A Phylogenetic Rate Parameter Indicates Different Sequence Divergence Patterns in Orthologs and Paralogs.

Heterotachy-the change in sequence evolutionary rate over time-is a common feature of protein molecular evolution. Decades of studies have shed light on the conditions under which heterotachy occurs, and there is evidence that site-specific evolutionary rate shifts are correlated with changes in protein function. Here, we present a large-scale, computational analysis using thousands of protein sequence alignments from animal and plant proteomes, representing genes related either by orthology (speciation events) or paralogy (gene duplication), to compare sequence divergence patterns in orthologous vs. paralogous sequence alignments. We use sequence-based phylogenetic analyses to infer overall sequence divergence (tree length/number of sequences) and to fit site-specific rates to a discrete gamma distribution with a shape parameter α. This inference method is applied to real protein sequence alignments, as well as alignments simulated under various models of protein sequence evolution. Our simulations indicate that sequence divergence and the α parameter are positively correlated when sequences evolve with heterotachy, meaning that inferred site rate distributions appear more uniform as sequences diverge. Divergence and α are also positively correlated in both orthologous and paralogous genes, but the average increase in α (as a function of divergence) is significantly higher in paralogous protein alignments than in orthologous alignments. This result is consistent with the widely held view that recently duplicated proteins initially evolve under relaxed selective pressure, promoting functional divergence by accumulation of amino acid replacements, and hence experience more evolutionary rate fluctuations than orthologous proteins. We discuss these findings in the context of the ortholog conjecture, a long-standing assumption in molecular evolution, which posits that protein sequences related by orthology tend to be more functionally conserved than paralogous proteins.

Exploring Evolutionary Constraints in the Proteomes of Zika, Dengue, and Other Flaviviruses to Find Fitness-Critical Sites.

Dengue virus (DENV) challenges vaccine design due to antibody-dependent enhancement (ADE) and evidence suggests that Zika virus (ZIKV) experiences ADE with DENV and West Nile virus (WNV) antibodies. Thus, multiple flaviviruses must be considered when developing novel therapies against ZIKV. We analyzed 42 flavivirus polyproteins in their evolutionary context to identify motifs conserved in sequence with low real-time and evolutionary conformational flexibility, thought to be fitness-critical sites. We also analyzed evolutionary rate-shifts between clades for insight on vector specificity. For mosquito-borne flaviviruses, two conserved motifs were identified within the RNA-dependent RNA polymerase (RdRP), critical for flavivirus genome replication. Clade-specific motifs were identified for the ZIKV+DENV and WNV clades, many of which were also in RdRP. Six sites in motifs for WNV experienced significant evolutionary rate-shifts, suggesting their importance for functional divergence. Overall, some of these motifs are prime candidates as broadly neutralizing antiviral drug targets across different mosquito-borne flaviviruses.

Large-Scale Analyses of Site-Specific Evolutionary Rates across Eukaryote Proteomes Reveal Confounding Interactions between Intrinsic Disorder, Secondary Structure, and Functional Domains.

Various structural and functional constraints govern the evolution of protein sequences. As a result, the relative rates of amino acid replacement among sites within a protein can vary significantly. Previous large-scale work on Metazoan (Animal) protein sequence alignments indicated that amino acid replacement rates are partially driven by a complex interaction among three factors: intrinsic disorder propensity; secondary structure; and functional domain involvement. Here, we use sequence-based predictors to evaluate the effects of these factors on site-specific sequence evolutionary rates within four eukaryotic lineages: Metazoans; Plants; Saccharomycete Fungi; and Alveolate Protists. Our results show broad, consistent trends across all four Eukaryote groups. In all four lineages, there is a significant increase in amino acid replacement rates when comparing: (i) disordered vs. ordered sites; (ii) random coil sites vs. sites in secondary structures; and (iii) inter-domain linker sites vs. sites in functional domains. Additionally, within Metazoans, Plants, and Saccharomycetes, there is a strong confounding interaction between intrinsic disorder and secondary structure-alignment sites exhibiting both high disorder propensity and involvement in secondary structures have very low average rates of sequence evolution. Analysis of gene ontology (GO) terms revealed that in all four lineages, a high fraction of sequences containing these conserved, disordered-structured sites are involved in nucleic acid binding. We also observe notable differences in the statistical trends of Alveolates, where intrinsically disordered sites are more variable than in other Eukaryotes and the statistical interactions between disorder and other factors are less pronounced.

Evolution of intrinsic disorder in eukaryotic proteins.

Conformational flexibility conferred though regions of intrinsic structural disorder allows proteins to behave as dynamic molecules. While it is well-known that intrinsically disordered regions can undergo disorder-to-order transitions in real-time as part of their function, we also are beginning to learn more about the dynamics of disorder-to-order transitions along evolutionary time-scales. Intrinsically disordered regions endow proteins with functional promiscuity, which is further enhanced by the ability of some of these regions to undergo real-time disorder-to-order transitions. Disorder content affects gene retention after whole genome duplication, but it is not necessarily conserved. Altered patterns of disorder resulting from evolutionary disorder-to-order transitions indicate that disorder evolves to modify function through refining stability, regulation, and interactions. Here, we review the evolution of intrinsically disordered regions in eukaryotic proteins. We discuss the interplay between secondary structure and disorder on evolutionary time-scales, the importance of disorder for eukaryotic proteome expansion and functional divergence, and the evolutionary dynamics of disorder.

The Nuanced Interplay of Intrinsic Disorder and Other Structural Properties Driving Protein Evolution.

Protein evolution often occurs at unequal rates in different sites along an amino acid chain. Site-specific evolutionary rates have been linked to several structural and functional properties of proteins. Previous analyses of this phenomenon have involved relatively small datasets and, in some cases, the interaction among multiple structural factors is not evaluated. Here, we present the results of a large-scale phylogenetic and statistical analysis, testing the effects and interactions of three structural properties on amino acid replacement rates. We used sequence-based computational methods to predict (i) intrinsic disorder propensity, (ii) secondary structure, and (iii) functional domain involvement across millions of amino acid sites in thousands of sequence alignments of metazoan proteins. Our results somewhat corroborate earlier findings that intrinsically disordered sites tend to be more variable than ordered sites, but there is considerable overlap among their rate distributions, and a significant confounding interaction exists between intrinsic disorder and secondary structure. Notably, protein sites that are consistently predicted to be both intrinsically disordered and involved in secondary structures tend to be the most conserved at the amino acid level, suggesting that they are highly constrained and functionally important. In addition, a significant interaction exists between functional domain involvement and secondary structure. These findings suggest that multiple structural drivers of protein evolution should be evaluated simultaneously in order to get a clear picture of their individual effects as well as any confounding interactions among them.

Regeneration of posterior segments and terminal structures in the bearded fireworm, Hermodice carunculata (Annelida: Amphinomidae).

Like many other annelids, bearded fireworms, Hermodice carunculata, are capable of regenerating posterior body segments and terminal structures lost to amputation. Although previous research has examined anterior regeneration in other fireworm species, posterior regenerative ability in fireworms remains poorly studied. As the morphology of the anal lobe (a small, fleshy terminal structure of unknown function) has been used to distinguish East and West Atlantic H. carunculata populations, there is a more imminent need to understand the morphology and organization of tissues in specimens undergoing posterior regeneration, and the timeframe in which significant developmental changes occur. To further investigate this phenomenon, we amputated the posterior segments of living H. carunculata specimens collected from the Gulf of Mexico and monitored posterior regeneration over a 6-month study period. Although many aspects of posterior regeneration in H. carunculata are consistent with the findings of other annelid regeneration studies, histological analysis revealed that once formed, anal lobe morphology remains relatively unchanged at all stages of posterior regeneration; East Atlantic morphotypes were not observed in the West Atlantic specimens studied here. Additionally, we found that the ventral nerve chord, which is partially responsible for the regeneration of lost body parts in polychaete annelids, terminates within the anal lobe, suggesting that this structure may play a role in the formation of new segments.

The curious case of Hermodice carunculata (Annelida: Amphinomidae): evidence for genetic homogeneity throughout the Atlantic Ocean and adjacent basins.

Over the last few decades, advances in molecular techniques have led to the detection of strong geographic population structure and cryptic speciation in many benthic marine taxa, even those with long-lived pelagic larval stages. Polychaete annelids, in particular, generally show a high degree of population divergence, especially in mitochondrial genes. Rarely have molecular studies confirmed the presence of 'cosmopolitan' species. The amphinomid polychaete Hermodice carunculata was long considered the sole species within its genus, with a reported distribution throughout the Atlantic and adjacent basins. However, recent studies have indicated morphological differences, primarily in the number of branchial filaments, between the East and West Atlantic populations; these differences were invoked to re-instate Hermodice nigrolineata, formerly considered a junior synonym of H. carunculata. We utilized sequence data from two mitochondrial (cytochrome c oxidase subunit I, 16S rDNA) markers and one nuclear (internal transcribed spacer) marker to examine the genetic diversity of Hermodice throughout its distribution range in the Atlantic Ocean, including the Mediterranean Sea, the Caribbean Sea, the Gulf of Mexico and the Gulf of Guinea. Our analyses revealed generally low genetic divergences among collecting localities and between the East and West Atlantic, although phylogenetic trees based on mitochondrial data indicate the presence of a private lineage in the Mediterranean Sea. A re-evaluation of the number of branchial filaments confirmed differences between East and West Atlantic populations; however, the differences were not diagnostic and did not reflect the observed genetic population structure. Rather, we suspect that the number of branchial filaments is a function of oxygen saturation in the environment. Our results do not support the distinction between H. carunculata in the West Atlantic and H. nigrolineata in the East Atlantic. Instead, they re-affirm the older notion that H. carunculata is a cohesive species with a broad distribution across the Atlantic Ocean.