Prokaryotic Diversity in the Antarctic: The Tip of the Iceberg

B.J. Tindall
DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany Received: 27 June 2003 / Accepted: 30 September 2003 / Online publication: 2 April 2004

Abstract

In contrast to the rather limited diversity of plants and animals to be found in the Antarctic, the microbial diversity of this continent has been shown to be surprisingly diverse. Apparently barren soil and rock landscapes, as well as the numerous and diverse lakes found at the edges of the continent, harbor a range of prokaryotes which indicate that the extremely low temperatures which prevail seasonally are no obstacle to microbial colonization. Both direct cultivation methods and modern molecular genetic methods have contributed to our understanding of the range of organisms to be found. Cultivation based studies are often hampered by constraints inherent in the methods selected for the isolation of organisms. Molecular-based approaches do not suffer from the same cultivation-based biases, but other problems need to be taken into consideration. It has rarely been possible to combine both techniques in a single study, nor has it usually been possible to take the results and conclusions drawn from the study of one environment and apply this knowledge to a further series of experiments on the same environment. The Antarctic may be considered to be a geographically well isolated area to study. Comparison with other environments that may also be isolated from their surroundings (i.e., hot springs or highly saline lakes) allows parallels to be drawn. The conclusions drawn provide important insights into the way the Antarctic may have been colonized and the microbiota diversied. Much work still needs to be done beyond the simple task of making an inventory. The functioning of complex communities, such as mat systems, requires an understanding of the ecology of the systems, not only at the level of the whole system, but also the role of localized environments within that system. Perhaps these ecosystems have, in the absence of plant and animal communities, a role to play in the

monitoring of polar climate change. The information available at present clearly indicates that the Antarctic is deserving of further study at the microbial level.

Introduction

Correspondence to: B.J. Tindall

DOI: 10.1007/s00248-003-1050-7
d

The polar regions of the earth, the Arctic or Antarctic, quickly conjure up pictures of snow- and ice-covered landscapes. In both regions the temperatures may sink to )40C or lower during the winter months and during the summer only the edges of the ice and snow masses melt. These regions proved to be challenges for the earlier explorers, with the history books littered with heroic battles with the elements, ships being caught in the ice, and life and death struggles being documented as recently as 100 years ago. The geographical surroundings of the Arctic and Antarctic contribute to some differences, but both regions show similar characteristics. The fact that major land masses border the Arctic means that land animals (such as caribou) may migrate from winter grazing areas in the south into the arctic tundra during the short ice-free summer. In contrast the only animals (apart from humans) that can populate the Antarctic have access only via the sea (penguins) or the air (other birds). In both regions the vegetation is not very diverse and trees are absent. Given the fact that the environment is hostile to humans and that the range of plants and animals to be found in the polar regions is limited, it is not surprising that they are considered to be extreme. However, we know that environments such as hydrothermal vents, saline lakes, or anoxic sediments, which are hostile to eukaryotes, may still harbor a wide range of prokaryotes. In the case of extreme thermophiles and extreme halophiles we know that the ability to grow under such conditions may be correlated with specic evolutionary groups, which are specialized to cope with the prevailing conditions. In the case of environments such as the Arctic or Antarctic, what can we say about the diversity of prokaryotes found there, and do we see similar patterns of localized evolutionary groups spe271

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cically adapted to a psychrophilic way of life similar to thermophiles and halophiles? Within the past 20 years a signicant amount of work has been undertaken on studying the microbiota of the polar regions, with more emphasis being placed on the Antarctic. Despite the fact that there is limited vegetation in the Antarctic there are more different environments to study than just rocks and snow. In particular there are a wide diversity of aquatic environments ranging from the sea and sea ice to a large number of lakes on the edge of the Antarctic which may range in salinity from freshwater to highly saline. Even then it is not simply a case of increasing the sodium chloride concentration, but each lake may have its own ionic and organic composition, as well as being affected by the degree to which the ice thaws in summer, etc. Studies on such environments have only just begun, but the results clearly indicate that the application of methods being developed in the study of prokaryote diversity in other environments will contribute quickly to a changing picture of microbial diversity in the Antarctic.
Cultivation-Based Studies
The Diversity of Microorganisms Isolated. Over the past two decades a wide variety of strains have been isolated from various environments associated with the Antarctic, either on the continent itself or directly in the vicinity, such as marine environments or sea ice. Within the past 5 years there has been an almost exponential rise in the number of species that have been isolated from the Antarctic, which would indicate that this environment is by far from devoid of prokaryotes. In many cases the species which have been isolated have proven to be previously unknown species [414, 17, 18, 30, 3234, 3647, 49, 52, 53, 5560, 63, 64, 66, 67, 7779, 86]. In other cases there is ample evidence for bacterial biomass in soil samples [31] or preliminary studies on aquatic environments indicate extensive, undescribed, isolated diversity [33, 90, 91]. While this may give the impression that there are taxa present which are unique to the Antarctic, it should also be remembered that studies conducted on the microbiota in the Antarctic have not been accompanied by comparable work in other parts of the globe, or in human-made environments that have characteristics similar to those of the Antarctic. Caution should, therefore, be exercised when extrapolating from the current data. The Role of Environmental Parameters and How The modern Much of the Diversity Can Be Cultivated.

approach to the isolation of prokaryotes has changed over past decades. In particular, the use of molecular genetic methods (whether gene probes or environmental clone libraries) has indicated that cultivation techniques may not recover the full diversity of species present, nor

does it always recover the dominant population [45, 73, 94]. Although there has been a trend toward treating strains that were identied by molecular genetic methods and were not recovered by cultivation-based techniques as noncultivatable, this viewpoint has also been modied in the past 5 years. The continued use of molecular genetic methods has clearly pointed to the potential prokaryote diversity in environments such as the Antarctic, but this also raises questions about access to that diversity. If we wish to recover the organisms themselves, then we must learn more about the ways in which we can improve the methods of recovering the strains present. In the rst instance it would seem appropriate to gain an insight into the natural microbial environment, in order to design methods that would be appropriate for the isolation of the strains present. Unfortunately it is not always easy to gain access to such data at the microbial level, with localized environments being formed, which cannot easily be studied by current methodology. In some cases the conclusions we may draw based on the study of strains isolated from an environment is the rst step toward understanding the environment and the selection pressures which prevail at the microbial level. In such cases it may be necessary to design a set of isolation strategies, which allow a group of strains to be isolated. The information gained from the study of those strains, and in conjunction with other data (such as that based on molecular genetic studies) may mean that modications need to be made to the original strategy and then implemented in a second series of isolation experiments. Naturally this information may then be correlated with the other existing data, and a cycle of experimental design, isolation, and interpretation may need to be carried out a number of times before we gain an idea of what factors are inuencing our results in which fashion. Unfortunately such approaches are potentially time consuming and cannot be carried out within the framework of projects with a limited time scale. The data available to date indicate that most Antarctic environments (whether terrestrial, aquatic, or offshore marine) contain a diverse range of prokaryotes and in many cases unicellular eukaryotes such as protozoa or yeasts. Consequently this means that both molecular genetic and isolation based studies should be based on a fairly large data set (i.e., isolates or clones). Thus, the use of 100 clones or 100 isolates (obtained from a single medium) is probably not representative [19, 90]. This report was limited to 100 strains because of limitations of the computer evaluation as well as the fact that the objective was to examine a methodological approach. The selection of 100 strains should not be taken to indicate that they were representative of the larger number of strains isolatedsee below. Recent work on aquatic environments in the Antarctic illustrate some of the problems and conclusions which may be drawn.

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Approaching the Problem of Cultivating the Diver-

Studies on a mat system from Lake Fryxell have been based on a coordinated cultivation and molecular geneticbased approach [19, 90]. Although it was not possible to isolate strains and DNA from exactly the same starting material, squares of mat material measuring about 10 cm 10 cm (approximately 0.51.0 cm thick) served as the starting material, with one such square being used as the source for both the cultivation and molecular geneticbased methods. Mat material was collected and sent as soon as possible by air to the DSMZ. The sample was stored cold during transport and processed as soon as possible on arrival. Given the prevailing temperatures in the Antarctic, the preparation of 10-fold dilution series was carried out on ice, and all media, including plates, were kept cold until they were placed at the relevant incubation temperature. The selection of 10 different media was based on a consideration of the potential selective effects of the media chosen. A fairly rich medium, for example, was chosen for the enrichment of actinomycetes, whereas media described by Schlesner [85] (supplemented with antibiotics) was used for the enrichment of members of the PlanctomycesPirellulaGemmata group. The same media were also used, but without antibiotics as a general isolation medium. Selective isolation of strains present in the form of heat-resistant spores was also carried out by heating samples for 5 min at 60C. Given the fact that cyanobacteria were known to be major constituents of the mats, and that cyanobacterial cultures are often difcult to free of heterotrophic bacteria, a cyanobacterium medium [82] was used in order to cocultivate bacteria and cyanobacteria. All media were inoculated in parallel from the same dilution series, and parallel batches (in triplicate) of plates were incubated at 5C, 15C, and 25C. The fact that the major screening program concentrated on aerobes was based on information which indicated that the mat system was aerobic, being located in shallow water at the edge of the lake, which is a result of melting of the ice cover during the summer months. Based on this isolation strategy different media were capable of supporting different numbers of colonies. In the case of the enrichments set up to isolate strains germinating from heat-resistant spores no colonies were detected, which would indicate that classical bacilli were not present in the samples. In the case of the medium to which antibiotics had been added, and were intended to be selective for members of the PlanctomycesPirellulaGemmata group, the diversity of colonies present was low (as was to be expected), but some of these also represented antibiotic-resistant strains not belonging to the PlanctomycesPirellulaGemmata group, which is also not unexpected (Schlesner, personal communication). It is interesting to note that although a
sity.

smaller number of isolates were picked from the medium designed to isolate cyanobacteria, growth of a range of strains was detected, albeit slowly on this medium to which the only organic constituent added was agar. Signicantly few cyanobacterial colonies were present on these plates, indicating that the source of the organic nutrients was probably due to impurities in or components released from the agar. In contrast few colonies were isolated from richer media, such as tryptic soy agar. In total >900 strains were picked and grouped, primarily according to their FT-IR spectra as outlined by Tindall et al. [90]. Colonies were picked according to differences in color and form, and no attempt was made to pick all strains, which would probably have (at least) doubled the number of isolates. Although this may not have contributed to the diversity of organism recovered from the mat system, it would have allowed a statistical evaluation of the distribution of the different FT-IR groups. Incubation at different temperatures showed that growth on plates at 5C was generally slower than at 15C, which was generally slower than growth at 25C. Despite the fact that the central part of Lake Fryxell is covered by ice in summer and only an outer, moated region is ice free, there was no indication that the majority of the isolates were psychrophiles with temperature optima <10C. Slow growth of many of the strains isolated at 5C appeared to be due to the fact that they had higher temperature optima. In fact many strains isolated at either 5C or 15C grew at the next highest temperature. Only in the case of certain strains, such as those that appeared to resemble Hymenobacter roseosalivarius [47], were temperature optima clearly below 15C. This is generally consistent with previous reports [92]. Given that the mat system is a tightly packed concentration of microscopic organisms one may, perhaps, conclude that it contains a large quantity of organic material. This would, indirectly, be reected by the ability of most isolates to grow on media such as tryptic soy broth. In contrast, many of the strains isolated on media with lower nutrient concentrations were not able to grow on such media. This would imply that there is a difference between the presence of nutrients and their availability. Thus, in an environment such as the mat system in Lake Fryxell, the presence of large numbers of prokaryotes might seem to imply that the environment is eutrophic, whereas the cultivation-based studies would seem to imply that organisms are present which are clearly oligotrophic. This is not restricted to the need to use oligotrophic conditions during isolation, but also for the subsequent transfer of the isolates. The methods of isolating organisms from the environment developed over the past 100 years has largely relied on the use of nutrient levels that the organism may only rarely nd in nature. There are several reports which now indicate that

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the organisms which may be isolated from aquatic environments may be inuenced by the choice of nutrient levels in the media or the addition of signal molecules [20, 24, 77]. The fact that isolates were obtained on a medium used for the isolation of photoautotrphic cyanobacteria, in which only impurities from the agar could serve as the nutrient source, further indicates the need to also study nutrient ux and availability in the natural environment. Given a dense population of actively growing cells there may also be strong competition for the nutrients which are available, effectively creating an environment in which nutrients are not readily available. This, in turn, mimics a situation which we would expect to nd where fewer nutrients and cells are present (i.e., an oligotrophic state).
Aerobes vs Anaerobes: Coupling Molecular and Cultivation-Based Studies

In addition to the isolation of aerobes from the Lake Fryxell mat system a number of enrichments were set up in liquid culture for anaerobes [19]. Thus the spectrum of anaerobes recovered in pure culture was potentially biased to a much lower number than the aerobes. However, the results of the molecular genetic analyses would seem to indicate that many of the clones could be assigned to taxonomic groups where the ability to grow anaerobically was widespread. However, a closer look at both methodology and the results illustrates a number of reasons for these differences. Although it is generally accepted that in certain taxa (such as the c-subclass of the Proteobacteria) it is difcult to predict whether a novel taxon will be an aerobe, facultative anaerobe, or obligate anaerobe based on its 16S rDNA sequence alone, in other cases (such as the clostridia) one may predict with a reasonable degree of certainty that a novel taxon will be an anaerobe. Given that the majority of the sequences from the 16S rDNA clone library may be attributed to taxa within groups of anaerobes, one may predict that anaerobes predominate in the mat system. This would appear to be in contradiction to the isolation of some 900 strains of aerobes from the same mat and comparatively few anaerobes. However, it is here that one should examine the methods used for the isolation of strains in more detail. The methods for isolating strains from the mat samples were designed under the premises that the algal mat system would be aerobic, since it lies in shallow water at the edge of the lake. Thus, the selection of some 10 different media and plating under aerobic conditions failed to take into consideration the fact that the mat may quickly become anaerobic below the rst few millimeters. This illustrates the need to examine factors such as the depth at which oxygenic photosynthesis occurs (indirectly also the depth of light penetration) as well as oxygen, sulde, and redox gradients within the mat, despite

the fact that it is only 0.51 cm thick. It should be noted that the limited number of isolates obtained by anaerobic enrichment included a number of strains which were clearly aerobes that were facultatively anaerobic. In contrast no attempt was made to test the ability of all 900+ isolates obtained under aerobic conditions for their ability to grow under anaerobic conditions. One may speculate that the lower layers of the mat are probably anaerobic, and that oxygen is effectively prevented from migrating down into this layer by the dense population of aerobes in the upper layer, which remove oxygen via respiration. Clearly the removal of oxygen would also lead to anoxic conditions in the upper layers of the mat, which would mean that the ability to grow aerobically and under reduced oxygen tensions would be a selective advantage. It is worth mentioning the fact that the mat system would be subjected to a different light regime in the Antarctic compared to that at higher latitudes. In the middle of the summer and winter the mat would not be subjected to a diurnal cycle, the light regime being 24 h of light or darkness, respectively, which would have a direct effect on photosynthetic oxygen production. During the winter months the lake is also completely frozen. Of course from a purely technical point of view, employing serial dilutions and plating, under aerobic conditions, of each serial dilution in triplicate, together with selecting three different growth temperatures, quickly results in several hundred plates which have to be examined for growth and colonies picked. In contrast, the anaerobic isolates were enriched in liquid media in a small number of bottles using a similar number of media. Given the anaerobic nature of the mat it would have been more appropriate to parallel the aerobic isolation methods with isolation under anaerobic conditions. However, working with several hundred anaerobes is considerably more time consuming. Thus the discrepancies between the two methods can largely be attributed to emphasis on aerobes rather than anaerobes. Studies on mat systems which are more easily accessible either in saline environments or in hot springs have provided interesting systems which demonstrate some of the approaches that may be adopted to study them [70, 71, 75, 95]. Factors such as temperature or salinity gradients are fairly easy to detect, since they occur over large distances. However examining the infrastructure of mat material in hot springs clearly indicated layering and associated strain stratication [75]. The application of modem methodologies, including microelectrode work and electron microscopy of the mat prole, is a technique that may be easily applied in Yellowstone National Park, but is rather more difcult to implement in the Antarctic. Despite this, the success of the work on hot spring mats indicates that vertical stratication plays an important role in mat systems and

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that this should also be taken into consideration when examining the microbial diversity of the mat system of Lake Fryxell. In the past it has been tempting to take the results of molecular genetic studies and simply indicate that the differences between those results and the results of the cultivation-dependent studies are due to the fact that the majority of prokaryotes cannot be isolated. However, the results of the combined molecular genetic and cultivation-based studies clearly indicate that this is an oversimplication and that careful examination of the results suggests where improvements may be made in the way the methods of cultivation are selected [20, 76]. It is now recognized that it is difcult to draw conclusion on the physiology and biochemistry of organism identied in such ecological systems based on the 16S rDNA sequence alone, although some generalisations may be made in some groups (i.e. isolates assigned to the clostridia are usually anaerobes). Furthermore the work on the mat system of Lake Fryxell also indicates how important it is to understand the environment in situ.
Molecular Characterization
The Question of Cultivatability.

A number of studies have been carried out making use of 16S rDNA derived clone libraries to characterize the microbial population present in a variety of aquatic environments. These studies are based on the ndings by Ward et al. [94] and Gioivannoni et al. [45] that the 16S rDNA sequences recovered from a hot spring mat system in Yellowstone National Park or from the Saragasso Sea did not reect the cultivated diversity known at the time. This, in turn, lead to the assumption that the majority of prokaryotes could not be cultivated. Although it is true that such studies do not rely on the results of cultivation, it is now accepted that novel clones indicate the presence of taxa which have not been isolated to date. This is certainly true of studies in the Antarctic where an increasing number of novel cloned I6S rDNA sequences have also been accompanied by an increasing number of novel isolates. Despite the parallels the currently published molecular studies have rarely been incorporated with studies dealing with the isolation of novel taxa. In the case of studies on Deep Lake, Organic Lake, and Ekho Lake, Bowman et al. [15] relied upon the relatively limited number of isolates reported in the literature [812, 17, 21, 33, 34, 3643, 53, 55, 56, 66, 86]. Certainly the diversity of taxa clearly related to extremely halophilic or halotolerant Archaea and Bacteria from Deep Lake reects its salinity, but the presence of sequences from taxonomic groups in which obligate anaerobes can be found is indicative of the fact that the in situ conditions may be anoxic. In spite of the fact that Deep Lake is highly saline and members of the genus Halorubrum [40,

65] may be isolated, the data suggest that this lake is rather different from the eutrophic salinas extensively studied on the eastern coast of Spain. In fact, not all such highly saline environments are eutrophic, so that care must be taken when comparing limited reports from the literature. The reports of clones grouping with clones from apparently novel halophilic Archaea from salt marshes [69] is also indicative that neither the large distances between the different environments nor the low temperatures are a signicant obstacle to colonization of the Antarctic by prokaryotes. In parallel to the studies of Bowman et al. [15], Labrenz [54] has characterized a diverse range of organisms isolated from Ekho Lake. In such cases it is unfortunate that sampling for the molecular and cultivation based techniques was not coordinated, since both studies leave a number of questions unanswered with respect to why a particular cloned sequence is not represented in the cultivated strains or why the sequences of cultivated strains are not represented among the cloned environmental sequences. Whereas Labrenz [54] reported on the recovery of isolates from the water column, Bowman et al. [15] relied on sediment samples from the deepest points of the lake. The recovery of clones belonging to anaerobic groups of organisms (members of the family Haloanaerobiaceae) together with organisms which may well be capable of aerobic growth (members of the a or c subclasses of the Proteobacteria) suggests that the sediment may not be uniformly aerobic or anaerobic. An alternative would be that organisms sink from the aerobic zone and are only slowly degraded. However, this would emphasize the fact that either dead organisms or DNA that has not been degraded may falsify the results. In such cases examining the organisms for their rRNA content may indicate their metabolic state and allow corrections to be made for such artefacts.
The Role of Perceived and Real Species Numbers in An increasing numAppreciating Microbial Diversity.

ber of novel taxa have been isolated from the Antarctic during the past two decades. While this may be coupled to increased interest in the Antarctic as a source of novel isolates, it is also probably a reection of the increasing accuracy with which modern methods allow us to quickly determine which taxa have not been characterized before. Both aspects certainly play a role in the current increase in novel taxa being described from the Antarctic, as well as the fact that only a comparatively small proportion of prokaryotic species diversity has been described. The application of molecular genetic methods for studying microbiota without having to isolate organisms drew our attention to the fact that there were two problems associated with a cultivation-based study [45, 94]. First, cultivation methods had not given us an accurate picture of the diversity of prokaryotes to be found

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in nature. At present only about 40005000 named species are known, which may represent anywhere between 0.1 and 10% of all extant prokaryotes. Few microbiologists have taken into consideration the fact that a signicant fraction of the data on all extant prokaryotes is missing and this may have a signicant inuence on the conclusions we are drawing based on a rather limited data set. Second, the fact that organisms could be detected in environments that were clearly different from those which had been isolated quickly led to the concept that most prokaryotes could not be cultivated. However, it is signicant that those groups which were the rst to report that clone libraries indicated that the isolation techniques had failed to isolate the dominant prokaryote populations were also those groups which have continued to attempt to isolate those organisms that are, in fact dominant in the environment and that it may be possible to isolate with suitable media and information on the physiology of the organisms concerned [76, 95].
Reliability and Accuracy of Molecular Genetic Meth-

Within the past decade our appreciation of the advantages and limitations of the molecular genetic approach have become apparent. It is now accepted that the 16S rDNA sequence alone does not provide information on the physiological and biochemical potential of the organisms identied by the 16S rDNA sequences alone. Although in some cases generalizations may be made, such as that anaerobic growth occurs in groups like the clostridia or methanogens, in groups such as the a subclass of the Proteobacteria prediction is more difcult. The latter is particularly true in groups such as members of the BradyrhizobiumNitrobacterRhodopseudomonas Apia group, where growth may be heterotrophic, anoxygenic phototrophic, or nitrite oxidizers, and the organisms all share 16S rDNA sequence similarities >95%. The detection of functional genes, such as those associated with bacteriochlorophyll production may not always be reliable indicators of anoxygenic photosynthetic activity, since it is becoming apparent that these genes may be fairly widely distributed within the alpha subclass of the Proteobacteria, but the genes may not be translated, or translation does not necessarily lead to growth associated with anoxygenic photosynthesis. Closer examination of the methodology associated with the way molecular genetic studies are carried out indicates that, although they may reveal organisms that have not been detected by conventional isolation techniques, these methods may not always accurately reect the composition of the microbial population. Quantitative measures rely on accurately reecting the number of organisms associated with a particular gene (usually 16S rDNA) sequence. Factors such as gene copy number and the growth phase of the organisms are two factors that are not easy to estimate in an unknown population, and
ods.

even in dened populations there are problems associated with the quantication of organisms using such methods [35, 61]. Similarly it has been shown that factors associated with the PCR amplication of environmental DNA may have an effect on the results obtained. This includes the annealing temperature of the primer, the number of PCR cycles, and the choice of primers [3, 48]. Primer choice is usually for universal primers, but such methods fail to detect organisms if the universal primer fails to bind [51]. Other factors that include the efciency of recovery of DNA are the methods used for the extraction of the DNA and the physiological state of the organisms; the ease with which cells may be disrupted may also play a role. It should be noted that more aggressive methods of isolating DNA from organisms that are difcult to lyse may also have the side effect of damaging the DNA isolated from organisms that are easy to lyse. Schmalenberger and Tebbe [84] have reported that a single DGGE band may harbor more than one sequence. Although this effect appears to be well known among scientists working in this area, this is rarely documented, nor have the causes been extensively examined and explained. A factor that is rarely taken into consideration when using 16S rDNA data alone to identify organisms from clone libraries is the inherent limitations of using a single parameter to assign organisms to known taxa. The most signicant factor is the fact that the accuracy of the interpretation is only as good as the taxonomy on which it is based. Thus, gene probes designed to be specic for members of the genus Azoarcus [50, 81] take on a wider specicity with the description of Azoarcus strains, included in the initial work, as members of new genera [80]. Similarly, the infrastructure of groupings obtained by 16S rDNA sequence data alone may change as more organisms are added to the data set, and in some cases organisms may appear to change their generic afliation, or alternatively it may be impossible to describe or delineate genera based on the 16S rDNA alone. Further problems lurk in the databases where the quality of sequences deposited may seriously hinder accurate comparisons or where the identity of strains associated with a particular sequence may be inaccurate, but only apparent to the experts or after careful comparative studies (see [1, 27, 89]). Despite the desire to delineate all prokaryote species based on 16S rDNA sequence data alone there is now ample evidence that the resolution of the 16S rDNA sequences is not sufcient. However, although this problem can be tackled with pure cultures by adding other data, the trend in molecular microbial ecology has been to create the concept of phylotypes or phylospecies, where the cutoff point is generally 98% 16S rDNA sequence similarity [15, 16, 19], although it is well documented that such values are fairly typical for strains belonging to different species. Thus using the concept of

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phylotypes or phylospecies currently underestimates the number of species (dened in a classical, genotypicphenotypic study [93]) present in the same or different environments. Taking such factors into consideration and learning to appreciate not only the benets of the molecular work, but also the limitations, is a process which will inherently contribute to our appreciation of microbial diversity.
The Source of the Prokaryotes
Indigenous Microbiota and Living Microbial Fossils.

As a result of the prolonged periods of cold during the winter months, the average annual growth rates of unicellular organisms can be taken to be fairly low. However, the modern Antarctic continent was not always a cold, ice-covered land mass. Continental drift has brought the present continent to its current position, although at one time the ora and fauna of the continent were typical of its more tropical location. In its current geographically isolated position it is tempting to speculate on the slow adaptation of the endemic microora as the continent slowly drifted to its present position versus colonization of the Antarctic from outside sources. The majority of studies on the microbiota of the Antarctic have concentrated on aquatic environments, although more recent studies have taken into account the diversity of prokaryotes that can be detected in glacial ice [2326, 98]. The application of the principle of the molecular clock should allow one to attempt a correlation between the geological age of various lakes or glacial ice and the observed genetic differences between strains detected in the Antarctic and strains isolated elsewhere. Two major problems with this approach relate to the fact that we currently only know a small fraction of all extant prokaryotes and the fact that most forms of sequence analysis automatically assume that all data are from organisms which may be treated as terminal taxa (i.e., occur at the ends of branches). The problem that relates to our limited knowledge of the diversity of all extant prokaryotes has a direct reection on how we may interpret the data currently available. If, for example, two strains show 10% 16S rDNA sequence divergence and one assumes a constant mutation rate of 12% divergence per 100 million years [68], then one may speculate that they diverged from their last common ancestor 10002000 million years ago. In contrast, two strains that show only 1% 16S rDNA sequence divergence would appear to have shared a last common ancestor 50100 million years ago. However, in the rst case the large degree of sequence divergence observed may simply be due to the fact that additional, more closely related strains or species are currently not known, making such speculation rather a risky undertaking. In the case of very closely related strains there is evidence that it may not be uncommon to nd 0.51.0%

16S rDNA sequence divergence between strains within a species (this assumes the acceptance of a bacterial species as currently dened based on a combination of phenotypic and genotypic parameters). Thus 1.0% sequence divergence may simply be a natural phenomenon within a species and not necessarily indicative of divergence from a common ancestor 50100 million years ago. Recent studies on either bacteria isolated from or 16S rDNA sequences amplied from glacial ice of known geological age suggest that it may be possible to study the microbiota which became entrapped at the time the ice rst froze. It is well known that ice cores serve as a window into the past climate of the Antarctic, so the extrapolation to include studies on bacteria, either by isolation or amplication, would seem reasonable. However, the common method of examining the 16S rDNA either from isolates or from DNA isolated from environmental samples suffers from the problem of limited resolution. If we take the quoted value of 12% 16S rDNA sequence divergence per 100 million years, then relatively large time scales would be needed for changes in the 16S rDNA sequences to become apparent if one could nd the genuine frozen dormant ancestor of a modern Antarctic bacterium. Christner et al. [25] have reported on the isolation of bacteria and amplied 16S rDNA sequences from glacial ice in China as old as 750,000 years and from accretion ice from above Lake Vostoc, which is assumed to have been isolated by an ice sheet for at least 420,000 years. However, such time scales are too short for there to be appreciable differences in 16S rDNA sequence between the ancestral strain frozen in ice and its modern descendant. It should be noted that studies on the ora and fauna of permafrost soil using DNA amplication have indicated a good correlation with sequences from genuine plant and animal fossils (i.e., from mammoths), although this was limited to samples no older than about 500,000 years [96]. These studies also recorded signicant DNA damage, which did not allow the recovery of large sections of amplied DNA.
Sympatric vs Allopatric Speciation. In the case of actively growing microbial communities where there is no speculation about whether they have been dormant over long periods of geological time, the way in which communities may have developed, either from a native prokaryotic biota or by input from outside, is not easy to dissect. The discovery of organisms (either as isolates or via the clone library) which show a relatively high degree of 16S rDNA sequence dissimilarity to other known organisms may simply be due to our inadequate data set, or it could indicate specialization of a particular group to life in the Antarctic to such a degree that closely related species from temperate regions will never be found. Only future investigations on microbial diversity will help to

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throw light on this question. In the case of strains which are clearly closely related to known species, it is easier to examine some of the possible ways in which the lakes have been colonized. A variety of lakes have been studied, which range in salinity from freshwater through brackish and marine to hypersaline lakes where the salinity approaches saturation with sodium chloride. In the case of lakes with a salinity close to that of seawater, the coastal waters surrounding the Antarctic are an obvious source of microbial inocula. Thus, it would not be surprising to nd those organisms reported from the marine environment also in the brackish to marine lakes. In some cases the salt tolerance of certain taxa is such that they may also thrive in freshwater as well as brackish to marine aquatic environments. At the other extreme, several bacilli, members of the genera Staphylococcus or Halomonas, are well known for their broad salt tolerance, being able to tolerate salinities of 10% NaCl or even greater. In contrast, organisms adapted to life in hypersaline lakes would seem to be cut off from a similar environment that is geographically close. Bowman et al. [15] reported on the recovery of 16S rDNA clones of various members of the Archaea usually found in salinas or salt marshes at higher latitudes, although they were not identical with the strains isolated by Franzmann et al. [40] from the same lake. Studies on some of the isolates from Lake Fryxell indicate that groups of strains that share a high degree of 16S rDNA similarity to themselves and to other, known species may be differentiated into subgroups on the basis of FT-IR spectra [90]. The ability to differentiate groups of organisms that are virtually indistinguishable based on the 16S rDNA sequences is consistent with data from other groups, who suggest that there may be ecological specialization of local populations [28, 72, 74, 95] which allow them to be distinguished at the molecular level based on different genes and perhaps even at the physiological level. Whether it would be wise to assign each of these strains, which are obviously distinguishable from similar strains found at different localities, the status of different species is something which is still debatable. Papke et al. [74] have used their data to call into question the widely held belief that everything is everywhere. Clearly there is gathering evidence which shows that bacterial species do not evolve on a globally sympatric scale, but that local differences in the environment may be a signicant driving force in strain differentiation. As a consequence this may lead to the evolution of different species in locally different environments (i.e., allopatric speciation). Clearly in the case of the Antarctic strains similar to members of the genus Janthinobacterium [90] these strains are virtually identical at the 16S rDNA level to the two known species in the genus, Janthinobacterium lividum and Janthinobacterium agaricidamnosum. However, unpublished data that have subsequently become available indicates not only that the

Antarctic isolates are distinct from members of these two species, but also that they form two subgroups. This would imply that the Janthinobacterium strains have evolved independently of populations in other parts of the global (allopatric evolution), but that there may also be a potenial for speciation within the local Antarctic population (sympatric evolution). Similar effects are to be seen in other groups of strains, which would indicate some interaction between the strains and the local environment. In such cases the relatively small degree of 16S rDNA sequence divergence clearly indicates that the Antarctic isolates have not separated from their parent populations hundreds of millions of years ago, but rather that a time scale of thousands of years may be more appropriate. In such cases one may speculate that lakes such as Lake Fryxell have been colonized by a population of prokaryote species which was initially not signicantly different either from strains present in other lakes in the Antarctic or from strains present in other parts of the globe. It is possible that this population then responded to differences in the local environment, with changes slowly making changes from the parent population apparent. However, in the case of the two subgroups within the Janthinobacterium species it is virtually impossible to draw denite conclusions as to whether a single parent population entered the lake, which further differentiated into two subgroups, or whether two different parent strains entered the lake and the current subgroupings are simply a reection of the colonization by two different subgroups. Similarly effects are to be seen in the different species and subspecies within the Clostridium strains isolated from this lake system [88]. Sikorski et al. [87] have carried out similar, but more detailed, work on Pseudomonas stutzeri strains and have illustrated the complexity of the problem. Speculation on the origin and dynamics of such systems is complicated by a number of problems. First, ecology has tended to concentrate on animals and plants, where the dynamics can be followed in cycles over decades. In the case of microbial systems the situation is more complex because of the small size of the organisms concerned and some of the methodological difculties associated with microbial ecological studies. In particular it is not easy to accurately analyze the distribution of organisms in a particular environment. Although it is now possible to gain a better insight into the distribution of organisms in the natural environment, the resolution of the 16S rDNA approach is such that one cannot easily determine whether one is dealing with a single species or several closely related species, or even to what extent there may be different subpopulations of the same species in the same environment. Second, even if it is possible to determine whether the dominant organisms present are limited to two or 20 different strains, it is evident that a single environment may be populated by several hundred

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strains, some of which would appear to be present in relatively low numbers. At present we certainly do not understand the ecological signicance of such complex microbial populations.Although there has been a tendency to study individual strains in the laboratory, different methods are needed to study the same organisms in complex populations as well as to study their interactions.
Conclusions

Despite the fact that a large number of taxa have been reported to have been isolated from the Antarctic it is still difcult to assess the signicance of the diversity reported relative to the global distribution and diversity of prokaryotes. One particular obstacle is the comparatively small number of prokaryotic species which have been described in detail. If only 0.110% of all extant prokaryotes have been named (about 40005000 species to date), then the total numbers of species may be between 40,000 and 4,000,000. Given the low numbers of known (named) species, it is hardly surprising that the modication of existing techniques quickly allows us to isolate novel species. However, there are two aspects of completing this inventory which are relevant to the prokaryotic diversity in the Antarctic: rst, recognizing the new species, and second, the distribution of these species. This is best illustrated by the example of members of the genus Psychrobacter and species closely related to Clostridium estherteticum. Psychrobacter urativorans served as a model psychrophile, and in the earlier literature it was referred to as Micrococcus cryophilus, where as Psychrobacter immobilis was initially isolated from chilled meat. Bowman et al. [7, 10] isolated a number of new species from the Antarctic, expanding the known habitats and diversity of members of this genus. Subsequently members of the genus Psychrobacter have been isolated from a number of marine environments where suitable temperatures prevail [32, 62, 83]. Similarly, Clostridium estherteticum [29] was initially isolated from chilled meat, but four new species have recently been reported from an algal mat system in Lake Fryxell, sharing no more than 3.5% 16S rDNA sequence divergence [88]. In both cases the rst isolates were obtained from environments very different from the Antarctic and one may not have initially concluded that similar species would be present in a natural, cold environment. Not only does the isolation of similar species from the Antarctic clearly indicate that a wider diversity of such species may be present in the natural environment, but the discovery of novel species in the Antarctic has also provided a nucleus around which even greater diversity is now being reported from similar environments. However, the description of novel taxa, coupled to some of the modern molecular methods, also has its drawbacks. In particular, if a study concen-

trates on the description of novel taxa, then modern screening methods allow us to quickly identify those strains which are potentially members of known taxa, with the consequence that the presence of these known species is not reported at the expense of describing novel taxa. Similarly, the use of molecular methods in the screening of isolates or natural populations has provided a valuable insight into the diversity of prokaryotes, but it has also meant that one may also be incorrectly estimating the true diversity if one relies on a single indicator gene, such as the 16S rRNA gene. In order either to prescreen isolates or to make a statement about species diversity in clone libraries, one often encounters a cutoff value of >98% 16S rDNA sequence similarity as delineating phylotypes, or presumptive species. Although it is now fairly well documented that two strains with <97% 16S rDNA sequence similarity do not meet the criteria which would include them in the same species, there is also ample evidence that strains sharing >98% 16S rDNA sequence similarity may also belong to different species. It should be noted that novel species should be delineated by a combination of genetic (including DNADNA hybridization) and phenotypic parameters. Two examples relevant to the Antarctic illustrate the point. Reddy et al. [79] have shown that Halomonas glaciei shares 99.8% 16S rDNA sequence similarity to Halomonas variabilis; applying the 98% 16S rDNA sequence similarity cutoff would have included both species in the same phylotype. Secondly, Spring et al. [88] have shown not only that three of the novel Clostridium species they isolated share >98% 16S rDNA sequence similarity, but that heterogeneity within a species or even between operons of the 16S rRNA gene within the same strain can contribute to misinterpretations. In the case of interoperon heterogeneity within the same strain, these may be mistakenly taken to indicate two different strains. However, microheterogeneity between multiple operons has been documented in the literature. In the absence of isolates on which to take studies further, it would not be possible to detect these problems in interpreting the data. It would appear that setting a cutoff point of 98% 16S rDNA sequence similarity may help to simplify interpretation of the data, but realistically the evidence indicates that such a group potentially contains one or more species. This problem is not limited to comparisons between clone libraries or isolates from different environments, but is obviously applicable to one sample where such closely related 16S rDNA sequences may be obtained both from novel taxa and from clone libraries [19, 88]. It should be noted that metagenomics has also recognized the significance of detectable differences in genomes of organisms sharing 16S rDNA sequences with a high degree of similarity [2]. It is evident that only a small proportion of prokaryotes have been detected that occur on or in the sur-

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roundings of the Antarctic. Various problems accompany the work of microbiologists, not least the isolated situation, where it is not possible to order chemicals that have run out on a 24-hour-service basis. In addition, all equipment must be present, which means that working with more demanding organisms such as anaerobes is more difcult. Alternatively, samples must be either xed or own out under suitable conditions which alter the natural conditions as little as possible. Even if we had a comprehensive database on the prokaryotic diversity in the Antarctic, it would be an even greater task to correlate this with the global distribution of similar (or identical species), where so many data are lacking. We know little about the detailed ecology of complex microbiological systems in the Antarctic, such as the mat systems, where nutrient cycling and microbial interactions remain to be studied. Here, too the role of individual species in situ may play an important aspect in the way these mats are formed and the evolution not only of the whole ecosystem, but also of individual strains or species. However, the recent work carried out on the microbiology of Antarctic environments clearly indicates that there is a rich diversity present in an environment which is hostile to all but a few multicellular eukaryotes. As work continues in this area it will be possible not only to document the diversity of prokaryotes present in the Antarctic, but also to approach some of the problems associated with studying microorganisms in this environment. Based on our current knowledge we may predict that prokaryotic diversity in the Antarctic is far greater than could have been predicted a decade ago and that such work is far from complete. In addition to providing access to genetic resources that may be of potential use, little is known about the way in which microbial populations may be used to monitor climate change or the effects of pollution. One aspect is fairly evident: our current knowledge of prokaryotic diversity in the Antarctic is just the tip of the iceberg.

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Acknowledgment

Work on the Lake Fryxell mat system was supported by a EU grant PL 970040 (MICROMAT). Cathy Welch is thanked for collecting the mat material, and the Long Term Ecosystem Research Program (LTER), under whose auspices the material was collected. Note Added in Press Taton A, Grubisic S, Brambilla E, Rutger De Wit Ri, Wilmotte A (2003) Cyanobacterial diversity in natural and articial microbial mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a morphological and molecular approach. Appl Environ Microbiol 69:51575169 have provided data on the cyanobacterial diversity of the mat system of Lake Frxyell using both molecular and morphological methods.

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