Scott Seamounts0?rsos.royalsocietypublishing.org R. Soc. open sci. 2:…………………………………………90?W80?S 70?S 60?S 50?S 40?SFigure 1. Map of the Southern Ocean with sampling sites of the specimens of Colossendeis megalonyx analysed in this study. Colours correspond to those in figures 3 and 4. For a detailed overview of samples and sampling sites, see electronic supplementary material, S1 and S2. Photo of Colossendeis megalonyx: Claudia P. Arango.Burdwood Bank). Here, we substantially expanded the dataset of Krabbe et al. [37] by adding COI data for over 300 specimens from the same areas as well as from other regions in South America, along the Scotia Arc, and from the West and East Antarctic shelf. We further included data from an additional locus, the nuclear ribosomal gene region internal transcribed Sch66336MedChemExpress Sch66336 spacer (ITS), for a subset of individuals. This region, which includes the gene for 5.8S rRNA as well as the non-coding ITS1 and ITS2, has been found to be useful to distinguish closely related species in many different animal groups (e.g. [38?0]), including pycnogonids [41]. With the new dataset, we tested (i) whether there are further overlooked mitochondrial clades additional to the six clades found by Krabbe et al. [37]; (ii) whether the proposed narrow distribution ranges of the clades were supported by the new data from many more regions; (iii) whether or not the nuclear data support the pattern revealed by the mitochondrial data; and (iv) whether C. megalonyx colonized the Antarctic from the Subantarctic or vice versa. We discuss the new findings in the context of marine Antarctic evolution during the Pleistocene glaciations.2. Material and methodsA 658 bp fragment of the mitochondrial COI gene was sequenced for a total of 418 putative C. megalonyx specimens from different parts of the Southern Ocean (see figure 1 for a map of the sampling sites) and for an additional 82 specimens belonging to other colossendeid species (table S1). Individuals were determined to species level with the keys of Child [34] and Pushkin [42] prior to completing any genetic analyses. DNA extractions were performed using the Qiagen DNeasy Blood purchase LM22A-4 Tissue Kit following the manufacturer’s protocol with the exception of using only 100 elution buffer (EB) to increase final DNA concentration. PCR for COI was performed as outlined by Krabbe et al. [33]. An approximate 1000 bp fragment of the ITS (18S TS1?.8S TS2?8S) was sequenced for a subset of 76 C. megalonyx specimens and 34 other colossendeids. PCR was performed as follows: 94 C for 2 min, followed by 37 cycles of 94 C for 20 s, 55 C for 30 s and 65 C for 80 s, with a final extension at 65 C for 10 min. Primers used for PCR were ITSRA2 and ITS2.2 [43]. For both gene regions, the PCR mix consisted of 2 10?HotMaster Taq Buffer (5Prime, Hilden, Germany), 2 of 2 mM dNTPs, 0.1 of 100 HCO or ITSRA2 primer, 0.1 of 100 LCO or ITS2.2 primer [43,44], 0.1 of 5 U -1 HotMaster Taq (5Prime, Hilden, Germany), 1 DNA (approx. 20 ng),90?Efilled up to 20 with sterile H2 O. PCR products were purified with a 1 : 2 mix of Exo and FastAP for 15 min at 37 C followed by inactivation for 15 min at 85 C. Sequencing was performed at GATC Biotech (Cologne, Germany). For COI, the colossendeid sequences from Krabbe et al. [37] (96 C. megalonyx, 19 from other species) and all sequences from GenBank that were identified as members of the Colossendeidae by BLASTn searches (37 C. megalonyx, 113 from.Scott Seamounts0?rsos.royalsocietypublishing.org R. Soc. open sci. 2:…………………………………………90?W80?S 70?S 60?S 50?S 40?SFigure 1. Map of the Southern Ocean with sampling sites of the specimens of Colossendeis megalonyx analysed in this study. Colours correspond to those in figures 3 and 4. For a detailed overview of samples and sampling sites, see electronic supplementary material, S1 and S2. Photo of Colossendeis megalonyx: Claudia P. Arango.Burdwood Bank). Here, we substantially expanded the dataset of Krabbe et al. [37] by adding COI data for over 300 specimens from the same areas as well as from other regions in South America, along the Scotia Arc, and from the West and East Antarctic shelf. We further included data from an additional locus, the nuclear ribosomal gene region internal transcribed spacer (ITS), for a subset of individuals. This region, which includes the gene for 5.8S rRNA as well as the non-coding ITS1 and ITS2, has been found to be useful to distinguish closely related species in many different animal groups (e.g. [38?0]), including pycnogonids [41]. With the new dataset, we tested (i) whether there are further overlooked mitochondrial clades additional to the six clades found by Krabbe et al. [37]; (ii) whether the proposed narrow distribution ranges of the clades were supported by the new data from many more regions; (iii) whether or not the nuclear data support the pattern revealed by the mitochondrial data; and (iv) whether C. megalonyx colonized the Antarctic from the Subantarctic or vice versa. We discuss the new findings in the context of marine Antarctic evolution during the Pleistocene glaciations.2. Material and methodsA 658 bp fragment of the mitochondrial COI gene was sequenced for a total of 418 putative C. megalonyx specimens from different parts of the Southern Ocean (see figure 1 for a map of the sampling sites) and for an additional 82 specimens belonging to other colossendeid species (table S1). Individuals were determined to species level with the keys of Child [34] and Pushkin [42] prior to completing any genetic analyses. DNA extractions were performed using the Qiagen DNeasy Blood Tissue Kit following the manufacturer’s protocol with the exception of using only 100 elution buffer (EB) to increase final DNA concentration. PCR for COI was performed as outlined by Krabbe et al. [33]. An approximate 1000 bp fragment of the ITS (18S TS1?.8S TS2?8S) was sequenced for a subset of 76 C. megalonyx specimens and 34 other colossendeids. PCR was performed as follows: 94 C for 2 min, followed by 37 cycles of 94 C for 20 s, 55 C for 30 s and 65 C for 80 s, with a final extension at 65 C for 10 min. Primers used for PCR were ITSRA2 and ITS2.2 [43]. For both gene regions, the PCR mix consisted of 2 10?HotMaster Taq Buffer (5Prime, Hilden, Germany), 2 of 2 mM dNTPs, 0.1 of 100 HCO or ITSRA2 primer, 0.1 of 100 LCO or ITS2.2 primer [43,44], 0.1 of 5 U -1 HotMaster Taq (5Prime, Hilden, Germany), 1 DNA (approx. 20 ng),90?Efilled up to 20 with sterile H2 O. PCR products were purified with a 1 : 2 mix of Exo and FastAP for 15 min at 37 C followed by inactivation for 15 min at 85 C. Sequencing was performed at GATC Biotech (Cologne, Germany). For COI, the colossendeid sequences from Krabbe et al. [37] (96 C. megalonyx, 19 from other species) and all sequences from GenBank that were identified as members of the Colossendeidae by BLASTn searches (37 C. megalonyx, 113 from.
