BACKGROUND
bacteria belonging to the phylum planctomycetes have revealed several remarkable features that set them apart from other bacteria.
their cryptic morphology led early microbiologists to mistake them for fungi, and the discovery of their cell compartmentalization, featuring membrane bounded organelles, raised fundamental questions about the evolution of eukaryotes  <cit> .
further, the unique anammox metabolism found in some planctomycetes has revolutionized the view of microbial nitrogen cycling  <cit> .
the planctomycetes also possess cell walls without peptidoglycan, a characteristic that they share only with the obligate intracellular bacteria within chlamydiae.
in addition to the interest sparked by these unusual and fascinating features, planctomycetes have in later years attracted considerable attention because of their presence in a wide variety of environments on earth.
by investigating bacterial communities using molecular methods , planctomycetes have been repeatedly detected in soil, sediments, marine and freshwater systems and in terrestrial hot springs to mention just a few .
however, their metabolic potential and function in these ecosystems is often unclear, as 16s rrna gene sequence investigations only rarely give clues to ecological roles.
in the marine environment, delong and co-workers  <cit>  found that planctomycetes were more abundant in clone libraries from marine aggregate  attached bacteria than from free-living bacteria.
since then, results from several studies suggest that planctomycetes favor a biofilm lifestyle, adhering to surfaces in aquatic environments including marine sediments  <cit>  , diatom cells  <cit> , seaweeds and other aquatic macrophytes  <cit> .
rhodopirellula baltica is an extensively studied marine particle-attached planctomycete.
its genome sequence reveals a large number of genes involved in the breakdown of sulfated polysaccharides  <cit> , a carbon source found in marine photosynthetic organisms such as microalgae and seaweeds, who's detrital material is thought to generate marine snow.
such genes are also encountered in other planctomycete genomes and planctomycete-derived metagenomic fosmid libraries from seawater collected in upwelling zones  <cit> .
the overrepresentation of such genes, and the association of r. baltica and other planctomycetes with marine snow has led to the hypothesis that heterotrophic planctomycetes are specialized degraders of sulfated polymeric carbon, for example in marine snow  <cit> .
given the significance of marine snow as part of the so-called "biological pump" of carbon in the oceans  <cit> , planctomycetes may thereby be playing a crucial role in global carbon turnover  <cit> .
still, quantitative data on the distribution of planctomycetes in the marine environment and elsewhere is still scarce, and very little is known about the yet uncultured planctomycete lineages that are assumed to carry out the bulk of these globally critical processes.
kelps are large brown seaweeds of the order laminariales.
they often form dense stands along rocky coastlines that are referred to as kelp forests.
kelp forest ecosystems are some of the most productive ecosystems in the world  <cit> .
their immense importance for coastal biodiversity, productivity and human economy has long been recognized in temperate regions of the world and is only beginning to be understood in the tropics  <cit> .
kelp forests along the atlantic coasts of europe are dominated by the large kelp laminaria hyperborea.
bacteria associated to kelp are believed to be important in the carbon and nitrogen turnover in kelp forest food webs  <cit> , but it is still not known what types of bacteria are involved in these processes.
recently, the seasonal dynamics of the cell density and bacterial community composition in biofilms on l. hyperborea were addressed.
in this study, planctomycetes were frequently detected throughout the year but their abundance and phylogenetic relationships were not considered  <cit> .
in order to address the importance of this group of bacteria in kelp forests, we therefore aimed to take an in-depth look at the abundance and phylogenetic diversity of planctomycete communities inhabiting l. hyperborea surface biofilms.
this was achieved by using fluorescence in situ hybridization  to quantify their abundance and visualize their distribution in the biofilm as well as 16s rrna gene clone library construction to elucidate their phylogenetic relationships, community composition and diversity at different times of the year.
in addition, a selective cultivation approach was used to assess the culturability of planctomycetes from kelp surfaces.
RESULTS
abundance of planctomycetes in kelp surface biofilms
quantification of planctomycetes in samples from july  <dig>  february  <dig> and september  <dig> using fish showed that they make up a large part of the kelp surface biofilm community in all three sampling occasions.
in july and september they dominated the community, with cells hybridizing with the planctomycetes-specific probe pla <dig>  <cit>  accounting for over 50% of the total dapi stained cells on average .
in february, the planctomycetes were less abundant; with pla <dig> hybridized cells corresponding to an average of 24% of total dapi stained cells.
samples that were also subjected to hybridization with the pir <dig>  <cit>  probe showed similar percentages  of hybridized cells as the with pla <dig> probe .
inspection of the cloned 16s rrna gene sequences revealed that the pir <dig> target sequence was present in all clones except those belonging to the om <dig> lineage  suggesting that the specificity of this probe needs to be reevaluated.
the different formamide concentrations  used in hybridization with the pla <dig> probe did not change the proportion of pla <dig> hybridized cells significantly .
the average proportion of the dapi stained cells that hybridized with the eub <dig> probes was 79% in july, 74% in september and 52% in february .
cell distribution of planctomycetes in the biofilms
fluorescence microscopy images of dapi and fish stained biofilm cells revealed a complex and variable microscopic landscape.
the microbial cells appeared to be unevenly distributed on the kelp surface, often occurring in clusters  or growing along straight lines .
planctomycetes cells were found within all these structures, and appeared to grow evenly intermingled with other cells .
fluorescence microscope images showed dapi and fish signals corresponding to different cell morphologies in the biofilm, ranging from long filaments, cocci of different sizes and small rods .
the planctomycete fish signals were always in the shape of small and medium sized cocci  and displayed the "ring" shape typical of planctomycete cell organization  <cit>  .
the eub <dig> fish signals included the whole range of morphologies  and were both ring-shaped and solid.
isolation and cultivation of planctomycetes from kelp surfaces
one strain, named "p1", belonging to planctomycetes was isolated from kelp surface biofilm material from september  <dig>  it displayed morphological features typical for rhodopirellula baltica, with ovoid cells and rosette formation .
it formed pink colonies on m <dig> solid media that were visible after approximately seven days of incubation in room temperature after inoculation.
it was closely related to the type strain of rhodopirellula baltica  and to rhodopirellula strain k <dig> isolated from seawater in iceland  <cit>  .
however, it was not closely related to any of the clone library sequences from kelp surface biofilms .
phylogenetic diversity of planctomycetes from kelp surface biofilms
three clone libraries, from february  <dig>  july  <dig> and september  <dig>  constructed with the planctomycetes-specific primer pla46f and the general bacterial primer 1542r were analyzed to gain insight into the phylogenetic diversity of the planctomycetes growing in kelp surface biofilms.
in total,  <dig> clones were sequenced in the forward direction from the three clone libraries, resulting in partial 16s rrna gene sequences of approximately  <dig> basepairs.
of these, only  <dig> sequences  did not classify as belonging to planctomycetes and were discarded from the further analyses.
these unspecific sequences classified as deltaproteobacteria , gammaproteobacteria , actinobacteria  and verrucomicrobia  while one remained unclassified using the greengenes g2chip classifier  <cit> .
the remaining  <dig> partial planctomycete 16s rrna gene sequences clustered into  <dig> otus at 98% sequence similarity.
other otu definitions  gave different numbers of otus, but the general trends observed in the dataset were the same.
one to six representative clones of each otu were selected for sequencing in the reverse direction in order to assemble near full-length 16s rrna gene sequences.
of the assembled sequences, three were removed from the analyses because of poor sequence quality and two because of indications of chimeric origin.
the remaining  <dig> near full-length planctomycete 16s rrna gene sequences have been deposited to genbank under the accession numbers hm <dig> to hm <dig>  and the sequence of the p <dig> isolate under hm <dig>
the clone libraries from february, july and september showed considerable overlap in otu composition .
the july library had the lowest otu richness and consisted of a subset of the otus detected in the other two libraries.
the highest otu richness and the most unique otus  were found in february.
september was intermediate in otu richness and the number of unique otus .
the diversity of the three clone libraries is illustrated in figure  <dig> using rarefaction curves showing the expected number of otus encountered with clone sampling effort.
july displays a near asymptotic curve, indicating low diversity, while september is intermediate and february displays the highest diversity.
the shannon diversity index and the chao <dig> richness estimates for the clone libraries  show the same relative diversity pattern.
the phylogenetic analysis of the near full-length sequences obtained in this study and other planctomycete sequences obtained from the silva reference database  <cit>  revealed that highly divergent lineages of the planctomycetes phylum are represented in kelp surface biofilms .
the kelp surface biofilm clone sequences appear to cluster within five major lineages that have been labeled as: "rb1" and "rb2" , rhodopirellula, planctomyces and "om190".
the "rb1" and "rb2" lineages appear more closely related to the rhodopirellula and blastopirellula genera than to the pirellula genus and were given their labels based on that .
yet the phylogenetic analyses do not place them consistently with either of the genera.
sequence similarities of 86-90% to rhodopirellula baltica and blastopirellula marina indicate that they probably represent distinct phylogenetic lineages that could correspond to new genera according to conventional taxonomical practice.
the "rb1" lineage was by far the most represented in all three clone libraries .
sequences that cluster within the "rb2", rhodopirellula and planctomyces lineages were only represented in september and february, indicating a seasonal difference, while om <dig> representatives were present at low numbers in all three clone libraries .
discussion
to our knowledge, the kelp surface biofilms investigated in this study display the highest proportion of bacteria belonging to planctomycetes reported in a natural bacterial community so far.
this observation is consistent with earlier results from a dgge based study on seasonal variation of laminaria hyperborea  surface biofilm communities  <cit> .
other habitats where a high abundance of planctomycetes has been reported include seawater during a diatom bloom where planctomycetes related to pirellula were detected attached to diatom cells and were among the dominant lineages in the bloom samples  <cit> .
in investigations of sandy sediments containing algal cells  <cit> , planctomycetes were also abundant, accounting for up to 20% of total cells, accompanied by cytophaga/flavobacteria.
gade and co-workers  <cit>  used order-, genus- and strain specific fish probes to detect planctomycetes in a range of aquatic habitats and recorded abundances up to 11% of total cells in some lakes.
peat bogs with sphagnum moss have also been reported to harbor abundant  planctomycete populations  <cit> .
similarly to kelp surfaces, these environments are all highly influenced by photosynthetic eukaryotes.
the studies mentioned above have all quantified planctomycetes using specific fish probes.
several other studies have detected planctomycetes using pcr based cloning and fingerprinting methods followed by sequencing.
two of the most frequently used general bacterial pcr primers, targeting the 16s rrna gene around e. coli positions 8- <dig> and 338- <dig>  contain mismatches against planctomycete sequences  <cit> .
this may have caused planctomycete abundances to be underestimated in many habitats, leading investigators to turn their attention towards bacterial groups that appear more abundant.
despite awareness of this problem, the literature and the sequence databases probably reflect a tradition of neglect towards the planctomycetes.
in the light of this, it is difficult to say whether the dominance of planctomycetes on laminaria hyperborea surface biofilms represents a unique feature of this habitat, or if other planctomycete-dominated bacterial communities have been overlooked until now.
for example, staufenberger and co-workers  <cit>  did not detect planctomycetes in surface biofilms of another species of kelp  using general bacterial primers for cloning and dgge analysis.
yet, use of different primers has let to the detection of planctomycetes on both the kelps s. latissima and laminaria digitata .
a possible explanation for the suitability of kelp as a habitat for planctomycetes is its content of sulfated polysaccharides, a class of molecules that some marine planctomycetes are known for being able to degrade  <cit> .
for example, laminaria hyperborea contains fucoidan, a class of complex brown algal sulfated polysaccharides.
these substances are secreted to the surface of l. hyperborea via mucilage channels  <cit> .
it is reasonable to assume that planctomycetes living on kelp surfaces utilize substances produced by the kelp, for example fucoidan, as carbon sources.
however, the presence of suitable carbon sources appears insufficient to explain the observed dominance of planctomycetes, as they must not only be able to grow and divide, but also outcompete other bacteria to be successful.
another contributing factor to the success of planctomycetes on kelp surfaces may be resistance to chemical antimicrobial defense compounds produced by the kelp.
antibacterial activity has been detected in extracts from many species of kelp, yet the substances responsible for the activity have often not been identified  <cit> .
the lack of peptidoglycan in planctomycete cell walls makes them resistant to conventional cell wall targeting antibiotics like ampicillin.
resistance to other antibiotics, targeting for example protein synthesis  has also been reported in some marine planctomycetes  <cit> .
in many cases the reference sequences that are the most closely related to kelp surface planctomycetes are obtained from other marine eukaryotes such as for example red and green seaweeds, corals, crustaceans and sponges .
the frequent association of planctomycetes to eukaryotes has previously been noted  <cit> .
this could point to a general lifestyle pattern of heterotrophic planctomycetes.
they may combine an affinity for sulfated polysaccharides and other polymeric carbon molecules  <cit>  produced by their eukaryote hosts with a resistance to eukaryote chemical defense molecules.
the resulting competitive advantage over other bacterial groups that are utilizing the same kind of substrates, for example the bacteroidetes  <cit>  might be one of the keys to the success of planctomycetes in a wide variety of environments on earth.
our results show differences between the different sampling times , in planctomycete abundance, otu composition and diversity.
for example, in february there is a relatively low abundance of planctomycetes  compared to july and september.
this may be linked to the age of the kelp tissue, as the kelp lamina is older in february compared to in july and september due to the seasonal growth cycle of the kelp.
aging of the kelp tissue could be associated with lowered antibacterial chemical defense by the kelp, as the old kelp lamina is to be shed soon after february, and does therefore not need to be defended against microbial colonization.
without the presence of chemical defense substances, the planctomycetes could loose their competitive advantage over other bacterial groups, explaining their lower abundance in february.
the senescence of the kelp tissue as it ages could also cause the appearance of new niches involved in degradation of different kelp constituents, thereby enabling the more diverse planctomycete communities that are observed in february compared to july and september .
among the different planctomycete lineages that are represented on the kelp, the lineage defined as "rb1" in this study appears to be the most abundant, accounting for a majority of the clones at all sampling times .
the high abundance of rb <dig> planctomycetes may thus be the cause of the observed dominance of planctomycetes on kelp surfaces .
their high abundance implies a lifestyle that makes them particularly successful on kelp surfaces.
yet the lineage also includes reference sequences from a variety of other marine habitats, indicating that rb <dig> is not a kelp-specific lineage.
the rb <dig> and rb <dig> lineages, defined in this study, are clearly related to the "pirellulae", a lineage including the genera pirellula, rhodopirellula and blastopirellula .
yet our phylogenetic analyses did not place them reliably with any of the described genera, indicated by the bootstrap support for the relevant branches in figure  <dig>  there are no sequences of cultured strains within the rb <dig> and rb <dig> lineages available in the databases.
another uncultured lineage, the so-called om <dig> planctomycetes  is also represented by clones from kelp surfaces at all sampling times, yet in low numbers.
this is a lineage that appears to branch off deeply in the planctomycete tree and representatives have been detected in a variety of environments including seawater  <cit> , soil  <cit>  and marine eukaryotes  <cit> .
these lineages have yet to be cultured and described and will reveal valuable information on planctomycete metabolism and evolution if cultivation is successful.
using conventional approaches, the rhodopirellula sp.
strain p <dig> could easily be isolated.
several closely related strains have been brought into culture earlier  <cit> .
however, the 16s rrna gene sequence of p <dig> does not correspond to any of the abundant otus detected on the kelp surfaces, for example within the rb <dig> lineage.
kelp surfaces are nevertheless a promising source for isolation of novel planctomycete strains, using more ambitious and creative approaches that take into account the environmental factors experienced by bacteria on kelp surfaces.
the rewards awaiting such attempts can be substantial, given the representation of highly divergent lineages of the planctomycete tree in kelp surface biofilms.
CONCLUSIONS
kelp  surface biofilms have a uniquely high relative abundance of planctomycetes.
several distinct lineages are represented, and the diversity and composition of the planctomycetes change during the year, probably influenced by aging of the kelp tissue.
the finding of abundant planctomycete populations in kelp surface biofilms agrees well with the view of heterotrophic planctomycetes as surface attached, specialized degraders of sulfated polysaccharides in the marine environment, as kelps are known to produce such substances.
furthermore, we wish to extend this view by hypothesizing that many heterotrophic planctomycetes share a preference of intimate coexistence with eukaryotes, which may be linked to antibiotic resistance.
the study addresses the urgent need for more detailed, quantitative knowledge on the diverse marine planctomycetes.

 bacteria belonging to the phylum planctomycetes have revealed several remarkable features that set them apart from other bacteria. their cryptic morphology led early microbiologists to mistake them for fungi, and the discovery of their cell compartmentalization, featuring membrane bounded organelles, raised fundamental questions about the evolution of eukaryotes. further, the unique anammox metabolism found in some planctomycetes has revolutionized the view of microbial nitrogen cycling. the planctomycetes also possess cell walls without peptidoglycan, a characteristic that they share only with the obligate intracellular bacteria within chlamydiae. in addition to the interest sparked by these unusual and fascinating features, planctomycetes have in later years attracted considerable attention because of their presence in a wide variety of environments on earth. by investigating bacterial communities using molecular methods, planctomycetes have been repeatedly detected in soil, sediments, marine and freshwater systems and in terrestrial hot springs to mention just a few. however, their metabolic potential and function in these ecosystems is often unclear, as 16s rrna gene sequence investigations only rarely give clues to ecological roles. in the marine environment, delong and co-workers found that planctomycetes were more abundant in clone libraries from marine aggregate attached bacteria than from free-living bacteria. since then, results from several studies suggest that planctomycetes favor a biofilm lifestyle, adhering to surfaces in aquatic environments including marine sediments, diatom cells, seaweeds and other aquatic macrophytes. rhodopirellula baltica is an extensively studied marine particle-attached planctomycete. its genome sequence reveals a large number of genes involved in the breakdown of sulfated polysaccharides, a carbon source found in marine photosynthetic organisms such as microalgae and seaweeds, who's detrital material is thought to generate marine snow. such genes are also encountered in other planctomycete genomes and planctomycete-derived metagenomic fosmid libraries from seawater collected in upwelling zones. the overrepresentation of such genes, and the association of r. baltica and other planctomycetes with marine snow has led to the hypothesis that heterotrophic planctomycetes are specialized degraders of sulfated polymeric carbon, for example in marine snow. given the significance of marine snow as part of the so-called "biological pump" of carbon in the oceans, planctomycetes may thereby be playing a crucial role in global carbon turnover. still, quantitative data on the distribution of planctomycetes in the marine environment and elsewhere is still scarce, and very little is known about the yet uncultured planctomycete lineages that are assumed to carry out the bulk of these globally critical processes. kelps are large brown seaweeds of the order laminariales. they often form dense stands along rocky coastlines that are referred to as kelp forests. kelp forest ecosystems are some of the most productive ecosystems in the world. their immense importance for coastal biodiversity, productivity and human economy has long been recognized in temperate regions of the world and is only beginning to be understood in the tropics. kelp forests along the atlantic coasts of europe are dominated by the large kelp laminaria hyperborea. bacteria associated to kelp are believed to be important in the carbon and nitrogen turnover in kelp forest food webs, but it is still not known what types of bacteria are involved in these processes. recently, the seasonal dynamics of the cell density and bacterial community composition in biofilms on l. hyperborea were addressed. in this study, planctomycetes were frequently detected throughout the year but their abundance and phylogenetic relationships were not considered. in order to address the importance of this group of bacteria in kelp forests, we therefore aimed to take an in-depth look at the abundance and phylogenetic diversity of planctomycete communities inhabiting l. hyperborea surface biofilms. this was achieved by using fluorescence in situ hybridization to quantify their abundance and visualize their distribution in the biofilm as well as 16s rrna gene clone library construction to elucidate their phylogenetic relationships, community composition and diversity at different times of the year. in addition, a selective cultivation approach was used to assess the culturability of planctomycetes from kelp surfaces.  abundance of planctomycetes in kelp surface biofilms quantification of planctomycetes in samples from july february and september using fish showed that they make up a large part of the kelp surface biofilm community in all three sampling occasions. in july and september they dominated the community, with cells hybridizing with the planctomycetes-specific probe pla accounting for over 50% of the total dapi stained cells on average. in february, the planctomycetes were less abundant; with pla hybridized cells corresponding to an average of 24% of total dapi stained cells. samples that were also subjected to hybridization with the pir probe showed similar percentages of hybridized cells as the with pla probe. inspection of the cloned 16s rrna gene sequences revealed that the pir target sequence was present in all clones except those belonging to the om lineage suggesting that the specificity of this probe needs to be reevaluated. the different formamide concentrations used in hybridization with the pla probe did not change the proportion of pla hybridized cells significantly. the average proportion of the dapi stained cells that hybridized with the eub probes was 79% in july, 74% in september and 52% in february. cell distribution of planctomycetes in the biofilms fluorescence microscopy images of dapi and fish stained biofilm cells revealed a complex and variable microscopic landscape. the microbial cells appeared to be unevenly distributed on the kelp surface, often occurring in clusters or growing along straight lines. planctomycetes cells were found within all these structures, and appeared to grow evenly intermingled with other cells. fluorescence microscope images showed dapi and fish signals corresponding to different cell morphologies in the biofilm, ranging from long filaments, cocci of different sizes and small rods. the planctomycete fish signals were always in the shape of small and medium sized cocci and displayed the "ring" shape typical of planctomycete cell organization. the eub fish signals included the whole range of morphologies and were both ring-shaped and solid. isolation and cultivation of planctomycetes from kelp surfaces one strain, named "p1", belonging to planctomycetes was isolated from kelp surface biofilm material from september it displayed morphological features typical for rhodopirellula baltica, with ovoid cells and rosette formation. it formed pink colonies on m solid media that were visible after approximately seven days of incubation in room temperature after inoculation. it was closely related to the type strain of rhodopirellula baltica and to rhodopirellula strain k isolated from seawater in iceland. however, it was not closely related to any of the clone library sequences from kelp surface biofilms. phylogenetic diversity of planctomycetes from kelp surface biofilms three clone libraries, from february july and september constructed with the planctomycetes-specific primer pla46f and the general bacterial primer 1542r were analyzed to gain insight into the phylogenetic diversity of the planctomycetes growing in kelp surface biofilms. in total, clones were sequenced in the forward direction from the three clone libraries, resulting in partial 16s rrna gene sequences of approximately basepairs. of these, only sequences did not classify as belonging to planctomycetes and were discarded from the further analyses. these unspecific sequences classified as deltaproteobacteria, gammaproteobacteria, actinobacteria and verrucomicrobia while one remained unclassified using the greengenes g2chip classifier. the remaining partial planctomycete 16s rrna gene sequences clustered into otus at 98% sequence similarity. other otu definitions gave different numbers of otus, but the general trends observed in the dataset were the same. one to six representative clones of each otu were selected for sequencing in the reverse direction in order to assemble near full-length 16s rrna gene sequences. of the assembled sequences, three were removed from the analyses because of poor sequence quality and two because of indications of chimeric origin. the remaining near full-length planctomycete 16s rrna gene sequences have been deposited to genbank under the accession numbers hm to hm and the sequence of the p isolate under hm the clone libraries from february, july and september showed considerable overlap in otu composition. the july library had the lowest otu richness and consisted of a subset of the otus detected in the other two libraries. the highest otu richness and the most unique otus were found in february. september was intermediate in otu richness and the number of unique otus. the diversity of the three clone libraries is illustrated in figure using rarefaction curves showing the expected number of otus encountered with clone sampling effort. july displays a near asymptotic curve, indicating low diversity, while september is intermediate and february displays the highest diversity. the shannon diversity index and the chao richness estimates for the clone libraries show the same relative diversity pattern. the phylogenetic analysis of the near full-length sequences obtained in this study and other planctomycete sequences obtained from the silva reference database revealed that highly divergent lineages of the planctomycetes phylum are represented in kelp surface biofilms. the kelp surface biofilm clone sequences appear to cluster within five major lineages that have been labeled as: "rb1" and "rb2", rhodopirellula, planctomyces and "om190". the "rb1" and "rb2" lineages appear more closely related to the rhodopirellula and blastopirellula genera than to the pirellula genus and were given their labels based on that. yet the phylogenetic analyses do not place them consistently with either of the genera. sequence similarities of 86-90% to rhodopirellula baltica and blastopirellula marina indicate that they probably represent distinct phylogenetic lineages that could correspond to new genera according to conventional taxonomical practice. the "rb1" lineage was by far the most represented in all three clone libraries. sequences that cluster within the "rb2", rhodopirellula and planctomyces lineages were only represented in september and february, indicating a seasonal difference, while om representatives were present at low numbers in all three clone libraries. discussion to our knowledge, the kelp surface biofilms investigated in this study display the highest proportion of bacteria belonging to planctomycetes reported in a natural bacterial community so far. this observation is consistent with earlier results from a dgge based study on seasonal variation of laminaria hyperborea surface biofilm communities. other habitats where a high abundance of planctomycetes has been reported include seawater during a diatom bloom where planctomycetes related to pirellula were detected attached to diatom cells and were among the dominant lineages in the bloom samples. in investigations of sandy sediments containing algal cells, planctomycetes were also abundant, accounting for up to 20% of total cells, accompanied by cytophaga/flavobacteria. gade and co-workers used order-, genus- and strain specific fish probes to detect planctomycetes in a range of aquatic habitats and recorded abundances up to 11% of total cells in some lakes. peat bogs with sphagnum moss have also been reported to harbor abundant planctomycete populations. similarly to kelp surfaces, these environments are all highly influenced by photosynthetic eukaryotes. the studies mentioned above have all quantified planctomycetes using specific fish probes. several other studies have detected planctomycetes using pcr based cloning and fingerprinting methods followed by sequencing. two of the most frequently used general bacterial pcr primers, targeting the 16s rrna gene around e. coli positions 8- and 338- contain mismatches against planctomycete sequences. this may have caused planctomycete abundances to be underestimated in many habitats, leading investigators to turn their attention towards bacterial groups that appear more abundant. despite awareness of this problem, the literature and the sequence databases probably reflect a tradition of neglect towards the planctomycetes. in the light of this, it is difficult to say whether the dominance of planctomycetes on laminaria hyperborea surface biofilms represents a unique feature of this habitat, or if other planctomycete-dominated bacterial communities have been overlooked until now. for example, staufenberger and co-workers did not detect planctomycetes in surface biofilms of another species of kelp using general bacterial primers for cloning and dgge analysis. yet, use of different primers has let to the detection of planctomycetes on both the kelps s. latissima and laminaria digitata. a possible explanation for the suitability of kelp as a habitat for planctomycetes is its content of sulfated polysaccharides, a class of molecules that some marine planctomycetes are known for being able to degrade. for example, laminaria hyperborea contains fucoidan, a class of complex brown algal sulfated polysaccharides. these substances are secreted to the surface of l. hyperborea via mucilage channels. it is reasonable to assume that planctomycetes living on kelp surfaces utilize substances produced by the kelp, for example fucoidan, as carbon sources. however, the presence of suitable carbon sources appears insufficient to explain the observed dominance of planctomycetes, as they must not only be able to grow and divide, but also outcompete other bacteria to be successful. another contributing factor to the success of planctomycetes on kelp surfaces may be resistance to chemical antimicrobial defense compounds produced by the kelp. antibacterial activity has been detected in extracts from many species of kelp, yet the substances responsible for the activity have often not been identified. the lack of peptidoglycan in planctomycete cell walls makes them resistant to conventional cell wall targeting antibiotics like ampicillin. resistance to other antibiotics, targeting for example protein synthesis has also been reported in some marine planctomycetes. in many cases the reference sequences that are the most closely related to kelp surface planctomycetes are obtained from other marine eukaryotes such as for example red and green seaweeds, corals, crustaceans and sponges. the frequent association of planctomycetes to eukaryotes has previously been noted. this could point to a general lifestyle pattern of heterotrophic planctomycetes. they may combine an affinity for sulfated polysaccharides and other polymeric carbon molecules produced by their eukaryote hosts with a resistance to eukaryote chemical defense molecules. the resulting competitive advantage over other bacterial groups that are utilizing the same kind of substrates, for example the bacteroidetes might be one of the keys to the success of planctomycetes in a wide variety of environments on earth. our results show differences between the different sampling times, in planctomycete abundance, otu composition and diversity. for example, in february there is a relatively low abundance of planctomycetes compared to july and september. this may be linked to the age of the kelp tissue, as the kelp lamina is older in february compared to in july and september due to the seasonal growth cycle of the kelp. aging of the kelp tissue could be associated with lowered antibacterial chemical defense by the kelp, as the old kelp lamina is to be shed soon after february, and does therefore not need to be defended against microbial colonization. without the presence of chemical defense substances, the planctomycetes could loose their competitive advantage over other bacterial groups, explaining their lower abundance in february. the senescence of the kelp tissue as it ages could also cause the appearance of new niches involved in degradation of different kelp constituents, thereby enabling the more diverse planctomycete communities that are observed in february compared to july and september. among the different planctomycete lineages that are represented on the kelp, the lineage defined as "rb1" in this study appears to be the most abundant, accounting for a majority of the clones at all sampling times. the high abundance of rb planctomycetes may thus be the cause of the observed dominance of planctomycetes on kelp surfaces. their high abundance implies a lifestyle that makes them particularly successful on kelp surfaces. yet the lineage also includes reference sequences from a variety of other marine habitats, indicating that rb is not a kelp-specific lineage. the rb and rb lineages, defined in this study, are clearly related to the "pirellulae", a lineage including the genera pirellula, rhodopirellula and blastopirellula. yet our phylogenetic analyses did not place them reliably with any of the described genera, indicated by the bootstrap support for the relevant branches in figure there are no sequences of cultured strains within the rb and rb lineages available in the databases. another uncultured lineage, the so-called om planctomycetes is also represented by clones from kelp surfaces at all sampling times, yet in low numbers. this is a lineage that appears to branch off deeply in the planctomycete tree and representatives have been detected in a variety of environments including seawater, soil and marine eukaryotes. these lineages have yet to be cultured and described and will reveal valuable information on planctomycete metabolism and evolution if cultivation is successful. using conventional approaches, the rhodopirellula sp. strain p could easily be isolated. several closely related strains have been brought into culture earlier. however, the 16s rrna gene sequence of p does not correspond to any of the abundant otus detected on the kelp surfaces, for example within the rb lineage. kelp surfaces are nevertheless a promising source for isolation of novel planctomycete strains, using more ambitious and creative approaches that take into account the environmental factors experienced by bacteria on kelp surfaces. the rewards awaiting such attempts can be substantial, given the representation of highly divergent lineages of the planctomycete tree in kelp surface biofilms.  kelp surface biofilms have a uniquely high relative abundance of planctomycetes. several distinct lineages are represented, and the diversity and composition of the planctomycetes change during the year, probably influenced by aging of the kelp tissue. the finding of abundant planctomycete populations in kelp surface biofilms agrees well with the view of heterotrophic planctomycetes as surface attached, specialized degraders of sulfated polysaccharides in the marine environment, as kelps are known to produce such substances. furthermore, we wish to extend this view by hypothesizing that many heterotrophic planctomycetes share a preference of intimate coexistence with eukaryotes, which may be linked to antibiotic resistance. the study addresses the urgent need for more detailed, quantitative knowledge on the diverse marine planctomycetes. 
biofilms on surfaces of l. hyperborea display an even higher proportion of planctomycetes compared to other investigated planctomycete-rich habitats such as open water, sandy sediments and peat bogs. the findings agree well with the hypothesis of the role of planctomycetes as degraders of sulfated polymeric carbon in the marine environment as kelps produce such substances. in addition, the abundant planctomycete populations on kelp surfaces and in association with other eukaryotes suggest that coexistence with eukaryotes may be a key feature of many planctomycete lifestyles. we aimed to make a detailed investigation of the abundance and phylogenetic diversity of planctomycetes inhabiting these kelp surfaces. one planctomycete strain that was closely related to rhodopirellula baltica was isolated using selective cultivation techniques. their ecological roles in these habitats are still poorly understood. planctomycetes accounted for 51-53% of the bacterial biofilm cells in july and september and 24% in february according to fluorescence in situ hybridization results. in contrast to the abundance, the diversity of the planctomycete populations increased from july to february and was probably influenced by the aging of the kelp tissue. bacteria belonging to planctomycetes display several unique morphological and genetic features and are found in a wide variety of habitats on earth.
 bacteria belonging to planctomycetes display several unique morphological and genetic features and are found in a wide variety of habitats on earth. their ecological roles in these habitats are still poorly understood. planctomycetes have previously been detected throughout the year on surfaces of the kelp laminaria hyperborea from southwestern norway. we aimed to make a detailed investigation of the abundance and phylogenetic diversity of planctomycetes inhabiting these kelp surfaces.  planctomycetes accounted for 51-53% of the bacterial biofilm cells in july and september and 24% in february according to fluorescence in situ hybridization results. several separate planctomycetes lineages within pirellulae, planctomyces and om were represented in 16s rrna gene clone libraries and the most abundant clones belonged to yet uncultured lineages. in contrast to the abundance, the diversity of the planctomycete populations increased from july to february and was probably influenced by the aging of the kelp tissue. one planctomycete strain that was closely related to rhodopirellula baltica was isolated using selective cultivation techniques.  biofilms on surfaces of l. hyperborea display an even higher proportion of planctomycetes compared to other investigated planctomycete-rich habitats such as open water, sandy sediments and peat bogs. the findings agree well with the hypothesis of the role of planctomycetes as degraders of sulfated polymeric carbon in the marine environment as kelps produce such substances. in addition, the abundant planctomycete populations on kelp surfaces and in association with other eukaryotes suggest that coexistence with eukaryotes may be a key feature of many planctomycete lifestyles. 