Marianne Ellegaard

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.

Variations in dinoflagellate cyst morphology under conditions of changing salinity during the last 2000 years in the Limfjord, Denmark

Morphological variations are examined in the dinoflagellate cysts Spiniferites spp., Lingulodinium polyedrum and Protoceratium reticulatum (=Operculodinium centrocarpum) from a core taken in the Bjørnsholm Bay, the Limfjord, Denmark. The fjord has a history of changing salinity, and unusual cyst morphotypes are found in the greatest numbers during periods of inferred low salinity. Variation occurs primarily in cyst process morphology, and the aberrant morphotypes have processes that are shorter, thicker and/or more membranous. The different morphotypes are described and compared with other varieties and forms of the three taxa and to other closely related taxa.

Phytoplankton growth after a century of dormancy illuminates past resilience to catastrophic darkness

Photosynthesis evolved in the oceans more than 3 billion years ago and has persisted throughout all major extinction events in Earths history. The most recent of such events is linked to an abrupt collapse of primary production due to darkness following the Chicxulub asteroid impact 65.5 million years ago. Coastal phytoplankton groups (particularly dinoflagellates and diatoms) appear to have been resilient to this biotic crisis, but the reason for their high survival rates is still unknown. Here we show that the growth performance of dinoflagellate cells germinated from resting stages is unaffected by up to a century of dormancy. Our results clearly indicate that phytoplankton resting stages can endure periods of darkness far exceeding those estimated for the Cretaceous-Paleogene extinction and may effectively aid the rapid resurgence of primary production in coastal areas after events of prolonged photosynthesis shut-down.

Morphological and LSU rDNA sequence variation within the Gonyaulax spinifera-Spiniferites group (Dinophyceae) and proposal of G-elongata comb. nov and G-membranacea comb. nov

Abstract Cultures were established from cysts of the cyst-based taxa Spiniferites elongatus and S. membranaceus. Motile cells and cysts from both cultures and sediment samples were examined using light and scanning electron microscopy. The cyst–theca relationship was established for S. elongatus. The motile cells have the tabulation pattern 2 pr, 4′, 6″, 6c, ≥ 4s, 6″′, 1p, 1″″, but they remain unattributable to previously described Gonyaulax species. There was large variation in process length and process morphology in cysts from both cultures and wild samples and there was variation in ornamentation and in the development of spines and flanges in motile cells. A new combination, G. elongata (Reid) Ellegaard et al. comb. nov. is proposed, following new rules of the International Code of Botanical Nomenclature that give genera based on extant forms priority over genera based on fossil forms. Extreme morphological variation in the cyst and motile stages of S. membranaceus is described and this species is also transferred to the genus Gonyaulax, as G. membranacea (Rossignol) Ellegaard et al. comb. nov. Approximately 1500 bp of large subunit (LSU) rDNA were determined for these two species and for G. baltica, G. cf. spinifera (= S. ramosus) and G. digitalis (= Bitectatodinium tepikiense). LSU rDNA showed sequence divergences similar to those estimated between species in other genera within the Gonyaulacales; a phylogeny for the Gonyaulacales was established, including novel LSU rONA sequences for Alexandrium margalefii, A. pseudogonyaulax and Pyrodinium bahamense var. compressum. Our results show that motile stages obtained from the germination of several cysts of the ‘fossil-based’ Spiniferites and B. tepikiense, which were previously attributed to ‘Gonyaulax spinifera group undifferentiated’, belong to distinct species of the genus Gonyaulax. These species show small morphological differences in the motile stage but relatively high sequence divergence. Moreover, this group of species is monophyletic, supported by bootstrap values of 100% in parsimony and maximum likelihood analyses.

TEMPERATURE AND SALINITY EFFECTS ON GROWTH OF A NON‐CHAIN‐FORMING STRAIN OF FYMNODINIUM CATENATUM (DINOOHYCEAE) ESTABLISHED FROM A CYST FROM RECENT SEDIMENTS IN THE SOUND (øRESUND), DENMARK1

The athecate, marine dinoflagellate Gymnodinium catenatum Graham 1943 was cultured from a resting cyst found in sediment samples from The Sound, Denmark. Gymnodinium catenatum has not previously been registered alive in Danish waters, although fossilized cysts have been found in old sediments. The description (morphology and ultrastructure) of the strain established from the cyst complied with earlier studies of G. catenatum with the exception that chains longer than two cells were never see. Fitting the growth rates of the motile stage of G. catenatum to a linear model showed a signified influence of temperature and salinity. Maximal division rate was 0.4 div‐day−1 at 20–25°C and 20% salinity. Gymnodinium catenatum causes paralytic shellfish poisoning in other parts of the world, but the toxicity of the Danish strain has not been determined. Possible explanations for the presence of G. catenatum in Danish waters ware discussed.

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 from Recent Danish marine sediments

Twenty-three different cyst types were found in a survey of dinoflagellate resting stages (cysts) in sediment samples from two sites in Danish waters: Oresund (The Sound) and Aarhus Bay. This is the first survey of its kind from Danish waters. The cyst types found were: Diplopsalis lenticula, Gymnodinium catenatum, Polykrikos schwartzii, Scrippsiella trochoidea, four species of Gonyaulax, ten species of Protoperidinium and five unidentified cyst types. Five species have not previously been reported from Danish waters. Where possible, the cysts were germinated and identification was based on characteristics of both cyst and motile stages. Among the germinated cysts was Gymnodinium catenatum, a naked dinoflagellate causing Paralytic Shellfish Poisoning (PSP) elsewhere in the world and not previously found living in Danish waters.

GYMNODINIUM COROLLARIUM SP. NOV. (DINOPHYCEAE)—A NEW COLD‐WATER DINOFLAGELLATE RESPONSIBLE FOR CYST SEDIMENTATION EVENTS IN THE BALTIC SEA1

A naked dinoflagellate with a unique arrangement of chloroplasts in the center of the cell was isolated from the northern Baltic proper during a spring dinoflagellate bloom (March 2005). Morphological, ultrastructural, and molecular analyses revealed this dinoflagellate to be undescribed and belonging to the genus Gymnodinium F. Stein. Gymnodinium corollarium A. M. Sundström, Kremp et Daugbjerg sp. nov. possesses features typical of Gymnodinium sensu stricto, such as nuclear chambers and an apical groove running in a counterclockwise direction around the apex. Phylogenetic analyses based on partial nuclear‐encoded LSU rDNA sequences place the species in close proximity to G. aureolum, but significant genetic distance, together with distinct morphological features, such as the position of chloroplasts, clearly justifies separation from this species. Temperature and salinity experiments revealed a preference of G. corollarium for low salinities and temperatures, confirming it to be a cold‐water species well adapted to the brackish water conditions in the Baltic Sea. At nitrogen‐deplete conditions, G. corollarium cultures produced small, slightly oval cysts resembling a previously unidentified cyst type commonly found in sediment trap samples collected from the northern and central open Baltic Sea. Based on LSU rDNA comparison, these cysts were assigned to G. corollarium. The cysts have been observed in many parts of the Baltic Sea, indicating the ecologic versatility of the species and its importance for the Baltic ecosystem.

The species concept in a marine diatom: LSU rDNA-based phylogenetic differentiation in Skeletonema marinoi /dohrnii (Bacillariophyceae) is not reflected in morphology

M. Ellegaard, A. Godhe, K. Härnström and M. McQuoid. 2008. The species concept in a marine diatom: LSU rDNA–based phylogenetic differentiation in Skeletonema marinoi/dohrnii (Bacillariophyceae) is not reflected in morphology. Phycologia 47: 156–167. DOI: 10.2216/07-09.1 The ubiquitous marine diatom genus Skeletonema includes several newly described species. This paper presents a study of morphology and LSU sequence diversity of one of these new species, Skeletonema marinoi. The 15 monoclonal strains (from the Swedish west coast, the Swedish east coast, Portugal and Canada) included in the study showed differences in LSU rDNA sequence within the morphospecies, with differences seen even among clones established from a single plankton net sample. Morphologically, all clones were indistinguishable from each other and from the closely related species Skeletonema dohrnii. In the original description of these two species, they were differentiated by the structure of the girdle bands. However, the girdle band types of both species were found within single samples of almost all clones of S. marinoi in this study. The LSU-based phylogeny is consistent with the split into two species, and there may be a difference in their biogeographical distribution. We therefore do not at present suggest that the two species be merged. Questions regarding species delimitation and cryptic species within protists often arise from such studies that include data from both morphological and DNA sequence analysis, and such questions are addressed here.

Protoperidinium minutum (Dinophyceae) from Portugal: cyst–theca relationship and phylogenetic position on the basis of single-cell SSU and LSU rDNA sequencing

Ribeiro S., Lundholm N., Amorim A. and Ellegaard M. 2010. Protoperidinium minutum (Dinophyceae) from Portugal: cyst–theca relationship and phylogenetic position on the basis of single-cell SSU and LSU rDNA sequencing. Phycologia 49: 48–63. DOI: 10.2216/09-11.1 Round brown spiny cysts are common elements of Recent and Quaternary dinoflagellate cyst records and are often used to infer past climate conditions. Echinidinium and Islandinium, two cyst-based genera composed of round brown spiny cysts, are believed to have affinities within the Protoperidiniaceae. However, their biological counterparts are still virtually unknown. In this study, we examined the cyst–theca relationship of an Echinidinium-like cyst isolated from recent sediments of the Portuguese coast. The cysts (25–34 µm) had an intercalary theropylic archeopyle and numerous processes (4–9 µm) with tapered stems and minutely expanded tips. Germinated cells were identified as Protoperidinium minutum on the basis of theca morphology and tabulation. This taxon has a complicated taxonomic history and most likely represents a complex of species with very similar thecae but different cyst morphologies. To provide a first step in elucidating the phylogeny of P. minutum and its evolutionary relationship among the Protoperidiniaceae, we undertook the first molecular study of this taxon on the basis of small-subunit (SSU) and large-subunit (LSU) ribosomal (r)DNA genetic sequences obtained through single-cell polymerase chain reaction. On the basis of SSU rDNA analysis, P. minutum formed a clade together with the Diplopsaloideae, not grouping together with the other Protoperidinium species. LSU rDNA-based phylogenies indicate P. minutum as early divergent within the Protoperidiniaceae. The evolutionary significance of round brown spiny cysts produced by P. minutum-like species and diplopsalids is discussed.