93 52 A F I G G Grsos.royalsocietypublishing.org R. Soc. open

93 52 A F I G G Grsos.royalsocietypublishing.org R. Soc. open sci. 2:…………………………………………IV0.531VVIE1 N3 G N3 N3 NIII1 94 NN2 N2 N2 E1 E1 D1 E1 C D1 D1 D11 94 E1 E1 E1 E1 E1 E1 E2 E2 E2 EDII1D1 E1 E1 1 100 0.96 61 B C CIoutgroups 0.03 substitutions per siteFigure 4. Bayesian phylogenetic tree of C. Larotrectinib web megalonyx ITS ARRY-470MedChemExpress Larotrectinib sequences. Numbers above branches are posterior probabilities, numbers below branches are bootstrap percentages for the ML analysis. Letters on branches refer to COI clade assignment of the respective specimen. Colours indicate geographical origin of samples. Numbers I to VI refer to the six ITS groups identified.clade Aclade Drsos.royalsocietypublishing.org R. Soc. open sci. 2:…………………………………………clade Eclade IBouvet Island S. Georgia S. Sandwich Islands Peninsula-South Orkneys Eastern Weddell Sea Terre Ad ie unknown106 specimens 10 specimens1 specimenFigure 5. Haplotype network for C. megalonyx clades A, D1, E1 and I based on the mitochondrial COI gene. Sizes of circles are proportional to number of individuals per haplotype. Colours indicate geographical origin of samples. Black dots represent hypothetical haplotypes.clades. These mostly involve those cases in which the distances are at an intermediate level falling into the barcode gap (about 2? ), i.e. the clades D1 + D2 + D3, E1 + E2 and N1 + N2 + N3. Interpretation of these as one, two or three distinct groupings should be considered ambiguous. In all other cases, the two methods agree, therefore showing a clear distinction between inter- and intraclade divergence levels, i.e. the presence of a barcoding gap.4.2. Geographical distribution of mitochondrial groupsSpecimens of some of the different clades recovered show a narrow distribution range, others are widely distributed and occur in sympatry. This stands in contrast to the findings of Krabbe et al. [37], who analysed only 96 specimens with more limited geographical sampling than in this study. The results here agree with several other studies on Antarctic benthic invertebrates (e.g. [9,20,22]) proposing circumpolar distributions of species based on molecular data. It should be noted that at least two clades exhibit obvious morphological differences from all others, namely clade C, which lacks pigmented eyes, and clade F, which includes animals significantly larger than all others examined. Lack of eyes has been previously reported for the (sub)species C. (megalonyx) orcadense [36,59] known from the South Orkneys, South Africa and Madagascar, while clade C is restricted to Bouvet Island and the Weddell Sea slope in our samples. If several species coexist sympatrically, it is to be expected that they exhibit ecological differences. Therefore, we expect that, if the mitochondrial clades are indeed distinct species, a detailed study would reveal noticeable morphological or physiological [23] differences.4.3. Implications of the nuclear dataAs shown in the results, there are several instances where the ITS data are incongruent with the COI data. This could be explained by retention of ancestral polymorphisms or by intragenomic polymorphism in ITS, as has been described for other arthropods [60,61]. However, the differences observed between different ITS groups in our study are generally higher than observed in those cases. Besides, we did not detect large amounts of conflicting signals in our sequence electropherograms, which would be expected in the case of polymorp.93 52 A F I G G Grsos.royalsocietypublishing.org R. Soc. open sci. 2:…………………………………………IV0.531VVIE1 N3 G N3 N3 NIII1 94 NN2 N2 N2 E1 E1 D1 E1 C D1 D1 D11 94 E1 E1 E1 E1 E1 E1 E2 E2 E2 EDII1D1 E1 E1 1 100 0.96 61 B C CIoutgroups 0.03 substitutions per siteFigure 4. Bayesian phylogenetic tree of C. megalonyx ITS sequences. Numbers above branches are posterior probabilities, numbers below branches are bootstrap percentages for the ML analysis. Letters on branches refer to COI clade assignment of the respective specimen. Colours indicate geographical origin of samples. Numbers I to VI refer to the six ITS groups identified.clade Aclade Drsos.royalsocietypublishing.org R. Soc. open sci. 2:…………………………………………clade Eclade IBouvet Island S. Georgia S. Sandwich Islands Peninsula-South Orkneys Eastern Weddell Sea Terre Ad ie unknown106 specimens 10 specimens1 specimenFigure 5. Haplotype network for C. megalonyx clades A, D1, E1 and I based on the mitochondrial COI gene. Sizes of circles are proportional to number of individuals per haplotype. Colours indicate geographical origin of samples. Black dots represent hypothetical haplotypes.clades. These mostly involve those cases in which the distances are at an intermediate level falling into the barcode gap (about 2? ), i.e. the clades D1 + D2 + D3, E1 + E2 and N1 + N2 + N3. Interpretation of these as one, two or three distinct groupings should be considered ambiguous. In all other cases, the two methods agree, therefore showing a clear distinction between inter- and intraclade divergence levels, i.e. the presence of a barcoding gap.4.2. Geographical distribution of mitochondrial groupsSpecimens of some of the different clades recovered show a narrow distribution range, others are widely distributed and occur in sympatry. This stands in contrast to the findings of Krabbe et al. [37], who analysed only 96 specimens with more limited geographical sampling than in this study. The results here agree with several other studies on Antarctic benthic invertebrates (e.g. [9,20,22]) proposing circumpolar distributions of species based on molecular data. It should be noted that at least two clades exhibit obvious morphological differences from all others, namely clade C, which lacks pigmented eyes, and clade F, which includes animals significantly larger than all others examined. Lack of eyes has been previously reported for the (sub)species C. (megalonyx) orcadense [36,59] known from the South Orkneys, South Africa and Madagascar, while clade C is restricted to Bouvet Island and the Weddell Sea slope in our samples. If several species coexist sympatrically, it is to be expected that they exhibit ecological differences. Therefore, we expect that, if the mitochondrial clades are indeed distinct species, a detailed study would reveal noticeable morphological or physiological [23] differences.4.3. Implications of the nuclear dataAs shown in the results, there are several instances where the ITS data are incongruent with the COI data. This could be explained by retention of ancestral polymorphisms or by intragenomic polymorphism in ITS, as has been described for other arthropods [60,61]. However, the differences observed between different ITS groups in our study are generally higher than observed in those cases. Besides, we did not detect large amounts of conflicting signals in our sequence electropherograms, which would be expected in the case of polymorp.

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