Genetic, physiological and ecological diversity of the diatom genus <i>Leptocylindrus</i>

Diatoms are among the most productive photoautotrophic organisms on Earth. Within the diatom genus Leptocylindrus, two species, L. danicus and L. minimus, are reported as abundant in coastal waters worldwide and in the Gulf of Naples (GoN). This thesis aimed at a closer characterisation of Leptocylindrus species through different approaches, including the study of their morphology, molecular phylogeny, metagenomics and biochemistry. Eighty-three strains from the GoN and one strain (CCMP 1856) from the Atlantic US coast were analysed. Based on the morphological, molecular and life cycle differences, the taxonomy of the genus was revised. The GoN species previously identified as L. minimus was in fact identified as L. belgicus Meunier and was placed in a new genus, Tenuicylindrus Nanjappa and Zingone which, along with Leptocylindrus Cleve, belongs to the family Leptocylindraceae. Five species in all were included in the genus Leptocylindrus: L. danicus Cleve and L. minimus Gran, two novel species L. hargravesii and L. convexus, and L. aporus (Hargraves) Nanjappa & Zingone, which was raised from the variety to the species status. The real Leptocylindrus minimus was not found in the GoN. To address the distribution of the 6 species outside the GoN, two metagenomic databases, BioMarKs (Europe) and Tara Oceans (worldwide) were explored. Sequences of L. aporus, L. convexus and L. danicus were recovered at many sites in European waters and across the world's seas, while those of L. minimus were retrieved only in the Oslo fjord and those of T. belgicus were only found in the GoN and Oslo fjord. Additional diversity was observed in the Tara Oceans dataset but, in lack of morphological information, whether this diversity is real remains to be clarified. All species except L. minimus were also categorised based on the diversity in their oxylipins pathways. Leptocylindrus danicus and L. hargravesii shared common lipoxygenase pathways, different from the ones shared by the species L. aporus and L. convexus. Tenuicylindrus belgicus exhibits a pathway distinct from that of Leptocylindrus species. Species-specific compounds produced in minor quantities were also observed. Physiological experiments show that L. aporus can withstand higher (26 "C) temperature but not lower (12 CC) temperature, while L. danicus can withstand low temperature but not high temperature. This corresponds to the species occurrence in the natural environment, where L. aporus blooms during summer and L. danicus is found in all seasons except summer. Altogether, through an interdisciplinary approach, the studies described in this thesis provide substantial information that may have important implications in the field of ecology, evolution, conservation biology and biotechnology

Diatom flagellar genes and their expression during sexual reproduction in Leptocylindrus danicus

Background: Flagella have been lost in the vegetative phase of the diatom life cycle, but they are still present in male gametes of centric species, thereby representing a hallmark of sexual reproduction. This process, besides maintaining and creating new genetic diversity, in diatoms is also fundamental to restore the maximum cell size following its reduction during vegetative division. Nevertheless, sexual reproduction has been demonstrated in a limited number of diatom species, while our understanding of its different phases and of their genetic control is scarce. Results: In the transcriptome of Leptocylindrus danicus, a centric diatom widespread in the world's seas, we identified 22 transcripts related to the flagella development and confirmed synchronous overexpression of 6 flagellum-related genes during the male gamete formation process. These transcripts were mostly absent in the closely related species L. aporus, which does not have sexual reproduction. Among the 22 transcripts, L. danicus showed proteins that belong to the Intra Flagellar Transport (IFT) subcomplex B as well as IFT-A proteins, the latter previously thought to be absent in diatoms. The presence of flagellum-related proteins was also traced in the transcriptomes of several other centric species. Finally, phylogenetic reconstruction of the IFT172 and IFT88 proteins showed that their sequences are conserved across protist species and have evolved similarly to other phylogenetic marker genes. Conclusion: Our analysis describes for the first time the diatom flagellar gene set, which appears to be more complete and functional than previously reported based on the genome sequence of the model centric diatom, Thalassiosira pseudonana. This first recognition of the whole set of diatom flagellar genes and of their activation pattern paves the way to a wider recognition of the relevance of sexual reproduction in individual species and in the natural environment

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract: Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Abstract: Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways

Genetic, physiological and ecological diversity of the diatom genus Leptocylindrus

Diatoms are among the most productive photoautotrophic organisms on Earth. Within the diatom genus Leptocylindrus, two species, L. danicus and L. minimus, are reported as abundant in coastal waters worldwide and in the Gulf of Naples (GoN). This thesis aimed at a closer characterisation of Leptocylindrus species through different approaches, including the study of their morphology, molecular phylogeny, metagenomics and biochemistry. Eighty-three strains from the GoN and one strain (CCMP 1856) from the Atlantic US coast were analysed. Based on the morphological, molecular and life cycle differences, the taxonomy of the genus was revised. The GoN species previously identified as L. minimus was in fact identified as L. belgicus Meunier and was placed in a new genus, Tenuicylindrus Nanjappa and Zingone which, along with Leptocylindrus Cleve, belongs to the family Leptocylindraceae. Five species in all were included in the genus Leptocylindrus: L. danicus Cleve and L. minimus Gran, two novel species L. hargravesii and L. convexus, and L. aporus (Hargraves) Nanjappa & Zingone, which was raised from the variety to the species status. The real Leptocylindrus minimus was not found in the GoN. To address the distribution of the 6 species outside the GoN, two metagenomic databases, BioMarKs (Europe) and Tara Oceans (worldwide) were explored. Sequences of L. aporus, L. convexus and L. danicus were recovered at many sites in European waters and across the world's seas, while those of L. minimus were retrieved only in the Oslo fjord and those of T. belgicus were only found in the GoN and Oslo fjord. Additional diversity was observed in the Tara Oceans dataset but, in lack of morphological information, whether this diversity is real remains to be clarified. All species except L. minimus were also categorised based on the diversity in their oxylipins pathways. Leptocylindrus danicus and L. hargravesii shared common lipoxygenase pathways, different from the ones shared by the species L. aporus and L. convexus. Tenuicylindrus belgicus exhibits a pathway distinct from that of Leptocylindrus species. Species-specific compounds produced in minor quantities were also observed. Physiological experiments show that L. aporus can withstand higher (26 "C) temperature but not lower (12 CC) temperature, while L. danicus can withstand low temperature but not high temperature. This corresponds to the species occurrence in the natural environment, where L. aporus blooms during summer and L. danicus is found in all seasons except summer. Altogether, through an interdisciplinary approach, the studies described in this thesis provide substantial information that may have important implications in the field of ecology, evolution, conservation biology and biotechnology.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Oxylipin Diversity in the Diatom Family Leptocylindraceae Reveals DHA Derivatives in Marine Diatoms

Marine planktonic organisms, such as diatoms, are prospective sources of novel bioactive metabolites. Oxygenated derivatives of fatty acids, generally referred to as oxylipins, in diatoms comprise a highly diverse and complex family of secondary metabolites. These molecules have recently been implicated in several biological processes including intra- and inter-cellular signaling as well as in defense against biotic stressors and grazers. Here, we analyze the production and diversity of C-20 and C-22 non-volatile oxylipins in five species of the family Leptocylindraceae, which constitute a basal clade in the diatom phylogeny. We report the presence of species-specific lipoxygenase activity and oxylipin patterns, providing the first demonstration of enzymatic production of docosahexaenoic acid derivatives in marine diatoms. The differences observed in lipoxygenase pathways among the species investigated broadly reflected the relationships observed with phylogenetic markers, thus providing functional support to the taxonomic diversity of the individual species

Oxylipin Diversity in the Diatom Family Leptocylindraceae Reveals DHA Derivatives in Marine Diatoms

Marine planktonic organisms, such as diatoms, are prospective sources of novel bioactive metabolites. Oxygenated derivatives of fatty acids, generally referred to as oxylipins, in diatoms comprise a highly diverse and complex family of secondary metabolites. These molecules have recently been implicated in several biological processes including intra- and inter-cellular signaling as well as in defense against biotic stressors and grazers. Here, we analyze the production and diversity of C20 and C22 non-volatile oxylipins in five species of the family Leptocylindraceae, which constitute a basal clade in the diatom phylogeny. We report the presence of species-specific lipoxygenase activity and oxylipin patterns, providing the first demonstration of enzymatic production of docosahexaenoic acid derivatives in marine diatoms. The differences observed in lipoxygenase pathways among the species investigated broadly reflected the relationships observed with phylogenetic markers, thus providing functional support to the taxonomic diversity of the individual species

Oxylipin Diversity in the Diatom Family Leptocylindraceae Reveals DHA Derivatives in Marine Diatoms

Marine planktonic organisms, such as diatoms, are prospective sources of novel bioactive metabolites. Oxygenated derivatives of fatty acids, generally referred to as oxylipins, in diatoms comprise a highly diverse and complex family of secondary metabolites. These molecules have recently been implicated in several biological processes including intra- and inter-cellular signaling as well as in defense against biotic stressors and grazers. Here, we analyze the production and diversity of C20 and C22 non-volatile oxylipins in five species of the family Leptocylindraceae, which constitute a basal clade in the diatom phylogeny. We report the presence of species-specific lipoxygenase activity and oxylipin patterns, providing the first demonstration of enzymatic production of docosahexaenoic acid derivatives in marine diatoms. The differences observed in lipoxygenase pathways among the species investigated broadly reflected the relationships observed with phylogenetic markers, thus providing functional support to the taxonomic diversity of the individual species