Oxytoxaceae are prorocentralean rather than peridinialean dinophytes and taxonomic clarification of heterotrophic Oxytoxum lohmannii (≡ "Amphidinium" crassum) by epitypification.

During evolution of Dinophyceae, size reduction of the episome has occurred in several lineages (including unarmoured Amphidiniales and armoured Prorocentrales). One such species is Amphidinium crassum, whose taxonomic identity is elusive though showing morphological similarities with Oxytoxaceae (currently placed in armoured Peridiniales). Plankton samples were taken at the type locality of A. crassum in Kiel Bight (Baltic Sea) in order to establish monoclonal strains. The protist material was examined in detail using light and electron microscopy, and a long (2984 bp) ribosomal RNA sequence gained was part of a taxon sample comprising 206 specimen vouchers and representing the known molecular diversity of Dinophyceae. Cells of A. crassum were ovoid and exhibited a plate pattern po, 4', 1a, 6'', 5c, 4s, 5''', 1''''. In the molecular phylogeny, the species seemed to belong neither to Amphidiniales nor to Peridiniales but to Prorocentrales and clustered with other representatives of Oxytoxaceae. The morphological diversity of Prorocentrales appears thus expanded, and the group may include a number of previously unrecognised representatives unusually having five postcingular and only a single antapical plate. The taxonomic identity of A. crassum is clarified by epitypification, and the species notably exhibits both an apical pore and an additional epithecal pore.

The second most abundant dinophyte in the ponds of a botanical garden is a species new to science.

In the microscopy realm, a large body of dark biodiversity still awaits to be uncovered. Unarmoured dinophytes are particularly neglected here, as they only present inconspicuous traits. In a remote German locality, we collected cells, from which a monoclonal strain was established, to study morphology using light and electron microscopy and to gain DNA sequences from the rRNA operon. In parallel, we detected unicellular eukaryotes in ponds of the Botanical Garden Munich-Nymphenburg by DNA-metabarcoding (V4 region of the 18S rRNA gene), weekly sampled over the course of a year. Strain GeoK*077 turned out to be a new species of Borghiella with a distinct position in molecular phylogenetics and characteristic coccoid cells of ovoid shape as the most important diagnostic trait. Borghiella ovum, sp. nov., was also present in artificial ponds of the Botanical Garden and was the second most abundant dinophyte detected in the samples. More specifically, Borghiella ovum, sp. nov., shows a clear seasonality, with high frequency during winter months and complete absence during summer months. The study underlines the necessity to assess the biodiversity, particularly of the microscopy realm more ambitiously, if even common species such as formerly Borghiella ovum are yet unknown to science.

DNA sequences from type specimens and type strains - how to increase their number and improve their annotation in NCBI GenBank and related databases.

Scientific names permit humans and search engines to access knowledge about the biodiversity that surrounds us, and names linked to DNA sequences are playing an ever-greater role in search-and-match identification procedures. Here, we analyze how users and curators of the National Center for Biotechnology Information (NCBI) are flagging and curating sequences derived from nomenclatural type material, which is the only way to improve the quality of DNA-based identification in the long run. For prokaryotes, 18,281 genome assemblies from type strains have been curated by NCBI staff and improve the quality of prokaryote naming. For Fungi, type-derived sequences representing over 21,000 species are now essential for fungus naming and identification. For the remaining eukaryotes, however, the numbers of sequences identifiable as type-derived are minuscule, representing only 1,000 species of arthropods, 8,441 vertebrates, and 430 embryophytes. An increase in the production and curation of such sequences will come from (i) sequencing of types or topotypic specimens in museum collections, (ii) the March 2023 rule changes at the International Nucleotide Sequence Database Collaboration requiring more metadata for specimens, and (iii) efforts by data submitters to facilitate curation, including informing NCBI curators about a specimen's type status. We illustrate different type-data submission journeys and provide best-practice examples from a range of organisms. Expanding the number of type-derived sequences in DNA databases, especially of eukaryotes, is crucial for capturing, documenting, and protecting biodiversity.

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.

Morphological and Phylogenetic Characterisation of Prorocentrum spinulentum, sp. nov. (Prorocentrales, Dinophyceae), a Small Spiny Species from the North Atlantic.

Prorocentrum comprises dinophytes with several unique traits, including the presence of two large thecal plates and apical insertion of flagella. Species delimitation for many small and similar planktonic species is challenging, as SEM analyses and DNA sequence information of type material are rarely available. Based on a strain from the North Atlantic Prorocentrum spinulentum, sp. nov. is described here. Cells were small (9.0-12.8 µm long, 8.5-11.9 µm deep), oval to almost round in lateral view and moderately compressed. The ovoid nucleus was in median or slightly sub-median position on the cells ventral side. The plate surface appeared spiny in light microscopy with thecal pores visible in empty thecae. Electron microscopy revealed plates densely covered by relatively long spines and two size classes of thecal pores. The periflagellar area consisted of 8 platelets, and there was a prominent wing (ca. 1 µm wide and long) on platelet 1. The new species is distinct in DNA trees and embedded in the Prorocentrum shikokuense species group. It differs from the protologues of other small species of Prorocentrum by the unique combination of cell size and shape, the presence of long spines on the thecal plate surface and scattered thecal pores. The thorough morphological description of this species, representing a previously uncharacterised lineage within Prorocentrum, increases and improves our knowledge of the diversity within this important group of planktonic organisms.

Fensomea setacea, gen. & sp. nov. (Cladopyxidaceae, Dinophyceae), is neither gonyaulacoid nor peridinioid as inferred from morphological and molecular data.

Dinophyte evolution is essentially inferred from the pattern of thecal plates, and two different labelling systems are used for the important subgroups Gonyaulacales and Peridiniales. The partiform hypotheca of cladopyxidoid dinophytes fits into the morphological concepts of neither group, although they are assigned to the Gonyaulacales. Here, we describe the thecate dinophyte Fensomea setacea, gen. & sp. nov., which has a cladopyxidoid tabulation. The cells displayed a Kofoidean plate formula APC, 3', 4a, 7″, 7C, 6S, 6''', 2'''', and slender processes were randomly distributed over the echinate or baculate surface. In addition, we obtained rRNA sequences of F. setacea, gen. & sp. nov., but dinophytes that exhibit a partiform hypotheca did not show a close relationship to Gonyaulacales. Character evolution of thecate dinophytes may have progressed from the ancestral state of six postcingular plates, and two more or less symmetrically arranged antapical plates, towards patterns of only five postcingular plates (Peridiniales) or more asymmetrical configurations (Gonyaulacales). Based on our phylogenetic reconsiderations the contact between the posterior sulcal plate and the first postcingular plate, as well as the contact between an antapical plate and the distalmost postcingular plate, do not represent a rare, specialized gonyaulacoid plate configuration (i.e., the partiform hypotheca of cladopyxidoid dinophytes). Instead, these contacts correspond to the common and regular configuration of peridinioid (and other) dinophytes.

Recommendations for epitypification of dinophytes exemplified by Lingulodinium polyedra and molecular phylogenetics of the Gonyaulacales based on curated rRNA sequence data.

Gonyaulacales include a considerable number of harmful algae and to understand their origin and rise, knowledge of the evolutionary relationships is necessary. Many scientific names of protists introduced prior to the availability of DNA analytics are ambiguous and impede communication about biological species and their traits in the microbial world. Strains of Lingulodinium polyedra were established from its type locality in the Kiel Fjord (Germany) to clarify its taxonomy. Moreover, the phylogeny of Gonyaulacales was inferred based on 329 rRNA sequence accessions compiled in a curated sequence data base, with as much as possible type material equivalents included. Gonyaulacales were monophyletic and segregated into seven lineages at high systematic level, of which †Lingulodiniaceae constituted the first branch of the Gonyaulacales. Their type species had a plate formula APC (Po, X, cp), 3', 3a, 6'' 6c, 6s, 6''', 2'''' and is taxonomically clarified by epitypification. Recommendations for this important taxonomic tool are provided, with a focus on microorganisms. Most gonyaulacalean taxa established at generic rank are monophyletic, with Alexandrium, Coolia and Gonyaulax as notable exceptions. From an evolutionary perspective, gonyaulacalean dinophytes with quinqueform hypotheca are monophyletic and derive from a paraphyletic group showing the sexiform configuration.

The Windblown: Possible Explanations for Dinophyte DNA in Forest Soils.

Dinophytes are widely distributed in marine- and fresh-waters, but have yet to be conclusively documented in terrestrial environments. Here, we evaluated the presence of these protists from an environmental DNA metabarcoding dataset of Neotropical rainforest soils. Using a phylogenetic placement approach with a reference alignment and tree, we showed that the numerous sequencing reads that were phylogenetically placed as dinophytes did not correlate with taxonomic assignment, environmental preference, nutritional mode, or dormancy. All the dinophytes in the soils are rather windblown dispersal units of aquatic species and are not biologically active residents of terrestrial environments.

Taxonomy and Molecular Phylogenetics of Ensiculiferaceae, fam. nov. (Peridiniales, Dinophyceae), with Consideration of their Life-history.

In the current circumscription, the Thoracosphaeraceae comprise all dinophytes exhibiting calcified coccoid cells produced during their life-history. Species hitherto assigned to Ensiculifera and Pentapharsodinium are mostly based on the monadoid stage of life-history, while the link to the coccoid stage (occasionally treated taxonomically distinct) is not always resolved. We investigated the different life-history stages and DNA sequence data of Ensiculifera mexicana and other species occurring in samples collected from all over the world. Based on concatenated ribosomal RNA gene sequences Ensiculiferaceae represented a distinct peridinalean branch, which showed a distant relationship to other calcareous dinophytes. Both molecular and morphological data (particularly of the coccoid stage) revealed the presence of three distinct clades within Ensiculiferaceae, which may include other dinophytes exhibiting a parasitic life-history stage. At a higher taxonomic level, Ensiculiferaceae showed relationships to parasites and endosymbionts (i.e., Blastodinium and Zooxanthella) as well as to dinophytes harbouring diatoms instead of chloroplasts. These unexpected phylogenetic relationships are corroborated by the presence of five cingular plates in all such taxa, which differs from the six cingular plates of most other Thoracosphaeraceae. We herein describe Ensiculiferaceae, emend the descriptions of Ensiculifera and Pentapharsodinium, erect Matsuokaea and provide several new combinations at the species level.

Dawn of the dinophytes: A first attempt to date origin and diversification of harmful algae.

Unicellular dinophytes include a considerable number of harmful algae and potent producers of toxins. The dinophyte fossil record is one of the richest among protists and indicates a geologically old origin of the group dating back to the Triassic. Besides of these records, very few molecular dating studies of dinophytes have been published to date, precluding an understanding of the diversification history of these organisms. In the present study, we used first appearances in the fossil record for the best-represented dinophyte lineages, namely Gonyaulacales and Peridiniales, to calibrate a molecular phylogeny. It is inferred from ribosomal RNA sequence data covering a representative taxon sampling of all currently recognised lineages. Dinophytes may have started diversifying during main tectonic events of the supercontinent Pangaea, witnessing and surviving some of the biggest mass extinction events on Earth. Groups including harmful dinophytes originated at different points in time, but they all predate the Cretaceous-Paleogene boundary. Our chronogram provides a first time frame and may stimulate studies in future bringing molecular phylogenetics of dinophytes and their impressive fossil record together in more detail.

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.

Resolving relationships in an exceedingly young Neotropical orchid lineage using Genotyping-by-sequencing data.

Poor morphological and molecular differentiation in recently diversified lineages is a widespread phenomenon in plants. Phylogenetic relationships within such species complexes are often difficult to resolve because of the low variability in traditional molecular loci. Furthermore, biological phenomena responsible for topological incongruence such as Incomplete Lineage Sorting (ILS) and hybridisation complicate the resolution of phylogenetic relationships among closely related taxa. In this study, we employ a Genotyping-by-sequencing (GBS) approach to disentangle evolutionary relationships within a species complex belonging to the Neotropical orchid genus Cycnoches. This complex includes seven taxa distributed through Central America and the Colombian Chocó, and is nested within a clade estimated to have first diversified in the early Quaternary. Previous phylogenies inferred from few loci failed to provide support for internal relationships within the complex. Our Neighbour-net and coalescent-based analyses inferred from ca. 13,000 GBS loci obtained from 31 individuals belonging to six of the seven traditionally accepted Cycnoches taxa provided a robust phylogeny for this group. The genus Cycnoches includes three main clades that are further supported by morphological traits and geographic distributions. Similarly, a topology reconstructed through maximum likelihood (ML) inference of concatenated GBS loci produced results that are comparable with those reconstructed through coalescence and network-based methods. Our comparative phylogenetic informativeness analyses suggest that the low support evident in the ML phylogeny might be attributed to the abundance of uninformative GBS loci, which can account for up to 50% of the total number of loci recovered. The phylogenomic framework provided here, as well as morphological evidence and geographical patterns, suggest that the six entities previously thought to be different species or subspecies might actually represent only three distinct segregates. We further discuss the limited phylogenetic informativeness found in our GBS approach and its utility to disentangle relationships within recent and rapidly evolving species complexes. Our study is the first to demonstrate the utility of GBS data to reconstruct relationships within young (~2 Ma) Neotropical plant clades, opening new avenues for studies of species complexes that populate the species-rich orchid family.

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.

High abundance of Amphidomataceae (Dinophyceae) during the 2015 spring bloom of the Argentinean Shelf and a new, non-toxigenic ribotype of Azadinium spinosum.

Azaspiracids (AZA) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin, and associated with human incidents of shellfish poisoning. They are produced by a few species of Amphidomataceae, but diversity and occurrence of the small-sized dinophytes remain poorly explored for many regions of the world. In order to analyze the presence and importance of Amphidomataceae in a highly productive area of Argentinean coastal waters (El Rincón area, SW Atlantic), a scientific cruise was performed in 2015 to sample the early spring bloom. In a multi-method approach, light microscopy was combined with real-time PCR molecular detection of Amphidomataceae, with chemical analysis of AZA, and with the establishment and characterization of amphidomatacean strains. Both light microscopy and PCR revealed that Amphidomataceae were widely present in spring plankton communities along the El Rincón area. They were particularly abundant offshore at the shelf front, reaching peak densities of 2.8 × 105 cells L-1, but no AZA were detected in field samples. In total, 31 new strains were determined as Az. dalianense and Az. spinosum, respectively. All Az. dalianense were non-toxigenic and shared the same rRNA sequences. The large majority of the new Az. spinosum strains revealed for the first time the presence of a non-toxigenic ribotype of this species, which is otherwise the most important AZA producer in European waters. One of the new Az. spinosum strains, with a particular slender shape and some other morphological peculiarities, clustered with toxigenic strains of Az. spinosum from Norway and, exceptionally for the species, produced only AZA-2 but not AZA-1. Results indicate a wide diversity within Az. spinosum, both in terms of sequence data and toxin profiles, which also will affect the qualitative and quantitative performance of the specific qPCR assay for this species. Overall, the new data provide a more differentiated perspective of diversity, toxin productivity and occurrence of Amphidomataceae in a poorly explored region of the global ocean.

Two Novel, Distantly Related Papillomaviruses Isolated from Healthy Skin of the Timor Deer (Rusa timorensis).

We report the complete genome sequences of Rusa timorensis papillomavirus 1 (RtimPV1) and Rusa timorensis papillomavirus 2 (RtimPV2), isolated from hair follicles of asymptomatic skin from the same Timor deer specimen. RtimPV1 and RtimPV2 are evolutionarily only distantly related. RtimPV1 lacks a canonical E2-binding site, and RtimPV2 does not carry an E6 gene.

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.

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.

Plate pattern clarification of the marine dinophyte Heterocapsa triquetra sensu Stein (Dinophyceae) collected at the Kiel Fjord (Germany).

One of the most common marine dinophytes is a species known as Heterocapsa triquetra. When Stein introduced the taxon Heterocapsa, he formally based the type species H. triquetra on the basionym Glenodinium triquetrum. The latter was described by Ehrenberg and is most likely a species of Kryptoperidinium. In addition to that currently unresolved nomenclatural situation, the thecal plate composition of H. triquetra sensu Stein (1883) was controversial in the past. To clarify the debate, we collected material and established the strain UTKG7 from the Baltic Sea off Kiel (Germany, the same locality as Stein had studied), which was investigated using light and electron microscopy, and whose systematic position was inferred using molecular phylogenetics. The small motile cells (18-26 µm in length) had a biconical through fusiform shape and typically were characterized by a short asymmetrically shaped, horn-like protuberance at the antapex. A large spherical nucleus was located in the episome, whereas a single pyrenoid laid in the lower cingular plane. The predominant plate pattern was identified as apical pore complex (Po, cp?, X), 4', 2a, 6'', 6c, 5s, 5''', 2''''. The triradiate body scales were 254-306 nm in diameter, had 6 ridges radiating from a central spine, 9 peripheral and 3 radiating spines, and 12 peripheral bars as well as a central depression in the basal plate. Our work provides a clarification of morphological characters and a new, validly published name for this important but yet formally undescribed species of Heterocapsa: H. steinii sp. nov.