Anna Godhe

Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data

Summary 1. The nuclear ribosomal internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi. Its two highly variable spacers (ITS1 and ITS2) are usually species specific, whereas the intercalary 5.8S gene is highly conserved. For sequence clustering and BLAST searches, it is often advantageous to rely on either one of the variable spacers but not the conserved 5.8S gene. To identify and extract ITS1 and ITS2 from large taxonomic and environmental data sets is, however, often difficult, and many ITS sequences are incorrectly delimited in the public sequence databases. 2. We introduce ITSx, a Perl-based software tool to extract ITS1, 5.8S and ITS2 – as well as full-length ITS sequences – from both Sanger and high-throughput sequencing data sets. ITSx uses hidden Markov models computed from large alignments of a total of 20 groups of eukaryotes, including fungi, metazoans and plants, and the sequence extraction is based on the predicted positions of the ribosomal genes in the sequences. 3. ITSx has a very high proportion of true-positive extractions and a low proportion of false-positive extractions. Additionally, process parallelization permits expedient analyses of very large data sets, such as a one million sequence amplicon pyrosequencing data set. ITSx is rich in features and written to be easily incorporated into automated sequence analysis pipelines. 4. ITSx paves the way for more sensitive BLAST searches and sequence clustering operations for the ITS region in eukaryotes. The software also permits elimination of non-ITS sequences from any data set. This is particularly useful for amplicon-based next-generation sequencing data sets, where insidious non-target sequences are often found among the target sequences. Such non-target sequences are difficult to find by other means and would contribute noise to diversity estimates if left in the data set.

Viability of phytoplankton resting stages in the sediments of a coastal Swedish fjord

Viable diatom and dinoflagellate resting stages were recovered from sediments in Koljö Fjord on the west coast of Sweden. To determine the maximum survival time of buried resting stages, samples from sediment depths down to 50 cm were incubated at temperatures of 3, 10 and 18 °C. Sediment cores were dated by 210Pb and the age of samples containing viable resting stages was determined using the constant rate of supply model. Dilution cultures of surface sediments allowed semiquantitative estimates of the potential seed bank. Dinoflagellate cysts from species such as Diplopsalis sp., Gymnodinium nolleri, Oblea rotunda and Protoceratium reticulatum were viable down to 15 cm depth, or 37 years old. Spores and resting cells of the diatoms Chaetoceros spp., Detonula confervacea and Skeletonema costatum were viable to over 40 cm depth, and may have been buried for many decades. The seed bank of living resting stages in surficial sediments was found to be rich (c. 57000 diatom resting stages g−1 wet weight and c. 200 dinoflagellate cysts g−1 wet weight), and the percentage of viable resting stages was higher for spore- and cyst-forming species. The oxygen-deficient sediments in Koljö Fjord appear to be a natural conservator of cell viability, a condition not easily simulated in laboratory studies. These results are ecologically important since spores and cysts are a repository of genetic material able to repopulate waters if resuspended and exposed to suitable light, temperature and nutrients.

Quantification of Diatom and Dinoflagellate Biomasses in Coastal Marine Seawater Samples by Real-Time PCR

ABSTRACT Two real-time PCR assays targeting the small-subunit (SSU) ribosomal DNA (rDNA) were designed to assess the proportional biomass of diatoms and dinoflagellates in marine coastal water. The reverse primer for the diatom assay was designed to be class specific, and the dinoflagellate-specific reverse primer was obtained from the literature. For both targets, we used universal eukaryotic SSU rDNA forward primers. Specificity was confirmed by using a BLAST search and by amplification of cultures of various phytoplankton taxa. Reaction conditions were optimized for each primer set with linearized plasmids from cloned SSU rDNA fragments. The number of SSU rDNA copies per cell was estimated for six species of diatoms and nine species of dinoflagellates; these were significantly correlated to the biovolumes of the cells. Nineteen field samples were collected along the Swedish west coast and subjected to the two real-time PCR assays. The linear regression of the proportion of SSU rDNA copies of dinoflagellate and diatom origin versus the proportion of dinoflagellate and diatom biovolumes or biomass per liter was significant. For diatoms, linear regression of the number of SSU rDNA copies versus biovolume or biomass per liter was significant, but no such significant correlation was detected in the field samples for dinoflagellates. The method described will be useful for estimating the proportion of dinoflagellate versus diatom biovolume or biomass and the absolute diatom biovolume or biomass in various aquatic disciplines.

Hundred years of genetic structure in a sediment revived diatom population

This paper presents research on the genetic structure and diversity of populations of a common marine protist and their changes over time. The bloom-forming diatom Skeletonema marinoi was used as a model organism. Strains were revived from anoxic discrete layers of a 210Pb-dated sediment core accumulated over more than 100 y, corresponding to >40,000 diatom mitotic generations. The sediment core was sampled from the highly eutrophic Mariager Fjord in Denmark. The genetic structure of S. marinoi was examined using microsatellite markers, enabling exploration of changes through time and of the effect of environmental fluctuations. The results showed a stable population structure among and within the examined sediment layers, and a similar genetic structure has been maintained over thousands of generations. However, established populations from inside the fjord were highly differentiated from open-sea populations. Despite constant water exchange and influx of potential colonizers into the fjord, the populations do not mix. One fjord population, accumulated in 1980, was significantly differentiated from the other groups of strains isolated from the fjord. This differentiation could have resulted from the status of Mariager Fjord, which was considered hypereutrophic, around 1980. There was no significant genetic difference between pre- and posteutrophication groups of strains. Our data show that dispersal potential and generation time do not have a large impact on the genetic structuring of the populations investigated here. Instead, the environmental conditions, such as the extreme eutrophication of the Mariager Fjord, are deemed more important.

Dinoflagellate Cysts in Recent Sediments from the West Coast of Sweden

Abstract This is the first study of dinoflagellate cysts in recent coastal sediments from the Swedish west coast. Sediments from 19 sites were investigated. Fifty-four types of cysts were encountered, of these 40 were identified to species level, representing 13 genera. The most common species were those of Lingulodinium polyedrum, Protoceratium reticulatum, Scrippsiella trochoidea, Pentapharsodinium dalei and Gonyaulax cf. spinifera. Cysts of the potentially toxic species Alexandrium minutum and Alexandrium tamarense were widely distributed as well as Gymnodinium nolleri, the non-toxic G. catenatum-like microreticulate cyst found in Northern Europe. Nine of the species found in this survey have not previously been reported from Sweden: Diplopelta parva, D. symmetrica, Diplopsalopsis latipeltata, Diplopsalis lebourae, Protoperidinium americanum, P. avellana, P. divaricatum, P. nudum and P. stellatum.

Intraspecific variability in the response of bloom-forming marine microalgae to changed climate conditions

Phytoplankton populations can display high levels of genetic diversity that, when reflected by phenotypic variability, may stabilize a species response to environmental changes. We studied the effects of increased temperature and CO2 availability as predicted consequences of global change, on 16 genetically different isolates of the diatom Skeletonema marinoi from the Adriatic Sea and the Skagerrak (North Sea), and on eight strains of the PST (paralytic shellfish toxin)-producing dinoflagellate Alexandrium ostenfeldii from the Baltic Sea. Maximum growth rates were estimated in batch cultures of acclimated isolates grown for five to 10 generations in a factorial design at 20 and 24°C, and present day and next century applied atmospheric pCO2, respectively. In both species, individual strains were affected in different ways by increased temperature and pCO2. The strongest response variability, buffering overall effects, was detected among Adriatic S. marinoi strains. Skagerrak strains showed a more uniform response, particularly to increased temperature, with an overall positive effect on growth. Increased temperature also caused a general growth stimulation in A. ostenfeldii, despite notable variability in strain-specific response patterns. Our data revealed a significant relationship between strain-specific growth rates and the impact of pCO2 on growth—slow growing cultures were generally positively affected, while fast growing cultures showed no or negative responses to increased pCO2. Toxin composition of A. ostenfeldii was consistently altered by elevated temperature and increased CO2 supply in the tested strains, resulting in overall promotion of saxitoxin production by both treatments. Our findings suggest that phenotypic variability within populations plays an important role in the adaptation of phytoplankton to changing environments, potentially attenuating short-term effects and forming the basis for selection. In particular, A. ostenfeldii blooms may expand and increase in toxicity under increased water temperature and atmospheric pCO2 conditions, with potentially severe consequences for the coastal ecosystem.

Linking the planktonic and benthic habitat: genetic structure of the marine diatom Skeletonema marinoi

Dormant life stages are important strategies for many aquatic organisms. The formation of resting stages will provide a refuge from unfavourable conditions in the water column, and their successive accumulation in the benthos will constitute a genetic reservoir for future planktonic populations. We have determined the genetic structure of a common bloom‐forming diatom, Skeletonema marinoi, in the sediment and the plankton during spring, summer and autumn two subsequent years (2007–2009) in Gullmar Fjord on the Swedish west coast. Eight polymorphic microsatellite loci were used to assess the level of genetic differentiation and the respective gene diversity of the two different habitats. We also determined the degree of genetic differentiation between the seed banks inside the fjord and the open sea. The results indicate that Gullmar Fjord has one dominant endogenous population of S. marinoi, which is genetically differentiated from the open sea population. The fjord population is encountered in the plankton and in the sediment. Shifts from the dominant population can happen, and in our study, two genetically differentiated plankton populations, displaying reduced genetic diversity, occurred in September 2007 and 2008. Based on our results, we suggest that sill fjords maintain local long‐lived and well‐adapted protist populations, which continuously shift between the planktonic and benthic habitats. Intermittently, short‐lived and mainly asexually reproducing populations can replace the dominant population in the water column, without influencing the genetic structure of the benthic seed bank.

COMPARISON OF THREE COMMON MOLECULAR TOOLS FOR DISTINGUISHING AMONG GEOGRAPHICALLY SEPARATED CLONES OF THE DIATOM SKELETONEMA MARINOI SARNO ET ZINGONE (BACILLARIOPHYCEAE)1

Skeletonema marinoi Sarno et Zingone is a planktonic marine diatom with a widespread geographic distribution. Different populations of this species may show distinct genetic signatures. We have evaluated the utility of three common molecular methods for distinguishing clones of S. marinoi from different geographic regions. Clonal cultures were isolated from the Canadian west coast, south west Portugal, and the east and west coasts of Sweden. All strains originated from resting stages in sediment. More than 90% of the individually isolated chains grew to densities suitable for DNA extraction. Genetic signatures of clones from each sample location were assessed by sequencing variable domains (D1–D3) of the nuclear large subunit (LSU) rRNA gene and internal transcriber spacer (ITS) (ITS‐1, 5.8S and ITS‐2) regions, and also by analysis of randomly amplified polymorphic DNA patterns. Analysis of molecular variance showed that strains from the four geographic areas were significantly separated by all three methods but that differences among European samples were best resolved by ITS 2 sequences.

Buried alive – germination of up to a century-old marine protist resting stages

Lundholm N., Ribeiro S., Andersen T.J., Koch T., Godhe A., Ekelund F. and Ellegaard M. 2011. Buried alive – germination of up to a century-old marine protist resting stages. Phycologia 50: 629–640. DOI: 10.2216/11-16.1 We report on the survival and germination of up to a century-old marine protist resting stages naturally preserved in sediments from Koljö Fjord on the west coast of Sweden. This work has focused on germination of dinoflagellate cysts, but diatom resting stages were also observed. We record the longest known survival of dormant dinoflagellate cells. We individually isolated more than 1200 cysts of the three most abundant dinoflagellate taxa: Pentapharsodinium dalei, Lingulodinium polyedrum and Scrippsiella spp. Germination success decreased with core depth, and all successful germinations took place within the first 2 wk of incubation. Pentapharsodinium dalei had the highest germination success rate, with a maximum of up to 80% in 28-yr-old sediment, and could successfully germinate from core sediments dated to 1920 ± 12. Scrippsiella spp. cysts with cell contents occurred down to c. 90-yr-old sediment and could germinate from down to ca. 40-yr-old sediments, with a maximum germination rate of 50–60% in recent sediments. Cysts of L. polyedrum germinated frequently down to 20 yr and rarely to c. 80 yr, with a maximum of 20–50% germination success in recent sediments. Cyst isolation under cooled conditions rather than at room temperature resulted in a significantly higher germination success in P. dalei, while no effect was observed for L. polyedrum. The time elapsed since slicing of the core affected survival of L. polyedrum cysts negatively, most likely due to the effect of oxygen. The long-term survival potential of benthic resting stages that we report here has important implications, as viable resting stages accumulated in bottom sediments can be transported back to the water column by, for example, bioturbation and human-mediated sediment dredging. Hence, the sediment may to a higher degree than previously considered play a role as seed bank. This is important in a changing climate and might have particularly severe impacts in the case of harmful species.

Dinoflagellate Cysts in Recent Marine Sediments from SW India

Abstract During the period February–June 1996, dinoflagellate cysts were sampled and examined from surface sediments from two different estuaries and three different offshore sites around Mangalore, SW India. Forty-three different types of cysts were recorded in the sediment, of which 38 cysts belonged to the orders Gonyaulacales, Gymnodinales and Peridiniales. Five cyst types could not be identified. Five types of cysts recorded belonged to the potentially toxic genera of Alexandrium Halim emend. Balech and Gymnodinium Stein. The toxic species Gymnodinium catenatum Graham was studied, for the first time, in Indian waters. The cyst flora from SW India is compared to earlier studies on cyst biodiversity and biogeography from the tropics. To our knowledge this is the first study of dinoflagellate cysts in recent coastal sediments from Indian waters.