4.1. Two genetically divergent species
Both molecular markers studied in this work (the mitochondrial COI and nuclear 18S gene) distinguished two congruent groups, therefore there was strong evidence for recognising two genetically divergent species within C. verrucosa sensu lato populations from the Antarctic Peninsula: C. verrucosa sp. A and C. verrucosa sp. B . Nuclear and mitochondrial genes evolve independently because they differ in the mode of inheritance, ploidy, amount of recombination, introns number, mutation rate, repair mechanisms and effective population size (Hill, 2015). Thus, studying only one type of marker, can lead to a systematic bias in the inference of evolutionary processes (Ballard & Whitlock, 2004). Such is the case of cichlid fish radiations in lakes of Africa, where different speciation patterns were addressed using only one type of marker (Seehausen et al., 2003). In this work both markers showed the same speciation pattern, which implies that both molecules were impacted in the same way by the same evolutionary mechanism, thus making strong the evidence for the mutually isolated gene pools.
Within the results obtained here, for some particular specimens no sequences were obtained of COI, while 18S nuclear gene could be sequenced. In this case, the phylogeny constructed with 18S resolved basal branches that comprised clades that would present deeper genetic divergence with the rest of the animals, than the divergence amongC. verrucosa sp. A and sp. B (see Fig. 2). Nuclear genes usually evolve at a slower rate compared to mitochondrial ones (Allio, Donega, Galtier, & Nabholz, 2017; Havird & Sloan, 2016). Furthermore, fast substitution rate and gene rearrangements were described for ascidians mitochondrial genome, and have been proposed to cause difficulties in standard polymerase chain reaction (PCR), because of mutations on the primer site (Delsuc et al., 2018; Denoeud et al., 2010; Gissi et al., 2010). Hereafter, the basal branches obtained in this study with 18S sequences, composed by individuals from Burdwood Bank/MPA Namucurá, Scotia Sea, Weddell Sea and Potter Cove (stations located in the tip of the Antarctic Peninsula and South America, see Fig. 3), could represent more distant species within C. verrucosa sensu lato,and be constituted by individuals in which the COI primer binding site has been mutated or rearranged.
Following with the effort of distinguishing genetic differentiated species, widely-adopted molecular markers, such as COI and 18S, are helpful to characterize unstudied groups (Hebert, Cywinska, Ball, & deWaard, 2003). Here, applying ABGD method in COI and 18S a barcode-gap, with no intermediate values, was found in the frequency distribution of the genetic differences between individuals of the putative C. verrucosa. This gap is observed when the divergence between organisms that belong to the same species is smaller than the divergence among organisms that belong to different species (Puillandre et al., 2012). Moreover, a robust approach for species delimitation is to compare genetic distances with related undisputed species pairs, given that the nucleotide substitution rate is quite homogeneous at interspecific level (Griggio et al., 2014; Held, 2003). In this study, the genetic distance between C. verrucosa sp. A and sp. B was >10.20% for COI, and >3% for 18S. The COI nucleotide divergence among ascidian species from the same genera range from 8.7% to 21.7% (Hirose, Oka, & Hirose, 2009; Nydam & Harrison, 2007; Pérez-Portela & Turon, 2008), and between species within Styelidae family range from 10.8% to 16.5% (Lacoursière-Roussel et al., 2012; Reem et al., 2017). Regarding 18S gene, it has been found 0 - 0.58% nucleotide divergence among samples from genera Diplosoma . Furthermore, it has been described smaller divergence among the suborder of Diplosoma (Phlebobranchia) than among the suborder Stolidobranchia, in which C. verrucosa is described (Yokobori, Kurabayashi, Neilan, Maruyama, & Hirose, 2006). Bock, Macisaac, and Cristescu (2012) found larger divergences (2.3 - 10.1%) in 18S gene among putative cryptic species of Botryllus schlosseri ; important discussion exists on the definition of cryptic species within B.schlosseri but no study has confirmed yet a genetic and/or morphological evidence, therefore B. schlosseri is currently being treated as a species complex (Lejeusne, Bock, Therriault, MacIsaac, & Cristescu, 2011; Nydam, Giesbrecht, & Stephenson, 2017b; Reem et al., 2017; Yund, Collins, & Johnson, 2015). On the other hand, Nydam and Harrison (2010) studied five nuclear loci in order to unravel the existence of two distinct forms ofCiona intestinalis, and found that Type A vs. Type B (recently defined by Brunetti et al. (2015) as two morphological and genetically different species) divergences range from 3.5 to 12.4 %. All this indicates that, within the samples studied here, the genetic differentiation was similar to those found in other species in the same family and other ascidians species pairs, therefore we can define two genetic divergent species based on mitochondrial as well as nuclear evidence.