1 British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 OET, UK
2 Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR, UK
3 Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
Correspondence
David A. Pearce
dpearce{at}bas.ac.uk
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ABSTRACT |
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Present address: NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK.
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INTRODUCTION |
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It is known from temperate and tropical areas that increased nutrient loading is often accompanied by a change in species composition (Hawes, 1990). Also, in polar environments, the character of freshwaters can be radically changed (e.g. switched from an oligotrophic to a eutrophic state), by quite minor events such as interference with an inflow or through the introduction of small amounts of inorganic or organic substances, especially if the volume is small (Fogg, 1998
). Despite these observations, however, the community composition and taxonomy of the bacterioplankton has not been considered in detail, even though these communities are sensitive to disturbance and have been shown using fingerprinting techniques, to change with environmental conditions (Crump et al., 2003
; Pearce, 2005
). In lower latitude limnetic systems, differences in the bacterioplankton community composition between lakes of different trophic status have already been observed (Lindström, 2000
), and it has been shown that the availability of nutrients and seasonal changes in nutrient concentrations can affect the bacterioplankton community composition (Donner et al., 1996
; Yannarell et al., 2003
). In this study, we examine the bacterioplankton community composition in Heywood Lake, Antarctica, and compare it to observed patterns in neighbouring Signy Island lakes with little (Sombre Lake) or no (Moss Lake) natural nutrient enrichment.
The significance of this particular system is that Heywood Lake is situated in a region of the globe subject to extremely rapid environmental change. Quayle et al. (2002) report data for the Signy Island lakes showing that mean lake temperatures in winter have increased by 0·9 °C between 1980 and 1995, and nutrient levels at some sites exhibit order of magnitude increases per decade. In addition, photographic estimates suggest that permanent ice cover on these lakes has receded by approx. 45 % since 1951, while lake ice records indicate that the open-water period has increased significantly. In this way, Heywood Lake might indicate the type of changes that could take place in analogous systems subjected to environmental change.
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METHODS |
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Heywood Lake is 427 m long and 137 m wide with an area of 41 730 m2. The lake comprises two basins separated by a shallow neck region. The larger north basin has a maximum depth of 6·4 m and a mean depth of 2 m. Its drainage basin is composed of approximately 1 % snow, 93 % rock, 5 % lichen and 1 % moss cover, so the ice-free terrain is composed of frost-shattered rock and rudimentary soils formed from glacial diamiction (Noon et al., 2002). The lake lies only 4 m above sea level and is separated from the sea by 200 m of gently sloping ground. This permits ready access to the catchment by Antarctic fur seals (Arctocephalus gazella) and Southern elephant seals (Mirounga leonina). So Heywood Lake, although adjacent and physically similar to the oligotrophic Moss and Sombre Lakes, is different principally because its catchment is accessible to seals, which contribute considerable amounts of organic matter to the water. Unlike its more oligotrophic neighbours, there is evidence of high levels of nutrient input from seal activity over 33 % of the catchment area, and up to 1 % from bird activity, so that the total annual carbon gain in Moss Lake is less than an eighth of that in Heywood Lake (catchment data from Heywood et al., 1980
; Ellis-Evans, 1982
, 1990
; and Fogg, 1998
). Butler (1999a)
obtained values of 0·75·1 mg dissolved organic carbon l1 and 0·86·4 mg total organic carbon l1 for Heywood Lake. A striking example of the impact on Signy Island moss communities is demonstrated by the presence of a seal exclusion fence (Fig. 1b
). The once widespread moss carpet has been compacted and is now largely moribund; it has been recolonized by a community dominated by the nitrophilous alga Prasiola crispa, which is characteristic of sites frequented by seals and birds (Ellis-Evans, 1990
).
Antarctic fur seal numbers in the Heywood Lake catchment area have increased markedly since 1974, and particularly since 1984, including a tenfold increase between 1976 and 1988 (see Fig. 2 and data in Ellis-Evans, 1990
). Peak numbers (in February) have been monitored on Signy Island and have been found to increase from only a few animals each year in the 1950s and 1960s, to a few dozen in the early 1970s, but then to 20003000 between 1976 and 1983, 800017 000 between 1984 and 1993, 20 500 in 1994 and 1995 (Smith, 1997
) until, most recently, 10 004 in 2003 (British Antarctic Survey, unpublished data). This expansion of the fur seal population has also been reported elsewhere in the Southern Ocean (Bester et al., 2003
). The presence of the fur seals has resulted in considerable nutrient input, biological change and perturbation of the lake sediments, which are between 5000 and 7000 years old. Seal impact has been severe and both physical disturbance and nutrient toxicity have resulted in significant or complete destruction of Antarctic plant communities surrounding the lake. In addition to fur seal excrement and fur wash, plant and organic accumulations are being compressed and eroded into the lakes (Smith, 1997
).
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Physical and chemical analyses.
Vertical profiles of temperature, oxygen concentration and conductivity were taken at 1 m intervals with a Solomat WP4007 water quality meter connected to a 803PS Sonde (Zellweger Analytics). Light penetration was measured using a Grant 1000 series Squirrel logger connected to three SKP 215 flat quantum sensors (Skye Instruments). Subsamples of lake water (500 ml) were filtered through Whatman GF/C filters using a Mityvac hand pump. Part of the 500 ml subsamples were used for fluorometric analysis of chlorophyll a concentrations, according to Welschmeyer (1994). Measurements of ammonium-N, nitrate-N, dissolved reactive phosphate (DRP), total dissolved phosphate (TDP) and chloride were measured using GF/C-filtered water and an Alpkem FS 3000 auto-analyser (Alpkem) with a model 5027 autosampler attached. Total dissolved nitrogen and dissolved reactive silicate were determined spectrophotometrically using the methods of D'Elia et al. (1977)
and Mackereth et al. (1989)
, respectively. An Orion 250A pH meter was used to measure pH and alkalinity by the Gran titration technique (Mackereth et al., 1989
).
Determination of total population density.
The total population density in each sample was determined by direct microscopical counts of DAPI stained cells, using the method described by Porter & Feig (1980).
Analysis of 16S rRNA gene fragments.
Enzymic amplification of 16S rDNA was performed on DNA extracted directly from Heywood Lake water using the method described by Pearce (2000). For these amplifications, each PCR mixture (50 µl) contained 10 ng extracted DNA as template, 10 pmol each primer, 20 nmol each dNTP, 1 U SuperTaq polymerase (HT Biotech) and the SuperTaq buffer supplied with the enzyme. Primers used were 8F, 5'-AGAGTTTGATCCTGGCTCAG-3' (Dunbar et al. 1999
), and 1500R, 5'-AGAAAGGAGGTGATCCAGCC-3' (Andrade et al. 2002
), generating a 1492 bp fragment. Amplification reactions were performed with a Genius thermocycler (Techne) using the following conditions: an initial denaturation step consisting of 94 °C for 5 min, 30 cycles consisting of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 70 s, and a final elongation step consisting of 72 °C for 5 min. Controls containing no DNA were also used to ensure that contaminants were not being amplified. The PCR products were cleaned using GFX PCR clean up columns (Pharmacia). Cleaned products were ligated into the pGEMT-Easy vector (Promega) and ligation mixtures were transformed into competent JM109 cells as recommended by the manufacturer. Transformants were screened using black/white selection on Luria agar containing S-Gal/IPTG and 50 µg ampicillin ml1 (Sigma). Putative positive colonies were transferred to individual tubes containing 50 µl sterile water. The cell suspensions were subjected to two freeze/thaw cycles and 1 µl aliquots were used as templates in a PCR reaction containing the M13F/M13R universal primers (M13F, 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'; M13R, 5'-GAGCGGATAACAATTTCACACAGG-3'). PCR conditions were as described above, except that the annealing temperature was raised to 58·5 °C. A length (760 bp) of each clone obtained was sequenced with the M13F primer using the Big Dye terminator kit v.2 (Applied Biosystems). Sequence reactions were carried out at the British Antarctic Survey using a Megabase 500 capillary sequencer. Clone sequences were compared with the GenBank nucleotide database using GAPPED-BLAST searches (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) (Altschul et al., 1997
) to determine their closest phylogenetic neighbours. Identification to the genus level was defined as a 16S rRNA gene sequence similarity of
97 % with that of a sequence deposited in the EMBL database (Drancourt et al., 2000
).
Direct culture of bacteria.
Cultures were obtained by making a dilution series of lake water and pipetting 200 µl onto solid casein-peptone-starch (CPS), tryptic soy broth agar (TSBA) and R2A agar plates in three replicates. Plates were incubated at 4 °C for 2 months.
Fatty acid methyl ester (FAME) analysis.
The phenotypic diversity and identity of individual strains isolated by direct culture was determined by FAME analysis, as described by Thompson et al. (1993) and van der Gast et al. (2001)
. The samples were injected into a Hewlett Packard model 5890 series II gas chromatograph. Fatty acid peaks were named by the Microbial Identification System (MIS) software (Microbial ID) and isolates were identified using the MIS Aerobe Library.
Fluorescence in situ hybridization (FISH).
A sample (10 ml) of each lake-water sample was filtered through a black polycarbonate 0·2 µm screen membrane filter (Poretics). Cells were fixed with 2 ml 4 % paraformaldehyde in PBS for 30 min. A gentle vacuum was then applied and cells were rinsed, initially in 5 ml PBS, then in 5 ml distilled water. Filters were removed from the filtration apparatus, air-dried, placed on a glass microscope slide and stored at 20 °C. Five different in situ hybridization probes were used specific for the domain Bacteria, the -,
- and
-subclasses of the Proteobacteria and the CytophagaFlavobacterium group as described by Pearce et al. (2003)
.
Denaturing gradient gel electrophoresis (DGGE).
Enzymic amplification of 16S rDNA was performed on extracted community DNA using the Muyzer primer pair (Muyzer et al., 1993) which amplifies corresponding Escherichia coli positions 341534 bp of the 16S rRNA gene. A volume of 25 µl was separated by DGGE as described by Pearce (2000)
. The gel was run for 80 min at 60 °C and 10 V cm1, before staining for 45 min in a solution containing 0·5 µg ethidium bromide ml1. This was visualized on a UV transilluminator (UVP). Photographs were taken with a Gelcam (Polaroid) using Polaroid 665 professional positive/negative instant pack film. Gel images were analysed using Gelcompar II software (Applied Maths) and bands were determined using band recognition software. Bands were then excised, reamplified and cleaned using GFX PCR clean up columns (Pharmacia), ligated into the pGEMT-Easy vector (Promega) and transformed into competent JM109 cells as described for the rDNA clone libraries.
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RESULTS |
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Direct 16S rDNA sequencing from environmental clones
DNA from 60 environmental clones could be identified to within 97 % of a named genus based on sequence comparison of 16S rRNA genes. Using this information, 73·3 % of the bacteria were affiliated with the
-Proteobacteria, 15·0 % with the
-Proteobacteria and 11·8 % with the Actinobacteria. The specific taxa found were the
-Proteobacteria genera Acidovorax, Alcaligenes, Bordetella, Janthinobacterium, Polaromonas, Polynucleobacter, Pseudomonas (mephitica), Rhodoferax and Variovorax, the
-Proteobacteria genera Caulobacter, Rhodobacter and Sphingomonas, and the Actinobacteria taxa Aeromicrobium, Cryobacterium, Frigoribacterium and Actinomycetales (Table 2
). Coverage was estimated to be 71·7 % (Good, 1953
).
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DISCUSSION |
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Similarities with adjacent oligotrophic lakes
Overall, the bacterioplankton community in Heywood Lake was dominated by genera with characteristics that included the ability to utilize a wide variety of organic compounds and involvement in the mineralization of organic matter, they were all aerobic (or facultatively anaerobic), they contained species which produced pigment and were widely distributed globally in fresh waters. All of these properties are normally associated with oligotrophic aerobic pelagic environments (Pearce, 2003; Pearce et al., 2003
). The genera identified in Heywood Lake through 16S rDNA sequencing or FAME analysis that were also present in oligotrophic Sombre Lake (Pearce et al., 2003
) were Acidovorax, Arcanobacterium, Bordetella, Cellulomonas, Flavobacterium, Janthinobacterium, Micrococcus, Pseudomonas, Rhodobacter, Sphingomonas and Staphylococcus. This represents a Sørensen coefficient of 0·20, or 42·3 % of the genera identified in this study.
Differences from adjacent oligotrophic lakes
Bacterioplankton taxa identified in nutrient-enriched Heywood Lake from direct 16S rDNA sequencing or FAME analysis included: Acinetobacter, Actinobacteria, Actinomycetales, Aeromicrobium, Caulobacter, Cryobacterium, Frigoribacterium, Pelistega, Polaromonas, Polynucleobacter, Rhodococcus, Rhodoferax, Stenotrophomonas, Tsukamurella and Variovorax. These groups were not found in the neighbouring oligotrophic lakes.
Heywood Lake bacterioplankton was dominated by bacteria most of which have yet to be cultivated (Table 3). Of the 13 dominant groups identified using DGGE, eight were closely related to uncultivated bacteria in the database, three were identified to genus level and only two could be identified to species level. This pattern is consistent with oligotrophic Moss and Sombre Lakes, and at least three of the dominant groups in Heywood Lake were identical to dominant groups in the oligotrophic systems. However, in nutrient-enriched Heywood Lake, there was a larger number of dominant groups: Moss Lake 9, Sombre Lake 8 and Heywood Lake 13.
Cyanobacteria
DGGE suggested the importance of the Actinobacteria and, unlike both of the oligotrophic lakes used for comparison, none of the dominant bacterioplankton were Cyanobacteria. This is unusual, as many of the microbial communities in non-marine ecosystems within polar regions are dominated by Cyanobacteria, with populations that are amongst the highest recorded in any natural environment (Vincent et al., 2000). This observation is supported by the dominance (as determined by DGGE) of Nostoc sp. in Moss Lake (Pearce, 2003
) and an uncultured cyanobacterium clone in Sombre Lake (Pearce et al., 2003
). The lack of cyanobacterial dominance might be linked to the high nitrogen concentration associated with fur seal enrichment, as relatively high N : P ratios in nutrient-rich lakes tend to favour non-cyanobacterial taxa (Smith, 1983
, 1990
). This might also be due to the observation that pico-cyanobacteria have a high nutrient-scavenging ability and can be an important part of the pelagic community in terms of biomass in more oligotrophic conditions (Stockner, 1988
; Stockner et al., 2000
).
Marine species
A particularly interesting feature of the Heywood Lake bacterioplankton was the presence of psychrophilic marine bacteria, including Polaromonas vacuolata (Irgens et al., 1996) and Rhodobacter sphaeroides (Pinhassi et al., 1997
). In addition, the
-Proteobacteria were significant and, though not exclusively, are more traditionally associated with marine environments. This is probably due to the close proximity of the lake to the sea and frequent visits by marine mammals and birds. Although we cannot be certain that the marine bacteria were active within the lake, the relatively high levels of
-Proteobacteria, as determined by FISH, suggested that they might be (as the probes target rRNA). A similar phenomenon was observed by Bell & Laybourn-Parry (1999)
in nutrient-enriched Rookery Lake, where the ciliate community resembled the marine community and was dissimilar to that found in older saline lakes within the Vesthold Hills.
Ubiquity of bacterioplankton genera
Although the Heywood Lake bacterioplankton community was not consistent with neighbouring oligotrophic lakes, many of the genera isolated from Heywood Lake represented ubiquitous aquatic bacteria and the bacteria most common in aquatic habitats, such as Gram-negative rods, particularly Pseudomonas and Flavobacterium. In addition, surface films formed during calm weather on lakes and ponds often comprise Pseudomonas, Caulobacter, Flavobacterium and Micrococcus. Significantly, many of the genera found have been identified in freshwater systems elsewhere (Table 4), including nutrient-enriched systems in temperate areas such as soil, marine, alkaline, sedimentary and estuarine systems, and as such it might be more appropriate, as suggested by Reynolds et al. (2002)
for the phytoplankton and by Yannarell et al. (2003)
for the bacterioplankton, to develop and use a functional classification of these aquatic micro-organisms.
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Reduction in species richness
Using the Simpson diversity index (Simpson, 1949), from 16S rRNA gene sequences Heywood Lake has a lower bacterioplankton biodiversity at 0·92 than Sombre Lake at 0·94. The probability of selecting a clone with a new sequence from a random sample was 10·3 % (1 : 10) for Heywood Lake and 16·0 % (1 : 6) for Sombre Lake.
Increasing evenness
Lindström & Leskinen (2002) cite several investigations of bacterioplankton diversity in aquatic environments with results indicating that although the species richness of these communities is high, only a few taxa (<20) dominate. In this study a larger number of dominant groups were detected than in the adjacent oligotrophic lakes, suggesting nutrient enrichment has led to greater evenness among dominant bacterioplankton. The use of FISH showed that the
-Proteobacteria was clearly the most abundant group, followed by the CytophagaFlavobacterium group, the
-Proteobacteria and the
-Proteobacteria at most depths studied (Fig. 4
). The
-Proteobacteria, however, were not as large a component of the Heywood Lake bacterioplankton as found in the Moss and Sombre Lake bacterioplankton and at a number of depths the CytophagaFlavobacterium were either equally or more abundant. In proportion to the overall population size, the
-Proteobacteria and the
-Proteobacteria were also relatively more numerous, suggesting that the species evenness of Heywood Lake is higher than that of Moss or Sombre Lakes (Pearce, 2003
; Pearce et al., 2003
).
Dominant bacterioplankton groups and methodology
The complementary results from each of the different methods stressed the importance of a polyphasic approach. 16S sequence analysis of two-thirds of the predicted diversity present suggested over 70 % of the sequences detected were from -Proteobacteria, 15 % from
-Proteobacteria and 11 % from Actinobacteria. FAME analysis indicated that 80 % of cultured isolates were Actinobacteria, but as this technique is culture-dependent, this may indicate the Actinobacteria are taken into culture more readily, suggesting that they might be more resistant to environmental changes or have wider environmental tolerance limits. FISH indicated a predominance of
-Proteobacteria, followed by CytophagaFlavobacterium,
-Proteobacteria and
-Proteobacteria (unfortunately we did not have access to an Actinobacteria-specific probe in the field), whilst over 50 % of DGGE bands were Actinobacteria, 23 % Bacteroidetes and 8 %
-Proteobacteria. Taken together, these results suggest that there is much greater biodiversity among the
-Proteobacteria, but that a small number of key Actinobacteria dominate the Heywood Lake bacterioplankton community.
Shift in dominance with trophic status
A key observation from this study was an apparent shift in dominance with trophic status. Moss Lake was dominated by the Proteobacteria (5/9 DGGE bands), Sombre Lake showed no clear dominance, with equal numbers of Proteobacteria, Bacteroidetes and Actinobacteria, whilst the Actinobacteria dominated in Heywood Lake (7/13 DGGE bands), indicating a potential shift in dominant groups from -Proteobacteria to Actinobacteria with increasing nutrient enrichment. The Actinobacteria are widespread and abundant in several lake systems.
Resistant groups
In Heywood Lake, three of the dominant groups were found to be dominant in neighbouring oligotrophic systems, suggesting that there are indeed important freshwater groups, or alternatively that there is a species-specific resistance to environmental perturbation in the form of nutrient enrichment. This is supported by the observation that Heywood Lake has a diversity of bacterioplankton which differs markedly from its neighbours, but this diversity does include some of the genera found in the oligotrophic systems, thus suggesting that specific groups may have a wider tolerance to environmental change.
Conclusion
Changes in the utilization of catchments by marine mammals or other perturbations of nutrient supply are likely to result in significant changes in the nutrient status and bacterioplankton communities of Antarctic lake systems. Nutrient-enriched Heywood Lake has a high bacterioplankton population density, reduced species richness and increasing evenness. Less than half of the genera found were also present in neighbouring oligotrophic lakes, including three of the dominant groups. A comparison of the three lakes with different nutrient levels suggested an apparent shift in dominance with trophic status. Other key observations included the absence of dominant Cyanobacteria and the presence of marine bacteria. Overall, the bacterioplankton community of Heywood Lake was distinct and differed markedly from its more oligotrophic neighbours. This significant impact of nutrient enrichment on Heywood Lake bacterioplankton communities, therefore, suggests that low-temperature oligotrophic freshwater lake systems may have low resistance to environmental change.
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ACKNOWLEDGEMENTS |
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Received 20 April 2004;
revised 14 July 2005;
accepted 18 July 2005.
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