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The dinoflagellates are a diverse lineage of microbial eukaryotes. Dinoflagellate monophyly and their position within the group Alveolata are well established. However, phylogenetic relationships between dinoflagellate orders remain unresolved. To date, only a limited of dinoflagellate studies have used a broad taxon sample with more than two concatenated markers. This lack of resolution makes it difficult to determine the evolution of major phenotypic characters such as morphological features or toxin production e.
Here we present an improved dinoflagellate phylogeny, based on eight genes, with the broadest taxon sampling to date.
Fifty-five sequences for eight phylogenetic markers from nuclear and mitochondrial regions were amplified from 13 species, four orders, and concatenated phylogenetic inferences were conducted with orthologous sequences. Phylogenetic resolution is increased with addition of support for the deepest branches, though can be improved yet further.
We show for the first time that the characteristic dinoflagellate thecal plates, cellulosic material that is present within the 518 nudes alveoli, appears to have had a single origin. In addition, the monophyly of most dinoflagellate orders is confirmed: the Dinophysiales, the Gonyaulacales, the Prorocentrales, the Suessiales, and the Syndiniales. Our improved phylogeny, along with of PCR to detect the sxtA gene in various lineages, allows us to suggest that this gene was probably acquired separately in Gymnodinium and the common ancestor of Alexandrium and Pyrodinium and subsequently lost in some descendent species of Alexandrium.
This is an open-access article distributed under the terms of the Creative Commons Attributionwhich permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study de, data collection and analysis, decision to publish, or 518 nudes of the manuscript. Competing interests: The authors have declared that no competing interests exist. Approximately species of living dinoflagellates are known, most of which are found in marine habitats . Species vary widely, in characteristics such as cell morphology and modes of nutrition e.
Dinoflagellate taxonomy is based on morphological characters such as the presence of a dinokaryon, and the arrangement and shape of thecal plate-containing amphiesmal vesicles. The Blastodiniales and Noctilucales lack a dinokaryon during particular life cycle stages. For this reason, it has been hypothesized that these lineages are basal although recent evidence suggests that the Blastodiniales may have diverged more recently . The arrangement of the thecal plate bearing amphiesmal vesicles is an important character in distinguishing clades of dinoflagellates .
The thecate armored orders Dinophysiales, Gonyaulacales, Peridiniales, Prorocentrales and Suessiales have comparatively fewer, large amphiesmal vesicles in distinctive patterns, with cellulosic material in the vesicles. Athecate unarmored or 518 nudes taxa, however Gymnodiniales, Noctilucales and Syndiniales often contain hundreds of alveoli lacking cellulosic material, and therefore relationships are determined 518 nudes on other features, such as the presence and shape of grooves on the cell surface or on the cell apex, and the shape of the epicone .
The monophyly of dinoflagellates and their sister relationships to the Apicomplexa have been established from dinoflagellate phylogenies, as well as global eukaryotic phylogenies . Fossil evidence suggests that these groups diverged earlier than Ma and that the species Alexandrium tamarense is a fairly recent dinoflagellate lineage, emerging between 23—45 Ma .
The phylogenetic relationship between the dinoflagellate orders, however, is unresolved, with a lack of statistical support for the phylogenetic backbone . Early molecular phylogenetic studies of dinoflagellate relationships were based on ribosomal rDNA, either partial large-subunit LSU or most frequently, small-subunit SSU . However, the low proportion of informative characters resulted in poor resolution despite broad taxon sampling .
Concatenated rDNA inferences have added more resolution, proving useful in the interpretation of genus 518 nudes relationships . However, inter-order relationships remain unclear, with deep branches receiving little or no statistical support, making trends difficult to infer . Recently, the use of protein genes for phylogenetic inference of dinoflagellates has increased, in particular actin, alpha- and beta-tubulin hsp90 and the mitochondrial cytochrome genes . However, as yet few have inferred a broad dinoflagellate phylogeny based on more than two concatenated genetic markers .
The identification of the marine alveolate lineages MALVgives an insight into the large parasitic Syndiniales diversity . A well-resolved dinoflagellate phylogeny is essential to understanding the evolution of toxin synthesis in this phylum. Approximately known species of dinoflagellates produce a variety of toxins, that can accumulate in the water column as Harmful Algal Blooms HABs .
Saxitoxin STXand its analogues, is one such toxin that can cause severe symptoms upon consumption of vector species . STX is synthesized by eukaryotic marine dinoflagellates and freshwater cyanobacteria . The toxins appear to be synthesized by similar processes in cyanobacteria and dinoflagellates .
The biosynthetic pathway and genes responsible for STX- synthesis are known from cyanobacterial species . One such core gene, sxtAthe unique starting gene of STX synthesis, has recently been identified in the dinoflagellates Gymnodinium catenatum and multiple species within the genus Alexandrium . The origin of this gene cluster within the dinoflagellates may have occurred by way of a horizontal gene transfer HGT event between an ancestral STX-producing bacterium and the dinoflagellates, before Alexandrium and Pyrodinium diverged .
Thus the ability to produce STX may have been secondarily lost for some descendent species. As the sxtA sequence of Gymnodinium catenatum, in the order Gymnodiniales, branches within Alexandrium, an independent acquisition of STX from a dinoflagellate-dinoflagellate transfer has been postulated . As the phylogenetic relationship between dinoflagellate species remains unresolved, trends in the evolution of the genetic basis for the synthesis of STX or other toxins cannot be established .
The aim of this study is to improve the resolution of the dinoflagellate phylogeny by sampling a broad range of both taxa and genes using concatenated alignments. This will allow us to address relationships between orders and identify possible phylogenetic trends in the evolution of STX production and other major phenotypic characters, such as morphological traits.
To achieve this, 55 sequences for eight molecular markers were amplified from 13 species, spanning four orders. A concatenated phylogenetic approach was used with all orthologous database sequences. Furthermore, we tested 20 species from five orders for presence of sxtA1 and sxtA4. Our use of order, family and genus-level names follows the taxonomic revision of Fensome et al. Woloszynskia halophila Pelagodinium beii syn. Gymnodinium beii and Protodinium simplex syn. Gymnodinum simplex .
Strains were not maintained axenically. Inclusion of Pyrodinium bahamense in the study would be desirable as it produces saxitoxin, however no culture was available to us. Thus, mRNA was utilized for cob and cox1, as only a single functional sequence is reported, in comparison to multiple genomic copies . Likewise, actin in dinoflagellates is present in a variable of copies in the genome, including pseudogenes; therefore mRNA was again favored .
Template was only PCR amplified for the genes and strains lacking Genbank sequence 518 nudes. The universal primers used in this study have been deed using Primaclade based on alignments constructed from multiple orthologous dinoflagellate sequences. Melting temperature T M was calculated using OligoCalc . Additional manual editing was performed in MacCladev4.
The presence of sxtA 1 and sxtA 4 genes were tested for all dinoflagellate strains following the protocol described in . A positive gDNA control from A. The three-rDNA genes 18S, 5. To increase phylogenetic al, allowing for synonymous substitutions, the nucleotide sequence 3 rd codon removed was used for subsequent inferences. Outgroup taxa Apicomplexa was established from dinoflagellate phylogenies as well as global eukaryotic phylogenies that concur in placing this as the closest extant relative to the dinoflagellates .
The resulting single gene alignments were subsequently checked manually using MacCladev4. The eight separate alignments were then checked with Gblocks v0. The supermatrix concatenated approach provides support not always apparent with fewer genes . To reduce missing data and improve phylogenetic placement, concatenated Hematodinium sequences were a composite in silico chimeric of closely related intra-genus species H.
The critical factor for 518 nudes placement is not character absence, but the quality and of those present . The most likely topology was established from separate searches and bootstrap analyses were performed with pseudoreplicates. Trees were generated from two independent runs with one heated and one cold chain in the Markov Chain Monte Carlo MCMC with 40, generations, sampling every Analyses ran until the average standard deviation of split frequencies were 0.
Burn-in trees were set based on the assessment of likelihood plots and convergence diagnostics implemented in MrBayes. The majority rule tree and posterior probabilities for each inference was constructed from a consensus of the sampled post burn-in trees. Noctiluca scintillans was excluded from the presented concatenated analyses as its cryptic and inconsistent placement reduced phylogenetic support.
phylogenies based on the mitochondrial cytochrome genes, cob and cox1place Heterocapsa basal within the dinoflagellate lineage a possible phylogenetic artifact as a result of a faster mutation rate . As 518 nudes position is inconsistent with morphological data and phylogenies without mitochondrial genes we investigated this further.
In this context, the comparative rate was defined as the ratio of the pairwise distances of Heterocapsa to the ingroup taxa, compared with the mean distance between the same ingroup taxa. Here we considered A.
The distance from Heterocapsa to the ingroup taxa was divided by the mean distance 518 nudes the ingroup taxa to each other. Subsequently, cob and cox1 solely for Heterocapsa were excluded from inferences. Fifty-five dinoflagellate sequences from 13 species and four orders were successfully amplified for 18S, 5. All sequences generated in this study have been deposited in Genbank under the accession s, 18S: JX and JX, 5. Alignments generated in this study represent the broadest taxon sampling and character inferred for the dinoflagellates to date. Also the comparison of topologies inferred from separate rDNA and protein coding gene datasets demonstrated good congruence Fig.518 nudes
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