Occurrence of Belonolaimus in Sinaloa, Northwestern Mexico: A New Report on Distribution and Host Range.

The present study reports the occurrence of the genus Belonolaimus in the state of Sinaloa, Mexico, associated with native plants (i.e., Ziziphus amole and Stenocereus alamosensis) in a natural coastal ecosystem. Both morphological and molecular approaches were employed to characterize the Sinaloa population. Notwithstanding of some morphological and morphometric variation between Belonolaimus from Sinaloa and other valid species, the characterization indicates that this population might belong to the Belonolaimus longicaudatus species complex. Molecular analyses based on the 28S gene and ITS1-5.8S-ITS2 regions of the ribosomal RNA (rRNA) identified four major clades within Belonolaimus; however, none of the species including B. longicaudatus, B. gracilis, and B. euthychilus were supported as monophyletic; yet monophyly is argued to be a basic requirement of species status. Sequence divergence among different Belonolaimus populations and species varied according to the rRNA dataset (i.e., ITS1-5.8S-ITS2 > 28S > 18S) used, thus showing the importance of using genes with different rates of evolution to estimate species relationships. The fact that Belonolaimus has not been found in other cultivated (including on suitable hosts) areas in Sinaloa and that this population is relatively distant from the common B. longicaudatus groups (i.e., clades A and B) suggests that its appearance was not due to a recent introduction associated with the local agriculture.

Microsporidia-nematode associations in methane seeps reveal basal fungal parasitism in the deep sea.

The deep sea is Earth's largest habitat but little is known about the nature of deep-sea parasitism. In contrast to a few characterized cases of bacterial and protistan parasites, the existence and biological significance of deep-sea parasitic fungi is yet to be understood. Here we report the discovery of a fungus-related parasitic microsporidium, Nematocenator marisprofundi n. gen. n. sp. that infects benthic nematodes at methane seeps on the Pacific Ocean floor. This infection is species-specific and has been temporally and spatially stable over 2 years of sampling, indicating an ecologically consistent host-parasite interaction. A high distribution of spores in the reproductive tracts of infected males and females and their absence from host nematodes' intestines suggests a sexual transmission strategy in contrast to the fecal-oral transmission of most microsporidia. N. marisprofundi targets the host's body wall muscles causing cell lysis, and in severe infection even muscle filament degradation. Phylogenetic analyses placed N. marisprofundi in a novel and basal clade not closely related to any described microsporidia clade, suggesting either that microsporidia-nematode parasitism occurred early in microsporidia evolution or that host specialization occurred late in an ancient deep-sea microsporidian lineage. Our findings reveal that methane seeps support complex ecosystems involving interkingdom interactions between bacteria, nematodes, and parasitic fungi and that microsporidia parasitism exists also in the deep-sea biosphere.

Perceptions of fishers to sea turtle bycatch, illegal capture and consumption in the San Ignacio-Navachiste-Macapule lagoon complex, Gulf of California, Mexico.

In this study, 10% of all registered fishermen in the coastal towns of Navachiste in Sinaloa, in northwestern Mexico, answered a survey designed to collect data on their perceptions of the following topics: the impact of turtle meat consumption; human health; bycatch; illegal turtle fishing; the illegal sea turtle market; the local economy; pollution; environmental education; the success of protective legislation; and sea turtle-based ecotourism. Perceptions were analyzed using the fuzzy logic method through classification into 5 fuzzy membership sets: VL, very low; L, low; M, moderate; H, high; VH, very high. The 9 topics generated decision areas upon applying fuzzy inference that revealed the membership level of the answers in each fuzzy set. The economic potential of sea turtle-based ecotourism and the economic profitability of the illegal turtle meat market were perceived as VL. Conservation legislation was perceived as H, although inefficiently applied due to corruption. Ecotourism and impacts on sea turtles were perceived as VL, because they were deemed unprofitable activities at the individual and community levels. Environmental education was perceived as L, because it centers on nesting, hatching and releasing turtles and is directed at elementary and middle-school students. While fishers perceive a serious negative impact of fishing activities on sea turtles in the San Ignacio-Navachiste-Macapule area, they do not see themselves individually as part of the problem. Achieving sea turtle conservation in this region requires: suitable ecotourism infrastructure, government investments in promotion, and studies to estimate the minimum number of tourists needed to assure profitability.

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.

Diversity of free-living marine nematodes (Enoplida) from Baja California assessed by integrative taxonomy.

We used morphological and molecular approaches to evaluate the diversity of free-living marine nematodes (order Enoplida) at four coastal sites in the Gulf of California and three on the Pacific coast of Baja California, Mexico. We identified 22 morphological species belonging to six families, of which Thoracostomopsidae and Oncholaimidae were the most diverse. The genus Mesacanthion (Thoracostomopsidae) was the most widespread and diverse. Five allopatric species, genetically and morphologically differentiated, were found in two localities in the Gulf of California (M. sp1 and M. sp2) and three in the Pacific coast (M. sp3, M. sp4 and M. sp5). Overall, we produced 19 and 20 sequences for the 18S and 28S genes, respectively. Neither gene displayed intraspecific polymorphisms, which allowed us to establish that some morphological variation was likely either ontogenetic or due to phenotypic plasticity. Although 18S and 28S phylogenies were topologically congruent (incongruence length difference test, P > 0.05), divergences between species were much higher in the 28S gene. Moreover, this gene possessed a stronger phylogenetic signal to resolve relationships involving Rhabdodemania and Bathylaimus. On the other hand, the close relationship of Pareurystomina (Enchilidiidae) with oncholaimids warrants further study. The 28S sequences (D2D3 domain) may be better suited for DNA barcoding of marine nematodes than those from the 18S rDNA, particularly for differentiating closely related or cryptic species. Finally, our results underline the relevance of adopting an integrative approach encompassing morphological and molecular analyses to improve the assessment of marine nematode diversity and advance their taxonomy.

The role of habitat heterogeneity in structuring the community of intertidal free-living marine nematodes.

The role of habitat complexity has been widely neglected in the study of meiofaunal community patterns. We studied the intertidal nematode community of a structurally complex macrotidal beach exhibiting contrasting microhabitats (sandbars and runnels) to understand the influence of environmental gradients and habitat heterogeneity in the community structure. We tested whether topographical complexity affected (1) the zonation pattern in terms of abundance and diversity, and (2) local diversity by promoting compartmentalization into distinct faunal groups. Our analyses revealed three major faunal assemblages along the exposure gradient associated to differences in mean grain size and chlorophyll a. Diversity patterns involved a mid-intertidal peak, consistent with the intermediate disturbance hypothesis, and another peak at the limit with the subtidal region, consistent with the transition zone. These results highlight the predominance of environmental gradients in establishing intertidal zonation. However, microhabitats differed in environmental conditions and possessed significantly distinct nematofaunal communities. Runnels featured higher levels of taxonomic and functional diversity, many unique genera, and the community differed from the assemblage at the limit to the subtidal, stressing their role as distinct microhabitats. The nematofauna of the structurally complex beach was more diverse than the one from a homogeneous beach nearby, supporting the hypothesis that structural heterogeneity promotes diversity by compartmentalization and highlighting the importance of microhabitats in the assessment of biodiversity. Contrary to previous predictions, our results indicate potentially high regional marine nematode diversity in the Upper Gulf of California.

A phylogenetic framework for root lesion nematodes of the genus Pratylenchus (Nematoda): Evidence from 18S and D2-D3 expansion segments of 28S ribosomal RNA genes and morphological characters.

The root lesion nematodes of the genus Pratylenchus Filipjev, 1936 are migratory endoparasites of plant roots, considered among the most widespread and important nematode parasites in a variety of crops. We obtained gene sequences from the D2 and D3 expansion segments of 28S rRNA partial and 18S rRNA from 31 populations belonging to 11 valid and two unidentified species of root lesion nematodes and five outgroup taxa. These datasets were analyzed using maximum parsimony and Bayesian inference. The alignments were generated using the secondary structure models for these molecules and analyzed with Bayesian inference under the standard models and the complex model, considering helices under the doublet model and loops and bulges under the general time reversible model. The phylogenetic informativeness of morphological characters is tested by reconstruction of their histories on rRNA based trees using parallel parsimony and Bayesian approaches. Phylogenetic and sequence analyses of the 28S D2-D3 dataset with 145 accessions for 28 species and 18S dataset with 68 accessions for 15 species confirmed among large numbers of geographical diverse isolates that most classical morphospecies are monophyletic. Phylogenetic analyses revealed at least six distinct major clades of examined Pratylenchus species and these clades are generally congruent with those defined by characters derived from lip patterns, numbers of lip annules, and spermatheca shape. Morphological results suggest the need for sophisticated character discovery and analysis for morphology based phylogenetics in nematodes.

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.

Morphology and Description of Bursaphelenchus platzeri n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Nitidulid Beetles.

Bursaphelenchus platzeri n. sp., an associate of nitidulid beetles in southern California, is described and illustrated. Adult males and females of B. platzeri n. sp. were examined by scanning electron microscopy for ultrastructural comparisons with other members of the genus. Bursaphelenchus cocophilus (red ring nematode) appears to be the closest related taxon to B. platzeri n. sp. based upon shared morphological features of the fused spicules, female tail shape, phoresy with non-scolytid beetles, and molecular analysis of the near full-length small subunit (SSU) rDNA. Unfortunately, sequence data from the D2D3 expansion segments of the large subunit (LSU) rDNA and partial mitochondrial DNA COI did not help resolve the relationship of nearest relative. In addition to significant molecular sequence differences in SSU, LSU, and COI, B. platzeri n. sp., which is an obligate fungal feeder, can be differentiated from B. cocophilus because it is an obligate parasite of palms. Bursaphelenchus platzeri n. sp. can be differentiated from all other species of Bursaphelenchus by the length and shape of the female tail and spicule morphology. The spicules are fused along the ventral midline and possess unfused cucullae; the fused unit appears to function as a conduit for sperm. Population growth of B. platzeri n. sp. was measured in a time-course experiment at 25 degrees C in the laboratory on cultures of the fungus Monilinia fructicola grown on 5% glycerol-supplemented potato dextrose agar (GPDA). Nematode population densities rapidly increased from 25 to approximately 200,000/culture within 14 d and then plateaued for up to 28 d.

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.

Molecular phylogenetics and diagnosis of soil and clinical isolates of Halicephalobus gingivalis (Nematoda: Cephalobina: Panagrolaimoidea), an opportunistic pathogen of horses.

Phylogenetic relationships among six isolates of Halicephalobus gingivalis (Stefanski, 1954), a species with pathogenic potential in horses and humans, were evaluated using DNA sequences from the nuclear large-subunit ribosomal RNA (LSU rDNA) gene. Sequences from nematodes obtained from in vitro cultures (soil or clinical sources), or isolated from infected horse tissues, were compared. Gene sequences from a fatal equine clinical case from southern California and a free-living isolate recovered from southern California soil showed no fixed differences. Sequences from isolates representing two fatal equine cases from North America, one from Ontario, Canada and another from Tennessee also showed no fixed differences. In contrast, two equine cases from Tennessee had 18 fixed differences for this LSU region, the greatest observed among isolates from horses. Phylogenetic analysis of six Halicephalobus sequences and four outgroup taxa by maximum parsimony yielded one tree with five well-supported clades. This phylogeny did not group isolates of Halicephalobus strictly by region of geographic isolation or source of sample, and depicted one clinical and one soil isolate as sister taxa. These results confirm that free-living environmental isolates are potential sources of infection for horses. The phylogeny also reveals that diverse isolates can cause infections in horses within a relatively limited geographic region, and conversely that genetically similar sister taxa can be recovered from geographically distant localities. PCR primers that selectively amplify Halicephalobus DNA were designed and tested based on comparison of closely related nematodes as inferred from phylogenetic analysis.