1. Introduction
Triggered by a steep decline in atmospheric CO2, the Antarctic Polar Front (APF) has been working as a geographic, climatic, thermal and oceanographic barrier that isolated the Southern Ocean since the Eocene/Oligocene boundary, while the Antarctic Circumpolar Current (ACC) has been playing a role as disperser force around Antarctica (DeConto & Pollard, 2003). Together with this, Antarctica and its Ocean suffered a series of glaciation cycles that fragmented its marine biota (Cristini, Grosfeld, Butzin, & Lohmann, 2012; Hewitt, 2004; Petit et al., 1999; Soler-Membrives, Linse, Miller, & Arango, 2017). In consequence, the Southern Ocean is one of the most diverse and rich marine ecosystems which presents a high level of endemism, even in comparison with temperate and tropical environments (Allcock & Strugnell, 2012; Halanych & Mahon, 2018; Rogers, 2007). Numerous cryptic species were discovered in this region, i.e. genetically distinct species that have been previously classified as a single species due to their similar phenotypes (Bickford et al., 2006; Held & Wägele, 2005; Held, 2003). Therefore, the real species number in Antarctica would be higher than the registered today, and species yet undescribed represent an important portion of biodiversity (Dömel et al., 2015; Galaska, Sands, Santos, Mahon, & Halanych, 2017; Havermans, Nagy, Sonet, De Broyer, & Martin, 2011; Riesgo, Taboada, & Avila, 2015; Wilson, Hunter, Lockhart, & Halanych, 2007). Ascidians are an important group in the Antarctic benthic communities, being even dominant in some assemblages (Gili et al., 2006; Sahade, Tatián, Kowalke, Kühne, & Esnal, 1998; Sahade et al., 2015). Cnemidocarpa verrucosa (Lesson, 1830) (Chordata, Tunicata) is the largest and most abundant Styelidae in the Antarctic Ocean, it shows circumpolar distribution in the Antarctic and is also distributed in the south of South America (Kott & Mather, 1969; Monniot & Monniot, 1983; Pineda, Turon, Pérez-Portela, & López-Legentil, 2016). It can inhabit muddy to hard bottoms and waters between five to more than 500 m deep (Ramos-esplá, Cárcel, & Varela, 2005; Tatián, Sahade, Doucet, & Esnal, 1998). Cnemidocarpa verrucosa is hermaphroditic, possesses lecithotrophic larvae and strong seasonality in reproduction (Bowden, Clarke, & Peck, 2009; Sahade, Tatián, & Esnal, 2004; Strathmann, Kendall, & Marsh, 2006).
The ascidian genome architecture has been demonstrated to be divergent from model organisms (small genome, transposon diversity, developmental gene stock, physical gene order, intron-exon organization, splicing patterns, and prevalence of single-exon 5’-genes operons), also there is circumstantial evidence that this group is characterized by an elevated rate of molecular and protein evolution (Dehal et al., 2002; Delsuc et al., 2018; Denoeud et al., 2010; Ganot, Kallesøe, Reinhardt, Chourrout, & Thompson, 2004; Haubold, Pfaffelhuber, & Lynch, 2010; Rubinstein et al., 2013; Satou et al., 2008). Furthermore, it has been demonstrated that hybridization and introgression take place among the closely related ascidians Ciona intestinalis and Ciona robusta . Hybridization was observed under field and lab conditions (Bouchemousse, Bishop, & Viard, 2016; Sato, Shimeld, & Bishop, 2014), and introgression was detected in sympatry zones, with nuclear (Jade) and also mitochondrial (COI) genes introgressed (Nydam, Giesbrecht, & Stephenson, 2017a; Nydam & Harrison, 2011). Hereafter, studying species delimitation in ascidians it is interesting since barriers to gene flow between species would be expected to be permeable.
Considering all the above mentioned, the goals of this work were, a) to determine if there are more than one genetically divergent species within the nominal C. verrucosa; b) to resolve if the presumable species are also morphologically distinguishable; c) to test if species within C. verrucosa co-occur; and if so, to test whether their co-occurrence can be explained by secondary contact and drifting by the ACC. To be able to recognize species in the face of high morphological variation, sympatry, potential hybridization, and introgression is the ultimate goal of this work. Furthermore, being able to discriminate species at the laboratory and also at the field may have implications in many research fields, especially in biodiversity and experimental studies.