Extended phylogeny and morphology of marine and freshwater choanoflagellates with additional methodological studies on heterotrophic flagellates

Heterotrophic flagellates (HF) are small, ubiquitously distributed protists. As main bacterial feeders, they play a significant role in aquatic and terrestrial food webs. To ensure reliable investigations of the diversity and phylogeny of HF and especially choanoflagellates, a short, user-friendly guide to common heterotrophic freshwater flagellates was developed in the present dissertation. It aims at facilitating an easier morphospecies identification using morphological characteristics of the living organisms. Due to the large diversity and specific properties of HF, a reliable quantification of these organisms is very challenging. Different quantification methods such as the live-counting technique, various fixation methods, a cultivation method and molecular tools were thus compared and optimized regarding various environmental studies. On the basis of this comparison different recommendations have been provided. Among the variety of different groups of HF, special attention was paid to the group of choanoflagellates in the present doctoral thesis. This group is particularly interesting regarding evolutionary aspects on the basis of animal origin as they are known to be the closest protistan relatives to the Metazoa within the group of Opisthokonta. Nonetheless, the internal systematics of choanoflagellates - especially of the order Craspedida - is still controversially discussed as no clear monophyletic clustering could be discovered up to now. Different morphological and life cycle forms exist, inter alia, the presence or absence of a theca (organic cell covering) and the ability to form colonies. Here, the sequencing of the 18S and 28S ribosomal DNA and morphological description of twelve choanoflagellate isolates (marine, brackish, and freshwater) from world-wide sampling points could reveal new insights into the taxonomy and systematics of the order Craspedida. Five of the isolates were additionally included in a six-gene phylogenetic analysis (18S rDNA, 28S rDNA, hsp90, tubA, EF-1A and EFL) to obtain an enhanced phylogenetic resolution and evolutionary understanding of choanoflagellates. Besides, it was also possible to characterize a completely new group of undescribed choanoflagellate sequences from suboxic/anoxic environments, closely related to the choanoflagellate order Acanthoecida. This group could be described by one isolate which was assigned to a new genus with a surprising morphological similarity to the order Craspedida. Taken together, this combination of both morphological and molecular data extended the existing choanoflagellate sequence database by about one third and might provide the potential basis for a complete taxonomic revision of choanoflagellates and for insights into their evolution and the evolution of multicellularity. Furthermore, the difficult isolation, cultivation and sequencing processes of choanoflagellates could be partially optimized. Additional ecological studies were carried out regarding salinity tolerances, lorica inducement (covering of acanthoecid choanoflagellates) and feeding modes

Potential contribution of surface-dwelling Sargassum algae to deep-sea ecosystems in the southern North Atlantic

Deep-sea ecosystems, limited by their inability to use primary production as a source of carbon, rely on other sources to maintain life. Sedimentation of organic carbon into the deep sea has been previously studied, however, the high biomass of sedimented Sargassum algae discovered during the VEMA Transit expedition in 2014/2015 to the southern North Atlantic, and its potential as a regular carbon input, has been an underestimated phenomenon. To determine the potential for this carbon flux, a literature survey of previous studies that estimated the abundance of surface water Sargassum was conducted. We compared these estimates with quantitative analyses of sedimented Sargassum appearing on photos taken with an autonomous underwater vehicle (AUV) directly above the abyssal sediment during the expedition. Organismal communities associated to Sargassum fluitans from surface waters were investigated and Sargassum samples collected from surface waters and the deep sea were biochemically analyzed (fatty acids, stable isotopes, C:N ratios) to determine degradation potential and the trophic significance within deep-sea communities. The estimated Sargassum biomass (fresh weight) in the deep sea (0.07 − 3.75 g/m2) was several times higher than that estimated from surface waters in the North Atlantic (0.024 – 0.84 g/m2). Biochemical analysis showed degradation of Sargassum occurring during sedimentation or in the deep sea, however, fatty acid and stable isotope analysis did not indicate direct trophic interactions between the algae and benthic organisms. Thus, it is assumed that components of the deep-sea microbial food web form an important link between the macroalgae and larger benthic organisms. Evaluation of the epifauna showed a diverse nano- micro-, meio, and macrofauna on surface Sargassum and maybe transported across the Atlantic, but we had no evidence for a vertical exchange of fauna components. The large-scale sedimentation of Sargassum forms an important trophic link between surface and benthic production and has to be further considered in the future as a regular carbon input to the deep-sea floor in the North Atlantic

A six-gene phylogeny provides new insights into choanoflagellate evolution

Recent studies have shown that molecular phylogenies of the choanoflagellates (Class Choanoflagellatea) are in disagreement with their traditional taxonomy, based on morphology, and that Choanoflagellatea requires considerable taxonomic revision. Furthermore, phylogenies suggest that the morphological and ecological evolution of the group is more complex than has previously been recognized. Here we address the taxonomy of the major choanoflagellate order Craspedida, by erecting four new genera. The new genera are shown to be morphologically, ecologically and phylogenetically distinct from other choanoflagellate taxa. Furthermore, we name five novel craspedid species, as well as formally describe ten species that have been shown to be either misidentified or require taxonomic revision. Our revised phylogeny, including 18 new species and sequence data for two additional genes, provides insights into the morphological and ecological evolution of the choanoflagellates. We examine the distribution within choanoflagellates of these two additional genes, EF-1A and EFL, closely related translation GTPases which are required for protein synthesis. Mapping the presence and absenc

Extended phylogeny of the Craspedida (Choanomonada)

Currently choanoflagellates are classified into two distinct orders: loricate Acanthoecida and non-loricate Craspedida. The morphologically based taxonomy of the order Craspedida is in need of a revision due to its controversial, paraphyletic and inconsistent systematics and nomenclature. In this study, we add molecular data (SSU and parts of the LSU rDNA) of six new Craspedida species isolated from saline, brackish and freshwater habitats to the existing knowledge. Four of these six organisms could be described as new species: Paramonosiga thecata, Salpingoeca euryoecia, Salpingoeca ventriosa, Sphaeroeca leprechaunica, whereas two are assigned to previous morphologically described species: Salpingoeca fusiformis Saville Kent, 1880 and Salpingoeca longipes Saville Kent, 1880. Paramonosiga is established as a new genus of the Craspedida based on its phylogenetic position. Extending the dataset by six additional sequences shows that the craspedid taxonomy is still unsolved as the type specimen Salpingoeca gracilis has not yet been sequenced and hence a clear assignment to the genus Salpingoeca is not possible. Trying to assign morphological and ecological data to phylogenetic clades is not successful. We give an improved/emended morphological diagnosis for the two redescribed species and add molecular data for all six species, shedding light on their phylogenetic position. (C) 2014 Published by Elsevier GmbH

Methodological Studies on Estimates of Abundance and Diversity of Heterotrophic Flagellates from the Deep-Sea Floor

Extreme environmental conditions in the deep sea hamper access to protist communities. In combination with the potentially highly diverse species composition, it demands a wide range of methods to be applied at the same time to guarantee a high resolution of quantitative and qualitative studies of deep-sea heterotrophic flagellates (HF). Within this study, we present a possible combination of several culture-independent and culture-dependent methods available for investigating benthic deep-sea HF communities. Besides live-counting and fixation of HF, we refer to cultivation methods and molecular surveys using next generation sequencing. Laboratory ecological experiments under deep-sea conditions (high pressure, low temperature) could allow the approval of the potential deep-sea origin of sampled HF. The combination of different methods offers a unique possibility to receive detailed information on nanofaunal life in the deep sea. Specific fixation techniques to preserve samples directly at the sampling depth must be applied in further studies to reflect the real biodiversity of the largest habitat on earth

A Comparison of Methods to Analyze Aquatic Heterotrophic Flagellates of Different Taxonomic Groups

Heterotrophic flagellates contribute significantly to the matter flux in aquatic and terrestrial ecosystems. Still today their quantification and taxonomic classification bear several problems in field studies, though these methodological problems seem to be increasingly ignored in current ecological studies. Here we describe and test different methods, the live-counting technique, different fixation techniques, cultivation methods like the liquid aliquot method (LAM), and a molecular single cell survey called aliquot PCR (aPCR). All these methods have been tested either using aquatic field samples or cultures of freshwater and marine taxa. Each of the described methods has its advantages and disadvantages, which have to be considered in every single case. With the live-counting technique a detection of living cells up to morphospecies level is possible. Fixation of cells and staining methods are advantageous due to the possible long-term storage and observation of samples. Cultivation methods (LAM) offer the possibility of subsequent molecular analyses, and aPCR tools might complete the deficiency of LAM in terms of the missing detection of non-cultivable flagellates. In summary, we propose a combination of several investigation techniques reducing the gap between the different methodological problems. (C) 2017 Elsevier GmbH. All rights reserved