Linda Medlin

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2009-30 September 2009

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross‐tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

Ribosomal DNA phylogenies and a morphological revision provide the basis for a revised taxonomy of the Prymnesiales (Haptophyta)

Nucleotide sequences of the nuclear-encoded small subunit (18S rDNA) and partial large subunit (28S rDNA) ribosomal DNA were determined in 30 different species of the haptophyte genera Prymnesium, Chrysocampanula, Chrysochromulina, Imantonia and Platychrysis, all belonging to the order Prymnesiales. Phylogenies based on these and other available haptophyte 18S, 28S and plastid 16S rDNA sequences were reconstructed, and compared with available morphological and ultrastructural data. The rDNA phylogenies indicate that the genus Chrysochromulina is paraphyletic and is divided into two major clades. This is supported by ultrastructural and morphological data. There is a major split between Chrysochromulina species with a saddle-shaped cell form (clade B2) and the remaining species in the genus (clade B1). Clade B2 includes the type species C. parva and taxa belonging to this clade thus retain the name Chrysochromulina. The non-saddle-shaped Chrysochromulina species analysed are closely related to Hyalolithus, Prymnesium and Platychrysis species. Imantonia species are sister taxa to these species within clade B1. An amendment to the classification of the order Prymnesiales and the genera Prymnesium, Platychrysis and Chrysochromulina is proposed with one new and one emended family (Chrysochromulinaceae and Prymnesiaceae, respectively), two new genera (Haptolina and Pseudohaptolina), and one new species (Pseudohaptolina arctica). We suggest a revision of the taxonomy of the Prymnesiales that is in accordance with available molecular evidence and supported by morphological data.

CONTRIBUTION OF THE CLASS CRYPTOPHYCEAE TO PHYTOPLANKTON STRUCTURE IN THE GERMAN BIGHT1

The class Cryptophyceae (Division Cryptophyta) contains ecologically relevant species, which are widespread in aquatic environments. However, classification, identification, and enumeration of cryptophytes are challenged by a morphology that must be usually examined with EM to permit species identification. The quantitative importance of this group has been revealed by HPLC data. But ecological information assessing the occurrence or seasonality of cryptophytes in the marine environment is still scarce. Molecular techniques allow for a refined assessment of taxonomically challenging taxa, such as the cryptophytes. In our laboratory, a Phylochip was developed to facilitate and refine the assessment of cryptophyte microalgae. Here, we present the results of an environmental study that took advantage of the Phylochip. The study was designed to elucidate the seasonality and contribution of cryptophytes to phytoplankton structure in the German Bight. The occurrence of cryptophytes in total plankton versus the picoplankton fraction was assessed with the Phylochip between the years 2004 and 2006. Our data indicate that cryptophytes are an important and constant contributor to phytoplankton structure of the German Bight, especially in the picoplankton fraction.

A new cell stage in the haploid-diploid life cycle of the colony-forming haptophyte Phaeocystis antarctica and its ecological implications.

Few members of the well‐studied marine phytoplankton taxa have such a complex and polymorphic life cycle as the genus Phaeocystis. However, despite the ecological and biogeochemical importance of Phaeocystis blooms, the life cycle of the major bloom‐forming species of this genus remains illusive and poorly resolved. At least six different life stages and up to 15 different functional components of the life cycle have been proposed. Our culture and field observations indicate that there is a previously unrecognized stage in the life cycle of P. antarctica G. Karst. This stage comprises nonmotile cells that range in size from ∼4.2 to 9.8 μm in diameter and form aggregates in which interstitial spaces between cells are small or absent. The aggregates (hereafter called attached aggregates, AAs) adhere to available surfaces. In field samples, small AAs, surrounded by a colony skin, adopt an epiphytic lifestyle and adhere in most cases to setae or spines of diatoms. These AAs, either directly or via other life stages, produce the colonial life stage. Culture studies indicate that bloom‐forming, colonial stages release flagellates (microzoospores) that fuse and form AAs, which can proliferate on the bottom of culture vessels and can eventually reform free‐floating colonies. We propose that these AAs are a new stage in the life cycle of P. antarctica, which we believe to be the zygote, thus documenting sexual reproduction in this species for the first time.

Genomic characterisation of the ichthyotoxic prymnesiophyte Chrysochromulina polylepis, and the expression of polyketide synthase genes in synchronized cultures

The widely distributed prymnesiophyte species Chrysochromulina polylepis is prominent and well known for occasional formation of ichthyotoxic blooms. The chemical structure of the C. polylepis toxin(s) has not yet been elucidated, but the associated haemolytic activity, potent membrane disruption interactions and toxicity to finfish and protists have led to the suggestion that they may be similar to the prymnesins of Prymnesium parvum. Such polyether toxins are presumably formed partially or completely via polyketide biosynthetic pathways. In this genetic study of C. polylepis, we generated and analysed a genomic DNA and a normalized cDNA library. We estimated a genome size of approximately 230 mbp based upon analysis of >1000 genomic library clones. Of the cDNA library, 3839 clones were partially sequenced and annotated, representing approximately 2900 unique contigs. We detected several genes putatively related to toxin synthesis. Thirteen putative polyketide synthase (PKS)-related gene sequences were identified and phylogenetic analysis identified two of these as containing ketoacyl domains of the modular type I PKS. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) was used to follow the expression of PKS genes over the light/dark cycle of synchronized C. polylepis cultures. This is the first study showing the expression of PKS genes in marine microalgae, in this case in the toxigenic C. polylepis.

Monitoring of freshwater toxins in European environmental waters by using novel multi-detection methods

Monitoring the quality of freshwater is an important issue for public health. In the context of the European project μAqua, 150 samples were collected from several waters in France, Germany, Ireland, Italy, and Turkey for 2 yr. These samples were analyzed using 2 multitoxin detection methods previously developed: a microsphere-based method coupled to flow-cytometry, and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. The presence of microcystins, nodularin, domoic acid, cylindrospermopsin, and several analogues of anatoxin-a (ATX-a) was monitored. No traces of cylindrospermopsin or domoic acid were found in any of the environmental samples. Microcystin-LR and microcystin-RR were detected in 2 samples from Turkey and Germany. In the case of ATX-a derivatives, 75% of samples contained mainly H2 -ATX-a and small amounts of H2 -homoanatoxin-a, whereas ATX-a and homoanatoxin-a were found in only 1 sample. These results confirm the presence and wide distribution of dihydro derivatives of ATX-a toxins in European freshwaters. Environ Toxicol Chem 2017;36:645-654.

Chapter Seven - Genomics and Genetics of Diatoms

Diatoms are unicellular eukaryotes with nano-patterned silica cell walls and they contribute about 20% of global primary production. Their beautiful shells and significance for life on our planet already caused scientific interest many centuries ago. However, the development of genetics and genomics-enabled technology about two decades ago and their application to diatom research has caused a step change in our understanding of diatom evolution, biology and ecology. In contrast to plants and green algae, which were derived from primary endosymbiosis, diatom evolution seems to be based on secondary endosymbiosis involving green and red algae as endosymbionts and a heterotrophic exosymbiont that is believed to have provided the ability to use silicate for an external cell wall made of silica. This review will discuss how results obtained by the application of genetics and genomics have impacted our understanding of diatoms. We will provide evidence for how their complex evolution has shaped key features of their biology and their global distribution by adaptive diversification to very different habitats.

A Review of the Evolution of the Diatoms from the Origin of the Lineage to Their Populations

The diatoms are, without doubt, one of the most successful groups of unicellular algae and contribute significantly to the global carbon cycle. They arose within the heterokont lineage no earlier than 250 Ma. The radiation of the pigmented heterokonts and that of the haptophytes and dinoflagellates was likely a response to the Permian–Triassic (PT) extinction event when host cells with a red algal endosymbiont had an adaptive advantage. There are three major clades of diatoms, which have been formally recognized at the class level, and their monophyly is clearly linked to the type of analysis done, the alignment by the secondary structure of the ribosomal RNA molecule, and the number of out-groups. The auxospore continues to be the defining feature of the deeper clades/classes of the diatoms; the three classes being the radial centrics, the bipolar centrics, which include the radial Thalassiosirales, and the pennate diatoms. Additional important defining features are the position of the cribrum in loculate areolae and the presence or absence of a central structure in the annulus. Sublineages within each class generally follow traditional orders of diatoms based on morphology. The araphid diatoms are shown to comprise two groups: the basal araphids that have both a properizonial auxospore like the bipolar, mediophycean diatoms and a perizonial auxospore like the raphid diatoms and the core araphids that have a perizonial auxospore like the raphid diatoms. Raphid diatoms are monophyletic with the Eunotiales as a basal lineage. Canal raphe diatoms have arisen twice. The Bacillariales are a basal divergence, whereas the Surirellales diverged more recently with the canal raphe evolving from amphoroid diatoms through Entomoneis to Surirella. Nearly all of the cosmopolitan diatom species that have been investigated with molecular techniques have been shown to be composed of cryptic species. Breeding studies help to confirm that the cryptic species conform to a biological species concept and underscore the premise that the diatoms are underclassified as a group at the species level. Genetic diversity studies have shown that the diatoms have strongly structured populations both spatially and temporally.

NEW MOLECULAR TOOLS: APPLICATION OF THE µAQUA PHYLOCHIP AND CONCOMITANT FISH PROBES TO STUDY FRESHWATER PATHOGENS FROM SAMPLES TAKEN ALONG THE TIBER RIVER, ITALY

Current knowledge about aquatic pathogens are scarce because bacteria, protozoans, algae and their toxins occur at low concentrations, making them difficult to measure directly or to filter sufficient volumes to facilitate detection. We developed and validated tools to detect pathogens in freshwater systems. To evaluate impacts on water quality, a phylogenetic microarray was developed in the EU project μAQUA to detect simultaneously numerous pathogens and was applied in MicroCoKit, to samples taken from four locations from two seasons for two years along the length of the Tiber River, Italy. The sites ranged from a pristine site near its source to ones contaminated by agricultural, industrial and anthropogenic waste moving downstream to near its mouth. Fifty litres were collected and concentrated using a hollow-fibre ultrafiltration, a rapid method with minimal cell loss to provide a concentrate for downstream analysis. The 60 Da cut-off ensures many organics, such as toxins, will be concentrated for analysis. Aliquots from the concentrate were preserved in TRI-Reagent and total RNA extracted, labelled and hybridised to the phylochip to detect pathogenic bacteria, protozoa and toxic cyanobacteria. The microarray results gave positive signals for all pathogens. Calibration curves enabled us to infer cell concentrations. Cross validation was performed using FISH probes for selected toxic cyanobacteria and hybridised to aliquots taken from the raw water prior to concentration by the hollow fibre filters.

PERMANENT GENETIC RESOURCES NOTE: Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2010 30 September 2010

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross‐tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.