First report of molecular characterization and phylogeny of Trichuris fossor Hall, 1916 (Nematoda: Trichuridae).

Because species of Trichuris are morphologically similar and ranges of host preference are variable, using molecular data to evaluate species delineations is essential for properly quantifying biodiversity of and relationships within Trichuridae. Trichuris fossor has been reported from Thomomys spp. (Rodentia: Geomyidae, 'pocket gophers') hosts based on morphological features alone. Partial 18S rRNA sequences for specimens identified as T. fossor based on morphology, along with sequences from 26 additional taxa, were used for a phylogenetic analysis. Evolutionary histories were constructed using maximum likelihood and Bayesian inference. In both analyses, the specimens fell within the Trichuris clade with 100% support and formed a distinct subclade with 100% support. These results confirm that T. fossor is a distinct species and represent the first molecular report for it. Relatedness among species within the family were well resolved in the BI tree. This study represents an initial effort to obtain a more comprehensive view of Trichuridae by including a new clade member, T. fossor. A better understanding of Trichuridae phylogeny could contribute to further characterization of host-associations, including species that infect livestock and humans.Because species of Trichuris are morphologically similar and ranges of host preference are variable, using molecular data to evaluate species delineations is essential for properly quantifying biodiversity of and relationships within Trichuridae. Trichuris fossor has been reported from Thomomys spp. (Rodentia: Geomyidae, 'pocket gophers') hosts based on morphological features alone. Partial 18S rRNA sequences for specimens identified as T. fossor based on morphology, along with sequences from 26 additional taxa, were used for a phylogenetic analysis. Evolutionary histories were constructed using maximum likelihood and Bayesian inference. In both analyses, the specimens fell within the Trichuris clade with 100% support and formed a distinct subclade with 100% support. These results confirm that T. fossor is a distinct species and represent the first molecular report for it. Relatedness among species within the family were well resolved in the BI tree. This study represents an initial effort to obtain a more comprehensive view of Trichuridae by including a new clade member, T. fossor. A better understanding of Trichuridae phylogeny could contribute to further characterization of host-associations, including species that infect livestock and humans.

Genome Announcement: The Draft Genomes of Two Radopholus similis populations from Costa Rica.

Radopholus similis is an economically important pest of both banana and citrus in tropical regions. Here we present draft genomes from two populations of R. similis from Costa Rica that were created and assembled using short read libraries from Illumina HiSeq technology.

Paternal Mitochondrial Transmission in Intra-Species Caenorhabditis briggsae Hybrids.

To study mitochondrial-nuclear genetic interactions in the nematode Caenorhabditis briggsae, our three laboratories independently created 38 intra-species cytoplasmic-nuclear hybrid (cybrid) lines. Although the cross design combines maternal mitotypes with paternal nuclear genotypes, eight lines (21%) unexpectedly contained paternal mitotypes. All eight share in common ancestry of one of two genetically related strains. This unexpected parallel observation of paternal mitochondrial transmission, undesirable given our intent of creating cybrids, provides a serendipitous experimental model and framework to study the molecular and evolutionary basis of uniparental mitochondrial inheritance.

The Draft Genome of Globodera ellingtonae.

Globodera ellingtonae is a newly described potato cyst nematode (PCN) found in Idaho, Oregon, and Argentina. Here, we present a genome assembly for G. ellingtonae, a relative of the quarantine nematodes G. pallida and G. rostochiensis, produced using data from Illumina and Pacific Biosciences DNA sequencing technologies.

Genome-wide polymorphism and signatures of selection in the symbiotic sea anemone Aiptasia.

BACKGROUND: Coral reef ecosystems are declining in response to global climate change and anthropogenic impacts. Yet patterns of standing genetic variation within cnidarian species, a major determinant of adaptive potential, are virtually unknown at genome-scale resolution. We explore patterns of genome-wide polymorphism and identify candidate loci under selection in the sea anemone Aiptasia, an important laboratory model system for studying the symbiosis between corals and dinoflagellate algae of the genus Symbiodinium. RESULTS: Low coverage genome sequencing revealed large genetic distances among globally widespread lineages, novel candidate targets of selection, and considerably higher heterozygosity than previously reported for Aiptasia. More than 670,000 single nucleotide polymorphisms were identified among 10 Aiptasia individuals including two pairs of genetic clones. Evolutionary relationships based on genome-wide polymorphism supported the current paradigm of a genetically distinct population from the US South Atlantic that harbors diverse Symbiodinium clades. However, anemones from the US South Atlantic demonstrated a striking lack of shared derived polymorphism. Heterozygosity was an important feature shaping nucleotide diversity patterns: at any given SNP site, more than a third of individuals genotyped were heterozygotes, and heterozygosity within individual genomes ranged from 0.37-0.58%. Analysis of nonsynonymous and synonymous sites suggested that highly heterozygous regions are evolving under relaxed purifying selection compared to the rest of the Aiptasia genome. Genes previously identified as having elevated evolutionary rates in Aiptasia compared to other cnidarians were found in our study to be under strong purifying selection within Aiptasia. Candidate targets of selection, including lectins and genes involved in Rho GTPase signalling, were identified based on unusual signatures of nucleotide diversity, Tajima's D, and heterozygosity compared to genome-wide averages. CONCLUSIONS: This study represents the first genome-wide analysis of Tajima's D in a cnidarian. Our results shed light on patterns of intraspecific genome-wide polymorphism in a model for studies of coral-algae symbiosis and present genetic targets for future research on evolutionary and cellular processes in early-diverging metazoans.

The mitochondrial genome of Globodera ellingtonae is composed of two circles with segregated gene content and differential copy numbers.

BACKGROUND: The evolution of animal mitochondrial (mt) genomes has resulted in a highly conserved structure: a single compact circular chromosome approximately 14 to 20 kb long. Within the last two decades exceptions to this conserved structure, such as the division of the genome into multiple chromosomes, have been reported in a diverse set of metazoans. We report on the two circle multipartite mt genome of a newly described cyst nematode, Globodera ellingtonae. RESULTS: The G. ellingtonae mt genome was found to be comprised of two circles, each larger than any other multipartite circular mt chromosome yet reported, and both were larger than the single mt circle of the model nematode Caenorhabditis elegans. The genetic content of the genome was disproportionately divided between the two circles, although they shared a ~6.5 kb non-coding region. The 17.8 kb circle (mtDNA-I) contained ten protein-coding genes and two tRNA genes, whereas the 14.4 kb circle (mtDNA-II) contained two protein-coding genes, 20 tRNA genes and both rRNA genes. Perhaps correlated with this division of genetic content, the copy number of mtDNA-II was more than four-fold that of mtDNA-I in individual nematodes. The difference in copy number increased between second-stage and fourth-stage juveniles. CONCLUSIONS: The segregation of gene types to different mt circles in G. ellingtonae could provide benefit by localizing gene functional types to independent transcriptional units. This is the first report of both two-circle and several-circle mt genomes within a single genus. The differential copy number associated with this multipartite mt organization could provide a model system for deconstructing mechanisms regulating mtDNA copy number both in somatic cells and during germline development.

The biocontrol nematode Phasmarhabditis hermaphrodita infects and increases mortality of Monadenia fidelis, a non-target terrestrial gastropod species endemic to the Pacific Northwest of North America, in laboratory conditions.

Inundative biological control (biocontrol) efforts in pest management lead to the mass distribution of commercialized biocontrol agents. Many 'biocontrol gone awry' incidents have resulted in disastrous biodiversity impacts, leading to increased scrutiny of biocontrol efforts. The nematode Phasmarhabditis hermaphrodita is sold as a biocontrol agent on three continents and targets pest gastropods such as Deroceras reticulatum, the Grey Field Slug; P. hermaphrodita is not presently approved for use in the United States. Investigations into the potential for P. hermaphrodita to infect non-target gastropod species of conservation relevance, however, are limited. We examined the effects of three strains of P. hermaphrodita on mortality in Monadenia fidelis, the Pacific Sideband, a snail species endemic to the Pacific Northwest of North America, in laboratory conditions. Across a 71-day laboratory infectivity assay, snails exposed to each of the three nematode strains, each analyzed at two doses, experienced a mean 50% mortality by days 20-42. All nematode-treated snails were dead by the end of the study. By contrast, 30/30 water-control snails experienced no mortality. Nematodes killed smaller, juvenile-stage snails significantly faster than those in larger and more developmentally advanced hosts. Our results provide direct evidence that the biocontrol nematode P. hermaphrodita infects and kills M. fidelis, a non-target gastropod species endemic to the Pacific Northwest, in laboratory conditions. This study suggests that introduction of P. hermaphrodita to new ecosystems might negatively impact endemic gastropod biodiversity and advocates for further investigation of non-target effects, including in conditions closer to the natural environments of non-target species.

Selective sweeps and parallel mutation in the adaptive recovery from deleterious mutation in Caenorhabditis elegans.

Deleterious mutation poses a serious threat to human health and the persistence of small populations. Although adaptive recovery from deleterious mutation has been well-characterized in prokaryotes, the evolutionary mechanisms by which multicellular eukaryotes recover from deleterious mutation remain unknown. We applied high-throughput DNA sequencing to characterize genomic divergence patterns associated with the adaptive recovery from deleterious mutation using a Caenorhabditis elegans recovery-line system. The C. elegans recovery lines were initiated from a low-fitness mutation-accumulation (MA) line progenitor and allowed to independently evolve in large populations (N ∼ 1000) for 60 generations. All lines rapidly regained levels of fitness similar to the wild-type (N2) MA line progenitor. Although there was a near-zero probability of a single mutation fixing due to genetic drift during the recovery experiment, we observed 28 fixed mutations. Cross-generational analysis showed that all mutations went from undetectable population-level frequencies to a fixed state in 10-20 generations. Many recovery-line mutations fixed at identical timepoints, suggesting that the mutations, if not beneficial, hitchhiked to fixation during selective sweep events observed in the recovery lines. No MA line mutation reversions were detected. Parallel mutation fixation was observed for two sites in two independent recovery lines. Analysis using a C. elegans interactome map revealed many predicted interactions between genes with recovery line-specific mutations and genes with previously accumulated MA line mutations. Our study suggests that recovery-line mutations identified in both coding and noncoding genomic regions might have beneficial effects associated with compensatory epistatic interactions.

A genome-wide view of Caenorhabditis elegans base-substitution mutation processes.

Knowledge of mutation processes is central to understanding virtually all evolutionary phenomena and the underlying nature of genetic disorders and cancers. However, the limitations of standard molecular mutation detection methods have historically precluded a genome-wide understanding of mutation rates and spectra in the nuclear genomes of multicellular organisms. We applied two high-throughput DNA sequencing technologies to identify and characterize hundreds of spontaneously arising base-substitution mutations in 10 Caenorhabditis elegans mutation-accumulation (MA)-line nuclear genomes. C. elegans mutation rate estimates were similar to previous calculations based on smaller numbers of mutations. Mutations were distributed uniformly within and among chromosomes and were not associated with recombination rate variation in the MA lines, suggesting that intragenomic variation in genetic hitchhiking and/or background selection are primarily responsible for the chromosomal distribution patterns of polymorphic nucleotides in C. elegans natural populations. A strong mutational bias from G/C to A/T nucleotides was detected in the MA lines, implicating oxidative DNA damage as a major endogenous mutagenic force in C. elegans. The observed mutational bias also suggests that the C. elegans nuclear genome cannot be at equilibrium because of mutation alone. Transversions dominate the spectrum of spontaneous mutations observed here, whereas transitions dominate patterns of allegedly neutral polymorphism in natural populations of C. elegans and many other animal species; this observation challenges the assumption that natural patterns of molecular variation in noncoding regions of the nuclear genome accurately reflect underlying mutation processes.

Evolution of Caenorhabditis mitochondrial genome pseudogenes and Caenorhabditis briggsae natural isolates.

Although most metazoan mitochondrial genomes are highly streamlined and encode little noncoding DNA outside of the "AT" region, the accumulation of mitochondrial pseudogenes and other types of noncoding DNA has been observed in a growing number of animal groups. The nematode species Caenorhabditis briggsae harbors two mitochondrial DNA (mtDNA) pseudogenes, named Psinad5-1 and Psinad5-2, presumably derived from the nad5 protein-coding gene. Here, we provide an in-depth analysis of mtDNA pseudogene evolution in C. briggsae natural isolates and related Caenorhabditis species. Mapping the observed presence and absence of the pseudogenes onto phylogenies suggests that Psinad5-1 originated in the ancestor to C. briggsae and its recently discovered outcrossing relative species Caenorhabditis sp. 5 and Caenorhabditis sp. 9. However, Psinad5-1 was not detected in Caenorhabditis sp. 9 natural isolates, suggesting a lineage-specific loss of this pseudogene in this species. Our results corroborated the previous finding that Psinad5-2 originated within C. briggsae. The observed pattern of mitochondrial pseudogene gain and loss in Caenorhabditis was inconsistent with predictions of the tandem duplication-random loss model of mitochondrial genome evolution and suggests that intralineage recombination-like mechanisms might play a major role in Caenorhabditis mtDNA evolution. Natural variation was analyzed at the pseudogenes and flanking mtDNA sequences in 141 geographically diverse C. briggsae natural isolates. Although phylogenetic analysis placed the majority of isolates into the three previously established major intraspecific clades of C. briggsae, two new and unexpected haplotypes fell outside of these conventional groupings. Psinad5-2 copy number variation was observed among C. briggsae isolates collected from the same geographic site. Patterns of nucleotide diversity were analyzed in Psinad5-1 and Psinad5-2, and confidence intervals were found to overlap values from synonymous sites in protein-coding genes, consistent with neutral expectations. Our findings provide new insights into the mode and tempo of mitochondrial genome and pseudogene evolution both within and between Caenorhabditis nematode species.

Genomic Analyses of Globodera pallida, A Quarantine Agricultural Pathogen in Idaho.

Globodera pallida is among the most significant plant-parasitic nematodes worldwide, causing major damage to potato production. Since it was discovered in Idaho in 2006, eradication efforts have aimed to contain and eradicate G. pallida through phytosanitary action and soil fumigation. In this study, we investigated genome-wide patterns of G. pallida genetic variation across Idaho fields to evaluate whether the infestation resulted from a single or multiple introduction(s) and to investigate potential evolutionary responses since the time of infestation. A total of 53 G. pallida samples (~1,042,000 individuals) were collected and analyzed, representing five different fields in Idaho, a greenhouse population, and a field in Scotland that was used for external comparison. According to genome-wide allele frequency and fixation index (Fst) analyses, most of the genetic variation was shared among the G. pallida populations in Idaho fields pre-fumigation, indicating that the infestation likely resulted from a single introduction. Temporal patterns of genome-wide polymorphisms involving (1) pre-fumigation field samples collected in 2007 and 2014 and (2) pre- and post-fumigation samples revealed nucleotide variants (SNPs, single-nucleotide polymorphisms) with significantly differentiated allele frequencies indicating genetic differentiation. This study provides insights into the genetic origins and adaptive potential of G. pallida invading new environments.

Mitochondrial DNA Variation and Selfish Propagation Following Experimental Bottlenecking in Two Distantly Related Caenorhabditis briggsae Isolates.

Understanding mitochondrial DNA (mtDNA) evolution and inheritance has broad implications for animal speciation and human disease models. However, few natural models exist that can simultaneously represent mtDNA transmission bias, mutation, and copy number variation. Certain isolates of the nematode Caenorhabditis briggsae harbor large, naturally-occurring mtDNA deletions of several hundred basepairs affecting the NADH dehydrogenase subunit 5 (nduo-5) gene that can be functionally detrimental. These deletion variants can behave as selfish DNA elements under genetic drift conditions, but whether all of these large deletion variants are transmitted in the same preferential manner remains unclear. In addition, the degree to which transgenerational mtDNA evolution profiles are shared between isolates that differ in their propensity to accumulate the nduo-5 deletion is also unclear. We address these knowledge gaps by experimentally bottlenecking two isolates of C. briggsae with different nduo-5 deletion frequencies for up to 50 generations and performing total DNA sequencing to identify mtDNA variation. We observed multiple mutation profile differences and similarities between C. briggsae isolates, a potentially species-specific pattern of copy number dysregulation, and some evidence for genetic hitchhiking in the deletion-bearing isolate. Our results further support C. briggsae as a practical model for characterizing naturally-occurring mtgenome variation and contribute to the understanding of how mtgenome variation persists in animal populations and how it presents in mitochondrial disease states.

Phylogenetic evidence for the invasion of a commercialized European Phasmarhabditis hermaphrodita lineage into North America and New Zealand.

Biological control (biocontrol) as a component of pest management strategies reduces reliance on synthetic chemicals, and seemingly offers a natural approach that minimizes environmental impact. However, introducing a new organism to new environments as a classical biocontrol agent can have broad and unanticipated biodiversity effects and conservation consequences. Nematodes are currently used in a variety of commercial biocontrol applications, including the use of Phasmarhabditis hermaphrodita as an agent targeting pest slug and snail species. This species was originally discovered in Germany, and is generally thought to have European origins. P. hermaphrodita is sold under the trade name Nemaslug®, and is available only in European markets. However, this nematode species was discovered in New Zealand and the western United States, though its specific origins remained unclear. In this study, we analyzed 45 nematode strains representing eight different Phasmarhabditis species, collected from nine countries around the world. A segment of nematode mitochondrial DNA (mtDNA) was sequenced and subjected to phylogenetic analyses. Our mtDNA phylogenies were overall consistent with previous analyses based on nuclear ribosomal RNA (rRNA) loci. The recently discovered P. hermaphrodita strains in New Zealand and the United States had mtDNA haplotypes nearly identical to that of Nemaslug®, and these were placed together in an intraspecific monophyletic clade with high support in maximum likelihood and Bayesian analyses. We also examined bacteria that co-cultured with the nematode strains isolated in Oregon, USA, by analyzing 16S rRNA sequences. Eight different bacterial genera were found to associate with these nematodes, though Moraxella osloensis, the bacteria species used in the Nemaslug® formulation, was not detected. This study provided evidence that nematodes deriving from the Nemaslug® biocontrol product have invaded countries where its use is prohibited by regulatory agencies and not commercially available.

Complex Transmission Patterns and Age-Related Dynamics of a Selfish mtDNA Deletion.

Despite wide-ranging implications of selfish mitochondrial DNA (mtDNA) elements for human disease and topics in evolutionary biology (e.g., speciation), the forces controlling their formation, age-related accumulation, and offspring transmission remain largely unknown. Selfish mtDNA poses a significant challenge to genome integrity, mitochondrial function, and organismal fitness. For instance, numerous human diseases are associated with mtDNA mutations; however, few genetic systems can simultaneously represent pathogenic mitochondrial genome evolution and inheritance. The nematode Caenorhabditis briggsae is one such system. Natural C. briggsae isolates harbor varying levels of a large-scale deletion affecting the mitochondrial nduo-5 gene, termed nad5Δ. A subset of these isolates contains putative compensatory mutations that may reduce the risk of deletion formation. We studied the dynamics of nad5Δ heteroplasmy levels during animal development and transmission from mothers to offspring in genetically diverse C. briggsae natural isolates. Results support previous work demonstrating that nad5Δ is a selfish element and that heteroplasmy levels of this deletion can be quite plastic, exhibiting high degrees of inter-family variability and divergence between generations. The latter is consistent with a mitochondrial bottleneck effect, and contrasts with previous findings from a laboratory-derived model uaDf5 mtDNA deletion in C. elegans. However, we also found evidence for among-isolate differences in the ability to limit nad5Δ accumulation, the pattern of which suggested that forces other than the compensatory mutations are important in protecting individuals and populations from rampant mtDNA deletion expansion over short time scales.

Variable Abundance and Distribution of Wolbachia and Cardinium Endosymbionts in Plant-Parasitic Nematode Field Populations.

The bacterial endosymbiont Wolbachia interacts with different invertebrate hosts, engaging in diverse symbiotic relationships. Wolbachia is often a reproductive parasite in arthropods, but an obligate mutualist in filarial nematodes. Wolbachia was recently discovered in plant-parasitic nematodes, and, is thus far known in just two genera Pratylenchus and Radopholus, yet the symbiont's function remains unknown. The occurrence of Wolbachia in these economically important plant pests offers an unexplored biocontrol strategy. However, development of Wolbachia-based biocontrol requires an improved understanding of symbiont-host functional interactions and the symbiont's prevalence among nematode field populations. This study used a molecular-genetic approach to assess the prevalence of a Wolbachia lineage (wPpe) in 32 field populations of Pratylenchus penetrans. Populations were examined from eight different plant species in Washington, Oregon, and California. Nematodes were also screened for the endosymbiotic bacterium Cardinium (cPpe) that was recently shown to co-infect P. penetrans. Results identified wPpe in 9/32 and cPpe in 1/32 of P. penetrans field populations analyzed. No co-infection was observed in field populations. Wolbachia was detected in nematodes from 4/8 plant-hosts examined (raspberry, strawberry, clover, and lily), and in all three states surveyed. Cardinium was detected in nematodes from mint in Washington. In the wPpe-infected P. penetrans populations collected from raspberry, the prevalence of wPpe infection ranged from 11 to 58%. This pattern is unlike that in filarial nematodes where Wolbachia is an obligate mutualist and occurs in 100% of the host. Further analysis of wPpe-infected populations revealed female-skewed sex ratios (up to 96%), with the degree of skew positively correlating with wPpe prevalence. Uninfected nematode populations had approximately equal numbers of males and females. Comparisons of 54 wPpe 16S ribosomal RNA sequences revealed high similarity across the geographic isolates, with 45 of 54 isolates being identical at this locus. The complete absence of wPpe among some populations and low prevalence in others suggest that this endosymbiont is not an obligate mutualist of P. penetrans. The observed sex ratio bias in wPpe-infected nematode populations is similar to that observed in arthropods where Wolbachia acts as a reproductive manipulator, raising the question of a similar role in plant-parasitic nematodes.

Sex and Mitonuclear Adaptation in Experimental Caenorhabditis elegans Populations.

To reveal phenotypic and functional genomic patterns of mitonuclear adaptation, a laboratory adaptation study with Caenorhabditis elegans nematodes was conducted in which independently evolving lines were initiated from a low-fitness mitochondrial electron transport chain (ETC) mutant, gas-1 Following 60 generations of evolution in large population sizes with competition for food resources, two distinct classes of lines representing different degrees of adaptive response emerged: a low-fitness class that exhibited minimal or no improvement compared to the gas-1 mutant ancestor, and a high-fitness class containing lines that exhibited partial recovery of wild-type fitness. Many lines that achieved higher reproductive and competitive fitness levels were also noted to evolve high frequencies of males during the experiment, consistent with adaptation in these lines having been facilitated by outcrossing. Whole-genome sequencing and analysis revealed an enrichment of mutations in loci that occur in a gas-1-centric region of the C. elegans interactome and could be classified into a small number of functional genomic categories. A highly nonrandom pattern of mitochondrial DNA mutation was observed within high-fitness gas-1 lines, with parallel fixations of nonsynonymous base substitutions within genes encoding NADH dehydrogenase subunits I and VI. These mitochondrial gene products reside within ETC complex I alongside the nuclear-encoded GAS-1 protein, suggesting that rapid adaptation of select gas-1 recovery lines was driven by fixation of compensatory mitochondrial mutations.

Muller's Ratchet and compensatory mutation in Caenorhabditis briggsae mitochondrial genome evolution.

BACKGROUND: The theory of Muller' Ratchet predicts that small asexual populations are doomed to accumulate ever-increasing deleterious mutation loads as a consequence of the magnified power of genetic drift and mutation that accompanies small population size. Evidence for Muller's Ratchet and knowledge on its underlying molecular mechanisms, however, are lacking for natural populations. RESULTS: We characterized mitochondrial genome evolutionary processes in Caenorhabditis briggsae natural isolates to show that numerous lineages experience a high incidence of nonsynonymous substitutions in protein-coding genes and accumulate unusual deleterious noncoding DNA stretches with associated heteroplasmic function-disrupting genome deletions. Isolate-specific deletion proportions correlated negatively with nematode fecundity, suggesting that these deletions might negatively affect C. briggsae fitness. However, putative compensatory mutations were also observed that are predicted to reduce heteroplasmy levels of deleterious deletions. Paradoxically, compensatory mutations were observed in one major intraspecific C. briggsae clade where population sizes are estimated to be very small (and selection is predicted to be relatively weak), but not in a second major clade where population size estimates are much larger and selection is expected to be more efficient. CONCLUSION: This study provides evidence that the mitochondrial genomes of animals evolving in nature are susceptible to Muller's Ratchet, suggests that context-dependent compensatory mutations can accumulate in small populations, and predicts that Muller's Ratchet can affect fundamental evolutionary forces such as the rate of mutation.