Evolution of Type II Antifreeze Protein Genes in Teleost Fish: A Complex Scenario Involving Lateral Gene Transfers and Episodic Directional Selection.

I examined hypotheses about lateral transfer of type II antifreeze protein (AFP) genes among "distantly" related teleost fish. The effects of episodic directional selection on amino acid evolution were also investigated. The strict consensus results showed that the type II AFP and type II antifreeze-like protein genes were transferred from Osmerus mordax to Clupea harengus, from the ancestral lineage of the Brachyopsis rostratus-Hemitripterus americanus clade to the ancestor of the Hypomesus nipponensis-Osmerus mordax group and from the ancestral lineage of Brachyopsis rostratus-Hemitripterus americanus-Siniperca chuatsi-Perca flavescens to Perca flavescens. At the present time, the available evidence is more consistent with the LGT hypothesis than with other alternative explanations. The overall results indicate that evolutionary history of the type II AFP gene is complex, and that episodic directional selection was instrumental in the evolution of this freeze-preventing protein from a C-type lectin precursor.

Phylogenetic analyses of a combined data set suggest that the Attheya lineage is the closest living relative of the pennate diatoms (Bacillariophyceae).

A Bayesian analysis of a seven gene data set was conducted to reconstruct phylogenetic relationships among a sample of centric and pennate diatoms and to test alternative hypotheses about the closest living relative of Bacillariophyceae. A lineage, composed of two Attheya species, was inferred to share the most recent common ancestor with Bacillariophyceae--a relationship that was also corroborated by the combined parsimony analysis. All competing hypotheses about the closest living relative of Bacillariophyceae were rejected because 100% of the trees in the post-burn-in sample in the Bayesian analysis supported the Attheya-Bacillariophyceae clade. According to a partitioned Bremer support analysis, the majority of the genes in the combined data matrix supported the Attheya--Bacillariophyceae relationship. The global topology of the phylogenetic tree indicated that a monophyletic group consisting of Thalassiosirales and Toxarium undulatum formed the deepest branch followed by a node uniting a clade composed of Bacillariophyceae/Attheya species and a lineage made up of Eucampia zoodiacus, Chaetocerotales, Lithodesmiales, Triceratiales, Biddulphiales and Cymatosirales. Except for the phylogenetic positions of Lithodesmiales, Thalassiosira sp and Skeletonema costatum, the optimal tree obtained from the combined parsimony analysis showed the same branching order of taxa as those seen in the consensus tree inferred from three independent Markov chain Monte Carlo analyses. Noteworthy findings are that Toxarium undulatum shares a strongly supported node with Thalassiosirales and that the genus Attheya is not a member of the Chaetocerotales lineage.

Evolution of antifreeze protein genes in the diatom genus fragilariopsis: evidence for horizontal gene transfer, gene duplication and episodic diversifying selection.

Hypotheses about horizontal transfer of antifreeze protein genes to ice-living diatoms were addressed using two different statistical methods available in the program Prunier. The role of diversifying selection in driving the differentiation of a set of antifreeze protein genes in the diatom genus Fragilariopsis was also investigated. Four horizontal gene transfer events were identified. Two of these took place between two major eukaryote lineages, that is from the diatom Chaetoceros neogracile to the copepod Stephos longipes and from a basidiomycete clade to a monophyletic group, consisting of the diatom species Fragilariopsis curta and Fragilariopsis cylindrus. The remaining two events included transfers from an ascomycete lineage to the proteobacterium Stigmatella aurantiaca and from the proteobacterium Polaribacter irgensii to a group composed of 4 proteobacterium species. After the Fragilariopsis lineage acquired the antifreeze protein gene from the basidiomycetes, it duplicated and went through episodic evolution, characterized by strong positive selection acting on short segments of the branches in the tree. This selection pattern suggests that the paralogs differentiated functionally over relatively short time periods. Taken together, the results obtained here indicate that the group of antifreeze protein genes considered here have a complex evolutionary history.

A molecular genetic timescale for the diversification of autotrophic stramenopiles (Ochrophyta): substantive underestimation of putative fossil ages.

BACKGROUND: Stramenopiles constitute a large and diverse eukaryotic clade that is currently poorly characterized from both phylogenetic and temporal perspectives at deeper taxonomic levels. To better understand this group, and in particular the photosynthetic stramenopiles (Ochrophyta), we analyzed sequence data from 135 taxa representing most major lineages. Our analytical approach utilized several recently developed methods that more realistically model the temporal evolutionary process. METHODOLOGY/PRINCIPAL FINDINGS: Phylogenetic reconstruction employed a Bayesian joint rate- and pattern-heterogeneity model to reconstruct the evolutionary history of these taxa. Inferred phylogenetic resolution was generally high at all taxonomic levels, sister-class relationships in particular receiving good statistical support. A signal for heterotachy was detected in clustered portions of the tree, although this does not seem to have had a major influence on topological inference. Divergence time estimates, assuming a lognormally-distributed relaxed molecular clock while accommodating topological uncertainty, were broadly congruent over alternative temporal prior distributions. These data suggest that Ochrophyta originated near the Proterozoic-Phanerozoic boundary, diverging from their sister-taxon Oomycota. The evolution of the major ochrophyte lineages appears to have proceeded gradually thereafter, with most lineages coming into existence by ∼200 million years ago. CONCLUSIONS/SIGNIFICANCE: The evolutionary timescale of the autotrophic stramenopiles reconstructed here is generally older than previously inferred from molecular clocks. However, this more ancient timescale nevertheless casts serious doubt on the taxonomic validity of putative xanthophyte/phaeophyte fossils from the Proterozoic, which predate by as much as a half billion years or more the age suggested by our molecular genetic data. If these fossils truly represent crown stramenopile lineages, then this would imply that molecular rate evolution in this group proceeds in a fashion that is fundamentally incompatible with the relaxed molecular clock model employed here. A more likely scenario is that there is considerable convergent morphological evolution within Heterokonta, and that these fossils have been taxonomically misdiagnosed.

Microevolution and speciation in Thalassiosira weissflogii (Bacillariophyta).

In this study five different molecular markers were used to: (1) infer the phylogeographic differentiation of Thalassiosira weissflogii in the Atlantic and Pacific Oceans; and (2) address the biological species status of the inferred geographic lineages. The results of the ribosomal RNA data analyses suggested that the Hawaiian isolate evolved first after which the Indonesian and the Atlantic/California strains diverged. In contrast, the tree derived from the partial sexually induced gene 1 (Sig1) data exhibited an initial divergence between the Eastern Atlantic/Western Atlantic/California and the Hawaiian/Indonesian groups after which the latter evolved into the Hawaiian and Indonesian lineages. The partial beta-tubulin phylogeny discerns an early "split" between an Eastern Atlantic/Western Atlantic clade and an Indonesia/California/Hawaii group which later differentiated into distinct Hawaiian and Indonesian/California "branches". The number of compensatory base changes (CBCs) in the ITS2 indicated that the "Atlantic group", including the California isolate, constituted a single reproductive unit and that the Indonesian and Hawaiian T. weissflogii are two different biological species with regard to each other and to the "Atlantic clade". The beta-tubulin tree contradicts the reproductive units recovered by the compensatory base change analysis due to the close affinity of the California and Indonesia strains whereas the sexually induced gene 1 phylogeny supports the existence of three biological species, despite exhibiting a temporal sequence of geographic diversification that is different from that seen in topologies derived from the ribosomal RNA data. It is hypothesized that the tree derived from the sexually induced gene 1 reflects the relative order of the evolution of reproductive isolation in the different T. weissflogii strains while the lineages in the other phylogenies depict time elapsed since common ancestry. The current investigation is the first to have shown concordance in the pattern of variation between a reasonably good biological species marker (CBCs in the ITS2) and a protein hypothesized to be involved in sperm-egg recognition/adhesion in diatoms (Sig1). It was further discussed that the Pacific/Indonesian T. weissflogii species complex most likely evolved in the presence of gene flow and "local" environmental selection. Three hypotheses which potentially can explain the genetic diversification/similarity between the Atlantic T. weissflogii samples and the isolate obtained off the coast of California were proposed and discussed.

Diversity dynamics of marine planktonic diatoms across the Cenozoic.

Diatoms are the dominant group of phytoplankton in the modern ocean. They account for approximately 40% of oceanic primary productivity and over 50% of organic carbon burial in marine sediments. Owing to their role as a biological carbon pump and effects on atmospheric CO(2) levels, there is great interest in elucidating factors that influenced the rapid rise in diatom diversity during the past 40 million years. Two biotic controls on diversification have been proposed to explain this diversity increase: (1) geochemical coupling between terrestrial grasslands and marine ecosystems through the global silicon cycle; and (2) competitive displacement of other phytoplankton lineages. However, these hypotheses have not been tested using sampling-standardized fossil data. Here we show that reconstructions of species diversity in marine phytoplankton reject these proposed controls and suggest a new pattern for oceanic diatom diversity across the Cenozoic. Peak species diversity in marine planktonic diatoms occurred at the Eocene-Oligocene boundary and was followed by a pronounced decline, from which diversity has not recovered. Although the roles of abiotic and biotic drivers of diversification remain unclear, major features of oceanic diatom evolution are decoupled from both grassland expansion and competition among phytoplankton groups.

Evidence for positive selection on a sexual reproduction gene in the diatom genus Thalassiosira (Bacillariophyta).

Single likelihood ancestor counting (SLAC), fixed effects likelihood (FEL), and several random effects likelihood (REL) methods were utilized to identify positively and negatively selected sites in sexually induced gene 1 (Sig1) of four different Thalassiosira species. The SLAC analysis did not find any sites affected by positive selection but suggested 13 sites influenced by negative selection. The SLAC approach may be too conservative because of low sequence divergence. The FEL and REL analyses revealed over 60 negatively selected sites and two positively selected sites that were unique to each method. The REL method may not be able to reliably identify individual sites under selection when applied to short sequences with low divergence. Instead, we proposed a new alignment-wide test for adaptive evolution based on codon models with variation in synonymous and nonsynonymous substitution rates among sites and found evidence for diversifying evolution without relying on site-by-site testing. The performance of the FEL and REL approaches was evaluated by subjecting the tests to a type I error rate simulation analysis, using the specific characteristics of the Sig1 data set. Simulation results indicated that the FEL test had reasonable Type I errors, while REL might have been too liberal, suggesting that the two positively selected sites identified by FEL (codons 94 and 174) are not likely to be false positives. The evolution of these codon sites, one of which is located in functional domain II, appears to be associated with divergence among the three major Thalassiosira lineages.

Diatom phylogenetics inferred based on direct optimization of nuclear-encoded SSU rRNA sequences.

Direct optimization (DO) of 126 nuclear-encoded SSU rRNA diatom sequences was conducted. The optimal phylogeny indicated several unique relationships with respect to those recovered from a maximum likelihood (ML) analysis of an alignment based on maximizing primary and secondary structural similarity between 126 nuclear-encoded SSU rRNA diatom sequences (Medlin and Kaczmarska, 2004). Dividing diatoms into the subdivisions Coscinodiscophytina and Bacillariophytina was not supported by the DO phylogeny, due to the paraphyly of the former. The same pertains to Coscinodiscophyceae, Mediophyceae, Thalassiosira, Fragilaria and Amphora. The ordinal-level classification of the diatoms proposed by Round et al. (1990) was for the most part found to be unsupported. The DO phylogeny represented a more rigorous hypothesis than the ML tree because DO maximized character congruence during the homology testing (i.e., alignment/tree search) process whereas the non-phylogenetic similarity-based alignment used in the ML analysis did not. The above statement is supported by "controlled" parsimony analyses of 35 sequences, which strongly suggested that dissimilarities in the DO and ML tree structure were due to the specific homology testing approach used. It could not be precluded that differences in taxon sampling and the use of a dissimilar optimality criteria contributed to discrepancies in the structure of the optimal ML and DO trees.

The effect of positive selection on a sexual reproduction gene in Thalassiosira weissflogii (Bacillariophyta): results obtained from maximum-likelihood and parsimony-based methods.

Maximum-Likelihood-based and parsimony-based methods were used to test for potential effects of positive selection on the sexually induced gene 1 (Sig1) in Thalassiosira weissflogii. The Sig proteins are thought to play a role in mediating sperm-egg recognition during the sexual reproduction phase. The results obtained from parsimony-based analyses showed that none of the amino acid sites were influenced by positive selection. Maximum-likelihood analyses indicated that positive selection was affecting a maximum of seven and a minimum of four amino acid sites in the polypeptide derived from Sig1. It was concluded that the results obtained from the maximum-likelihood-based method are more reliable than those obtained from the parsimony-based approach. This is apparently the first study that has shown that reproductive proteins in unicellular eukaryotes are influenced by positive selection.

RpoA: a useful gene for phylogenetic analysis in diatoms.

The aim of this study was to compare the usefulness of two chloroplast-encoded genes (rpoA and rbcL) and the nuclear-encoded small subunit (SSU) ribosomal RNA for reconstructing phylogenetic relationships among diatoms at lower taxonomic levels. To this end, the rpoA and rbcL genes for selected centric and pennate diatoms were sequenced. The new rpoA and rbcL sequences, and an existing nuclear-encoded SSU rRNA data set, were subjected to weighted/unweighted parsimony, maximum likelihood, minimum evolution, and Bayesian analyses. All of the tree-building methods employed showed, based on the support values, that the rpoA gene was the most useful, relative to the rbcL and SSU rRNA genes, in determining phylogenetic relationships among the sampled diatoms. The support values for the relationships among the pennate lineages were, in many instances, greater in the rpoA trees than in the SSU rRNA trees. These results suggest that rpoA might be of value in determining phylogenetic relationships among pennate lineages.

Codon adaptation and synonymous substitution rate in diatom plastid genes.

Diatom plastid genes are examined with respect to codon adaptation and rates of silent substitution (Ks). It is shown that diatom genes follow the same pattern of codon usage as other plastid genes studied previously. Highly expressed diatom genes display codon adaptation, or a bias toward specific major codons, and these major codons are the same as those in red algae, green algae, and land plants. It is also found that there is a strong correlation between Ks and variation in codon adaptation across diatom genes, providing the first evidence for such a relationship in the algae. It is argued that this finding supports the notion that the correlation arises from selective constraints, not from variation in mutation rate among genes. Finally, the diatom genes are examined with respect to variation in Ks among different synonymous groups. Diatom genes with strong codon adaptation do not show the same variation in synonymous substitution rate among codon groups as the flowering plant psbA gene which, previous studies have shown, has strong codon adaptation but unusually high rates of silent change in certain synonymous groups. The lack of a similar finding in diatoms supports the suggestion that the feature is unique to the flowering plant psbA due to recent relaxations in selective pressure in that lineage.

A "Total Evidence" Analysis of the Phylogenetic Relationships among the Photosynthetic Stramenopiles.

Phylogenetic relationships among the nine major autotrophic stramenopile taxa were inferred in a combined analysis of the rbcL, SSU rDNA, partial LSU rRNA, carotenoid, and ultrastructural data sets. The structure of the shortest combined tree is: (Outgroup, ((((Bacillariophyceae, (Pelagophyceae, Dictyochophyceae)),((Phaeophyceae, Xanthophyceae), Raphidophyceae)), Eustigmatophyceae),(Chrysophyceae, Synurophyceae))). The Synurophyceae/Chrysophyceae is the best supported group followed by the Phaeophyceae/Xanthophyceae and the Pelagophyceae/Dictyochophyceae clades. The monophyletic groups composed of Bacillariophyceae/Pelagophyceae/Dictyochophyceae and Phaeophyceae/Xanthophyceae/Raphidophyceae received the lowest Bremer support values. The optimal combined tree suggests that the diatom frustule is derived from the siliceous "skeleton" in Dictyochophyceae, that the reduced flagellar apparatus arose once in the Bacillariophyceae/Dictyochophyceae/Pelagophyceae clade, and that the specific photoreceptor-eyespot apparatus in Chrysophyceae and the Phaeophyceae/Xantophyceae clade originated independently within the autotrophic stramenopiles. Despite conflicts in tree structure between the most-parsimonious combined phylogeny and the optimal tree(s) of each data partition, it cannot be concluded that extensive incongruence exists between the data sets.