Experimental Evolution Studies in Φ6 Cystovirus.

Experimental evolution studies, in which biological populations are evolved in a specific environment over time, can address questions about the nature of spontaneous mutations, responses to selection, and the origins and maintenance of novel traits. Here, we review more than 30 years of experimental evolution studies using the bacteriophage (phage) Φ6 cystovirus. Similar to many lab-studied bacteriophages, Φ6 has a high mutation rate, large population size, fast generation time, and can be genetically engineered or cryogenically frozen, which facilitates its rapid evolution in the laboratory and the subsequent characterization of the effects of its mutations. Moreover, its segmented RNA genome, outer membrane, and capacity for multiple phages to coinfect a single host cell make Φ6 a good non-pathogenic model for investigating the evolution of RNA viruses that infect humans. We describe experiments that used Φ6 to address the fitness effects of spontaneous mutations, the consequences of evolution in the presence of coinfection, the evolution of host ranges, and mechanisms and consequences of the evolution of thermostability. We highlight open areas of inquiry where further experimentation on Φ6 could inform predictions for pathogenic viruses.

Genomic diversity and demographic history of the endangered Sichuan hill-partridge (Arborophila rufipectus).

Species conservation can be improved by knowledge of genetic diversity and demographic history. The Sichuan hill-partridge (Arborophila rufipectus, SP) is an endangered species endemic to the mountains in southwestern China. However, little is known about this species' genomic variation and demographic history. Here, we present a comprehensive whole-genome analysis of six SP individuals from the Laojunshan National Nature Reserve in Sichuan Province, China. We observe a relatively high genetic diversity and low level of recent inbreeding in the studied SP individuals. This suggests that the current population carries genetic variability that may benefit the long-term survival of this species, and that the present population may be larger than currently recognized. Analyses of demographic history showed that fluctuations in the effective population size of SP are inconsistent with changes of the historical climate. Strikingly, evidence from demographic modeling suggests SPs population decreased dramatically 15,100 years ago after the Last Glacial Maximum, possibly due to refugial isolation and later human interference. These results provide the first detailed and comprehensive genomic insights into genetic diversity, genomic inbreeding levels, and demographic history of the Sichuan hill-partridge, which are crucial for the conservation and management of this endangered species.

Empirical Evidence That Complexity Limits Horizontal Gene Transfer.

Horizontal gene transfer (HGT) is a major contributor to bacterial genome evolution, generating phenotypic diversity, driving the expansion of protein families, and facilitating the evolution of new phenotypes, new metabolic pathways, and new species. Comparative studies of gene gain in bacteria suggest that the frequency with which individual genes successfully undergo HGT varies considerably and may be associated with the number of protein-protein interactions in which the gene participates, that is, its connectivity. Two nonexclusive hypotheses have emerged to explain why transferability should decrease with connectivity: the complexity hypothesis (Jain R, Rivera MC, Lake JA. 1999. Horizontal gene transfer among genomes: the complexity hypothesis. Proc Natl Acad Sci U S A. 96:3801-3806.) and the balance hypothesis (Papp B, Pál C, Hurst LD. 2003. Dosage sensitivity and the evolution of gene families in yeast. Nature 424:194-197.). These hypotheses predict that the functional costs of HGT arise from a failure of divergent homologs to make normal protein-protein interactions or from gene misexpression, respectively. Here we describe genome-wide assessments of these hypotheses in which we used 74 existing prokaryotic whole genome shotgun libraries to estimate rates of horizontal transfer of genes from taxonomically diverse prokaryotic donors into Escherichia coli. We show that 1) transferability declines as connectivity increases, 2) transferability declines as the divergence between donor and recipient orthologs increases, and that 3) the magnitude of this negative effect of divergence on transferability increases with connectivity. These effects are particularly robust among the translational proteins, which span the widest range of connectivities. Whereas the complexity hypothesis explains all three of these observations, the balance hypothesis explains only the first one.

Leishmania parasites exchange drug-resistance genes through extracellular vesicles.

Leishmania are eukaryotic parasites that have retained the ability to produce extracellular vesicles (EVs) through evolution. To date, it has been unclear if different DNA entities could be associated with Leishmania EVs and whether these could constitute a mechanism of horizontal gene transfer (HGT). Herein, we investigate the DNA content of EVs derived from drug-resistant parasites, as well as the EVs' potential to act as shuttles for DNA transfer. Next-generation sequencing and PCR assays confirm the enrichment of amplicons carrying drug-resistance genes associated with EVs. Transfer assays of drug-resistant EVs highlight a significant impact on the phenotype of recipient parasites induced by the expression of the transferred DNA. Recipient parasites display an enhanced growth and better control of oxidative stress. We provide evidence that eukaryotic EVs function as efficient mediators in HGT, thereby facilitating the transmission of drug-resistance genes and increasing the fitness of cells when encountering stressful environments.

Experimental Hybridization in Leishmania: Tools for the Study of Genetic Exchange.

Despite major advances over the last decade in our understanding of Leishmania reproductive strategies, the sexual cycle in Leishmania has defied direct observation and remains poorly investigated due to experimental constraints. Here, we summarize the findings and conclusions drawn from genetic analysis of experimental hybrids generated in sand flies and highlight the recent advances in generating hybrids in vitro. The ability to hybridize between culture forms of different species and strains of Leishmania should invite more intensive investigation of the mechanisms underlying genetic exchange and provide a rich source of recombinant parasites for future genetic analyses.

Microevolution of Trypanosoma cruzi reveals hybridization and clonal mechanisms driving rapid genome diversification.

Protozoa and fungi are known to have extraordinarily diverse mechanisms of genetic exchange. However, the presence and epidemiological relevance of genetic exchange in Trypanosoma cruzi, the agent of Chagas disease, has been controversial and debated for many years. Field studies have identified both predominantly clonal and sexually recombining natural populations. Two of six natural T. cruzi lineages (TcV and TcVI) show hybrid mosaicism, using analysis of single-gene locus markers. The formation of hybrid strains in vitro has been achieved and this provides a framework to study the mechanisms and adaptive significance of genetic exchange. Using whole genome sequencing of a set of experimental hybrids strains, we have confirmed that hybrid formation initially results in tetraploid parasites. The hybrid progeny showed novel mutations that were not attributable to either (diploid) parent showing an increase in amino acid changes. In long-term culture, up to 800 generations, there was a variable but gradual erosion of progeny genomes towards triploidy, yet retention of elevated copy number was observed at several core housekeeping loci. Our findings indicate hybrid formation by fusion of diploid T. cruzi, followed by sporadic genome erosion, but with substantial potential for adaptive evolution, as has been described as a genetic feature of other organisms, such as some fungi.

Metagenomic and metatranscriptomic analyses reveal that biobed systems can enrich for antibiotic resistance and genetic mobility genes.

Antibiotic resistance gene pollution in the environment has been identified as a potential contributor to the global issue of antibiotic resistance prevalence, creating a need to identify and characterize environmental reservoirs for antibiotic resistance genes. Because many polluted environments have been shown to contain elevated levels of antibiotic resistance genes, agriculturally based pesticide bioremediation systems called 'biobeds' could serve as environmental reservoirs for antibiotic resistance genes, although this has never been extensively explored. Metagenomic and metatranscriptomic analyses of an on-farm biobed system sampled before and after a season of pesticide use demonstrated that in situ pesticide applications applied to biobeds can enrich for multidrug, sulphonamide, aminoglycoside and beta-lactam resistance genes. Additionally, this study demonstrated an enrichment for genes associated with gene mobilization, such as genes involved in horizontal gene transfer and plasmid mobility, as well as transposons and integrases.

Sex under pressure: stress facilitates Leishmania in vitro hybridization.

The selection of Leishmania hybrids in axenic culture was considered rare until recently, when Louradour and Ferreira et al., demonstrated that induced DNA damage facilitates genetic exchange, resulting in full genome tetraploid progenies in vitro. Meiosis-related gene homologues HAP2, GEX1, and RAD51 were found to be involved, opening new avenues for functional genomic studies.

Mammalian Orthoreovirus Reassortment Proceeds with Little Constraint on Segment Mixing.

Segmentation of viral genomes provides the potential for genetic exchange within coinfected cells. However, for this potential to be realized, coinfecting genomes must mix during the viral life cycle. The efficiency of reassortment, in turn, dictates its potential to drive evolution. The opportunity for mixing within coinfected cells may vary greatly across virus families, such that the evolutionary implications of genome segmentation differ as a result of core features of the viral life cycle. To investigate the relationship between viral replication compartments and genetic exchange, we quantified reassortment in mammalian orthoreovirus (reovirus). Reoviruses carry a 10-segmented, double-stranded RNA genome, which is replicated within proteinaceous structures termed inclusion bodies. We hypothesized that inclusions impose a barrier to reassortment. We quantified reassortment between wild-type (wt) and variant (var) reoviruses that differ by one nucleotide per segment. Studies of wt/var systems in both T1L and T3D backgrounds revealed frequent reassortment without bias toward particular genotypes. However, reassortment was more efficient in the T3D serotype. Since T1L and T3D viruses exhibit different inclusion body morphologies, we tested the impact of this phenotype on reassortment. In both serotypes, reassortment levels did not differ by inclusion morphology. Reasoning that the merging of viral inclusions may be critical for genome mixing, we then tested the effect of blocking merging. Reassortment proceeded efficiently even under these conditions. Our findings indicate that reovirus reassortment is highly efficient despite the localization of many viral processes to inclusion bodies, and that the robustness of this genetic exchange is independent of inclusion body structure and fusion. IMPORTANCE Quantification of reassortment in diverse viral systems is critical to elucidate the implications of genome segmentation for viral evolution. In principle, genome segmentation offers a facile means of genetic exchange between coinfecting viruses. In practice, there may be physical barriers within the cell that limit the mixing of viral genomes. Here, we tested the hypothesis that localization of the various stages of the mammalian orthoreovirus life cycle within cytoplasmic inclusion bodies compartmentalizes viral replication and limits genetic exchange. Contrary to this hypothesis, our data indicate that reovirus reassortment occurs readily within coinfected cells and is not strongly affected by the structure or dynamics of viral inclusion bodies. We conclude that the potential for reassortment to contribute to reovirus evolution is high.

Finding common ground: Toward comparable indicators of adaptive capacity of tree species to a changing climate.

Adaptive capacity, one of the three determinants of vulnerability to climate change, is defined as the capacity of species to persist in their current location by coping with novel environmental conditions through acclimation and/or evolution. Although studies have identified indicators of adaptive capacity, few have assessed this capacity in a quantitative way that is comparable across tree species. Yet, such multispecies assessments are needed by forest management and conservation programs to refine vulnerability assessments and to guide the choice of adaptation measures. In this paper, we propose a framework to quantitatively evaluate five key components of tree adaptive capacity to climate change: individual adaptation through phenotypic plasticity, population phenotypic diversity as influenced by genetic diversity, genetic exchange within populations, genetic exchange between populations, and genetic exchange between species. For each component, we define the main mechanisms that underlie adaptive capacity and present associated metrics that can be used as indices. To illustrate the use of this framework, we evaluate the relative adaptive capacity of 26 northeastern North American tree species using values reported in the literature. Our results show adaptive capacity to be highly variable among species and between components of adaptive capacity, such that no one species ranks consistently across all components. On average, the conifer Picea glauca and the broadleaves Acer rubrum and A. saccharinum show the greatest adaptive capacity among the 26 species we documented, whereas the conifers Picea rubens and Thuja occidentalis, and the broadleaf Ostrya virginiana possess the lowest. We discuss limitations that arise when comparing adaptive capacity among species, including poor data availability and comparability issues in metrics derived from different methods or studies. The breadth of data required for such an assessment exemplifies the multidisciplinary nature of adaptive capacity and the necessity of continued cross-collaboration to better anticipate the impacts of a changing climate.

A sugar utilization phenotype contributes to the formation of genetic exchange communities in lactic acid bacteria.

In prokaryotes, a major contributor to genomic evolution is the exchange of genes via horizontal gene transfer (HGT). Areas with a high density of HGT networks are defined as genetic exchange communities (GECs). Although some phenotypes associated with specific ecological niches are linked to GECs, little is known about the phenotypic influences on HGT in bacterial groups within a taxonomic family. Thanks to the published genome sequences and phenotype data of lactic acid bacteria (LAB), it is now possible to obtain more detailed information about the phenotypes that affect GECs. Here, we have investigated the relationship between HGT and internal and external environmental factors for 178 strains from 24 genera in the Lactobacillaceae family. We found a significant correlation between strains with high utilization of sugars and HGT bias. The result suggests that the phenotype of the utilization of a variety of sugars is key to the construction of GECs in this family. This feature is consistent with the fact that the Lactobacillaceae family contributes to the production of a wide variety of fermented foods by sharing niches such as those in vegetables, dairy products and brewing-related environments. This result provides the first evidence that phenotypes associated with ecological niches contribute to form GECs in the LAB family.

Reproduction in Trypanosomatids: Past and Present.

Diseases caused by trypanosomatids (Sleeping sickness, Chagas disease, and leishmaniasis) are a serious public health concern in low-income endemic countries. These diseases are produced by single-celled parasites with a diploid genome (although aneuploidy is frequent) organized in pairs of non-condensable chromosomes. To explain the way they reproduce through the analysis of natural populations, the theory of strict clonal propagation of these microorganisms was taken as a rule at the beginning of the studies, since it partially justified their genomic stability. However, numerous experimental works provide evidence of sexual reproduction, thus explaining certain naturally occurring events that link the number of meiosis per mitosis and the frequency of mating. Recent techniques have demonstrated genetic exchange between individuals of the same species under laboratory conditions, as well as the expression of meiosis specific genes. The current debate focuses on the frequency of genomic recombination events and its impact on the natural parasite population structure. This paper reviews the results and techniques used to demonstrate the existence of sex in trypanosomatids, the inheritance of kinetoplast DNA (maxi- and minicircles), the impact of genetic exchange in these parasites, and how it can contribute to the phenotypic diversity of natural populations.

The sexual side of parasitic protists.

Much of the vast evolutionary landscape occupied by Eukaryotes is dominated by protists. Though parasitism has arisen in many lineages, there are three main groups of parasitic protists of relevance to human and livestock health: the Apicomplexa, including the malaria parasite Plasmodium and coccidian pathogens of livestock such as Eimeria; the excavate flagellates, encompassing a diverse range of protist pathogens including trypanosomes, Leishmania, Giardia and Trichomonas; and the Amoebozoa, including pathogenic amoebae such as Entamoeba. These three groups represent separate, deep branches of the eukaryote tree, underlining their divergent evolutionary histories. Here, I explore what is known about sex in these three main groups of parasitic protists.

Prevalence of Genetically Complex Leishmania Strains With Hybrid and Mito-Nuclear Discordance.

Approximately 20 Leishmania species are known to cause cutaneous, mucocutaneous, and visceral disorders in humans. Identification of the causative species in infected individuals is important for appropriate treatment and a favorable prognosis because infecting species are known to be the major determinant of clinical manifestations and may affect treatments for leishmaniasis. Although Leishmania species have been conventionally identified by multilocus enzyme electrophoresis, genetic analysis targeting kinetoplast and nuclear DNA (kDNA and nDNA, respectively) is now widely used for this purpose. Recently, we conducted countrywide epidemiological studies of leishmaniasis in Ecuador and Peru to reveal prevalent species using PCR-RFLP targeting nDNA, and identified unknown hybrid parasites in these countries together with species reported previously. Furthermore, comparative analyses of kDNA and nDNA revealed the distribution of parasites with mismatches between these genes, representing the first report of mito-nuclear discordance in protozoa. The prevalence of an unexpectedly high rate (~10%) of genetically complex strains including hybrid strains, in conjunction with the observation of mito-nuclear discordance, suggests that genetic exchange may occur more frequently than previously thought in natural Leishmania populations. Hybrid Leishmania strains resulting from genetic exchanges are suggested to cause more severe clinical symptoms when compared with parental strains, and to have increased transmissibility by vectors of the parental parasite species. Therefore, it is important to clarify how such genetic exchange influences disease progression and transmissibility by sand flies in nature. In addition, our aim was to identify where and how the genetic exchange resulting in the formation of hybrid and mito-nuclear discordance occurs.

Study on the Occurrence of Genetic Exchange Among Parasites of the Leishmania mexicana Complex.

In Leishmania, genetic exchange has been experimentally demonstrated to occur in the sand fly vector and in promastigote axenic cultures through a meiotic-like process. No evidence of genetic exchange in mammalian hosts have been reported so far, possibly due to the fact that the Leishmania species used in previous studies replicate within individual parasitophorous vacuoles. In the present work, we explored the possibility that residing in communal vacuoles may provide conditions favorable for genetic exchange for L. mexicana and L. amazonensis. Using promastigote lines of both species harboring integrated or episomal drug-resistance markers, we assessed whether genetic exchange can occur in axenic cultures, in infected macrophages as well as in infected mice. We obtained evidence of genetic exchange for L. amazonensis in both axenic promastigote cultures and infected macrophages. However, the resulting products of those putative genetic events were unstable as they did not sustain growth in subsequent sub-cultures, precluding further characterization.

Sequence analysis of the Petunia inflata S-locus region containing 17 S-Locus F-Box genes and the S-RNase gene involved in self-incompatibility.

Self-incompatibility in Petunia is controlled by the polymorphic S-locus, which contains S-RNase encoding the pistil determinant and 16-20 S-locus F-box (SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of the S2 -haplotype of the S-locus in Petunia inflata using bacterial artificial chromosome clones collectively containing all 17 SLF genes, SLFLike1, and S-RNase. Two SLF pseudogenes and 28 potential protein-coding genes were identified, 20 of which were also found at the S-loci of both the S6a -haplotype of P. inflata and the SN -haplotype of self-compatible Petunia axillaris, but not in the S-locus remnants of self-compatible potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Comparative analyses of S-locus sequences of these three S-haplotypes revealed potential genetic exchange in the flanking regions of SLF genes, resulting in highly similar flanking regions between different types of SLF and between alleles of the same type of SLF of different S-haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at the S-loci of all three S-haplotypes and in the flanking regions of all S-locus genes examined. We also found evidence of the association of transposable elements with SLF pseudogenes. Based on the hypothesis that SLF genes were derived by retrotransposition, we identified 10 F-box genes as putative SLF parent genes. Our results shed light on the importance of non-coding sequences in the evolution of the S-locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion of SLF genes.

Patterns and impacts of nonvertical evolution in eukaryotes: a paradigm shift.

Evolution of eukaryotic species and their genomes has been traditionally understood as a vertical process in which genetic material is transmitted from parents to offspring along a lineage, and in which genetic exchange is restricted within species boundaries. However, mounting evidence from comparative genomics indicates that this paradigm is often violated. Horizontal gene transfer and mating between diverged lineages blur species boundaries and challenge the reconstruction of evolutionary histories of species and their genomes. Nonvertical evolution might be more restricted in eukaryotes than in prokaryotes, yet it is not negligible and can be common in certain groups. Recognition of such processes brings about the need to incorporate this complexity into our models, as well as to conceptually reframe eukaryotic diversity and evolution. Here, I review the recent work from genomics studies that supports the effects of nonvertical modes of evolution including introgression, hybridization, and horizontal gene transfer in different eukaryotic groups. I then discuss emerging patterns and effects, illustrated by specific examples, that support the conclusion that nonvertical processes are often at the root of important evolutionary transitions and adaptations. I will argue that a paradigm shift is needed to naturally accommodate nonvertical processes in eukaryotic evolution.

Genetic structure, gene flow pattern, and association analysis of superior germplasm resources in domesticated upland cotton (Gossypium hirsutum L.).

Gene flow patterns and the genetic structure of domesticated crops like cotton are not well understood. Furthermore, marker-assisted breeding of cotton has lagged far behind that of other major crops because the loci associated with cotton traits such as fiber yield and quality have scarcely been identified. In this study, we used 19 microsatellites to first determine the population genetic structure and patterns of gene flow of superior germplasm resources in upland cotton. We then used association analysis to identify which markers were associated with 15 agronomic traits (including ten yield and five fiber quality traits). The results showed that the upland cotton accessions have low levels of genetic diversity (polymorphism information content = 0.427), although extensive gene flow occurred among different ecological and geographic regions. Bayesian clustering analysis indicated that the cotton resources used in this study did not belong to obvious geographic populations, which may be the consequence of a single source of domestication followed by frequent genetic introgression mediated by human transference. A total of 82 maker-trait associations were examined in association analysis and the related ratios for phenotypic variations ranged from 3.04% to 47.14%. Interestingly, nine SSR markers were detected in more than one environmental condition. In addition, 14 SSR markers were co-associated with two or more different traits. It was noteworthy that NAU4860 and NAU5077 markers detected at least in two environments were simultaneously associated with three fiber quality traits (uniformity index, specific breaking strength and micronaire value). In conclusion, these findings provide new insights into the population structure and genetic exchange pattern of cultivated cotton accessions. The quantitative trait loci of domesticated cotton identified will also be very useful for improvement of yield and fiber quality of cotton in molecular breeding programs.

A New Pneumococcal Capsule Type, 10D, is the 100th Serotype and Has a Large cps Fragment from an Oral Streptococcus.

Streptococcus pneumoniae (pneumococcus) is a major human pathogen producing structurally diverse capsular polysaccharides. Widespread use of highly successful pneumococcal conjugate vaccines (PCVs) targeting pneumococcal capsules has greatly reduced infections by the vaccine types but increased infections by nonvaccine serotypes. Herein, we report a new and the 100th capsule type, named serotype 10D, by determining its unique chemical structure and biosynthetic roles of all capsule synthesis locus (cps) genes. The name 10D reflects its serologic cross-reaction with serotype 10A and appearance of cross-opsonic antibodies in response to immunization with 10A polysaccharide in a 23-valent pneumococcal vaccine. Genetic analysis showed that 10D cps has three large regions syntenic to and highly homologous with cps loci from serotype 6C, serotype 39, and an oral streptococcus strain (S. mitis SK145). The 10D cps region syntenic to SK145 is about 6 kb and has a short gene fragment of wciNα at the 5' end. The presence of this nonfunctional wciNα fragment provides compelling evidence for a recent interspecies genetic transfer from oral streptococcus to pneumococcus. Since oral streptococci have a large repertoire of cps loci, widespread PCV usage could facilitate the appearance of novel serotypes through interspecies recombination.IMPORTANCE The polysaccharide capsule is essential for the pathogenicity of pneumococcus, which is responsible for millions of deaths worldwide each year. Currently available pneumococcal vaccines are designed to elicit antibodies to the capsule polysaccharides of the pneumococcal isolates commonly causing diseases, and the antibodies provide protection only against the pneumococcus expressing the vaccine-targeted capsules. Since pneumococci can produce different capsule polysaccharides and therefore reduce vaccine effectiveness, it is important to track the appearance of novel pneumococcal capsule types and how these new capsules are created. Herein, we describe a new and the 100th pneumococcal capsule type with unique chemical and serological properties. The capsule type was named 10D for its serologic similarity to 10A. Genetic studies provide strong evidence that pneumococcus created 10D capsule polysaccharide by capturing a large genetic fragment from an oral streptococcus. Such interspecies genetic exchanges could greatly increase diversity of pneumococcal capsules and complicate serotype shifts.