Structure,development, and evolutive patterns of spermatozoa in rhabditid nematodes (Nematoda: Rhabditida)

Spermatogenesis of five rhabditid nematodes was studied using transmission electron microscopy and is described herein. Structure and development of nematode sperm in all available representatives of the extensive order Rhabditida have been analysed and the main characteristics of each infraorder are discussed. The ancestral sperm of the order Rhabditida was reconstructed using maximum likelihood and Bayesian methods based on 44 ultrastructural sperm characters. The hypothetical ancestral spermatogenesis of the order Rhabditida agrees with the previously suggested “rhabditid” pattern and appears to be conserved throughout the order Rhabditida. Despite the enormous variation of rhabditid nematodes, few groups deviate from the ancestral pattern. This conserved pattern can be informative within the phylum Nematoda at order level, but poses limitations when used in taxonomic and phylogenetic analysis within Rhabditida.     

Moving towards a complete molecular framework of the Nematoda: a focus on the Enoplida and early-branching clades

Background  

The subclass Enoplia (Phylum Nematoda) is purported to be the earliest branching clade amongst all nematode taxa, yet the deep phylogeny of this important lineage remains elusive. Free-living marine species within the order Enoplida play prominent roles in marine ecosystems, but previous molecular phylogenies have provided only the briefest evolutionary insights; this study aimed to firmly resolve internal relationships within the hyper-diverse but poorly understood Enoplida. In addition, we revisited the molecular framework of the Nematoda using a rigorous phylogenetic approach in order to investigate patterns of early splits amongst the oldest lineages (Dorylaimia and Enoplia).     

MOTUs, Morphology, and Biodiversity Estimation: A Case Study Using Nematodes of the Suborder Criconematina and a Conserved 18S DNA Barcode

DNA barcodes are increasingly used to provide an estimate of biodiversity for small, cryptic organisms like nematodes. Nucleotide sequences generated by the barcoding process are often grouped, based on similarity, into molecular operational taxonomic units (MOTUs). In order to get a better understanding of the taxonomic resolution of a 3' 592-bp 18S rDNA barcode, we have analyzed 100 MOTUs generated from 214 specimens in the nematode suborder Criconematina. Previous research has demonstrated that the primer set for this barcode reliably amplifies all nematodes in the Phylum Nematoda. Included among the Criconematina specimens were 25 morphologically described species representing 12 genera. Using the most stringent definition of MOTU membership, where a single nucleotide difference is sufficient for the creation of a new MOTU, it was found that an MOTU can represent a subgroup of a species (e.g. Discocriconemella limitanea), a single species (Bakernema inaequale), or a species complex (MOTU 76). A maximum likelihood phylogenetic analysis of the MOTU dataset generated four major clades that were further analyzed by character-based barcode analysis. Fourteen of the 25 morphologically identified species had at least one putative diagnostic nucleotide identified by this character-based approach. These diagnostic nucleotides could be useful in biodiversity assessments when ambiguous results are encountered in database searches that use a distance-based metric for nucleotide sequence comparisons. Information and images regarding specimens examined during this study are available online.     

A Position Paper on the Electronic Publication of Nematode Taxonomic Manuscripts

Several nematode species have now attained ‘model organism’ status, yet there remain many niches in basic biological inquiry for which nematodes would be ideal model systems of study. However, furthering the model system approach is hindered by lack of information on nematode biodiversity. The shortage of taxonomic resources to inventory and characterize biodiversity hinders research programs in invasion biology, ecosystem functioning, conservation biology, and many others. The disproportion between numbers of species to be described and numbers of available taxonomic specialists is greater for Nematoda than for any other metazoan phylum. A partial solution to the taxonomic impediment is the adoption of recent advances in electronic publishing. Electronic publishing has the potential to increase the rate at which taxonomic papers are published, the breadth of their distribution, and the type, quantity, quality, and accessibility of data. We propose that the Journal of Nematology implement the advantageous aspects of electronic publication as a means to help ameliorate the limitations of an underdeveloped taxonomy and empower the nematological disciplines currently hindered by it.     

The Evolution of Tyrosine-Recombinase Elements in Nematoda

Transposable elements can be categorised into DNA and RNA elements based on their mechanism of transposition. Tyrosine recombinase elements (YREs) are relatively rare and poorly understood, despite sharing characteristics with both DNA and RNA elements. Previously, the Nematoda have been reported to have a substantially different diversity of YREs compared to other animal phyla: the Dirs1-like YRE retrotransposon was encountered in most animal phyla but not in Nematoda, and a unique Pat1-like YRE retrotransposon has only been recorded from Nematoda. We explored the diversity of YREs in Nematoda by sampling broadly across the phylum and including 34 genomes representing the three classes within Nematoda. We developed a method to isolate and classify YREs based on both feature organization and phylogenetic relationships in an open and reproducible workflow. We also ensured that our phylogenetic approach to YRE classification identified truncated and degenerate elements, informatively increasing the number of elements sampled. We identified Dirs1-like elements (thought to be absent from Nematoda) in the nematode classes Enoplia and Dorylaimia indicating that nematode model species do not adequately represent the diversity of transposable elements in the phylum. Nematode Pat1-like elements were found to be a derived form of another Pat1-like element that is present more widely in animals. Several sequence features used widely for the classification of YREs were found to be homoplasious, highlighting the need for a phylogenetically-based classification scheme. Nematode model species do not represent the diversity of transposable elements in the phylum.     

The ultrastructure of the cuticle of adult female Mermis nigrescens (Nematoda)

The ultrastructure of the cuticle of the adult female nematode Mermis nigrescens has been described. There is an epicuticle and three-layered membrane covering the cuticle. The cortex is penetrated by canals which extend from the surface of the cuticle to the matrix of the layer beneath the cortex. Beneath the cortex are two layers of giant fibres which spiral around the nematode, a thick layer containing a network of fibres and a basal layer containing a vacuolated matrix material. it is thought that the epicuticle is secreted from the canals in the cortex. The possible functions of the layers in the cuticle have been discussed and similarities with the cuticle of the Acanthocephala have been noted.     

Ultrastructure of the Pharyngeal Cuticle and Lectin Labelling with Wheat Germ Agglutinin-gold Conjugate Indicating Chitin in the Pharyngeal Cuticle of Oesophagostomum dentatum(Strongylida, Nematoda)

The ultrastructure of the pharynx of the first juvenile stage and of adult Oesophagostomum dentatum (Nematoda, Strongylida) is investigated by TEM. Additional light microscopical and SEM data are presented for the anterior alimentary canal of all life-history stages and for adult O. quadrispinulatum . Ultrathin sections of adult and the first juvenile stage O. dentatum are labelled with the lectin wheat germ agglutinin coupled to colloidal gold. The pharyngeal cuticle consists of a trilaminate epicuticle and an amorphous basal layer, which also exhibits individual fibres in adult worms. The pharyngeal cells of the first juvenile stage are already arranged as in the adult worm. In comparison with data for additional nematodes taken from the literature, a trilaminate epicuticle and an amorphous basal layer are postulated for the pharynx in the ground pattern of the Nematoda. Lectin-gold labelling reveals a strong binding to the pharyngeal cuticle of O. dentatum . Competitive inhibition experiments with N -acetyl-D-glucosamine and triacetylchitotriose, as well as treatment with chitinase, neuraminidase and sulphuric acid, indicate that the pharyngeal cuticle contains chitin. This finding is supported by staining of the pharyngeal cuticle by the fluorescent brightener calcofluor white in cryosections of O. dentatum . The occurrence of chitin is interpreted as an original character of the Nematoda. © 1997 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.     

Molecular approach for assessing responses of microbial-feeding nematodes to burning and chronic nitrogen enrichment in a native grassland

A substantial proportion of the primary productivity in grassland ecosystems is allocated belowground, sustaining an abundant and diverse community of microbes and soil invertebrates. These belowground communities drive many important ecosystem functions and are responsive to a variety of environmental changes. Nematodes, an abundant and diverse component of grassland soil communities, are particularly responsive to altered environmental conditions, such as those associated with reduced fire frequency and nitrogen enrichment, with the most consistent responses displayed by microbial-feeding nematodes. However, much of the available research characterizing nematode responses to environmental change has been carried out at the taxonomic level of family or by broad trophic categories (e.g. fungivores, bacterivores). The extent to which differential responses to environmental change occurs at the genus level or below is unclear. Therefore, the objective of this study was to use molecular methods to quantify the response of microbial-feeding nematodes, at the lowest levels of taxonomic resolution, to nitrogen enrichment and changes in fire frequency. Using sequencing and quantitative polymerase chain reaction (PCR) probes for the 18S ribosomal RNA gene and the ITS1 region, we identified 19 microbial-feeding nematode taxa across four families. When nematodes were sampled across treatments, we found that some nematode taxa within a family responded similarly to nitrogen and burning treatments, while other taxa within the same family respond quite differently. Additionally, although nematodes from different families on average responded differently to nitrogen enrichment and burning, similar responses were seen in nematode taxa that span three taxonomic families. Thus, if nematodes are to be used as indicators of environmental change, care should be taken to assess the response at the lowest taxonomic level possible.     

Ascaris lumbricoides: zonal localization of immunoreactive collagen in the cuticle

Monoclonal antibodies that were raised against cuticular components from the free-living nematode Panagrellus silusiae were found to react with a cuticular collagenous domain from Ascaris lumbricoides. One of these monoclonal antibodies was used to localize the collagenous epitope within sectioned Ascaris cuticles. By indirect immunofluorescence, accessible binding sites were observed in the basal zone of the cuticle. Immunological staining occurred in the innermost lamella of the basal zone, i.e., basal lamella, in which the fibrillar palisade gave a strong response. The three layers of the spiral fiber system of the basal zone exhibited a distinctive immunofluorescence pattern. In each of these layers, irregular shaped blocks, often quadrangular, were immunostained; whereas, adjacent blocks were immunonegative. Immunostaining was, for the most part, absent from the cortical and medial zones of the cuticle as well as from other tissues within the worm.     

Agriculture erases climate constraints on soil nematode communities across large spatial scales

Anthropogenic conversion of natural to agricultural land reduces aboveground biodiversity. Yet, the overall consequences of land‐use changes on belowground biodiversity at large scales remain insufficiently explored. Furthermore, the effects of conversion on different organism groups are usually determined at the taxonomic level, while an integrated investigation that includes functional and phylogenetic levels is rare and absent for belowground organisms. Here, we studied the Earth's most abundant metazoa—nematodes—to examine the effects of conversion from natural to agricultural habitats on soil biodiversity across a large spatial scale. To this aim, we investigated the diversity and composition of nematode communities at the taxonomic, functional, and phylogenetic level in 16 assemblage pairs (32 sites in total with 16 in each habitat type) in mainland China. While the overall alpha and beta diversity did not differ between natural and agricultural systems, all three alpha diversity facets decreased with latitude in natural habitats. Both alpha and beta diversity levels were driven by climatic differences in natural habitats, while none of the diversity levels changed in agricultural systems. This indicates that land conversion affects soil biodiversity in a geographically dependent manner and that agriculture could erase climatic constraints on soil biodiversity at such a scale. Additionally, the functional composition of nematode communities was more dissimilar in agricultural than in natural habitats, while the phylogenetic composition was more similar, indicating that changes among different biodiversity facets are asynchronous. Our study deepens the understanding of land‐use effects on soil nematode diversity across large spatial scales. Moreover, the detected asynchrony of taxonomic, functional, and phylogenetic diversity highlights the necessity to monitor multiple facets of soil biodiversity in ecological studies such as those investigating environmental changes.     

Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences

Background

The orders Ascaridida, Oxyurida, and Spirurida represent major components of zooparasitic nematode diversity, including many species of veterinary and medical importance. Phylum-wide nematode phylogenetic hypotheses have mainly been based on nuclear rDNA sequences, but more recently complete mitochondrial (mtDNA) gene sequences have provided another source of molecular information to evaluate relationships. Although there is much agreement between nuclear rDNA and mtDNA phylogenies, relationships among certain major clades are different. In this study we report that mtDNA sequences do not support the monophyly of Ascaridida, Oxyurida and Spirurida (clade III) in contrast to results for nuclear rDNA. Results from mtDNA genomes show promise as an additional independently evolving genome for developing phylogenetic hypotheses for nematodes, although substantially increased taxon sampling is needed for enhanced comparative value with nuclear rDNA. Ultimately, topological incongruence (and congruence) between nuclear rDNA and mtDNA phylogenetic hypotheses will need to be tested relative to additional independent loci that provide appropriate levels of resolution.

Results

For this comparative phylogenetic study, we determined the complete mitochondrial genome sequences of three nematode species, Cucullanus robustus (13,972 bp) representing Ascaridida, Wellcomia siamensis (14,128 bp) representing Oxyurida, and Heliconema longissimum (13,610 bp) representing Spirurida. These new sequences were used along with 33 published nematode mitochondrial genomes to investigate phylogenetic relationships among chromadorean orders. Phylogenetic analyses of both nucleotide and amino acid sequence datasets support the hypothesis that Ascaridida is nested within Rhabditida. The position of Oxyurida within Chromadorea varies among analyses; in most analyses this order is sister to the Ascaridida plus Rhabditida clade, with representative Spirurida forming a distinct clade, however, in one case Oxyurida is sister to Spirurida. Ascaridida, Oxyurida, and Spirurida (the sampled clade III taxa) do not form a monophyletic group based on complete mitochondrial DNA sequences. Tree topology tests revealed that constraining clade III taxa to be monophyletic, given the mtDNA datasets analyzed, was a significantly worse result.

Conclusion

The phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3^rd positions) indicates that nematodes representing Ascaridida, Oxyurida and Spirurida do not share an exclusive most recent common ancestor, in contrast to published results based on nuclear ribosomal DNA. Overall, mtDNA genome data provides reliable support for nematode relationships that often corroborates findings based on nuclear rDNA. It is anticipated that additional taxonomic sampling will provide a wealth of information on mitochondrial genome evolution and sequence data for developing phylogenetic hypotheses for the phylum Nematoda.

     

Embryogenesis and the first-stage larva of Thelazia lacrymalis

The female reproductive system of Thelazia lacrymalis (Nematoda: Thelaziidae) was investigated by light and scanning electron microscopy (SEM) with regard to the developmental stages and the stage deposited by the gravid nematode. Female T. lacrymalis have a didelphic and opisthodelphic type of reproductive system with paired ovaries, oviducts and uteri and a single vagina and vulva. Round and spindle-shaped primary oocytes are documented within the ovaries and oviducts, respectively. The distal part of each uterus provides a fertilization chamber filled with spermatozoa, followed by a sphincter-like part. Further anterior, the uteri broaden gradually containing dividing zygotes, small and large morulae, tadpole-stage embryos and horseshoe-shaped embryos which increase in length and become slimmer forming pretzel-stage embryos and larvae rolled up. The larvae stretch gradually and finally lie straight but still covered with their egg membrane in the vagina. The egg membrane encloses the whole larva and is enlarged at the pointed tail of the larva forming a bulb. At the SEM level, the first-stage larva is shown to have a terminal mouth and three hooks directed posteriorly and a striated cuticle. As morphologically identical larvae were also found in lavages of the conjunctival sac of horses infected with T. lacrymalis, this nematode species can be described as ovoviviparous.     

Phylogeny of the Zoroasteridae (Zorocallina; Forcipulatida): evolutionary events in deep-sea Asteroidea displaying Palaeozoic features

The Zoroasteridae comprise a small but widespread family of asteroids distributed throughout the deep sea. Although poorly understood, they are often collected in the hundreds, suggesting that they occupy important ecological roles. A phylogenetic analysis including 24 terminal taxa and 70 morphological characters was performed, resulting in a single most-parsimonious tree. The tree separated zoroasterids with open, reticulate skeletons (e.g. Myxoderma ) as more basal than those with more heavily armored, imbricate skeletons (e.g. Zoroaster ), which were more derived. In addition to agreement with established genera, a new genus is supported by the phylogeny as the sister taxon to Myxoderma . The cladistic analysis was performed in conjunction with a revisionary survey of zoroasterid species, resulting in taxonomic changes to species in nearly every genus. Bathymetric and physiographic shifts were observed between the reticulate and imbricate zoroasterid clades. Zoroasterids possess a single marginal plate series, which occurs in basal sister-group neoasteroids (crown-group asteroids). Phylogenetic results suggest that the morphololgical resemblance between zoroasterids and Palaeozoic taxa, such as Calliasterella , is convergent but a paraphyletic Zoroasteride cannot be rejected and remains consistent with basal crown-group affinities. Although the phylogenetic position of the Eocene Zoroaster aff. fulgens was not strongly supported, its presence within a derived cluster of Zoroaster spp. suggests a relatively recent (i.e. Cenozoic) diversification into the deep sea. Taxonomic revisions, and geographical and bathymetric range extensions are also included.  © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society , 2007, 150 , 177–210.     

Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders

The trans-splicing of short spliced leader (SL) RNAs onto the 5' ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda.     

DIAGNOSTIC ANATOMICAL CHARACTERS IN THE PLEUROTHALLIDINAE (ORCHIDACEAE)

An anatomical survey of 200 species in 22 genera of subtribe Pleurothallidinae (Orchidaceae) indicates which vegetative characters are of diagnostic value and at what taxonomic level. Most systematically useful characters are associated with the leaf, and include features of trichomes, cuticle, epidermis, hypodermis, spiral thickenings, and number of vein series. The secondary stem is of limited usefulness at higher levels. Number of velamen layers and protoxylem poles in the root may be reliable taxonomic indicators with certain limitations and precautions. The established subtribal phylogenetic trends of reduction in number of pollinia and specialization of the perianth may be correlated with particular anatomical trends, which are either reductionary also or involve specializations directly related to the water relations of the epiphytic habit.     

The mitochondrial genome sequence of Enterobius vermicularis (Nematoda: Oxyurida)--an idiosyncratic gene order and phylogenetic information for chromadorean nematodes

The complete mitochondrial genome sequence was determined for the human pinworm Enterobius vermicularis (Oxyurida: Nematoda) and used to infer its phylogenetic relationship to other major groups of chromadorean nematodes. The E. vermicularis genome is a 14,010-bp circular DNA molecule that encodes 36 genes (12 proteins, 22 tRNAs, and 2 rRNAs). This mtDNA genome lacks atp8, as reported for almost all other nematode species investigated. Phylogenetic analyses (maximum parsimony, maximum likelihood, neighbor joining, and Bayesian inference) of nucleotide sequences for the 12 protein-coding genes of 25 nematode species placed E. vermicularis, a representative of the order Oxyurida, as sister to the main Ascaridida+Rhabditida group. Tree topology comparisons using statistical tests rejected an alternative hypothesis favoring a closer relationship among Ascaridida, Spirurida, and Oxyurida, which has been supported from most studies based on nuclear ribosomal DNA sequences. Unlike the relatively conserved gene arrangement found for most chromadorean taxa, E. vermicularis mtDNA gene order is very unique, not sharing similarity to any other nematode species reported to date. This lack of gene order similarity may represent idiosyncratic gene rearrangements unique to this specific lineage of the oxyurids. To more fully understand the extent of gene rearrangement and its evolutionary significance within the nematode phylogenetic framework, additional mitochondrial genomes representing a greater evolutionary diversity of species must be characterized.     

New approaches to a better understanding of nematode phylogeny: molecular and developmental studies*

Recent studies making use of ribosomal DNA sequences have led to a wealth of new information that can be used to construct phylogenetic trees and compare them with those derived from more traditional approaches. Here, some examples are presented where the molecular data have stimulated alternative views. To determine whether differences in developmental processes can be used as markers for intraphyletic relationships, comparative studies of nematode embryogenesis have been performed. These results indicate that considerable deviations from the standard pattern found in the model system Caenorhabditis elegans exist which appear to be characteristic for certain subgroups within the taxon Nematoda.     

Review: Taxonomy of Nitella (Charales, Charophyceae) based on comparative morphology of oospores and multiple DNA marker phylogeny using cultured material

The genus Nitella is the largest group in the Charales and has the highest diversity of vegetative and oospore morphologies. In his worldwide monograph on the Charales, R. D. Wood characterized the sections and species of Nitella mainly on the basis of vegetative morphology and treated oospore wall morphology as diagnostic at the infraspecific level. Therefore, many species of Nitella exhibiting distinct external morphology of the oospore wall (EMOW) were reduced to infraspecific rank and only 53 of 204 species previously described were recognized within Nitella . However, recent morphological and molecular phylogenetic studies have demonstrated the phylogenetic validity of using EMOW for diagnosing some species of Nitella . More recently, a scanning electron microscopy (SEM) study of internal morphology of the oospore wall (IMOW) and multiple DNA marker analyses using both nuclear and chloroplast gene sequences were conducted to improve our understanding of the taxonomy of Nitella at the species level, on the basis of cultured material of a large number of species. Multiple DNA marker analyses resolved detailed and robust phylogenetic relationships within the genus and demonstrated the taxonomic and phylogenetic significance of IMOW. In addition, they supported the taxonomic decisions based on differences in oospore morphology, suggesting that an integrated approach, involving both SEM studies of the EMOW and IMOW and multiple DNA marker analyses, is appropriate to address problems at lower taxonomic levels within the genus, as well as to construct a natural taxonomic system for the genus. In this paper, recent morphological and molecular phylogenetic studies are reviewed and recent improvements in taxonomy of Nitella are summarized. Moreover, the evolution of morphological features and phylogenetic relationships within Nitella are discussed, focusing especially on oospore morphology.     

Caenorhabditis elegans as a model for parasitic nematodes: a focus on the cuticle

The phylum Nematoda consists of over half a million species of worms that inhabit astoundingly diverse environments. Nematodes can live as obligatory parasites of plants and animals, or alternate a parasitic with a free-living life style. The fact that the vast majority of species are strictly free living often surprises parasitology students, for obviously the highest research priorities in this field have involved parasites of medical, veterinary and agricultural importance. Here Samuel Politz and Mario Philipp contend that some basic questions concerning the biology of the parasite cuticle can be investigated more easily and in greater depth in the free-living nematode Caenorhabditis elegans than in the parasites themselves.