Comparative mitochondrial genomics in Nematoda reveal astonishing variation in compositional biases and substitution rates indicative of multi-level selection.

BACKGROUND: Nematodes are the most abundant and diverse metazoans on Earth, and are known to significantly affect ecosystem functioning. A better understanding of their biology and ecology, including potential adaptations to diverse habitats and lifestyles, is key to understanding their response to global change scenarios. Mitochondrial genomes offer high species level characterization, low cost of sequencing, and an ease of data handling that can provide insights into nematode evolutionary pressures. RESULTS: Generally, nematode mitochondrial genomes exhibited similar structural characteristics (e.g., gene size and GC content), but displayed remarkable variability around these general patterns. Compositional strand biases showed strong codon position specific G skews and relationships with nematode life traits (especially parasitic feeding habits) equal to or greater than with predicted phylogeny. On average, nematode mitochondrial genomes showed low non-synonymous substitution rates, but also high clade specific deviations from these means. Despite the presence of significant mutational saturation, non-synonymous (dN) and synonymous (dS) substitution rates could still be significantly explained by feeding habit and/or habitat. Low ratios of dN:dS rates, particularly associated with the parasitic lifestyles, suggested the presence of strong purifying selection. CONCLUSIONS: Nematode mitochondrial genomes demonstrated a capacity to accumulate diversity in composition, structure, and content while still maintaining functional genes. Moreover, they demonstrated a capacity for rapid evolutionary change pointing to a potential interaction between multi-level selection pressures and rapid evolution. In conclusion, this study helps establish a background for our understanding of the potential evolutionary pressures shaping nematode mitochondrial genomes, while outlining likely routes of future inquiry.

Nematode mitochondrial metagenomics: A new tool for biodiversity analysis.

DNA barcoding approaches have greatly increased our understanding of biodiversity on the planet, and metabarcoding is widely used for classifying members of the phylum Nematoda. However, loci typically utilized in metabarcoding studies are often unable to resolve closely related species or are unable to recover all taxa present in a sample due to inadequate PCR primer binding. Mitochondrial metagenomics (mtMG) is an alternative approach utilizing shotgun sequencing of total DNA to recover the mitochondrial genomes of all species present in samples. However, this approach requires a comprehensive reference database for identification and currently available mitochondrial sequences for nematodes are highly dominated by sequences from the order Rhabditida, and excludes many clades entirely. Here, we analysed the efficacy of mtMG for the recovery of nematode taxa and the generation of mitochondrial genomes. We first developed a curated reference database of nematode mitochondrial sequences and expanded it with 40 newly sequenced taxa. We then tested the mito-metagenomics approach using a series of nematode mock communities consisting of morphologically identified nematode species representing various feeding traits, life stages, and phylogenetic relationships. We were able to identify all but two species through the de novo assembly of COX1 genes. We were also able to recover additional mitochondrial protein coding genes (PCGs) for 23 of the 24 detected species including a full array of 12 PCGs from five of the species. We conclude that mtMG offers a potential for the effective recovery of nematode biodiversity but remains limited by the breadth of the reference database.

Ecological and Morphological Differentiation Among COI Haplotype Groups in the Plant Parasitic Nematode Species Mesocriconema Xenoplax.

DNA barcoding with the mitochondrial COI gene reveals distinct haplotype subgroups within the monophyletic and parthenogenetic nematode species, Mesocriconema xenoplax. Biological attributes of these haplotype groups (HG) have not been explored. An analysis of M. xenoplax from 40 North American sites representing both native plant communities and agroecosystems was conducted to identify possible subgroup associations with ecological, physiological, or geographic factors. A dataset of 132 M. xenoplax specimens was used to generate sequences of a 712 bp region of the cytochrome oxidase subunit I gene. Maximum-likelihood and Bayesian phylogenies recognized seven COI HG (≥99/0.99 posterior probability/bootstrap value). Species delimitation metrics largely supported the genetic integrity of the HG. Discriminant function analysis of HG morphological traits identified stylet length, total body length, and stylet knob width as the strongest distinguishing features among the seven groups, with stylet length as the strongest single distinguishing morphological feature. Multivariate analysis identified land cover, ecoregion, and maximum temperature as predictors of 53.6% of the total variation (P = 0.001). Within land cover, HG categorized under "herbaceous," "woody wetlands," and "deciduous forest" were distinct in DAPC and RDA analyses and were significantly different (analysis of molecular variance P = 0.001). These results provide empirical evidence for molecular, morphological, and ecological differentiation associated with HG within the monophyletic clade that represents the species Mesocriconema xenoplax.

Context-dependent role of abiotic and biotic factors structuring nematode communities along two environmental gradients.

Although abiotic environmental factors have been historically regarded as the dominant deterministic process in microbial community assembly, recent studies indicate that biotic interactions may be equally significant. However, the extent to which both processes are important in assembly of belowground communities is unknown. Along two environmental gradients: alkalinity (ranging from pH ~7 to ~11) and habitat type (lakes, shorelines, and prairies around lakes) present in the Western Nebraska Sandhills, we used 18S rRNA gene marker metabarcoding and statistical analyses, including generalized dissimilarity modelling (GDM), to evaluate the dynamics between abiotic and biotic factors that might play a role in nematode community assembly. Lakes supported the least diverse and prairies the most diverse communities with completely distinct compositions. We also observed a potential role of alkalinity in shaping these communities but only in lakes. Generally, GDMs indicated the influence of both abiotic and biotic factors. However, their relative importance in explaining community variability was dependent on the habitat. Biotic factors influenced the lake communities most, followed by shorelines and prairies, explaining ~47%, 27% and 8% of the variation, respectively. In contrast, the role of abiotic factors was relatively similar in lakes, shorelines and prairies (~15%, 18% and 14% of the variation, respectively). Most variation in the shorelines (62%) and prairies (82%) remained unexplained, suggesting the potential importance of factors associated with specific traits or a stronger role of stochastic processes. Nevertheless, our findings suggest both deterministic processes are important in nematode community assembly, but their specific contributions are context-dependent.

Nematode biodiversity assessments need vouchered databases: A BOLD reference library for plant-parasitic nematodes in the superfamily Criconematoidea.

Nematodes are frequently cited as underrepresented in faunistic surveys using DNA barcoding with COI. This underrepresentation is generally attributed to a limited presence of nematodes in DNA databases which, in turn, is often ascribed to structural variability and high evolutionary rates in nematode mitochondrial genomes. Empirical evidence, however, indicates that many taxa are readily amplified with primer sets specifically targeted to different nematode families. Here we report the development of a COI reference library of 1726 specimens in the terrestrial plant parasitic nematode superfamily Criconematoidea. Specimens collected during an ecoregion survey of North America were individually photographed, measured, and PCR amplified to produce a 721 bp region of COI for taxonomic analysis. A neighbor-joining tree structured the dataset into 179 haplotype groups that generally conformed to morphospecies in traditional analysis or Barcode Index Numbers (BINs) in the BOLD system, although absent formal BIN membership due to insufficient overlap with the Folmer region of COI. Approximately one-third of the haplotype groups could be associated with previously described species. The geographic distribution of criconematid nematode species suggests a structure influenced by the major habitat types in the United States and Canada. All sequences collected in the ecoregion survey are deposited in BOLD.

Description of Gracilacus paralatescens n. sp. (Nematoda:Paratylenchinae) found from the rhizosphere of Bamboo in Zhejiang, China.

Gracilacus paralatescens n. sp., isolated in Anji County, Zhejiang Province, China from the rhizospheric soil of bamboo. The new species can be characterized by the female lateral field with three incisures, stylet 71.5 to 78.8 µm long, lip region truncated, excretory pore located anterior to basal knobs. Vulval lips non-protruding and without vulval flap, spermatheca large, elongated squarish shaped filled with sperms. Tail slender, relatively straight having wedge shape terminus. The spicule slender, slightly curved and 17.5 to 18.9 µm long. In the phylogenetic analysis based on 18S, D2-D3 of 28S and ITS regions of rDNA, the new species is clustered with Paratylenchid species having longer stylet length. Morphologically, the new species belongs to Group 9 of Paratylenchus sensu lato and is most similar to G. latescens.Gracilacus paralatescens n. sp., isolated in Anji County, Zhejiang Province, China from the rhizospheric soil of bamboo. The new species can be characterized by the female lateral field with three incisures, stylet 71.5 to 78.8 µm long, lip region truncated, excretory pore located anterior to basal knobs. Vulval lips non-protruding and without vulval flap, spermatheca large, elongated squarish shaped filled with sperms. Tail slender, relatively straight having wedge shape terminus. The spicule slender, slightly curved and 17.5 to 18.9 µm long. In the phylogenetic analysis based on 18S, D2-D3 of 28S and ITS regions of rDNA, the new species is clustered with Paratylenchid species having longer stylet length. Morphologically, the new species belongs to Group 9 of Paratylenchus sensu lato and is most similar to G. latescens.

Species Delimitation and Description of Mesocriconema nebraskense n. sp. (Nematoda: Criconematidae), a Morphologically Cryptic, Parthenogenetic Species from North American Grasslands.

Nematode surveys of North American grasslands conducted from 2010 to 2015 frequently recovered a species of criconematid nematode morphologically resembling Mesocriconema curvatum. These specimens were recovered from remnant native prairies in the central tallgrass ecoregion of North America, and not from surrounding agroecosystems. Historical records indicate that M. curvatum is a cosmopolitan species feeding on a wide range of agronomic and native plants. DNA barcoding indicates North American grasslands contain at least 10 phylogenetically distinct lineages of Mesocriconema that resemble, but are not, M. curvatum. Analysis of the two most common lineages reveals two distinctly different population structures. The variation in population structure suggests unique evolutionary histories associated with their diversification. These two major lineages share a sympatric distribution and their slight morphological differences contrast with a high level of genetic separation. Based on their genetic divergence, fixed diagnostic nucleotides, population structure, species delimitation metrics, and a sympatric distribution, we believe that one of these distinct lineages warrants formal nomenclatural recognition. Herein, we provide formal recognition for Mesocriconema nebraskense n. sp. and discuss its relationship to other Mesocriconema lineages discovered in native North American grasslands.

Description and Distribution of Three Criconematid Nematodes from Hangzhou, Zhejiang Province, China.

Populations of Criconemoides parvus , Discocriconemella hengsungica , and Discocriconemella limitanea , isolated in Hangzhou, China from the rhizosphere soil of woody perennials were characterized morphologically and molecularly. The morphometric data of the Chinese populations were compared with populations from other regions of the world. DNA barcoding with the mitochondrial COI gene confirmed conspecificity of Chinese and Costa Rican populations of D. limitanea . Phylogenetic assessment using a near full-length 18S ribosomal DNA sequence provided weak support for a grouping of Criconemoides parvus from China and C. annulatus from western North America. The phylogenetic position of D. hengsungica from China and an unknown species of Discocriconemella from Thailand relative to D. limitanea suggests that the genus Discocriconemella is not monophyletic. The study provides the first record of D. hengsungica in China and confirms the presence of C. parvus previously reported from China. Biogeographic implications of these nematode distributions are discussed.

Diversity of bacteria carried by pinewood nematode in USA and phylogenetic comparison with isolates from other countries.

Pine wilt disease (PWD) is native to North America and has spread to Asia and Europe. Lately, mutualistic relationship has been suggested between the pinewood nematode (PWN), Bursaphelenchus xylophilus the causal nematode agent of PWD, and bacteria. In countries where PWN occurs, nematodes from diseased trees were reported to carry bacteria from several genera. However no data exists for the United States. The objective of this study was to evaluate the diversity of the bacterial community carried by B. xylophilus, isolated from different Pinus spp. with PWD in Nebraska, United States. The bacteria carried by PWN belonged to Gammaproteobacteria (79.9%), Betaproteobacteria (11.7%), Bacilli (5.0%), Alphaproteobacteria (1.7%) and Flavobacteriia (1.7%). Strains from the genera Chryseobacterium and Pigmentiphaga were found associated with the nematode for the first time. These results were compared to results from similar studies conducted from other countries of three continents in order to assess the diversity of bacteria with associated with PWN. The isolates from the United States, Portugal and China belonged to 25 different genera and only strains from the genus Pseudomonas were found in nematodes from all countries. The strains from China were closely related to P. fluorescens and the strains isolated from Portugal and USA were phylogenetically related to P. mohnii and P. lutea. Nematodes from the different countries are associated with bacteria of different species, not supporting a relationship between PWN with a particular bacterial species. Moreover, the diversity of the bacteria carried by the pinewood nematode seems to be related to the geographic area and the Pinus species. The roles these bacteria play within the pine trees or when associated with the nematodes, might be independent of the presence of the nematode in the tree and only related on the bacteria's relationship with the tree.

Nematode spatial and ecological patterns from tropical and temperate rainforests.

Large scale diversity patterns are well established for terrestrial macrobiota (e.g. plants and vertebrates), but not for microscopic organisms (e.g. nematodes). Due to small size, high abundance, and extensive dispersal, microbiota are assumed to exhibit cosmopolitan distributions with no biogeographical patterns. This assumption has been extrapolated from local spatial scale studies of a few taxonomic groups utilizing morphological approaches. Recent molecularly-based studies, however, suggest something quite opposite. Nematodes are the most abundant metazoans on earth, but their diversity patterns are largely unknown. We conducted a survey of nematode diversity within three vertical strata (soil, litter, and canopy) of rainforests at two contrasting latitudes in the North American meridian (temperate: the Olympic National Forest, WA, U.S.A and tropical: La Selva Biological Station, Costa Rica) using standardized sampling designs and sample processing protocols. To describe nematode diversity, we applied an ecometagenetic approach using 454 pyrosequencing. We observed that: 1) nematode communities were unique without even a single common species between the two rainforests, 2) nematode communities were unique among habitats in both rainforests, 3) total species richness was 300% more in the tropical than in the temperate rainforest, 4) 80% of the species in the temperate rainforest resided in the soil, whereas only 20% in the tropics, 5) more than 90% of identified species were novel. Overall, our data provided no support for cosmopolitanism at both local (habitats) and large (rainforests) spatial scales. In addition, our data indicated that biogeographical patterns typical of macrobiota also exist for microbiota.

Reflections on plant and soil nematode ecology: past, present and future.

The purpose of this review is to highlight key developments in nematode ecology from its beginnings to where it stands today as a discipline within nematology. Emerging areas of research appear to be driven by crop production constraints, environmental health concerns, and advances in technology. In contrast to past ecological studies which mainly focused on management of plant-parasitic nematodes, current studies reflect differential sensitivity of nematode faunae. These differences, identified in both aquatic and terrestrial environments include response to stressors, environmental conditions, and management practices. Methodological advances will continue to influence the role nematodes have in addressing the nature of interactions between organisms, and of organisms with their environments. In particular, the C. elegans genetic model, nematode faunal analysis and nematode metagenetic analysis can be used by ecologists generally and not restricted to nematologists.

Ecometagenetics confirm high tropical rainforest nematode diversity.

The general patterns of increasing biodiversity from the poles to the equator have been well documented for large terrestrial organisms such as plants and vertebrates but are largely unknown for microbiota. In contrast to macrobiota, microbiota have long been assumed to exhibit cosmopolitan, random distributions and a lack of spatial patterns. To evaluate the assumption, we conducted a survey of nematode diversity within the soil, litter and canopy habitats of the humid lowland tropical rainforest of Costa Rica using an ultrasequencing ecometagenetic approach at a species-equivalent taxonomic level. Our data indicate that both richness and diversity of nematode communities in the tropical rainforests of Costa Rica are high and exceed observed values from temperate ecosystems. The majority of nematode species were unknown to science, providing evidence for the presence of highly endemic (not cosmopolitan) species of still completely undiscovered biodiversity. Most importantly, the greater taxonomic resolution used here allowed us to reveal predictable habitat associations for specific taxa and thus gain insights into their nonrandom distribution patterns.

Morphological and molecular characterization of Discocriconemella inarata, an endemic nematode from north american native tallgrass prairies.

Discocriconemella inarata, a plant parasitic nematode species originally discovered in a virgin tallgrass prairie in northwest Iowa, was re-examined by molecular and morphological analyses of topotype material. This species has never been recorded in cultivated fields and could potentially serve as an indicator for high quality prairie habitats. DNA sequence from a conserved 3' portion of the 18S ribosomal gene exhibited an identical match between D. inarata topotype specimens and topotype specimens of Mesocriconema xenoplax from Fresno, California. Higher resolution sequence analyses using the internal transcribed spacer 1 (ITS1) and a portion of the mitochondrial gene cytochrome b (cytb) allowed discrimination of D. inarata apart from M. xenoplax. This pair of species formed a well-supported clade with other Mesocriconema species exclusive of tropical Discocriconemella species. Scanning electron microscopy confirmed the absence of submedian lobes on D. inarata, suggesting a secondary loss of this defining morphological characteristic for Mesocriconema. Observations and measurements of D. inarata juveniles were added for the first time. Surveys of other prairies within the Great Plains expanded the known distribution of this species.

Vittatidera zeaphila (Nematoda: Heteroderidae), a new genus and species of cyst nematode parasitic on corn (Zea mays).

A new genus and species of cyst nematode, Vittatidera zeaphila, is described from Tennessee. The new genus is superficially similar to Cactodera but is distinguished from other cyst-forming taxa in having a persistent lateral field in females and cysts, persistent vulval lips covering a circumfenestrate vulva, and subventral gland nuclei of the female contained in a separate small lobe. Infective juveniles (J2) are distinguished from all previously described Cactodera spp. by the short stylet in the second-stage juvenile (14-17 µm); J2 of Cactodera spp. have stylets at least 18 µm long. The new species also is unusual in that the females produce large egg masses. Known hosts are corn and goosegrass. DNA analysis suggests that Vittatidera forms a separate group apart from other cyst-forming genera within Heteroderinae.

Evaluating high-throughput sequencing as a method for metagenomic analysis of nematode diversity.

Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time-consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400-bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community.