At clade, suggesting that they do not belong to the C. order Y-27632 megalonyx complex. The K2P genetic distances showed a clear bimodal distribution, with a barcoding gap at approximately 4 (figure 2). GMYC analysis showed that a single-threshold GMYC model was better than a single population model (p = 5.5 ?10-10 ) and maximum-likelihood (ML) resulted in a number of 20 ML entities (confidence interval: 19?3), including the six clades already recognized by Krabbe et al. [37]. Material was available for 17 of the ML entities, while three clades (J, K, L) were only based on GenBank specimens. The number of samples in each clade ranged from one (clades J, M) to 161 (clade A). Average intraclade distances ranged from 0 to 1.9 , while interclade distances ranged from 2.7 to 12.5 . The same clades are also distinguished in the bGMYC analysis. These groupings are shown in table 1.1200 1000 frequency 800 600 400 200rsos.royalsocietypublishing.org R. Soc. open sci. 2:…………………………………………4 5 6 7 8 9 uncorrected pairwise distances ( )Figure 2. Histogram of uncorrected pairwise genetic distances for the COI fragment within C. megalonyx (all clades). Only unique haplotypes are used for calculating pairwise distances.The ABGD analysis resulted in only 15 clades, here termed A . Clades D and N correspond, respectively, to three clades in the GMYC analysis, while clade E corresponds to two clades. Here these clades are named D1/D2/D3, E1/E2 and N1/N2/N3 (see table 2 for clade delimitations resulting from different approaches). Bayesian phylogenetic analysis recovered most of the single-threshold GMYC groupings as monophyletic, although B was Anisomycin molecular weight paraphyletic with respect to M (figure 3). While many interclade relationships are poorly resolved, several clades formed strongly supported monophyletic groups, namely A + F + G + (H + I), (B + M) + C, D + E and L + O. Interestingly, the clades B (Falkland Islands) and M (Chile) formed a South American group clustering inside the mostly Antarctic C. megalonyx complex as sister to clade C (Bouvet, Eastern Weddell Sea). Similar results were found in the maximum-likelihood analysis, except that clade D1 was found to be paraphyletic with respect to D2 and D3.3.1.2. Internal transcribed spacer dataThe ITS phylogenetic tree showed a differentiation into six distinct monophyletic groups named I I (figure 4), which mostly correspond to larger groupings of different COI clades. The analysis of the dataset cropped with GBLOCKS resulted in a slightly different phylogenetic tree, but the differentiation into six groups did not change. There was no differentiation between specimens belonging to different COI clades within these groups. As an example, group IV included individuals belonging to COI clades A, H and I, but those clades could not be distinguished by ITS sequences. Group II showed strong intragroup variation, but no division into the COI clades D1, E1 and E2 was found. In some cases, there were also discrepancies in assignment to larger groups between COI and ITS. Group II mostly included individuals from clades D and E, but also one clade C individual. Group III included individuals from clades N2 and N3 as well as one each from clades E and G. Individuals from COI clade I were found in both groups IV and V.3.2. Phylogeographic analysis3.2.1. Cytochrome c oxidase subunit I dataMost of the clades are geographically widely distributed, and eight of them are found in both East and West Antarctica (tabl.At clade, suggesting that they do not belong to the C. megalonyx complex. The K2P genetic distances showed a clear bimodal distribution, with a barcoding gap at approximately 4 (figure 2). GMYC analysis showed that a single-threshold GMYC model was better than a single population model (p = 5.5 ?10-10 ) and maximum-likelihood (ML) resulted in a number of 20 ML entities (confidence interval: 19?3), including the six clades already recognized by Krabbe et al. [37]. Material was available for 17 of the ML entities, while three clades (J, K, L) were only based on GenBank specimens. The number of samples in each clade ranged from one (clades J, M) to 161 (clade A). Average intraclade distances ranged from 0 to 1.9 , while interclade distances ranged from 2.7 to 12.5 . The same clades are also distinguished in the bGMYC analysis. These groupings are shown in table 1.1200 1000 frequency 800 600 400 200rsos.royalsocietypublishing.org R. Soc. open sci. 2:…………………………………………4 5 6 7 8 9 uncorrected pairwise distances ( )Figure 2. Histogram of uncorrected pairwise genetic distances for the COI fragment within C. megalonyx (all clades). Only unique haplotypes are used for calculating pairwise distances.The ABGD analysis resulted in only 15 clades, here termed A . Clades D and N correspond, respectively, to three clades in the GMYC analysis, while clade E corresponds to two clades. Here these clades are named D1/D2/D3, E1/E2 and N1/N2/N3 (see table 2 for clade delimitations resulting from different approaches). Bayesian phylogenetic analysis recovered most of the single-threshold GMYC groupings as monophyletic, although B was paraphyletic with respect to M (figure 3). While many interclade relationships are poorly resolved, several clades formed strongly supported monophyletic groups, namely A + F + G + (H + I), (B + M) + C, D + E and L + O. Interestingly, the clades B (Falkland Islands) and M (Chile) formed a South American group clustering inside the mostly Antarctic C. megalonyx complex as sister to clade C (Bouvet, Eastern Weddell Sea). Similar results were found in the maximum-likelihood analysis, except that clade D1 was found to be paraphyletic with respect to D2 and D3.3.1.2. Internal transcribed spacer dataThe ITS phylogenetic tree showed a differentiation into six distinct monophyletic groups named I I (figure 4), which mostly correspond to larger groupings of different COI clades. The analysis of the dataset cropped with GBLOCKS resulted in a slightly different phylogenetic tree, but the differentiation into six groups did not change. There was no differentiation between specimens belonging to different COI clades within these groups. As an example, group IV included individuals belonging to COI clades A, H and I, but those clades could not be distinguished by ITS sequences. Group II showed strong intragroup variation, but no division into the COI clades D1, E1 and E2 was found. In some cases, there were also discrepancies in assignment to larger groups between COI and ITS. Group II mostly included individuals from clades D and E, but also one clade C individual. Group III included individuals from clades N2 and N3 as well as one each from clades E and G. Individuals from COI clade I were found in both groups IV and V.3.2. Phylogeographic analysis3.2.1. Cytochrome c oxidase subunit I dataMost of the clades are geographically widely distributed, and eight of them are found in both East and West Antarctica (tabl.
