Vitamin B₁₂-dependent algae are unable to grow in medium made with natural filter-sterilized seawater or freshwater unless a vitamin B₁₂ supplement is added to the culture, suggesting that the concentration of free vitamin B₁₂ in the natural environment in generally too low to support the growth of vitamin B₁₂-dependent algae.  The source of cobalamin for algae appears to be vitamin B₁₂-synthesizing bacteria, because when these organisms are grown in co-culture, the algae no longer require the vitamin. In return for vitamin B₁₂, the bacteria are able to use the products of algal photosynthesis to grow, indicating an important and unsuspected symbiosis. Using the green alga <i>Chlamydomonas reinhardtii </i>as a model organism, RT-PCR analysis has demonstrated that vitamin B₁₂ regulates the expression of the gene encoding the vitamin B₁₂-independent methionine synthase. Cobalamin is likely to regulate the expression of this gene by binding directly to the mRNA and influencing its secondary structure, since affinity binding studies suggest that the mRNAs that encode for the methionine synthase proteins have a high affinity for cobalamin. Further work in <i>C. reinhardtii </i>and <i>Euglena gracilis </i>has shown that a cell wall protein is involved in the high affinity uptake of vitamin B₁₂, since mutants deficient in cell wall biosynthesis are impaired in their ability to take up the vitamin. The <i>C. reinhardtii </i>protein has been purified by affinity chromatography, and identified by mass spectrometry. The complete cDNA encoding the <i>C. reinhardtii </i>vitamin B₁₂ binding protein has been cloned, and the protein shown to be a member of the pherophorin family, a well-known group of green algal cell wall proteins. A combination of techniques has indicated that the protein is glycosylated, and that the sugar moieties are attached to a central domain that contains a significant number of hydroxyproline residues.

The focus of this PhD project is the functional and molecular diversity of Leptocylindraceae diatom species, the study of which can lead to a better understanding of long standing questions regarding the ecology and evolution of phytoplankton. A wide range of tools, spanning from microscopical observations and physiological measurements to molecular techniques and high throughput sequencing, is utilized during this attempt. The genus <i>Leptocylindrus</i> has been chosen as the main target species due its worldwide and at the same time local importance in the Gulf of Naples and also because of the already extended study of the species in Stazione Zoologica Anton Dohrn (SZN) towards the direction mentioned above. Leptocylindraceae are centric diatoms that occupy a basal position in the diatom phylogeny and are abundant in marine plankton worldwide. In the Gulf of Naples (GoN), five out of the six species are found; <i>L. minimus</i> is known to be absent from the Mediterranean environment. The family shows a morphological conservation but the seasonal patterns between the species differ considerably. Indeed, physiological experiments of two <i>Leptocylindrus</i> species that show contrasting seasonality confirmed their opposed preferences regarding temperature as well as a high intraspecific phenological variability. In addition, the analysis of transcriptomes acquired for the three temperatures of one of them (<i>L. aporus</i>) indicated the possibly important role of transposable elements in response to stress and diatom adaptation. Furthermore, the transcriptomes of all <i>Leptocylindrus</i> species were explored in order to detect basic intra- and interspecific similarities and/or differences. HTS sequencing data from the MareChiara station in GoN and from the Tara expedition in the world’s seas were analyzed in order to assess the actual diversity of this important diatom family. A significant level of intraspecific variability was detected while the distribution of species and populations at spatial and temporal scale supported the functional differences among them that account for their distinct seasonality and their adaptation to different environmental conditions.

The number and abundance of diatom species in environmental samples are counted traditionally by means of light microscopy (LM). However, recognizing –let alone, counting- species is often challenging because of the existence of cryptic species and intraspecific phenotypic plasticity. Proper characterization requires isolation of cells, growing them into monoclonal cultures, and characterizing the cultures genetically and morphologically. However, not all species grow in culture, featureless ones are less likely to be isolated, and the procedure is laborious. High-throughput sequencing (HTS) metabarcoding bypasses morphology; DNA is collected from environmental samples, a particular marker sequenced, and the resulting sequences sorted into clusters or terminal clades assumed to represent species. Yet, reference barcodes of taxonomically validated species are needed to identify these clades. This exercise is the main aim of my thesis. Since it is impossible to do this for all the diversity within a PhD thesis project, we selected Chaetocerotaceae, an abundant and diverse family of marine planktonic diatoms, containing two genera: <i>Chaetoceros</i> and <i>Bacteriastrum</i>. Its members uniquely share setae; thin siliceous tubes emerging from the valve corners, facilitating detection in samples. Strains were obtained from the Gulf of Naples (GoN), from Central Chile and Roscoff – at sites for which LTER time series data are available. A total of 270 strains were obtained from these sites, and their 18S- and partial 28S rDNA sequences and morphological information gathered. The strains grouped into 60 genetically distinct species, thus providing a dataset of validated Chaetocerotacean 18S reference barcodes. Inferred molecular phylogenies showed monophyletic Chaetocerotaceae as well as monophyletic <i>Bacteriastrum</i> inside paraphyletic <i>Chaetoceros</i>, and the presence of cryptic diversity. To start with taxonomic updates, the species <i>C. sporotruncatus</i> and <i>C. dichatoensis</i> were described within the <i>C. socialis</i> species-complex based on spore morphology and sequence differences. Several rDNA sequences contained spliceosomal introns (ca. 100bp) and/or group-I introns (ca. 400bp). Phylogenies inferred from the introns did not corroborate rDNA phylogenies, suggesting horizontal gene transfer. Presence/absence of introns in conspecific strains sampled in different seasons suggests population differentiation between these seasons. A HTS dataset consisting of V4-sequences (part of 18S) from 48 seawater samples taken over the seasons in the GoN revealed 76 terminal clades of which 46 grouped with a reference barcode. Some of these species occur year-round whereas most others are seasonal. Surprisingly, of the 30 clades belonging to unknown Chaetocerotacean species, two appear to be among the most abundant in the GoN.

Charophytes playa significant role in ecological succession and the community structure of many water bodies. Thirty three species have been recorded in the UK; however the future existence of 17 species. is now considered threatened due to habitat degradation and loss. The aim of this thesis was to investigate limits of environmental tolerance for British charophytes with particular reference to water quality and rarer species. The work was carried out in four stages: 1. Field surveys at 49 major British charophyte sites (2004-2007) to record (a) the abundance of all macrophyte species, (b) charophyte perfonnance as indicated by the efficiency of photosystem IT - (Fv/Fm), and (c) the chemical and physical properties of waters and sediment. 2. Intensive studies at three nationally important charophyte sites: Slapton Ley (Devon), Orton Pits (Cambridgeshire) and Hickling Broad (Norfolk) to include analysis of interstitial water chemistry. 3. Probabilistic detennination of environmental ranges for charophytes as derived via pair wise and multivariate statistical analysis of collected field data. 4. Hypotheses generated from analysis ofthe field data to demonstrate that environmental concentrations of copper, inorganic phosphate and salinity were key limiting factors of charophyte survival were tested via a series of laboratory investigations. The survey results indicate that the probability of charophytes occupying a site is dependent upon minimum concentrations of filterable calcium, chloride, magnesium, sodium, and sulphate; it was also limited by maximum environmental concentrations of filterable cobalt (0.36 ,ugr1 ), copper (4.9 ,ugr1 ), manganese (16 mgr1 ), nitrate (2.6 mgr1 0.58 mgr1 N), inorganic phosphate (56 ,ugr1 , 18.3 ,ugr1 P) and silicate (6.2 mgr1 ) • The probability of finding charophytes decreased significantly in waters of high lightextinction coefficient (Kd > 4.4), in fluid sediments or under competitive canopies. The probability of charophyte presence at a site is greatly reduced by· competitive succession pressure from Phragmites australis. Charophyte species may be grouped according to tolerance of the conductivity of open and interstitial waters and also according to interstitial water Eh and pH. Charophytes are also sensitive to low open water Eh, which is reflected as photosystem-IT stress. Environmental nitrate concentrations pose the widest threat to charophyte habitats, whilst dissolved cobalt lowers probability of presence at the lowest concentrations; copper, manganese and inorganic phosphate were also shown to limit the probability of charophyte survival at concentrations commonly recorded in the field. Of three SSSI sites investigated, none was in favourable condition: Slapton Ley was in danger of succeeding to Carr and reed-swamp, Hickling Broad contained no charophytes by 2007 and Orton Pits (Cambridgeshire) was suffering from diffuse nitrate and phosphorus pollution, with point-source pollution from copper. The results of the laboratory experiments re-enforced the field survey findings showing that copper caused reduced growth at 50 ,ugrl in all species tested, with a reduction of photosynthetic function at 100 ,ugrl . This concentration also caused a loss of rhizoids in Chara intermedia, and is similar to the mean copper concentrations (97 ,ugrl ) recorded at 70 sample points in the interstitial waters ofHickling Broad in August 2006. Growth rates of Charophytes cultured in a phosphate rich solution were significantly reduced at 100 ,ugrl P04 (32.6 ,ugrl P), however photosynthetic function remained unstressed suggesting alternative mechanisms of interference. Changes in salinity were shown to affect growth and photosynthetic function in three species previously common to Hickling Broad in different ways; Chara intermedia performed best at 12.5-25% addition of seawater to the recent median salinities, however Chara connivens and Nitellopsis obtusa performed better at a 12.5% reduction from recent median salinities. The results suggest that should recovery ever take place, changes in salinity are likely to affect the charophyte species assemblage in Hickling Broad and that such a recovery is likely to be hindered by deep sediments, interstitial heavy metal concentrations, internal phosphorus reserves and saline ingress.

Perturbations to environmental conditions experienced by any one species, can invoke a physiological and/or behavioural response that may be expressed morphologically and ecologically. This has important implications for diatom taxonomy and the use of diatoms as environmental indicators. The reliable recognition of a taxon with a particular life history, morphology, ecology and physiology may necessitate the adoption of narrower species concepts than those in current floras. This would have the advantage of clarifying the ecological ranges of taxa used in biological monitoring programmes. Gomphonema parvulum has long been a taxonomic problem. The species exhibit considerable is found over a wide range of environmental conditions. lt has also been used as an indicator of &quot;pollution&quot; in diatom indices of water quality. Clones of G. parvulum, exhibiting a range of morphologies, were isolated from different sites into unialgal culture. Experimental investigations on the clones, tested against different environmental variables to determine ecological tolerances of different isolates, occasionally produced auxospores. This rare opportunity allowed the morphology of particular clones to be studied over their full size range (initial cell through to mother cells). Additionally, aspects of diatom behaviour, including sexual reproduction could be observed and recorded. Results indicate that not only are there differences in cell shape and behaviour with size and environmental condition, but in some G. parvulum clones, heteropolarity is not determined in the auxospore or initial cell, but becomes established after a series of vegetative divisions. This increases the likelihood that specimens may have been incorrectly identified as different species or varieties (G. parvulum complex encompasses two previously described species, G. gracile and G. hebridense), underlining the need for experimental studies and culturing. These observations argue strongly against reliance on diatom valve morphology as the sole criterion on which to delimit taxa, and provide a compelling argument for the benefits of algal culturing and observation of live material. There are serious issues for the sampling of waters and the use of diatom indices of water quality. Community analyses, water quality indices and taxonomic studies will be invalid, especially if different parts of the life cycle are shown to be ecologically as well as morphological variable.

The relationships between photosynthesis and acetylene reduction (nitrogen fixation) have been examined, mainly in the unicellular blue-green alga Gloeocapsa, though for comparative purposes, a second unicellular alga, Aphanothece pellida, and the filamentous heterocystous alga, Anabaena cylindrica, have also been studied. Glococapsa can fix nitrogen in the dark, so there is no obligatory link between photosynthesis and nitrogen fixation in this organism. However, studies using inhibitors of photosynthesis suggest that, in the light, electrons derived photosynthetically from water can be used, directly for nitrogen fixation by Gloeocapsa. This suggestion is supported by the action spectra and Emerson enhancements of photosynthesis and nitrogen fixation. Photosynthesis apparently also supplies ATP for nitrogen fixation in the light, though in the dark both oxidative and substrate-level phosphorylation can support nitrogen fixation. Finally, possible mechanisms by which the oxygen-sensitive nitrogenase of Gloeocapsa might be protected from photosynthetically produced oxygen were also examined. Gloeocapsa can remove toxic products derived from oxygen, such as superoxide and hydrogen peroxide, but it proved difficult to assess the role of this system under normal conditions of growth. Indeed, it is possible that it is a combination of several mechanisms which prevents nitrogenase from inactivation by oxygen.

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