They are young, and they are many: dating freshwater lineages in unicellular dinophytes.

Dinophytes are one of few protist groups that have an extensive fossil record and are therefore appropriate for time estimations. However, insufficient sequence data and strong rate heterogeneity have been hindering to put dinophyte evolution into a time frame until now. Marine-to-freshwater transitions within this group are considered geologically old and evolutionarily exceptional due to strong physiological constraints that prevent such processes. Phylogenies based on concatenated rRNA sequences (including 19 new GenBank entries) of two major dinophyte lineages, Gymnodiniaceae and Peridiniales, were carried out using an uncorrelated molecular clock and five calibration points based on fossils. Contrarily to previous assumptions, marine-to-freshwater transitions are more frequent in dinophytes (i.e. five marine-freshwater transitions in Gymnodiniaceae, up to ten but seven strongly supported transitions in Peridiniales), and none of them occurred as early as 140 MYA. Furthermore, most marine-to-freshwater transitions, and the followed diversification, took place after the Cretaceous-Paleogene boundary. Not older than 40 MYA, the youngest transitions within Gymnodiniaceae and Peridiniales occurred under the influence of the Eocene climate shift. Our evolutionary scenario indicates a gradual diversification of dinophytes without noticeable impact of catastrophic events, and their freshwater lineages have originated several times independently at different points in time.

Molecular phylogenetics of dinophytes harboring diatoms as endosymbionts (Kryptoperidiniaceae, Peridiniales), with evolutionary interpretations and a focus on the identity of Durinskia oculata from Prague.

Peridinialean dinophytes include a unique evolutionary group of algae harboring a diatom as an endosymbiont (Kryptoperidiniaceae), whose phylogenetic origin and internal relationships are not fully resolved at present. Several interpretations of the thecal plate pattern present in Durinskia oculata currently compete and lead to considerable taxonomic confusion. Moreover, it is unclear at present whether the species is restricted to freshwater habitats, or occurs in the marine environment as well. We collected material at the type locality of D. oculata in the Czech Republic and established monoclonal strains. Dinophyte cells were studied using light and electron microscopy, and we also determined DNA sequences of several rRNA regions (including the Internal Transcribed Spacers) for molecular characterization and phylogenetics. The morphology of strain GeoM∗662 indicated a plate formula of Po, X, 4', 2a, 6″, 5c, 5s, 5‴, 2⁗, which was sustained also in form of a microscopic slide serving as an epitype. In the molecular DNA tree based on a matrix composed of concatenated rRNA sequences, strain GeoM∗662 showed a close relationship to other species of Durinskia, and the freshwater species clearly differs from the marine members. Two independent colonization events from the marine into the freshwater environment can be inferred within the Kryptoperidiniaceae. We provide a summarizing cladogram of dinophytes harboring a diatom as endosymbiont with evolutionary novelties indicated as well as a morphological key to the 6 species of Durinskia that are currently accepted.

Spatial fragmentation in the distribution of diatom endosymbionts from the taxonomically clarified dinophyte Kryptoperidinium triquetrum (= Kryptoperidinium foliaceum, Peridiniales).

Among the photosynthetically active dinophytes, the Kryptoperidiniaceae are unique in having a diatom as endosymbiont instead of the widely present peridinin chloroplast. Phylogenetically, it is unresolved at present how the endosymbionts are inherited, and the taxonomic identities of two iconic dinophyte names, Kryptoperidinium foliaceum and Kryptoperidinium triquetrum, are also unclear. Multiple strains were newly established from the type locality in the German Baltic Sea off Wismar and inspected using microscopy as well as molecular sequence diagnostics of both host and endosymbiont. All strains were bi-nucleate, shared the same plate formula (i.e., po, X, 4', 2a, 7'', 5c, 7s, 5''', 2'''') and exhibited a narrow and characteristically L-shaped precingular plate 7''. Within the molecular phylogeny of Bacillariaceae, endosymbionts were scattered over the tree in a highly polyphyletic pattern, even if they were gained from different strains of a single species, namely K. triquetrum. Notably, endosymbionts from the Baltic Sea show molecular sequences distinct from the Atlantic and the Mediterranean Sea, which is the first report of such a spatial fragmentation in a planktonic species of dinophytes. The two names K. foliaceum and K. triquetrum are taxonomically clarified by epitypification, with K. triquetrum having priority over its synonym K. foliaceum. Our study underlines the need of stable taxonomy for central questions in evolutionary biology.

Dinastridium verrucosum Baumeister from Bavaria (Germany) is a Borghiellacean Dinophyte (†Suessiales).

Phytodinialean dinophytes are poorly known at present and their phylogenetic relationships largely elusive. Historical names of microscopic species are frequently ambiguous, and a reliable application is impeded although crucial to fully explore the biology of organisms. We collected material close to the type locality of a historical species, namely Dinastridium verrucosum, and established eight strains for morphological and molecular studies. The motile cells showed an obovate shape in outline and were dorso-ventrally slightly flattened. They were orange-brown in colour and had a descending cingulum. In light microscopy, an eyespot was discerned in a few monadoid cells in the central region of the sulcus. Furthermore, a morphologically characteristic, 4-6µm long apical furrow was observed on the episome of the cells in SEM. Older cultivated material further exhibited coccoid cells of irregular shape, with wart-like protuberances and covered by a more or less extensive mucilage. This morphology is indistinguishable from the lectotype of D. verrucosum. In a molecular phylogeny, the species was placed in the Borghiellaceae (†Suessiales). As taxonomic result, we epitypify the historical name, D. verrucosum, and perform the necessary combination to Borghiella.

Zero Intercalary Plates in Parvodinium (Peridiniopsidaceae, Peridiniales) and Phylogenetics of P. elpatiewskyi, comb. nov.

Parvodinium elpatiewskyi, comb. nov., is a common freshwater dinophyte without intercalary plates and with various spines on hypothecal sutures. However, the taxonomy of the species has had a complex history, and its systematic placement remained unclear. The conserved type of P. elpatiewskyi, comb. nov., illustrated here for the first time using electron microscopy, is an environmental sample. Based on the newly collected material from Berlin (Germany) we provide a morphological description using light and electron microscopy as well as new molecular rRNA sequence data to specify the phylogenetic position of P. elpatiewskyi, comb. nov. This species belongs to Peridiniopsidaceae, more precisely to Parvodinium, which usually possesses two intercalary plates. However, evolutionary inference indicates the loss of such plates in P. elpatiewskyi, comb. nov. Other traits that are of taxonomic importance and have not received enough attention in the past are the large Sd plate converging the second antapical plate and the presence of cellular hypocystal opening during replication.

The Hot Spot in a Cold Environment: Puzzling Parvodinium (Peridiniopsidaceae, Peridiniales) from the Polish Tatra Mountains.

Because of a great variety of remote localities and cold habitats, the Tatra Mountains are home to many freshwater protist lineages. Dinophytes have been subjected to a number of studies from this area dating mostly to the first half of the 20th century, but their true diversity remains elusive until today. We collected water tow samples at five lakes in the Tatra Mountains in order to establish monoclonal strains. We found four lineages that were distinctive in terms of morphology and DNA sequence data and that could be assigned to peridinialean Parvodinium. These four species can be readily distinguished based on a general shape, size, thecal plate tabulation pattern and presence or absence of an antapical protuberance. The plate overlap pattern is considered conserved at higher taxonomic levels, and the divergent keystone Plate 3' in Parvodinium marciniakii, sp. nov., thus appears as a striking diagnostic character. For taxonomic conclusion, we describe two species new to science and validate three old scientific names (i.e., one species and two varieties). Our study underlines that the biodiversity assessment, particularly of species adapted to cold environments, is anything but completed as shown from remote and unexplored European landscapes such as the Tatra Mountains.

Taxonomic Clarification of the Unusual Dinophyte Gymnodinium limneticum Wolosz. (Gymnodiniaceae) from the Tatra Mountains.

The Gymnodiniaceae -even in a strict sense- comprise a vast diversity of dinophytes regarding morphology and ecology. Taxonomy and nomenclature of their constituent species remain problematic, although crucial to fully explore the biology of the group. Here, we present the rarely documented dinophyte Gymnodinium limneticum from its type locality at Lake Morskie Oko in Poland, for which we established strains and made extensive morphological studies. The species was unusual in exhibiting capsoid cells as predominant life-history stage, which were embedded and dividing in extensive mucilage leading to an enkaptic pseudocolonial system. We also generated ribosomal RNA sequences that were included in a comprehensive molecular phylogeny. Our species was clearly identified as a member of the Gymnodiniaceae s.str. but within the lineage, it was only distantly related to the type species of Gymnodinium, G. fuscum. Rather, it constituted a monophyletic group together with species assigned to Spiniferodinium and as a nomenclatural result, we propose two new combinations (i.e., Sp. limneticum comb. nov., Sp. palustre comb. nov.). As Spiniferodinium now includes dinophyte species inhabiting marine or freshwater environments as well, our investigations may provide evidence for an evolutionary scenario with corresponding transitions being more frequent than considered before.