Genetic and morphological analyses of tub gurnard Chelidonichthys lucerna populations in Turkish marine waters

Genetic and morphological structure of tub gurnard Chelidonichthys lucerna populations in Turkish marine waters were investigated with mtDNA sequencing of 16S rRNA and morphological characters. C. lucerna samples were collected from the Black Sea, Marmara, Aegean and northeastern Mediterranean coasts of Turkey. The lowest genetic diversity was found in the northeastern Mediterranean (Iskenderun Bay) population, while the highest was in the Marmara population with overall average value of genetic diversity within populations. A total of 14 haplotypes was found, and the highest haplotype diversity was in the Black Sea whereas the lowest was in the northeastern Mediterranean population (Iskenderun Bay). The Black Sea and Iskenderun Bay populations showed the least genetic divergence (0.001081), while the highest was between the Marmara Sea and northeastern Mediterranean (Antalya Bay) populations (0.002067). Pairwise comparisons of genetic distance revealed statistically significant differences (P < 0.05) between the Marmara and both the Aegean and northeastern Mediterranean (Antalya Bay) samples. Neighbour joining tree analyses clustered the northeastern Mediterranean populations (Antalya Bay and Iskenderun Bay) as genetically more interrelated populations, whereas the Aegean Sea population was clustered as most isolated one. Discriminant function analysis of morphological characters showed that only the Black Sea population is differentiated from the other populations.     

<Emphasis Type="Italic">Salinispora</Emphasis><Emphasis Type="Italic">arenicola</Emphasis> from temperate marine sediments: new intra-species variations and atypical distribution of secondary metabolic genes

The obligate marine actinobacterium Salinispora arenicola was successfully cultured from temperate sediments of the Pacific Ocean (Tosa Bay, offshore Kochi Prefecture, Japan) with the highest latitude of 33°N ever reported for this genus. Based on 16S rRNA gene sequence analysis, the Tosa Bay strains are of the same phylotype as the type strain S. arenicola NBRC105043. However, sequence analysis of their 16S-23S rRNA intergenic spacer (ITS) revealed novel sequence variations. In total, five new ITS sequences were discovered and further phylogenetic analyses using gyrase B and rifamycin ketosynthase (KS) domain sequences supported the phylogenetic diversity of the novel Salinispora isolates. Screening of secondary metabolite genes in these strains revealed the presence of KS1 domain sequences previously reported in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. Moreover, salinosporamide biosynthetic genes, which are highly homologous to those of Bahamas-endemic S. tropica, were detected in several Tosa Bay isolates, making this report the first detection of salinosporamide genes in S. arenicola. The results of this study provide evidence of a much wider geographical distribution and secondary metabolism diversity in this genus than previously projected.     

Comparative study of two typing methods, hsp65 PRA and ITS sequencing, revealed a possible evolutionary link between Mycobacterium kansasii type I and II isolates

One hundred and ninety-eight clinical isolates of Mycobacterium kansasii collected between 2003 and 2004 in Japan were genotyped by PCR and restriction enzyme analysis (PRA) and 16S-23S internal transcribed spacer (ITS) sequencing. The results demonstrated that clinical isolates of M. kansasii in Japan are almost exclusively of the type I PRA genotype, as is the case in other countries. Although the results of subtyping using the 16S-23S ITS sequence were generally consistent with subtyping using hsp65 PRA, four strains showed a discrepancy between the two methods. Sequence analysis of the hsp65, gyrB and 16S rRNA genes and the ITS sequence of the four strains suggests that they branched from type II and could be considered an ancestral strain of the type I strain. The newly recognized strains were designated as intermediate type I.     

Molecular genetic delineation of Phaeocystis species (Prymnesiophyceae) using coding and non-coding regions of nuclear and plastid genomes

Sequence variation among 22 isolates representing a global distribution of the prymnesiophyte genus Phaeocystis has been compared using nuclear-encoded 18S rRNA genes and two non-coding regions: the ribosomal DNA internal transcribed spacer 1 (ITS1) separating the 18S rRNA and 5.8S rRNA genes and the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) spacer flanked by short stretches of the adjacent large and small subunits (rbcL and rbcS). 18S rRNA can only resolve major species complexes. The analysis suggests that an undescribed unicellular Phaeocystis sp. (isolate PLY 559) is a sister taxon to the Mediterranean unicellular Phaeocystis jahnii; this clade branched prior to the divergence of all other Phaeocystis species, including the colonial ones. Little divergence was seen among the multiple isolates sequenced from each colonial species complex. RUBISCO spacer regions are even more highly conserved among closely related colonial Phaeocystis species and are identical in Phaeocystis antarctica, Phaeocystis pouchetii and two warm-temperate strains of Phaeocystis globosa, with a single base substitution in two cold-temperate strains of P. globosa. The RUBISCO spacer sequences from two predominantly unicellular Phaeocystis isolates from the Mediterranean Sea and PLY 559 were clearly different from other Phaeocystis strains. In contrast, ITS1 exhibited substantial inter- and intraspecific sequence divergence and showed more resolution among the taxa. Distinctly different copies of the ITS1 region were found in P. globosa, even among cloned DNA from a single strain, suggesting that it is a species complex and making this region unsuitable for phylogenetic analysis in this species. However, among nine P. antarctica strains, four ITS1 haplotypes could be separated. Using the branching order in the ITS1 tree we have attempted to trace the biogeographic history of the dispersal of strains in Antarctic coastal waters.     

Genetic differentiation of Mediterranean horse mackerel (Trachurus mediterraneus) populations as revealed by mtDNA PCR-RFLP analysis

The genetic population structure of Mediterranean horse mackerel, Trachurus mediterraneus , from seven locations throughout the Black, Marmara, Aegean and eastern Mediterranean seas was investigated using restriction fragment length polymorphism (RFLP) analysis of the mtDNA 16S rDNA region. An approximately 2000-bp segment was screened in 280 individuals using six restriction enzymes, resulting in 10 composite haplotypes. The most common haplotype was present in 56.42% individuals; the next most frequent haplotype was present in 22.85% individuals. Average haplotype diversity within samples was moderate (0.38), and nucleotide diversity was low (0.00435). Mean nucleotide divergence for the seven sampling sites was 0.0028. Nucleotide divergence among samples was moderate, with the highest value detected between the Aegean Sea (Izmir) and the eastern Black Sea (Trabzon) populations (0.007055), and the lowest (−0.000043) between the Marmara Sea (Adalar) and the western Black Sea (Sile) populations. In Monte Carlo pairwise comparisons of haplotype frequencies, the Sinop from the middle Black Sea, Trabzon from the eastern Black Sea, and Iskenderun Bay from the north-eastern Mediterranean Sea exhibited highly significant (P   <   0.001) geographical differentiation from each other and from all other populations. Mantel's test indicated that the nucleotide divergence among populations of T. mediterraneus was not significantly associated with their geographical isolation ( r  = −0.2963; P   >   0.05). Consequently, the mtDNA 16S rDNA region provided evidence for the existence of three distinct T. mediterraneus populations (Sinop, Trabzon and Iskenderun Bay) in the Black and north-eastern Mediterranean seas.     

Diversity of environmental Mycobacterium isolates from hemodialysis water as shown by a multigene sequencing approach

Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1, gyrB, hsp65, recA, rpoB, and sodA). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus.     

Resolution of Prochlorococcus and Synechococcus ecotypes by using 16S-23S ribosomal DNA internal transcribed spacer sequences

Cultured isolates of the marine cyanobacteria Prochlorococcus and Synechococcus vary widely in their pigment compositions and growth responses to light and nutrients, yet show greater than 96% identity in their 16S ribosomal DNA (rDNA) sequences. In order to better define the genetic variation that accompanies their physiological diversity, sequences for the 16S-23S rDNA internal transcribed spacer (ITS) region were determined in 32 Prochlorococcus isolates and 25 Synechococcus isolates from around the globe. Each strain examined yielded one ITS sequence that contained two tRNA genes. Dramatic variations in the length and G+C content of the spacer were observed among the strains, particularly among Prochlorococcus strains. Secondary-structure models of the ITS were predicted in order to facilitate alignment of the sequences for phylogenetic analyses. The previously observed division of Prochlorococcus into two ecotypes (called high and low-B/A after their differences in chlorophyll content) were supported, as was the subdivision of the high-B/A ecotype into four genetically distinct clades. ITS-based phylogenies partitioned marine cluster A Synechococcus into six clades, three of which can be associated with a particular phenotype (motility, chromatic adaptation, and lack of phycourobilin). The pattern of sequence divergence within and between clades is suggestive of a mode of evolution driven by adaptive sweeps and implies that each clade represents an ecologically distinct population. Furthermore, many of the clades consist of strains isolated from disparate regions of the world's oceans, implying that they are geographically widely distributed. These results provide further evidence that natural populations of Prochlorococcus and Synechococcus consist of multiple coexisting ecotypes, genetically closely related but physiologically distinct, which may vary in relative abundance with changing environmental conditions.     

Ecotypes of planktonic actinobacteria with identical 16S rRNA genes adapted to thermal niches in temperate, subtropical, and tropical freshwater habitats

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6 degrees C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.     

Diversity of culturable actinobacteria isolated from marine sponge <Emphasis Type="Italic">Haliclona</Emphasis> sp.

This study describes actinobacteria isolated from the marine sponge Haliclona sp. collected in shallow water of the South China Sea. A total of 54 actinobacteria were isolated using media selective for actinobacteria. Species diversity and natural product diversity of isolates from marine sponge Haliclona sp. were analysed. Twenty-four isolates were selected on the basis of their morphology on different media and assigned to the phylum Actinobacteria by a combination of 16S rRNA gene based restriction enzymes digestion and 16S rRNA gene sequence analysis. The 16S rRNA genes of 24 isolates were digested by restriction enzymes TaqI and MspI and assigned to different groups according to their restriction enzyme pattern. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Streptomyces, Nocardiopsis, Micromonospora and Verrucosispora; one other isolate was recovered that does not belong to known genera based on its unique 16S rRNA gene sequence. To our knowledge, this is the first report of a bacterium classified as Verrucosispora sp. that has been isolated from a marine sponge. The majority of the strains tested belong to the genus Streptomyces and three isolates may be new species. All of the 24 isolates were screened for genes encoding polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). PKS and NRPS sequences were detected in more than half of the isolates and the different "PKS-I-PKS-II-NRPS" combinations in different isolates belonging to the same species are indicators of their potential natural product diversity and divergent genetic evolution.     

Phylogenetic diversity of Synechococcus strains isolated from the East China Sea and the East Sea

Phylogenetic relationships among 33 Synechococcus strains isolated from the East China Sea (ECS) and the East Sea (ES) were studied based on 16S rRNA gene sequences and 16S–23S rRNA gene internal transcribed spacer (ITS) sequences. Pigment patterns of the culture strains were also examined. Based on 16S rRNA gene and ITS sequence phylogenies, the Synechococcus isolates were clustered into 10 clades, among which eight were previously identified and two were novel. Half of the culture strains belonged to clade V or VI. All strains that clustered into novel clades exhibited both phycoerythrobilin and phycourobilin. Interestingly, the pigment compositions of isolates belonging to clades V and VI differed from those reported for other oceanic regions. None of the isolates in clade V showed phycourobilin, whereas strains in clade VI exhibited both phycourobilin and phycoerythrobilin, which is in contrast to previous studies. The presence of novel lineages and the different pigment patterns in the ECS and the ES suggests the possibility that some Synechococcus lineages are distributed only in geographically restricted areas and have evolved in these regions. Therefore, further elucidation of the physiological, ecological, and genetic characteristics of the diverse Synechococcus strains is required to understand their spatial and geographical distribution.     

Molecular characterization of Hysterothylacium fabri (Nematoda: Anisakidae) from Zeus faber (Pisces: Zeidae) caught off the Mediterranean coasts of Turkey based on nuclear ribosomal and mitochondrial DNA sequences

In the present study, Hysterothylacium fabri was found in the coasts of the Mediterranean Sea, Turkey and characterized by sequencing of nuclear (internal transcribed spacer, ITS) and mitochondrial (cytochrome c oxidase subunit 2, cox2) markers. Pairwise comparison between the entire ITS fragment including ITS-1, 5.8S, ITS-2 sequences of the H. fabri isolates from the Mediterranean Sea (Turkey, KC852206) and other H. fabri isolates from the South China Sea (JQ520158), the South Korea waters (JX974558) showed differences ranged from 0.1 and 1.1%. With the present study, H. fabri from the Mediterranean Sea was characterized for the first time by sequencing of the cox2 gene.     

Ecotypes of Planktonic Actinobacteria with Identical 16S rRNA Genes Adapted to Thermal Niches in Temperate, Subtropical, and Tropical Freshwater Habitats

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6°C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.     

天津地区气单胞菌分离株的鉴定与多位点序列分型

[目的]研究气单胞菌菌株分类情况,并分析其致病性.[方法]采集环境样品和鱼类标本,分离并鉴定气单胞菌菌株,并运用多位点序列分型(Multilocus sequence typing,MLST)方法进行分类研究,利用PCR和测序方法分析毒力基因Aera、Hly、Aha1、GCAT和Nuc的分布.[结果]通过对分离菌株的16S rRNA基因进行分析,确认属于4种不同气单胞菌的7个分离株.发现所有菌株至少有1种毒力基因阳性,其中3株具有4种毒力基因.药物敏感实验显示,6株分离株对3种或3种以上抗菌素具有多重耐药性.最后,对看家基因gyrB、groL、gltA、metG、ppsA和recA进行分析,与MLST数据库中的等位基因序列比对,发现7株分离株均为新的不同的序列型(Sequence type,ST).[结论]气单胞菌具有较高的遗传多样性.     

Diversity of Salmonella strains isolated from the aquatic environment as determined by serotyping and amplification of the ribosomal DNA spacer regions

Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater. Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonella strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186-194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates.     

Characterization by genotypic methods of symbiotic Nostoc strains isolated from five species of Gunnera

The genetic diversity of ten symbiotic Nostoc strains isolated from different Gunnera species was investigated. The strains were analyzed using molecular methods with different taxonomic resolutions, including restriction fragment length polymorphisms (RFLP) of the PCR-amplified 16S ribosomal gene and the 16S-23S internal transcribed spacer (ITS) region combined with computer-assisted analyses. The functional gene hetR, assigned to heterocyst differentiation, was used for denaturing gradient gel electrophoresis. A high genetic diversity was observed among the isolates even in the conserved gene coding for the small ribosomal unit. No correlation was observed between clustering of cyanobacteria and the host species of Gunnera.     

The gyrB gene is a useful phylogenetic marker for exploring the diversity of Flavobacterium strains isolated from terrestrial and aquatic habitats in Antarctica

Within the phylum Bacteroidetes, the gyrB gene, encoding for the B subunit of the DNA gyrase, has been used as a phylogenetic marker for several genera closely related to Flavobacterium. The phylogenies of the complete 16S rRNA gene and the gyrB gene were compared for 33 Antarctic Flavobacterium isolates and 23 type strains from closely related Flavobacterium species. gyrB gene sequences provided a higher discriminatory power to distinguish between different Flavobacterium groups than 16S rRNA gene sequences. The gyrB gene is therefore a promising molecular marker for elucidating the phylogenetic relationships among Flavobacterium species and should be evaluated for all the other type strains of described Flavobacterium species. Combining the phylogeny of both genes, the new Antarctic Flavobacterium strains constitute 15 Flavobacterium groups, including at least 13 potentially new species together with one group of isolates probably belonging to the species Flavobacterium micromati and one group close to Flavobacterium gelidilacus.     

Prevalence and microdiversity of Alteromonas macleodii-like microorganisms in different oceanic regions

The presence, prevalence and variability of microorganisms related to the species Alteromonas macleodii, a well known culturable gamma-Proteobacterium, has been studied in different seawater samples from diverse geographical locations, in both the Northern and Southern hemispheres, and tested with two molecular techniques (rRNA hybridization and gene cloning and sequencing). Results show that A. macleodii-like microorganisms are present in high proportions in North Atlantic and, especially, Mediterranean waters, being higher at deep samples and particle-associated fractions, in agreement with previous findings. In contrast, Southern samples (all from very cold areas near Antarctica) presented no significant hybridization signals. The analysis of the ribosomal ITS (16S-23S internal transcribed spacers) revealed that A. macleodii-like microorganisms from Mediterranean, North Atlantic, Caribbean and Red Sea waters differed in both size and sequence, mostly depending on their geographical origin, with Mediterranean and North Atlantic clones clustering into two main groups whereas Caribbean and Red Sea clones appeared separated.     

Combined Analyses of the ITS Loci and the Corresponding 16S rRNA Genes Reveal High Micro- and Macrodiversity of SAR11 Populations in the Red Sea

Bacteria belonging to the SAR11 clade are among the most abundant prokaryotes in the pelagic zone of the ocean. 16S rRNA gene-based analyses indicate that they constitute up to 60% of the bacterioplankton community in the surface waters of the Red Sea. This extremely oligotrophic water body is further characterized by an epipelagic zone, which has a temperature above 24°C throughout the year, and a remarkable uniform temperature (∼22°C) and salinity (∼41 psu) from the mixed layer (∼200 m) to the bottom at over 2000 m depth. Despite these conditions that set it apart from other marine environments, the microbiology of this ecosystem is still vastly understudied. Prompted by the limited phylogenetic resolution of the 16S rRNA gene, we extended our previous study by sequencing the internal transcribed spacer (ITS) region of SAR11 in different depths of the Red Sea’s water column together with the respective 16S fragment. The overall diversity captured by the ITS loci was ten times higher than that of the corresponding 16S rRNA genes. Moreover, species estimates based on the ITS showed a highly diverse population of SAR11 in the mixed layer that became diminished in deep isothermal waters, which was in contrast to results of the related 16S rRNA genes. While the 16S rRNA gene-based sequences clustered into three phylogenetic subgroups, the related ITS fragments fell into several phylotypes that showed clear depth-dependent shifts in relative abundances. Blast-based analyses not only documented the observed vertical partitioning and universal co-occurrence of specific phylotypes in five other distinct oceanic provinces, but also highlighted the influence of ecosystem-specific traits (e.g., temperature, nutrient availability, and concentration of dissolved oxygen) on the population dynamics of this ubiquitous marine bacterium.     

The distribution and molecular diversity of the Eastern Atlantic and Mediterranean chthamalids (Crustacea, Cirripedia)

The three chthamalids Chthamalus stellatus , C. montagui and Euraphia depressa are common inhabitants of the intertidal zone in the Eastern Atlantic, Mediterranean Sea and Black Sea. In this study, we investigated the occurrence of these barnacles in a wide range of their distribution. Population divergences of these two species have been inferred using three molecular markers — internal transcribed spacer (ITS), elongation factor 1α (EF-1α) and cytochrome oxidase subunit I (COI). ITS sequences of C. stellatus were identical throughout the species range, whereas ITS sequences of C. montagui indicated that the Black Sea and Mediterranean populations are isolated from the Atlantic population. The COI and EF-1α sequences were the most variable and informative. They indicated a high genetic divergence between Atlantic, Mediterranean and Black Sea populations for C. montagui . In addition significant genetic structure was found among the populations of C. stellatus based on EF-1α but not COI. Interestingly, our molecular dating analysis correlated the pattern of diversification in C. montagui to major geological changes that occurred in the Mediterranean during the end of the Messinian and Pleiocene periods. We suggest that palaeohistory shaped the divergences between Chthamalus populations that have probably been maintained by current hydrographic conditions. Finally, COI phylogenetic analysis placed the genus Euraphia within the Chthamalus clade, suggesting the need for a taxonomic revision of Euraphia . This study represents the most detailed phylogeographical analysis of intertidal Mediterranean species to date, and shows that geological events have strongly shaped the current diversity pattern of this fauna.     

Low intraspecific diversity in a polynucleobacter subcluster population numerically dominating bacterioplankton of a freshwater pond

Cultivation-dependent and -independent methods were combined to investigate the microdiversity of a Polynucleobacter subcluster population (Betaproteobacteria) numerically dominating the bacterioplankton of a small, humic freshwater pond. Complete coverage of the population by cultivation allowed the analysis of microdiversity beyond the phylogenetic resolution of ribosomal markers. Fluorescent in situ hybridization with two probes specific for the narrow subcluster C (PnecC bacteria) of the Polynucleobacter cluster revealed that this population contributed up to 60% to the total number of bacterioplankton cells. Microdiversity was investigated for a date at which the highest relative numbers of PnecC were observed. A clone library of fragments of the ribosomal operon (16S rRNA genes, complete 16S-23S internal transcribed spacer 1 [ITS1], partial 23S rRNA genes) amplified with universal bacterial primers was constructed. The library was stepwise screened for fragments from PnecC bacteria and for different ITS genotypes of PnecC bacteria. The isolated PnecC strains were characterized by sequencing of the 16S rRNA genes and the ITS1. Both the clone library and the established culture collection contained only the same three ITS genotypes, and one of them contributed 46% to the entire number of clones. Genomic fingerprinting of the isolates with several methods always resulted in the detection of only one fingerprint per ITS genotype. We conclude that a Polynucleobacter population with an extremely low intraspecific diversity and an uneven structure numerically dominated the bacterioplankton community in the investigated habitat. This low intraspecific diversity is in strong contrast to the high intraspecific diversities found in marine bacterial populations.