Extensive Sequence Variation Research Articles

Genomic characterization of circulating human respiratory syncytial viruses A and B in Kuwait using whole-genome sequencing.

The human respiratory syncytial virus (RSV) is considered one of the most common viruses that infect children globally. The virus is known to have extensive gene sequence variability within and between RSV groups A and B globally; however, there is no information on the whole-genome characterization and diversity of RSV in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with acute respiratory tract infection (ARTI) in Kuwait. Therefore, this study aimed to sequence the entire genome of RSV strains isolated from patients with ARTI in Kuwait. Between January 2020 and September 2022, 7,093 respiratory samples were collected from hospitalized infants, children, and adults and were analyzed for respiratory viruses by multiplex real-time PCR. Whole-genome sequencing using the Oxford Nanopore sequencing technology was performed on 84 RSV-positive samples. The results revealed a higher prevalence of group A (76%) than group B (24%) RSV isolates. Phylogenetic analysis showed that RSV-A strains clustered with the GA2.3.5 sub-genotype and RSV-B strains clustered with the GB5.0.5a sub-genotype; however, forming new lineages of RSV-A and RSV-B circulated in Kuwait during this period. Genetic variability was higher among the group A viruses than group B viruses, and the rate of synonymous and missense mutations was high in genes other than the G protein-coding gene. We also detected several known and unique molecular markers in different protein-coding genes. This is the first study in Kuwait to characterize the whole genomes of RSV A and B to identify the circulating genotypes, comprehend the genetic diversity and the evolution of the virus, and identify important genetic markers associated with specific genotypes.IMPORTANCEWhole-genome sequencing of respiratory syncytial virus (RSV) strains in Kuwait using MinION Nanopore technology was used to characterize and analyze the genotypes and sub-genotypes of the RSV circulating among patients with acute respiratory tract infections in Kuwait. This study also identified known and unknown gene mutations and imported genetic markers associated with specific genotypes. These results will assist in establishing a framework for RSV classification and allow for a better consideration of the mechanisms leading to the generation of diversity of RSV. In addition, these data will allow a comparison of vaccine viruses with those in Kuwait, providing useful insights into future vaccine and therapy strategies for RSV in Kuwait.

A FLOWERING LOCUS T ortholog is associated with photoperiod-insensitive flowering in hemp (Cannabis sativa L.).

Hemp (Cannabis sativa L.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products. Cannabis sativa is a short-day plant, and its flowering is highly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity in C. sativa, and photoperiod-insensitive ("autoflower") cultivars are available. Using a bi-parental mapping population and bulked segregant analysis, we identified Autoflower2, a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp. Autoflower2 contains an ortholog of the central flowering time regulator FLOWERING LOCUS T (FT) from Arabidopsis thaliana which we termed CsFT1. We identified extensive sequence divergence between alleles of CsFT1 from photoperiod-sensitive and insensitive cultivars of C. sativa, including a duplication of CsFT1 and sequence differences, especially in introns. Furthermore, we observed higher expression of one of the CsFT1 copies found in the photoperiod-insensitive cultivar. Genotyping of several mapping populations and a diversity panel confirmed a correlation between CsFT1 alleles and photoperiod response, affirming that at least two independent loci involved in the photoperiodic control of flowering, Autoflower1 and Autoflower2, exist in the C. sativa gene pool. This study reveals the multiple independent origins of photoperiod insensitivity in C. sativa, supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novel C. sativa cultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.

Effects of intrinsically disordered regions in gp120 underlying HIV neutralization phenotypes

HIV envelope protein gp120 is considered a primary molecular determinant of viral neutralization phenotype due to its critical role in viral entry and immune evasion. The intrinsically disordered regions (IDRs) in gp120 are responsible for their extensive sequence variations and significant structural rearrangements. Despite HIV neutralization phenotype and sequence/structural information of gp120 have been experimentally characterized, there remains a gap in our understanding of the correlation between the viral phenotype and IDRs in gp120. Here, we combined machine learning (ML) techniques and molecular dynamics (MD) simulations to gain data-driven and molecule-mechanism insights into relationships between viral sequence, structure, and phenotypes from the perspective of IDRs in gp120. ML models, trained only on the length and disorder score of IDRs, achieved equivalent performance to the best baseline model using amino acid sequences to discriminate HIV neutralization phenotype, indicating that the lengths or disorder of specific IDRs are strongly related to HIV neutralization phenotypes. Comparative MD analysis reveals that gp120 with extreme neutralization phenotypes in multiple conformational states, especially some IDRs, exhibit significantly distinct structural dynamics, conformational flexibility, and thermodynamic distributions. Taken together, our study provided insights into the role of IDRs in gp120 responding to HIV neutralization phenotypes, which will advance the understanding of molecular mechanisms underlying viral function associated with HIV neutralization phenotype and help develop antiviral vaccines or drugs.

Decoupled evolution of the Sex Peptide gene family and Sex Peptide Receptor in Drosophilidae

Across internally fertilising species, males transfer ejaculate proteins that trigger wide-ranging changes in female behaviour and physiology. Much theory has been developed to explore the drivers of ejaculate protein evolution. The accelerating availability of high-quality genomes now allows us to test how these proteins are evolving at fine taxonomic scales. Here, we use genomes from 264 species to chart the evolutionary history of Sex Peptide (SP), a potent regulator of female post-mating responses in Drosophila melanogaster. We infer that SP first evolved in the Drosophilinae subfamily and has since followed markedly different evolutionary trajectories in different lineages. Outside of the Sophophora-Lordiphosa, SP exists largely as a single-copy gene with independent losses in several lineages. Within the Sophophora-Lordiphosa, the SP gene family has repeatedly and independently expanded. Up to seven copies, collectively displaying extensive sequence variation, are present in some species. Despite these changes, SP expression remains restricted to the male reproductive tract. Alongside, we document considerable interspecific variation in the presence and morphology of seminal microcarriers that, despite the critical role SP plays in microcarrier assembly in D. melanogaster, appears to be independent of changes in the presence/absence or sequence of SP. We end by providing evidence that SP's evolution is decoupled from that of its receptor, Sex Peptide Receptor, in which we detect no evidence of correlated diversifying selection. Collectively, our work describes the divergent evolutionary trajectories that a novel gene has taken following its origin and finds a surprisingly weak coevolutionary signal between a supposedly sexually antagonistic protein and its receptor.

Advancements in copy number variation screening in herbivorous livestock genomes and their association with phenotypic traits.

Copy number variations (CNVs) have garnered increasing attention within the realm of genetics due to their prevalence in human, animal, and plant genomes. These structural genetic variations have demonstrated associations with a broad spectrum of phenotypic diversity, economic traits, environmental adaptations, epidemics, and other essential aspects of both plants and animals. Furthermore, CNVs exhibit extensive sequence variability and encompass a wide array of genomes. The advancement and maturity of microarray and sequencing technologies have catalyzed a surge in research endeavors pertaining to CNVs. This is particularly prominent in the context of livestock breeding, where molecular markers have gained prominence as a valuable tool in comparison to traditional breeding methods. In light of these developments, a contemporary and comprehensive review of existing studies on CNVs becomes imperative. This review serves the purpose of providing a brief elucidation of the fundamental concepts underlying CNVs, their mutational mechanisms, and the diverse array of detection methods employed to identify these structural variations within genomes. Furthermore, it seeks to systematically analyze the recent advancements and findings within the field of CNV research, specifically within the genomes of herbivorous livestock species, including cattle, sheep, horses, and donkeys. The review also highlighted the role of CNVs in shaping various phenotypic traits including growth traits, reproductive traits, pigmentation and disease resistance etc., in herbivorous livestock. The main goal of this review is to furnish readers with an up-to-date compilation of knowledge regarding CNVs in herbivorous livestock genomes. By integrating the latest research findings and insights, it is anticipated that this review will not only offer pertinent information but also stimulate future investigations into the realm of CNVs in livestock. In doing so, it endeavors to contribute to the enhancement of breeding strategies, genomic selection, and the overall improvement of herbivorous livestock production and resistance to diseases.

Massive expansion of sex-specific SNPs, transposon-related elements, and neocentromere formation shape the young W-chromosome from the mosquitofish Gambusia affinis

BackgroundThe Western mosquitofish, Gambusia affinis, is a model for sex chromosome organization and evolution of female heterogamety. We previously identified a G. affinis female-specific marker, orthologous to the aminomethyl transferase (amt) gene of the related platyfish (Xiphophorus maculatus). Here, we have analyzed the structure and differentiation of the G. affinis W-chromosome, using a cytogenomics and bioinformatics approach.ResultsThe long arm of the G. affinis W-chromosome (Wq) is highly enriched in dispersed repetitive sequences, but neither heterochromatic nor epigenetically silenced by hypermethylation. In line with this, Wq sequences are highly transcribed, including an active nucleolus organizing region (NOR). Female-specific SNPs and evolutionary young transposable elements were highly enriched and dispersed along the W-chromosome long arm, suggesting constrained recombination. Wq copy number expanded elements also include female-specific transcribed sequences from the amt locus with homology to TE. Collectively, the G. affinis W-chromosome is actively differentiating by sex-specific copy number expansion of transcribed TE-related elements, but not (yet) by extensive sequence divergence or gene decay.ConclusionsThe G. affinis W-chromosome exhibits characteristic genomic properties of an evolutionary young sex chromosome. Strikingly, the observed sex-specific changes in the genomic landscape are confined to the W long arm, which is separated from the rest of the W-chromosome by a neocentromere acquired during sex chromosome evolution and may thus have become functionally insulated. In contrast, W short arm sequences were apparently shielded from repeat-driven differentiation, retained Z-chromosome like genomic features, and may have preserved pseudo-autosomal properties.

Structural basis for breadth development in the HIV-1 V3-glycan targeting DH270 antibody clonal lineage

Antibody affinity maturation enables adaptive immune responses to a wide range of pathogens. In some individuals broadly neutralizing antibodies develop to recognize rapidly mutating pathogens with extensive sequence diversity. Vaccine design for pathogens such as HIV-1 and influenza has therefore focused on recapitulating the natural affinity maturation process. Here, we determine structures of antibodies in complex with HIV-1 Envelope for all observed members and ancestral states of the broadly neutralizing HIV-1 V3-glycan targeting DH270 antibody clonal B cell lineage. These structures track the development of neutralization breadth from the unmutated common ancestor and define affinity maturation at high spatial resolution. By elucidating contacts mediated by key mutations at different stages of antibody development we identified sites on the epitope-paratope interface that are the focus of affinity optimization. Thus, our results identify bottlenecks on the path to natural affinity maturation and reveal solutions for these that will inform immunogen design aimed at eliciting a broadly neutralizing immune response by vaccination.

Evidence of a Set of Core-Function Genes in 16 Bacillus Podoviral Genomes with Considerable Genomic Diversity.

Bacteriophage genomes represent an enormous level of genetic diversity and provide considerable potential to acquire new insights about viral genome evolution. In this study, the genome sequences of sixteen Bacillus-infecting bacteriophages were explored through comparative genomics approaches to reveal shared and unique characteristics. These bacteriophages are in the Salasmaviridae family with small (18,548-27,206 bp) double-stranded DNA genomes encoding 25-46 predicted open reading frames. We observe extensive nucleotide and amino acid sequence divergence among a set of core-function genes that present clear synteny. We identify two examples of sequence directed recombination within essential genes, as well as explore the expansion of gene content in these genomes through the introduction of novel open reading frames. Together, these findings highlight the complex evolutionary relationships of phage genomes that include old, common origins as well as new components introduced through mosaicism.

Disulfide-Directed Multicyclic Peptide Libraries for the Discovery of Peptide Ligands and Drugs

Multicyclic peptides with stable 3D structures are a kind of novel and promising peptide formats for drug design and discovery as they have the potential to combine the best characteristics of small molecules and proteins. However, the development of multicyclic peptides is largely limited to naturally occurring products. It remains a big challenge to develop multicyclic peptides with new structures and functions without recourse to the existing natural scaffolds. Here, we report a general and robust method relying on the utility of new disulfide-directing motifs for designing and discovering diverse multicyclic peptides with potent protein-binding capability. These peptides, referred to as disulfide-directed multicyclic peptides (DDMPs), are tolerant to extensive sequence manipulations and variations of disulfide-pairing frameworks, enabling the development of de novo DDMP libraries useful for ligand and drug discovery. This study opens a new avenue for creating a new generation of multicyclic peptides in sequence and structure space inaccessible by natural scaffolds, thus would greatly benefit the field of peptide drug discovery.

Group A Streptococcus Infections: Their Mechanisms, Epidemiology, and Current Scope of Vaccines.

Group A streptococci (GAS) are gram-positive, cocci-shaped bacteria that cause a wide variety of infections and are a cause of significant health burden, particularly in lower- and middle-income nations. The GAS genome contains a number of virulence factors such as the M-protein, hyaluronic acid, C5a peptidase, etc. Despite its significant health burden across the globe, a proper vaccine against GAS infections is not yet available. Various candidates for an effective GAS vaccine are currently being researched. These are based on various parts of the streptococcal genome. These include candidates based on the N-terminal region of the M protein, the conserved C-terminal region of the M protein, and other parts of the streptococcal genome. The development of a vaccine against GAS infections is hampered by certain challenges, such as extensive genetic heterogeneity and high protein sequence variation. This review paper sheds light on the various virulence factors of GAS, their epidemiology, the different vaccine candidates currently being researched, and the challenges associated with M-protein and non-M-protein-based vaccines. This review also sheds light on the current scenario regarding the status of vaccine development against GAS-related infections.

Independent evolution of sex chromosomes and male pregnancy-related genes in two seahorse species.

Unlike birds and mammals, many teleosts have homomorphic sex chromosomes and changes in the chromosome carrying the sex-determining locus, termed "turnovers", are common. Recent turnovers allow studies of several interesting questions. One question is whether the new sex-determining regions evolve to become completely non-recombining, and if so, how and why. Another is to test the prediction that evolutionary changes that benefit one sex will accumulate in the newly sex-linked region. To study these questions, we analyzed the genome sequences of two seahorse species of the Syngnathidae, a fish group in which many species evolved a unique structure, the male brood pouch. We find that both seahorse species have XY sex chromosome systems, and their sex chromosome pairs are not homologs, implying that at least one turnover event has occurred. The Y-linked regions respectively occupy 63.9% and 95.1% of the entire chromosome of the two species, and do not exhibit extensive sequence divergence with their X-linked homologs. We find evidence for occasional recombination between the extant sex chromosomes that may account for their homomorphic pattern. We argue that these Y-linked regions did not evolve by recombination suppression after the turnover. Instead, it can be explained by the ancestral nature of low crossover rate at the corresponding chromosome location. With such an ancestral crossover landscape, a turnover can instantly create an extensive Y-linked region. Finally, we investigate the adaptive evolution of male pouch related genes after they become Y-linked in the seahorse.

Contryphan sequence diversity: Messy N-terminus processing, effects on chromatographic behaviour and mass spectrometric fragmentation

Contryphans, peptides containing a single disulfide bond, are found abundantly in cone snail venom. The analysis of a large dataset of available contryphan sequences permits a classification based on the occurrence of proline residues at positions 2 and 5 within the macrocyclic 23-membered disulfide loop. Further sequence diversity is generated by variable proteolytic processing of the contryphan precursor proteins. In the majority of contryphans, presence of Pro at position 2 and a D-residue at position 3 leads to a slow conformational dynamics, manifesting as anomalous chromatographic profiles during LC analysis. LC-MS analysis of diverse contryphans suggests that elution profiles may be used as a rapid diagnostic for the presence of the Pro2-DXxx3 motif. Natural sequences from C.inscriptus and C.frigidus together with synthetic analogs permit the delineation of the features necessary for abnormal chromatographic behaviour. A diagnostic for the presence of Pro at position 5 is obtained by the observation of non-canonical fragment ions, generated by N-Cα bond cleavage at the dehydroalanine residue formed by disulfide cleavage. Anomalous LC profiles supports Pro at position 2, while non-canonical mass spectral fragments established Pro at position 5, providing a rapid method for contryphan analysis from LC-ESI-MS/MS profiles of crude Conus venom. SignificanceContryphans are peptides, widely distributed in cone snail venom, which display extensive sequence diversity. Heterogeneity of proteolytic processing of contryphan precursor proteins, together with post-translational modifications contributes to contryphan diversity. Contryphans, identified by a combination of mass spectrometry and transcriptomic analysis, are classified on the basis of sequence features, primarily the number of proline residues within the disulfide loop. Conformational diversity arises in contryphans by cis-trans isomerization of Cys-Pro bonds, resulting in characteristic chromatographic profiles, permitting identification even in crude venom mixtures. Rapid identification of contryphans in cone snail peptide libraries is also facilitated by diagnostic mass spectral fragments arising by non-canonical cleavage of the N-Cα bond at Cys(7).

Two-component vaccine consisting of virus-like particles displaying hepatitis C virus envelope protein 2 oligomers

Development of B-cell-based hepatitis C virus (HCV) vaccines that induce broadly neutralizing antibodies (bNAbs) is hindered by extensive sequence diversity and low immunogenicity of envelope glycoprotein vaccine candidates, most notably soluble E2 (sE2). To overcome this, we employed two-component approaches using self-assembling virus-like particles (cVLPs; component 1), displaying monomeric or oligomeric forms of HCV sE2 (sE2mono or sE2oligo; component 2). Immunization studies were performed in BALB/c mice and the neutralizing capacity of vaccine-induced antibodies was tested in cultured-virus-neutralizations, using HCV of genotypes 1–6. sE2-cVLP vaccines induced significantly higher levels of NAbs (p = 0.0065) compared to corresponding sE2 vaccines. Additionally, sE2oligo-cVLP was superior to sE2mono-cVLP in inducing bNAbs. Interestingly, human monoclonal antibody AR2A had reduced binding in ELISA to sE2oligo-cVLP compared with sE2mono-cVLP and competition ELISA using mouse sera from vaccinated animals indicated that sE2oligo-cVLP induced significantly less non-bNAbs AR2A (p = 0.0043) and AR1B (p = 0.017). Thus, cVLP-displayed oligomeric sE2 shows promise as an HCV vaccine candidate.

VSGs Expressed during Natural T. b. gambiense Infection Exhibit Extensive Sequence Divergence and a Subspecies-Specific Bias towards Type B N-Terminal Domains.

Trypanosoma brucei gambiense is the primary causative agent of human African trypanosomiasis (HAT), a vector-borne disease endemic to West and Central Africa. The extracellular parasite evades antibody recognition within the host bloodstream by altering its variant surface glycoprotein (VSG) coat through a process of antigenic variation. The serological tests that are widely used to screen for HAT use VSG as one of the target antigens. However, the VSGs expressed during human infection have not been characterized. Here, we use VSG sequencing (VSG-seq) to analyze the VSGs expressed in the blood of patients infected with T. b. gambiense and compared them to VSG expression in Trypanosoma brucei rhodesiense infections in humans as well as Trypanosoma brucei brucei infections in mice. The 44 VSGs expressed during T. b. gambiense infection revealed a striking bias toward expression of type B N termini (82% of detected VSGs). This bias is specific to T. b. gambiense, which is unique among T. brucei subspecies in its chronic clinical presentation and anthroponotic nature. The expressed T. b. gambiense VSGs also share very little similarity to sequences from 36 T. b. gambiense whole-genome sequencing data sets, particularly in areas of the VSG protein exposed to host antibodies, suggesting the antigen repertoire is under strong selective pressure to diversify. Overall, this work demonstrates new features of antigenic variation in T. brucei gambiense and highlights the importance of understanding VSG repertoires in nature. IMPORTANCE Human African trypanosomiasis is a neglected tropical disease primarily caused by the extracellular parasite Trypanosoma brucei gambiense. To avoid elimination by the host, these parasites repeatedly replace their variant surface glycoprotein (VSG) coat. Despite the important role of VSGs in prolonging infection, VSG expression during human infections is poorly understood. A better understanding of natural VSG gene expression dynamics can clarify the mechanisms that T. brucei uses to alter its VSG coat. We analyzed the expressed VSGs detected in the blood of patients with trypanosomiasis. Our findings indicate that there are features of antigenic variation unique to human-infective T. brucei subspecies and that natural VSG repertoires may vary more than previously expected.

Genetic Sequence Variation in the Plasmodium falciparum Histidine-Rich Protein 2 Gene from Field Isolates in Tanzania: Impact on Malaria Rapid Diagnosis.

Malaria rapid diagnosis test (RDT) is crucial for managing the disease, and the effectiveness of detection depends on parameters such as sensitivity and specificity of the RDT. Several factors can affect the performance of RDT. In this study, we focused on the pfhrp2 sequence variation and its impact on RDTs targeted by antigens encoded by Plasmodium falciparum histidine-rich protein 2 (pfhrp2). Field samples collected during cross-sectional surveys in Tanzania were sequenced to investigate the pfhrp2 sequence diversity and evaluate the impact on HRP2-based RDT performance. We observed significant mean differences in amino acid repeats between current and previous studies. Several new amino acid repeats were found to occur at different frequencies, including types AAY, AHHAHHAAN, and AHHAA. Based on the abundance of types 2 and 7 amino acid repeats, the binary predictive model was able to predict RDT insensitivity by about 69% in the study area. About 85% of the major epitopes targeted by monoclonal antibodies (MAbs) in RDT were identified. Our study suggested that the extensive sequence variation in pfhrp2 can contribute to reduced RDT sensitivity. The correlation between the different combinations of amino acid repeats and the performance of RDT in different malaria transmission settings should be investigated further.

Conformational buffering underlies functional selection in intrinsically disordered protein regions.

Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif-linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.

Extensive non-coding sequence divergence between the major human pathogen Aspergillus fumigatus and its relatives.

Invasive aspergillosis is a deadly fungal disease; more than 400,000 patients are infected worldwide each year and the mortality rate can be as high as 50-95%. Of the ~450 species in the genus Aspergillus only a few are known to be clinically relevant, with the major pathogen Aspergillus fumigatus being responsible for ~50% of all invasive mold infections. Genomic comparisons between A. fumigatus and other Aspergillus species have historically focused on protein-coding regions. However, most A. fumigatus genes, including those that modulate its virulence, are also present in other pathogenic and non-pathogenic closely related species. Our hypothesis is that differential gene regulation - mediated through the non-coding regions upstream of genes' first codon - contributes to A. fumigatus pathogenicity. To begin testing this, we compared non-coding regions upstream of the first codon of single-copy orthologous genes from the two A. fumigatus reference strains Af293 and A1163 and eight closely related Aspergillus section Fumigati species. We found that these non-coding regions showed extensive sequence variation and lack of homology across species. By examining the evolutionary rates of both protein-coding and non-coding regions in a subset of orthologous genes with highly conserved non-coding regions across the phylogeny, we identified 418 genes, including 25 genes known to modulate A. fumigatus virulence, whose non-coding regions exhibit a different rate of evolution in A. fumigatus. Examination of sequence alignments of these non-coding regions revealed numerous instances of insertions, deletions, and other types of mutations of at least a few nucleotides in A. fumigatus compared to its close relatives. These results show that closely related Aspergillus species that vary greatly in their pathogenicity exhibit extensive non-coding sequence variation and identify numerous changes in non-coding regions of A. fumigatus genes known to contribute to virulence.

Alignment-Free Analysis of Whole-Genome Sequences From Symbiodiniaceae Reveals Different Phylogenetic Signals in Distinct Regions.

Dinoflagellates of the family Symbiodiniaceae are predominantly essential symbionts of corals and other marine organisms. Recent research reveals extensive genome sequence divergence among Symbiodiniaceae taxa and high phylogenetic diversity hidden behind subtly different cell morphologies. Using an alignment-free phylogenetic approach based on sub-sequences of fixed length k (i.e. k-mers), we assessed the phylogenetic signal among whole-genome sequences from 16 Symbiodiniaceae taxa (including the genera of Symbiodinium, Breviolum, Cladocopium, Durusdinium and Fugacium) and two strains of Polarella glacialis as outgroup. Based on phylogenetic trees inferred from k-mers in distinct genomic regions (i.e. repeat-masked genome sequences, protein-coding sequences, introns and repeats) and in protein sequences, the phylogenetic signal associated with protein-coding DNA and the encoded amino acids is largely consistent with the Symbiodiniaceae phylogeny based on established markers, such as large subunit rRNA. The other genome sequences (introns and repeats) exhibit distinct phylogenetic signals, supporting the expected differential evolutionary pressure acting on these regions. Our analysis of conserved core k-mers revealed the prevalence of conserved k-mers (>95% core 23-mers among all 18 genomes) in annotated repeats and non-genic regions of the genomes. We observed 180 distinct repeat types that are significantly enriched in genomes of the symbiotic versus free-living Symbiodinium taxa, suggesting an enhanced activity of transposable elements linked to the symbiotic lifestyle. We provide evidence that representation of alignment-free phylogenies as dynamic networks enhances the ability to generate new hypotheses about genome evolution in Symbiodiniaceae. These results demonstrate the potential of alignment-free phylogenetic methods as a scalable approach for inferring comprehensive, unbiased whole-genome phylogenies of dinoflagellates and more broadly of microbial eukaryotes.

The dimerization mechanism of the N-terminal domain of spider silk proteins is conserved despite extensive sequence divergence

The N-terminal (NT) domain of spider silk proteins (spidroins) is crucial for their storage at high concentrations and also regulates silk assembly. NTs from the major ampullate spidroin (MaSp) and the minor ampullate spidroin are monomeric at neutral pH and confer solubility to spidroins, whereas at lower pH, they dimerize to interconnect spidroins in a fiber. This dimerization is known to result from modulation of electrostatic interactions by protonation of well-conserved glutamates, although it is undetermined if this mechanism applies to other spidroin types as well. Here, we determine the solution and crystal structures of the flagelliform spidroin NT, which shares only 35% identity with MaSp NT, and investigate the mechanisms of its dimerization. We show that flagelliform spidroin NT is structurally similar to MaSp NT and that the electrostatic intermolecular interaction between Asp 40 and Lys 65 residues is conserved. However, the protonation events involve a different set of residues than in MaSp, indicating that an overall mechanism of pH-dependent dimerization is conserved but can be mediated by different pathways in different silk types.

Recurrent Duplication and Diversification of Acrosomal Fertilization Proteins in Abalone.

Reproductive proteins mediating fertilization commonly exhibit rapid sequence diversification driven by positive selection. This pattern has been observed among nearly all taxonomic groups, including mammals, invertebrates, and plants, and is remarkable given the essential nature of the molecular interactions mediating fertilization. Gene duplication is another important mechanism that facilitates the generation of molecular novelty through functional divergence. Following duplication, paralogs may partition ancestral gene function (subfunctionalization) or acquire new roles (neofunctionalization). However, the contributions of duplication followed by sequence diversification to the molecular diversity of gamete recognition genes has been understudied in many models of fertilization. The marine gastropod mollusk abalone is a classic model for fertilization. Its two acrosomal proteins (lysin and sp18) are ancient gene duplicates with unique gamete recognition functions. Through detailed genomic and bioinformatic analyses we show how duplication events followed by sequence diversification has played an ongoing role in the evolution of abalone acrosomal proteins. The common ancestor of abalone had four members of its acrosomal protein family in a tandem gene array that repeatedly experienced positive selection. We find that both sp18 paralogs contain positively selected sites located in different regions of the paralogs, suggestive of functional divergence where selection acted upon distinct binding interfaces in each paralog. Further, a more recent species-specific duplication of both lysin and sp18 in the European abalone H. tuberculata is described. Despite clade-specific acrosomal protein paralogs, there are no concomitant duplications of egg coat proteins in H. tuberculata, indicating that duplication of egg proteins per se is not responsible for retention of duplicated acrosomal proteins. We hypothesize that, in a manner analogous to host/pathogen evolution, sperm proteins are selected for increased diversity through extensive sequence divergence and recurrent duplication driven by conflict mechanisms.