Computational protocol: Poles Apart: The “Bipolar” Pteropod Species Limacina helicina Is Genetically Distinct Between the Arctic and Antarctic Oceans

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Protocol publication

[…] Specimens of the Antarctic Limacina helicina antarctica were obtained from the Amundsen Sea and the vicinity of South Georgia Island (). The forma of these specimens was not determined. Specimens of the Arctic Limacina helicina helicina were identified as forma helicina and were obtained from the Beaufort Sea. Full locations and station data are available on Barcode of Life Data systems (BOLD)/GenBank. Extraction, amplification and sequencing followed standard DNA barcoding protocols , . DNA was also extracted using the high salt method . PCR amplifications were performed using the standard Folmer primers and sequencing was carried out by Macrogen (Korea). New sequences have been deposited in BOLD/GenBank (Accession numbers GQ861824–861832, GU7328230). The length of L. helicina sequences varied from 528 bp to 618 bp. This variation reflects difficulty in amplifying the fragments. Alternative COI primers, a combination of standard primers and mini-barcode primers yielding two overlapping fragments , had to be used in addition to recover these shorter sequences. The published sequences of Remigio and Hebert for single specimens of L. h. helicina and L. h. antarctica were included in subsequent calculations of genetic distance.The K2P model of sequence evolution was used to calculate the genetic distance for L. h. helicina and L. h. antarctica both within and between regions, (i.e., Arctic and Antarctic) using PAUP 4.0b10 . The genetic distance between COI sequences of five individuals collected from the Arctic was 0.15±0.06%, whilst the genetic distance between COI sequences of six individuals collected from the Antarctic was 0.60±0.07%. Genetic distance between individuals collected from the two regions was 33.56±0.09%.Bayesian analyses were conducted using BEAST v1.4.8 , using a SRD06 nucleotide model . Analyses were run with both strict clock and uncorrelated log-normal relaxed clock models, with the mean substitution rate fixed to 1.0. A Yule prior on branching rates was employed . Gymnosomata and Thecosomata were assumed to be reciprocally monophyletic . Two independent MCMC analyses were run for each parameter set. Acceptable mixing and an appropriate ‘burnin’ was determined using Tracer v1.4.1 . Each analysis was conducted for 20 million generations sampling every 1000 generations. The Bayes factor was used to compare strict and relaxed clock models as implemented in Tracer v1.4.1. The uncorrelated log-normal relaxed clock model was preferred with a Bayes Factor (natural log) of 20.9±0.2.Phylogenetic maximum likelihood analyses were performed with RAxML v.7.0.4 . All searches were completed with the GTRMIX option and bootstraps were calculated with 1000 replicates. To obtain a minimum divergence time estimate of L. helicina from Arctic and Antarctic regions we also analysed the data within BEAST v1.4.8 (using a SRD06 nucleotide model and an uncorrelated log-normal relaxed clock model) using a fixed calibration date of 58.7 Ma on the divergence of Limacina (Limacinidae) and Hyalocylis (Creseinae) . L. mercinensis is the oldest known limacinoid fossil from the Thanetian (58.7±0.2-55.8±0.2 Ma) . The oldest known Creseinae fossils are from the Ypresian (55.8±0.2-48.6±0.2 Ma) . Therefore the Limacinoidea and Cresinae lineages must have diverged prior to the Thanetian. The age of Thecosomata was constrained to be less than 65 Ma as the group is understood to have evolved in the Cenozoic .In recent classification the family Cavoliniidae contains the subfamilies Cavoliinae, Clioinae, Cuvierininae and Creseinae. In contrast, in our topology the Cavoliniidae is paraphyletic, with a sister taxon relationship between Hyalocylis (Creseinae) and Limacina. This relationship was further supported by the possession of a shared indel by both taxa not present in any of the other species sequenced. […]

Pipeline specifications

Software tools PAUP*, BEAST, RAxML
Application Phylogenetics
Chemicals Calcium Carbonate