Micractinium tetrahymenae (Trebouxiophyceae, Chlorophyta), a new endosymbiont isolated from ciliates

Endosymbiosis between coccoid green algae and ciliates are widely distributed and occur in various phylogenetic lineages among the Ciliophora. Most mixotrophic ciliates live in symbiosis with different species and genera of the so-called Chlorella clade (Trebouxiophyceae). The mixotrophic ciliates can be differentiated into two groups: (i) obligate, which always live in symbiosis with such green algae and are rarely algae-free and (ii) facultative, which formed under certain circumstances such as in anoxic environments an association with algae. A case of the facultative endosymbiosis is found in the recently described species of Tetrahymena, T. utriculariae, which lives in the bladder traps of the carnivorous aquatic plant Utricularia reflexa. The green endosymbiont of this ciliate belonged to the genus Micractinium. We characterized the isolated algal strain using an integrative approach and compared it to all described species of this genus. The phylogenetic analyses using complex evolutionary secondary structure-based models revealed that this endosymbiont represents a new species of Micractinium, M. tetrahymenae sp. nov., which was further confirmed by the ITS2/CBC approach

Endosymbiotic Green Algae in Paramecium bursaria: A New Isolation Method and a Simple Diagnostic PCR Approach for the Identification

Paramecium bursaria is a single-celled model organism for studying endosymbiosis among ciliates and green algae. Most strains of P. bursaria bear either Chlorella variabilis or Micractinium conductrix as endosymbionts. Both algal genera are unicellular green algae characterized by cup-shaped chloroplasts containing a single pyrenoid and reproduction by autospores. Due to their size and only few morphological characteristics, these green algae are very difficult to discriminate by microscopy only. Their cultivation is laborious and often unsuccessful, but we developed a three-step isolation method, which provided axenic cultures of endosymbionts. In addition to the time-consuming isolation, we developed a simple diagnostic PCR identification method using specific primers for C. variabilis and M. conductrix that provided reliable results. One advantage of this approach was that the algae do not have to be isolated from their host. For a comparative study, we investigated 19 strains of P. bursaria from all over the world (new isolates and available laboratory strains) belonging to the five known syngens (R1–R5). Six European ciliate strains belonging to syngens R1 and R2 bore M. conductrix as endosymbiont whereas C. variabilis was discovered in syngens R1–R5 having worldwide origins. Our results reveal the first evidence of C. variabilis as endosymbiont in P. bursaria in Europe

Molecular Phylogeny of Unicellular Marine Coccoid Green Algae Revealed New Insights into the Systematics of the Ulvophyceae (Chlorophyta)

Most marine coccoid and sarcinoid green algal species have traditionally been placed within genera dominated by species from freshwater or soil habitats. For example, the genera Chlorocystis and Halochlorococcum contain exclusively marine species; however, their familial and ordinal affinities are unclear. They are characterized by a vegetative cell with lobated or reticulated chloroplast, formation of quadriflagellated zoospores and living epi- or endophytically within benthic macroalgae. They were integrated into the family Chlorochytriaceae which embraces all coccoid green algae with epi- or endophytic life phases. Later, they were excluded from the family of Chlorococcales based on studies of their life histories in culture, and transferred to their newly described order, Chlorocystidales of the Ulvophyceae. Both genera form a “Codiolum”-stage that serves as the unicellular sporophyte in their life cycles. Phylogenetic analyses of SSU and ITS rDNA sequences confirmed that these coccoid taxa belong to the Chlorocystidales, together with the sarcinoid genus Desmochloris. The biflagellated coccoid strains were members of the genus Sykidion, which represented its own order, Sykidiales, among the Ulvophyceae. Considering these results and the usage of the ITS-2/CBC approach revealed three species of Desmochloris, six of Chlorocystis, and three of Sykidion. Three new species and several new combinations were proposed

Widespread Occurrence of Two Planktonic Ciliate Species (<i>Urotricha</i>, Prostomatida) Originating from High Mountain Lakes

Ciliates of the genus Urotricha are widely distributed and occur in almost any freshwater body. Thus far, almost all species have been described from morphology only. Here, we applied an integrative approach on the morphology, molecular phylogeny and biogeography of two species isolated from high mountain lakes in the Central Alps, Austria. As these remote lakes are known to have water temperatures Urotricha species resembled morphological features of several congeners. An accurate species assignment was difficult due to several overlapping characteristics. However, we tentatively attributed the investigated species to Urotricha nais and Urotricha globosa. The biogeographic analyses revealed their occurrence in Europe, Africa and Asia, and no correlations to (cold) temperatures were found. Our findings suggest that these two urotrichs, originating from two cold and remote habitats, are probably cryptic species well adapted to their harsh environment

Consensus ITS-2 secondary structure of <i>Coccomyxa</i> based on comparison of the structures of each strain (see S2 Fig).

<p>The 73 base-pairs used for the ITS-2 DNA Barcode are numbered and marked in black and grey circles.</p

Morphology and its plasticity of the new species of <i>Coccomyxa</i>.

<p><b>A</b>. <i>Coccomyxa polymorpha</i>, <b>B</b>. <i>Coccomyxa vinatzeri</i> (vegetative cells in the bottom row were stained with India Ink), and <b>C.</b><i>Coccomyxa galuniae</i> (vegetative cells in the upper right corner were stained with India Ink). Scale bar = 10 μm.</p

Evaluating the Species Boundaries of Green Microalgae (<i>Coccomyxa</i>, Trebouxiophyceae, Chlorophyta) Using Integrative Taxonomy and DNA Barcoding with Further Implications for the Species Identification in Environmental Samples

<div><p>Integrative taxonomy is an approach for defining species and genera by taking phylogenetic, morphological, physiological, and ecological data into account. This approach is appropriate for microalgae, where morphological convergence and high levels of morphological plasticity complicate the application of the traditional classification. Although DNA barcode markers are well-established for animals, fungi, and higher plants, there is an ongoing discussion about suitable markers for microalgae and protists because these organisms are genetically more diverse compared to the former groups. To solve these problems, we assess the usage of a polyphasic approach combining phenotypic and genetic parameters for species and generic characterization. The application of barcode markers for database queries further allows conclusions about the ‘coverage’ of culture-based approaches in biodiversity studies and integrates additional aspects into modern taxonomic concepts. Although the culture-dependent approach revealed three new lineages, which are described as new species in this paper, the culture-independent analyses discovered additional putative new species. We evaluated three barcode markers (V4, V9 and ITS-2 regions, nuclear ribosomal operon) and studied the morphological and physiological plasticity of <i>Coccomyxa</i>, which became a model organism because its whole genome sequence has been published. In addition, several biotechnological patents have been registered for <i>Coccomyxa</i>. <i>Coccomyxa</i> representatives are distributed worldwide, are free-living or in symbioses, and colonize terrestrial and aquatic habitats. We investigated more than 40 strains and reviewed the biodiversity and biogeographical distribution of <i>Coccomyxa</i> species using DNA barcoding. The genus <i>Coccomyxa</i> formed a monophyletic group within the Trebouxiophyceae separated into seven independent phylogenetic lineages representing species. Summarizing, the combination of different characteristics in an integrative approach helps to evaluate environmental data and clearly identifies microalgae at generic and species levels.</p></div

Phenotypic plasticity of various <i>Coccomyxa</i> strains under different salinities.

<p>A-F <i>C</i>. <i>polymorpha</i> (CAUP H5101), G-L <i>C</i>. <i>subellipsoidea</i> (CCAP 812/3), M-R <i>C</i>. <i>simplex</i> (CCAP 216/24), and S-X <i>C</i>. <i>vinatzeri</i> (ASIB V16). A, G, M and S 3BBM+V (0.2% NaCl; control); B, H, N, and T 3N-BBM+V with 0.6% NaCl; C, I, O, and U with 1.2% NaCl, D, J, P, and V with 1.8% NaCl, E, K, Q, and W with 2.5% NaCl, F, L, R, and X with 3.3% NaCl. Scale bar = 20 μm.</p