4.5. Timing and mode of speciation
In order to estimate time since speciation, data from crabs, shrimps, and urchins were used as proxy (mutation rate range of 0.016 to 0.026 substitutions per site, per million years) (Nydam & Harrison, 2011). Using this rates, we obtained the speciation of C. verrucosasp. A and sp. B at 3.58 - 2.20 million years ago (MYA). Several estimations of whole-genome mutation rates have been calculated in ascidians pointing out the rapid evolution of this group (Berna & Alvarez-Valin, 2014; Denoeud et al., 2010), but not specifically for mitochondrial genome, which typically evolves faster than the nuclear genome (Havird & Sloan, 2016). On average ascidian evolutionary whole-genome rate in terms of number of substitutions per million years is 6.25 times faster than vertebrates, and 2.08 faster than cephalochordates (Delsuc et al., 2018). This indicates that, even though we can estimate the speciation time based on other marine invertebrate taxa data, we should bear in mind that we may be overestimating the time since divergence and that the range of time since speciation proposed here would be an older limit with the actual speciation time likely being more recent. We can hypothesize that speciation took place after the Miocene or later, when Antarctica already experienced the cooling process (Zachos, Pagani, Sloan, Thomas & Billups, 2001). A summary of the evolutionary patterns in Antarctic organisms based on other studies reported radiation and speciation processes by 8-5 MYA; and cycles of population concentration, isolation in refugia and expansion, speciation and transoceanic dispersal by 1 MYA (Rogers, 2007). Some examples of these processes in Antarctic taxa are arthropods, annelids, echinoderms and molluscs (Baird, Miller, & Stark, 2011; Hemery et al., 2012; Linse, Cope, Lörz, & Sands, 2007; Raupach et al., 2010; Riesgo et al., 2015; Wilson et al., 2007). Then, a similar pattern of allopatric speciation followed by secondary contact can be attributed to C. verrucosa(Mayr, 1963). On the other hand, speciation in response to ecological opportunity (Simpson, 1953) can also be hypothesized with our results. Under this type of speciation, a new trait evolves and affects the ecological versatility of the specimens (Givnish et al., 2014; Liem, 1973). In this case, the development of a basal disc could represent an adaptive character for colonizing different substrates.