Improved phylogenomic sampling of free-living nematodes enhances resolution of higher-level nematode phylogeny.

BACKGROUND: Nematodes are among the most diverse and abundant metazoans on Earth, but research on them has been biased toward parasitic taxa and model organisms. Free-living nematodes, particularly from the clades Enoplia and Dorylaimia, have been underrepresented in genome-scale phylogenetic analyses to date, leading to poor resolution of deep relationships within the phylum. RESULTS: We supplemented publicly available data by sequencing transcriptomes of nine free-living nematodes and two important outgroups and conducted a phylum-wide phylogenomic analysis including a total of 108 nematodes. Analysis of a dataset generated using a conservative orthology inference strategy resulted in a matrix with a high proportion of missing data and moderate to weak support for branching within and placement of Enoplia. A less conservative orthology inference approach recovered more genes and resulted in higher support for the deepest splits within Nematoda, recovering Enoplia as the sister taxon to the rest of Nematoda. Relationships within major clades were similar to those found in previously published studies based on 18S rDNA. CONCLUSIONS: Expanded transcriptome sequencing of free-living nematodes has contributed to better resolution among deep nematode lineages, though the dataset is still strongly biased toward parasites. Inclusion of more free-living nematodes in future phylogenomic analyses will allow a clearer understanding of many interesting aspects of nematode evolution, such as morphological and molecular adaptations to parasitism and whether nematodes originated in a marine or terrestrial environment.

Assessing the Role of Environmental Conditions on Efficacy Rates of Heterorhabditis indica (Nematoda: Heterorhabditidae) for Controlling Aethina tumida (Coleoptera: Nitidulidae) in Honey Bee (Hymenoptera: Apidae) Colonies: a Citizen Science Approach.

Certain species of entomopathogenic nematodes, such as Heterorhabditis indica Poinar, Karunakar & David, have the potential to be effective controls for Aethina tumida (Murray), or small hive beetles, when applied to the soil surrounding honey bee (Apis mellifera L.) hives. Despite the efficacy of H. indica, beekeepers have struggled to use them successfully as a biocontrol. It is believed that the sensitivity of H. indica to certain environmental conditions is the primary reason for this lack of success. Although research has been conducted to explore the impact of specific environmental conditions--such as soil moisture or soil temperature-on entomopathogenic nematode infectivity, no study to date has taken a comprehensive approach that considers the impact of multiple environmental conditions simultaneously. In exploring this, a multivariate logistic regression model was used to determine what environmental conditions resulted in reductions of A. tumida populations in honey bee colonies. To obtain the sample sizes necessary to run a multivariate logistic regression, this study utilized citizen scientist beekeepers and their hives from across the mid-Atlantic region of the United States. Results suggest that soil moisture, soil temperatures, sunlight exposure, and groundcover contribute to the efficacy of H. indica in reducing A. tumida populations in A. mellifera colonies. The results of this study offer direction for future research on the environmental preferences of H. indica and can be used to educate beekeepers about methods for better utilizing H. indica as a biological control.

Tegumental ultrastructure of adult Quinqueserialis quinqueserialis (Trematoda: Notocotylidae): an intestinal parasite of muskrat (Ondatra zibethicus).

Ten adult Quinqueserialis quinqueserialis specimens were removed from the intestine of a naturally infected muskrat, and scanning electron microscopy was used to study the morphological characteristics of the trematodes. The mature trematode, which was easy to recognize by the monostome holdfast organ, with no anterior cone, measured 2200-2500 µm in length by 900-1050 µm in width. The body was elongated and tapering at the anterior end, but the posterior end was rounded, and in some specimens was slightly truncated. The mouth opening lay at the anterior end and was surrounded by the oral sucker, which was round, small to medium in size, and subterminal. The tegument of the rim and inside of the oral sucker was smooth and had two types of papillae, domed and rosette papillae. Around the oral sucker, tegument was covered with sharp, pointed spines. The common genital pore was located on the median line of the body, posterior to the oral sucker. The cirrus had smooth tegument at the base and was armed with numerous conical spines throughout its length. The ventral surface was concave and provided with five distinct longitudinal rows of ventral papillae, which extended from the anterior to the posterior end of the body. Each row consisted of 15 to 20 papillae, making 81 to 88 papillae in all. These papillae were variable in size. In most specimens, the papillae were simple knob-like structures, but in some cases, they appeared to be bi- or trifurcate. The tegument at the base of each ventral papilla showed minute spiny pattern, but it was smooth or folded on top and had small rosette and ciliated papillae. Tegument at the edges of the worm was smooth in the mid-parts, spiny on lateral parts, and included rosette papillae. The dorsal surface of the worm was smooth and slightly convex, and the tegument was provided with two large domed papillae in one third of the anterior end of the dorsal part, few thick spines in the mid-part, and excretory pore at the level just posterior to the end. No spines or papillae were seen around the excretory pore.

Evolution of Feeding Structures in the Marine Nematode Order Enoplida.

Marine nematodes of the order Enoplida may represent the earliest lineage of nematodes and have a variety of fixed and movable feeding structures in their stomas. This study used an 18S ribosomal RNA phylogeny of the orders Enoplida and Triplonchida (subclass Enoplia) to explore the evolution of these feeding structures in light of previous hypotheses based solely on morphology. The Enoplida and Triplonchida were found to be paraphyletic, as several taxa currently classified as Triplonchida, such as Rhabdodemania, were found to be part of the Enoplida clade. The position of Rhabdodemania within Enoplida was unclear, but a close relation to Enoplidae and Thoracostomopsidae was not supported, making it unlikely that its movable odontia are homologous with the mandibles of these families. A member of Anticomidae was well-supported as the base of the clade containing Phanodermatidae, Enoplidae, and Thoracostomopsidae, suggesting that taxa with buccal rods and mandibles evolved from nematodes with unarmed stomas. The Phanodermatidae were shown to be more closely related to the Enoplidae and Thoracostomopsidae than were the Leptosomatidae, suggesting that the buccal rods of the phanoderms (rather than the mandibular ridge/odontia complex of the Leptosomatidae), may be the origin of the mandibles.

Untangling the Ecology, Taxonomy, and Evolution of Chaetogaster limnaei (Oligochaeta: Naididae) Species Complex.

Within Oligochaeta, Chaetogaster limnaei is unusual in exhibiting a parasitic relationship with freshwater pulmonate snails. Taxonomic confusion has been caused by differences in what have been considered 2 subspecies of this worm: Chaetogaster limnaei limnaei is an ectosymbiont and is present inside the mantle cavity of the snail, whereas Chaetogaster limnaei vaghini is parasitic and lives in the kidney of the snail. This study explored the distribution of these annelids in central New York and used mitochondrial DNA sequence data from the COI locus to examine the relationship, evolution, and species status of the ectosymbiotic and parasitic forms of C. limnaei. Snails ( Physa gyrina ) were collected from 6 streams and lakes in central New York, with additional specimens collected from a lake in Massachusetts for comparison. One hundred and forty snails were examined, and at least 1 form of Chaetogaster was present in 88 specimens, a prevalence of 62.9%. COI sequence data from New York and Massachusetts did not reveal separate ectosymbiotic and parasitic lineages. Instead, all parasitic forms were part of a mixed clade that included both ectosymbiotic and parasitic forms. This mixed clade was nested within clades of ectosymbiotic forms only, suggesting that a plastic lineage of C. limnaei, able to be both ectosymbionts and parasites, evolved from ectoparasitic ancestors.

Using mitogenomic and nuclear ribosomal sequence data to investigate the phylogeny of the Xiphinema americanum species complex.

Nematodes within the Xiphinema americanum species complex are economically important because they vector nepoviruses which cause considerable damage to a variety of agricultural crops. The taxonomy of X. americanum species complex is controversial, with the number of putative species being the subject of debate. Accurate phylogenetic knowledge of this group is highly desirable as it may ultimately reveal genetic differences between species. For this study, nematodes belonging to the X. americanum species complex, including potentially mixed species populations, were collected from 12 geographically disparate locations across the U.S. from different crops and in varying association with nepoviruses. At least four individuals from each population were analyzed. A portion of the 18S nuclear ribosomal DNA (rDNA) gene was sequenced for all individuals while the internal transcribed spacer region 1 (ITS1) of rDNA was cloned and 2 to 6 clones per individual were sequenced. Mitochondrial genomes for numerous individuals were sequenced in parallel using high-throughput DNA sequencing (HTS) technology. Phylogenetic analysis of the 18S rDNA revealed virtually identical sequences across all populations. Analysis of ITS1 rDNA sequences revealed several well-supported clades, with some degree of congruence with geographic location and viral transmission, but also numerous presumably paralogous sequences that failed to form clades with other sequences from the same population. Analysis of mitochondrial DNA (mtDNA) indicated the presence of three distinct monophyletic clades of X. americanum species complex nematodes. Two clades contained nematodes found in association with nepovirus and the third contained divergent mtDNA sequences from three nematode populations from the western U.S. where nepovirus was absent. The inherent heterogeneity in ITS1 rDNA sequence data and lack of informative sites in 18S rDNA analysis suggests that mtDNA may be more useful in sorting out the taxonomic confusion of the X. americanum species complex.

Nematode small subunit phylogeny correlates with alignment parameters.

The number of nuclear small subunit (SSU) ribosomal RNA (rRNA) sequences for Nematoda has increased dramatically in recent years, and although their use in constructing phylogenies has also increased, relatively little attention has been given to their alignment. Here we examined the sensitivity of the nematode SSU data set to different alignment parameters and to the removal of alignment ambiguous regions. Ten alignments were created with CLUSTAL W using different sets of alignment parameters (10 full alignments), and each alignment was examined by eye and alignment ambiguous regions were removed (creating 10 reduced alignments). These alignment ambiguous regions were analyzed as a third type of data set, culled alignments. Maximum parsimony, neighbor-joining, and parsimony bootstrap analyses were performed. The resulting phylogenies were compared to each other by the symmetric difference distance tree comparison metric (SymD). The correlation of the phylogenies with the alignment parameters was tested by comparing matrices from SymD with corresponding matrices of Manhattan distances representing the alignment parameters. Differences among individual parsimony trees from the full alignments were frequently correlated with the differences among alignment parameters (580/1000 tests), as were trees from the culled alignments (403/1000 tests). Differences among individual parsimony trees from the reduced alignments were less frequently correlated with the differences among alignment parameters (230/1000 tests). Differences among majority-rule consensus trees (50%) from the parsimony analysis of the full alignments were significantly correlated with the differences among alignment parameters, whereas consensus trees from the reduced and culled analyses were not correlated with the alignment parameters. These patterns of correlation confirm that choice of alignment parameters has the potential to bias the resultant phylogenies for the nematode SSU data set, and suggest that the removal of alignment ambiguous regions reduces this effect. Finally, we discuss the implications of conservative phylogenetic hypotheses for Nematoda produced by exploring alignment space and removing alignment ambiguous regions for SSU rDNA.

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.

Molecular population genetics of Cucumber mosaic virus in California: evidence for founder effects and reassortment.

The structure and genetic diversity of a California Cucumber mosaic virus (CMV) population was assessed by single-strand conformation polymorphism and nucleotide sequence analyses of genomic regions 2b, CP, MP, and the 3' nontranslated region of RNA3. The California CMV population exhibited low genetic diversity and was composed of one to three predominant haplotypes and a large number of minor haplotypes for specific genomic regions. Extremely low diversity and close evolutionary relationships among isolates in a subpopulation suggested that founder effects might play a role in shaping the genetic structure. Phylogenetic analysis indicated a naturally occurring reassortant between subgroup IA and IB isolates and potential reassortants between subgroup IA isolates, suggesting that genetic exchange by reassortment contributed to the evolution of the California CMV population. Analysis of various population genetics parameters and distribution of synonymous and nonsynonymous mutations revealed that different coding regions and even different parts of coding regions were under different evolutionary constraints, including a short region of the 2b gene for which evidence suggests possible positive selection.