Foraminiferal analysis

2.3.1 Extraction and identification of live foraminifera by pseudopodial activity

The live foraminiferal specimens utilised in Chapters 3, 5 and 6 were identified by pseudopodial activity. The identification of live specimens from these samples was a collaborative effort between this researcher and others from the larger NERC project. Initially, surface sediment scrapes were sieved at 63 μm in ambient seawater (where available) in order to concentrate the foraminifera and to remove smaller particulate matter. The sieved residues were then stored in seawater in the fridge or a cold room at a constant temperature of 4o_C.

In order to extract potential live specimens from the samples, a small amount of the sediment was transferred into a small seawater filled petri dish using a pipette. The petri dish was placed onto an insulated ice block to keep the samples at a constant temperature. The ice block was insulated with bubble wrap where available or paper towelling to prevent the samples from super cooling (i.e. becoming too cold). Initially, potentially live foraminifera were differentiated from the dead (empty) tests within the sediments based on their cytoplasm test colouration. Live foraminifera commonly exhibit green, orange or pink test colouration (Murray, 2006); an example of cytoplasm colouration is exhibited in Figure 2.3.

These potentially living foraminifera were extracted from the sediment using a 0000-gauge paintbrush and were placed into a separate seawater filled petri dish, which was kept cold by an insulated ice block. These picked specimens were then observed for any signs of pseudopodial activity to determine if they were live. The first protocol employed by this thesis, investigated pseudopodial activity by foraminiferal ‘racing’. This technique is effective for foraminifera taken from dynamic environments e.g. from intertidal areas. Specimens were organised into lines

drawn on the base of the petri dish and were left for a couple of hours. Specimens were determined live if they departed from their predefined position on the lines. If no movement was observed, the second protocol for recognising pseudopodial activity was employed, which is outlined below.

Live foraminiferal specimens can also be identified by detecting the overnight formation of a sediment cocoon. Potential live specimens were thoroughly cleaned and placed into a petri dish filled with seawater and fine sediment. These petri dishes were then carefully placed into the fridge or cold store overnight. Live foraminifera were identified by the presence of a sediment cocoon, which was formed by pseudopodial activity which draws in and sifts through fine sediment in search of food. Examples of sediment cocoons are illustrated in Figure 2.3.

Figure 2.3 Light microscope images illustrating the overnight formation of a sediment cocoon. A) Distinctive cytoplasm colouration B) An example of a fully formed sediment cocoon, C) An example of a half-formed sediment cocoon.

Once the live specimens were identified from the surface sediment samples, these specimens were cleaned and placed onto micropalaeontological slides to dry, so that they were ready for SEM imaging prior to rRNA extraction. If the specimens did not exhibit any pseudopodial activity overnight, the petri dishes were returned to a fridge or a cold room and were checked daily for signs of pseudopodial activity over a course of 3-5 days.

2.3.2 Extraction and identification of ‘live’ Rose Bengal stained foraminifera

Prior to processing the samples collected by the scuba divers (NFSD) used in Chapter 4, the volume of the sediment in the sample pot was calculated. This enabled the standardisation of foraminiferal abundances per 100ml in later proceedings. The sediment surface in the sample bottle was marked with a water resistant permanent marker. The sediments were then wet

sieved at 63μm using a fine water spray until the effluent water ran clear. These samples were dried at 40o_{c degrees and the dry weight of each sediment sample was calculated. Between}

sieving each sediment sample, the sieves were washed and then submerged in methyl blue solution. This solution stains calcium carbonate blue identifying any residual contaminant specimens within the sample. Following the sieving, the marked sample containers were refilled with water up to the mark to detect the settled sample volume.

Small proportions of the sediment were then evenly distributed across a gridded brass-picking tray and ‘live’ stained individuals were identified based on their Rose Bengal staining (distinctive pink/red colouration). Rose Bengal staining of foraminifera is an important technique as it is an inexpensive and easy method for identifying live foraminifera, as it stains the protoplasm within the test (Walton, 1952). However, this staining technique has in recent years came under scrutiny, because it can also stain specimens which have died weeks or even months prior to sampling but still retain undecayed protoplasm (Bernhard, 1988; Murray and Bowser, 2002). Therefore, in order to minimise errors assigning ‘live’ stained specimens a strict protocol was enforced, whereby only individuals that were stained pink/ red across all of the test were considered alive. In situations where foraminifera exhibited pale or inconsistent staining, these specimens were considered dead at the time of collection (Goldstein and Harben, 1993). However, it should be noted that the degree of staining in foraminifera is often species-specific. For example, the porcellanous wall structure of some species obscures the colour of staining; therefore, a small amount of water was applied to these specimens in order to help identify the presence of staining within these taxa (as per recommendations of Schönfeld et al., 2012). Once specimens were identified as ‘live’, these specimens were then extracted from the sediments using a 0000-gauge paintbrush. In order to avoid bias of preferential picking towards more visible individuals, every foraminiferal specimen was picked from each square before moving onto the next square. Where possible, 300 specimens were extracted from each sample. This is because the target number of specimens needed for a reliable estimation of foraminiferal abundance is 300 specimens (Patterson and Fishbein, 1989). In samples with low abundances, a minimum target of 100 specimens was made (when possible). This provides a 99% confidence interval that species making up >5% of the assemblage are captured (Fatela and Taborda, 2002) and that species as rare as 3% of the assemblage are captured at a 95% confidence interval (Dennison and Hay, 1967).

2.3.2.1 Presentation of foraminiferal assemblage data

The foraminiferal assemblage counts presented in Chapter 4 were expressed as different measures of the community structure outlined below.

Relative abundance: percentage of each foraminiferal species in relation to all other counted foraminiferal specimens in a sample.

Absolute abundance (or standing crop): the number of live (Rose Bengal stained) specimens per standardised volume. Historically benthic foraminiferal standing crop is normally expressed as foraminifera per cm3 _{(Murray, 2006). However, as the area and depth of sediment at time of}

sampling is unknown, the absolute abundance (standing crop) in this thesis is expressed as ‘live’ foraminifera per 100ml.

Diversity indices: Measures of taxonomic diversity and community structure are outlined below. These measures of diversity were calculated using the PAST software v.2.17 (Hammer et al., 2001).

Species richness was calculated as the total number of species identified per sample.

Measures of Species diversity were calculated using both the Shannon Weiner diversity index (H’) and the Fisher’s alpha diversity index (Fisher et al., 1943). The Shannon Weiner diversity index (H’) provides a measure of species abundance and relative richness (Shannon, 1948). The Shannon Weiner index takes into account the number of individuals and the number of taxa; consequently, this index does not place much significance upon the chance occurrence of rare species (Hammer, 2006). The Fisher’s alpha species index (Fisher et al., 1943) was included as it is one of the most common measures of foraminiferal taxonomic diversity employed within the NE Atlantic, particularly in the NW Scottish shelf seas (Hannah and Rogerson, 1997; Murray, 2002; Austin and Cage, 2010). Fisher’s alpha diversity index (Fisher et al., 1943) attempts to understand patterns of relative abundance of species in a community. This statistic is calculated using a log series model to predict the number of different species at different levels of abundance.

Species Evenness was calculated using the Pielou species Evenness index, whereby the distribution of individual densities between different species was analysed (Pielou, 1966). A high Evenness index number equates to a relatively diverse sample.