3.3. Phylogenetic analysis and divergence time
The phylogenetic trees based on both molecular markers (nuclear and mitochondrial), using the Maximum Likelihood and Bayesian Inference approach show two well-supported, reciprocally monophyletic and congruent groups of samples (posterior probability, PP ≥ 0.99; bootstrap probability, BP = 99) (Fig. 2). Group “A” was more widely distributed along the Antarctic Peninsula, whereas group “B” was distributed in Weddell Sea, Potter Cove, Palmer Station, Paradise Bay, and Rothera Station. Moreover, the phylogeny built with the nuclear gene separated four extra groups; one cluster comprises exclusively individuals from Burdwood Bank/MPA Namuncurá (PP 1, BP 100), a second group with two samples from Potter Cove (PP 1, BP 98), a third branch with only one sample from Burdwood Bank/MPA Namuncurá, and a fourth group constituted by samples from Scotia Sea (PP 1, BP 99).
The two congruent clusters defined by the nuclear and mitochondrial phylogenetic trees were grouping the sequences in the same way as ABGD analysis and the haplotype network. However, two individuals were assigned to different groups depending on which marker was used, mitochondrial or nuclear. On one hand, the individual 291 from Potter Cove, was assigned to group A in 18S species delimitation, but to group B for COI. On the other hand, the individual 116 from Palmer Station was assigned to group B in 18S species delimitation, but to group A for COI. This crossed pattern is incompatible with congruent diversification of mitochondrial and nuclear genes, some hypothetical explanations for this result are discussed below (see discussion 4.4 section).
The estimation of divergence time among groups A and B, using COI sequences, was calculated between 3.58 Ma (95% High Posterior Density [HPD]: 2.331 - 4.935 Ma) and 2.20 Ma (95% High Posterior Density [HPD]: 1.423 - 3.028Ma).