Massane meiofauna records

Study Extent

Samples were collected covering as many microhabitats as possible in the Massane Forest Reserve to obtain a large diversity of freshwater and limno-terrestrial meiofauna. Freshwater samples covered running and standing waters, focusing on submerged mosses, wet sediments, layers of fallen leaves on the bottom of scours in the stream bed, macrophytes, epilithic biofilms, and plankton samples. Samples for limno-terrestrial taxa included moss and lichen patches on different substrates (e.g. rock, tree trunk, soil), leaf litter, dry soils, fungi, tree holes, ivy rootlets, and other available tree-related microhabitats (TreMs) as defined in Larrieu et al. (2018).

Sampling

Samples were collected in the field, stored in plastic containers, envelopes, or ziplock bags, brought to the lab of the reserve, and extracted within a few days or prepared for long-term storage. Dry samples for limno-terrestrial meiofauna were stored for longer periods in envelopes and studied in the following few months. We also used anesthetics, fixatives and stains for optimal microscopic observation of taxonomically relevant structures. Taxonomic identifications were performed to species-level whenever possible, or to the nearest reliable rank by the expert taxonomists involved in the faunistic survey.

Quality Control

Quality control for geographic data: Georeferenced data and elevation were obtained directly in the field using various GPS tools. Quality control was performed using Google maps identification of sites. Geographic coordinate format and absence of ASCII anomalous characters in the dataset were additionally controlled.
Quality control for taxonomic data: Species identification was performed by taxonomic experts involved in the project. Nomenclature validation and cleaning were based on WoRMs (Horton et al., 2017) and on the GBIF taxonomic backbone (GBIF Backbone Taxonomy, 2023).
Taxonomic remarks: All species of Gastrotricha found in the various habitats of La Massane belong to the sub-taxon Paucitubulatina. A comprehensive taxonomic discussion of every encountered gastrotrich species along with morphometric data will be provided in the course of a subsequent publication.

Method steps

1. The applied methods for meiofauna extraction from the samples and identification under the microscope varied depending on the selected group: For Gastrotricha, sedimentary substrata (sediment, organic debris, leaf litter etc.) were suspended in a bucket with ambient water in the field and initially pre-filtered through a coarse sieve in order to get rid of large particles and macrofauna. In a second step, this filtrate was sieved through a fine gauze (40 µm mesh size) and the captured meiofauna was rinsed into the sampling jar using a squirt bottle filled with ambient water. Patches of aquatic mosses (Fontinalis antipyretica Hedw.), floating plants or submerged roots of ferns were hand-picked or sampled in stream pools with a plankton net (65 µm mesh size) mounted to a bar.
2. Qualitative sampling procedures mostly follow the methods described in Balsamo et al. (2014) or Todaro et al. (2019). Back in the laboratory, subsamples of the filtrates were poured into petri dishes and screened under stereo microscopes using different illumination modes (see Rotifer section) and magnifications. Single gastrotrich specimens were picked from the petri dish using either a mouth pipette or a 2 µL micropipette. For microscopic investigation and documentation, single specimens were placed on glass slides with a drop of ambient water and covered with a cover slip. Some specimens were anesthetized with few microliters of 0.25% Buccain (PUREN Pharma GmbH & Co. KG, München, Germany) prior to microscopic observation, alternatively the specimen was gently clamped between slide and coverslip by removing excess water carefully from the edge of the coverslip using a snippet of filter paper. Observation and live digital recording of specimens was carried out with a Olympus BX53 microscope equipped with high resolution objectives and differential interference contrast. An euromex HD-Ultra digital microscope camera VC.3036-HDS was adapted to the camera port of the microscope and every specimen was recorded with a series of still and video images. Taxonomic identification was mostly carried out after the expedition and was based on current monographs and taxonomic revisions (Balsamo 1983, Schwank 1990, Kisielewski 1991, Balsamo et al. 2014), original species descriptions and with the aid of the Gastrotricha World Portal (GWP, Todaro & Tongiorgi 2023).
3. For Nematoda, 3 categories of samples were distinguished: (1) The first 5-cm of stream sediments were collected using a PVC corer (diam. 9 cm), and the sediment was fixed in a solution of 4% buffered formaldehyde. The nematodes were further extracted quantitatively from the sediment samples using a density-centrifugation procedure following Schenk & Traunspurger (2021). Briefly, the organic supernatant, containing the nematodes, was poured through 20 µm meshes. After extraction, the organisms were stained with a few drops of Rose Bengal and counted under a stereo-microscope (40x magnification). When available, the first 50 nematodes encountered while counting were removed from each sample, transferred to anhydrous glycerol and mounted on slides following the method of Seinhorst (1959). (2) Stream epilithic biofilms were collected by scraping with a toothbrush the superior face of three cobbles, washing off the detached biofilm over 20 µm meshes. Biofilm-dwelling organisms retained on meshes were preserved, stained, counted, and mounted on slides as in (1). (3) The nematodes colonizing TreMs associated with beech (Fagus sylvatica L.) were also sampled. After prior knowledge from field monitoring campaigns, we selected the most accessible and widespread TreM-types available in Massane forest after Larrieu et al.'s (2018) TreM typology. Approximately 100 g of TreM substrate was collected by hand, with a shovel, or with a spoon when they were easy to dislodge. In other cases, we used a knife, a small axe, or a hand-saw to sample. TreM samples were then slid in an airtight bag, and transported to the laboratory within 24h. In the laboratory, we followed the protocol described by Travé et al. (1954): Briefly, the samples were placed in a fine gauze cloth (openings ca. 500 μm), positioned on a steel grid placed in the upper part of a large funnel (largest diam. 40 cm, so-called “Baermann funnels”). The funnels were set with a water phase, expanding slightly over the steel grid, so the TreM samples in the gauze were slightly soaking at the interface with water. The funnels were positioned under a lamp, so that small (usually lucifugous) hydrophilic organisms (such as nematodes, rotifers and tardigrades) were expected to quickly migrate in the water phase through the gauze's openings and steel grid, finally sinking into the bottom of the funnel. After 48 hours of migration – a period deemed long-enough to allow significant migration, and short enough to prevent substantial reproduction or predation within the funnel, (Travé et al. 1954) – we poured the entire water phase through a 20 μm sieve. The content of the sieve was preserved in 4% buffered formaldehyde and nematodes were further stained with Rose Bengal, counted, and mounted on slides as in (1).
4. For Rotifera, lotic samples like Fontinalis moss patches growing in rocky riffles and cascades were directly collected into a screw-cap tube while submerged and further inspected in the laboratory. Water in dendrotelms and puddles was sampled by a hose coupled to a 50 mL syringe. Water in stream pools was concentrated over 20 μm sieve. Aquatic samples were kept in the dark at 5°C and processed within a week after their collection. Terrestrial samples (moss and lichen patches) were kept dry and inspected in the laboratory after rewetting within 2 months. The samples were screened at a stereo-microscope with magnification between 6x and 80x, using bright, oblique, and dark field to avoid biases in the description of species diversity, given differential abilities of different species to stick to the substrate particles when treated with various extraction techniques.
5. For Platyhelminthes, ca. 1 L of soil, river sediment, leaf litter, moss, woody detritus was scraped and handpicked in zip-lock bags and directly returned to the laboratory where they were stored at 5°C in the dark. For some waterlogged samples, we employed an oxygen-depletion method (overnight stagnation in a wide-mouthed glass jar) to drive larger microturbellaria to the surface where they could be handpicked and concentrated. For most samples however, microturbellarians were extracted following a modified version of the Whitehead & Hemming (1965) tray method (which coincidentally also proved an efficient extraction technique for all meiofaunal taxa studied in this paper). Briefly, the sample was evenly spread onto a fine gauze tissue set onto a ~2 mm polypropylene sieve stacked within a seed sprouting tray filled with water to cover the surface of the substrate, and let to sit for up to 24 h. After that, the water in the tray, containing minimal substrate, was poured on 20 and 62 μm meshes, and a squirt bottle was used to concentrate the contents into petri dishes, which were then inspected for flatworms under a binocular. Specimens were further directly identified or wet-mounted on slides, semi-squeezing animals under a cover slip using tissue paper to wick away excess water. These were then microscopically observed in a Nikon Ni-U microscope equipped with DIC. Photos and/or videos of all specimens, emphasizing reproductive anatomy, were recorded as voucher data using a Nikon Digital Sight 10 microscope camera, and these were used to guide identification using primary literature and the Turbellarian Taxonomic Database Tyler et al (2023). Note that we use the term “microturbellaria” to refer to free-living Platyhelminthes (excluding Acoelomorpha) of microscopic size - a non-monophyletic group, which is nonetheless coherent from an ecological perspective, and distinct as well from their parasitic relatives in terms of the communities of researchers studying these animals.
6. For Tardigrada, samples of mosses and lichens were examined using standard methods as described in Stec et al. (2015). All specimens were mounted on microscope slides in a small drop of Hoyer’s medium and secured with a cover slip, following the protocol by Morek et al. (2016). Slides were then dried for five to seven days at 60 °C. Dried slides were sealed with a transparent nail polish and examined under an Leica DMLB light microscope with phase contrast, associated with digital camera. Taxonomic identification was carried out with the use of taxonomic keys and recent taxonomic revisions (Maucci (1986), Pilato & Binda (2010), Kaczmarek & Michalczyk (2017), Gąsiorek et al. (2019), Stec (2022)), and original species descriptions. All slides are deposited in the Tardigrada collection at the Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016 Kraków, Poland.

The dataset covers only animals considered as meiofauna, defined as microscopic invertebrates, belonging to the phyla Gastrotricha, Nematoda, Platyhelminthes, Rotifera, and Tardigrada.
Taxonomic ranks: All identified organisms during the survey, with data from variety and subspecies levels to species, genus, and family rank were included in the dataset.
Taxonomic methods: All reported names are provided according to the currently (August 2023) most updated nomenclature of WoRMs (Horton et al., 2017) and checked against the taxonomic backbone of GBIF (GBIF Backbone Taxonomy, 2023). For Rotifera, the rotifer List of Available Names, LAN (Segers et al., 2012), was used for all scientific names published before the year 2000. For Gastrotricha, validity of generic, sub-generic and species names was checked against Balsamo et al. (2009) and Todaro & Tongiorgi (2023).
Taxon specialists: for Gastrotricha, Thiago Quintao Araujo, Nicolas Bekkouche, Alexander Kieneke, Axell Kou Minowa; for Nematoda, Nabil Majdi, Walter Traunspurger; for Platyhelminthes, Christopher Laumer; for Rotifera Monogononta, Thiago Quintao Araujo; for Rotifera Bdelloidea, Diego Fontaneto; for Tardigrada, Daniel Stec.

1. Gastrotricha
   rank: phylum
2. Nematoda
   rank: phylum
3. Platyhelminthes
   rank: phylum
4. Rotifera
   rank: phylum
5. Tardigrada
   rank: phylum

Western Palearctic region, between Pyrenean and Mediterranean areas. Country: France, Municipality of Argelès s/Mer. min Longitude: 3.0310236 – min Latitude: 42.4917080 – max Longitude: 3.0366200 – max Latitude: 42.4981389. The data are georeferenced according to WGS 84.