05 Substrate/Soil Sampling & Processing

The study area was subdivided into 20 x 20 metre quadrants. At each quadrant at least one 10cm deep soil core was collected beside the location where unconsolidated soil depth was measured. If the quadrant contained the boundary between two distinctive tesserae, each tessera was sampled separately one metre from the boundary. Soil sampling was restricted to a day after a rain event of at least 2.5cm of precipitation to ensure the soil was naturally wet. (Table 5 and  6).

Unconsolidated soil depth: Each location was first assessed for the depth of unconsolidated soil using a flat end 3/8 inch wide by 3 feet long screw fitted in one side with two, three inch long nuts to serve as a handle. The screw was hand driven into the ground (applying about a 20kg force) until it was not possible to penetrate the ground any further. The depth was reported as unconsolidated soil depth.

Collecting soil cores: Prior to soil sampling, the sampling site was photographed from breast height covering an area of approximately 30 x 30 cm2; the plant material at the sampling location was trimmed down to about 1cm off the ground leaving the litter layer intact to form part of the sample. A 5cm in diameter soil core about 10cm deep was extracted using a bulb planting tool, carefully released into a plastic bag, inspected for layering, invertebrates, root layer, etc. and any measurements and observations annotated on the bag. The core was then sealed and stored in ice until further processing.

In the lab, The litter, organic layers, and soil were then reinspected visually for size ranges of litter, soil and root – soil agglomerates, organic thickness, root depth, horizons etc., as well as, for living arthropods, molluscs, and annelids – specimens were collected for later identification (see below for details).

Processing of dry litter and organic layers: The litter and organic layers samples, if present, were separated from the core and inspected under the dissecting microscope for live meso- and microfauna (arthropods, molluscs, and annelids) – specimens were collected for later identification (see below for details). Subsequently, the litter and organic layers were allow to air dry and reexamined for mesofauna (see below for details).

Processing of soil samples. The soil core was examined for discernable colour layers and root structure (depth of root balls and extent of long roots or long root bundles). Most of the soils examined had a natural cleavage at the bottom of the root ball that extended in the order of 4 to 6cm deep. This layer was noted as Top Layer.  The other section of the core usually consisted of soil of uniform appearance and was noted as Bottom Layer. In some cases three distinct layers were discernable the middle layer consisting of a fine gray clay (Middle Layer). Each layer was separated from the rest and broken into smaller pieces by squizzing it in the hand while still moist. Except for the Middle gray clay layers, Top and Bottom layers broke into distinctive aggregates.

Top Soil Layer.  After air drying, the roots of this layer were removed carefully dislodging the soil aggregates attached to the roots or root ball. The dry separated roots were collected and weight. The soil sample was then sieved through a 0.5cm, and a 0.2cm mesh. Each fraction collected was weight and examined under the dissecting microscope for evidence of mesofauna and animal activity (shells, insect remnants (mainly ants), cacoons, rodent scat, etc. – all findings collected and photographed). After each fraction was examined and processed the three sieved fractions were macerated using a ceramic mortar. Depending on the sample the soil would contain different amounts of small rocks. These rocks would be removed by sieving the soil again through the 0.2cm mesh, collected and weight.

Middle and Bottom Soil Layers.   After air drying, the roots of these layers were removed carefully dislodging the soil aggregates attached to the roots. Each of the soil layers were then processed as described above.

Processing of soil specimens: Specimens were collected in labelled 500ìL conical centrifuge test tubes until further use. Each soil layer sample would generate 3 collecting vials identified as <.2cm, .2 to .5cm, and > .5 cm. Most of the snail specimens were collected from the <.2cm  and .2 to .5 cm fractions.  Handling of specimens was done using NO Inox tweezers, or  00, 3/0, 20/0, and 3 paint brushes made of camel hair and white synthetic fibres.  White fibre brushes were used for cleaning and general handling, while camel hair brushes were used for delicate items and transfer of small specimens. Any live mites or larvae were collected separately in a vial with 70% isopropanol mixture.

The contents of each vial was layered over a cotton pad inside a petri dish. Any water sensitive specimens (such as earthworm casts, fine plant material, scats, etc.) were dry stored separately. The rest of the sample on the cotton pad – which consisted mostly of shells, seeds, and arthropod remains, was soaked in distilled water for a minute or two before gently scrubbing off the soil adhered to the specimens using a 00 white fibre paint brush. During cleaning specimens were sorted into four categories: “shells”, “seeds”, “arthropods” and “other”.

Dry snail shells were laid out on the cotton pad and separated into distinguishable species and counted. Identification of the snails was done based on the keys by Burch1, Grimm et al.2, and Pearce et al.3

Similarly, seeds were grouped into different species or types and photographed with a scale.

Lastly, arthropods or any other animal remains were grouped into recognizable body parts (e.g. heads, pronota, elytra, etc.) , scats, cacoons, etc. and photographed with a scale. Specimens in 70% isopropanol were also photographed with a scale.

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