Description: Morphometric measurements of Fragilariopsis kerguelensis valves from a two-year time series covering the period from November 2002 to October 2004 collected by a sediment trap at 800m below the ocean surface which was moored near 54° S 140° E in ca. 2300 m water depth, close to the top of the Australia-Antarctica mid-ocean ridge.

Description: Light microscopy analysis of diatom frustules is widely used in basic and applied research, notably taxonomy, morphometrics, water quality monitoring and paleo-environmental studies. Although there is a need for automation in these applications, various developments in image processing and analysis methodology supporting these tasks have not become widespread in diatom-based analyses. We have addressed this issue by combining our automated diatom image analysis software SHERPA with a commercial slide-scanning microscope. The resulting workflow enables mass-analyses of a broad range of morphometric features from individual frustules mounted on permanent slides. Extensive automation and internal quality control of the results helps to minimize user intervention, but care was taken to allow the user to stay in control of the most critical steps (exact segmentation of valve outlines and selection of objects of interest) using interactive functions for reviewing and revising results. In this contribution, we describe our workflow and give an overview of factors critical for success, ranging from preparation and mounting through slide scanning and autofocus finding to final morphometric data extraction. To demonstrate the usability of our methods we finally provide an example application by analysing Fragilariopsis kerguelensis valves originating from a sediment core, which substantially extends the size range reported in the literature.

Description: Valves of the diatom Fragilariopsis kerguelensis, sampled along four North-South transects across the Southern Ocean, were imaged in high resolution, extended depth-of focus light microscopic images. The images were segmented and morphometric features extracted using the software SHERPA.

Description: Fragilariopsis kerguelensis is one of the most abundant diatoms in the sediments of the Southern Ocean. Its morphometric features have been proposed as proxies, based on links with, for example, iron availability, sea surface temperature and glacial/interglacial conditions. We investigated morphometric changes in F. kerguelensis valves in a well-studied sediment core record, focusing on transition periods between glacials and interglacials. By applying a high-throughput diatom imaging and morphometry workflow, we found two clearly distinct morphotypes which were differentiated by their rectangularity. One of them occurred preferentially in glacial samples, whilst the other persisted throughout. This indicates that their relative abundances depend on environmental conditions and thereby points to the possibility that paleo-proxies based on F. kerguelensis valve morphometric features might in the future benefit from differentiating these two morphotypes. As an initial exploration of this idea, we show that the abundance ratio of both morphotypes correlates well with paleo-temperatures which had been reconstructed using independent data from the same core. Distinguishing between the two morphotypes only became possible by image analysis for precisely measuring diatom valve outlines and area, highlighting the potential of such methods for diatom analyses.

Description: Background: Light microscopic analysis of diatom frustules is widely used both in basic and applied research, notably taxonomy, morphometrics, water quality monitoring and paleo-environmental studies. In these applications, usually large numbers of frustules need to be identified and / or measured. Although there is a need for automation in these applications, and image processing and analysis methods supporting these tasks have previously been developed, they did not become widespread in diatom analysis. While methodological reports for a wide variety of methods for image segmentation, diatom identification and feature extraction are available, no single implementation combining a subset of these into a readily applicable workflow accessible to diatomists exists. Results: The newly developed tool SHERPA offers a versatile image processing workflow focused on the identification and measurement of object outlines, handling all steps from image segmentation over object identification to feature extraction, and providing interactive functions for reviewing and revising results. Special attention was given to ease of use, applicability to a broad range of data and problems, and supporting high throughput analyses with minimal manual intervention. Conclusions: Tested with several diatom datasets from different sources and of various compositions, SHERPA proved its ability to successfully analyze large amounts of diatom micrographs depicting a broad range of species. SHERPA is unique in combining the following features: application of multiple segmentation methods and selection of the one giving the best result for each individual object; identification of shapes of interest based on outline matching against a template library; quality scoring and ranking of resulting outlines supporting quick quality checking; extraction of a wide range of outline shape descriptors widely used in diatom studies and elsewhere; minimizing the need for, but enabling manual quality control and corrections. Although primarily developed for analyzing images of diatom valves originating from automated microscopy, SHERPA can also be useful for other object detection, segmentation and outline-based identification problems.

Description: While attempting to assemble a reference image set of Southern Ocean diatoms for training automatic classification algorithms, we encountered numerous specimens which we were unable to classify unequivocally into one of three highly similar Fragilariopsis species. Problems about the delimitation of these species were also raised at the last Polar Marine Diatom Workshop in 2015 in Salamanca. The present study originated from these two sources. Using semi-automated microscopy and image analyses, we assembled a set of 501 specimen images and accompanying morphometric data, and 12 members of the polar marine diatomist community contributed their identification of these specimens independently from each other. After comparing the identification results themselves, we used the morphometric features extracted in an attempt to clarify the nature of morphometric distinction of the three taxa in uni-and bivariate analyses, and performed multivariate classification experiments and tested their agreement with expert consensus opinion. Beyond the specific insights into morphometric distinction of the studied taxa, our study also highlights some of the more generic challenges and possibilities of research at the interface between automatic identification and traditional taxonomy.