Finding the actual needle in a virtual haystack

by Christoph
 

For ecologists, there are two approaches to assessing biodiversity of a sampling site, be it solid rock, soil, fresh water, marine water, air, etc. The traditional approach is to record in the field, observe and count, and take samples for further study in the laboratory, including cultivation and DNA sequencing. In the other approach, recently introduced by Mechas, biodiversity is assessed by global sequencing of environmental DNA (eDNA) collected from a sampling site and its taxonomic classification and quantification by bioinformatics (keyword: metabarcoding). When an­alyzing the resulting voluminous datasets, opera­tional taxonomic units (OTUs) often pop up that correspond to known taxa/species never be­fore found at the sampling site − the topic that brought you here − in addition to unknown taxa/species, of course.
 

Figure 1. (C) View of the Bassa Nera from an adjacent mountain (click image for a geographical overview by Garcés-Pastor et al. (2019)). Frontispiece: View of the Bassa Nera (on the right). Source

Recently, Garcés-Pastor et al. (2019) reported micrognathozoan eDNA in a metabarcoding study of a high-elevation peat bog in the Pyrenees, Spain. Using universal 18S rRNA primers, the authors found micrognathozoan eDNA as one of the "20 most abundant 18S MOTUs [molecular operational taxonomic units] for the fen microhabitat". The micrognathozoan in question was Limnognathia maerski, discovered in submerged mosses at a cold freshwater spring in Disko Island, Greenland, in the mid-90s and formally described by Kristensen & Funch (2000). L. maerski is so far the only species in the phylum Micrognatho­zoa, a sister clade of the Rotifera, and has only been found on Île de la Possession, sub-Antarctic Indian Ocean and a few other disparate places (a bit more on Limnognathia biology below).
 

Despite the high number of reads Garcés-Pastor et al. (2019) took their discovery with caution, as its location lay far from known micrognathozoan localities and there were no specimens. But based on this eDNA evidence from the Pyrenees an expedition there was planned to find the actual needle in the virtual haystack, and carried out.
 

During this expedition, Giribet et al. (2023) − two of the co‑authors were also involved in the previous eDNA study − sampled 15 sites along the edge of the Bassa Nera peat bog (Figure 1) over four days of repeated sampling. They found Micrognathozoa in samples of only one of the sites, 62 specimens in total (Figure 2B). "Finding" meant squeezing submerged Sphagnum mosses − typical inhabitants of peat bogs and preferred source for collecting tardigrades − and concentrating the fauna on a 30 μm mesh before transferring it to multiple small containers (50–250 ml). Then, the filtered squeezed samples were brought to an improvised lab and examined alive under the stereomicroscope. They do not say how many hours they spent at the microscope(s), nor how many organisms they had to look through in total. But even without exact quantification it is clear that the micrognathozoan found in low numbers only at one of the 15 not far apart sampling sites is a rare find. It is thus the first docu­mented case of a micrognathozoan detected with eDNA and later found in the wild and the first confirmed micrognathozoan outside Greenland and Île de la Possession (they later learned that micrognathozoan eDNA was also found in a 2020 survey of the Danube river, Europe (PDF)). Micrognathozoa apparently have a worldwide but highly patchy distribution. The adage 'Everything is everywhere: but the environment selects' comes to mind...
 

(click to enlarge)
Figure 2. (B) Specimen of Limnognathia sp. collected in the water samples (anterior end is on top). Source. (C) Anatomical diagram of Limnognathia maerski. The cilia on the forehead create a current that moves food particles towards the mouth. The ventral cilia move the animal. Adapted from a diagram on the alchetron.com website.

Limnognathia maerski and the anatomically very similar Limnognathia sp. found in the Bassa Nera peat bog are multi‑cellular animals belonging to the phylum Micrognathozoa. Their bi­laterial body plan is readily seen in Figure 2C. Note the symmetrically arranged eyes, eggs, and proto‑nephridia (kidneys). Figure 2A unfortunately lacks a scale, but it can be assumed that the animal is ~100 µm long like Limno­gna­thia maerski. As in the rotifers ('wheel animals' after the corona around the mouth), the mouth parts of the micrognathozoans (jaw animals) are par­ticularly conspicuous and the jaws structurally very complex (see details here). They are quite dif­fer­ent from the "mouth parts" of single‑celled ciliates (oral groove/vestibulum, buccal overture, cell mouth/cyto­stome).
 

Are you surprised to find a metazoan here at Small Things Considered ? First, Limnognathia has a body length of ~100 µm, which is truly microscopic or "microbial," and thus much the same size as the large, single‑celled protist Paramecium bursaria (80−150 µm cell length), and just a tad smaller than the Kikiki huna fairyfly wasp (~150 µm body length). On the other hand, we know of large single-celled bacteria that we consider Small Things, for example Thiomarga­rita namibiensis (100–300 µm ⌀) or Achromatium oxaliferum (30×125 µm).
 

Second, we at STC have never been too picky about the term "microbes" in the past, and Merry once wrote fondly of the phylogenetic sister clan of the micrognathozoans, the Scandalous Bdelloid Rotifers (0.1–0.5 mm body length). Only the other much beloved tiny animals, the tardi­grades (0.5‒1 mm body length), have not made it to "honorary microbe" status with us, they are simply too big somehow. But then there are the myxozoans, which Roberto pointed out to me while editing this post. These minute animals(!) have hardly more than 10 cells and reach lengths of ~10 µm (see here in STC). In comparison, Limnognathia is a real monster! And it is really not very clever to classify "microbes" by their size alone, under the microscope you will find all life forms.
 

Third, the tiny creatures that Antonie van Leeuwenhoek saw in his microscope, he called kleijne dierkens. Henry Oldenburg, the secretary of the Royal Society in London, UK who took care of Antonie's letters, translated kleijne dierkens as animalcules, little animals. Among them were, as we know today, rotifers, that is, animals. If that is not sufficient reason to treat Limnognathia here!
 

Giribet et al. (2023) comment on the ever-increasing difficulty of obtaining data sets from published studies for meta‑studies by others, and they are not alone with these complaints: "Thus, unexpected findings in metabarcoding studies, rather than being dismissed as artifactual or question­able, can provide a map to locate and discover rare specimens, including the discovery of novel taxa in new regions. These treasure maps may, however, be hidden in the unfiltered data sets or supplementary materials of published studies or scientific reports, and thus improved data visibility and collaboration across disciplines are key factors for these treasures to be found and to begin the treasure hunt." Fortunately, their treasure hunt for the living Limnognathia needle was successful in the peat bog and it did not remain as just a couple of files in the haystack of sequence data.
 

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