ITS samples from the air and snow of Livingston Island

Study Extent

Air and snow samples were collected at Punta Polaca (62°40′16″ S; 60°22′43″ W), Hurd Peninsula, Livingston Island, South Shetland Islands, near to the Spanish station Juan Carlos I in March 2019.

Sampling

Two air samples were collected with a high flow glass impinger following Šantl-Temkiv et al. The chamber was filled with 2 L of sampling liquid (ddH2O) and the sampler was run for 5 min, so that the liquid came in contact with the entire chamber, after which 0.5 L of the sampling liquid was removed, stored as a control, and analyzed along with the samples. The resulting solution was filtered directly on the Sterivex filter units for the air. Air was collected over c. 5 h on March 11th 2019. In addition, the two separate air DNA extractions were combined together in order to increase DNA yield. Two freshly deposited snow samples were collected on March 20th 2019 at the same site using a sterilized shovel. Both pairs of samples were separately combined in order to increase DNA yield. Snow was melted at room temperature, under strictly sterile conditions, for 24 h in the laboratory at Juan Carlos I Station and then filtered using Sterivex filters.

Quality Control

The control represented a field blank to certify that the samples were not contaminated by external organisms.

Method steps

1. Total DNA was extracted from environmental samples using the Qiagen Power Soil Kit (Qiagen, USA) following the manufacturer’s instructions. Extracted DNA was used as template for generating PCR amplicons. The internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA was used as a DNA barcode for molecular species identification. PCR amplicons were generated using the universal primers ITS3 and ITS4 and were sequenced by high-throughput sequencing at Macrogen Inc. (South Korea) on an Illumina MiSeq sequencer, using the MiSeq Reagent Kit v3 (600-cycle) following the manufacturer’s protocol.

Antarctica: Livingston Island, near Spanish Juan Carlos I station