Cyanophage diversity, inferred from g20 gene analyses, in the largest natural lake in France, Lake Bourget

The genetic diversity of the natural freshwater community of cyanophages and its variations over time have been investigated for the first time in the surface waters of the largest natural lake in France. This was done by random screening of clone libraries for the g20 gene and by denaturing gradient gel electrophoresis (DGGE). Nucleotide sequence analysis revealed 35 distinct cyanomyovirus g20 genotypes among the 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). The distances between these OTUs were comparable to those reported between marine clusters. Moreover, some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Both approaches for the g20 gene (sequencing and DGGE analysis) showed that there was a clear seasonal pattern of variation in the composition of the cyanophage community that could reflect changes in its biological, chemical, and/or physical environment.     

Microbial Community Structure and Dynamics in the Largest Natural French Lake (Lake Bourget)

We investigated the dynamics and diversity of heterotrophic bacteria, autotrophic and heterotrophic flagellates, and ciliates from March to July 2002 in the surface waters (0–50 m) of Lake Bourget. The heterotrophic bacteria consisted mainly of “small” cocci, but filaments (>2 μm), commonly considered to be grazing-resistant forms under increased nanoflagellate grazing, were also detected. These elongated cells mainly belonged to the Cytophaga-Flavobacterium (CF) cluster, and were most abundant during spring and early summer, when mixotrophic or heterotrophic flagellates were the main bacterial predators. The CF group strongly dominated fluorescent in situ hybridization–detected cells from March to June, whereas clear changes were observed in early summer when Beta-proteobacteria and Alpha-proteobacteria increased concomitantly with maximal protist grazing pressures. The analysis of protist community structure revealed that the flagellates consisted mainly of cryptomonad forms. The dynamics of Cryptomonas sp. and Dinobryon sp. suggested the potential importance of mixotrophs as consumers of bacteria. This point was verified by an experimental approach based on fluorescent microbeads to assess the potential grazing impact of all protist taxa in the epilimnion. From the results, three distinct periods in the functioning of the epilimnetic microbial loop were identified. In early spring, mixotrophic and heterotrophic flagellates constituted the main bacterivores, and were regulated by the availability of their resources mainly during April (phase 1). Once the “clear water phase” was established, the predation pressure of metazooplankton represented a strong top-down force on all microbial compartments. During this period only mixotrophic flagellates occasionally exerted a significant bacterivory pressure (phase 2). Finally, the early summer was characterized by the highest protozoan grazing impact and by a rapid shift in the carbon pathway transfer, with a fast change-over of the main predators contribution, i.e., mixotrophic, heterotrophic flagellates and ciliates in bacterial mortality. The high abundance of ciliates during this period was consistent with the high densities of resources (heterotrophic nanoflagellates, algae, bacteria) in deep layers containing the most chlorophyll. Bacteria, as ciliates, responded clearly to increasing phytoplankton abundance, and although bacterial grazing impact could vary largely, bacterial abundance seemed to be primarily bottom-up regulated (phase 3).     

Phylogenetic Diversity of Marine Cyanophage Isolates and Natural Virus Communities as Revealed by Sequences of Viral Capsid Assembly Protein Gene g20

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.     

Dispersal and Phylogenetic Diversity of Nonmarine Picocyanobacteria, Inferred from 16S rRNA Gene and cpcBA-Intergenic Spacer Sequence Analyses

More than 20 Synechococcus and Cyanobium isolates were obtained from central European subalpine lakes and sequenced for their 16S rRNA gene and part of the phycocyanin operon (cpc), specifically the intergenic spacer (IGS) between cpcB and cpcA, and corresponding flanking regions (cpcBA-IGS). Maximum-likelihood analyses revealed the existence of at least six to seven clusters of nonmarine picocyanobacteria within the picophytoplankton clade and support the conjecture of global dispersal for some closely related picocyanobacterial genotypes.     

Cyanobacterial bloom termination: the disappearance of Planktothrix rubescens from Lake Bourget (France) after restoration

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Estimates of protozoan- and viral-mediated mortality of bacterioplankton in Lake Bourget (France)

1. We performed three, 1‐week in situ experiments in March‐April (expt 1), May (expt 2) and August (expt 3) 2003 in order to assess protozoan and virus‐induced mortality of heterotrophic bacteria in a French lake. Viral and bacterial abundances were obtained using flow cytometry (FCM) while protozoa were counted using epifluorescence microscopy (EFM). 2. A dilution approach, applied to pretreated grazer‐free samples, allowed us to estimate that viral lysis could be responsible for 60% (expt 1), 35% (expt 2) and 52% (expt 3) of daily heterotrophic bacterial mortality. Flagellate (both mixotrophic and heterotrophic) grazing in untreated samples, was responsible for 56% (expt 1), 63% (expt 2) and 18% (expt 3) of daily heterotrophic bacteria removal. 3. These results therefore suggest that both viral lysis and flagellate grazing had a strong impact on bacterial mortality, and this impact varied seasonally. 4. From parallel transmission electron microscopy (TEM) analysis, we found that the burst size (i.e. the number of viruses potentially released per lysed cell) ranged from nine to 25 (expt 1), 10 to 35 (expt 2) and eight to 25 (expt 3). The percentage of infected heterotrophic bacteria was 5.7% (expt 1), 3.4% (expt 2) and 5.7% (expt 3) so that the calculated percentage of bacterial mortality induced by viruses was 6.3% (expt 1), 3.7% (expt 2) and 6.3% (expt 3). 5. It is clear that the dilution‐FCM and TEM methods yielded different estimates of viral impact, although both methods revealed an increased impact of viruses during summer.     

Contribution of mathematical modeling to lake ecosystem understanding: Lake Bourget (Savoy,France)

This paper demonstrates how mathematical modeling can contribute to improve understanding of lake behavior. Since the 60's Lake Bourget, one of the largest in France, had been suffering from eutrophication which was checked in 1980 by the diversion of the main sewers entering the lake. A research program was implemented between 1987 and 1990, including an on-site sampling campaign conducted concurrently with thermal and biogeochemical modeling of lake behavior. The model helped provide a better understanding of the ecosystem, displaying some processes hitherto misunderstood: (1) Winter overturn does not reach the bottom of the water column when the weather is mild. This leads to a incomplete reoxygenation of the hypolimnion and to redox conditions inducing the release of orthophosphate from the sediment, (2) Grazing by herbivorous zooplankton is getting more important in the control of spring algal growth as eutrophication of the lake regresses, (3) Settling of particulate phosphorus seems a complex and very important process in Lake Bourget, showing high sedimentation rates for particulate mineral phosphorus.     

Phylogenetic Diversity of Sequences of Cyanophage Photosynthetic Gene psbA in Marine and Freshwaters

Many cyanophage isolates which infect the marine cyanobacteria Synechococcus spp. and Prochlorococcus spp. contain a gene homologous to psbA, which codes for the D1 protein involved in photosynthesis. In the present study, cyanophage psbA gene fragments were readily amplified from freshwater and marine samples, confirming their widespread occurrence in aquatic communities. Phylogenetic analyses demonstrated that sequences from freshwaters have an evolutionary history that is distinct from that of their marine counterparts. Similarly, sequences from cyanophages infecting Prochlorococcus and Synechococcus spp. were readily discriminated, as were sequences from podoviruses and myoviruses. Viral psbA sequences from the same geographic origins clustered within different clades. For example, cyanophage psbA sequences from the Arctic Ocean fell within the Synechococcus as well as Prochlorococcus phage groups. Moreover, as psbA sequences are not confined to a single family of phages, they provide an additional genetic marker that can be used to explore the diversity and evolutionary history of cyanophages in aquatic environments.     

Marine and Freshwater Cyanophages in a Laurentian Great Lake: Evidence from Infectivity Assays and Molecular Analyses of g20 Genes

While it is well established that viruses play an important role in the structure of marine microbial food webs, few studies have directly addressed their role in large lake systems. As part of an ongoing study of the microbial ecology of Lake Erie, we have examined the distribution and diversity of viruses in this system. One surprising result has been the pervasive distribution of cyanophages that infect the marine cyanobacterial isolate Synechococcus sp. strain WH7803. Viruses that lytically infect this cyanobacterium were identified throughout the western basin of Lake Erie, as well as in locations within the central and eastern basins. Analyses of the gene encoding the g20 viral capsid assembly protein (a conservative phylogenetic marker for the cyanophage) indicate that these viruses, as well as amplicons from natural populations and the ballast of commercial ships, are related to marine cyanophages but in some cases form a unique clade, leaving questions concerning the native hosts of these viruses. The results suggest that cyanophages may be as important in freshwater systems as they are known to be in marine systems.     

Phylogenetic Analyses of Trichodinids (Ciliophora, Oligohymenophora) Inferred from 18S rRNA Gene Sequence Data

Partial 18S rRNA gene sequences of the three trichodinids, namely Trichodina modesta Lom, 1970, Trichodina paraheterodentata Tang and Zhao 2012. and Trichodinella epizootica (Raabe 1950) ?rámek-Hu?ek, 1953, were acquired and used to construct phylogenetic trees. The results revealed that Trichodinella epizootica clustered with Trichodinella sp.; Trichodina paraheterodentata Tang and Zhao 2012 was sister to the clade composed of Trichodina heterodentata Duncan, 1977 and Trichodina nobilis Chen, 1963; Trichodina modesta Lom, 1970 clustered with Trichodina reticulata Hirschman and Partsch, 1955. The branching order of species within the Mobilia clade was closely correlated with GC content. Furthermore, blade morphology was also found to be the primary morphological character in determining the phylogenetic relationships among members of the genus Trichodina. The present findings suggest that the genus Trichodina is paraphyletic when species of Trichodinella are included in the analyses.     

Viral abundance, production, decay rates and life strategies (lysogeny versus lysis) in Lake Bourget (France)

We have investigated the ecology of viruses in Lake Bourget (France) from January to August 2008. Data were analysed for viral and bacterial abundance and production, viral decay, frequency of lysogenic cells, the contribution of bacteriophages to prokaryotic mortality and their potential influence on nutrient dynamics. Analyses and experiments were conducted on samples from the epilimnion (2 m) and the hypolimnion (50 m), taken at the reference site of the lake. The abundance of virus‐like particles (VLP) varied from 3.4 × 10⁷ to 8.2 × 10⁷ VLP ml^?1; with the highest numbers and virus‐to‐bacterium ratio (VBR = 69) recorded in winter. Viral production varied from 3.2 × 10⁴ VLP ml^?1 h^?1 (July) to 2 × 10⁶ VLP ml^?1 h^?1 (February and April), and production was lower in the hypolimnion. Viral decay rate reached 0.12–0.15 day^?1, and this parameter varied greatly with sampling date and methodology (i.e. KCN versus filtration). Using transmission electron microscopy (TEM) analysis, viral lysis was responsible for 0% (January) to 71% (February) of bacterial mortality, while viral lysis varied between 0% (April) and 53% (January) per day when using a modified dilution approach. Calculated from viral production and burst size, the virus‐induced bacterial mortality varied between 0% (January) and 68% (August). A weak relationship was found between the two first methods (TEM versus dilution approach). Interestingly, flow cytometry analysis performed on the dilution experiment samples revealed that the viral impact was mostly on high DNA content bacterial cells whereas grazing, varying between 8.3% (June) and 75.4% (April), was reflected in both HDNA and LDNA cells equally. The lysogenic fraction varied between 0% (spring/summer) and 62% (winter) of total bacterial abundance, and increased slightly with increasing amounts of mitomycin C added. High percentages of lysogenic cells were recorded when bacterial abundance and activity were the lowest. The calculated release of carbon and phosphorus from viral lysis reached up to 56.5 µgC l^?1 day^?1 (assuming 20 fgC cell^?1) and 1.4 µgP l^?1 day^?1 (assuming 0.5 fgP cell^?1), respectively, which may represent a significant fraction of bacterioplankton nutrient demand. This study provides new evidence of the quantitative and functional importance of the virioplankton in the functioning of microbial food webs in peri‐alpine lakes. It also highlights methodologically dependent results.     

Long-term temperature evolution in a deep sub-alpine lake,Lake Bourget,France: how a one-dimensional model improves its trend assessment

Water temperature and the alternation of stratification and mixing count amongst key drivers of lake ecology. Reliable long time-series of water temperature are rare. Here, we investigated how a numerical model can fill the gaps in heterogeneous time-series and make it possible to identify a significant trend in the lake thermal regime. We computed the mean water temperature and the Schmidt stability, an indicator of the stratification strength, in a deep and well-stratified lake (Lake Bourget, France), between 1976 and 2008. We first used temperature measurements and then a one-dimensional vertical model, which we describe here. The model performs as well as the best existing models. During the 1976–2008 periods, whereas no statistically significant trend came out of the measurements, we found in the simulation results an increase of 0.12 °C per decade in the water temperature and of 5.9 days per decade in the stratification duration. This shows that the temperature history of a lake can be reconstructed and a reliable long-term trend computed when weather data and a calibrated temperature model are available. Our results also suggest that different local climatic influences cause a slower warming in this lake of the south-western Alps than in other sub-alpine lakes.     

黄河上游花斑裸鲤Cyt b基因的序列变异和遗传多样性

花斑裸鲤（Gymncypris eckloni）主要分布于黄河上游高原宽谷河段深水缓流处或静水湖泊中,在高原淡水生态系统的食物链中具有重要的地位。本文获得了黄河上游花斑裸鲤5个种群共68个个体线粒体DNA（mtDNA）细胞色素b基因的全序列（1 140 bp）,分析了序列变异和种群遗传多样性。68个序列经比对后,发现30个（2.63%）多态性位点,共定义了18个单倍型。结果显示,花斑裸鲤种群单倍型多样度和核苷酸多样度均低于其它鲤科鱼类,这可能与青藏高原经所经历的地质变迁和古气候环境的改变有关,由此生活在高原水域中的鱼类或多或少经历过瓶颈效应。AMOVA分析结果显示,遗传差异主要发生在种群之内,而不是来自不同地理组群间或组群内种群间。单倍型网络图和系统发育分析均没有显示出单倍型与地理位置的对应关系,提示黄河上游花斑裸鲤自然种群未出现分化,应作为一个整体进行保护。单倍型歧点分布呈现为单峰以及中性检验Fu’s Fs（&#8722;15.3400, P<0.001）和Tajima’s D（&#8722;0.6254, P =0.3080）结果综合表明,花斑裸鲤可能经历过近期的种群扩张事件。     

黄河上游花斑裸鲤Cytb基因的序列变异和遗传多样性

花斑裸鲤(Gymncypris eckloni)主要分布于黄河上游高原宽符河段深水缓流处或静水湖泊中,在高原淡水生态系统的食物链中具有重要的地位.本文获得了黄河上游仡斑裸鲤5个种群共68个个体线粒体DNA(mtDNA)细胞色素b基因的全序列(1140bp),分析了序列变异和种群遗传多样性.68个序列经比对后,发现30个(2.63%)多态性位点,共定义了18个单倍型.结果显示,花斑裸鲤种群单倍型多样度和核苷酸多样度均低于其它鲤科鱼类,这可能与青藏高原经所经历的地质变迁和古气候环境的改变有关,由此生活在高原水域中的鱼类或多或少经历过瓶颈效应.AMOVA分析结果显示,遗传差异主要发生在种群之内,而不是来自不同地理组群问或组群内种群间.单倍型网络图和系统发育分析均没有显示出单倍型与地理位置的对应关系,提示黄河上游花斑裸鲤自然种群未出现分化,应作为一个整体进行保护.单倍犁歧点分布呈现为单峰以及中性检验Fu's Fs (-15.3400,P＜0.001)和Tajima's D(-0.6254.P=0.3080)结果综合表明,花斑裸鲤可能经历过近期的种群扩张事件.     

Spatial variability in the abundance and composition of the free-living bacterioplankton community in the pelagic zone of Lake Bourget (France)

Spatial variations in the abundance and diversity of the free-living bacterioplankton community of a large Alpine lake, Lake Bourget (France), were investigated in the pelagic zone by means of two two-dimensional samplings taken in 2003. Lake-water samples were collected in winter during water mixing, and in early summer during stratification. The population abundance in each sample was determined by flow cytometry. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments from organisms measuring less than 2 mum was used to assess eubacterioplankton community composition. In winter, no obvious differences were observed in either the abundance or the diversity of the bacterial community, on either the horizontal or the vertical scales. The only influence detected was that of river water input, but this was at a very minor scale relative to the surface area of the lake. In early summer, changes were found in the community composition on the vertical scale related to the thermal stratification of the water column. There were also marked differences on the horizontal scale at 15 m depth due to internal waves. The implications of these findings for sampling strategies are very important from the perspective of comparative studies of free-living bacterial community diversity and functioning in large and deep lakes.     

A 2-year assessment of the main environmental factors driving the free-living bacterial community structure in Lake Bourget (France)

Despite the considerable attention that has been paid to bacterioplankton over recent decades, the dynamic of aquatic bacterial community structure is still poorly understood, and long-term studies are particularly lacking. Moreover, how the environment governs diversity patterns remains a key issue in aquatic microbial ecology. In this study, we used denaturing gradient gel electrophoresis of PCR-amplified partial 16S rRNA gene fragments and multivariable statistical approaches to explore the patterns of change in the free-living bacterial community in the mesotrophic and mono-meromictic Lake Bourget (France). A monthly sampling was conducted over two consecutive years (2007 and 2008) and at two different depths characterizing the epi- and hypolimnion of the lake (2 and 50 m, respectively). Temporal shifts in the bacterial community structure followed different patterns according to depth, and no seasonal reproducibility was recorded from 1 year to the next. Our results showed that the bacterial community structure displayed lower diversity at 2 m (22 bands) compared to 50 m (32 bands) and that bacterial community structure dynamics followed dissimilar trends between the two depths. At 2 m, five shifts in the bacterial community structure occurred, with the temporal scale varying between 2 and 8 months whereas, at 50 m, four shifts in the bacterial community structure took place at 50 m, with the temporal scale fluctuating between 3 and 13 months. More than 60% of the bacterial community structure variance was explained by seven variables at 2 m against eight at 50 m. Nutrients (PO₄-P, NH₄-N and NO₃-N) and temperature were responsible for 49.6% of the variance at 2 m whereas these nutrients, with dissolved oxygen and chlorophyll a accounting for 59.6% of the variance at 50 m. Grazing by ciliates played also a critical role on the bacterial community structure at both depths. Our results suggest that the free-living bacterial community structure in the epi- and hypolimnion of Lake Bourget is mainly driven by combined, but differently weighted, top-down and bottom-up factors at 2 and 50 m.     

若尔盖湿地青海沙蜥红原亚种线粒体ND4-tRNAleu基因的序列变异和遗传多样性

研究了青海沙蜥红原亚种(Phrynocephalus vlangalii hongyuanensis)分布于四川若尔盖辖曼(XM)、红原(HY)和甘肃玛曲(MQ)这3个地理单元7个居群的ND4-tRNAleu基因的序列变异和遗传多样性。72个序列比对得到785bp的片断,含变异位点7个,定义了9种单倍型。结果显示7个居群总的核苷酸多样性较低(0.00231±0.00016),单倍型多样性较高(0.806±0.024);就单个居群而言,MQa、MQb、XMb的遗传多样性低,XMc相对较高。各居群间的Kimura双参数遗传距离小(0.001—0.005),最大遗传距离出现在MQa和XMa之间,XM单元各居群之间遗传距离最小。但分子变异分析(AMOVA)显示3个单元间出现了显著差异(P<0.01),遗传变异主要存在于地理单元间,占62.61%。除MQ单元形成一支,单倍型网络图没有显示出单倍型和地理位置的对应关系,XM各居群以及HY混杂在一起。XM单元单倍型的不配对分布(Mismatch distribution)为明显左移的单峰,同时Fu’sFstest得到负值(Fs=-2.21937),这都暗示XM单元可能经历了近期种群扩张,这是其单倍型多样性较高和核苷酸多样性较低的原因。MQ单元遗传多样性低而与其他单元显著分化,推测与黄河在若尔盖玛曲之间贯通有关。近期沼泽的形成对XMb的隔离时间短,使得其遗传多样性低但还不足以形成大的遗传差异。     

Phylogenetic Diversity of Archaea and Bacteria in the Anoxic Zone of a Meromictic Lake (Lake Pavin, France)

The compositions of archaeal and bacterial populations at different depths (60 m [mixolimnion-chemocline interface], 70 m [chemocline-subchemocline interface], 90 m, and 92 m [the water-sediment interface]) in the anoxic zone of the water column in Lake Pavin, a freshwater permanently stratified mountain lake in France, were determined. Phylogenetic trees were constructed from sequences to assess archaeal and bacterial diversity at the four sites.     

小孢盘菌属种间关系初探（英文）

3个编码蛋白的基因RPB1、RPB2和TEF1以及1个非编码序列28S rD NA的联合分析结果表明,小孢盘菌属位于系统树的上端,形成一个单系群,其中长春小孢盘菌和北京小孢盘菌为姐妹群,它们进一步与该属另3个种(包括该属的模式种)聚类在一起。序列分析结果支持将子囊孢子大小作为小孢盘菌属区分物种的依据,还表明火丝菌科可能不是一个单系群。在参试属中,小孢盘菌属与侧盘菌属的关系较为密切,它们具有极其相似的薄壁子囊,这表明在属的等级上子囊形态可能携带系统发育方面的信息。