatellite level, a complete lack of contemporary gene flow between clades, suggestive of reproductive isolation ( Bock et al., 2012). Later on, studies on the entire mitochondrial genome (mtDNA) identified three surprisingly distant taxa/subclades within clade A, that have been explained as further cryptic species or as the outcome of ongoing speciation events ( Griggio et al., 2014). A Pacific specimen “sc6ab” from clade A has been the target of a genome sequencing project, thus leading to the publication of both the nuclear and mitochondrial genomes ( Voskoboynik et al., 2013). Moreover, a Mediterranean clade A specimen having the same cox1 haplotype of the mtDNA of the “VE” specimen ( Griggio et al., 2014) has been designated as the neotype for Botryllus schlosseri when it was redescribed by Brunetti et al. (2017). The sc6ab and the VE specimens belong to two of the three divergent taxa/subclades identified by the mtDNA within clade A ( Griggio et al., 2014).

The exact phylogenetic relationships between the five clades remain elusive, because the few published cox1 trees are not fully resolved, due to the presence of unreliably supported nodes ( Lopez-Legentil et al., 2006; Yund et al., 2015) and/or were reconstructed with unusual and potentially inappropriate methodologies for the cox1 nucleotide sequences (i.e. assuming a strict molecular clock ( Yund et al., 2015) or analysing the translated sequences ( Lopez-Legentil et al., 2006)). However, without considering node reliability/ methodology anomalies, all trees agree in recognizing clade E as sister to all other clades ( Lopez-Legentil et al., 2006; Bock et al., 2012; Yund et al., 2015).

Population genetic studies have also started investigating the geographic distribution, origin and dispersal history of the two widespread cryptic species, i.e. clade A and clade E ( Bock et al., 2012; Yund et al., 2015; Nydam et al., 2017; Reem et al., 2017). Although the picture is far from complete, these studies have revealed that clades A and E are both successful invaders and have shown traces of recent range expansion of clade E ( Nydam et al., 2017). Moreover, hypotheses on the geographic origin and dispersal history of the species complex/clades have also been presented ( Berrill, 1950; Carlton, 2005; Nydam et al., 2017; Reem et al., 2017).

Due to the relevance of this species complex, the need for a resolution on the taxonomic crypsis of this organism is growing. In particular, the Botryllus schlosseri taxonomic re-evaluation should lead not only to a more accurate discrimination of this species from other botryllids, but also to the identification of clear morphological and molecular diagnostic characters able to distinguish the various cryptic species. An accurate taxonomic description will help the studies on this model ascidian, with a better interpretation of possible differences in the outcomes of experiments carried out on different clades, as well as the finding of possible differences in the invasive potential of the various cryptic species, with consequences in the setting of the most appropriate monitoring and management strategies. For this purpose, and considering the lack of a type specimen and the previous unclear morphological descriptions, in 2017 we designated a neotype of Botryllus schlosseri , as reference point for future studies (Brunetti et al., 2017). Following an integrative taxonomy approach, this neotype was described both at the morphological and molecular level, allowing to associate a precise morphology to clade A (Brunetti et al., 2017) and in particular to a cox1 haplotype identical to that of the previously published mtDNA of the VE specimen (European Nucleotide Archive [ENA] Accession Number: FM177702 View Materials ) ( Griggio et al., 2014). We believe that an integrative taxonomy approach should be applied to all genetically identified clades of the Botryllus schlosseri species complex with the aim of elucidating their taxonomic status.

Here, in order to clarify the taxonomic status of clade E and its relationship to clade A, we analysed clade E specimens according to an integrative taxonomy approach that accounts for morphological and molecular characters. Then, we compared the clade E features to those of the Botryllus schlosseri neotype (Brunetti et al., 2017). In particular, we analysed the colony and zooid morphology, sequenced the entire mtDNA of a clade E specimen and compared several genome-level mitochondrial features (i.e. sequence divergence, gene order, and non-coding regions). The differences observed between clade E and clade A / Botryllus schlosseri sensu Brunetti (2017) at both the mitogenomic and morphological level show that clade E sensu Bock et al. (2012) can be recognized as a distinct species, which is described below.