Both-strand gene coding in a plastome-like mitogenome of an enoplid nematode.

The phylum Nematoda remains very poorly sampled for mtDNA, with a strong bias toward parasitic, economically important or model species of the Chromadoria lineage. Most chromadorian mitogenomes share a specific order of genes encoded on one mtDNA strand. However, the few sequenced representatives of the Dorylaimia lineage exhibit a variable order of mtDNA genes encoded on both strands. While the ancestral arrangement of nematode mitogenome remains undefined, no evidence has been reported for Enoplia, the phylum's third early divergent major lineage. We describe the first mitogenome of an enoplian nematode, Campydora demonstrans, and contend that the complete 37-gene repertoire and both-strand gene encoding are ancestral states preserved in Enoplia and Dorylaimia versus the derived mitogenome arrangement in some Chromadoria. The C. demonstrans mitogenome is 17,018 bp in size and contains a noncoding perfect inverted repeat with 2013 bp-long arms, subdividing the mitogenome into two coding regions. This mtDNA arrangement is very rare among animals and instead resembles that of chloroplast genomes in land plants. Our report broadens mtDNA taxonomic sampling of the phylum Nematoda and adds support to the applicability of cox1 gene as a phylogenetic marker for establishing nematode relationships within higher taxa.

Insights into dryland biocrust microbiome: geography, soil depth and crust type affect biocrust microbial communities and networks in Mojave Desert, USA.

Biocrusts are the living skin of drylands, comprising diverse microbial communities that are essential to desert ecosystems. Despite there being extensive knowledge on biocrust ecosystem functions and lichen and moss biodiversity, little is known about factors structuring diversity among their microbial communities. We used amplicon-based metabarcode sequencing to survey microbial communities from biocrust surface and subsurface soils at four sites located within the Mojave Desert. Five biocrust types were examined: Light-algal/Cyanobacteria, Cyanolichen, Green-algal lichen, Smooth-moss and Rough-moss crust types. Microbial diversity in biocrusts was structured by several characteristics: (i) central versus southern Mojave sites displayed different community signatures, (ii) indicator taxa of plant-associated fungi (plant pathogens and wood saprotrophs) were identified at each site, (iii) surface and subsurface microbial communities were distinct and (iv) crust types had distinct indicator taxa. Network analysis ranked bacteria-bacteria interactions as the most connected of all within-domain and cross-domain interaction networks in biocrust surface samples. Actinobacteria, Proteobacteria, Cyanobacteria and Ascomycota functioned as hubs among all phyla. The bacteria Pseudonocardia sp. (Pseudonocardiales, Actinobacteria) and fungus Alternaria sp. (Pleosporales, Ascomycota) were the most connected had the highest node degree. Our findings provide crucial insights for dryland microbial community ecology, conservation and sustainable management.

Alloionema similis n. sp., a genetically divergent sibling species of A. appendiculatum Schneider, 1859 (Rhabditida: Alloionematidae) from invasive slugs in California, USA.

A new species of Alloionema Schneider, 1859, A. similis n. sp., and the known species A. appendiculatum Schneider, 1859 were isolated from cadavers of invasive slugs in California. Both species are described based on morphology, morphometrics and molecular data. Alloionema similis n. sp. is morphologically very similar to A. appendiculatum but can be distinguished by a more posterior position of the excretory pore in the Kleinform females and longer tail in the Kleinform males. Substantial differences between the two species are, however, found in both 18S and 28S rDNA sequences. Sequence analysis revealed unambiguous autapomorphies in nucleotide sequence and secondary structure of rRNA genes, separating A. appendiculatum and A. similis n. sp. Molecular phylogenies were inferred from concatenated secondary-structure based multiple sequence alignments of nearly complete 18S and the D1-D3 domains of the 28S rRNA genes. Phylogenetic analyses placed these two species as sister taxa in a monophyletic clade, separately from Neoalloionema tricaudatum Ivanova, Pham Van Luc & Spiridonov, 2016 and N. indicum Nermut, Puza & Mrácek, 2016.

Culture-independent investigation of the microbiome associated with the nematode Acrobeloides maximus.

BACKGROUND: Symbioses between metazoans and microbes are widespread and vital to many ecosystems. Recent work with several nematode species has suggested that strong associations with microbial symbionts may also be common among members of this phylu. In this work we explore possible symbiosis between bacteria and the free living soil bacteriovorous nematode Acrobeloides maximus. METHODOLOGY: We used a soil microcosm approach to expose A. maximus populations grown monoxenically on RFP labeled Escherichia coli in a soil slurry. Worms were recovered by density gradient separation and examined using both culture-independent and isolation methods. A 16S rRNA gene survey of the worm-associated bacteria was compared to the soil and to a similar analysis using Caenorhabditis elegans N2. Recovered A. maximus populations were maintained on cholesterol agar and sampled to examine the population dynamics of the microbiome. RESULTS: A consistent core microbiome was extracted from A. maximus that differed from those in the bulk soil or the C. elegans associated set. Three genera, Ochrobactrum, Pedobacter, and Chitinophaga, were identified at high levels only in the A. maximus populations, which were less diverse than the assemblage associated with C. elegans. Putative symbiont populations were maintained for at least 4 months post inoculation, although the levels decreased as the culture aged. Fluorescence in situ hybridization (FISH) using probes specific for Ochrobactrum and Pedobacter stained bacterial cells in formaldehyde fixed nematode guts. CONCLUSIONS: Three microorganisms were repeatedly observed in association with Acrobeloides maximus when recovered from soil microcosms. We isolated several Ochrobactrum sp. and Pedobacter sp., and demonstrated that they inhabit the nematode gut by FISH. Although their role in A. maximus is not resolved, we propose possible mutualistic roles for these bacteria in protection of the host against pathogens and facilitating enzymatic digestion of other ingested bacteria.

Neotobrilus nicsmolae n. sp. (Tobrilidae: Nematoda) and Chronogaster carolinensis n. sp. (Chronogasteridae: Nematoda) from Lake Phelps, North Carolina.

Two new species, Neotobrilus nicsmolae n. sp. and Chronogaster carolinensis n. sp. are described from a small, acidic, temperate, natural lake in North Carolina. N. nicsmolae n. sp. comes close to three members of the genus reported from North America, N. filipjevi, N. longus, and N. hopei. However, N. nicsmolae is unique with in the genus in having a combination of characters: size smaller than 1,700 µm, shorter outer labial and cephalic setae, tail shorter than 250 µm, last ventromedian supplement close (about 5 µm) to cloacal opening, spicule length of 61 to 85 µm, flagelloid sperm, and possession of subterminal setae. Assessment of relationships among clades within the Triplonchida using DNA sequences of the D2D3 expansion segment of the LSU rDNA showed that the family Trichodoridae and the genus Tripyla were recovered as monophyletic. The genus Tobrilus was recovered as monophyletic in the neighbor-joining and maximum likelihood trees, but that was not so in the maximum-parsimony tree. The separation among genera of the Trichodoridae, i.e., Trichodorus and Paratrichodorus, was not clear-cut in all phylograms. Chronogaster carolinensis n. sp. in having one ventral mucro with no spine and vacuolated lateral glandular bodies comes close to C. typica and C. ethiopica but differs from all hitherto known species in a combination of characteristics: in having long cephalic setae, long stoma, crystalloid bodies, vacuolated lateral glandular bodies, and a tail terminus with blunt ventral mucro, and its lack of lateral line.

Metagenetic community analysis of microbial eukaryotes illuminates biogeographic patterns in deep-sea and shallow water sediments.

Microbial eukaryotes (nematodes, protists, fungi, etc., loosely referred to as meiofauna) are ubiquitous in marine sediments and probably play pivotal roles in maintaining ecosystem function. Although the deep-sea benthos represents one of the world's largest habitats, we lack a firm understanding of the biodiversity and community interactions amongst meiobenthic organisms in this ecosystem. Within this vast environment, key questions concerning the historical genetic structure of species remain a mystery, yet have profound implications for our understanding of global biodiversity and how we perceive and mitigate the impact of environmental change and anthropogenic disturbance. Using a metagenetic approach, we present an assessment of microbial eukaryote communities across depth (shallow water to abyssal) and ocean basins (deep-sea Pacific and Atlantic). Within the 12 sites examined, our results suggest that some taxa can maintain eurybathic ranges and cosmopolitan deep-sea distributions, but the majority of species appear to be regionally restricted. For Operationally Clustered Taxonomic Units (OCTUs) reporting wide distributions, there appears to be a taxonomic bias towards a small subset of taxa in most phyla; such bias may be driven by specific life history traits amongst these organisms. In addition, low genetic divergence between geographically disparate deep-sea sites suggests either a shorter coalescence time between deep-sea regions or slower rates of evolution across this vast oceanic ecosystem. While high-throughput studies allow for broad assessment of genetic patterns across microbial eukaryote communities, intragenomic variation in rRNA gene copies and the patchy coverage of reference databases currently present substantial challenges for robust taxonomic interpretations of eukaryotic data sets.

An improved molecular phylogeny of the Nematoda with special emphasis on marine taxa.

Phylogenetic reconstructions of relations within the phylum Nematoda are inherently difficult but have been advanced with the introduction of large-scale molecular-based techniques. However, the most recent revisions were heavily biased towards terrestrial and parasitic species and greater representation of clades containing marine species (e.g. Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, Enoplida, and Monhysterida) is needed for accurate coverage of known taxonomic diversity. We now add small subunit ribosomal DNA (SSU rDNA) sequences for 100 previously un-sequenced species of nematodes, including 46 marine taxa. SSU rDNA sequences for >200 taxa have been analysed based on Bayesian inference and LogDet-transformed distances. The resulting phylogenies provide support for (i) the re-classification of the Secernentea as the order Rhabditida that derived from a common ancestor of chromadorean orders Araeolaimida, Chromadorida, Desmodorida, Desmoscolecida, and Monhysterida and (ii) the position of Bunonema close to the Diplogasteroidea in the Rhabditina. Other, previously controversial relationships can now be resolved more clearly: (a) Alaimus, Campydora, and Trischistoma belong in the Enoplida, (b) Isolaimium is placed basally to a big clade containing the Axonolaimidae, Plectidae, and Rhabditida, (c) Xyzzors belongs in the Desmodoridae, (d) Comesomatidae and Cyartonema belongs in the Monhysterida, (e) Globodera belongs in the Hoplolaimidae and (f) Paratylenchus dianeae belongs in the Criconematoidea. However, the SSU gene did not provide significant support for the class Chromadoria or clear evidence for the relationship between the three classes, Enoplia, Dorylaimia, and Chromadoria. Furthermore, across the whole phylum, the phylogenetically informative characters of the SSU gene are not informative in a parsimony analysis, highlighting the short-comings of the parsimony method for large-scale phylogenetic modelling.

Phylogeny of Cephalobina (Nematoda): molecular evidence for recurrent evolution of probolae and incongruence with traditional classifications.

Nematodes of the suborder Cephalobina include an ecologically and morphologically diverse array of species that range from soil-dwelling microbivores to parasites of vertebrates and invertebrates. Despite a long history of study, certain of these microbivores (Cephaloboidea) present some of the most intractable problems in nematode systematics; the lack of an evolutionary framework for these taxa has prevented the identification of natural groups and inhibited understanding of soil biodiversity and nematode ecology. Phylogenetic analyses of ribosomal (LSU) sequence data from 53 taxa revealed strong support for monophyly of taxa representing the Cephaloboidea, but do not support the monophyly of most genera within this superfamily. Historically these genera have primarily been recognized based on variation in labial morphology, but molecular phylogenies show the same general labial (probolae) morphotype often results from recurrent similarity, a result consistent with the phenotypic plasticity of probolae previously observed for some species in ecological time. Phylogenetic analyses of LSU rDNA also recovered strong support for some other groups of cephalobs, including taxa representing most (but not all) Panagrolaimoidea. In addition to revealing homoplasy of probolae, molecular trees also imply other unexpected patterns of character evolution or polarity, including recurrent similarity of offset spermatheca presence, and representation of complex probolae as the ancestral condition within Cephaloboidea. For Cephalobidae, molecular trees do not support traditional genera as natural groups, but it remains untested if deconstructing probolae morphotypes or other structural features into finer component characters may reveal homologies that help delimit evolutionary lineages.

A quick tour of nematode diversity and the backbone of nematode phylogeny.

Contrary to textbook dogma, nematodes are not only highly diverse, but often also complex and biologically specialized metazoans. Just a few of the many fascinating adaptations are reviewed in this chapter, as a prelude to a quick tour through phylogenetic relationships within the phylum. Small Subunit rDNA sequences have confirmed several controversial prior hypotheses, as well as revealing some unexpected relationships, resulting in a recent proposal for revised classification. Three major lineages exist within the phylum: Chromadoria, Enoplia and Dorylaimia. The exact order of appearance of these lineages is not yet resolved, which also leaves room for uncertainty about the biology and morphology of the exclusive common ancestor of nematodes. Enoplia and Dorylaimia differ considerably in many respects from C. elegans, which is a member of Chromadoria. The latter group is extremely diverse in its own right, for example in ecological range, in properties of the cuticle and in structure of the pharynx. The formerly relatively widely accepted class Secernentea is deeply nested within Chromadoria, and has therefore recently been relegated to the rank and name of order Rhabditida. Within this order, closer relatives of C. elegans include strongylids, diplogasterids and bunonematids. Tylenchs, cephalobs and panagrolaimids are also members of Rhabditida, albeit probably more distantly related to C. elegans.

An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding.

Molecular surveys of meiofaunal diversity face some interesting methodological challenges when it comes to interstitial nematodes from soils and sediments. Morphology-based surveys are greatly limited in processing speed, while barcoding approaches for nematodes are hampered by difficulties of matching sequence data with traditional taxonomy. Intermediate technology is needed to bridge the gap between both approaches. An example of such technology is video capture and editing microscopy, which consists of the recording of taxonomically informative multifocal series of microscopy images as digital video clips. The integration of multifocal imaging with sequence analysis of the D2D3 region of large subunit (LSU) rDNA is illustrated here in the context of a combined morphological and barcode sequencing survey of marine nematodes from Baja California and California. The resulting video clips and sequence data are made available online in the database NemATOL (http://nematol.unh.edu/). Analyses of 37 barcoded nematodes suggest that these represent at least 32 species, none of which matches available D2D3 sequences in public databases. The recorded multifocal vouchers allowed us to identify most specimens to genus, and will be used to match specimens with subsequent species identifications and descriptions of preserved specimens. Like molecular barcodes, multifocal voucher archives are part of a wider effort at structuring and changing the process of biodiversity discovery. We argue that data-rich surveys and phylogenetic tools for analysis of barcode sequences are an essential component of the exploration of phyla with a high fraction of undiscovered species. Our methods are also directly applicable to other meiofauna such as for example gastrotrichs and tardigrades.

Intraspecific variation in Radopholus similis isolates assessed with restriction fragment length polymorphism and DNA sequencing of the internal transcribed spacer region of the ribosomal RNA cistron.

Restriction fragment length polymorphism and direct sequencing of the internal transcribed spacer rDNA region of 19 isolates of Radopholus similis yielded significant diversity, both among isolates and within some individuals. Restriction fragment length polymorphism with HaeIII, AluI and Tru9I yielded two sets of patterns. Digestion with RsaI revealed one or two supernumerary bands in single nematodes from five isolates, and sequencing confirmed microheterogeneity in four of these. Phylogenetic analysis grouped most isolates closely together, except for the five isolates with additional bands for RsaI. Our data reveal more population structure than previously found and lend further support to the synonymy of R. similis and 'Radopholus citrophilus'.

Video capture and editing as a tool for the storage, distribution, and illustration of morphological characters of nematodes.

Morphological identification and detailed observation of nematodes usually requires permanent slides, but these are never truly permanent and often prevent the same specimens to be used for other purposes. To efficiently record the morphology of nematodes in a format that allows easy archiving, editing, and distribution, we have assembled two micrographic video capture and editing (VCE) configurations. These assemblies allow production of short video clips that mimic multifocal observation of nematode specimens through a light microscope. Images so obtained can be used for training, management, and online access of "virtual voucher specimens" in taxonomic collections, routine screening of fixed or unfixed specimens, recording of ephemeral staining patterns, or recording of freshly dissected internal organs prior to their decomposition. We provide an overview of the components and operation of both of our systems and evaluate their efficiency and image quality. We conclude that VCE is a highly versatile approach that is likely to become widely used in nematology research and teaching.

Phylogenetic Analyses of Meloidogyne Small Subunit rDNA.

Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species.