Alga-derived substrates select for distinct Betaproteobacterial lineages and contribute to niche separation in Limnohabitans strains

We examined the proportions of major Betaproteobacteria subgroups within bacterial communities in diverse nonaxenic, monospecific cultures of algae or cyanobacteria: four species of cryptophyta (genera Cryptomonas and Rhodomonas), four species of chlorophyta (genera Pediastrum, Staurastrum, and Chlamydomonas), and two species of cyanobacteria (genera Dolichospermum and Aphanizomenon). In the cryptophyta cultures, Betaproteobacteria represented 48 to 71% of total bacteria, the genus Limnohabitans represented 18 to 26%, and the Polynucleobacter B subcluster represented 5 to 16%. In the taxonomically diverse chlorophyta group, the genus Limnohabitans accounted for 7 to 45% of total bacteria. In contrast, cyanobacterial cultures contained significantly lower proportions of the Limnohabitans bacteria (1 to 3% of the total) than the cryptophyta and chlorophyta cultures. Notably, largely absent in all of the cultures was Polynucleobacter necessarius (Polynucleobacter C subcluster). Subsequently, we examined the growth of Limnohabitans strains in the presence of different algae or their extracellular products (EPP). Two strains, affiliated with Limnohabitans planktonicus and Limnohabitans parvus, were separately inoculated into axenic cultures of three algal species growing in an inorganic medium: Cryptomonas sp., Chlamydomonas noctigama, and Pediastrum boryanum. The Limnohabitans strains cocultured with these algae or inoculated into their EPP consistently showed (i) pronounced population growth compared to the control without the algae or EPP and (ii) stronger growth stimulation of L. planktonicus than of L. parvus. Overall, growth responses of the Limnohabitans strains cultured with algae were highly species specific, which suggests a pronounced niche separation between two closely related Limnohabitans species likely mediated by different abilities to utilize the substrates produced by different algal species.     

Complete genome sequence of Polynucleobacter necessarius subsp. asymbioticus type strain (QLW-P1DMWA-1(T))

Polynucleobacter necessarius subsp. asymbioticus strain QLW-P1DMWA-1(T) is a planktonic freshwater bacterium affiliated with the family Burkholderiaceae (class Betaproteobacteria). This strain is of interest because it represents a subspecies with cosmopolitan and ubiquitous distribution in standing freshwater systems. The 16S-23S ITS genotype represented by the sequenced strain comprised on average more than 10% of bacterioplankton in its home habitat. While all strains of the subspecies P. necessarius asymbioticus are free-living freshwater bacteria, strains belonging to the only other subspecies, P. necessarius subsp. necessarius are obligate endosymbionts of the ciliate Euplotes aediculatus. The two subspecies of P. necessarius are the instances of two closely related subspecies that differ in their lifestyle (free-living vs. obligate endosymbiont), and they are the only members of the genus Polynucleobacter with completely sequenced genomes. Here we describe the features of P. necessarius subsp. asymbioticus, together with the complete genome sequence and annotation. The 2,159,490 bp long chromosome with a total of 2,088 protein-coding and 48 RNA genes is the first completed genome sequence of the genus Polynucleobacter to be published and was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2006.     

Ubiquity of Polynucleobacter necessarius subspecies asymbioticus results from ecological diversification

The subspecies Polynucleobacter necessarius asymbioticus (> 99% 16S rRNA similarity) has a cosmopolitan distribution and a ubiquitous occurrence in lentic freshwater habitats. We tested if the observed ubiquity of these free-living planktonic freshwater bacteria results from a euryoecious (generalist) adaptation of P. n. asymbioticus strains, or from ecological diversification within the subspecies. We developed a reverse line blot hybridization assay enabling the cultivation-independent detection of 13 groups within the subspecies in environmental samples. A set of 121 lentic freshwater habitats, spanning a broad variety of habitat types (e.g. pH levels ranging from 3.8 to 8.5) was investigated for the presence of these 13 P. n. asymbioticus groups. Statistical analyses of the reverse line blot hybridization detections revealed pronounced differences in habitat preferences of several of the groups. Their preferences differed regarding pH, conductivity, dissolved organic carbon and oxygen concentration of habitats. For some groups, differences in environmental preferences resulted even in complete niche separation between them. The revealed differences in habitat preferences suggest that the previously reported ubiquity of P. n. asymbioticus results from ecological diversification within the taxon and not from generalist adaptation of strains.     

Isolation of strains belonging to the cosmopolitan Polynucleobacter necessarius cluster from freshwater habitats located in three climatic zones

More than 40 bacterial strains belonging to the cosmopolitan Polynucleobacter necessarius cluster (Betaproteobacteria) were isolated from a broad spectrum of freshwater habitats located in three climatic zones. Sequences affiliated with the freshwater P. necessarius cluster are among the most frequently detected in studies on bacterial diversity in freshwater ecosystems. Despite this frequent detection with culture-independent techniques and the cosmopolitan occurrence of members affiliated with this cluster, no isolates have been reported thus far. The isolated strains have been obtained from lakes, ponds, and rivers in central Europe, the People's Republic of China, and East Africa by use of the filtration-acclimatization method. The 16S rRNA gene sequences of the isolates are 98.8 to 100% identical to reference sequences obtained by various authors by use of culture-independent methods. The isolates, aerobic heterotrophs, grew on a wide range of standard complex media and formed visible colonies on agar plates. Thus, the previous lack of isolates cannot be explained by a lack of appropriate media. Most of the isolates possess, under a wide range of culture conditions, very small cells (<0.1 micro m(3)), even when grown in medium containing high concentrations of organic substances. Thus, these strains are obligate ultramicrobacteria. The obtained strains have a C-shaped cell morphology which is very similar to that of recently isolated ultramicrobacterial Luna cluster strains (Actinobacteria) and the SAR11 cluster strains (Alphaproteobacteria).     

Substrate incorporation patterns of bacterioplankton populations in stratified and mixed waters of a humic lake

Bacterial incorporation of glucose, leucine, acetate and 4-hydroxybenzoic acid (HBA) was investigated in an artificially divided humic lake (Grosse Fuchskuhle, Germany). Two basins with contrasting influx of allochthonous organic carbon were sampled during late summer stratification (oxic and anoxic layers) and after autumn mixing. High total and cell-specific incorporation rates were observed for glucose and HBA in stratified and mixed waters respectively, but only a small fraction of bacteria visibly incorporated HBA. The oxic layer of the more humic-rich basin featured a significantly lower fraction of glucose incorporating cells and substantially higher proportions of acetate assimilating bacteria. Niche differentiation was observed in two betaproteobacterial populations: cells affiliated with the Polynucleobacter C subcluster efficiently incorporated acetate but little glucose, whereas the opposite was found for members of the R-BT065 clade. By contrast, leucine incorporation was variable in both taxa. Considering the high concentrations and rapid photochemical generation of organic acids in humic waters our results may help to explain the success of the Polynucleobacter C lineage in such habitats. Specific substrate or habitat preferences were also present in three subgroups of the actinobacterial acI lineage: The numerically dominant clade in oxic waters (acI-840-1) was absent in the anoxic zone and did not incorporate acetate. A second group (acI-840-2) was found both in the epi- and hypolimnion, whereas the third one (acI-840-3) only occurred in anoxic waters. Altogether our results suggest a constitutive preference for some substrates versus an adaptive utilization of others in the studied microbial groups.     

Polynucleobacter necessarius, an obligate bacterial endosymbiont of the hypotrichous ciliate Euplotes aediculatus, is a member of the β-subclass of Proteobacteria

Abstract An almost full length 16S rRNA gene of the obligate bacterial endosymbiont Polynucleobacter necessarius was amplified using the polymerase chain reaction in combination with site-specific primers. The amplified DNA was directly sequenced and compared with other bacterial 16S rRNA sequences. P. necessarius belongs to the β-subclass of Proteobacteria and shows the closest relationship to Alcaligenes eutrophus, Burkholderia solanacearum , and B. pickettii . In situ hybridization with a specific oligonucleotide probe corroborated the assignment of the retrieved sequence to P. necessarius .     

Betaproteobacterial symbionts of the ciliate Euplotes: origin and tangled evolutionary path of an obligate microbial association

The Polynucleobacter-Euplotes association is an obligatory symbiotic system between a monophyletic group of ciliate species belonging to the genus Euplotes and bacteria of the species Polynucleobacter necessarius (Betaproteobacteria). Both organisms are unable to survive independently. Several studies revealed the existence of free-living populations of Polynucleobacter bacteria which are phylogenetically closely related to the endosymbiotic ones, but never share associations with Euplotes in the natural environment. Hence, following the most parsimonious explanation on the origin of the association, this symbiosis should represent a synapomorphic character for the hosts' clade. Nevertheless, phylogenetic analyses performed on an increased number of strains here presented suggest that Euplotes species, during their evolution, recruited Polynucleobacter bacteria as symbionts more than once. Moreover, in three cases, we observed different bacteria as obligate symbionts. These symbionts are the first characterized representatives of a phylogenetic lineage branching in a basal position with respect to the genus Polynucleobacter. The hypothesis that the original obligate symbionts belonged to this newly discovered clade and that, only subsequently, in most cases they have been replaced by Polynucleobacter bacteria recruited from the environment is proposed and discussed. The evolutionary path of this association seems anyway to have been more complex than so far supposed.     

Ubiquity of Polynucleobacter necessarius ssp. asymbioticus in lentic freshwater habitats of a heterogenous 2000 km2 area

We present a survey on the distribution and habitat range of Polynucleobacter necessarius ssp. asymbioticus (PnecC), a numerically and functionally important taxon in the plankton of freshwater systems. We systematically sampled stagnant freshwater habitats in a heterogeneous 2000 km² area, together with ecologically different habitats outside this area. In total, 137 lakes, ponds and puddles were investigated, which represent an enormous diversity of habitats differing, e.g. in depth (< 10 cm – 171 m) and pH (3.9–8.5). PnecC bacteria were detected by cultivation‐independent methods in all investigated habitats, and their presence was confirmed by cultivation of strains from selected habitats representing the whole studied ecological range. The determined relative abundance of the subspecies ranged from values close to the detection limit of FISH (0.2%) to 67% (average 14.5%), and the highest observed absolute abundance was 5.3 × 10⁶ cells ml^?1. Statistical analyses revealed that the abundance of PnecC bacteria was partially controlled by factors linked to concentrations of humic substances, which support the hypothesis that these bacteria utilize photodegradation products of humic substances. Based on the revealed statistical relationships, an average relative abundance of this subspecies of 20% in global freshwater habitats was extrapolated. Our study provides important implications for the current debate on ubiquity and biogeography in microorganisms.     

Temporal and spatial variability in nearshore bacterioplankton communities of Lake Michigan

The spatial and temporal variability of bacterial communities were determined for the nearshore waters of Lake Michigan, an oligotrophic freshwater inland sea. A freshwater estuary and nearshore sites were compared six times during 2006 using denaturing gradient gel electrophoresis (DGGE). Bacterial composition clustered by individual site and date rather than by depth. Seven 16S rRNA gene clone libraries were constructed, yielding 2717 bacterial sequences. Spatial variability was detected among the DGGE banding patterns and supported by clone library composition. The clone libraries from deep waters and the estuary environment revealed highest overall bacterial diversity. Betaproteobacteria sequence types were the most dominant taxa, comprising 40.2–67.7% of the clone libraries. BAL 47 was the most abundant freshwater cluster of Betaproteobacteria , indicating widespread distribution of this cluster in the nearshore waters of Lake Michigan. Incertae sedis 5 and Oxalobacteraceae sequence types were prevalent in each clone library, displaying more diversity than previously described in other freshwater environments. Among the Oxalobacteraceae sequences, a globally distributed freshwater cluster was determined. The nearshore waters of Lake Michigan are a dynamic environment that experience forces similar to the coastal ocean environment and share common bacterial diversity with other freshwater habitats.     

Differences in structure and dynamics of Polynucleobacter communities in a temperate and a subtropical lake, revealed at three phylogenetic levels

The results of ecological studies investigating bacteria by cultivation-independent methods are expected to be influenced by the phylogenetic resolution of the applied molecular tools. This potential influence was investigated in a comparative community study on Polynucleobacter cluster bacteria (Betaproteobacteria) inhabiting the pelagic of the large, shallow, partially hypertrophic Taihu Lake located in subtropical East Asia, and the deep oligo-mesotrophic Lake Mondsee located in temperate Central Europe. The two contrasting habitats were sampled over the same period of 12 months. The community dynamics were investigated at three phylogenetic levels by fluorescent in situ hybridizations with a set of nested probes specific for the beta II clade (including the Polynucleobacter cluster), the genus-like monophyletic Polynucleobacter cluster, and four species-like subclusters. Subcluster B was the numerically dominating subcluster in both lakes over the investigation periods, but demonstrated different population dynamics in the two habitats. Interhabitat comparisons of the Polynucleobacter community structure in the two lakes in the study, and a previously investigated acidic pond indicated ecological diversification within the phylogenetically narrow Polynucleobacter cluster. These results could be obtained by help of the subcluster-specific probes, but would have been missed with probes of a lower phylogenetic resolution.     

Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia)

We have found a Polynucleobacter bacterium in the cytoplasm of Euplotes harpa, a species living in a brackish-water habitat, with a cirral pattern not corresponding to that of the freshwater Euplotes species known to harbor this type of bacteria. The symbiont has been found in three strains of the species, obtained by clonal cultures from ciliates collected in different geographic regions. The 16S rRNA gene sequence of this bacterium identifies it as a member of the beta-proteobacterial genus Polynucleobacter. This sequence shares a high similarity value (98.4-98.5%) with P. necessarius, the type species of the genus, and is associated with 16S rRNA gene sequences of environmental clones and bacterial strains included in the Polynucleobacter cluster (>95%). An oligonucleotide probe was designed to corroborate the assignment of the retrieved sequence to the symbiont and to detect similar bacteria rapidly. Antibiotic experiments showed that the elimination of the bacteria stops the reproductive cycle in E. harpa, as has been shown for the freshwater Euplotes species.     

Ecophysiological differences of betaproteobacterial populations in two hydrochemically distinct compartments of a subtropical lagoon

We studied the population sizes and substrate incorporation patterns of three phylogenetic groups of Betaproteobacteria in a coastal subtropical lagoon that is characterized by a sharp transition from humic freshwater to turbid brackish water. Various cellular processes were addressed by short-term incubations with four radiolabelled compounds and microautoradiographic assessment of substrate incorporation. Group-specific differences in the abundances and the respective physiological state of the three populations were observed upon transfer from the humic-rich compartment to the main body of the lagoon (estimated at 1–2 days). Members of the clade B of Polynucleobacter (PnecB) experienced only an insignificant change in cell numbers, but displayed a general metabolic downshift, carbon metabolism (glucose incorporation) being most affected. By contrast, bacteria from the closely related Polynucleobacter C clade (PnecC) clearly differed in total abundances and in the numbers of DNA-synthesizing or glucose incorporating cells. At the same time, PnecC bacteria maintained comparable levels of protein synthesis (leucine uptake) in both lagoon compartments, and the proportion of cells incorporating N -acetylglucosamine was even higher in the main body of the lagoon. Members of the R-BT lineage showed little changes in cell numbers, DNA synthesis and carbon metabolism. Altogether, the observed patterns of substrate metabolism suggest that different bacterial populations in the lagoon undergo specific physiological adjustments in response to changing environmental conditions.     

Spatio-temporal niche separation of planktonic Betaproteobacteria in an oligo-mesotrophic lake

We investigated the diversity of planktonic Betaproteobacteria and the seasonal population changes of betaproteobacterial taxa in an oligo-mesotrophic lake (Piburger See, Austria). Focus was put on the vertical distribution of the investigated populations and on differences between their respective cell fractions with apparent amino acid incorporation. On average, 66% of betaproteobacterial cells and 73% of their diversity could be attributed to four clades within three lineages that were further analysed by fluorescence in situ hybridization. The numbers of bacteria from the R-BT subclade of the beta I lineage and from the PnecB subgroup of the beta II lineage were rather constant throughout the water column. In contrast, members of another subgroup of beta II (PnecC) and bacteria related to Methylophilus (beta IV) were particularly numerous in the oxygen-depleted zone. In general, only moderate seasonal changes in abundance were observed in the upper water layers, whereas there was a clear relationship between decreasing oxygen levels and the rise of bacteria from the PnecC and beta IV clades in deeper strata. On average, almost 80% of beta I bacteria, but < 15% of cells from the beta IV clade, showed amino acid incorporation. Our results suggest that the studied populations occupy distinct vertical and ecophysiological niches in Piburger See.     

Suboptimal light conditions negatively affect the heterotrophy of Planktothrix rubescens but are beneficial for accompanying Limnohabitans spp

We examined the effect of light on the heterotrophic activity of the filamentous cyanobacterium Planktothrix rubescens and on its relationship with the accompanying bacteria. In situ leucine uptake by bacteria and cyanobacteria was determined in a subalpine mesotrophic lake, and natural assemblages from the zone of maximal P. rubescens abundances were incubated for 2 days at contrasting light regimes (ambient, 100× increased, dark). Planktothrix rubescens from the photic zone of the lake incorporated substantially more leucine, but some heterotrophic activity was maintained in filaments from the hypolimnion. Exposure of cyanobacteria to increased irradiance or darkness resulted in significantly lower leucine incorporation than at ambient light conditions. Highest abundances and leucine uptake of Betaproteobacteria from the genus Limnohabitans were found in the accompanying microflora at suboptimal irradiance levels for P. rubescens or in dark incubations. Therefore, two Limnohabitans strains (representing different species) were co-cultured with axenic P. rubescens at different light conditions. The abundances and leucine incorporation rates of both strains most strongly increased at elevated irradiance levels, in parallel to a decrease of photosynthetic pigment fluorescence and the fragmentation of cyanobacterial filaments. Our results suggest that Limnohabitans spp. in lakes might profit from the presence of physiologically stressed P. rubescens.     

环梅山岛海域春季浮游古菌群落空间分布特征研究

[目的]海洋浮游古菌是生物地球化学循环的关键驱动者,但其在近岸海域的水平空间分布特征还未被充分了解.本研究以与陆地紧密相连的环梅山岛海域为例研究浮游古菌在海陆过渡带的水平分布模式.[方法]利用16S rRNA基因扩增子测序,以期从优势类群分布、群落组成变化和物种共现模式3个层面揭示梅山湾潟湖区和临近海域春季浮游古菌的空...     

Imported fire ants near the edge of their range: disturbance and moisture determine prevalence and impact of an invasive social insect

1.?Habitat disturbance and species invasions interact in natural systems, making it difficult to isolate the primary cause of ecosystem degradation. A general understanding requires case studies of how disturbance and invasion interact across a variety of ecosystem - invasive species combinations. 2.?Dramatic losses in ant diversity followed the invasion of central Texas by red imported fire ants (Solenopsis invicta). However, recent manipulative studies in Florida revealed no effect on ant diversity following the removal of S.?invicta from a disturbed pasture habitat, but moderate loss of diversity associated with their introduction into undisturbed habitat and no invasion occurred without disturbance. Thus, the importance of S.?invicta in driving diversity loss and its ability to invade undisturbed systems is unresolved. 3.?We examine the distribution and abundance of a large monogyne S.?invicta population and its association with the co-occurring ant assemblage at a site in south Texas close to the aridity tolerance limit of S.?invicta. 4.?We document that moisture modulates S.?invicta densities. Further, soil disturbing habitat manipulations greatly increase S.?invicta population densities. However, S.?invicta penetrates all habitats regardless of soil disturbance history. In contrast, controlled burns depress S.?invicta densities. 5.?In habitats where S.?invicta is prevalent, it completely replaces native fire ants. However, S.?invicta impacts native ants as a whole less strongly. Intriguingly, native ants responded distinctly to S.?invicta in different environments. In wet, undisturbed environments, high S.?invicta abundance disrupts the spatial structure of the ant assemblage by increasing clumping and is associated with reduced species density, while in dry-disturbed habitats, sites with high S.?invicta abundance possess high numbers of native species. Analyses of co-occurrence indicate that reduced species density in wet-undisturbed sites arises from negative species interactions between native ants and S.?invicta. However, these same data suggest that the high native species density of abundant S.?invicta sites in dry-disturbed environments does not result from facilitation. 6.?Monogyne S.?invicta populations play different roles in different environments, driving ant diversity loss in some, but being largely symptomatic of habitat disturbance in others.     

Parallel evolutionary patterns in multiple lineages of arctic Artemisia L. (Asteraceae)

Early observers of plant evolution in the Arctic have noted a floristic similarity with temperate alpine regions and a predominance of high ploidy levels. The aim of our study was to survey these and other traits in multiple closely related but independently evolved lineages of Artemisia. Our phylogenetic study was based on 133 taxa using 3'-ETS and ITS, and on data on morphology, karyology, distribution, and ecological preferences. We compared Arctic lineages with sister groups and tested whether patterns were significantly different. We found: (1) Artemisia has independently adapted to Arctic habitats 13-18 times; (2) There were no ecological preferences of putative progenitors that might determine the colonization success in the Arctic, although most sister groups were centered in steppe habitats; (3) Plant height was distinctly reduced in Arctic lineages; (4) Arctic lineages contained no more polyploids than their respective sister groups or taxa from other habitats; (5) Enlarged flower heads have evolved repeatedly, probably for better pollinator attraction. This strategy could be a substitute for polyploidy, which is typical in other Arctic taxa. Stronger pollinator attraction should result in better outcrossing and higher heterozygosity in the offspring, which is among the main effects of polyploidy.     

The micro-distribution of aquatic macroinvertebrates in the Wye river system: the result of abiotic or biotic factors?

SUMMARY. 1. Macroinvertebrates were sampled from three habitats at each of forty-five sites in the catchment of the Welsh River Wye. Micro-distribution patterns between three habitat types were assessed for those species which occurred most commonly.
2. Of the fifty-three taxa for which analysis was possible, twenty showed a significant association with habitat type. Eleven occur mostly in margins, eight mostly in riffles, and one in flats (depositing reaches).
3. Twenty-one taxa, including eleven additional to those show significant associations, showed. significant differences between habitats in their relative abundance. Ten were most abundant in margins, eight in riffles and three in flats.
4. Factors possibly responsible for these habitat preferences are discussed.     

Factors affecting the distribution of stream macroinvertebrates in geothermal areas: Taupo Volcanic Zone,New Zealand

The distribution of macroinvertebrates was investigated among sites within five geothermally influenced and two non- or minimally-influenced streams in the Taupo Volcanic Zone, New Zealand, to examine the responses of communities to broad environmental gradients within and among habitats. To date, examination of geothermal stream macroinvertebrates has typically been from single habitats, and has not been examined over a regional scale. Sites within and among streams represented a range of sizes, depths, water velocities and substrate types. Sites with little or no geothermal influence typically had temperatures less than 15°C and pH between 5 and 8. Four of the geothermally influenced streams had temperatures greater than 25°C, and ranged from alkaline (pH 9.1) to highly acidic (pH 3.0). Most taxa recorded were typical inhabitants of non-geothermal streams that are tolerant to elevated temperatures, extreme pH conditions and/or high toxicant levels. Diptera, Coleoptera and Mollusca dominated geothermal sites, and Ephemeroptera, Plecoptera and Trichoptera were absent. In addition, an obligate dweller of geothermal habitats, Ephydrella thermarum, and a nonindigenous tropical gastropod, Melanoides tuberculata, were recorded. Canonical correspondence analysis implicated multiple factors in determining the distribution of invertebrates over the region. Overall, distribution was most strongly associated with temperature gradients, particularly longitudinally within streams. This distribution was likely directly related to species temperature tolerances, but also temperature effects on algal abundance and composition, its differential effects on species’ potential competitors and predators, and its effects on toxicant availability. Differences in invertebrate composition among streams were associated with major differences in pH and substrate. Increased acidity was associated with a significant decrease in invertebrate taxa richness, with acidic sites having a limited fauna dominated by dipterans (e.g., Naonella sp., Polypedilum sp.). Stability of flow and environmental conditions may enhance competitive interactions among taxa, enhancing the importance of substrate type in these systems. The presence of non-indigenous species (e.g., M. tuberculata, Poecilia reticulata), currently limited in distribution, also affected species composition. Overall, communities become less speciose, although more unusual in composition, with increased geothermal influence. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: K. Martens