Bindhu Verghese

Molecular diversity and evolution of myticin-C antimicrobial peptide variants in the Mediterranean mussel, Mytilus galloprovincialis

Mussels have diverse groups of cysteine rich, cationic antimicrobial peptides (AMPs) (defensins, mytilins, myticins, and mytimycin) that constitute an important component of their innate immune defence. Despite the identification and characterization of these AMPs in mussels, the underlying genetic mechanisms that maintain high diversity among multiple variants of the myticin-C isoform are poorly understood. Using phylogeny-based models of sequence evolution and several site-by-site frequency spectrum statistical tests for neutrality, herein we report that positive selection has been the major driving force in maintaining high diversity among the allelic-variants of the myticin-C AMP of Mytilus galloprovincialis. The statistical tests rejected the hypothesis that all polymorphism within myticin-C loci is neutral. Although a majority of the codons constrained to purifying selection (rate of amino acid replacement to the silent substitution, omega < 1), approximately 8% of the codons with omega approximately equal to 5.5 are under positive selection (omega > 1), thus indicating adaptive evolution of certain amino acids. Direct interaction of these peptides with the surrounding pathogens and/or altered/new pathogens in the changing environment is the likely cause of molecular adaptation of certain amino acid sites in myticin-C variants.

HYPERSALINE SOIL SUPPORTS A DIVERSE COMMUNITY OF DUNALIELLA (CHLOROPHYCEAE)1

Numerous isolates of the green halophile Dunaliella were studied as part of a survey of microbial diversity at the Great Salt Plains (GSP) in Oklahoma, USA. The GSP is a large (∼65 km2) salt flat with extreme temporal and spatial fluctuations in salinity and temperature. Although the flagellate halophile Dunaliella is common worldwide, nearly all cultured isolates are from saline habitats that are primarily aquatic rather than primarily terrestrial. The diverse GSP Dunaliella strains exhibit three morphotypes: a predominantly motile form, a motile form with a prominent palmelloid phase (nonmotile, mucilage rich), and a palmelloid form with a weakly motile phase. All had broad salinity optima well below typical in situ salinities at the GSP, and two of the palmelloid isolates grew as well in freshwater as in highly saline media. Molecular phylogenetic and evolutionary analyses revealed that Dunaliella from the GSP (and two similar habitats in the Great Basin, USA) are allied with D. viridis Teodor. but possess phylogenetic diversity in excess of existing global isolates from aquatic habitats. In addition, isolates from primarily terrestrial habitats exhibit statistically higher rates of nucleotide substitution than the phylogenetically homogeneous set of primarily aquatic Dunaliella taxa. We hypothesize that dynamically extreme saline soil habitats may select for different and more diverse Dunaliella lineages than more stable saline aquatic habitats. We also propose Dunaliella as a tractable microbial model for in situ testing of evolutionary and phylogeographic hypotheses.

AN UNRECOGNIZED ANCIENT LINEAGE OF GREEN PLANTS PERSISTS IN DEEP MARINE WATERS 1

We provide molecular phylogenetic evidence that the obscure genera Palmophyllum Kütz. and Verdigellas D. L. Ballant. et J. N. Norris form a distinct and early diverging lineage of green algae. These palmelloid seaweeds generally persist in deep waters, where grazing pressure and competition for space are reduced. Their distinctness warrants recognition as a new order, the Palmophyllales. Although phylogenetic analyses of both the 18S rRNA gene and two chloroplast genes (atpB and rbcL) are in agreement with a deep‐branching Palmophyllales, the genes are in conflict about its exact phylogenetic placement. Analysis of the nuclear ribosomal DNA allies the Palmophyllales with the prasinophyte genera Prasinococcus and Prasinoderma (Prasinococcales), while the plastid gene phylogeny placed Palmophyllum and Verdigellas as sister clade to all other Chlorophyta.

Positive natural selection in the evolution of human metapneumovirus attachment glycoprotein

Abstract Human metapneumovirus (hMPV), a newly discovered virus of the family Paramyxoviridae, has been associated with upper and lower respiratory tract infections in different age groups in many countries. The putative attachment (G) glycoprotein of this virus was previously reported to have shown more extensive nucleotide and deduced amino acid sequence polymorphism than any other genomic regions of this virus, leading to four sub-lineages. Using a maximum likelihood-based codon substitution model of sequence evolution, here we report that sequences of extracellular domain of 8 amino acid sites in lineage 1a, and 3 amino acid sites each in lineage 1b, 2a, and 2b have a higher rate of nonsynonymous substitutions (dN) than the synonymous substitutions (dS) with a posterior probability above 0.95, thus suggesting the evidence of adaptive evolution driven by Darwinian selection. Although it is unclear whether these amino acid adaptations are driven by differential immune pressure or some other factors, identification of these positively selected amino acid sites would help in better screening using epitope mapping technology to identify and localize the sites that can be recognized by the immune system. We also observed surprisingly higher nucleotide substitution rates per site, per year for each lineage of hMPV than the rates that were previously reported for the human respiratory syncytial virus, suggesting rapid evolutionary dynamics of hMPV.

Detecting molecular adaptation at individual codons in the glycoprotein gene of the geographically diversified infectious hematopoietic necrosis virus, a fish rhabdovirus

Salmonid fishes, the principal hosts of the infectious hematopoietic necrosis virus (IHNV), are a candidate species for aquaculture in many countries. IHNV causes an acute disease resulting in severe economic loss in salmonid fish farming. Previous phylogenetic analyses revealed the existence of multiple genogroups of this virus throughout the geographical range of its host. Here, we report the importance of natural selection in shaping the evolution of certain codons at the surface glycoprotein (G-protein) gene of this virus. Maximum likelihood (ML)-based codon substitution analyses revealed that approximately 2.8% of the codons for the entire G-protein are shown to have higher nonsynonymous substitution per nonsynonymous site (dn) than the synonymous substitutions per synonymous site (ds) (dn/ds=omega>4.335). Thus, the data suggest that positive selection (omega>1) is the major driving force in the evolution of certain codons. However, majority of these positively selected sites cannot be mapped to the regions of antigenic determinants of IHNV. Based on the reports of previous studies, epitopes with positively selected sites are immunodominant and viruses can escape from immune responses by producing antigenic variation at positively selected sites, therefore, vaccines directed against these neutralizing epitopes of IHNV that consist of no positively selected sites will be more effective. Some of the positively selected sites showed radical change in amino acids with respect to their charge and polarity; however, it is unclear how these changes affect the fitness of the virus.

Dynamic evolutionary pattern of α2-macroglobulin in a model organism, the zebrafish (Danio rerio)

Studies of alpha-2-macroglobulin (alpha(2)M), a universal protease inhibitor, have indicated that it plays a unique and critical role in the innate immune system of vertebrate and invertebrate animals. The distinctive mechanism of pathogen inhibition--through physical entrapment of the pathogen-derived protease--makes alpha(2)M an ideal candidate for molecular evolutionary analysis. Furthermore, recent studies revealed that the Osteichthyes are characterized by levels of alpha(2)M diversification that exceed those recorded in other animal groups. Our study of Danio rerio (zebrafish) indicated that (1) two distinct lineages of alpha(2)M and alpha(2)M-like isoforms exist and (2) at least some codons in the functional domains of alpha(2)M have been subjected to positive Darwinian selection. The findings of several hot-spots for nonsynonymous substitutions in the two functional domains such as bait region and receptor binding domain, suggest that host-immune selection have played a dominant role in these two genomic regions of alpha(2)M. The presence of two, non-monophyletic alpha(2)M lineages in zebrafish provides compelling evidence of an ancient gene duplication event. The accelerated rate of nucleotide substitution in the functional domains of alpha(2)M is consistent with similar observations of other immune system components.

Detecting molecular adaptation at individual codons in the pattern recognition protein, lipopolysaccharide- and β-1,3-glucan-binding protein of decapods

Pattern recognition proteins play an important role in the innate immune response of invertebrates. Herein we report the evolutionary relationships among Gram-negative bacteria binding proteins (GNBPs) that were previously identified and characterized from a wide array of invertebrates. Our results, together with those obtained in previous studies, indicate that decapod lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP/BGBP) has retained the crucial components for glucanase activity, and shares a common ancestor with GNBPs, as well as with the glucanase proteins of a wide range of invertebrates, rather than with GNBPs of some arthropods. However, experimental evidence of earlier studies suggested a lack of glucanase activity by these proteins, thus implying that during evolutionary time these proteins might have lost their glucan binding protein, but retained their glucan binding activity. The present results have also revealed that although a vast majority of the decapod LGBP/BGBP codons are constrained to purifying selection, certain codons are shown to have a higher rate of nonsynonymous substitutions per nonsynonymous site (dN) than synonymous substitutions per synonymous site (dS), indicating these codons have evolved adaptively (dN/dS>1). Although purifying selection (dN/dS<1) appears to be the major driving force in the evolution of a vast majority of LGBP/BGBP codons in decapods, the findings of several hotspots for nonsynonymous substitutions in this protein indicate host immune selection might play an important role in maintaining diversity among these ecologically diversified decapod species.