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1.  Descriptions of Two New Species of Tylenchorhynchus Cobb, 1913 (Nematoda: Tylenchida), with Details on Morphology and Variation of T. claytoni 
Journal of Nematology  1987;19(1):58-68.
Two new species of plant parasitic nematodes (Tylenchorhynchus quaidi n. sp. and T. tritici n. sp.) from Pakistan are described and illustrated. Tylenchorhynchus quaidi n. sp., from soil around roots of potato (Solanum tuberosum) from an experimental field of NNRC, Karachi, Pakistan, is distinguishable from other species by its peculiar sunken dome-shaped head. Although similar to T. goffarti, it differs by head shape, areolation of lateral field, ratios a (23-28 vs. 29-37) and c (11-14 vs. 13-20), and a vagina that is half sclerotized and half unsclerotized. Tylenchorhynchus tritici n. sp., from soil around roots of wheat (Triticum aestivum) from Campbellpur, Pakistan, is similar to T. ventrosignatus and T. nordiensis. It differs from T. ventrosignatus by a continuous lip region, number of head annules (2-3 vs. 4), coarse body annulation, absence of a wave-like structure near the vulva, and by tail shape and number of tail annules (15-23 vs. 28-32). It differs from T. nordiensis by stylet length (12.4-14.6 vs. 11-13 μm), shape of stylet knobs, number of head annules (2-3 vs. 4), non-areolated lateral field in region of phasmids, and not fusing in posterior third of tail. Morphometrics of Tylenchorhynchus claytoni from soil around stunted maize (Zea mays L.), in Muscatine County, Iowa, and several other populations are given. Detailed morphometric data on T. claytoni based on topotypes collected from type locality and several other populations revealed that this species shows variations in the shape of tail in females, number of tail annules (and sometimes annules extending further back on the terminus, almost being an annulated terminus), position of phasmid, and shape of lip region. The subgenus Bitylenchus is proposed as a new synonym of Tylenchorhynchus and its species referred to the latter genus.
PMCID: PMC2618616  PMID: 19290107
taxonomy; morphology; Tylenchorhynchus; new species; potato; Solanum tuberosum; wheat; Triticum aestivum; maize; Zea mays L.
2.  Description of Trilineellus clathrocutis n.g., n.sp. (Tylenchorhynchinae: Tylenchida Thorne, 1949) with a Key to Species and Observations on Tylenchorhynchus sensu stricto 
Journal of Nematology  1981;13(2):135-141.
TrilineeIlus clathrocutis n.g., n.sp. is described and illustrated. It was found as an associate of corn (Zea mays) in Stockton, Georgia, USA, and is related to a group of Tylenchorhynchus sensu lato species having three lines in nonareolated lateral fields. This new species is closely related to Tylenehorhynehus divittatus Siddiqi 1961, T. sculptus Seinhorst 1963, and T. triglyphus Seinhorst 1963 (syn. T. chonai Sethi & Swarup 1968) Tarjan 1973. It differs from these species primarily by having longitudinal striae on the body. These four species are differentiated from Tylenchorhynchus sensu stricto by having three lateral lines instead of four. They differ from Uliginotylenchus Siddiqi 1971 by having nonareolated lateral fields, fewer than 25 annules on conoid rounded tails, differently shaped gubernacula, nonattenuated stylets, and other distinctive characters. They differ from Triversus Sher 1973 by having the male tail enclosed by the bursa and by having rounded female tails. SEM observations of T. clathrocutis reveal a cuticle deeply cut by longitudinal and horizontal striae and bearing wide (> 2.0 μm) annules. Trilineellus is proposed to accommodate the new species and the three-incisured species still within Tylenchorhynchus. Tylenchorhynchus is thereby the repository for species within Tylenchorhynchinae having four lines in the lateral field, no conspicuous labial disc, and bursa enclosing the male tail.
PMCID: PMC2618075  PMID: 19300734
taxonomy; morphology; Tylenchorhynchus; Trilineellus; Uliginotylenchus; Quinisulcius; Triversus; stunt nematode; new species; new genus; ryegrass; corn; SEM ultrastructure; Lolium perenne; Zea mays
3.  Plant Parasitic Nematodes Associated with Leatherleaf Fern 
Journal of Nematology  1986;18(1):26-30.
Seven species of plant parasitic nematodes were found to be associated with leatherleaf fern (Rumohra adiantiformis) in central Florida. Of these, Pratylenchus penetrans, Tylenchorhynchus claytoni, and Criconemoides curvatum were commonly encountered. Nematode communities generally included two or three species of plant parasitic nematodes, with greatest diversity in nematode species occurring in ferneries shaded by oak trees. Species diversity was not correlated with fernery age. Leatherleaf fern was tolerant of P. penetrans and T. claytoni in microplot tests.
PMCID: PMC2618512  PMID: 19294134
species diversity; tolerance; survey; pathogenicity
4.  Nematodes Parasitic on Forest Trees: III. Reproduction on Selected Hardwoods 
Journal of Nematology  1971;3(2):170-173.
The host-parasite relationships of 13 species of plant parasitic nematodes and five species of hardwoods native to the southeastern United States were tested on greenhouse-grown tree seedlings for 6-10 months. Criteria for parasitism were completion o f life cycle and population increase of nematodes. Belonolaimus longicaudatus, Helicotylenchus dihystera, Scutellonema brachyurum and Tylenchorhynchus claytoni parasitized and reproduced on three or more of the species tested. Hoplolaimus galeatus and Pratylenchus brachyurus parasitized two species, Trichodorus christiei and Criconemoides xenoplax parasitized only red maple. Meloidogyne javanica/Liriodendron tulipifera combination was the only positive root-knot nematode/hardwood host-parasite relationship. Hemicycliophora silvestris, Meloidogyne arenaria, M. incognita, and M. hapla were not parasites of the tree species tested.
PMCID: PMC2619869  PMID: 19322363
Host-parasite relations; Liquidambar styraciflua; Acer rubrum; Liriodendron tulipifera; Platanus occidentalis; Populus heterophylla
5.  Monophyly of clade III nematodes is not supported by phylogenetic analysis of complete mitochondrial genome sequences 
BMC Genomics  2011;12:392.
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.
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.
The phylogenetic hypotheses from comparative analysis of the complete mitochondrial genome data (analysis of nucleotide and amino acid datasets, and nucleotide data excluding 3rd 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.
PMCID: PMC3163570  PMID: 21813000
Mitochondrial genome; Molecular phylogeny; Ascaridida; Spirurida; Oxyurida; Chromadorea; Nematoda
6.  Hoplolaimoidea (Nematoda: Tylenchida) from the Aleutian Islands with Descriptions of Four New Species 
Journal of Nematology  1984;16(2):194-203.
Four new species of hoplolaimoid nematodes (Merlinius adakensis, Pratylenchoides megalobatus, Pratylenchus pratensisobrinus, and Pratylenchus ventroprojectus) are described from Adak Island in the Aleutian chain. M. adakensis n. sp. is separated from other species by body length (0.96-1.3 mm), stylet length (32-36 μm), number of tail annules (49-68), and c' (3.1-4.1). P. megalobatus n. sp. differs from all known Pratylenchoides spp. by having a very long esophageal gland lobe (b' = 2.4-3.3, overlap 3-6 times the body width). P. pratensisobrinus n. sp. closely resembles P. pratensis (de Man) Filipjev, but has a longer stylet (15-17 μm), a longer tail (c = 12-15; c' = 2.8-3.7), and more tail annules (23-37). P. ventroprojectus n. sp. is distinguished by body length (392-475 μm), three lip annules, low and flattened cephalic capsule, and presence of terminal subventral projection. Pratylenchoides variabilis Sher, Helicotylenchus amplius Anderson &Eveleigh, and H. spitsbergensis Loof are also reported from Adak and Amchitka Islands.
PMCID: PMC2618363  PMID: 19295900
stunt nematodes; root-lesion nematodes; spiral nematodes; taxonomy; ectoparasites; endoparasites; new species
7.  Lineage-specific variations of congruent evolution among DNA sequences from three genomes, and relaxed selective constraints on rbcL in Cryptomonas (Cryptophyceae) 
Plastid-bearing cryptophytes like Cryptomonas contain four genomes in a cell, the nucleus, the nucleomorph, the plastid genome and the mitochondrial genome. Comparative phylogenetic analyses encompassing DNA sequences from three different genomes were performed on nineteen photosynthetic and four colorless Cryptomonas strains. Twenty-three rbcL genes and fourteen nuclear SSU rDNA sequences were newly sequenced to examine the impact of photosynthesis loss on codon usage in the rbcL genes, and to compare the rbcL gene phylogeny in terms of tree topology and evolutionary rates with phylogenies inferred from nuclear ribosomal DNA (concatenated SSU rDNA, ITS2 and partial LSU rDNA), and nucleomorph SSU rDNA.
Largely congruent branching patterns and accelerated evolutionary rates were found in nucleomorph SSU rDNA and rbcL genes in a clade that consisted of photosynthetic and colorless species suggesting a coevolution of the two genomes. The extremely accelerated rates in the rbcL phylogeny correlated with a shift from selection to mutation drift in codon usage of two-fold degenerate NNY codons comprising the amino acids asparagine, aspartate, histidine, phenylalanine, and tyrosine. Cysteine was the sole exception. The shift in codon usage seemed to follow a gradient from early diverging photosynthetic to late diverging photosynthetic or heterotrophic taxa along the branches. In the early branching taxa, codon preferences were changed in one to two amino acids, whereas in the late diverging taxa, including the colorless strains, between four and five amino acids showed changes in codon usage.
Nucleomorph and plastid gene phylogenies indicate that loss of photosynthesis in the colorless Cryptomonas strains examined in this study possibly was the result of accelerated evolutionary rates that started already in photosynthetic ancestors. Shifts in codon usage are usually considered to be caused by changes in functional constraints and in gene expression levels. Thus, the increasing influence of mutation drift on codon usage along the clade may indicate gradually relaxed constraints and reduced expression levels on the rbcL gene, finally correlating with a loss of photosynthesis in the colorless Cryptomonas paramaecium strains.
PMCID: PMC1285359  PMID: 16232313
8.  A Key and Diagnostic Compendium to the Species of the Genus Tylenchorhynchus Cobb, 1913 (Nematoda: Belonolaimidae) 
Journal of Nematology  2000;32(1):20-34.
An identification key to 111 species of Tylenchorhynchus is given. Tylenchorhynchus is defined as containing only those species with four incisures in the lateral field. A compendium providing the most important diagnostic characters for use in identification of species is included as a supplement to the key. Some species in a related but unnecessary genus were placed in the genus Tylenchorhynchus, based on morphological structures and logical groupings of the species. Bitylenchus iphilus is transferred to Tylenchorhynchus. The diagnosis of Tylenchorhynchus is emended, and a list of all the valid species of the genus is given. The characters most useful for separating species are the stylet length, shape of lip region, number of lip annules, shape of tail and tail terminus, number of tail annules, and position of vulva (V%). As defined in the paper, the genus currently is composed of 111 valid species.
PMCID: PMC2620427  PMID: 19270946
Compendium; diagnosis; identification; key; morphology; nematode; stunt nematodes; taxonomy; Tylenchorhynchus
9.  Effect of Foliar Application of Carbofuran and a Related Compound on Plant-parasitic Nematodes under Greenhouse and Growth Chamber Conditions 
Journal of Nematology  1981;13(1):20-24.
Greenhouse and growth chamber studies were made to investigate the downward systemic nematicidal activity of carbofuran and its analog 2,3-dihydro-2,2-dimethyl-7-benzofuranyl [(di-n-butyl)-4-aminosulfenyl] carbamate against Meloidogyne incognita, Tylenchorhynchus claytoni, and Pratylenchus penetrans. Oxamyl was used as standard in tests with T. claytoni. Both carbofuran and its analog reduced all three plant-parasitic species when plant foliage was sprayed with chemical suspension of 1,200, 2,400 or 4,800 ppm. The studies show that fewer chemical applications were required to reduce populations of P. penetrans than to reduce populations of T. claytoni. Oxamyl was somewhat less active than either carbofuran or its analog.
PMCID: PMC2618044  PMID: 19300716
nonfumigants; nematicides; Meloidogyne incognita; Tylenchorhynchus claytoni; Pratylenchus penetrans; systemic nematicides; oxamyl; carbofuran analog
10.  Xiphinema llanosum and Trophurus vultus, Two New Plant Nematode Species from Pasture Soils in Colombia 
Journal of Nematology  1990;22(3):262-267.
Xiphinema llanosum n. sp. and Trophurus vultus n. sp. are described and illustrated from grass soils in Llanos Orientales, Colombia. Xiphinema llanosum is a bisexual species. The female body length is 2.3-2.7 mm, odontostyle 86-96 μm, and odontophore 58-65 μm long; vulva at 42-47%; anterior ovary is absent; the anterior uterus and oviduct are similar to the posterior branch but slightly reduced; and the tail is dorsally convex-conoid with a blunt hemispherical terminus. Male body length is 2.06-2.96 mm; spicules are 40-44 μm long; and four (rarely three or five) anterior ventromedian supplementary papillae are present. Trophurus vultus females are 0.52-0.67 mm long; vulva at 56-60%; stylet is 10.5-13.5 μm long; isthmus is as long as the basal esophageal bulb; the tail is subclavate, 1.6-2.2 times anal body width long; and the terminal cuticle thickness is about one-sixth of the tail length.
PMCID: PMC2619053  PMID: 19287719
Andropogon gayanus; Colombia; new species; pasture grass; taxonomy; Trachypogon sp.; Trophurus vultus; Xiphinema llanosum
11.  Seasonal Population Dynamics of Selected Plant-parasitic Nematodes as Measured by Three Extraction Procedures 
Journal of Nematology  1969;1(3):232-239.
Seasonal fluctuations in field populations of Meloidogyne spp. (M. incognita and M. hapla), Pratylenchus zeae, Criconemoides ornatum, Tylenchorhynchus claytoni, Belonolaimus longicaudatus, and Helicotylenchus dihystera were determined monthly for 1 year by three extraction procedures. Baermann funnel method (BF) gave highest recoveries of Meloidogyne spp. and P. zeae during summer and fall, but centrifugal-flotation (CF) and sugar-flotation-sieving (SFS) usually yielded higher numbers of these nematodes during winter and spring. CF was t h e only effective method for recovery of C. ornatum with maximum numbers occurring in September. Recoveries of T. claytoni were similar with all methods in summer and fall. However, BF gave low numbers in winter and spring, whereas population peaks with the flotation methods occurred in January and February. All methods gave similar recoveries of B. longicaudatus with highest numbers occurring in November and December. This species declined drastically in late winter and spring. Yields of H. dihystera were similar for all three methods with CF consistently higher and the major peaks occurring in August.
PMCID: PMC2617831  PMID: 19325683
12.  Molecular Systematics of the Deep-Sea Hydrothermal Vent Endemic Brachyuran Family Bythograeidae: A Comparison of Three Bayesian Species Tree Methods 
PLoS ONE  2012;7(3):e32066.
Brachyuran crabs of the family Bythograeidae are endemic to deep-sea hydrothermal vents and represent one of the most successful groups of macroinvertebrates that have colonized this extreme environment. Occurring worldwide, the family includes six genera (Allograea, Austinograea, Bythograea, Cyanagraea, Gandalfus, and Segonzacia) and fourteen formally described species. To investigate their evolutionary relationships, we conducted Maximum Likelihood and Bayesian molecular phylogenetic analyses, based on DNA sequences from fragments of three mitochondrial genes (16S rDNA, Cytochrome oxidase I, and Cytochrome b) and three nuclear genes (28S rDNA, the sodium–potassium ATPase a-subunit ‘NaK’, and Histone H3A). We employed traditional concatenated (i.e., supermatrix) phylogenetic methods, as well as three recently developed Bayesian multilocus methods aimed at inferring species trees from potentially discordant gene trees. We found strong support for two main clades within Bythograeidae: one comprising the members of the genus Bythograea; and the other comprising the remaining genera. Relationships within each of these two clades were partially resolved. We compare our results with an earlier hypothesis on the phylogenetic relationships among bythograeid genera based on morphology. We also discuss the biogeography of the family in the light of our results. Our species tree analyses reveal differences in how each of the three methods weighs conflicting phylogenetic signal from different gene partitions and how limits on the number of outgroup taxa may affect the results.
PMCID: PMC3293879  PMID: 22403623
13.  Molecular phylogenetics of Ruscaceae sensu lato and related families (Asparagales) based on plastid and nuclear DNA sequences 
Annals of Botany  2010;106(5):775-790.
Previous phylogenetics studies of Asparagales, although extensive and generally well supported, have left several sets of taxa unclearly placed and have not addressed all relationships within certain clades thoroughly (some clades were relatively sparsely sampled). One of the most important of these is sampling within and placement of Nolinoideae (Ruscaceae s.l.) of Asparagaceae sensu Angiosperm Phylogeny Group (APG) III, which subfamily includes taxa previously referred to Convallariaceae, Dracaenaaceae, Eriospermaceae, Nolinaceae and Ruscaceae.
A phylogenetic analysis of a combined data set for 126 taxa of Ruscaceae s.l. and related groups in Asparagales based on three nuclear and plastid DNA coding genes, 18S rDNA (1796 bp), rbcL (1338 bp) and matK (1668 bp), representing a total of approx. 4·8 kb is presented. Parsimony and Bayesian inference analyses were conducted to elucidate relationships of Ruscaceae s.l. and related groups, and parsimony bootstrap analysis was performed to assess support of clades.
Key Results
The combination of the three genes results in the most highly resolved and strongly supported topology yet obtained for Asparagales including Ruscaceae s.l. Asparagales relationships are nearly congruent with previous combined gene analyses, which were reflected in the APG III classification. Parsimony and Bayesian analyses yield identical relationships except for some slight variation among the core asparagoid families, which nevertheless form a strongly supported group in both types of analyses. In core asparagoids, five major clades are identified: (1) Alliaceae s.l. (sensu APG III, Amarylidaceae–Agapanthaceae–Alliaceae); (2) Asparagaceae–Laxmanniaceae–Ruscaceae s.l.; (3) Themidaceae; (4) Hyacinthaceae; (5) Anemarrhenaceae–Behniaceae–Herreriaceae–Agavaceae (clades 2–5 collectively Asparagaceae s.l. sensu APG III). The position of Aphyllanthes is labile, but it is sister to Themidaceae in the combined maximum-parsimony tree and sister to Anemarrhenaceae in the Bayesian analysis. The highly supported clade of Xanthorrhoeaceae s.l. (sensu APG III, including Asphodelaceae and Hemerocallidaceae) is sister to the core asparagoids. Ruscaceae s.l. are a well-supported group. Asparagaceae s.s. are sister to Ruscaceae s.l., even though the clade of the two families is weakly supported; Laxmanniaceae are strongly supported as sister to Ruscaceae s.l. and Asparagaceae. Ruscaceae s.l. include six principal clades that often reflect previously named groups: (1) tribe Polygonateae (excluding Disporopsis); (2) tribe Ophiopogoneae; (3) tribe Convallarieae (excluding Theropogon); (4) Ruscaceae s.s. + Dracaenaceae + Theropogon + Disporopsis + Comospermum; (5) Nolinaceae, (6) Eriospermum.
The analyses here were largely conducted with new data collected for the same loci as in previous studies, but in this case from different species/DNA accessions and greater sampling in many cases than in previously published analyses; nonetheless, the results largely mirror those of previously conducted studies. This demonstrates the robustness of these results and answers questions often raised about reproducibility of DNA results, given the often sparse sampling of taxa in some studies, particularly the earliest ones. The results also provide a clear set of patterns on which to base a new classification of the subfamilies of Asparagaceae s.l., particularly Ruscaceae s.l. (= Nolinoideae of Asparagaceae s.l.), and examine other putatively important characters of Asparagales.
PMCID: PMC2958784  PMID: 20929900
Aphyllanthes; Asparagaceae; Convallariaceae; Dracaenaceae; Eriospermum; monocot phylogenetics; Nolinaceae; Nolinoideae
14.  Molecular phylogeny of the higher and lower taxonomy of the Fusarium genus and differences in the evolutionary histories of multiple genes 
Species of the Fusarium genus are important fungi which is associated with health hazards in human and animals. The taxonomy of this genus has been a subject of controversy for many years. Although many researchers have applied molecular phylogenetic analysis to examine the taxonomy of Fusarium species, their phylogenetic relationships remain unclear only few comprehensive phylogenetic analyses of the Fusarium genus and a lack of suitable nucleotides and amino acid substitution rates. A previous stugy with whole genome comparison among Fusairum species revealed the possibility that each gene in Fusarium genomes has a unique evolutionary history, and such gene may bring difficulty to the reconstruction of phylogenetic tree of Fusarium. There is a need not only to check substitution rates of genes but also to perform the exact evaluation of each gene-evolution.
We performed phylogenetic analyses based on the nucleotide sequences of the rDNA cluster region (rDNA cluster), and the β-tubulin gene (β-tub), the elongation factor 1α gene (EF-1α), and the aminoadipate reductase gene (lys2). Although incongruence of the tree topologies between lys2 and the other genes was detected, all genes supported the classification of Fusarium species into 7 major clades, I to VII. To obtain a reliable phylogeny for Fusarium species, we excluded the lys2 sequences from our dataset, and re-constructed a maximum likelihood (ML) tree based on the combined data of the rDNA cluster, β-tub, and EF-1α. Our ML tree indicated some interesting relationships in the higher and lower taxa of Fusarium species and related genera. Moreover, we observed a novel evolutionary history of lys2. We suggest that the unique tree topologies of lys2 are not due to an analytical artefact, but due to differences in the evolutionary history of genomes caused by positive selection of particular lineages.
This study showed the reliable species tree of the higher and lower taxonomy in the lineage of the Fusarium genus. Our ML tree clearly indicated 7 major clades within the Fusarium genus. Furthermore, this study reported differences in the evolutionary histories among multiple genes within this genus for the first time.
PMCID: PMC3270093  PMID: 22047111
15.  CORE: A Phylogenetically-Curated 16S rDNA Database of the Core Oral Microbiome 
PLoS ONE  2011;6(4):e19051.
Comparing bacterial 16S rDNA sequences to GenBank and other large public databases via BLAST often provides results of little use for identification and taxonomic assignment of the organisms of interest. The human microbiome, and in particular the oral microbiome, includes many taxa, and accurate identification of sequence data is essential for studies of these communities. For this purpose, a phylogenetically curated 16S rDNA database of the core oral microbiome, CORE, was developed. The goal was to include a comprehensive and minimally redundant representation of the bacteria that regularly reside in the human oral cavity with computationally robust classification at the level of species and genus. Clades of cultivated and uncultivated taxa were formed based on sequence analyses using multiple criteria, including maximum-likelihood-based topology and bootstrap support, genetic distance, and previous naming. A number of classification inconsistencies for previously named species, especially at the level of genus, were resolved. The performance of the CORE database for identifying clinical sequences was compared to that of three publicly available databases, GenBank nr/nt, RDP and HOMD, using a set of sequencing reads that had not been used in creation of the database. CORE offered improved performance compared to other public databases for identification of human oral bacterial 16S sequences by a number of criteria. In addition, the CORE database and phylogenetic tree provide a framework for measures of community divergence, and the focused size of the database offers advantages of efficiency for BLAST searching of large datasets. The CORE database is available as a searchable interface and for download at
PMCID: PMC3081323  PMID: 21544197
16.  Comparing the Usefulness of Distance, Monophyly and Character-Based DNA Barcoding Methods in Species Identification: A Case Study of Neogastropoda 
PLoS ONE  2011;6(10):e26619.
DNA barcoding has recently been proposed as a promising tool for the rapid species identification in a wide range of animal taxa. Two broad methods (distance and monophyly-based methods) have been used. One method is based on degree of DNA sequence variation within and between species while another method requires the recovery of species as discrete clades (monophyly) on a phylogenetic tree. Nevertheless, some issues complicate the use of both methods. A recently applied new technique, the character-based DNA barcode method, however, characterizes species through a unique combination of diagnostic characters.
Methodology/Principal Findings
Here we analyzed 108 COI and 102 16S rDNA sequences of 40 species of Neogastropoda from a wide phylogenetic range to assess the performance of distance, monophyly and character-based methods of DNA barcoding. The distance-based method for both COI and 16S rDNA genes performed poorly in terms of species identification. Obvious overlap between intraspecific and interspecific divergences for both genes was found. The “10× rule” threshold resulted in lumping about half of distinct species for both genes. The neighbour-joining phylogenetic tree of COI could distinguish all species studied. However, the 16S rDNA tree could not distinguish some closely related species. In contrast, the character-based barcode method for both genes successfully identified 100% of the neogastropod species included, and performed well in discriminating neogastropod genera.
This present study demonstrates the effectiveness of the character-based barcoding method for species identification in different taxonomic levels, especially for discriminating the closely related species. While distance and monophyly-based methods commonly use COI as the ideal gene for barcoding, the character-based approach can perform well for species identification using relatively conserved gene markers (e.g., 16S rDNA in this study). Nevertheless, distance and monophyly-based methods, especially the monophyly-based method, can still be used to flag species.
PMCID: PMC3200347  PMID: 22039517
17.  Molecular Phylogeny of the Small Ermine Moth Genus Yponomeuta (Lepidoptera, Yponomeutidae) in the Palaearctic 
PLoS ONE  2010;5(3):e9933.
The small ermine moth genus Yponomeuta (Lepidoptera, Yponomeutidae) contains 76 species that are specialist feeders on hosts from Celastraceae, Rosaceae, Salicaceae, and several other plant families. The genus is a model for studies in the evolution of phytophagous insects and their host-plant associations. Here, we reconstruct the phylogeny to provide a solid framework for these studies, and to obtain insight into the history of host-plant use and the biogeography of the genus.
Methodology/Principal Findings
DNA sequences from an internal transcribed spacer region (ITS-1) and from the 16S rDNA (16S) and cytochrome oxidase (COII) mitochondrial genes were collected from 20–23 (depending on gene) species and two outgroup taxa to reconstruct the phylogeny of the Palaearctic members of this genus. Sequences were analysed using three different phylogenetic methods (parsimony, likelihood, and Bayesian inference).
Roughly the same patterns are retrieved irrespective of the method used, and they are similar among the three genes. Monophyly is well supported for a clade consisting of the Japanese (but not the Dutch) population of Yponomeuta sedellus and Y. yanagawanus, a Y. kanaiellus–polystictus clade, and a Rosaceae-feeding, western Palaearctic clade (Y. cagnagellus–irrorellus clade). Within these clades, relationships are less well supported, and the patterns between the different gene trees are not so similar. The position of the remaining taxa is also variable among the gene trees and rather weakly supported. The phylogenetic information was used to elucidate patterns of biogeography and resource use. In the Palaearctic, the genus most likely originated in the Far East, feeding on Celastraceae, dispersing to the West concomitant with a shift to Rosaceae and further to Salicaceae. The association of Y. cagnagellus with Euonymus europaeus (Celastraceae), however, is a reversal. The only oligophagous species, Y. padellus, belongs to the derived western Palaearctic clade, evidence that specialisation is reversible.
PMCID: PMC2847947  PMID: 20360968
18.  Morphology and molecular phylogeny of a marine interstitial tetraflagellate with putative endosymbionts: Auranticordis quadriverberis n. gen. et sp. (Cercozoa) 
BMC Microbiology  2008;8:123.
Comparative morphological studies and environmental sequencing surveys indicate that marine benthic environments contain a diverse assortment of microorganisms that are just beginning to be explored and characterized. The most conspicuous predatory flagellates in these habitats range from about 20–150 μm in size and fall into three major groups of eukaryotes that are very distantly related to one another: dinoflagellates, euglenids and cercozoans. The Cercozoa is a diverse group of amoeboflagellates that cluster together in molecular phylogenies inferred mainly from ribosomal gene sequences. These molecular phylogenetic studies have demonstrated that several enigmatic taxa, previously treated as Eukaryota insertae sedis, fall within the Cercozoa, and suggest that the actual diversity of this group is largely unknown. Improved knowledge of cercozoan diversity is expected to help resolve major branches in the tree of eukaryotes and demonstrate important cellular innovations for understanding eukaryote evolution.
A rare tetraflagellate, Auranticordis quadriverberis n. gen. et sp., was isolated from marine sand samples. Uncultured cells were in low abundance and were individually prepared for electron microscopy and DNA sequencing. These flagellates possessed several novel features, such as (1) gliding motility associated with four bundled recurrent flagella, (2) heart-shaped cells about 35–75 μm in diam., and (3) bright orange coloration caused by linear arrays of muciferous bodies. Each cell also possessed about 2–30 pale orange bodies (usually 4–5 μm in diam.) that were enveloped by two membranes and sac-like vesicles. The innermost membrane invaginated to form unstacked thylakoids that extended towards a central pyrenoid containing tailed viral particles. Although to our knowledge, these bodies have never been described in any other eukaryote, the ultrastructure was most consistent with photosynthetic endosymbionts of cyanobacterial origin. This combination of morphological features did not allow us to assign A. quadriverberis to any known eukaryotic supergroup. Thus, we sequenced the small subunit rDNA sequence from two different isolates and demonstrated that this lineage evolved from within the Cercozoa.
Our discovery and characterization of A. quadriverberis underscores how poorly we understand the diversity of cercozoans and, potentially, represents one of the few independent cases of primary endosymbiosis within the Cercozoa and beyond.
PMCID: PMC2500021  PMID: 18647416
19.  Heterorhabditidoides rugaoensis n. sp. (Rhabditida: Rhabditidae), a Novel Highly Pathogenic Entomopathogenic Nematode Member of Rhabditidae 
Journal of Nematology  2012;44(4):348-360.
A novel entomopathogenic nematode species, Heterorhabditidoides rugaoensis n. sp. RG081015, collected from Rugao, China, is described. The new species is morphologically very similar to H. chongmingensis but can be distinguished from it on the basis of some morphological characteristics, combined with molecular data and a cross-hybridization test. Males of the new species can be recognized on the basis of body length averaging 1396.2 μm; lateral field with one ridge; metastome isoglottoid with one hemispherical swellings comprised of two to three well-developed warts; asymmetric spicules; peloderan bursa. In IJs, EP = 134.5 μm; ES = 149.3 μm; tail length = 82.5 μm; and a = 20.5. Hermaphroditic females have four to five lateral ridges. The 18S rDNA and ITS sequences of the two nematodes share 99% and 98% identity, respectively. Phylogenetic trees of 18S rDNA and ITS indicate that the new species is most closely related to H. chongmingensis; thus, the two nematodes belong to the same genus. Failure of cross-hybridization between them indicates that nematode strain RG081015 is a novel species and is described herein as H. rugaoensis n. sp. The LC50 of the novel species against Galleria mellonella were 24.35 IJs / ml within 48 hours of infection. Morphological characteristics, genetic similarity analyses, and phylogenetic relationships provide strong evidence that some species of Oscheius/Insectivora-group should be reassigned to the genus Heterorhabditidoides.
PMCID: PMC3592371  PMID: 23482845
Entomopathogenic nematode; Heterorhabditidoides rugaoensis n. sp.; Insectivora-group; new nematode species; pathogenicity; phylogeny; Oscheius; taxonomy
20.  Phylogeographic patterns, genetic affinities and morphological differentiation between Epipactis helleborine and related lineages in a Mediterranean glacial refugium 
Annals of Botany  2010;107(3):427-436.
Background and Aims
In the Mediterranean basin, the Italian peninsula has been suggested to be one of the most important glacial refugia for temperate tree species. The orchid genus Epipactis is widely represented in the Italian peninsula by widespread species and several endemic, localized taxa, including selfing and outcrossing taxa. Here the phylogenetic and phylogeographic relationships in a group of closely related taxa in Epipactis are investigated with the aim of understanding the role of this refugial area for cladogenesis and speciation in herbaceous species, such as terrestrial orchids.
Ribosomal DNA (rDNA) was employed to assess phylogenetic relationships, and plastid sequence variation in the rbcL–accD spacer was used to reveal phylogeographic patterns among plastid haplotypes using a parsimony network.
Key Results
Low genetic variation and shared ribotypes were detected in rDNA, whereas high levels of sequence variation and a strong phylogeographic structure were found in the examined plastid region. The parsimony plastid haplotype network identified two main haplotype groups, one including E. atrorubens/microphylla/muelleri/leptochila and the other including all accessions of E. helleborine and several localized and endemic taxa, with a combination of widespread and rare haplotypes detected across the Italian peninsula. A greater genetic divergence separated the Italian and other European accessions of E. helleborine.
Phylogenetic and phylogeographic patterns support a working hypothesis in which the Italian peninsula has only recently been colonized by Epipactis, probably during the most recent phase of the Quaternary age and, nevertheless, it acted as a remarkable centre of diversification for this orchid lineage. Changes in pollination strategy and recurrent shifts in mating system (from allogamy to autogamy) could have represented the mechanism promoting this rapid diversification and the observed high taxonomic complexity detected in the E. helleborine species complex.
PMCID: PMC3043932  PMID: 21193482
Epipactis; chloroplast DNA; genetic variation; plastid haplotype network; mating systems; orchids; plastid spacer; ribosomal spacer
21.  Molecular phylogeny of brachiopods and phoronids based on nuclear-encoded small subunit ribosomal RNA gene sequences 
Brachiopod and phoronid phylogeny is inferred from SSU rDNA sequences of 28 articulate and nine inarticulate brachiopods, three phoronids, two ectoprocts and various outgroups, using gene trees reconstructed by weighted parsimony, distance and maximum likelihood methods. Of these sequences, 33 from brachiopods, two from phoronids and one each from an ectoproct and a priapulan are newly determined. The brachiopod sequences belong to 31 different genera and thus survey about 10% of extant genus-level diversity. Sequences determined in different laboratories and those from closely related taxa agree well, but evidence is presented suggesting that one published phoronid sequence (GenBank accession UO12648) is a brachiopod-phoronid chimaera, and this sequence is excluded from the analyses. The chiton, Acanthopleura, is identified as the phenetically proximal outgroup; other selected outgroups were chosen to allow comparison with recent, non-molecular analyses of brachiopod phylogeny. The different outgroups and methods of phylogenetic reconstruction lead to similar results, with differences mainly in the resolution of weakly supported ancient and recent nodes, including the divergence of inarticulate brachiopod sub-phyla, the position of the rhynchonellids in relation to long- and short-looped articulate brachiopod clades and the relationships of some articulate brachiopod genera and species. Attention is drawn to the problem presented by nodes that are strongly supported by non-molecular evidence but receive only low bootstrap resampling support. Overall, the gene trees agree with morphology-based brachiopod taxonomy, but novel relationships are tentatively suggested for thecideidine and megathyrid brachiopods. Articulate brachiopods are found to be monophyletic in all reconstructions, but monophyly of inarticulate brachiopods and the possible inclusion of phoronids in the inarticulate brachiopod clade are less strongly established. Phoronids are clearly excluded from a sister-group relationship with articulate brachiopods, this proposed relationship being due to the rejected, chimaeric sequence (GenBank UO12648). Lineage relative rate tests show no heterogeneity of evolutionary rate among articulate brachiopod sequences, but indicate that inarticulate brachiopod plus phoronid sequences evolve somewhat more slowly. Both brachiopods and phoronids evolve slowly by comparison with other invertebrates. A number of palaeontologically dated times of earliest appearance are used to make upper and lower estimates of the global rate of brachiopod SSU rDNA evolution, and these estimates are used to infer the likely divergence times of other nodes in the gene tree. There is reasonable agreement between most inferred molecular and palaeontological ages. The estimated rates of SSU rDNA sequence evolution suggest that the last common ancestor of brachiopods, chitons and other protostome invertebrates (Lophotrochozoa and Ecdysozoa) lived deep in Precambrian time. Results of this first DNA-based, taxonomically representative analysis of brachiopod phylogeny are in broad agreement with current morphology-based classification and systematics and are largely consistent with the hypothesis that brachiopod shell ontogeny and morphology are a good guide to phylogeny.
PMCID: PMC1692429
22.  Phylogeography of the neotropical Anopheles triannulatus complex (Diptera: Culicidae) supports deep structure and complex patterns 
Parasites & Vectors  2013;6:47.
The molecular phylogenetic relationships and population structure of the species of the Anopheles triannulatus complex: Anopheles triannulatus s.s., Anopheles halophylus and the putative species Anopheles triannulatus C were investigated.
The mitochondrial COI gene, the nuclear white gene and rDNA ITS2 of samples that include the known geographic distribution of these taxa were analyzed. Phylogenetic analyses were performed using Bayesian inference, Maximum parsimony and Maximum likelihood approaches.
Each data set analyzed septely yielded a different topology but none provided evidence for the seption of An. halophylus and An. triannulatus C, consistent with the hypothesis that the two are undergoing incipient speciation. The phylogenetic analyses of the white gene found three main clades, whereas the statistical parsimony network detected only a single metapopulation of Anopheles triannulatus s.l. Seven COI lineages were detected by phylogenetic and network analysis. In contrast, the network, but not the phylogenetic analyses, strongly supported three ITS2 groups. Combined data analyses provided the best resolution of the trees, with two major clades, Amazonian (clade I) and trans-Andean + Amazon Delta (clade II). Clade I consists of multiple subclades: An. halophylus + An. triannulatus C; trans-Andean Venezuela; central Amazonia + central Bolivia; Atlantic coastal lowland; and Amazon delta. Clade II includes three subclades: Panama; cis-Andean Colombia; and cis-Venezuela. The Amazon delta specimens are in both clades, likely indicating local sympatry. Spatial and molecular variance analyses detected nine groups, corroborating some of subclades obtained in the combined data analysis.
Combination of the three molecular markers provided the best resolution for differentiation within An. triannulatus s.s. and An. halophylus and C. The latest two species seem to be very closely related and the analyses performed were not conclusive regarding species differentiation. Further studies including new molecular markers would be desirable to solve this species status question. Besides, results of the study indicate a trans-Andean origin for An. triannulatus s.l. The potential implications for malaria epidemiology remain to be investigated.
PMCID: PMC3606328  PMID: 23433428
Anopheles triannulatus s.s.; Anopheles halophylus; Anopheles triannulatus C; phylogeography; COI gene; White gene; ITS2
23.  Dinoflagellate Phylogeny as Inferred from Heat Shock Protein 90 and Ribosomal Gene Sequences 
PLoS ONE  2010;5(10):e13220.
Interrelationships among dinoflagellates in molecular phylogenies are largely unresolved, especially in the deepest branches. Ribosomal DNA (rDNA) sequences provide phylogenetic signals only at the tips of the dinoflagellate tree. Two reasons for the poor resolution of deep dinoflagellate relationships using rDNA sequences are (1) most sites are relatively conserved and (2) there are different evolutionary rates among sites in different lineages. Therefore, alternative molecular markers are required to address the deeper phylogenetic relationships among dinoflagellates. Preliminary evidence indicates that the heat shock protein 90 gene (Hsp90) will provide an informative marker, mainly because this gene is relatively long and appears to have relatively uniform rates of evolution in different lineages.
Methodology/Principal Findings
We more than doubled the previous dataset of Hsp90 sequences from dinoflagellates by generating additional sequences from 17 different species, representing seven different orders. In order to concatenate the Hsp90 data with rDNA sequences, we supplemented the Hsp90 sequences with three new SSU rDNA sequences and five new LSU rDNA sequences. The new Hsp90 sequences were generated, in part, from four additional heterotrophic dinoflagellates and the type species for six different genera. Molecular phylogenetic analyses resulted in a paraphyletic assemblage near the base of the dinoflagellate tree consisting of only athecate species. However, Noctiluca was never part of this assemblage and branched in a position that was nested within other lineages of dinokaryotes. The phylogenetic trees inferred from Hsp90 sequences were consistent with trees inferred from rDNA sequences in that the backbone of the dinoflagellate clade was largely unresolved.
The sequence conservation in both Hsp90 and rDNA sequences and the poor resolution of the deepest nodes suggests that dinoflagellates reflect an explosive radiation in morphological diversity in their recent evolutionary past. Nonetheless, the more comprehensive analysis of Hsp90 sequences enabled us to infer phylogenetic interrelationships of dinoflagellates more rigorously. For instance, the phylogenetic position of Noctiluca, which possesses several unusual features, was incongruent with previous phylogenetic studies. Therefore, the generation of additional dinoflagellate Hsp90 sequences is expected to refine the stem group of athecate species observed here and contribute to future multi-gene analyses of dinoflagellate interrelationships.
PMCID: PMC2951904  PMID: 20949037
24.  Novel Evolutionary Lineages Revealed in the Chaetothyriales (Fungi) Based on Multigene Phylogenetic Analyses and Comparison of ITS Secondary Structure 
PLoS ONE  2013;8(5):e63547.
Cyphellophora and Phialophora (Chaetothyriales, Pezizomycota) comprise species known from skin infections of humans and animals and from a variety of environmental sources. These fungi were studied based on the comparison of cultural and morphological features and phylogenetic analyses of five nuclear loci, i.e., internal transcribed spacer rDNA operon (ITS), large and small subunit nuclear ribosomal DNA (nuc28S rDNA, nuc18S rDNA), β-tubulin, DNA replication licensing factor (mcm7) and second largest subunit of RNA polymerase II (rpb2). Phylogenetic results were supported by comparative analysis of ITS1 and ITS2 secondary structure of representatives of the Chaetothyriales and the identification of substitutions among the taxa analyzed. Base pairs with non-conserved, co-evolving nucleotides that maintain base pairing in the RNA transcript and unique evolutionary motifs in the ITS2 that characterize whole clades or individual taxa were mapped on predicted secondary structure models. Morphological characteristics, structural data and phylogenetic analyses of three datasets, i.e., ITS, ITS-β-tubulin and 28S-18S-rpb2-mcm7, define a robust clade containing eight species of Cyphellophora (including the type) and six species of Phialophora. These taxa are now accommodated in the Cyphellophoraceae, a novel evolutionary lineage within the Chaetothyriales. Cyphellophora is emended and expanded to encompass species with both septate and nonseptate conidia formed on discrete, intercalary, terminal or lateral phialides. Six new combinations in Cyphellophora are proposed and a dichotomous key to species accepted in the genus is provided. Cyphellophora eugeniae and C. hylomeconis, which grouped in the Chaetothyriaceae, represent another novel lineage and are introduced as the type species of separate genera.
PMCID: PMC3665825  PMID: 23723988
25.  Genome-level homology and phylogeny of Vibrionaceae (Gammaproteobacteria: Vibrionales) with three new complete genome sequences 
BMC Microbiology  2013;13:80.
Phylogenetic hypotheses based on complete genome data are presented for the Gammaproteobacteria family Vibrionaceae. Two taxon samplings are presented: one including all those taxa for which the genome sequences are complete in terms of arrangement (chromosomal location of fragments; 19 taxa) and one for which the genome sequences contain multiple contigs (44 taxa). Analyses are presented under the Maximum Parsimony and Maximum Likelihood optimality criteria for total evidence datasets, the two chromosomes separately, and individual analyses of locally collinear blocks. Three of the genomes included in the 44 taxon dataset, those of Vibrio gazogenes, Salinivibrio costicola, and Aliivibrio logei have been newly sequenced and their genome sequences are documented here.
Phylogenetic results for the 19-taxon datasets show similar levels of collinear subset of dataset incongruence as a previous study of 22 taxa from the sister family Shewanellaceae, while also echoing the strong phylogenetic performance of random subsets of data also shown in this study. Phylogenetic results for both the 19-taxon and 44-taxon datasets corroborate previous hypotheses about the placement of Photobacterium and Aliivibrio within Vibrionaceae and also highlight problems with how Photobacterium is delimited and indicate that it likely should be dissolved into Vibrio to produce a phylogenetic taxonomy. The 19-taxon and 44-taxon trees based on the large chromosome are congruent for the majority of taxa that are present in both datasets. Analyses of the 44-taxon sampling based on the second, small chromosome are quite different from those based on the large chromosome, which is not surprising given the dramatically divergent nature of the small chromosome and the difficulty in postulating primary homologies.
The phylogenetic analyses presented here represent the most comprehensive genome-level phylogenetic analyses in terms of taxa and data. Based on the availability of genome data for many bacterial species on GenBank, many other bacterial groups would also be amenable to similar genome-scale phylogenetic analyses even when present in multiple contigs. The result that collinear subsets of data are incongruent with the concatenated dataset and with each other while random data subsets show very little incongruence echoes the result of previous work on Shewanellaceae. The 44-taxon phylogenetic analysis presented here thus represents the future of phylogenomic analyses in scope and complexity.
PMCID: PMC3663674  PMID: 23578061

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