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Nematodes from galls on Myrtaceae. VIII. Fergusobia from small galls on shoot buds, with descriptions of four new species.
Zootaxa
PUBLISHED: 08-26-2014
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Small shoot bud galls induced by the Fergusobia (Nematoda: Neotylenchidae)/Fergusonina (Diptera: Fergusoninidae) mutualism occur on various Eucalyptus spp. Four new species of Fergusobia, collected from small shoot bud galls on Eucalyptus camaldulensis, E. gomphocephala and E. leucoxylon, are described. Fergusobia gomphocephalae Davies n. sp. is morphologically characterized by a combination of a small C-shaped parthenogenetic female with a variable, conoid tail, a small C-shaped infective female with a hemispherical tail tip, and an arcuate or J-shaped male with a broad tail, angular spicule and short peloderan bursa. Fergusobia leucoxylonae Davies n. sp. has a C-shaped parthenogenetic female with a conoid tail with a narrowly rounded tip, an arcuate infective female with a broadly rounded tail tip, and an almost straight to barely J-shaped male with angular (not heavily sclerotised) spicule and short bursa. Fergusobia schmidti Davies & Bartholomaeus n. sp. has an arcuate to open C-shaped parthenogenetic female with a relatively large body diameter, relatively long stylet and small tail with a broadly rounded tail tip, an open C-shaped infective female with a broadly rounded to hemispherical tail tip, and an arcuate to barely J-shaped male with spicules angular at about 33% of their length and peloderan bursa arising at about half body length. Fergusobia sporangae Davies n. sp. has an arcuate to open C-shaped parthenogenetic female with a relatively long stylet and a broadly rounded tail tip, an arcuate infective female with a short tail with a broadly rounded to hemispherical tip, and an arcuate to barely J-shaped male with angular (not heavily sclerotised) spicule and short peloderan bursa. Various forms of small shoot bud galls are described. From phylogenetic analyses based on sequences of the D2/D3 expansion segment of the large subunit rRNA gene, the four new species belong to two sister clades of Fergusobia. The larval shield morphology of their associated fly species and possible genetic relationships are discussed. 
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Nematodes from galls on Myrtaceae. VII. Fergusobia from 'leafy' leaf bud galls in Australia, with re-description of Fergusobia tumifaciens (Currie 1937) Wachek 1955 and descriptions of Fergusobia planchonianae n. sp. and Fergusobia viminalisae n. sp.
Zootaxa
PUBLISHED: 08-25-2014
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Fergusobia tumifaciens (Currie 1937) Wachek 1955, the type species for the genus Fergusobia, is re-described from specimens collected from 'leafy' leaf bud galls on Eucalyptus bridgesiana near Albury in New South Wales, Australia. It is morphologically characterized by the combination of an open C-shaped parthenogenetic female with a small broadly conoid tail, a C-shaped infective female with a bluntly rounded tail tip, and an arcuate to J-shaped male with angular spicules, not heavily sclerotised, and short to mid-length peloderan bursa. Two new species of Fergusobia, collected from 'leafy' leaf bud galls on, respectively, Eucalyptus planchoniana in Queensland, and E. viminalis in South Australia, Australia, are described. Fergusobia planchonianae Davies n. sp. is characterised by the combination of a C-shaped parthenogenetic female with a conoid tail, an arcuate infective female with an hemispherical tail tip, and an almost straight to arcuate to C-shaped male with an angular spicule, a long peloderan bursa and a narrow tail. Fergusobia viminalisae Davies n. sp. is characterised by the combination of an open C-shaped parthenogenetic female with a broadly conoid tail, a C-shaped infective female with a bluntly rounded tail tip, and an arcuate to J-shaped male with an angular (not heavily sclerotised) spicule and short to mid-length peloderan bursa. The shield morphologies of the fly larvae associated with the 'leafy' leaf bud galls and their possible relationships are outlined. Possible evolutionary relationships of the Fergusobia nematodes from these galls are discussed, considering their morphology, DNA sequences, and the relationships of the associated Fergusonina flies and host plants. 
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Nematodes from galls on Myrtaceae. VI. Fergusobia from galls on Angophora in Australia, with description of F. colbrani n. sp. and key.
Zootaxa
PUBLISHED: 08-22-2014
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Collection data and biological information is presented on the Fergusobia (Nematoda: Neotylenchidae)/ Fergusonina (Diptera: Fergusoninidae) mutualism inducing galls on Angophora in Australia. Three species and two morphospecies have been recognised. Fergusobia colbrani Davies n. sp. is described from soft spheroid leaf galls on Angophora floribunda. It is characterised by a combination of morphological characters including a small C-shaped parthenogenetic female with a short broadly conoid tail, an arcuate infective female with an almost hemispherical tail tip, and an arcuate to barely J-shaped male with an angular spicule having a notched tip and mid-length leptoderan bursa. A key to the species and morphospecies of nematodes collected from Angophora is presented. Possible relationships of these organisms are discussed based on evidence from the nematode morphology, gall forms, and the morphology of the dorsal shield of the associated Fergusonina fly larvae. 
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Phylogenetic Status and Morphological Characters of Rhabditolaimus anoplophorae (Rhabditida: Diplogastridae).
J. Nematol.
PUBLISHED: 03-20-2014
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Rhabditolaimus anoplophorae Kanzaki and Futai was re-isolated from its type host (carrier), the cerambycid beetle Anoplophora malasiaca, collected in an experimental field of the Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan. The nematode was cultured on nematode growth medium plates seeded with Escherichia coli OP50, and its morphological characters and molecular profile were examined to modernize the description. Scanning electron microscopic and light microscopy revealed the presence of four stomatal flaps, a very long gymnostom, a single ventral papilla in males, and a horizontal slit-like vulval opening in females. The positions of the deirids, hemizonids, phasmids, and rectal glands are additionally described, and the absence of a male bursa was confirmed. Phylogenetically, the genus forms a well-supported clade in the family Diplogastridae. Rhabditolaimus anoplophorae is a member of the monophyletic Rhabditolaimus clade and is closely related to R. leuckarti and several undescribed species.
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Revision of the paraphyletic genus Koerneria Meyl, 1960 and resurrection of two other genera of Diplogastridae (Nematoda).
Zookeys
PUBLISHED: 01-01-2014
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Recent inferences of phylogeny from molecular characters, as well as a reexamination of morphological and biological characters, reject the monophyly of the nematode genus Koerneria Meyl, 1960 (Diplogastridae). Here, Koerneria sensu lato is revised. The genus, which previously consisted of 40 species, is separated into three genera. Almost all of the transferred species are moved to the resurrected genus Allodiplogaster Paramonov & Sobolev in Skrjabin et al. (1954). Koerneria and Allodiplogaster are distinguished from each other by a weakly vs. clearly striated body surface, an undivided vs. divided stomatal cheilostom, and arrangement of the terminal ventral triplet of male genital papillae, namely in that v5 and v6 are paired and separated from v7 vs. v5-v7 being close to each other. Allodiplogaster is further divided into two groups of species, herein called the henrichae and striata groups, based on both morphological and life-history traits. The henrichae group is characterized by papilliform labial sensilla and male genital papillae, a conical tail in both males and females, and an association with terrestrial habitats and insects, whereas the striata group is characterized by setiform labial sensilla and male genital papillae, an elongated conical tail in both sexes, and an association with aquatic habitats. A second genus, Anchidiplogaster Paramonov, 1952, is resurrected to include a single species that is characterized by its miniscule stoma and teeth, unreflexed testis, and a distinct lack of male genital papillae or stomatal apodemes. Lastly, one further species that was previously included in Koerneria sensu lato is transferred to the genus Pristionchus Kreis, 1932. The revision of Koerneria sensu lato is necessitated by the great variability in its subordinate taxa, which occupy a variety of habitats, in addition to the increased attention to Diplogastridae as a model system for comparative mechanistic biology.
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New plant-parasitic nematode from the mostly mycophagous genus Bursaphelenchus discovered inside figs in Japan.
PLoS ONE
PUBLISHED: 01-01-2014
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A new nematode species, Bursaphelenchus sycophilus n. sp. is described. The species was found in syconia of a fig species, Ficus variegata during a field survey of fig-associated nematodes in Japan. Because it has a well-developed stylet and pharyngeal glands, the species is considered an obligate plant parasite, and is easily distinguished from all other fungal-feeding species in the genus based upon these characters. Although B. sycophilus n. sp. shares an important typological character, male spicule possessing a strongly recurved condylus, with the "B. eremus group" and the "B. leoni group" of the genus, it was inferred to be monophyletic with the "B. fungivorus group". The uniquely shaped stylet and well-developed pharyngeal glands is reminiscent of the fig-floret parasitic but paraphyletic assemblage of "Schistonchus". Thus, these morphological characters appear to be an extreme example of convergent evolution in the nematode family, Aphelenchoididae, inside figs. Other characters shared by the new species and its close relatives, i.e., lack of ventral P1 male genital papilla, female vulval flap, and papilla-shaped P4 genital papillae in males, corroborate the molecular phylogenetic inference. The unique biological character of obligate plant parasitism and highly derived appearance of the ingestive organs of Bursaphelenchus sycophilus n. sp. expands our knowledge of the potential morphological, physiological and developmental plasticity of the genus Bursaphelenchus.
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Molecular Characterization and Development of Real-Time PCR Assay for Pine-Wood Nematode Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae).
PLoS ONE
PUBLISHED: 01-01-2013
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Bursaphelenchus xylophilus, the pine-wood nematode (PWN), is the causal agent of pine wilt disease, one of the most damaging emerging pest problems to forests around the world. It is native to North America where it causes relatively minor damage to native conifers but is labeled an EPPO-A-2 pest and a quarantine nematode for many countries outside of the United States because of its potential for destruction to their native conifers. Exports of wood logs and commodities involving softwood packaging materials now require a lab test for the presence/absence of this regulated nematode species. We characterized the DNA sequences on the ribosomal DNA small subunit, large subunit D2/D3, internal transcribed spacer (ITS) and mitochondrial DNA cytochrome oxidase subunit one on the aphelenchid species and described the development of a real-time-PCR method for rapid and accurate identification of PWN targeting the ITS-1. A total of 97 nematode populations were used to evaluate the specificity and sensitivity of this assay, including 45 populations of B. xylophilus and 36 populations of 21 other species of Bursaphelenchus which belong to the abietinus, cocophilus, eggersi, fungivorus, hofmanni, kevini, leoni, sexdentati, and xylophilus groups and one unassigned group from a total of 13 groups in the genus Bursaphelenchus; 15 populations of Aphelenchoides besseyi, A. fragariae, Aphelenchoides species and Aphelenchus avenae; and one population of mixed nematode species from a soil sample. This assay proved to be specific to B. xylophilus only and was sensitive to a single nematode specimen regardless of the life stages present. This approach provides rapid species identification necessary to comply with the zero-tolerance export regulations.
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Field Responses of Bermudagrass and Seashore paspalum Cultivars to Sting and Spiral Nematodes.
J. Nematol.
PUBLISHED: 08-16-2011
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Belonolaimus longicaudatus and Helicotylenchus spp. are damaging nematode species on bermudagrass (Cynodon spp.) and seashore paspalum (Paspalum vaginatum) in sandy soils of the southeastern United States. Eight bermudagrass and three seashore paspalum cultivars were tested for responses to both nematode species in field plots for two years in Florida. Soil samples were taken every three months and nematode population densities in soil were quantified. Turfgrass aboveground health was evaluated throughout the growing season. Results showed that all bermudagrass cultivars, except TifSport, were good hosts for B. longicaudatus, and all seashore paspalum cultivars were good hosts for H. pseudorobustus. Overall, bermudagrass was a better host for B. longicaudatus while seashore paspalum was a better host for H. pseudorobustus. TifSport bermudagrass and SeaDwarf seashore paspalum cultivars supported the lowest population densities of B. longicaudatus. Seashore paspalum had a higher percent green cover than bermudagrass in the nematode-infested field. Nematode intolerant cultivars were identified.
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Evaluation of Econem™, a formulated Pasteuria sp. Bionematicide, for management of Belonolaimus longicaudatus on golf course turf.
J. Nematol.
PUBLISHED: 03-16-2011
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In 2010, a turfgrass bionematicide containing in vitro produced Pasteuria sp. for management of Belonolaimus longicaudatus was launched under the tradename Econem(™). Greenhouse pot studies and field trials on golf course fairways and tee boxes evaluated Econem at varied rates and application frequencies. Trials on putting greens compared efficacy of three applications of Econem at 98 kg/ha to untreated controls and 1,3-dichloropropene at 53 kg a.i/ha. Further putting green trials evaluated the ability of three applications of Econem at 98 kg/ha to prevent resurgence of population densities of B. longicaudatus following treatment with 1,3-dichloropropene at 53 kg a.i./ha. None of the Econem treatments in pot studies were effective at reducing B. longicaudatus numbers (P ? 0.05). Econem was associated with reduction in population densities of B. longicaudatus (P ? 0.1) on only a single sampling date in one of the eight field trials and did not improve turf health in any of the trials (P > 0.1). These results did not indicate that Econem is an effective treatment for management of B. longicaudatus on golf course turf.
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Ecometagenetics confirm high tropical rainforest nematode diversity.
Mol. Ecol.
PUBLISHED: 11-04-2010
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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.
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Effects of formulation and host nematode density on the ability of in vitro-produced pasteuria endospores to control its host Belonolaimus longicaudatus.
J. Nematol.
PUBLISHED: 09-17-2010
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The effect of nematode population density at the time of application and formulations of in vitro-produced Pasteuria spp. endospores on the final population density of Belonolaimus longicaudatus was studied in an 84-d-long pot bioassay. The experiment utilized a factorial design consisting of 30 or 300 B. longicaudatus /100 cm(3) of sandy soil and three formulations of in vitro-produced Pasteuria spp. endospores (nontreated, granular, or liquid). No differences were observed in percent endospore attachment between nematode inoculum levels during either trial. Granular and liquid formulations of in vitro-produced endospores suppressed nematode population densities by 22% and 59% in the first trial and 20% and 63% in the second, respectively compared with the nontreated control. The liquid formulation increased percent endospore attachment by 147% and 158%, respectively, compared with the granular formulation. The greatest root retention by the host plant was observed at the lower B. longicaudatus inoculation level following application of the liquid formulation. While both the granular and liquid formulations reduced B. longicaudatus population densities in the soil, the liquid spore suspension was most effective.
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Reproducibility of read numbers in high-throughput sequencing analysis of nematode community composition and structure.
Mol Ecol Resour
PUBLISHED: 12-15-2009
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Although nematodes are the most abundant metazoan animals on Earth, their diversity is largely unknown. To overcome limitations of traditional approaches (labour, time, and cost) for assessing biodiversity of nematode species in environmental samples, we have previously examined the suitability of high-throughput sequencing for assessing species level diversity with a set of control experiments employing a mixture of nematodes of known number and with known sequences for target diagnostic loci. Those initial experiments clearly demonstrated the suitability of the approach for identification of nematode taxa but lacked the replicate experiments necessary to evaluate reproducibility of the approach. Here, we analyze reads generated from three different PCR amplifications and three different sequencing reactions to examine the differential PCR amplification, the possibility of emulsion PCR artefacts, and differences between sequencing reactions. Our results suggest that both qualitative and quantitative data are consistent and highly reproducible. Variation associated with in-house PCR amplification or emPCR and sequencing are present but the representation of each nematode is very consistent from experiment to experiment and supports the use of read counts to estimate relative abundance of taxa in a metagenetic sample.
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Teratodiplogaster fignewmani gen. nov., sp. nov. (Nematoda: Diplogastridae) from the syconia of Ficus racemose in Australia.
Zool. Sci.
PUBLISHED: 09-02-2009
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During the course of a survey of fig-associated nematodes in eastern Australia, we discovered an unusual diplogastrid nematode that we describe herein as Teratodiplogaster fignewmani gen. nov., sp. nov. This nematode was isolated as adults and juveniles from the syconia of Ficus racemose in Queensland and Western Australia. It is presumed to be associated with the agaonid fig wasp, Ceratosolen fusciceps, for dispersion to new phase-B sycones. Teratodiplogaster fignewmani gen. nov., sp. nov. was inferred to be the sister taxon to the genus Parasitodiplogaster based upon molecular phylogeny using nearly full-length sequences of the SSU and D2/D3 LSU ribosomal RNA genes. It is sufficiently distinct in morphology (with many autapomorphies) to justify a new monotypic genus with the hypothesis that further species of Teratodiplogaster gen. nov. will be discovered when sycones from other Ficus from tropical Australasia and possibly Africa are sampled.
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The first report of a putative "entomoparasitic adult form" in Bursaphelenchus.
J. Parasitol.
PUBLISHED: 04-18-2009
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A putative "entomoparasitic adult form" of Bursaphelenchus luxuriosae was found in the tracheal system and body cavity of its cerambycid vector beetle, Acalolepta luxuriosa. Morphologically, this form is intermediate between the usual mycophagous adult and the phoretic dauer juvenile, i.e., it shares the primary and secondary reproductive features with the mycophagous phase but shares a degenerate digestive tract with JIV dauer juveniles. In addition, the "parasitic form" has specialized characters relative to the mycophagous phase analogous to dicyclic Hexatylina, e.g., vacuole-like dots (assumed to be sensory organs) at the anterior end, a very long vulval flap, conical female tail, and elongated male spicules. The presence of insect-parasitic juveniles has been reported in several Bursaphelenchus species, but this is the first confirmed report of a putative "entomoparasitic adult form" in the genus. Thus, the "parasitic adult form" of B. luxuriosae is hypothesized to be an autapomorphic character of this species in the genus. The physiological impact of the parasitism on the host beetle is assumed to be weak because no clear symptoms were observed in the infested beetles. Also, no nematode eggs or propagative juveniles were observed in the beetle host, suggesting that more research is needed to confirm the nature of the association (parasitic vs. endophoretic).
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Evaluating high-throughput sequencing as a method for metagenomic analysis of nematode diversity.
Mol Ecol Resour
PUBLISHED: 03-05-2009
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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.
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The nature and frequency of chimeras in eukaryotic metagenetic samples.
J. Nematol.
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Pyrosequencing of an artificially assembled nematode community of known nematode species at known densities allowed us to characterize the potential extent of chimera problems in multi-template eukaryotic samples. Chimeras were confirmed to be very common, making up to 17% of all high quality pyrosequencing reads and exceeding 40% of all OCTUs (operationally clustered taxonomic units). Typically, chimeric OCTUs were made up of single or double reads, but very well covered OCTUs were also present. As expected, the majority of chimeras were formed between two DNA molecules of nematode origin, but a small proportion involved a nematode and a fragment of another eukaryote origin. In addition, examples of a combination of three or even four different template origins were observed. All chimeras were associated with the presence of conserved regions with 80% of all recombinants following a conserved region of about 25bp. While there was a positive influence of species abundance on the overall number of chimeras, the influence of specific-species identity was less apparent. We also suggest that the problem is not nematode exclusive, but instead applies to other eukaryotes typically accompanying nematodes (e.g. fungi, rotifers, tardigrades). An analysis of real environmental samples revealed the presence of chimeras for all eukaryotic taxa in patterns similar to that observed in artificial nematode communities. This information warrants caution for biodiversity studies utilizing a step of PCR amplification of complex DNA samples. When unrecognized, generated abundant chimeric sequences falsely overestimate eukaryotic biodiversity.
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Nematode spatial and ecological patterns from tropical and temperate rainforests.
PLoS ONE
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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.
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Reverse taxonomy for elucidating diversity of insect-associated nematodes: a case study with termites.
PLoS ONE
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The molecular operational taxonomic unit (MOTU) has recently been applied to microbial and microscopic animal biodiversity surveys. However, in many cases, some of the MOTUs cannot be definitively tied to any of the taxonomic groups in current databases. To surmount these limitations, the concept of "reverse taxonomy" has been proposed, i.e. to primarily list the MOTUs with morphological information, and then identify and/or describe them at genus/species level using subsamples or by re-isolating the target organisms. Nevertheless, the application of "reverse taxonomy" has not been sufficiently evaluated. Therefore, the practical applicability of "reverse taxonomy" is tested using termite-associated nematodes as a model system for phoretic/parasitic organisms which have high habitat specificity and a potential handle (their termite host species) for re-isolation attempts.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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