Samples were collected and preserved as part of routine McMurdo Dry Valley Long Term Ecological Research (LTER) core limnological sampling. Samples were collected along a 4-16 m depth profile in Lake Fryxell and 4-25 m depth profile in Lake Hoare.
Samples preserved in 1 % Lugol’s solution at 5 °C were homogenized on a shaker table (150 RPM for 5 mins) before subsampling 150 mL and settling for 24 hrs. The supernatant was then aspirated off to concentrate the sample by 5-fold. 1-5 mL of homogenized subsample was imaged on FlowCam model VS-IV with a 10X lens and 100 µl flow cell. Images were captured using AutoImage Mode at 18-20 frames sec-1 at a flow rate ranging 0.15-0.20 mL min-1. The fluid volume that was imaged ranged from 5-20 %. Files containing the imaged plankton from each sample were then used to sort and build statistical filters based on particle morphologies.
The morphotypes of the imaged plankton were sorted and classified using the Visual Spreadsheet Software (version 4.17.14). From a select number samples that covered a range of times and depths throughout the record, images of like-morphotypes, such as a specific taxon or class of phytoplankton, were manually grouped into taxon- and morph-based libraries. For a total of 27 libraries that covered the range of morphotypes found in the plankton, we built statistical filters based on the specific geometries (e.g., length, width, aspect ratio) of the given particles in each library. The statistical filters were then built into a classification template of seven classes based on broad morphologic characterizations of the plankton. This classification template, based on statistical filters generated by particle attributes, was then applied to all the samples of interest to automatically sort and classify the image-based data.
We chose to assign taxonomic identifiers to only the classes in the classification template with high enough image resolutions for sufficient identification. This condition was met only for the haptorian ciliates of the genera Askenasia and Monodinium because these taxa are known to occur in these lakes and likely not to be confused with other protists. Additional classifications were made for broad algal groups but are not included in this dataset due to poor data quality. Separate classes with filters based on detritus, sediment, and air-bubbles were used to control for unwanted image data. Using our classification template, we were able to automatically classify haptorian ciliates from other microplankton and calculate attribute data (area-based diameter (ABD) and ABD biovolume) for each imaged particle.
