Retention of duplicated long-wavelength opsins in mosquito lineages by positive selection and differential expression.

BACKGROUND: Opsins are light sensitive receptors associated with visual processes. Insects typically possess opsins that are stimulated by ultraviolet, short and long wavelength (LW) radiation. Six putative LW-sensitive opsins predicted in the yellow fever mosquito, Aedes aegypti and malaria mosquito, Anopheles gambiae, and eight in the southern house mosquito, Culex quinquefasciatus, suggest gene expansion in the Family Culicidae (mosquitoes) relative to other insects. Here we report the first detailed molecular and evolutionary analyses of LW opsins in three mosquito vectors, with a goal to understanding the molecular basis of opsin-mediated visual processes that could be exploited for mosquito control. RESULTS: Time of divergence estimates suggest that the mosquito LW opsins originated from 18 or 19 duplication events between 166.9/197.5 to 1.07/0.94 million years ago (MY) and that these likely occurred following the predicted divergence of the lineages Anophelinae and Culicinae 145-226 MY. Fitmodel analyses identified nine amino acid residues in the LW opsins that may be under positive selection. Of these, eight amino acids occur in the N and C termini and are shared among all three species, and one residue in TMIII was unique to culicine species. Alignment of 5' non-coding regions revealed potential Conserved Non-coding Sequences (CNS) and transcription factor binding sites (TFBS) in seven pairs of LW opsin paralogs. CONCLUSIONS: Our analyses suggest opsin gene duplication and residues possibly associated with spectral tuning of LW-sensitive photoreceptors. We explore two mechanisms - positive selection and differential expression mediated by regulatory units in CNS - that may have contributed to the retention of LW opsin genes in Culicinae and Anophelinae. We discuss the evolution of mosquito LW opsins in the context of major Earth events and possible adaptation of mosquitoes to LW-dominated photo environments, and implications for mosquito control strategies based on disrupting vision-mediated behaviors.

Evolution of the Telomere-Associated Protein POT1a in Arabidopsis thaliana Is Characterized by Positive Selection to Reinforce Protein-Protein Interaction.

Gene duplication is a major driving force in genome evolution. Here, we explore the nature and origin of the POT1 gene duplication in Arabidopsis thaliana. Protection of Telomeres (POT1) is a conserved multifunctional protein that modulates telomerase activity and its engagement with telomeres. Arabidopsis thaliana encodes two divergent POT1 paralogs termed AtPOT1a and AtPOT1b. AtPOT1a positively regulates telomerase activity, whereas AtPOT1b is proposed to negatively regulate telomerase and promote chromosome end protection. Phylogenetic analysis uncovered two independent POT1 duplication events in the plant kingdom, including one at the base of Brassicaceae. Tests for positive selection implemented in PAML revealed that the Brassicaceae POT1a lineage experienced positive selection postduplication and identified three amino acid residues with signatures of positive selection. A sensitive and quantitative genetic complementation assay was developed to assess POT1a function in A. thaliana. The assay showed that AtPOT1a is functionally distinct from single-copy POT1 genes in other plants. Moreover, for two of the sites with a strong signature of positive selection, substitutions that swap the amino acids in AtPOT1a for residues found in AtPOT1b dramatically compromised AtPOT1a function in vivo. In vitro-binding studies demonstrated that all three sites under positive selection specifically enhance the AtPOT1a interaction with CTC1, a core component of the highly conserved CST (CTC1/STN1/TEN1) telomere protein complex. Our results reveal a molecular mechanism for the role of these positively selected sites in AtPOT1a. The data also provide an important empirical example to refine theories of duplicate gene retention, as the outcome of positive selection here appears to be reinforcement of an ancestral function, rather than neofunctionalization. We propose that this outcome may not be unusual when the duplicated protein is a component of a multisubunit complex whose function is in part specified by other members.

Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae).

PREMISE OF THE STUDY: Complete chloroplast genome studies can help resolve relationships among large, complex plant lineages such as Asteraceae. We present the first whole plastome from the Madieae tribe and compare its sequence variation to other chloroplast genomes in Asteraceae. METHODS: We used high throughput sequencing to obtain the Lasthenia burkei chloroplast genome. We compared sequence structure and rates of molecular evolution in the small single copy (SSC), large single copy (LSC), and inverted repeat (IR) regions to those for eight Asteraceae accessions and one Solanaceae accession. KEY RESULTS: The chloroplast sequence of L. burkei is 150 746 bp and contains 81 unique protein coding genes and 4 coding ribosomal RNA sequences. We identified three major inversions in the L. burkei chloroplast, all of which have been found in other Asteraceae lineages, and a previously unreported inversion in Lactuca sativa. Regions flanking inversions contained tRNA sequences, but did not have particularly high G + C content. Substitution rates varied among the SSC, LSC, and IR regions, and rates of evolution within each region varied among species. Some observed differences in rates of molecular evolution may be explained by the relative proportion of coding to noncoding sequence within regions. CONCLUSIONS: Rates of molecular evolution vary substantially within and among chloroplast genomes, and major inversion events may be promoted by the presence of tRNAs. Collectively, these results provide insight into different mechanisms that may promote intramolecular recombination and the inversion of large genomic regions in the plastome.

Structure and evolution of the plant cation diffusion facilitator family of ion transporters.

BACKGROUND: Members of the cation diffusion facilitator (CDF) family are integral membrane divalent cation transporters that transport metal ions out of the cytoplasm either into the extracellular space or into internal compartments such as the vacuole. The spectrum of cations known to be transported by proteins of the CDF family include Zn, Fe, Co, Cd, and Mn. Members of this family have been identified in prokaryotes, eukaryotes, and archaea, and in sequenced plant genomes. CDF families range in size from nine members in Selaginella moellendorffii to 19 members in Populus trichocarpa. Phylogenetic analysis suggests that the CDF family has expanded within plants, but a definitive plant CDF family phylogeny has not been constructed. RESULTS: Representative CDF members were annotated from diverse genomes across the Viridiplantae and Rhodophyta lineages and used to identify phylogenetic relationships within the CDF family. Bayesian phylogenetic analysis of CDF amino acid sequence data supports organizing land plant CDF family sequences into 7 groups. The origin of the 7 groups predates the emergence of land plants. Among these, 5 of the 7 groups are likely to have originated at the base of the tree of life, and 2 of 7 groups appear to be derived from a duplication event prior to or coincident with land plant evolution. Within land plants, local expansion continues within select groups, while several groups are strictly maintained as one gene copy per genome. CONCLUSIONS: Defining the CDF gene family phylogeny contributes to our understanding of this family in several ways. First, when embarking upon functional studies of the members, defining primary groups improves the predictive power of functional assignment of orthologous/paralogous genes and aids in hypothesis generation. Second, defining groups will allow a group-specific sequence motif to be generated that will help define future CDF family sequences and aid in functional motif identification, which currently is lacking for this family in plants. Third, the plant-specific expansion resulting in Groups 8 and 9 evolved coincident to the early primary radiation of plants onto land, suggesting these families may have been important for early land colonization.

A guardian of grasses: specific origin and conservation of a unique disease-resistance gene in the grass lineage.

The maize Hm1 gene provides protection against a lethal leaf blight and ear mold disease caused by Cochliobolus carbonum race 1 (CCR1). Although it was the first disease-resistance (DR) gene to be cloned, it remains a novelty because, instead of participating in the plant recognition and response system as most DR genes do, Hm1 disarms the pathogen directly. It does so by encoding an NADPH-dependent reductase, whose function is to inactivate Helminthosporium carbonum (HC) toxin, an epoxide-containing cyclic tetrapeptide, which the pathogen produces as a key virulence factor to colonize maize. Although CCR1 is strictly a pathogen of maize, orthologs of Hm1 and the HC-toxin reductase activity are present in the grass family, suggesting an ancient and evolutionarily conserved role of this DR trait in plants. Here, we provide proof for such a role by demonstrating its involvement in nonhost resistance of barley to CCR1. Barley leaves in which expression of the Hm1 homologue was silenced became susceptible to infection by CCR1, but only if the pathogen was able to produce HC toxin. Phylogenetic analysis indicated that Hm1 evolved exclusively and early in the grass lineage. Given the devastating ability of CCR1 to kill maize, these findings imply that the evolution and/or geographical distribution of grasses may have been constrained if Hm1 did not emerge.

Conservation of B class gene expression in the second whorl of a basal grass and outgroups links the origin of lodicules and petals.

Studies of flower development in core eudicot species have established a central role for B class MADS-box genes in specifying petal and stamen identities. Similarly in maize and rice, B class genes are essential for lodicule and stamen specification, suggesting homology of petals and lodicules and conservation of B class gene activity across angiosperms. However, lodicules are grass-specific organs with a morphology distinct from petals, thus their true homology to eudicot and nongrass monocot floral organs has been a topic of debate. To understand the relationship of lodicules to the sterile floral organs of nongrass monocots we have isolated and observed the expression of B class genes from a basal grass Streptochaeta that diverged before the evolution of lodicules, as well as the outgroups Joinvillea and Elegia, which have a typical monocot floral plan. Our results support a conserved role for B function genes across the angiosperms and provide additional evidence linking the evolution of lodicules and second whorl tepal/petals of monocots. The expression data and morphological analysis suggest that the function of B class genes should be broadly interpreted as required for differentiation of a distinct second floral whorl as opposed to specifying petal identity per se.

Phylogenetic relationships among early-diverging eudicots based on four genes: were the eudicots ancestrally woody?

Based on analyses of combined data sets of three genes (18S rDNA, rbcL, and atpB), phylogenetic relationships among the early-diverging eudicot lineages (Ranunculales, Proteales, Trochodendraceae, Sabiaceae, and Buxaceae) remain unclear, as are relationships within Ranunculales, especially the placement of Eupteleaceae. To clarify relationships among these early-diverging eudicot lineages, we added entire sequences of 26S rDNA to the existing three-gene data set. In the combined analyses of four genes based on parsimony, ML, and Bayesian analysis, Ranunculales are strongly supported as a clade and are sister to other eudicots. Proteales appear as sister to the remaining eudicots, which are weakly (59%) supported as a clade. Relationships among Trochodendraceae, Buxaceae (including Didymeles), Sabiaceae, and Proteales remain unclear. Within Ranunculales, Eupteleaceae are sister to all other Ranunculales, with bootstrap support of 70% in parsimony analysis and with posterior probability of 1.00 in Bayesian analysis. Our character reconstructions indicate that the woody habit is ancestral, not only for the basal angiosperms, but also for the eudicots. Furthermore, Ranunculales may not be ancestrally herbaceous, as long maintained. The woody habit appears to have been ancestral for several major clades of eudicots, including Caryophyllales, and asterids.

Gunnerales are sister to other core eudicots: implications for the evolution of pentamery.

Phylogenetic relationships among many lineages of angiosperms have been clarified via the analysis of large molecular data sets. However, with a data set of three genes (18S rDNA, rbcL, and atpB), relationships among lineages of core eudicots (Berberidopsidales, Caryophyllales, Gunnerales, Santalales, Saxifragales, asterids, rosids) remain essentially unresolved. We added 26S rDNA sequences to a three-gene matrix for 201 eudicots (8430 base pair aligned nucleotides per taxon). Parsimony analyses provided moderate (84%) jackknife support for Gunnerales, which comprise the two enigmatic families Gunneraceae and Myrothamnaceae, as sister to all other core eudicots. This position of Gunnerales has important implications for floral evolution. A dimerous or trimerous perianth is frequently encountered in early-diverging eudicots (e.g., Buxaceae, Proteales, Ranunculales, Trochodendraceae), whereas in core eudicots, pentamery predominates. Significantly, dimery is found in Gunneraceae and perhaps Myrothamnaceae (the merosity of the latter has also been interpreted as labile). Parsimony reconstructions of perianth merosity demonstrate lability among early-diverging eudicots and further indicate that a dimerous perianth could be the immediate precursor to the pentamerous condition characteristic of core eudicots. Thus, the developmental canalization that yielded the pentamerous condition of core eudicots occurred after the node leading to Gunnerales.

The root of the angiosperms revisited.

Most recent phylogenetic analyses of basal angiosperms have converged on the placement of Amborella as sister to all other extant angiosperms. However, certain recent studies suggest that Amborella and Nymphaeales (water lilies) form a clade sister to all remaining angiosperms or that Nymphaeales alone are the sister to the remaining angiosperms. We report here (i) maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses of 11 genes (>15,000 bp per taxon) for 16 taxa, (ii) maximum parsimony analysis for a subset of these genes for 104 taxa, and (iii) tests of alternative rootings with the nonparametric bootstrap and the likelihood ratio test with the parametric bootstrap. In addition, we use simulation analyses to examine the amount of bias that may be present in our methods of phylogeny estimation. Amborella continues to receive strong bootstrap support as the sister to all other extant angiosperms, and three of four tests reject alternative hypotheses of the angiosperm root. Although we cannot conclusively choose between Amborella vs. Amborella + Nymphaeales as sister to all other angiosperms, most analyses favor the former rooting.

Phylogeny of seed plants based on evidence from eight genes.

Relationships among the five groups of extant seed plants (cycads, Ginkgo, conifers, Gnetales, and angiosperms) remain uncertain. To explore relationships among groups of extant seed plants further and to attempt to explain the conflict among molecular data sets, we assembled a data set of four plastid (cpDNA) genes (rbcL, atpB, psaA, and psbB), three mitochondrial (mtDNA) genes (mtSSU, coxI, and atpA), and one nuclear gene (18S rDNA) for 19 exemplars representing the five groups of living seed plants. Analyses of the combined eight-gene data set (15 772 base pairs/taxon) with maximum parsimony (MP), maximum likelihood (ML), and Bayesian approaches reveal a gymnosperm clade that is sister to angiosperms. Within the gymnosperms, a conifer clade includes Gnetales as sister to Pinaceae. Cycads and Ginkgo are either successive sisters to this conifer clade (including Gnetales) or a clade that is sister to conifers and Gnetales. All analyses of the mtDNA partition and ML analyses of the nuclear partition yield very similar topologies. However, MP analyses of the combined cpDNA genes place Gnetales as sister to all other seed plants with strong bootstrap support, whereas ML and Bayesian analyses of the cpDNA data set place Gnetales as sister to Pinaceae. Maximum parsimony and ML analyses of first and second codon positions of the cpDNA partiation also place Gnetales as sister to Pinaceae. In contrast, MP analyses of third codon positions place Gnetales as sister to other seed plants, although ML analyses of third codon positions place Gnetales with Pinaceae. Thus, most of the discrepancies in seed plant topologies involve third codon positions of cpDNA genes. The likelihood ratio (LR) and Shimodaira-Hasegasa (SH) tests were applied to the cpDNA data. The preferred topology based on the LR test is that Gnetales are sister to Pseudotsuga. The SH test based on first and second codon and all three codon positions indicated that there is no significant difference between the best topology (Gnetales sister to Pseudotsuga) and Gnetales sister to a conifer clade. However, there is a significant difference between the best topology and topologies in which Gnetales are sister to the rest of the seed plants or Gnetales sister to angiosperms.

Molecular data place Hydnoraceae with Aristolochiaceae.

Utilization of molecular phylogenetic information over the past decade has resulted in clarification of the position of most angiosperms. In contrast, the position of the holoparasitic family Hydnoraceae has remained controversial. To address the question of phylogenetic position of Hydnoraceae among angiosperms, nuclear SSU and LSU rDNA and mitochondrial atp1 and matR sequences were obtained for Hydnora and Prosopanche. These sequences were used in combined analyses that included the above four genes as well as chloroplast rbcL and atpB (these plastid genes are missing in Hydnoraceae and were hence coded as missing). Three data sets were analyzed using maximum parsimony: (1) three genes with 461 taxa; (2) five genes with 77 taxa; and (3) six genes with 38 taxa. Analyses of separate and combined data partitions support the monophyly of Hydnoraceae and the association of that clade with Aristolochiaceae sensu lato (s.l.) (including Lactoridaceae). The latter clade is sister to Piperaceae and Saururaceae. Despite over 11 kilobases (kb) of sequence data, relationships within Aristolochiaceae s.l. remain unresolved, thus it cannot yet be determined whether Aristolochiaceae, Hydnoraceae, and Lactoridaceae should be classified as distinct families. In contrast to most traditional classifications, molecular phylogenetic analyses do not suggest a close relationship between Hydnoraceae and Rafflesiaceae. A number of morphological features is shared by Hydnoraceae and Aristolochiaceae; however, a more resolved phylogeny is required to determine whether these represent synapomorphies or independent acquisitions.