What are pelagic and benthic

Investigation of the pelagic and benthic protozoan communities in geologically young lakes of West Greenland

Table of Contents

1 Introduction

2. Material and methods
2.1. Sampling area
2.2. Sampling from lakes in West Greenland
2.3. Cultivation of Protozoa
2.4. Goose droppings samples
2.5. Animal fur swabs
2.6. Morphological examination
2.7. statistical evaluation

3. Results
3.1. Morphological analysis of the protozoal community
3.1.1. Species distribution in the lakes
3.1.2. Meltwater samples
3.1.3. Goose droppings samples
3.1.4. Animal fur swabs
3.2. Correlations
3.3. Comparative analysis of species / genera compositions

4. Discussion

5. Summary

6. Acknowledgments

7. References

8. Appendix
8.1. Tables: Raw data from cultivated samples, complete lists of species (Tables I-III), aliquot samples, goose faeces samples, animal skin swabs
8.2. Raw data on biotic and abiotic factors (Table IV)
8.3. Composition toilet medium
8.4. Images of the investigated waters
8.5. Raw data correlations and significance

Note

*1) Unfortunately, these and other parts of the work contain data (raw data, images) whose copyright does not belong to the author. Since the rights holders have not given their consent to their publication in this context, they must not be included here. In order to view this information, not only the consent of the author but also of the other rights holders is required. You can contact the responsible departments at the University of Aarhus (Professor Erik Jeppensen) and the University of Cologne (Professor Hartmut Arndt). Corresponding text sections have been deleted and marked with * 1) if they violate third-party rights, or written in italics if they are related to the relevant data.

1 Introduction

This study includes the first investigation of the pelagic and benthic protozoan communities in geologically young lakes of western Greenland under the influence of global warming. Among other things, an attempt is made to assess the role of hydrological and biotic factors, including conductivity, depth, size, temperature, pH value, organic load, bacterial count and fish population. It is also of interest to what extent the species composition differs with increasing age of the lakes and whether the biodiversity found increases due to increasing nutrient enrichment and organism migration. In addition, it should be checked whether there are indications of a bottom-up or top-down control in the composition of the food tissue, since both options were considered in previous approaches (Jeppesen et al., 2003, Worm et al., 2002).

It has long been known that the surroundings of snow, ice lakes and glaciers, which used to be perceived as completely hostile to life, represent a biome that provides a livelihood for free-living, single-celled organisms of all eight major eukaryotic groups (Adl et al., 2012) play a crucial role (Anesio & Laybourn-Parry, 2011). Particularly heterotrophic unicellular organisms have an important influence on the internal dynamics of terrestrial and aquatic ecosystems due to their bacteriophageous way of life (Adl & Grupta, 2006, Arndt et al., 2000, Azam et al., 1983). Together with bacteria, they decisively shape the food web, especially in oligotrophic and ultra-oligotrophic waters, such as those found in the Arctic (Rautio et al., 2011). In view of the rapidly advancing glacier retreat and the particularly intense local warming, a survey of the protozoal community seems particularly urgent. This work offers the rare opportunity to examine very young Arctic water systems (Briner et al., 2011) and to compare their successive colonization by protozoa.

The lakes chosen are located in the retreat area of ​​the rapidly melting Sermeq Kujalleq glacier in western Greenland, a few kilometers from Disko Bay (Thomas et al., 2011). They are 1-150 years old and were covered with ice before the glacier retreated (Briner et al., 2011). These are closely adjacent limnic waters with a depth of 1.0m to 50m and, at the time of sampling, a temperature between 0 ° C and a maximum of 12 ° C, depending on the depth. The environment is characterized by low mammalian diversity (Bee & Hall, 1956) and pronounced fragmentation of vegetation (Sitte et al., 2002), as well as limited productivity (Laybourn-Parry, 2009). Due to their geographical location, surface waters are frozen over for most of the year and predominantly show pronounced oligotrophy (Jeppesen et al., 2003). Determining the diversity of a habitat is one of the central aspects in ecological research (Johnson et al., 1996), with protozooplankton playing an important role. For this reason, samples were taken from a total of 30 of the lakes described in order to assess their composition of flagellate and ciliate species as well as amoeba and to identify possible differences in the context of the aging process. Vertical mixed samples from benthic and pelagic freshwater as well as enriched, Bouin-fixed samples were examined.

More recent research results suggest that in such cases it is not a question of a linearly predictable development, as previously assumed, but that much more complex relationships have to be considered in detail (Hulot et al., 2000, Jeppesen et al., 2010). This resulted in the following questions for this study:

- Is this process slower than in other bodies of water due to the low productivity and the short vegetation period due to the latitude? Or is it short because these factors are less significant for the colonization by protozoa than colonization routes such as "Long Distance Dispersal" (LDD), which can lead to rapid colonization of a biotope due to the ability to form light, resistant cysts (Brown & Hovmoller, 2002)? Do the low fluctuations lead to higher diversity than at other locations, or are the protozoan communities subject to the same dynamics as higher organisms in the Arctic, so that the extreme conditions lead to the dominance of fewer specialists (Hillebrand, 2004)? Are the food webs poor in trophic levels or can complex relationships be found, and if so, from what age of the waters?
- Another focus is on the determination of settlement routes. Which alternatives to the development of new areas will turn out to be the most important, how do communities develop? The faunistic parallels between the protozoan diversity of the lakes and that of snow (Akbayir, 2009, Al-Maarri, 2010) and ice cores (Jullmann, 2010) from the same region, as well as air from different locations (Alder, 2010, Feldmann, 2007) are examined , Fluess, 2009, Selse, 2010). Global wind currents in particular have long been known as a distribution vector, as they can also carry organisms and cysts with them in addition to large clouds of dust and pollen. They are able to transport particles several kilometers above land masses and oceans and at the same time enter organisms and nutrients as a basis for settlement (Kellogg & Griffin, 2006).
- Animals are already known from earlier studies as transport vectors not only for bacteria but also for protozoa, which is why studies have been carried out on their occurrence in fur, plumage and in the digestive tract of various species (Maguire 1959, 1963, Figuerola & Green, 2002, Finlay & Clarke, 1999, Schlichting 1978). For this reason, protozoa were made from the hair of resident animals such as musk ox (Ovibos moschatus), as well as the droppings of the flocks of Greenland white-fronted geese, which are very often found on the banks (Anser albifrons flavirostris), cultivated and determined (Kurm, unpublished). Protozoa could persist in their digestive system in the form of cysts and be introduced into new areas over long distances. In addition, their droppings could represent an important eutrophication influence for the nearby waters. This raises the question of which protozoa species are found in each case and, related to this, which of the entry possibilities play a role in the repopulation.
- Last but not least, the melting ice leaves behind inorganic nutrients, including nitrogen and phosphorus, through abrasion of the subsurface and the dust frozen inside. These previously unavailable or barely available minerals can now be used by bacteria for growth (Mindl et al. 2007), so it would be possible that they form the basis for a bottom-up controlled food web (Wollrab, 2012). The ice itself is by no means sterile, but also contains bacteria and protozoic cysts, as various analyzes have shown (Akbayir, 2009, Jullmann, 2010, Sazhin, 2004), and should therefore not be disregarded as a way of colonization.
- Ultimately, the sequencing of individual species offers the possibility of comparing them with occurrences at other locations in order to uncover differences or similarities. Due to the diverse forms that can also occur within a species, it is not sufficient to distinguish them solely on the basis of external characteristics such as morphotypes (Bass et al. 2007, Weisse 2008). Are the species found more endemic, highly adapted specialists, or are there mainly cosmopolitans, as is often assumed (Rogerson & Detwiler, 1999)? The examined ecosystems are simply structured, so that the formation of small habitats and different communities is only possible to a limited extent. This, as well as the lack of restrictions and the diverse range of distribution possibilities, could provide an explanation for the so far only small number of species described (Bass et al., 2007). Can the so-called “Ubiquitous Dispersal Hypothesis” (Baas-Becking 1931, Finlay 1998) also be maintained at such an extreme location? Is it true that protozoa are universally distributed due to their easily transportable cysts and live wherever they find favorable conditions (Bass et al., 2007), or are there limits to this in such a remote location? This is because the thesis that, in addition to the globally distributed species, there are also fewer endemic species (Foissner, 2007), which have only limited distribution areas, also exists (Bass et al., 2007).

1*)

Subsequent analyzes with molecular biological methods going beyond the scope of this work will be necessary in order to determine more precisely the number and affiliation of the species found. This includes in particular the sequencing of the rDNA of individual species and their classification in phylogenetic relationships to clarify the relationships. Whether and how many new species can then be identified may also offer a glimpse of how great our ignorance of the actual number of species of protozoa, especially the flagellates, is.

2. Material and methods

2.1. Sampling area

The sampling location is in West Greenland, east of Disko Bay and around 50 kilometers from the city of Ilulissat (at 69 ° N 49 ° W). The 40 km long ice fjord named after her runs there, as well as the Sermeq Kujalleq glacier, which is one of the most active glaciers in the world and had a flow rate of around 20 m / d until the beginning of this century. Since 2004, however, recent measurements have shown a significant increase in speed to around 40m / d, as well as a strong retreat of the glacier tongue inland (Maas et al., 2006). In contrast to before, when the increase in mass due to snowfall and the evacuation of water via fjords and calving glaciers were balanced, since 1990 scientists have recorded a significant loss of mass and a thinning of the ice layer in this region (Krabill et al., 1999). In the case of the glacier relevant for this work, the balanced discharge or the snowfall necessary to maintain the mass is 30 km3 / a (Thomas et al., 2011). The lakes selected here are particularly suitable for a study, as this process has only made them ice-free for a short time. The cause of this is climate change, which is leading to significantly greater warming here than in other parts of the world (Huntington & Fox, 2004). Because of this, the already very high speed of the ice fjord has increased significantly in recent decades. It also ensures a continuous decline in the ice sheet by around 35 km in the last 150 years, which in turn leads to the formation of countless freshwater lakes (Briner et al., 2010).

The 30 selected lakes are just such residual lakes, with an age between 1-2 and 50 years (lakes 1-20) and 50-150 years (from lake 21), and are located in an area of ​​approximately 150 km2 (see Figure 1). The age estimate was made based on the location of the lakes and the glacier retreat. Lakes 1-27 are located on a wall, 254-389 meters above sea level, the other waters are located a little to the west of it, deeper in a plain.

The lake and sampling numbers correspond to each other, non-existent numbers belong to lakes that could not be sampled due to the terrain or time conditions. They show different trophic states and visibility depths and have a depth between 1.0 and 50 meters. The closer environment is shown as described in the introduction, the colonization by geese is dealt with in a separate subsection (see 2.4. Geese).

Figure not included in this excerpt

Illustration 1: Large section: Satellite image of the test area near Ilulissat

(google, graphics © 2013 TerraMetrics), small excerpt: cartographic overview of the test site, individual lakes numbered in red (Jeppesen et al., 2012)

2.2. Sampling from lakes in West Greenland

The sampling took place between August 10, 2012 and August 28, 2012 at the specified locations (see 2.1. Sampling area). At that time, the maximum daily air temperature was 17.2 and the minimum daily air temperature 0.0 ° C, with occasional precipitation (National Climatic Data Center). As far as possible, the water samples were taken as a single 50 l vertical mixed sample from the boat with a Friediger collector and then 100 sterile Eppendorf vessels (Eppendorf AG, Hamburg, Germany) were filled to 1 ml. A sterilized grain of amaranth (Amaranthus) was added as a carbon source for the natural bacterial population. If sampling from the boat was not possible, it was carried out from the bank. Two bodies of water (lakes with the number 15 & number 20) were fed by meltwater rivulets, so 100 x 1ml samples of meltwater were taken from each of them. For this purpose, 5ml sediment with liquid was removed from each of the first 27 lakes in order to gain an insight into the settlement of the interstitial. In addition, a hand-held centrifuge (VWR International, Radnor, PA, USA) with 1000 revolutions / min was used, in which 90 ml of seawater were centrifuged for 1 minute in order to concentrate ciliates, later to fix and determine them with Boulin solution (Kurm , unpublished). In addition, data on the occurrence of fish (Salvelinus alpinus), Daphnia and other macroinvertebrates, algae, macrophytes and mosses, and the parameters water temperature, depth, visibility depth, oxygen content, conductivity, and the concentration of inorganic nutrients (phosphorus, nitrogen) of each lake are determined (Jeppesen et al., Unpublished, 1 * ). Finally, the bacterial density of the individual bodies of water was recorded (Jürgens, unpublished, 1 *).

The samples were transported to the lakes and cooled in thermoboxes in order to keep the temperature and light fluctuations caused by external influences as low as possible. Intermediate storage at the respective locations took place at 10 ° C.

2.3. Cultivation of Protozoa

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Figure 2: Tissue Culture Plates, culture bottles (Sarstedt) and Eppendorf vessels (Eppendorf).

First of all, immediately after their arrival in Germany, the 1ml samples were sorted according to lakes on the sterile bench in 24 tissue culture plates together with 1ml of autoclaved toilet fresh water medium (Guillard & Lorenzen, 1972, see Appendix 8.3. Composition of toilet medium) (Sarstedt, Nümbrecht, Germany) transferred (see Figure 2). When they were first examined under the microscope, they were divided into the categories “Empty”, “Only cysts” and “Living protozoa”. Samples from the last two categories were used to prepare 10 µl of liquid under sterile conditions together with 20 ml of toilet medium and a grain of wheat to stimulate bacterial growth in 50 ml culture bottles (Sarstedt, Nümbrecht, Germany) (see Figure 2). This method, known as the “Liquid Aliquot Method” (LAM), is more suitable for assessing the biodiversity of a sampling area than the observation of fixed samples, as it allows the observation of a development over a longer period of time.In order to come as close as possible to the conditions of their natural location, the aliquot samples were stored in a cooling chamber at 10 ° C. The culture bottles used have air-permeable sealing caps that ensure the supply of oxygen and nonetheless avoid external contamination. The samples were examined at regular intervals under the microscope and the organisms found were determined. In the case of the flagellates, the size, number, position and length of the flagella and the type of movement were evaluated for this purpose. In addition, specialists for the respective groups were consulted in dubious cases.

2.4. Goose droppings samples

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Figure 3: Anser albifrons (Omar Runolfsson, Wikipedia, Creative Commons Attribution 2.0 Generic license), Habitus and excrement pellets

In the summer months, various species of geese populate the lakes and shores of Arctic waters to breed there. In the municipality of Qaasuitsup, to which the sampling area also belongs, it is almost exclusively Greenland white-fronted geese (Anser albifrons flavirostris), whose excrement was found in varying amounts in the vicinity of the lakes (Malecki et al. 2000, Sharony, 2008) (see Figure 3). Since these could make a contribution to the eutrophication of water bodies that should not be underestimated, and also represent a vector for the spread of protozoa (Bull, 2004), several fecal samples were taken from the first two lakes. These were prepared and cultivated with WC medium at room temperature in order to determine the qualitative composition of the protozoal community and to compare it with that of the lakes.

Because of the great distances that wild geese cover in their capacity as migratory birds, two samples of excrement from Canada geese (Branta canadensis) collected from Cologne, a species that also occurs seasonally in Greenland. This is to determine whether there are overlaps in the protozoal communities of the geese depending on the species and location, or whether there are completely different compositions. These samples were grown under the same conditions as the goose droppings from Greenland.

2.5. Animal fur swabs

Apart from geese, other animals are also vectors for protozoal entry (Green & Figuerola, 2005), for this reason samples of the fur of freshly hunted musk oxen were also taken during an excursion in the same area in 2010 (Ovibos moschatus) and a reindeer (Rangifer tarandus) taken (see Figure 4). For this purpose, sterile disposable gloves and cellulose tissues freshly removed from the pack were used. With these, a swab was taken from the fur in order to transport it to Germany in sterile packaging. There they were frozen and later rinsed with toilet medium (Guillard & Lorenzen, 1972). The resulting liquid was cultivated under the same conditions as the water samples described above and additionally at room temperature, and the protozoa found were determined.

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Figure 4: Rangifer tarandus (Alexandre Buisse, CC BY-SA 3.0 http://creativecommons.org/licenses/by-sa/3.0/)

2.6. Morphological examination

All protozoa found were first viewed with the aid of an inverted microscope (Zeiss Axiovert S100 with objectives 100, 200, 400 and 630 times magnification, Carl Zeiss AG, Oberkochen, Germany) using phase contrast optics, and then so far determined as possible according to morphological criteria. The microscope used makes opening the culture bottles superfluous and thus prevents possible contamination. The protozoa found were assigned to the classification according to Adl (2012) following morphotypes. Jeuck & Arndt (2013), Streble & Krauter (2006) and Smirnov & Goodkov (1999) were used as identification literature and keys and, as far as possible, specific genera and species were determined.

2.7. statistical evaluation

The programs Microsoft Excel 2002 (Microsoft Corporation, Redmond, USA), PAST (Palaeontological Statistics, Geological Museum Copenhagen), R 3.0.1 (The R Foundation for Statistical Computing) and R-Commander 1.9-6 ( John Fox) used.

First, the rank correlation coefficient Spearmans Rho (ρ) was calculated for selected data sets. Correlation Coefficients | ρ | <0.2 were used as an indicator of no detectable, 0.2 ≤ | ρ | <0.4 for weak, 0.4 ≤ | ρ | <0.6 for moderate and 0.6 ≤ | ρ | considered for high correlation. The standard value 0.05 was chosen as the significance level. The significance was then determined in each case, with correlation coefficients whose p-values ​​were above the significance level were not taken into account for the further evaluation. These correlations were carried out again with the values ​​adjusted for statistical outliers, unless fewer than 10 values ​​of a variable were available. Each value was considered an outlier that was 1.5 times the 0.25-0.75 quartile distance outside of it. Then, using the minimum-square method, a regression line was calculated for selected pairs of variables that showed at least a moderate or high correlation, and the corresponding formula and the coefficient of determination were specified (cf. 3.2. Correlations). 1*)

3. Results

3.1. Morphological analysis of the protozoal community

3.1.1. Species distribution in the lakes

Due to the pressure and temperature differences during transport by helicopter and aircraft, some individual samples were lost, so that ultimately 2957 1ml aliquot samples were available for evaluation (see Appendix 8.1.Tables: Raw data from cultivated samples, complete lists of species (Tables I- III) Aliquot samples, goose faeces samples, animal skin swabs [Table I is not included in this version due to its size]). In 1664 of these, i.e. 56.3%, living protozoa could be detected either immediately or from cysts that could be cultured. In only 7.4% of the samples (218) cysts were found that could not be cultured later. Samples with one as well as with several cultivable species were found. A total of 25 Morphotypes or Morphospecies could be identified with the microscope, whereby the group of the colorless Chrysomonads of the genus Spumella most frequently, Paraphysomonas sp. was the second most common, followed by the kinetoplast Rhynchomonas nasuta (see Figure 5). 93.7% of the protozoa found were flagellates, 5.0% amoebas, and only 1.3% of the cultivated organisms were ciliates. Among the amoebas were those of Vannella -Type most common, the only existing ciliate was called Cinetochilum sp. identified. Morphospecies, which could not be determined with sufficient reliability, are from now on with “? Species name sp. “. Given as a percentage, the distribution of morphotypes and species in the sampled lakes was as follows:

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Figure 5: Shows the distribution of cultivable morphotypes and species in the total number of lakes.

If one calculated the constancy (relative proportion of the samples, i.e. lakes in which the taxon occurs) of the morphotypes across the lakes, a slightly different picture emerged, in which the dominant organisms partly changed places because Rhynchomonas nasuta in relation to the total number of lakes more often than Paraphysomonas sp. occurred (see Figure 6). The following formula was used for this (Tümpling & Friedrich, 1999):

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