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1.  Molecular Analysis of the Lance Nematode, Hoplolaimus spp., Using the First Internal Transcribed Spacer and the D1-D3 Expansion Segments of 28S Ribosomal DNA1  
Journal of Nematology  2008;40(3):201-209.
DNA sequence analyses of the nuclear ribosomal ITS1 region of the ribosomal DNA and D1-D3 expansion segments of the 28S gene were conducted to characterize the genetic variation of six amphimictic Hoplolaimus species, including H. magnistylus, H. concaudajuvencus, H. galeatus, Hoplolaimus sp. 1, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, and two closely related parthenogenetic species, H. columbus and H. seinhorsti. PCR amplifications of the combined D1-D3 expansion segments and the ITS1 region each yielded one distinct amplicon. In the D1-D3 region, there was no nucleotide sequence variation between populations of H. columbus, H. magnistylus, Hoplolaimus sp. 2 and Hoplolaimus sp. 3, whereas the ITS1 sequences had nucleotide variation among species. We detected conserved ITS1 regions located at the 3’ and 5’ end of ITS1 and also in the middle of the ITS1 among Hoplolaimus species. These regions were compared with sequences of distantly related Heterodera and Globedera. PCR-RFLP and sequence analysis of ITS1 and 28S PCR products revealed that several haplotypes existed in the same genome of H. columbus, H. magnistylus, H. seinhorsti, H. concaudajuvencus and Hoplolaimus sp. 1. Maximum likelihood and maximum parsimony analysis using the combined ITS1 and D1-D3 expansion segment sequences always produced trees with similar topology; H. columbus and H. seinhorsti grouped in one clade and the other six species (H. galeatus, H. concaudajuvencus, H. magnistylus, Hoplolaimus sp. 1, Hoplolaimus sp. 2, Hoplolaimus sp. 3) grouped in another. Molecular analysis supports morphological schemes for this genus to be divided into two groups based on several phenotypic traits derived from morphological evolution.
PMCID: PMC2664666  PMID: 19440260
28S gene; clades; D1-D3 region genome; haplotypes; Hoplolaimus, lance; ITS1; nematode
2.  Phylogenetic Analysis of the Hoplolaiminae Inferred from Combined D2 and D3 Expansion Segments of 28S rDNA1 
Journal of Nematology  2009;41(1):28-34.
DNA sequences of the D2-D3 expansion segments of the 28S gene of ribosomal DNA from 23 taxa of the subfamily Hoplolaiminae were obtained and aligned to infer phylogenetic relationships. The D2 and D3 expansion regions are G-C rich (59.2%), with up to 20.7% genetic divergence between Scutellonema brachyurum and Hoplolaimus concaudajuvencus. Molecular phylogenetic analysis using maximum likelihood and maximum parsimony was conducted using the D2-D3 sequence data. Of 558 characters, 254 characters (45.5%) were variable and 198 characters (35.4%) were parsimony informative. All phylogenetic methods produced a similar topology with two distinct clades: One clade consists of all Hoplolaimus species while the other clade consists of the rest of the studied Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consisted of Aorolaimus, Helicotylenchus, Rotylenchus, and Scutellonema species. Phylogenetic analysis using the outgroup species Globodera rostocheinsis suggests that Hoplolaiminae is paraphyletic. In this study, the D2-D3 region had levels of DNA sequence divergence sufficient for phylogenetic analysis and delimiting species of Hoplolaiminae.
PMCID: PMC3365300  PMID: 22661775
28S; analysis; Aorolaimus; clade; D2-D3; Helicotylenchus; Hoplolaiminae; Hoplolaimus; lance; nematode; phylogenetic; Rotylenchus; species; spiral; Scutellonema; taxonomy
4.  Survey of Plant-parasitic Nematodes in Missouri Cotton Fields 
Journal of Nematology  1992;24(4S):779-782.
During September 1990, 30 cotton fields in each of three Missouri counties were surveyed for plant-parasitic nematodes. Soil samples for nematode analysis consisted of a composite of 20 cores collected in a zig-zag pattern within a 1-ha block in each field. Cores were taken from within weed-free cotton rows. Nine genera of plant-parasitic nematodes were found (Rotylenchulus, Helicotylenchus, Hoplolaimus, Meloidogyne, Paratylenchus, Pratylenchus, Tylenchorhynchus, Heterodera, and Trichodorus), and five species were identified: Meloidogyne incognita, Rotylenchulus reniformis, Hoplolaimus galeatus, Pratylenchus vulnus, and P. scribneri. This is the first report of R. reniformis, H. galeatus, P. vulnus, and P. scribneri in Missouri cotton fields and the first report of R. reniformis and P. vulnus in Missouri. The known cotton pathogens M. incognita, R. reniformis, and H. galeatus were found in 30%, 3%, and 2% of the fields sampled, respectively. The correlation between sand content of the soil sample and the number of vermiform M. incognita in the sample was not significant, with r² = 0.13. Select fields where H. galeatus and R. reniformis were found in 1990 were sampled more intensely in 1991. The 1-ha block sampled in 1990 was sampled in 1991, along with three other 1-ha blocks uniformly distributed within the field. In addition, a 1-ha block was sampled in each of eight nearby fields, within 2 km of the first field. The nine plant-parasitic nematode genera identified in the 1990 survey were observed again in 1991. Within-field distribution of M. incognita, R. reniformis, and H. galeatus was not uniform. When M. incognita, R. reniformis, or H. galeatus were present in a field, the same species was found in 38%, 25%, or 50% of nearby fields, respectively.
PMCID: PMC2629866  PMID: 19283062
cotton; Gossypium hirsutum; Meloidogyne incognita; Missouri; nematode; Rotylenchulus reniformis; Hoplolaimus galeatus; survey
5.  Description and Larval Heteromorphism of Hoplolaimus concaudajuvencus, n. sp. (Nematoda: Hoplolain-lidae) 
Journal of Nematology  1970;2(2):161-166.
Hoplolaimus concaudajuvencus n. sp., of the genus Hoplolaimus Daday, 1905, characterized by larval heteromorphism, is described and illustrated as recovered from ryegrass/bermudagrass golf green turf in Florida. Females and males are closely related to H. galeatus (Cobb, 1913) Thorne, 1935, but have longer stylets with more definitely tulip-shaped stylet knobs which anteriorly tend to close upon the stylet shaft more than in H. galeatus. First and second-stage larvae have a conically-pointed tail unlike any known species of the genus. Subsequent stages, including females, have rounded tails essentially similar to other species of the genus and males possess the typical hopolaimid tail and bursa. The first molt was found to occur within the egg.
PMCID: PMC2618730  PMID: 19322290
Taxonomy; Hoplolaimus concaudajuvencus n. sp.; Morphology; Larval heteromorphism
6.  Morphological Comparison and Taxonomic Utility of Copulatory Structures of Selected Nematode Species 
Journal of Nematology  1987;19(3):314-323.
Spicules of 9 Meloidogyne, 2 Heterodera, 3 Globodera, and 12 other plant-parasitic, insect-parasitic, and free-living nematodes were excised and examined using scanning electron microscopy (SEM). Gubernacula of some of the species were also excised, and their structure was determined. The two spicules of all species examined were symmetrically identical in morphology. The spicule typically consisted of three parts: head, shaft, and blade with dorsal and ventral vela. The spicular nerve entered through the cytoplasmic core opening on the lateral outer surface of the spicule head and generally communicated with the exterior through one or two pores at the spicule tip. Spicules of Xiphinema sp. and Aporcelaimellus sp. were not composed of three typical parts, were less sclerotized, and lacked a cytoplasmic core opening and distal pores. Spicules of Aphelenchoides spp. had heads expanded into apex and rostrum and had very arcuate blades with thick dorsal and ventral edges (limbs). Gubernaculum shapes were stable within a species, but differed among species examined. The accessory structures of Hoplolaimus galeatus consisted of a tongue-shaped gubernaculum with two titillae at its distal end and a plate-like capitulum terminating distally in two flat, wing-like structures. A comparison of spicules of several species of Meloidogyne by SEM and light microscopy revealed no striking morphological differences.
PMCID: PMC2618657  PMID: 19290150
spicule; gubernaculum; capitulum; titillae; scanning electron microscopy; light microscopy; Aphelenchoides; Aporcelaimellus; Belonolaimus; Dolichodorus; Globodera; Heterorhabditis; Heterodera; Hoplolaimus; Meloidogyne; Mesorhabditis; Panagrellus; Tylenchorhynchus; Xiphinema
7.  Occurrence, Parasitism, and Pathogenicity of Nematodes Associated with Sycamore (Platanus occidentalis L.) 
Journal of Nematology  1971;3(2):189-196.
Ten species of stylet-bearing nematodes were recovered in a survey of sycamore (Platanus occidentalis L. ) stands in Georgia. Helicotylenchus, Xiphinema, and Criconemoides were the genera found most frequently. Populations of Hoplolaimus galeatus, Scutellonema brachyurum, Helicotylenchus dihystera and H. pseudorobustus increased on greenhouse-grown sycamore, but Trichodorus christiei, Xiphinema americanum, Meloidogyne hapla, M. arenaria and M. incognita did not. Hoplolaimus galeatus and S. brachyurum are semi-endoparasites; H. dihystera and H. pseudorobustus are migratory endoparasites. Hoplolaimus galeatus caused extensive root necrosis and marked decrease of fresh weights of seedling roots and tops. Helicotylenchus dihystera and S. brachyurum produced only qualitatively different sparse and unhealthy root growth. Helicotylenchus pseudorobustus caused only a reduction in root surface area.
PMCID: PMC2619866  PMID: 19322367
Helicotylenchus; Xiphinema; Hoplolaimus; Trichodorus; Meloidogyne
8.  Host Status of 'SeaIsle 1' Seashore Paspalum (Paspalum vaginatum) to Belonolaimus longicaudatus and Hoplolaimus galeatus 
Journal of Nematology  2004;36(4):493-498.
Belonolaimus longicaudatus and Hoplolaimus galeatus are considered among the most damaging pathogens of turfgrasses in Florida. However, the host status of seashore paspalum (Paspalum vaginatum) is unknown. Glasshouse experiments were performed in 2002 and 2003 to determine the tolerance of 'SeaIsle 1' seashore paspalum to a population of B. longicaudatus and a population of H. galeatus, and to compare to 'Tifdwarf' bermudagrass for differences. Both nematode species reproduced well on either grass, but only B. longicaudatus consistently reduced root growth as measured by root length. Belonolaimus longicaudatus reduced root growth (P ≤ 0.05) by 35% to 45% at 120 days after inoculation on both grasses. In 2003, higher inoculum levels of H. galeatus reduced root growth (P ≤ 0.05) by 19.4% in seashore paspalum and by 14% in bermudagrass after 60 and 120 days of exposure, respectively. Percentage reductions in root length caused by H. galeatus and B. longicaudatus indicated no differences between grass species, although Tifdwarf bermudagrass supported higher soil population densities of both nematodes than SeaIsle 1 seashore paspalum.
PMCID: PMC2620796  PMID: 19262830
Belonolaimus longicaudatus; bermudagrass; Hoplolaimus galeatus; host status; lance nematode; Paspalum vaginatum; seashore paspalum; sting nematode; tolerance
9.  Evaluation of a Seed-Treatment Method with Acetone for Delivering Systemic Nematicides with Wheat and Rye 
Journal of Nematology  1977;9(4):323-325.
Seeds of 'Coker 68-15' wheat and 'Maton' rye were immersed for 5 min in acetone solutions of oxamyl, carbofuran, or phenamiphos containing 0, 0.25, 0.5, 1.25, 2.5, or 5.0% (w/v) nematicide; after drying, seeds were planted in pots containing 500 gm of sandy loam naturally infested with Hoplolaimus galeatus and Tylenchorhynchus claytoni. In sterilized soil, only the 5% concentrations of all nematicides were toxic to rye, whereas both the 2.5 and 5% concentrations were damaging to wheat. Phenamiphos was generally the most phytotoxic compound. Numbers of T. claytoni in soil declined sharply in response to seed treatment with all nematicides. In soil planted with wheat, numbers were reduced 80% by the 1.25% treatment; little additional control was shown with higher concentrations. Soil with rye showed a 40-60% reduction in numbers of T. claytoni with the 1.25% solutions and little change at higher concentrations. Hoplolaimus galeatus developed only in pots with rye; root populations were suppressed (30-50%) by treatment with 1.25% or higher concentrations of all nematicides.
PMCID: PMC2620259  PMID: 19305616
control; oxamyl; carbofuran; phenamiphos; Secale; cereale; Triticum aestivum; Hoplolaimus galeatus; Tylenchorhynchus claytoni
10.  Avermectin B1, Isazofos, and Fenamiphos for Control of Hoplolaimus galeatus and Tylenchorhynchus dubius Infesting Poa annua 
Journal of Nematology  1996;28(4S):687.
Avermectin B₁, isazofos, and fenamiphos were evaluated in greenhouse experiments for efficacy against two common turfgrass parasites, Hoplolaimus galeatus and Tylenchorhynchus dubius. Treatments in all experiments were arranged in a completely randomized design and replicated four times. In the first experiment, avermectin B₁ at rates of 0.2 and 0.4 kg a.i./ha and isazofos at rates of 2.3 and 23 kg a.i./ha significantly reduced populations of both species of parasitic nematodes compared to controls at 14 and 28 days after treatment (P ≤ 0.01). In the second experiment, the greatest reductions in both nematode populations occurred at 28 and 56 days after treatment, where 23 kg a.i./ha of isazofos was applied (P ≤ 0.01). These reductions, however, were not different from reductions of H. galeatus at 28 and 56 days after treatment (P ≤ 0.01) or T. dubius at 56 days after treatment (P ≤ 0.01), where 0.2- and 0.4-kg a.i./ha rates of avermectin B₁ were mixed throughout the soil. In the third experiment, the greatest population reduction of H. galeatus was observed with a 0.4-kg a.i./ha treatment of avermectin B₁ at 56 days after treatment (P ≤ 0.05). T. dubius populations were reduced by the 0.4-kg a.i./ha rate of avermectin B₁ at 28 (P ≤ 0.01), 56 (P ≤ 0.05), and 70 (P ≤ 0.01) days after treatment. In the fourth and fifth experiments, avermectin B₁ at rates of 7.5 and 15.2 kg a.i./ha consistently reduced nematode populations compared to controls and performed as well or better than fenamiphos (P ≤ 0.01).
PMCID: PMC2619729  PMID: 19277196
avermectin; chemical control; fenamiphos; Hoplolaimus galeatus; isazofos; nematode; Poa annua; turfgrass; Tylenchorhynchus dubius
11.  Correlations of Field Populations of Nematodes with Crop Growth Responses for Determining Relative Involvement of Species 
Journal of Nematology  1975;7(2):193-198.
Treated and nontreated field plots were assayed, and the population density of each plant-parasitic nematode present was compared with crop growth and yield and with the population densities of other nematode species in the same plots. The strongest correlations between nematode population densities and growth responses occurred when soil assays for nematodes were made 55-73 days after planting. Belonolairnus longicaudatus was the most damaging parasite on peanut, Arachis hypogaea, as evidenced by high negative correlations between population densities and plant growth responses. Criconemoides ornatus, Meloidogyne hapla, Helicotylenchus dihystera, Trichodorus christie, Tylenchorhynchus claytoni, and Pratylenchus brachyurus were involved to varying degrees, depending on previous crop and initial densities of these nematodes. Hoplolaimus galeatus and Xiphinema americanum did not appear to affect crop response. The negative correlation of Trichodorus christiei to yield of soybean, Glycine max, was higher than that of Belonolairnus longicaudatus, although both contributed to yield losses. Similar correlation analyses showed that apparent antagonistic or synergistic population-density relationships among nematodes under field conditions depend on the time of sampling and the composition of the nematode community under study.
PMCID: PMC2620102  PMID: 19308153
12.  PCR-based molecular characterization and insilico analysis of food-borne trematode parasites Paragonimus westermani, Fasciolopsis buski and Fasciola gigantica from Northeast India using ITS2 rDNA 
Bioinformation  2011;6(2):64-68.
Food-borne fluke infections/trematodiases are emerging as a major public health problem worldwide with over 40 million people affected and over 10% of world population at risk of infection. The major concentration of these infections is in Southeast Asian and Western Pacific Regions, where the epidemiological factors (including the prevalent socio-cultural food habits) are conducive for transmission of these infections. The preponderance of these infections is usually in food deficit poor communities that lack access to proper sanitary infrastructure. While targeting health for all, especially the poor rural tribal communities, it is imperative to take these infections into account. Bayesian analysis phylogeny of food-borne trematode parasites under study showed that they are closely related phylogenetic groups. To focus the control strategies at the target populations, the aim of the present study was to establish molecular methods for accurate discrimination between common food-borne trematodes parasites Paragonimus (lung fluke), Fasciolopsis (giant intestinal fluke) and Fasciola (liver fluke), the infections of which commonly prevail in NE India. In the first step, we amplified and sequenced the second internal transcribed spacer (ITS2) region of ribosomal DNA, utilizing nucleotide differences between the multiple sequence alignments of the parasites under study. Based upon the differences in nucleotide sequences of conserved regions, we designed species-specific primers that can unequivocally discriminate one species from another. ITS2 sequence motifs allowed an accurate in-silico distinction of the trematodes. The data indicate that ITS2 motifs (≤ 50 bp in size) can be considered promising tool for trematode species identification. Using molecular morphometrics that is based on ITS2 secondary structure homologies, phylogenetic relationships with various isolates of several trematode species have been discussed. The present results suggest that the ITS2 specific primers can be used for epidemiological investigations of the prevalence of trematodiasis.
PMCID: PMC3082855  PMID: 21544167
Paragonimus; Fasciolopsis; Fasciola; trematodes; ITS rDNA; species-specific primer
13.  Isolates of the Pasteuria penetrans Group from Phytoparasitic Nematodes in Bermudagrass Turf 
Journal of Nematology  1990;22(4S):750-762.
A survey was conducted between 1985 and 1989 of isolates of the Pasteuria penetrans group on phytoparasitic nematodes in bermudagrass (Cynodon spp.) turf in southern Florida. Six different isolates of the P. penetrans group were observed from five different species of phytoparasitic nematode hosts. Five of the bacterial isolates were different (P ≤ 0.01) in sporangium diameter, endospore width, and ratio of sporangium diameter to endospore width. All locations surveyed had one or more isolates present, suggesting that the Pasteuria penetrans group is widespread in its distribution in southern Florida. Three survey sites had high densities of Belonolaimus longicaudatus, with more than 60% of the host population encumbered with a large-spored isolate of Pasteuria (mean sporangium diameter = 6.10 μm). One of these sites was monitored for 16 months during which the proportion of nematodes encumbered with this Pasteuria isolate remained constant. Soil infested with this isolate was not suppressive to Pasteuria-free populations of B. longicaudatus grown on bermudagrass for 6 months after controlled soil inoculation. However, the proportion of spore-encumbered and parasitized B. longicaudatus after 6 months was 73%, which was similar to the 74% level observed at the field site. The uhrastructure of mature sporangia of the large-spored isolates of Pasteuria from B. longicaudatus and Hoplolaimus galeatus is described and compared with ultrastructural descriptions of P. penetrans sensu strictu and P. thornei from the literature. These B. longicaudatus and H. galeatus isolates of Pasteuria appear to be distinct from the known species and may warrant new species status.
PMCID: PMC2619098  PMID: 19287792
bacterial parasite; Belonolaimus longicaudatus; bermudagrass; biological control; Helicotylenchus microlobus; Hoplolaimus galeatus; Meloidogyne spp.; Pasteuria penetrans group; Tylenchorhynchus annulatus; ultrastructure
14.  Molecular detection and species identification of Alexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline 
AoB Plants  2012;2012:pls033.
Misleading morphological observations assigned Alexandrium catenella as local dinoflagellate responsible for HABs in Southern Chilean coasts. Our work based on molecular methods found that local Alexandrium belongs to group I of the tamarensis complex composed mainly of A. tamarense.
Background and aims
On the basis of morphological evidence, the species involved in South American Pacific coast harmful algal blooms (HABs) has been traditionally recognized as Alexandrium catenella (Dinophyceae). However, these observations have not been confirmed using evidence based on genomic sequence variability. Our principal objective was to accurately determine the species of Alexandrium involved in local HABs in order to implement a real-time polymerase chain reaction (PCR) assay for its rapid and easy detection on filter-feeding shellfish, such as mussels.
For species-specific determination, the intergenic spacer 1 (ITS1), 5.8S subunit, ITS2 and the hypervariable genomic regions D1–D5 of the large ribosomal subunit of local strains were sequenced and compared with two data sets of other Alexandrium sequences. Species-specific primers were used to amplify signature sequences within the genomic DNA of the studied species by conventional and real-time PCR.
Principal results
Phylogenetic analysis determined that the Chilean strain falls into Group I of the tamarensis complex. Our results support the allocation of the Chilean Alexandrium species as a toxic Alexandrium tamarense rather than A. catenella, as currently defined. Once local species were determined to belong to Group I of the tamarensis complex, a highly sensitive and accurate real-time PCR procedure was developed to detect dinoflagellate presence in Mytilus spp. (Bivalvia) samples after being fed (challenged) in vitro with the Chilean Alexandrium strain. The results show that real-time PCR is useful to detect Alexandrium intake in filter-feeding molluscs.
It has been shown that the classification of local Alexandrium using morphological evidence is not very accurate. Molecular methods enabled the HAB dinoflagellate species of the Chilean coast to be assigned as A. tamarense rather than A. catenella. Real-time PCR analysis based on A. tamarense primers allowed the detection of dinoflagellate DNA in Mytilus spp. samples exposed to this alga. Through the specific assignment of dinoflagellate species involved in HABs, more reliable preventive policies can be implemented.
PMCID: PMC3526335  PMID: 23259043
15.  Characterization and Physical Mapping of Ribosomal RNA Gene Families in Plantago 
Annals of Botany  2006;97(4):541-548.
• Background and Aims The organization of rRNA genes in cultivated Plantago ovata Forsk. and several of its wild allies was analysed to gain insight into the phylogenetic relationships of these species in the genus which includes some 200 species.
• Methods Specific primers were designed to amplify the internal transcribed spacer (ITS1 and ITS2) regions from seven Plantago species and the resulting fragments were cloned and sequenced. Similarly, using specific primers, the 5S rRNA genes from these species were amplified and subsequently cloned. Fluorescence in-situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal RNA genes.
• Results The ITS1 region is 19–29 bp longer than the ITS2 in different Plantago species. The 5S rRNA gene-repeating unit varies in length from 289 to 581 bp. Coding regions are highly conserved across species, but the non-transcribed spacers (NTS) do not match any database sequences. The clone from the cultivated species P. ovata was used for physical mapping of these genes by FISH. Four species have one FISH site while three have two FISH sites. In P. lanceolata and P. rhodosperma, the 5S and 45S (18S-5·8S-25S) sites are coupled.
• Conclusions Characterization of 5S and 45S rRNA genes has indicated a possible origin of P. ovata, the only cultivated species of the genus and also the only species with x = 4, from a species belonging to subgenus Psyllium. Based on the studies reported here, P. ovata is closest to P. arenaria, although on the basis of other data the two species have been placed in different subgenera. FISH mapping can be used as an efficient tool to help determine phylogenetic relationships in the genus Plantago and show the interrelationship between P. lanceolata and P. lagopus.
PMCID: PMC2803661  PMID: 16481363
Plantago; Psyllium; rRNA genes; internal transcribed spacers; FISH mapping; phylogeny
16.  Cryptic species within Anopheles longipalpis from southern Africa and phylogenetic comparison with members of the An. funestus group 
House-resting Anopheles mosquitoes are targeted for vector control interventions; however, without proper species identification, the importance of these Anopheles to malaria transmission is unknown. Anopheles longipalpis, a non-vector species, has been found in significant numbers resting indoors in houses in southern Zambia, potentially impacting on the utilization of scarce resources for vector control. The identification of An. longipalpis is currently based on classical morphology using minor characteristics in the adult stage and major ones in the larval stage. The close similarity to the major malaria vector An. funestus led to investigations into the development of a molecular assay for identification of An. longipalpis. Molecular analysis of An. longipalpis from South Africa and Zambia revealed marked differences in size and nucleotide sequence in the second internal transcribed spacer (ITS2) region of ribosomal DNA between these two populations, leading to the conclusion that more than one species was being analysed. Phylogenetic analysis showed the Zambian samples aligned with An. funestus, An. vaneedeni and An. parensis, whereas the South African sample aligned with An. leesoni, a species that is considered to be more closely related to the Asian An. minimus subgroup than to the African An. funestus subgroup. Species-specific primers were designed to be used in a multiplex PCR assay to distinguish between these two cryptic species and members of the An. funestus subgroup for which there is already a multiplex PCR assay.
PMCID: PMC4118299  PMID: 18715522
Anopheles longipalpis; Anopheles funestus; South Africa; Zambia; internal transcribed spacer (ITS2); PCR; phylogenetics
17.  Intraspecific diversity in Sinningia speciosa (Gesneriaceae: Sinningieae), and possible origins of the cultivated florist's gloxinia 
AoB Plants  2012;2012:pls039.
This research examines the relationships between 24 accessions (17 wild, 7 cultivars) of Sinningia speciosa, the florist’s gloxinia. Phenetic and phylogenetic methods together suggest that distinct geographic lineages exist within this taxon, which will be important for future conservation efforts.
Background and aims
The florist's gloxinia is a familiar houseplant in the Gesneriaceae, the botanical family that includes the African violet (Saintpaulia) and other ornamental species. The gloxinia's wild progenitor is Sinningia speciosa (Lodd.) Hiern, a Brazilian endemic. Although it has been cultivated for almost 200 years, little is known about the genetic diversity in S. speciosa, how the wild populations relate to one another or even where the cultivated forms originated. Using available wild collections, preliminary phenetic and phylogenetic investigations were conducted to elucidate the interspecific relationships within S. speciosa and to infer the origins of the cultivars.
Amplified fragment length polymorphism (AFLP) analysis was applied to 24 accessions of S. speciosa (17 wild collections, seven cultivars) and one accession each of Sinningia guttata and Sinningia macrophylla. A maximum likelihood (ML) tree was also calculated from an alignment of the nuclear ribosomal internal transcribed spacer sequence from the same 26 accessions.
Principal results
Dice/UPGMA and principal coordinates analysis of the AFLP data partitioned S. speciosa into several distinct clusters, one of which included S. macrophylla. All cultivated ‘gloxinias’ grouped together in a major cluster with plants from Rio de Janeiro. The AFLP results were compared with a phylogenetic analysis of the ribosomal spacer region, which was informative in S. speciosa. The ML tree generally supported the AFLP results, although several clades lacked strong statistical support.
Independent analyses of two different data sets show that S. speciosa is a diverse species comprised of several lineages. Genetic distance estimates calculated from the AFLP data were positively correlated with geographic distances between populations, indicating that reproductive isolation could be driving speciation in this taxon. Molecular markers are under development for population genetic studies in S. speciosa, which will make it possible to define evolutionarily significant units for purposes of conservation.
PMCID: PMC3676262  PMID: 23755350
18.  Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA 
Journal of Nematology  1994;26(4):412-421.
The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other.
PMCID: PMC2619530  PMID: 19279910
Aphelenchoides; Ditylenchus; fungi; nematode; PCR; rDNA
19.  Molecular characterization and identification of members of the Anopheles subpictus complex in Sri Lanka 
Malaria Journal  2013;12:304.
Anopheles subpictus sensu lato is a major malaria vector in South and Southeast Asia. Based initially on polytene chromosome inversion polymorphism, and subsequently on morphological characterization, four sibling species A-D were reported from India. The present study uses molecular methods to further characterize and identify sibling species in Sri Lanka.
Mosquitoes from Sri Lanka were morphologically identified to species and sequenced for the ribosomal internal transcribed spacer-2 (ITS2) and the mitochondrial cytochrome c oxidase subunit-I (COI) genes. These sequences, together with others from GenBank, were used to construct phylogenetic trees and parsimony haplotype networks and to test for genetic population structure.
Both ITS2 and COI sequences revealed two divergent clades indicating that the Subpictus complex in Sri Lanka is composed of two genetically distinct species that correspond to species A and species B from India. Phylogenetic analysis showed that species A and species B do not form a monophyletic clade but instead share genetic similarity with Anopheles vagus and Anopheles sundaicus s.l., respectively. An allele specific identification method based on ITS2 variation was developed for the reliable identification of species A and B in Sri Lanka.
Further multidisciplinary studies are needed to establish the species status of all chromosomal forms in the Subpictus complex. This study emphasizes the difficulties in using morphological characters for species identification in An. subpictus s.l. in Sri Lanka and demonstrates the utility of an allele specific identification method that can be used to characterize the differential bio-ecological traits of species A and B in Sri Lanka.
PMCID: PMC3766661  PMID: 24001126
Anopheles subpictus; Anopheles sundaicus; Cytochrome c oxidase subunit-I; ITS2; Malaria; Sibling species; Sri Lanka
20.  Genetic variation of Hoplolaimus columbus populations in the United States using PCR-RFLP analysis of nuclear rDNA ITS regions1 
Journal of Nematology  2009;41(3):187-193.
Hoplolaimus columbus is an important nematode pest which causes economic loss of crops including corn, cotton, and soybean in the Southeastern United States. DNA sequences of the ITS1-5.8S-ITS2 region of ribosomal DNA from H. columbus were aligned and analyzed to characterize intraspecific genetic variation between eleven populations collected from Georgia, Louisiana, North Carolina, and South Carolina. In comparative sequence analysis with clones from either one or two individuals obtained from the eleven populations, we found variability existed among clones from an individual and that clonal diversity observed from within individuals was verified by PCR-RFLP. PCR-RFLP analysis with Rsa I and Msp I restriction enzymes yielded several fragments on 3.0% agarose gel that corresponded to different haplotypes in all populations and the sum of digested products exceeded the length of undigested PCR products, which revealed that ITS heterogeneity existed in a genome of H. columbus. This indicates that heterogeneity may play a role in the evolution of this parthenogenetic species.
PMCID: PMC3380495  PMID: 22736813
ITS region; PCR-RFLP; haplotypes; Hoplolaimus columbus; lance; nematode
21.  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
22.  Phylogeny of Sinojackia (Styracaceae) Based on DNA Sequence and Microsatellite Data: Implications for Taxonomy and Conservation 
Annals of Botany  2008;101(5):651-659.
Background and Aims
The genus Sinojackia consists of eight species, all endemic to China. All species of Sinojackia are endangered or threatened owing to poor recruitment within populations. Information on molecular phylogenetics is critical for developing successful conservation strategies for this genus.
Combined DNA sequence data from the nuclear ribosomal internal transcribed spacer regions and plastid psbA–trnH intergenic spacer and microsatellite data were used to infer a phylogeny of the genus.
Key Results
Parsimony analysis of the combined sequence data and multivariate analysis based on fruit characters indicated that Sinojackia dolichocarpa is monophyletic and genetically well separated from the other Sinojackia species, thus supporting its rank at the generic level as Changiostyrax. Phylogenetic relationships within Sinojackia sensu stricto are unresolved from the combined sequence data. A UPGMA dendrogram based on seven microsatellite loci of 96 individual plants yielded a first-diverging cluster of all individuals of S. microcarpa. The remaining species form another cluster without any definitive patterns corresponding to current species circumscriptions, suggesting either extensive hybridization or incipient speciation.
The results suggest that there are too many species recognized within Sinojackia sensu stricto, but this must be further assessed with comprehensive morphological and taxonomic revisionary work. The implications of the phylogenetic data for conservation are discussed.
PMCID: PMC2710174  PMID: 18245106
Changiostyrax; conservation; phylogeny; Sinojackia; Styracaceae
23.  Susceptibility of Japanese Holly to Criconemoides xenoplax, Tylenchorhynchus claytoni, and Certain Other Plant-Parasitic Nematodes 
Journal of Nematology  1976;8(1):26-31.
Three cultivars of Ilex crenata: 'Helleri', 'Convexa', and 'Rotundifolia' were inoculated with either Criconemoides xenoplax, Helicotylenchus dihystera, Hoplolaimus galeatus, Trichodorus cltristiei, or Tylenchorhynchus claytoni at 0, 200 (low), or 2,000 (high) nematodes/ 15-cm diam pot. Plants were kept in the greenhouse 10 mo prior to transplanting into 2.25 m² field plots. Helleri was severely stunted by C. xenoplax. Criconemoides xenoplax and T. claytoni caused lower plant vigor and top weights of Rotundifolia after 3 years. Above-ground symptoms included stunting, chlorosis, and leaf drop. Convexa was not susceptible to the nematodes tested. Low and high initial populations of the five nematodes tended to reach equilibria over the 3-year sampling period. Helicotylenchus dihystera and C. xenoplax occurred in the greatest densities regardless of host. None of the test plants were damaged by H. dihystera. Convexa was the least suitable host for nematode reproduction. Hoplolaimus galeatus, which was originally isolated from cotton, failed to reproduce or survive on any plant tested. Nematode densities over the 3-year sampling period did not always lit a linear regression model.
PMCID: PMC2620155  PMID: 19308191
24.  Species Identification and Strain Differentiation of Dermatophyte Fungi by Analysis of Ribosomal-DNA Intergenic Spacer Regions 
Journal of Clinical Microbiology  1999;37(4):931-936.
Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of T. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease MvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.
PMCID: PMC88627  PMID: 10074504
25.  Use of sequence motifs as barcodes and secondary structures of Internal Transcribed spacer 2 (ITS2, rDNA) for identification of the Indian liver fluke, Fasciola (Trematoda: Fasciolidae) 
Bioinformation  2009;3(7):314-320.
Most phylogenetic studies using current methods have focused on primary DNA sequence information. However, RNA secondary structures are particularly useful in systematics because they include characteristics that give “morphological” information which is not found in the primary sequence. Also DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat are useful for identification of trematodes. The species of liver flukes of the genus Fasciola (Platyhelminthes: Digenea: Fasciolidae) are obligate parasitic trematodes residing in the large biliary ducts of herbivorous mammals. While Fasciola hepatica has a cosmopolitan distribution, the other major species, i.e., F. gigantica is reportedly prevalent in the tropical and subtropical regions of Africa and Asia. To determine the Fasciola sp. of Assam (India) origin based on rDNA molecular data, ribosomal ITS2 region was sequenced (EF027103) and analysed. NCBI databases were used for sequence homology analysis and the phylogenetic trees were constructed based upon the ITS2 using MEGA and a Bayesian analysis of the combined data. The latter approach allowed us to include both primary sequence and RNA molecular morphometrics and revealed a close relationship with isolates of F. gigantica from China, Indonesia and Japan, the isolate from China with significant bootstrap values being the closest. ITS2 sequence motifs allowed an accurate in silico distinction of liver flukes. The data indicate that ITS2 motifs (≤ 50 bp in size) can be considered promising tool for trematode species identification. Using the novel approach of molecular morphometrics that is based on ITS2 secondary structure homologies, phylogenetic relationships of the various isolates of fasciolid species have been discussed.
PMCID: PMC2655052  PMID: 19294000
Fasciola hepatica; Fasciola gigantica; secondary structure; internal transcribed spacer; bar coding; motifs

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