2.2.1 Experimental setup
Jn situ experiments to investigate colonization patterns were conducted in the Kiel Fjord (Fig. 2.1) at the pier of the lnstitut fur Meereskunde. Artificial substrates were suspended from the outermost, unused part of the pier to a mean depth of 1.5 m.
Although the Western Baltic Sea is virtually non-tidal, wind-induced variations in water level occur. However, the artificial substrates remained at least 0.5 m below the water level.
Experiments with daily, weekly and monthly sampling intervals were performed (Table 2.1 ). The substrates for weekly and monthly sampled experiments consisted of kieselgur aquaria stones (50 x 25 x 25 mm3). The substrates for monthly aspects of colonization (in total 24 stones) were suspended in the water column in December 1996. Beginning with January 1997, two stones were collected in monthly intervals. For daily intervals, wood-substrates (25 x 15 x 15 mm3) were suspended in May 1998 and 3-4 substrates were harvested on each of 6 sampling dates.
Table 2.1: List of colonization experiments conducted in the Inner Kiel Fjord, giving information on names, duration, substrates, sampling procedure, and treatments.
experiment time substrate sampling method treatment
season 97 5 Dec 1996-9 Dec 1997 kieselgur monthly intervals none autumn 96 26 Sep-12 Dec 1996 kieselgur weekly intervals + 150 µMN
+ 10 µM P spring 97 11 Feb-6 May 1997 kieselgur weekly intervals + 150 µMN summer 97 6 May-8 Jui 1997 kieselgur weekly intervals none
summer98 25 May-3 Jun 1998 wood daily intervals none
For each of the three experiments with weekly sampling intervals, 12 experimental units were used. These experiments were designed to allow a direct comparison of colonization and enrichment experiments, for which only final harvest was analyzed (Chapter 4). Therefore, both experiments were performed with a similar setup.
Darkened 10 I PE-flasks were installed on top of the pier of the institute in Kiel and filled with medium (Fig. 2.2). The medium was based on seawater from the
surrounding of the pier. This was filtered (0.2 µm cellulose-acetate filters) and enriched with different concentrations of nutrients. Nitrogen was added as NaNQ3, phosphate as KH2P04. The concentrations of the other nutrients were not changed, i.e. remained at background levels (cf. Fig. 2.3). This liquid medium flowed through silicon tubes (inner diameter 4 mm) and trickled out through the artificial substrates.
With a precise mechanic regulation device, which was invented for intravenous infusion (Angiokard AK 5505), the flow rate was adjusted to 1 I d-1. Twice a week the supply bottle was refilled, the flow-rate was controlled, and readjusted if it deviated by > 10% from 1 I d-1. In the colonization experiments, all bottles contained the same nutrient concentrations (Table 2.1 ). By sampling one stone per week, the colonization process could be followed. Each stone sampled and taken out was replaced by a new one and at the end of the experiment all treatments were harvested again. In this way two series of samples were obtained. In the first series, the different incubation times had equal starting points and unequal harvesting points. In the second series (replacement stones) the starting points were unequal, but the harvesting points equal. Substrates for monthly and daily aspects were not replaced.
2.2.2 Sample preparation and analysis
Immediately after collecting, the substrates were transported to the laboratory, and the algae were scraped off quantitatively until no pigment colour could be detected on the stones. The biomass was suspended in organism-free filtered seawater (0.2 µm cellulose-acetate-filters). Subsamples were fixed with Lugol's iodine (10 g Kl + 5 g I per 100 ml) and counted within five weeks. Algal cells were counted with an inverted microscope (Leitz DMIRB) at 400x magnification with standard Utermohl-counting chambers (Hydrobios) (Utermohl 1958). 1 OOO cells were counted at minimum per sample. To compare the different species, which spanned several orders of magnitude in size, biovolume was calculated by fitting nearest geometric models (Hillebrand et al. 1999). For this, linear dimensions were measured from 20 specimens of each species (except for rare species, which were present with fewer individuals). Measurements were done with an ocular scale, calibrated to an object micrometer.
Further subsamples were used for species determination. The species were determined alive during the first days following the sampling. Additionally, diatoms were analyzed on permanent slides. For these, a subsample was washed with bidest. H20, and subsequently oxidized in 30% H202 for 3-5 days. An aliquot was dropped on a slide and the liquid was vaporized by heating. Afterwards the sample was mounted in Naphrax (Biological Supplies Ltd.). Taxonomy of diatoms follows the nomenclature of Round et al. (1990) and Snoeijs (with co-editors 1993-98), beyond these Kuylenstierna (1989-90), Krammer & Lange-Bertalot (1986-91) and Pankow (1990) were used for determination. Taxonomy of other algae followed the nomenclature of Pankow (1990).
In order to know the planktonic species composition, pelagic samples were taken throughout the study period at the surface by a simple scoop. They were fixed by Lugol's iodine, counted and the species composition was determined as described above. Dissolved inorganic nutrients were analyzed from these samples with a Continuous Flow Analyzer using the methods of Grasshoff et al. (1983) for silicate, nitrate, ammonium and phosphate. All nutrient concentrations and ratios in this text are molar. Surface water temperature and mid-day irradiance level were read from continuous measurement devices of the institute.
2.2.3 Analysis of diversity and statistical analysis
Diversity comprises the number of taxa present and the equitability of the distribution of abundances among the different taxa. Diversity indices are proposed as univariate measures composed of both characteristics. The statistical behaviour of several diversity indices has widely been discussed and this discussion is far from being settled (Hurlbert 1971, Peet 197 4, Pielou 1977, Robinson & Sandgren 1984, Krebs 1985, 1989; Valiela 1995). Therefore I employed two unrelated indices to show the robustness of my results. Since any two diversity indices are differently weighted regarding their sensitivity to species richness or equitability, they often show discrepancies in their response to changes in community composition (Hurlbert 1971 ). I adopted two statistics in common use, the Shannon-Weaver index H' and Simpson's index D', together with the species richness S and the evenness index J'. It was noted that H' is more sensitive to changes in rare species compared to D', which responds most strongly to changes in the most abundant species
(Krebs 1989). The insensitivity to the addition of rare species has been a major criticism against diversity indices (Sager & Hasler 1969, Brown 1973). However, rare species indeed make a minor contribution to communities and thus should have minor influences on community parameters like diversity (Hurlbert 1971 ). The insensitivity to minor changes enhances the reliability and objectivity of the indices, since the sampling effort is less influential.
Both diversity indices, H' and D', depend on contributions Pi of the ith species to the community or sample (Equ. 2.1 and 2.2). This can be expressed either in terms of contribution to total number of organisms or to total biomass. The calculation of H' based on biomass proportions was repeatedly recommended (Wilhm 1968, Hallegraeff & Ringelberg 1978, Cousins 1991 ). Some studies have used biovolume as biomass equivalent for benthic microalgae (Hill & Knight 1988, Carrick et al.
1988), since biovolume is an accessible way to measure the biomass of microbial species (Hillebrand et al. 1999) and includes size as a dominant denominator of biological processes in microbial communities (Steinman et al. 1992, Reynolds 1997, Sommer 1998). Since benthic microalgal species comprise several orders of magnitude in size, I decided to calculate the diversity indices on the basis of biovolume proportions throughout this study.
• Shannon-Weaver information theory index H'
H , = - -"-'
""'i=s i= 1I n p; · p;
(Equ. 2.1) with pi : contribution of the ith species to the total biovolume of the community.• complement of Simpson's index D
D' = 1 - D = 1 - L 1:1 Pl
(Equ. 2.2)Simpson's original index D measured the probability that two randomly depicted individuals represent one species. By using the complement D', the diversity of these organisms is analyzed (Krebs 1989).
• evenness J' (Pielou 1977)
, H' H'
J =
Hmax=
lnS (Equ. 2.3)This evenness index has recently been criticized for being dependent on species number (Smith & Wilson 1996). However, J' decreased with decreasing species number only if species numbers were <15. This threshold of species richness is exceeded in all microbial communities analyzed here.
• species richness S
The exact species number of a community cannot be estimated from samples.
However, S can easily be compared if it is based on a standardized sample size, which was the case since I counted 3000 cells in the enrichment experiments and
1 OOO cells in the colonization experiments.
The development of diversity during colonization was analyzed with linear and non-linear model-I regression. In order to compare the species composition of the pelagic and benthic community, a multivariate graphic approach was used. Relative abundances according to Table A2 were used in order to avoid the separation of benthic and pelagic samples just by the different biomass magnitudes. For all experiments with benthic samples (n=30) and monthly pelagic samples (n=24), Euclidean distances were calculated and used to create a multidimensional scaling (MDS) plot.