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

A story from the Miocene: Clock‐dated phylogeny of Sisymbrium L. (Sisymbrieae, Brassicaceae)

Morphological variability and imprecise generic boundaries have hindered systematic, taxonomical, and nomenclatural studies of Sisymbrium L. (Brassicaceae, Sisymbrieae DC.). The members of this almost exclusively Old‐World genus grow mostly on highly porous substrates across open steppe, semidesert, or ruderal habitats in the temperate zone of the Northern Hemisphere and African subtropics. The present study placed the biological history of Sisymbrium L. into time and space and rendered the tribus Sisymbrieae as monotypic. Five nuclear‐encoded and three chloroplast‐encoded loci of approximately 85% of all currently accepted species were investigated. Several accessions per species covering their whole distribution range allowed for a more representative assessment of intraspecific genetic diversity. In the light of fossil absence, the impact of different secondary calibration methods and taxon sets on time spans was tested, and we showed that such a combinatorial nested dating approach is beneficial. Multigene phylogeny accompanied with a time divergence estimation analysis placed the onset and development of this tribus into the western Irano‐Turanian floristic region during the Miocene. Continuous increase in continentality and decrease in temperatures promoted the diversity of the Sisymbrieae, which invaded the open grasslands habitats in Eurasia, Mediterranean, and South Africa throughout the Pliocene and Pleistocene. Our results support the assumption of the Irano‐Turanian region as a biodiversity reservoir for adjacent regions

Global distribution, climatic preferences and photosynthesis‐related traits of C4 eudicots and how they differ from those of C4 grasses

Abstract C₄ is one of three known photosynthetic processes of carbon fixation in flowering plants. It evolved independently more than 61 times in multiple angiosperm lineages and consists of a series of anatomical and biochemical modifications to the ancestral C3 pathway increasing plant productivity under warm and light‐rich conditions. The C4 lineages of eudicots belong to seven orders and 15 families, are phylogenetically less constrained than those of monocots and entail an enormous structural and ecological diversity. Eudicot C4 lineages likely evolved the C4 syndrome along different evolutionary paths. Therefore, a better understanding of this diversity is key to understanding the evolution of this complex trait as a whole. By compiling 1207 recognised C4 eudicots species described in the literature and presenting trait data among these species, we identify global centres of species richness and of high phylogenetic diversity. Furthermore, we discuss climatic preferences in the context of plant functional traits. We identify two hotspots of C4 eudicot diversity: arid regions of Mexico/Southern United States and Australia, which show a similarly high number of different C4 eudicot genera but differ in the number of C4 lineages that evolved in situ. Further eudicot C4 hotspots with many different families and genera are in South Africa, West Africa, Patagonia, Central Asia and the Mediterranean. In general, C4 eudicots are diverse in deserts and xeric shrublands, tropical and subtropical grasslands, savannas and shrublands. We found C4 eudicots to occur in areas with less annual precipitation than C4 grasses which can be explained by frequently associated adaptations to drought stress such as among others succulence and salt tolerance. The data indicate that C4 eudicot lineages utilising the NAD‐ME decarboxylating enzyme grow in drier areas than those using the NADP‐ME decarboxylating enzyme indicating biochemical restrictions of the later system in higher temperatures. We conclude that in most eudicot lineages, C4 evolved in ancestrally already drought‐adapted clades and enabled these to further spread in these habitats and colonise even drier areas

Still curling after all these years: <i>Glenodinium apiculatum</i> Ehrenb. (Peridiniales, Dinophyceae) repeatedly found at its type locality in Berlin (Germany)

<p>The contemporary occurrence of dinophytes at their type localities has not been intensely studied so far, despite the type locality's crucial importance for any reliable scientific name application. The microscopist and phycologist Ch.G. Ehrenberg described a number of dinophyte species more than 150 years ago, many of which are currently taxonomically ambiguous. We collected water tow and sediment samples at those same localities in Berlin that Ch.G. Ehrenberg may have visited as well. We isolated and established several strains of <i>Glenodinium apiculatum</i> that we investigated by applying contemporary microscopic and molecular methods. The plate formula of the species was 4′, 2a, 7″, 6c, 5s, 5′″, 2″″, without an apical pore complex, and the most distinctive morphological trait of <i>Glenodinium apiculatum</i> was the spiny hypotheca. The spines were irregularly scattered over hypothecal plate surface and arranged in raised edges between thecal plates. As inferred from molecular phylogenetics, <i>Glenodinium apiculatum</i> is assigned to <i>Palatinus</i>, which is an element of the Peridiniopsidaceae as a part of the Peridiniales. For taxonomic purposes, we epitypified Ch.G. Ehrenberg's taxon with newly collected material to ensure a reliable determination in the future. <i>Palatinus apiculatus</i> is not a fleeting star, and a number of dinophytes show a remarkably high fidelity to the sites from which they were originally described, even if the description was carried out a long time ago.</p

The many faces of <i>Peridinium cinctum</i> (Peridiniaceae, Peridiniales): morphological and molecular variability in a common dinophyte

<p><i>Peridinium cinctum</i> is a common freshwater dinophyte with a long history of research. Erich Lindemann was the first to assess intraspecific variability in this species focusing on plate pattern variation. Since then, this issue has been neglected but with the application of DNA sequence diagnostics, a combination of morphological and molecular characters may enable taxonomic delimitations. Our aim was to identify distinct morphotypes using plate pattern as the main characteristic and then compare them to the geographic occurrence of particular ribotypes (as inferred from sequences of the Internal Transcribed Spacer: ITS) in samples from Central Europe. Approximately 200 observations were carried out under the inverse light microscope for each of a total of 15 strains. We observed two main variations from the abundant plate pattern in <i>P. cinctum</i>, namely an unusual position of the 2a plate and the irregular shape of the 1a plate. In 88 (predominantly clonal) strains, we identified five different ribotypes (submitted as 71 new GenBank entries) which had no clear correlation to the defined morphotypes and/or spatial occurrences. In four cases, we detected two distinct ribotypes at the same locality. However, samples collected south of the Danube River presented a different predominant morphotype from the rest of the samples, thus implying a potential biogeographic signal as inferred from morphology. In general, there is morphological and molecular variability in <i>P. cinctum</i>, which is under-studied and which may uncover geographic or ecological correlations or even the existence of cryptic species.</p

Assessing dinophyte biodiversity in Bavarian lakes (Germany) by 18S v4 amplicon sequencing

Seventeen surface plankton tow samples were collected from piers at thirteen localities in Upper Bavaria (Germany) in April 2017 using a plankton net (mesh size 20µm). The localities included eleven lakes (one lake was sampled at two sites) and one subsidiary river, to cover standing and flowing bodies of water as well. Four sites had been sampled twice. Environmental DNA was extracted using the Genomic DNA from Soil kit (Machery-Nagel; Düren, Germany) following the manufacturer's protocol. The small subunit (SSU or 18S) of the ribosomal RNA (rRNA) operon V4 region (~410 bp) was the amplification target. Due to PCR biases or PCR errors that may artificially increase diversity, each PCR reaction was performed in triplicates. Forward and reverse primers were those used by Xiao et al. (2017) (DOI: 10.1007/s12010-016-2358-3). DNA amplification (PCR) for subsequent Illumina amplicon sequencing (Illumina; San Diego, USA-CA) was carried out using 5ng/µl template DNA, 1 µM of each primer and 2x KAPA Hifi HotStart Ready Mix (Roche; Penzberg, Germany). Resulting PCR products were visualised in 1% agarose gels and were purified using AMPure XP Beads (Beckman Coulter; Brea, USA-CA). Dual indices and Illumina sequence adapters were attached by means of an Index PCR using the Nextera XT Index Kit (Illumina), and final PCR products were again purified using AMPure XP Beads. The library was validated using an Agilent 2100 Bioanalyzer Software and a DNA 1000 Chip (Agilent Technologies; Santa Clara, USA-CA) to verify the size of the resulting fragments. The final DNA libraries were equimolarly pooled and run in a MiSeq System (Illumina) after combining the denatured PhiX control library (15%) and the denatured amplicon library. Some 6.5 million 2 x 300 bp paired-end reads were produced and demultiplexed into seventeen samples from thirteen sites. Using Trimmomatic (v0.38), 3'-ends of the reads were trimmed based on read quality information. PEAR (v0.9.10) with default settings was used to merge the paired-end reads. Sequences, which could not be merged, were discarded. Primer-matching sequence segments were truncated from the amplicons by cutadapt (v1.9) and amplicons were only kept in the sequence pool, if both, the segment of the forward and of the reverse primer could be found. Remaining sequences were filtered for further quality features by vsearch (v2.3.0). Sequences were discarded, if they were outside a 50 bp radius above or below the median length of the primer-truncated amplicon (~387 bp), if they carry any ambiguity or if the expected number of miscalled bases of a sequence (sum of all base error probabilities of a sequence) was above 1. Chimera were predicted also by vsearch utilising the UCHIME algorithm with default settings in de-novo mode for each sample separately and removed from the sample files. About 4 million sequences passed all filtering steps and were used as input for the OTU-clustering, which was done by the tool Swarm (v2.1.8) with default settings. The most abundant amplicon of each OTU-cluster was used as an OTU representative. These sequences were annotated by the RDP classifier implemented in mothur (v1.38.1) using the Ref_NR99 version of release 128 of the SILVA SSU sequence set using a reference with a confidence cutoff of 90. The annotation of each representative sequence was used as annotation of the OTU cluster as well and added to the corresponding line of the OTU table

Pleistocene dynamics of the Eurasian steppe as a driving force of evolution: Phylogenetic history of the genus Capsella (Brassicaceae)

Capsella is a model plant genus of the Brassicaceae closely related to Arabidopsis. To disentangle its biogeographical history and intrageneric phylogenetic relationships, 282 individuals of all five currently recognized Capsella species were genotyped using a restriction digest-based next-generation sequencing method. Our analysis retrieved two main lineages within Capsella that split c. one million years ago, with western C. grandiflora and C. rubella forming a sister lineage to the eastern lineage consisting of C. orientalis. The split was attributed to continuous latitudinal displacements of the Eurasian steppe belt to the south during Early Pleistocene glacial cycles. During the interglacial cycles of the Late Pleistocene, hybridization of the two lineages took place in the southwestern East European Plain, leading to the allotetraploid C. bursa-pastoris. Extant genetic variation within C. orientalis postdated any extensive glacial events. Ecological niche modeling showed that suitable habitat for C. orientalis existed during the Last Glacial Maximum around the north coast of the Black Sea and in southern Kazakhstan. Such a scenario is also supported by population genomic data that uncovered the highest genetic diversity in the south Kazakhstan cluster, suggesting that C. orientalis originated in continental Asia and migrated north- and possibly eastwards after the last ice age. Post-glacial hybridization events between C. bursa-pastoris and C. grandiflora/rubella in the southwestern East European Plain and the Mediterranean gave rise to C. thracica. Introgression of C. grandiflora/rubella into C. bursa-pastoris resulted in a new Mediterranean cluster within the already existing Eurasian C. bursa-pastoris cluster. This study shows that the continuous displacement and disruption of the Eurasian steppe belt during the Pleistocene was the driving force in the evolution of Capsella