Thomas Pröschold

Chloroidium, a common terrestrial coccoid green alga previously assigned to Chlorella (Trebouxiophyceae, Chlorophyta)

Ellipsoidal Chlorella-like species are very common in all kinds of aquatic and terrestrial habitats, and often identified as Chlorella saccharophila or C. ellipsoidea. However, the taxonomic status of these species remains unclear, because they are not related to the type species of the genus, Chlorella vulgaris. In this study, 23 strains isolated from different habitats, were investigated using a polyphasic approach, i.e. morphology and reproduction, ecophysiology, and combined SSU and ITS rDNA sequences. Phylogenetic analyses clearly demonstrated that these isolates formed a monophyletic lineage within the green algal class Trebouxiophyceae. All strains were characterized by ellipsoidal cell shape, unequal autospores during reproduction, and parietal chloroplasts, as well as by the biochemical capability to synthesize and accumulate the rather unusual polyol, ribitol. Although ribitol is a typical stress metabolite involved in osmotic acclimation, it can also be used as a chemotaxonomic marker. Comparative growth measurements under different temperature regimes indicated similar optimum growth temperatures and maximum growth rates in all studied Chlorella-like species. However, these were different from those of C. vulgaris. We therefore propose to transfer all Chlorella-like strains related to Chlorella saccharophila and C. ellipsoidea to the genus Chloroidium Nadson and to emend its diagnosis. We propose four new combinations: Chloroidium saccharophilum comb. nov., Chloroidium ellipsoideum comb. nov., Chloroidium angusto-ellipsoideum comb. nov. and Chloroidium engadinensis comb. nov. In contrast, Chlorella ellipsoidea sensu Punčochárová, which has other morphological and ecophysiological characters, should be assigned to the genus Pseudochlorella (P. pringsheimii comb. nov.).

UPDATING THE GENUS DICTYOSPHAERIUM AND DESCRIPTION OF MUCIDOSPHAERIUM GEN. NOV (TREBOUXIOPHYCEAE) BASED ON MORPHOLOGICAL AND MOLECULAR DATA

Recent molecular analyses of Dictyosphaerium strains revealed a polyphyletic origin of this morphotype within the Chlorellaceae. The type species Dictyosphaerium ehrenbergianum Nägeli formed an independent lineage within the Parachlorella clade, assigning the genus to this clade. Our study focused on three different Dictyosphaerium species to resolve the phylogenetic position of remaining species. We used combined analyses of morphology; molecular data based on SSU and internally transcribed spacer region (ITS) rRNA sequences; and the comparison of the secondary structure of the SSU, ITS‐1, and ITS‐2 for species and generic delineation. The phylogenetic analyses revealed two lineages without generic assignment and two distinct clades of Dictyosphaerium‐like strains within the Parachlorella clade. One clade comprises the lineages with the epitype strain of D. ehrenbergianum Nägeli and two additional lineages that are described as new species (Dictyosphaerium libertatis sp. nov. and Dictyosphaerium lacustre sp. nov.). An emendation of the genus Dictyosphaerium is proposed. The second clade comprises the species Dictyosphaerium sphagnale Hindák and Dictyosphaerium pulchellum H. C. Wood. On the basis of phylogenetic analyses, complementary base changes, and morphology, we describe Mucidosphaerium gen. nov with the four species Mucidosphaerium sphagnale comb. nov., Mucidosphaerium pulchellum comb. nov., Mucidosphaerium palustre sp. nov., and Mucidosphaerium planctonicum sp. nov.

Two new Dictyosphaerium-morphotype lineages of the Chlorellaceae (Trebouxiophyceae): Heynigia gen. nov. and Hindakia gen. nov.

Previous studies on the colonial coccoid green algal genus Dictyosphaerium have shown a polyphyletic origin of this morphotype within the Chlorellaceae. Recent molecular analyses assigned the type species D. ehrenbergianum to the Parachlorella clade of the Chlorellaceae. In the present study we focused on strains of D. tetrachotomum, one of the most frequent morphospecies of the genus in inland waters. Analyses of combined SSU and ITS rRNA gene sequences revealed that strains with D. tetrachotomum morphology do not share a close phylogenetic relationship with the type species D. ehrenbergianum. These strains are part of a subclade of the Chlorella clade of the Chlorellaceae, nested between members of Didymogenes and a second new lineage of different Dictyosphaerium-like strains. Due to the unique position of these strains in the tree, we here establish the new genera Hindakia and Heynigia. Morphological analysis and phylogenetic and secondary structure analyses of the SSU, ITS1 and ITS2 of Hindakia strains have revealed considerable differences within this subclade, leading to the separation of two distinct species: H. tetrachotoma and H. fallax. Closely related to this new genus are two colony-forming strains with spherical cells, constituting a distinct lineage consisting of Heynigia dictyosphaerioides, sp. nov., and Heynigia riparia, sp. nov. Further, our results have confirmed the polyphyletic origin of the Dictyosphaerium morphotype within the Chlorellaceae.

POLYPHYLETIC ORIGIN OF THE DICTYOSPHAERIUM MORPHOTYPE WITHIN CHLORELLACEAE (TREBOUXIOPHYCEAE)

The green algal Dictyosphaerium morphotype is characterized by spherical or oval cells connected by gelatinized strands to microscopic colonies, which are covered by prominent mucilaginous envelopes. Combined SSU and ITS rRNA gene sequence analyses revealed that this morphotype evolved independently both in the Chlorella and Parachlorella clades of the Chlorellaceae. It was shown that strains exhibiting the morphology of the type species Dictyosphaerium ehrenbergianum Nägeli established a sister lineage to Parachlorella. The strain D. ehrenbergianum CCAP 222/1A was designated as an authentic strain for establishing the epitype of the genus Dictyosphaerium. The comparison of this strain with the authentic strain of Parachlorella beijerinckii Krienitz, E. Hegewald, Hepperle, V. Huss, T. Rohr et M. Wolf (SAG 2046) showed considerable differences in the secondary structure of the ITS region. Within the whole ITS‐1 and ITS‐2 region, 27 compensatory base changes (CBCs) were recognized. In the conserved Helix III of the ITS‐2, five CBCs/HemiCBCs were detected. This is a conclusive argument for separation of these two species. The clear definition of Dictyosphaerium is intended to be the necessary starting point of taxonomic reevaluation of Dictyosphaerium‐like algae within different evolutionary lineages of the Chlorellaceae.

Polyphyletic distribution of bristle formation in Chlorellaceae: Micractinium, Diacanthos, Didymogenes and Hegewaldia gen. nov. (Trebouxiophyceae, Chlorophyta)

In the traditional sense, several families of Chlorococcales sensu lato, such as Golenkiniaceae, Micractiniaceae, and Scenedesmaceae contained taxa with bristle formation, whereas the Chlorellaceae contained only genera without bristles. However, phylogenetic analyses of the small subunit (SSU) and internal transcribed spacer (ITS) rDNA sequences have shown that the genera Micractinium, Diacanthos (formerly Micractiniaceae) and Didymogenes (formerly Scenedesmaceae) are closely related to the genus Chlorella. The bristle formation within the Chlorella‐clade is originated independently in four lineages: Micractinium pusillum, Diacanthos belenophorus, Didymogenes anomala, and Micractinium parvulum (also known as Golenkinia minutissima). The latter species is to exclude from the genus Micractinium. Consequently, we proposed the new genus Hegewaldia and transferred M. parvulum to this genus. In contrast, Diacanthos belenophorus is closely related to Micractinium pusillum. As a result, the new combination (Micractinium belenophorum comb. nov.) is proposed. Comparisons of the secondary structure of ITS‐1 and ITS‐2 rDNA sequences among the strains of Didymogenes and Hegewaldia are provided to support the species concept in these genera.

Phylogenetic relationship and divergence among planktonic strains of Arthrospira (Oscillatoriales, Cyanobacteria) of African, Asian and American origin deduced by 16S-23S ITS and phycocyanin operon sequences

Dadheech P.K., Ballot A., Casper P., Kotut K., Novelo E., Lemma B., Pröschold T. and Krienitz L. 2010. Phylogenetic relationship and divergence among planktonic strains of Arthrospira (Oscillatoriales, Cyanobacteria) of African, Asian and American origin deduced by 16S–23S ITS and phycocyanin operon sequences. Phycologia 49: 361–372. DOI: 10.2216/09-71.1 Arthrospira comprises multicellular, cylindrical, usually screwlike coiled trichomes and is cultivated commercially. In this study, 33 new strains of Arthrospira isolated from plankton samples collected in Mexico, East Africa and India were investigated and compared with 53 strains or samples of earlier considerations. The study included observations of morphological features and molecular phylogenetic analyses on the basis of nucleotide sequences of internal transcribed spacer (ITS) between 16S rRNA and 23S rRNA genes and partial sequences of beta and alpha subunits including intergenic spacer (cpcBA-IGS) of phycocyanin operon. Morphological traits of Arthrospira such as trichome width, type of coiling and apical cell were not always consistent in culture conditions. It was revealed that Arthrospira phylogeny on the basis of cpcBA-IGS locus was broadly comparable with the ITS region as both phylogenetic trees derived from nucleotide sequences could be divided into two main clusters. Cluster I comprised sequences from American strains mainly, whereas cluster II contained the sequences of the strains originating from Africa and Asia chiefly. Both genetic regions of the strains investigated in the present study coincidently showed a significant sequence divergence among Arthrospira strains from East Africa, India and Mexico indicating possible distinct evolutionary lineages.

MOLECULAR PHYLOGENY, ULTRASTRUCTURE, AND TAXONOMIC REVISION OF CHLOROGONIUM (CHLOROPHYTA): EMENDATION OF CHLOROGONIUM AND DESCRIPTION OF GUNGNIR GEN. NOV. AND RUSALKA GEN. NOV.(1).

We examined the molecular phylogeny and ultrastructure of Chlorogonium and related species to establish the natural taxonomy at the generic level. Phylogenetic analyses of 18S rRNA and RUBISCO LSU (rbcL) gene sequences revealed two separate clades of Chlorogonium from which Chlorogonium (Cg.) fusiforme Matv. was robustly separated. One clade comprised Cg. neglectum Pascher and Cg. kasakii Nozaki, whereas the other clade included the type species Cg. euchlorum (Ehrenb.) Ehrenb., Cg. elongatum (P. A. Dang.) Francé, and Cg. capillatum Nozaki, M. Watanabe et Aizawa. On the basis of unique ultrastructural characteristics, we described Gungnir Nakada gen. nov. comprising three species: G. neglectum (Pascher) Nakada comb. nov., G. mantoniae (H. Ettl) Nakada comb. nov., and G. kasakii (Nozaki) Nakada comb. nov. We also emended Chlorogonium as a monophyletic genus composed of Cg. euchlorum, Cg. elongatum, and Cg. capillatum. Because Cg. fusiforme was distinguished from the redefined Chlorogonium and Gungnir by the structure of its starch plate, which is associated with pyrenoids, we reclassified this species as Rusalka fusiformis (Matv.) Nakada gen. et comb. nov.

Species diversity in European Haematococcus pluvialis (Chlorophyceae, Volvocales)

Abstract: Haematococcus pluvialis has received much interest because it can accumulate large quantities of the pigment astaxanthin. While different isolates of H. pluvialis seem to differ considerably in their physiology, their phylogenetic diversity has not yet been thoroughly studied. We studied the species diversity in a set of temperate European strains of H. pluvialis based on internal transcribed spacer (ITS) rDNA and rbcL molecular phylogenies and determined their congruence with morphology and temperature preferences. The ITS rDNA phylogeny resolved three lineages with the European strains. Three additional lineages were not represented by our European strains. Both statistical parsimony network analysis and the general mixed Yule coalescent recovered these six lineages as different species. An ITS1 structure analysis revealed up to three compensatory base changes (CBCs) between each of the three European lineages, while one CBC was found between lineages in the ITS2 secondary structure. Yet the CBCs were not always present in all strains of each lineage. The rbcL was much less variable and separated only two of the three ITS lineages. We propose an epitype for H. pluvialis and describe the two lineages containing European strains as new species, Haematococcus rubicundus and Haematococcus rubens. Although there is broad morphological overlap, strains of H. pluvialis and H. rubens have, on average, more elongated cells, more noticeable cytoplasmic strands and a more (extreme) pear-shaped protoplast apex than H. rubicundus. Optimal temperatures for growth were similar for the three species and varied between 17°C and 23°C, which is lower than reported earlier from other strains. This study provides a robust phylogenetic framework for further ecophysiological study of Haematococcus.

Identification of Cyanobacteria in a Eutrophic Coastal Lagoon on the Southern Baltic Coast

Cyanobacteria are found worldwide in various habitats. Members of the picocyanobacteria genera Synechococcus and Prochlorococcus dominate in oligotrophic ocean waters. Other picocyanobacteria dominate in eutrophic fresh or brackish waters. Usually, these are morphologically determined as species of the order Chroococcales/clade B2. The phytoplankton of a shallow, eutrophic brackish lagoon was investigated. Phytoplankton was dominated by Aphanothece-like morphospecies year-round for more than 20 years, along a trophy and salinity gradient. A biphasic approach using a culture-independent and a culture-dependent analysis was applied to identify the dominant species genetically. The 16S rRNA gene phylogeny of clone sequences and isolates indicated the dominance of Cyanobium species (order Synechococcales sensu Komárek/clade C1 sensu Shih). This difference between morphologically and genetically based species identifications has consequences for applying the Reynolds functional-groups system, and for validity long-term monitoring data. The literature shows the same pattern as our results: morphologically, Aphanothece-like species are abundant in eutrophic shallow lagoons, and genetically, Cyanobium is found in similar habitats. This discrepancy is found worldwide in the literature on fresh- and brackish-water habitats. Thus, most Aphanothece-like morphospecies may be, genetically, members of Cyanobium.