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.